New Growth Spurt by inhibiting DHEA?

DHEA suppresses longitudinal bone growth by acting directly at growth plate through estrogen receptors.

"Dehydroepiandrosterone (DHEA) is produced by the adrenal cortex and is the most abundant steroid in humans. Although in some physiological and pathological conditions the increased secretion of DHEA and its sulfated form is associated with accelerated growth rate and skeletal maturation[note increased adult height is not stated]. In our study, DHEA suppressed metatarsal growth, growth plate chondrocyte proliferation, and hypertrophy/differentiation. In addition, DHEA increased the number of apoptotic chondrocytes in the growth plate. In cultured chondrocytes, DHEA reduced chondrocyte proliferation and induced apoptosis. The DHEA-induced inhibition of metatarsal growth and growth plate chondrocyte proliferation and hypertrophy/differentiation was nullified by culturing metatarsals with DHEA in the presence of ICI 182,780, an inhibitor of estrogen receptor, but not in the presence of Casodex, an inhibitor of androgen receptor. Lastly, nuclear factor-κB DNA binding activity was inhibited by the addition of DHEA in the medium of cultured chondrocyte. Our findings indicate that DHEA suppressed bone growth by acting directly at growth plate through estrogen receptor. Such growth inhibition is mediated by decreased chondrocyte proliferation and hypertrophy/differentiation and by increased chondrocyte apoptosis."

So reducing DHEA to a certain point likely increases growth.  Although likely some levels of DHEA are needed.

"The secretion of DHEA and DHEA-S by the adrenals increases during the adrenarche in children at the age of 6–8 yr, and elevated values of circulating DHEA-S and DHEA are maintained throughout adult life, providing high levels of substrates for conversion into potent androgens and estrogens in peripheral tissues "

"The addition of letrozole did not change the inhibition of DHEA on longitudinal bone growth, indicating that such suppression induced by DHEA was not through its conversion to estrogen but through itself"

Orteronel (TAK-700) according to Orteronel (TAK-700), a novel non-steroidal 17,20-lyase inhibitor: effects on steroid synthesis in human and monkey adrenal cells and serum steroid levels in cynomolgus monkeys., suppresses DHEA but it suppresses testosterone and estrogen as well.

According to Correlation of circulating dehydroepiandrosterone with activated protein C generation and carotid intima-media thickness in male patients with Type 2 diabetes., DHEA levels are lower in people with diabetes and that DHEA levels are correlated with c-protein inhibitor complex.  So inhibiting this complex may be a way to lower DHEA levels.

According to ACE inhibitor use was associated with lower serum dehydroepiandrosterone concentrations in older men., ACE inhibitor's may be an effective way to lower DHEA.  Unfortunately, ACE inhibitors like perendopril are only available by prescription.

Cheese which contains Lactobacillus helveticus may inhibit ACE and thus reduce DHEA.  American Swiss cheese usually contains this probiotic.  Fermented milk may have lactic acid bacteria that also reduce ACE.  So another possibility for how the dutch diet increases height.

Grow Taller with Tetramethylpyrazine?

Rhizoma Chuanxiong comes from the plant Ligusticum chinensis.Chuan Xiong,-MW4801sf- unsulfured herb Ligusticum chuanxiong rhizome Ligusticum / Szechuan lovage rhizome<- The herb is available for sale at places like Amazon.

Tetramethylpyrazine (TMP) promotes chondrocyte proliferation via pushing the progression of cell cycle

"Tetramethylpyrazine (TMP) is the major bioactive constituent of Rhizoma Chuanxiong which has long been used as an important component in several Chinese medicine formulations. We investigated the cellular effects of TMP in cultured primary chondrocytes. Chondrocytes isolated from the knee articular cartilage of SD rats were cultured and identified using toluidine blue staining. The second generation of chondrocytes was treated with or without TMP. TMP treatment could promote chondrocyte proliferation via pushing the progression of cell cycle.  the mRNA and protein levels of Cyclin D1 and CDK4 were significantly enhanced after TMP treatment[this could apply to MSCs as well and to growth plate chondrocytes, it depends whether TMP enhances Cyclin D1 via Beta-Catenin to how effective it is though], whereas those of p21 were significantly decreased."

"G1/S transition, which is one of the two main checkpoints, is a rate-limiting step in the cell cycle and regulates the cell proliferation. G1/S progression is highly regulated by Cyclin D1 and Cyclin-dependent kinase 4 (CDK4).  Cyclin D1, which is the first cyclin produced in the cell cycle, binds to existing CDK4, forming the active Cyclin D1-CDK4 complex, which in turn phosphorylates the retinoblastoma susceptibility protein (Rb). The phosphorylated Rb dissociates from the E2F/DP1/Rb complex, activating E2F. Activation of E2F{a couple E2F forms were downregulated by LSJL} results in transcription of various genes, such as Cyclin E and Cyclin A, regulating other cell cycle phases. p21 is a inhibitory protein of cell cycle, which prevents cell cycle progression by binding to and inactivating, Cyclin D1-CDK4 complex[so inhibiting p21 is another potential method to grow taller and TMP inhibits p21]."

TMP should increase chondrocyte hypertrophy as well and thus also adult height but since they were studying osteoarthritis they were likely not interested in this effect.

The Study Effects of tetramethylpyrazine on nitric oxide/cGMP signaling after cerebral vasospasm in rabbits. suggests that the effects of TMP involve NO/cGMP which would mean the effects of TMP are partially Beta-Catenin dependent.  And too much Beta-Catenin can result in apoptosis.  However the decrease of p21 may be independent of Beta-Catenin.

However in the study The protective effect of tetramethylpyrazine on cartilage explants and chondrocytes., no apoptotic effects were found on chondrocytes at 200 microM of TMP.

In the study Alleviation of CCl4-induced cirrhosis in rats by tetramethylpyrazine is associated with downregulation of leptin and TGF-beta1 pathway., TMP was found to downregulate the TGF-Beta1 and leptin pathways.  TGF-Beta1 increases Sox9 thus decreasing Beta-Catenin.  Thus TMP may upregulate Beta-Catenin in this and this increases Cyclin D1.  Leptin stimulates MAPK pathway.  MAPK pathway antagonizes the formulation of the Beta-Catenin/LEF complex.  Since TMP downregulates the Leptin pathway, TMP increases the formation of the Beta-Catenin/LEF complex thus increasing Cyclin D1 levels.

Thus, the effects on Cyclin D1 by TMP are likely mediated by Beta-Catenin which makes it a less promising treatment than Beta-Catenin independent methods.

According to the study Wnt/β-catenin signaling induces the aging of mesenchymal stem cells through the DNA damage response and the p53/p21 pathway., Beta-Catenin signaling increases p21 levels that means that effects of TMP on p21 are likely Beta-Catenin independent so it may be worth taking a supplement that contains TMP for that effect.

So it's worth taking TMP to decrease p21 levels independent of Beta-Catenin signaling.

TMP decreases TGF-Beta1 and Leptin pathways.  Thus you would want to compensate by stimulating those pathways you can take Leptin and increase TGF-Beta1 via LSJL and other supplements. TMP also stimulates the NO pathway which stimulates cGMP which decreases FGFR3 which inhibits cyclin D1 expression.  So in this way TMP increases Cyclin D1 independently of Beta-Catenin.

So TMP is a worthwhile supplement but you'll want to take Leptin & TGF-Beta1 supplements to keep Beta-Catenin in check. 

Tetramethylpyrazine: A review on its mechanisms and functions

"TMP has many physiological functions, including anti-oxidant, anti-inflammatory, and anti-apoptosis properties; autophagy regulation; vasodilation; angiogenesis regulation; mitochondrial damage suppression; endothelial protection; reduction of proliferation and migration of vascular smooth muscle cells; and neuroprotection"

Couldn't find any specific anti-bone effects.

EIF2alpha can help with height increase

Hiroki Yokota and Ping Zhang are focused highly on Salubrinal.  Salubrinal increases EIF2alpha phosphorylation.  Joint Loading reduces EIF2alpha phosphorylation. One of the preventative mechanisms of Salubrinal is reduced apoptosis.  Beta-Catenin increases Cyclin D1 levels and when Beta-Catenin degradation was inhibited, thus increasing levels of Beta Catenin to the extreme, apoptosis occurs.  Cyclin D1 affects the cell cycle and if a cell does not exit a cell cycle at a certain point apoptosis may occur.  In a situation like wound healing where there are many growth factors at play cyclin D1 may be high in a number of cells, salubrinal may lower these levels sufficiently such that they don't apoptose.

PKR and PKR-like endoplasmic reticulum kinase induce the proteasome-dependent degradation of cyclin D1 via a mechanism requiring eukaryotic initiation factor 2alpha phosphorylation.

"Cyclin D1 plays a critical role in controlling the G(1)/S transition via the regulation of cyclin-dependent kinase activity. Cyclin D1 translation [may be] decreased upon activation of the eukaryotic initiation factor 2alpha (eIF2alpha) kinases. We examined the effect of activation of the eIF2alpha kinases PKR and PKR-like endoplasmic reticulum kinase (PERK) on cyclin D1 protein levels and translation and determined that cyclin D1 protein levels decrease upon the induction of PKR and PERK catalytic activity but that this decrease is not due to translation. Inhibition of the 26 S proteasome with MG132 rescued cyclin D1 protein levels, indicating that rather than inhibiting translation, PKR and PERK act to increase cyclin D1 degradation[so inhibit PKR and PEKR to grow taller]. Interestingly, this effect still requires eIF2alpha phosphorylation at serine 51, as cyclin D1 remains unaffected in cells containing a non-phosphorylatable form of the protein[so if eIF2alpha is not phosphorylated PKR and PERK cannot degrade cyclin D1]. This proteasome-dependent degradation of cyclin D1 requires an intact ubiquitination pathway, although the ubiquitination of cyclin D1 is not itself affected. Furthermore, this degradation is independent of phosphorylation of cyclin D1 at threonine 286, which is mediated by the glycogen synthase kinase 3beta and mitogen-activated protein kinase pathways. Functional cross-talk [occurs] between eIF2alpha phosphorylation and the proteasomal degradation of cyclin D1 and this degradation is dependent upon eIF2alpha phosphorylation during short, but not prolonged, periods of stress."

"By working in conjunction with the cyclin E-cyclin-dependent kinase 2 (CDK2) complex, cyclin D1-CDK complexes phosphorylate and inactivate the cell-cycle inhibitory function of the retinoblastoma protein. Via this inhibition, cyclin D1 can release the repression of E2F transcriptional activity by retinoblastoma protein and the associated histone deacetylases and facilitate the transcription of genes active during the S phase of the cell cycle."

"Transcription of the cyclin D1 gene is increased upon many different types of stimuli, including insulin-like growth factor-1 (IGF-1) and IGF-II, amino acids, androgens, and retinoic acid" "Cyclin D1 gene expression is induced by many oncogenic signaling pathways, including Ras, Src, Her2/neu, β-catenin, and members of the signal transducer and activator of transcription (STAT) family"

"A decrease in cyclin D1 levels [is] coincident with increased eIF2α phosphorylation"

The beneficial effects of salubrinal on height on development depend on the current levels of Cyclin D1 translation versus equilibrium.  If a stimulus like say LSJL increases Cyclin D1 above equilibrium in existing growth plates(but has other pro-chondrogenic effects that increase height) then salubrinal will add to height growth by keeping Cyclin D1 under equilibrium.  If translation of Cyclin D1 is below equilibrium then Salubrinal will reduce height.

The study Overexpression of cyclin D1 promotes tumor cell growth and confers resistance to cisplatin-mediated apoptosis in an elastase-myc transgene-expressing pancreatic tumor cell line. suggests that overexpression of Cyclin D1 confers apoptosis resistance.  Although it was overexpression of Beta-Catenin that caused apoptosis and growth plate closure and not Cyclin D1 directly.  Maybe the apoptosis and growth plate closure was due to another effect of Beta Catenin other than Cyclin D1 stimulation. Also, it's possible that the effects of Cyclin D1 are different for chondrocytes than other cell types.

This study states that overexpression of Beta Catenin increases apoptosis independently of Cyclin D1:

Overexpression of beta-catenin induces apoptosis independent of its transactivation function with LEF-1 or the involvement of major G1 cell cycle regulators.

"beta-Catenin promotes epithelial architecture by forming cell surface complexes with E-cadherin and also interacts with TCF/LEF-1 in the nucleus to control gene expression. By DNA transfection, we overexpressed beta-catenin and/or LEF-1 in NIH 3T3 fibroblasts, corneal fibroblasts, corneal epithelia, uveal melanoma cells, and several carcinoma cell lines. In all cases (with or without LEF-1), the abundant exogenous beta-catenin localizes to the nucleus and forms distinct nuclear aggregates that are not associated with DNA. Surprisingly, we found that with time (5-8 d after transfection) cells overexpressing beta-catenin all undergo apoptosis. LEF-1 does not need to be present. Moreover, LEF-1 overexpression in the absence of exogenous beta-catenin does not induce apoptosis, even though some endogenous beta-catenin moves with the exogenous LEF-1 into the nucleus.  Full-length beta-catenin is able to induce LEF-1-dependent transactivation, whereas Arm beta-catenin totally abolishes the transactivating function. However, Arm beta-catenin, containing deletions of known LEF-1-transactivating domains, has the same apoptotic effects as full-length beta-catenin. Overexpressed beta-catenin also induces apoptosis in cells transfected with nuclear localization signal-deleted LEF-1 that localizes only in the cytoplasm. Thus, the apoptotic effects of overexpressed exogenous beta-catenin do not rely on its transactivating function with nuclear LEF-1. Overexpressed delta-catenin, containing 10 Arm repeats, induces only minor apoptosis, suggesting that the major apoptotic effect may be due to domains specific to beta-catenin as well as to Arm repeats. The absence of p53, Rb, cyclin D1, or E2F1 does not affect the apoptotic effect of overexpressed beta-catenin, but Bcl-x(L) reduces it. In vivo apoptosis of cells overexpressing beta-catenin might be a physiological mechanism to eliminate them from the population."

So the goal would be to increase cyclin D1 independent of Beta-Catenin. Beta-Catenin may increase apoptosis by it's activation of PS1.

The effect of Salubrinal on apoptosis must be independent of EIF2alpha and CHOP which salubrinal increases and which increases apoptosis.

Mechanical stimulation suppresses phosphorylation of eIF2alpha and PERK-mediated responses to stress to the endoplasmic reticulum.

"Mechanical stimulation was applied to mouse ulnae, MC3T3 cells, and mesenchymal stem cells. Mechanical stimulation reduces phosphorylation of eIF2alpha through inactivation of Perk. Furthermore, flow pre-treatment reduces thapsigargin-induced cell mortality through suppression of phosphorylation of Perk. However, H(2)O(2)-driven cell mortality, which is not mediated by Perk, is not suppressed by mechanical stimulation."

"First, the loaded ulnae exhibited a lower level of eIF2α-p than the contralateral counterpart with no alteration in the level of eIF2α. Second, in response to flow treatment the level of eIF2α–p decreased in both MSCs and MC3T3 cells."<-the contralateral length increase of limbs to LSJL does not involve alterations in EIF2a-p and this provides further evidence that LSJL provides a fluid flow stimulus to MSCs.

"thapsigargin, a pharmacological inducer of stress to the ER, alters expression of transcription factors such as ATF4, Runx2, and Osterix in MC3T3 osteoblast-like cells"

Fluid flow increased ATF3 levels but did not alter ATF4 levels.  Flow increased ATF6.

"Using C57/BL6 mice (female, ∼12 weeks; Harlan Sprague-Dawley, Inc.), elbow loading was conducted[LSJL]. In brief, the mouse was anesthetized and loads were applied to the left elbow for 3 min in the lateral-medial direction with 0.5 N force at 5 Hz. The right forelimb was used as a contralateral control. The pairs of ulnae were harvested at 1, 3, and 5 h after loading."

"mechanical stimulation down-regulated the protein levels of eIF2α-p and CHOP"

Conclusion: Increase Cyclin D1 as much as possible independent of Beta-Catenin(Sox 9 can keep Beta-Catenin in check). EIF2alpha increases Cyclin D1 independent of Beta-Catenin by reducing Cyclin D1 degradation. Reduce EIF2alpha phosphorylation by joint loading.

Regulation of eIF2α phosphorylation in hindlimb-unloaded and STS-135 space-flown mice

"Various environmental stresses elevate the phosphorylation level of eukaryotic translation initiation factor 2 alpha (eIF2α) and induce transcriptional activation of a set of stress responsive genes such as activating transcription factors 3 and 6 (ATF3 and ATF6) {LSJL induces ATF3}, CCAAT/enhancer-binding protein homologous protein (CHOP), and Xbp1 (X-box binding protein 1){downregulated by LSJL}. These stress sources include radiation, oxidation, and stress to the endoplasmic reticulum, and it is recently reported that unloading by hindlimb unloading is such a stress source. No studies, however, have examined the phosphorylation level of eIF2α (eIF2α-p) using skeletal samples that have experienced microgravity in space. In this study we addressed a question: Does a mouse tibia flown in space show altered levels of eIF2α-p? To address this question, we obtained STS-135 flown samples that were harvested 4–7 h after landing. The tibia and femur isolated from hindlimb unloaded mice were employed as non-flight controls. The effects of loading were also investigated in non- flight controls. The level of eIF2α-p of the non-flight controls was elevated during hindlimb unloading[however the levels of eif2a-p returned to control values over time naturally at 13 days] and reduced after being released from unloading. Second, the eIF2α-p level of space-flown samples was decreased, and mechanical loading to the tibia caused the reduction of the eIF2α-p level. Third, the mRNA levels of ATF3, ATF6, and CHOP were lowered in space-flown samples as well as in the non-flight samples 4–7 h after being released from unloading. A release from hindlimb unloading and a return to normal weight environment from space provided a suppressive effect to eIF2α-linked stress responses and that a period of 2–4 h is sufficient to induce this suppressive outcome."

"axial loading to the left tibia reduced the level of eIF2α-p compared to the non-loaded right tibia"<-Both axial loading and lateral loading reduce EIF2a-p so that is not likely to play a role in their differential changes in gene expression.

" In the femur, the level was lowered at 1 h (85 ±12% of the control level) after removing the hindlimb unloading"<-after unloading there is an overcompensation in the reduction of eif2a phosphorylation.  This could be due to an increase in mechanosensativity.  The level returned to normal after only 4-8 hours.

"This phosphorylation event [of eIF2alpha] is critically important for stressed cells to make a decision to survival or apoptosis."

"ATF3, ATF6, CHOP, and Xbp1 are activated in response to various environmental stresses, and their activation in general coincides with the elevation of eIF2α-p. ATF3 and ATF6 are transcription factors that are involved in growth and development of skeletal tissues, while the genes such as CHOP and Xbp1 are linked to apoptosis. In this study, these genes were downregulated together with the reduction in the eIF2α-p, suggesting that cellular apoptosis is suppressed."<-so since salubrinal enhances eIF2alpha-phosphorylation shouldn't it enhance apoptosis?

Heres a study linking Xbp1s to chondrocyte hypertrophy:

XBP1S Associates with RUNX2 and Regulates Chondrocyte Hypertrophy.

"Bone morphogenetic protein 2 (BMP2) is known to activate unfolded protein response (UPR) signaling molecules, including XBP1S and ATF6{BMP2 activates 2 similar molecules as EIF2alpha-phosphorylation}. BMP2 mediates mild ER stress-activated ATF6 and directly regulates XBP1S splicing in the course of chondrogenesis. XBP1S is differentially expressed during BMP2-stimulated chondrocyte differentiation, and exhibits prominent expression in growth plate chondrocytes. This expression is probably due to the activation of XBP1 gene by ATF6 and splicing by IRE1a. ATF6 directly binds to the 5'-flanking regulatory region of XBP1 gene at its consensus binding elements. Overexpression of XBP1S accelerates chondrocyte hypertrophy, as revealed by enhanced expression of type II Collagen, type X Collagen and Runx2; however, knockdown of XBP1S via the RNA interference(RNAi) approach abolishes hypertrophic chondrocyte differentiation. In addition, XBP1S associates with Runx2 and enhances Runx2-induced chondrocyte hypertrophy. Altered expression of XBP1S in chondrocyte hypertrophy was accompanied by altered levels of Indian hedgehog (IHH) and parathyroid hormone-related peptide (PTHrP). XBP1S may be a novel regulator of hypertrophic chondrocyte differentiation by (1) acting as a cofactor of Runx2 and (2) affecting IHH/PTHrP signaling."

"IgH chain binding protein (BiP), C/EBP homologous protein (CHOP), activating transcription factor 4 (ATF4), and ER degradation enhancing α-mannosidase-like protein (EDEM), were up-regulated by BMP2 stimulation."

"XBP1S protein was not detected until day 5 in BMP2-induced chondrocyte differentiation of ATDC5 cells"<-So XBP1S is likely not chondroinductive.

"XBP1S enhanced the expression of IHH, whereas it clearly suppressed the expression of PTHrP"<-IHH may have chondroinductive properties.

Microarray analysis of thapsigargin-induced stress to the endoplasmic reticulum of mouse osteoblasts.

"Activating transcription factor 4 (ATF4) protein has a dual role in osteoblasts. It functions as a responder to stress to the endoplasmic reticulum (ER) as well as a transcription factor for bone formation.  Based on microarray-derived mRNA expression data for mouse osteoblasts (MC3T3 E1 cells, clone 4), we analyzed the ER-stress responses in the presence of 10 nM Thapsigargin.  A strong linkage to an expression pattern [was] observed in the responses to hypoxia, and two molecular pathways [were identified]: ATF4-unlinked connective tissue development and ATF4-linked organ morphology. Real-time polymerase chain reaction (PCR) and Western blot analyses validated eIF2alpha-driven translational regulation as well as ATF4-linked transcriptional activation of transcription factors and growth factors including FOS, FGF-9, and BMP-2. Consistent with the role of p38 MAPK in hypoxia, phosphorylation of p38 MAPK was activated in nonapoptotic osteoblasts under surviving ER stress.  The level of phosphorylated PERK was elevated. These results support cross-talk between p38 MAPK and ER kinase."

"Thapsigargin is an inhibitor of Ca2+ ATPase in the ER"

Ppp1r15 gene knockout reveals an essential role for translation initiation factor 2 alpha (eIF2alpha) dephosphorylation in mammalian development.

"Diverse cellular stress responses are linked to phosphorylation of serine 51 on the alpha subunit of translation initiation factor 2. The resultant attenuation of protein synthesis and activation of gene expression figure heavily in the adaptive response to stress, but dephosphorylation of eIF2(alphaP), which terminates signaling in this pathway, is less well understood. GADD34 and CReP, the products of the related mammalian genes Ppp1r15a and Ppp1r15b, can recruit phosphatase catalytic subunits of the PPP1 class to eIF2(alphaP).  Unlike Ppp1r15a mutant mice, which are superficially indistinguishable from wild type, Ppp1r15b(-/-) mouse embryos survive gestation but exhibit severe growth retardation and impaired erythropoiesis, and loss of both Ppp1r15 genes leads to early embryonic lethality. These loss-of-function phenotypes are rescued by a mutation, Eif2a(S51A), that prevents regulated phosphorylation of eIF2alpha. eIF2(alphaP) dephosphorylation is the predominant role of PPP1R15 proteins in mammalian development."

So LSJL's role of EIF2a dephosphorylation may help with growth but axial loading may do the same thing.

"GADD34 (PPP1R15a) and CReP (PPP1R15b), share the ability to associate with the catalytic phosphatase PPP1 subunit and repress eIF2α phosphorylation when overexpressed"

But " inadequate signaling by eIF2(αP)—exemplified by the deficiency in ATF4, a translationally induced target of eIF2α phosphorylation" can be bad as well.

" eIF2(αP) must be regulated within a narrow range for normal fetal erythropoiesis"

This study could provide a clue as to whether axial loading induces an increase in EIF2a dephosphorylation:

Cyclic mechanical load causes global translational arrest in articular chondrocytes: a process which is partially dependent upon PKR phosphorylation.

"[Porcine articular cartilage explants were] cyclically (0.5 Hz, 0.1 Hz and 0.05 Hz) and statically loaded. Messenger RNA was extracted and newly synthesised proteins were measured by their incorporation of radiolabelled 35S[methionine/cysteine] or 35SO4. Some medium from loaded and unloaded explants was immunoblotted for type II collagen, CTGF and TIMP3. The pathways that control protein translation were investigated by immunoblotting explant lysates for PKR, PERK (PKR like endoplasmic reticulum kinase), eIF2a (eukaryotic initiation factor 2a), eEFs (eukaryotic elongation factors), and AMP-dependent kinase. Explants were also loaded in the presence of inhibitors of PKR, the fibroblast growth factor (FGF) receptor and PI3 kinase. Cyclic loading caused complete global translational arrest as evidenced by a total suppression of new protein synthesis whilst maintaining mRNA levels. Translational arrest did not occur following static loading and was partly dependent upon the load frequency. There was a rebound increase in protein synthesis when labelling was performed after load had been withdrawn. Phosphorylation of PKR occurred in explants following cyclic load and inhibition of PKR modestly reversed suppression of newly synthesised proteins suggesting that PKR, at least in part, was responsible for loading induced translational arrest. Translational control provides a rapid and potentially important mechanism for controlling the synthetic responses of articular chondrocytes in response to different types of mechanical load."

"type II collagen, aggrecan, MMP3, tissue inhibitor of metalloproteinase (TIMP)-1, and the house keeping gene GAPDH" were changed non-significantly to mechanical load.  MMP3 and Col2a1 decreased immediately after load.

Loading did not induce phosphorylation of EIF2a.  Loading may have induced slight dephosphorylation of EIF2a but it's hard to tell.  Basal levels of EIF2a-p were very low.

Suppression of osteoclastogenesis through phosphorylation of eukaryotic translation initiation factor 2 alpha.

"Using two chemical agents (salubrinal and guanabenz) that selectively inhibit de-phosphorylation of eIF2α, we evaluated the effects of phosphorylation of eIF2α on osteoclastogenesis of RAW264.7 pre-osteoclasts as well as development of MC3T3 E1 osteoblast-like cells. Salubrinal and guanabenz stimulated matrix deposition of osteoblasts through upregulation of activating transcription factor 4 (ATF4){Since it does this in general the upregulation of ATF4 may have pro-chondrogenic effects}. These agents reduced expression of the nuclear factor of activated T cells c1 (NFATc1) and inhibited differentiation of RAW264.7 cells to multi-nucleated osteoclasts. Partial silencing of eIF2α with RNA interference reduced suppression of salubrinal/guanabenz-driven downregulation of NFATc1. The elevated phosphorylation level of eIF2α not only stimulates osteoblastogenesis but also inhibit osteoclastogenesis through regulation of ATF4 and NFATc1."

"Administration of 5 μM salubrinal to MC3T3 E1-14 cells elevated phosphorylation of eIF2α, followed by an increase in ATF4 expression. Furthermore, the level of osteocalcin mRNA was increased 3.3 ± 0.5 fold (24 h) and 3.3 ± 0.3 fold (32 h) "

"Addition of RANKL increased the mRNA levels of NFATc1, c-Fos{up in LSJL}, TRAP, and OSCAR, and administration of 20 μM salubrinal significantly reduced their mRNA levels. On day 2, for instance, the RANKL-driven increase was 9.4 ± 0.5 fold (NFATc1), 1.9 ± 0.1 fold (c-fos), 165 ± 4.2 fold (TRAP), and 467 ± 22 fold (OSCAR). The reduction by 20 μM salubrinal was 46 % (NFATc1), 32 % (c-fos), 35 % (TRAP), and 21 % (OSCAR)"

"Administration of 20 μM salubrinal did not significantly alter the phosphorylation level of ERK, p38 MAPK, and NFκB"

"Both salubrinal and guanabenz interact with PP1 and inhibit its activity of de-phosphorylating p-eIF2α. "

Growing taller by taking Selenium?

Now Foods Selenium 100mcg, Yeast Free, 250 Tablets.

Selenium promotes proliferation of chondrogenic cell ATDC5 by increment of intracellular ATP content under serum deprivation.

"[Low] Selenium (Se) [can result in] chondronecrosis in growth plate and articular cartilage. Se stimulated ATDC5 cell proliferation under serum deprivation but not routine culture.  Se promoted G1-phase cell cycle progression along with induction of cyclin D1 expression at the mRNA and protein level{Beta-Catenin increases cyclin D1 and Cyclin D1 increases height up to a point.  Cyclin D1 makes cells hypertrophy longer before exiting the cell cycle.  If they take too long they just apoptose.  So Cyclin D1 works a little like a game of chicken.  One key area for future height increase will be determining the optimal level of Cyclin D1 before you get hit by the apoptosis train}. Se increased intracellular ATP content and decreased intracellular superoxide anion concentration without affecting intracellular redox status as estimated by ratio of the reduced and oxidized glutathione. Suppression of intracellular ATP synthesis by glycolysis inhibitor or mitochondrial uncoupler both abrogated Se-mediated cyclin D1 induction{So maybe it's the increase in ATP synthesis that enhances cyclin D1 and not Selenium itself}. Se stimulates proliferation of chondrogenic cell ATDC5 through acceleration of cell cycle progression accompanied with cyclin D1 induction by enhancement of intracellular ATP content."

The question is why Selenium only increased cell proliferation under serum deprivation. A possibility is that Cylin D1 has different effects based on a cells differentiation state. Under a more mature state Cyclin D1 has more an effect on hypertrophy rather than proliferation which could explain the lack of proliferation observed in less serum deprived media. Another explanation is "serum already contains sufficiency trace amounts (about 500 nmol·l–1) of Se"

"supplementation of Se significantly decreased G0/G1 phase cell distribution (59·86 ± 0·76% vs 65·09 ± 0·67% of the control group). In contrast, there were no significant change in both S and G2/M phase cell distribution from the cells supplemented with Se versus control. Se promotes cell cycle progression through acceleration of G0/G1 phase. " "Both mRNA and protein expression of cyclin D1 significantly increased at Se concentrations of 25–250 nmol·l–1 for 12-h incubation (increase 1·2, 1·71, 1·62 and 1·5 fold at protein level at concentration of 25, 50, 100 and 250 nmol·l–1 Se, respectively). "<-Note these are both for serum free media. Note cyclin D1 expression increased maximally at 50 nanomol per L.

Now we now that 50 nanomol per L of Selenium maximizes Cyclin D1 but we don't know what levels of Cyclin D1 maximize height growth.

But it's clear that since Selenium stimulated chondrocyte proliferation at 50 nanomol that a 1.71 fold increase in Cyclin D1 is not sufficient for train derailment. But I have no idea how that translates to human doses.

It's possible that the levels of Cyclin D1 needed for train derailment are very high as in the Beta-Catenin study Beta-Catenin never degraded as it was constitutionally active. I was not able to find other studies that show levels of Cyclin D1 necessary to induce apoptosis.

If you want to help growing taller research then researching Cyclin D1 would be a worthwhile use of your time as Cyclin D1 is involved in a number of height increase pathways. The NO and CNP pathway both increase Cyclin D1. FGFR3 decreases Cyclin D1 via Stat1. Inhibiting GSK3-Beta increases Cyclin D1 levels.

Effects of selenium and iodine deficiency on bone, cartilage growth plate and chondrocyte differentiation in two generations of rats.

"Sprague-Dawley rats were randomly divided into selenium deficiency (-Se+I), iodine deficiency (+Se-I), combined selenium and iodine deficiency (-Se-I), and selenium and iodine sufficient (+Se+I) groups. Growth of bone and cartilage, and the expression of type X collagen (ColX) and parathyroid hormone-related peptide (PTHrP) were measured in two generations of rats (F(0) and F(1)).

The tibial length in -Se-I rats was significantly shorter in F(1) generation. In +Se-I of F(1) rats, the thickness of the growth plate cartilage, and the proliferative zone was smaller, while in -Se-I rats the growth plate, and the proliferative and hypertrophic zones were also thinner in F(1) generation. In articular cartilage, ColX expression was increased in the deep zone in -Se-I rats of F(0) generation, and in -Se+I, +Se-I and -Se-I rats of F(1) generation. PTHrP expression was increased in the middle zone of -Se+I, +Se-I and -Se-I rats of both F(0) and F(1) generations. In the growth plate cartilage, ColX and PTHrP were expressed in the hypertrophic zone. ColX expression was significantly weaker in -Se+I and -Se-I rats in both F(0) and F(1) generations, while PTHrP expression was stronger in -Se+I, +Se-I and -Se-I rats in both F(0) and F(1) animals."

"There were no significant differences between the groups in F0 rats with respect to tibial length, half frontal plane diameter of tibia, and frontal articular cartilage diameters. However, in F1 rats the tibial length was significantly decreased in −Se−I group"<-surprisingly though in F0 +Selenium +Iodine had the shortest tibial length whereas in F1 +Se+I had the longest Tibial length.

"combined selenium and iodine deficiency only caused a moderate hypothyroidism in rats. It decreased T3 and T4 concentration in F0 and T3 concentration in F1 moderately."

"iodine deficiency starting from the embryo period may retard the chondrocyte differentiation in the growth plate cartilage. Iodine is an essential element of T4 which plays an important role in regulating bone growth and chondrocyte differentiation"

[Effects of selenium and/or iodine deficiency on chondrocyte apoptosis in rats].

"Spraguee-Dawley rats were randomly divided into selenium deficiency group, iodine deficiency group, combined selenium and iodine deficiency group, and control group.

In articular cartilage, the positive rate of apoptotic chondrocytes stained by TUNEL in the upper and middle zones in selenium deficiency group, iodine deficiency group, and combined selenium and iodine deficiency group were significantly higher than that in control group. The apoptotic chondrocytes were prominent in the middle zone. The positive percentage of chondrocytes apoptosis was not significantly different among these three groups. Compared with the control group, the expressions of both Bcl-2 and Bax were significantly higher in the upper and middle zone in the selenium deficiency group, iodine deficiency group, and combined selenium and iodine deficiency group; however, the expressions of Bcl-2 and Bax were not significantly different among these three groups."

Structural changes in femoral bone tissue of rats after subchronic peroral exposure to selenium.

"Twenty one-month-old male Wistar rats were randomly divided into two experimental groups. In the first group (Se group) young males were exposed to 5 mg Na2SeO3/L in drinking water, for 90 days. Ten one-month-old males without Se administration served as a control group.
The body weight, femoral length[by about 0.2 cm which was approx. a bone length difference of 5%] and cortical bone thickness were significantly decreased in Se group rats. These rats also displayed different microstructure in the middle part of the femur, both in medial and lateral views, where vascular canals expanded into the central area of the bone while, in control rats, these canals occurred only near the endosteal surfaces. Additionally, a smaller number of primary and secondary osteons was identified in Se group rats. Histomorphometric analyses revealed significant increases for area, perimeter, maximum and minimum diameters of primary osteons' vascular canals but significant reductions for all measured variables of Haversian canals and secondary osteons."

Selenium may result in decreased secretion of growth hormone (GH) and somatomedin C

Selenium effect on selenoprotein transcriptome in chondrocytes.

"Selenium is an essential micronutrient and exerts its biological functions predominantly through selenoproteins. Selenium deficiency is associated with cartilage function. This study demonstrated that all 24 selenoprotein transcripts in mouse genome were detectable in ATDC5 chondrocytes except deiodinase 1 (DIO1), DIO2, and selenoprotein V (Sel V), while all 25 selenoprotein{LSJL downregulates D5Wsu178e which encodes for a selenoprotein} transcripts in human genome were detectable in C28/I2 chondrocytes except glutathione peroxidase 6 (GPx6) and DIO1. In addition, gene expression of five selenoproteins (GPx1, Sel H, Sel N, Sel P, and Sel W) was up-regulated and two selenoproteins (SPS2 and Sel O) was down-regulated by sodium selenite (Se) in both ATDC5 and C28/I2 cells. Gene expression of six selenoproteins (TrxR1, Sel I, Sel M, Sel R, Sel S, Sel T) and one selenoprotein (GPx3) was up-regulated by Se in ATDC5 and C28/I2 cells, respectively. Gene expression of one selenoprotein (TrxR2) was down-regulated by Se only in ATDC5 cells. Further transcription inhibition assay showed that both transcriptional and posttranscriptional mechanisms involved in Se-regulated gene expression of GPx1, TrxR1, TrxR2, SPS2, Sel O, and Sel S. However, Se-regulated gene expression of Sel H, Sel I, Sel M, Sel N, Sel P, Sel R, Sel T, and Sel W mainly at posttranscriptional level. Moreover, new protein synthesis inhibition assay indicated that Se-mediated new protein synthesis also played roles in Se-regulated gene expression of GPx1, TrxR1, TrxR2, Sel H, Sel O, Sel P, Sel R, and Sel W."

LSJL also downregulates Selenium binding related gene Sephs1 and Hrpll.

"In mammals, the incorporation of Sec into selenoproteins occurs via a stop codon UGA and requires recoding by means of a Sec-insertion sequence (SECIS) with a specific selenocysteyl-tRNA (tRNA-Sec) and a number of trans-acting proteins, including the SECIS binding protein 2 (SBP2), the specialized translation elongation factor EFSec, ribosomal protein L30, and others"

" C-28/I2 cells express high levels of matrix-anabolic and matrix-catabolic genes and thus are suitable for investigation of chondrocyte anabolic and catabolic activity"

The key is if any of these seleproteins affect height.

Grow Taller by Inhibiting GSK3Beta?

GSK3Beta is inhibited by phosphorylating it. Lithium inhibits GSK3Beta.  cGKII from the NO or CNP pathway can inhibit GSK3Beta.  Serotonin inhibits GSK3Beta.  GSK3Beta degrades Beta-Catenin.  Insufficient Beta-Catenin results in dwarfism.

Deletion of glycogen synthase kinase-3β in cartilage results in up-regulation of glycogen synthase kinase-3α protein expression.

"Glycogen synthase kinase-3 (GSK-3) is a negative regulator of several signaling pathways that govern bone growth, such as insulin/IGF and Wnt/β-catenin. The two GSK-3 proteins, GSK-3α and GSK-3β, display both overlapping and distinct roles in different tissues. Pharmacological inhibition of GSK-3 signaling in a mouse tibia organ culture system results in enhanced bone growth[i.e. growing taller], accompanied by increased proliferation of growth plate chondrocytes and faster turnover of hypertrophic cartilage to bone. GSK-3 inhibition rescues some effects of phosphatidylinositide 3-kinase inhibition in this system, in agreement with the antagonistic role of these two kinases in response to signals such as IGF. Cartilage-specific deletion of the Gsk3b gene in mice has minimal effects on skeletal growth or development. Compensatory up-regulation of GSK-3α protein levels in cartilage is the likely cause for this lack of effect."

"GSK-3α and GSK-3β [have both overlapping and distinct roles]"

"Tibia treated with [an inhibitor that inhibits both GSK3alpha and Beta] grew 31% more than controls over the 6 d of organ culture"

"Inhibition of GSK-3 promotes long bone longitudinal growth by increasing bone formation."<-But is this only growth rate or adult height?

"GSK-3α, which is more widely expressed in the growth plate, is the predominate GSK-3 form targeted by inhibition of the PI3K pathway through the resting and proliferating zones, whereas GSK-3β is the main target in the prehypertrophic zone."

"The effect of the PI3K inhibitor was partially recovered by the combination with the GSK-3 inhibitor"<-Meaning some of the benefits of the PI3K pathway on height growth are mediated by it's inhibition of GSK-3.

"The cyclin-dependent kinase inhibitor p57 is both a marker of prehypertrophic chondrocytes and promotes cell cycle exit in the growth plate. In tibia organ culture, inhibition of GSK-3 or dual treatments of GSK-3 and PI3K inhibitors greatly increased the zone of p57 protein expression in the prehypertrophic zone"

"up-regulation of GSK-3α compensates for the loss of GSK-3β in cartilage."<-So we have to inhibit GSK-3alpha in addition to GSK-3Beta.

According to Inhibition of GSK3 by lithium, from single molecules to signaling networks., Lithium inhibits both forms of GSK3.  I'm not sure about the NO/CNP pathway or serotonin.  It's very likely that they do inhibit both.

Gain temporary height with spinal traction?

Because sometimes you need a little bit of height now...

Immediate changes in spinal height and pain after aquatic vertical traction in patients with persistent low back symptoms: a crossover clinical trial.

"Ninety-eight subjects were recruited using consecutive sampling, with 28 men and 32 women of a mean ± standard deviation (SD) age of 59.6 ± 11.6 years completing testing.
Each subject participated in 2 sessions that consisted of loaded walking for 15 minutes, followed by either 15 minutes of land-based supine position or 15 minutes of aquatic vertical traction.
Spinal height change, measured using a commercial stadiometer, was determined after completing loaded walking and after each intervention.
The mean ± SD height change of 4.99 ± 2.88 mm after aquatic vertical traction was similar to that of 4.21 ± 2.53 mm after the land-based supine flexion (P = .0969)[Not very significant increase in height but traction was only applied for 15 minutes what really matters is if the maximal spinal height is higher after aquatic vertical traction versus supine flexion]. Paired t-test indicated that both interventions resulted in significant increased height (P < .0001). Decreases in pain after aquatic intervention (2.7 ± 2.1 cm) were significantly greater than decreases after land intervention (1.7 ± 1.7 cm; P = .0034), and centralization of symptoms was more pronounced after aquatic vertical suspension compared with the supine land-based flexion condition (P < .0001). A significant correlation between height change and both pain reduction (r = 0.39; P = .001) and centralization (r = 0.29; P = .013) was observed for the aquatic intervention only.
Although both the aquatic and land interventions produced significant increases in overall spinal height, the aquatic intervention produced greater pain relief and centralization response in subjects with low back pain and signs of nerve root compression."

"Hyperextension sustained at a 20° angle for 20 minutes resulted in the largest height increase when compared with other extension angles and duration"

"Despite the fact that the deep-water runners had no contact with the ground, limited spinal shrinkage still occurred (−2.9 mm). The shrinkage in the deep water was thought to occur because of the muscular forces applied to the lumbar disks from action of the back, hip, and abdominal muscles. Such shrinkage did not occur after the aquatic intervention for any subject in our experiment, which suggests that small motions of the subjects and the addition of ankle weights may have assisted in decreasing muscular activity and allowed the subjects to quietly hang, which resulted in some spinal distraction"

"The results indicated that, without ankle weights, the average change per individual lumbar segment was 0.73-0.87 mm and that the addition of 5 kg to each ankle resulted in an average change in each segment that ranged from 0.84 to 1.41 mm."<-Would more ankle weights produce more results?  Remember you want no muscular activity.

[Acute effects of mechanical lumbar traction with different intensities on stature].<-This study is in portugese.

"Thirty apparently healthy subjects (age: 20.9 ± 1.7 years old), 14 male and 16 female, were submitted to two protocols of 15 minutes of continuous traction (with intensity of 10% and 50% of body weight), in two sessions one week apart. The protocol order was determined randomly. Stature was assessed before and immediately after the traction and every five minutes for 30 minutes after traction ceased.

Immediately after the traction both protocols induced a significant increase in stature, however the magnitude of the increase was significantly superior in the traction with 50% of body weight (0.567 ± 0.049 vs. 0.298 ± 0.041 cm, p>0.001)[maybe traction with higher percentage of body weight would be more significant, 0.5 cm is about 1/4 of an inch]. After the traction with 50% of body weight the increase in the stature was maintained until ten minutes after the end of the traction, while after the traction with 10% of body weight the effects on stature disappeared after five minutes.

The mechanical vertebral traction of the lumbar region performed continuously with 50% of body weight during 15 minutes induced an increase in stature superior and longer in time than that observed with a traction force of 10% of body weight."

50% body weight traction resulted in the greatest increase in height amongst all the studies.  Perhaps greater traction would be more beneficial.  The subjects were younger in the second study which could have made a difference. This device applies up to 200lbs of traction: Saunders Lumbar HomeTrac - Deluxe.  So up to 100% of body weight for most people.  And up to 50% of bodyweight up to extremes of weight.  So this device may give you a 1/4" when applied for 15 minutes for up to ten minutes afterwards.  Different amounts of traction may provide different results.

This is actually a study that can be performed by non-scientists.  Do 10% and 50% of bodyweight to compare your height gain to the study to establish a baseline.  Then do 75%, see how much height you gain after 15 minutes and see how long the height lasts.

Grow Taller with Testosterone

Testosterone inhibits myostatin and myostatin inhibits cell differentiation which can inhibit chondrogenesis.  Thus, testosterone definitely can lead to height increase that way but are there any other ways testosterone can increase height?

Testosterone and the male skeleton: a dual mode of action.

"Testosterone is an important hormone for both bone gain and maintenance in men. Hypogonadal men have accelerated bone turnover and increased fracture risk. In these men, administration of testosterone inhibits bone resorption and maintains bone mass. Testosterone, however, is converted into estradiol via aromatization in many tissues including male bone. The importance of estrogen receptor alpha activation as well of aromatization of androgens into estrogens was highlighted by a number of cases of men suffering from an inactivating mutation in the estrogen receptor alpha or in the aromatase enzyme. All these men typically had low bone mass, high bone turnover and open epiphyses{open epiphyses doesn't always mean tall stature it just means failure to complete endochondral ossification}. Estradiol contributes to the maintenance of bone mass after reaching peak bone mass, with an association between estradiol and fractures in elderly men. Estrogen receptor activation, but not androgen receptor activation, is involved in the regulation of male longitudinal appendicular skeletal growth in mice. Both the androgen and the estrogen receptor can independently mediate the cancellous bone-sparing effects of sex steroids in male mice. Selective KO studies of the androgen receptor in osteoblasts in male mice suggest that the osteoblast in the target cell for androgen receptor mediated maintenance of trabecular bone volume and coordination of bone matrix synthesis and mineralization. Testosterone has a dual mode of action on different bone surfaces with involvement of both the androgen and estrogen receptor."

In the studies description of aromatase deficiency and estrogen resistance both involve persistent growth into adulthood but this could merely mean a decrease in growth rate and not an increase in adult height as delayed bone age was involved in both.

In the information regarding removal of testes, the researchers found either equal or greater amount of length increase after additional testosterone.

"ERα is also involved in longitudinal bone formation but its action on periosteal surface as well as growth plate may be mediated indirectly by the GH-IGF-I axis"

This study found that testosterone had no effect on the growth plate when adrogen receptor was blocked:

Androgen receptor modulation does not affect longitudinal growth of cultured fetal rat metatarsal bones.

"Systemic administration of the nonaromatizable androgen oxandrolone{oxandrolone does not suppress testosterone in low dosage but it does in high dosages} stimulates growth in girls with Turner syndrome and boys with a constitutional delay of growth and puberty

Metatarsal bones from female and male rat fetuses (day E20) were cultured for 14 days in the presence of oxandrolone, testosterone or the androgen receptor (AR) antagonist flutamide with/without insulin-like growth-factor-I (IGF-I) or charcoal-treated serum.

The AR was found to be expressed in both male and female fetal rat metatarsal bones. Neither oxandrolone nor testosterone had any effect on metatarsal bone growth when tested at a wide concentration range (1 nM to 10 microM), not even in the presence of IGF-I (100 ng/ml) or charcoal-treated serum (10%). Bone growth was also unaffected when the AR was blocked by flutamide. Metatarsal bone growth was significantly stimulated by IGF-I.

Modulation of AR activity in the fetal rat growth plate does not affect linear bone growth. Extrapolating from these in vitro data, it could be speculated that oxandrolone stimulates longitudinal bone growth in treated children by acting indirectly{likely through myostatin inhibition} rather than directly through AR activation in growth plate chondrocytes."

So Testosterone stimulates linear bone growth but not through direct receptor activation.

"More controversial is the effect of oxandrolone on adult height in these boys. Some studies show no effect on adult height, while others suggest that there might be a positive effect also on adult height in boys who start the treatment before puberty"<-so maybe testosterone requires an equilibrium value and if you're above it there's no height increasing benefit but if you're below it like before puberty it increases height.

"On day 14 of culture, bone lengths compared to control were 96 ± 1% in male and 98 ± 2% in female bones treated with oxandrolone"<-So bone length decreased when testosterone was added above equilibrium and bone length increased(by 102% in testosterone challenged males) when testosterone was added below equilibrium.

"Chondrocyte mineralization [is] impaired by oxandrolone and testosterone"

"Our finding that testosterone, in contrast to oxandrolone, a nonaromatizable androgen, promotes chondrocyte proliferation, suggests that local aromatization of testosterone to estrogens may occur in the growth plate."

Thus Testosterone may only increase height by increasing IGF-1 levels and inhibiting myostatin, it likely does not have direct effects in the growth plate other than converting into estrogen.   Too much testosterone even when not converted to estrogen may also have negative effects on growth.

According to Analysis of testosterone effects on sonic hedgehog signaling in juvenile, adolescent and adult sprague dawley rat penis., testosterone in adults decreases Shh protein levels but increases Shh transcription.  "Testosterone treatment did not alter SHH signaling in juvenile rats. Shh mRNA increased 3.2-fold and SHH protein increased 1.2-fold in rats castrated during puberty. In adult rats, castration decreased Shh mRNA 3.2-fold but did not alter SHH protein. Testosterone supplement in adult rats increased Shh mRNA 2.3-fold and decreased SHH protein 1.3-fold."  Since SHH is pro-chondrogenic this can be a negative effect.  The fact that Shh protein increased 1.2 fold in rats after castration indicates that there may be an equilibrium level of testosterone.

Rapid membrane responses to dihydrotestosterone are sex dependent in growth plate chondrocytes.

"17β-Estradiol regulates proliferation and differentiation of female chondrocytes via a membrane-associated signaling pathway in addition to its estrogen receptor (ER) mediated effects. In contrast, testosterone does not elicit a similar membrane response, either in male or female cells. Whereas female rat growth plate chondrocytes convert testosterone to 17β-estradiol, male chondrocytes produce 5α-dihydrotestosterone (DHT). DHT was found to mediate sex-specific effects of testosterone in male cells. In this study, we hypothesized that DHT can induce sex-specific rapid membrane effects similar to other steroid hormones. Confluent cultures of chondrocytes isolated from resting zones of growth plates of both male and female rats were treated with 10(-10)-10(-7)M testosterone or DHT for 3, 9, 90 and 270min and protein kinase C (PKC) and phospholipase A2 (PLA2) activities were measured. To examine potential signaling pathways involved in PKC activation, male chondrocytes were treated with 10(-7)M DHT for 9min in the presence or absence of the phospholipase C (PLC) inhibitor U73122, the secretory PLA2 inhibitor quinacrine or the cytosolic PLA2 inhibitor AACOCF3; the Gαi inhibitor pertussis toxin (PTX) or Gαs activator cholera toxin (CTX), and the general G-protein inhibitor GDPβS; thapsigargin, an inhibitor of a Ca-ATPase pump in the endoplasmic reticulum; verapamil and nifedipine, inhibitors of specific L type Ca2+ channels on the cell membrane; and cyproterone acetate (CPA), which is an inhibitor of the classical androgen receptor (AR); as well as the transcription inhibitor actinomycin D, or the translation inhibitor cycloheximide. DHT induced a dose-dependent increase in PKC and PLA2 activity in male cells with the highest increase at 10(-7)M DHT{highest dose}, whereas testosterone had no effect. PKC activity was augmented at 9 and 90 min, and then decreased to baseline at 270min. Neither testosterone nor DHT affected PKC in female cells. U73122, quinacrine, and AACOCF3 inhibited DHT-induced activation of PKC. DHT treatment for 9 min had no effect in [(3)H]-thymidine incorporation in quiescent confluent cultures but caused a dose dependent increase in alkaline phosphatase specific activity. Inhibition of PLC reduced the response of to DHT in a dose dependent manner, indicating that PLC is involved. In conclusion, our study indicates that DHT, but not testosterone, has sex-specific rapid membrane effects in male growth plate chondrocytes involving PLC and PLA2-mediated PKC signaling pathways."

LSJL gene expression data was done on female rats.

"E2 causes a rapid activation of Ca2+ influx and activation of PKC only in cells from female rats"

"PKCα [is likely involved] in the mechanism [of DHT response]"

"inhibition of Ca2+ influx blocks the stimulatory effect of DHT on PKC. Gα proteins have also been shown to activate PI-PLC and PLC was required for DHT-dependent PKC activation, since specific inhibition of PI-PLC blocked activation of PKC by DHT. Moreover, inhibition of release of Ca2+ ions from the endoplasmic reticulum also reduced DHT‘s effect. PI-PLC catalyzes the release of IP3, which opens these Ca2+ ion channels and resulting DAG translocates the active PKC to the plasma membrane. Although Gαq is generally associated with PI-PLC activation, Gαi has also been shown to do so"

"Gαi is activating PLA2, and that is upstream of PI-PLC, as we have shown for 1α,25-dihydroxyvitamin D3-dependent activation of PKC in growth plate chondrocytes. DHT activated PKC via a PLA2-dependent pathway. DHT caused a rapid increase in PLA2 activity and inhibition of PLA2 blocked the stimulatory effect of DHT on PKC. Production of lysophospholipid and release of arachidonic acid are catalyzed by PLA2 via phospholipid hydrolysis. Arachidonic acid can act as co-factor for PKCα and also provides a substrate for constitutively active cyclooygenase-1 (Cox-1) in growth plate chondrocytes, which leads to prostaglandin production. In addition, lysophospholipid can activate PLC, and can then initiate the downstream PLC-PKC pathway"

C2C12 myoblastoma cell differentiation and proliferation is stimulated by androgens and associated with a modulation of myostatin and Pax7 expression.

"Androgens are modulators of skeletal muscle adaptation and regeneration processes. The control of satellite cell activity is a key mechanism during this process. In this study, we analyzed the ability of dihydrotestosterone (DHT) and anabolic steroids to induce and modulate the differentiation of C2C12 myoblastoma[muscle cell tumor] cells toward myotubes. C2C12 cells were dose-dependently treated with DHT and anabolic steroids. The treatment with DHT and anabolic steroids resulted in a stimulation of C2C12 cell proliferation and CK[creatine kinase] activity. The antiandrogen flutamide was able to antagonize this effect. The expression of the androgen receptor, SOX8, SOX9{up}, Delta, Notch, myostatin, and paired box gene7 (Pax7){LSJL downregulates Pax7a} was modulated by androgens. The treatment with DHT and anabolic steroids resulted in a strong stimulation of myostatin expression not only in undifferentiated cells{since MSCs are undifferentiated cells DHT will affect LSJL too} but also in myotubes. The stimulation could be antagonized by flutamide. The expression of Pax7 was detectable in C2C12 cells early after treatment with DHT. Our results demonstrate that the key mechanisms of satellite cell differentiation are modulated by androgens. Androgens stimulate the proliferation of C2C12 cells, accelerate the process of differentiation, and increase the expression of myostatin in undifferentiated and differentiated cells."

Both DHT-6 and DHT-9 tended to decrease Sox9 expression.

Lateral Joint Loading for the Jaw?

People have asked before if it's possible to do LSJL for the spine.  So far LSJL has only been done for the arms and legs and there's more research done on this field.  Here's a study on lateral loading for the jaw that predates the LSJL longitudinal growth studies that may provide some insight on how to grow taller via the spine or how to get a bigger jaw.

Influence of extraoral lateral force loading on the mandible in the mandibular development of growing rats.

"Thirty growing Wistar rats were divided into 3 groups: control, sham, and experimental. To determine longitudinal developmental changes, each animal was placed under anesthesia and immobilized in a custom-built body retainer. In the experimental group, a fixing device of aluminum was fitted to the zygomatic arch. Lateral force was then applied to the mandible with an open coil for 2 weeks. Absolute lengths and perpendicular heights from the baseline of the lower border of the mandible were measured.
Lateral force induced the mandible to shift toward the nonloaded side; absolute mandibular length at menton-condylion on the side where the load was applied was greater than that in the control group. No difference from the control group was noted on the nonloaded side, and there were no significant differences in perpendicular heights. Lateral loading on the mandible resulted in histopathologic changes: (1) on the side where the load was applied, the cartilaginous zone hypertrophied in the highest margin of the condylar head, the erosive zone expanded, and the width of the mandibular neck decreased; (2) the chondrocyte layer shifted to the medial side on the nonloaded side, and cartilaginous ossification occurred in the lateral direction immediately below the chondrocyte layer, which deformed the mandibular neck toward the medial side and caused asymmetric development of the mandible."

"A fixation device for loading lateral force onto the mandible was attached to the zygomatic arch. It was made of aluminum and was 7 mm wide, 50 mm long, and 0.5 mm thick. The device was fixed bilaterally to the cheek bones (horizontal to the Frankfort plane and immediately below the orbit) with a stainless steel wire with a diameter of 0.3 mm, so that the device passed under the lower border of the mandible. In the experimental group, a hole was made in the left mandibular incisor cervical area, and an open coil spring was ligated to the tooth by using a 0.25 mm stainless steel wire. The device and the coil spring were bound with a stainless screw. The force was loaded to displace the mandibular central incisor 2 mm to the right of the maxillary central incisor. The open coil spring, made of stainless steel containing nickel and chromium, measured 0.25 mm thick, 1.02 mm in diameter, and 7.0 mm long. In the load-shift curve obtained in a preliminary experiment with this coil spring, 2 mm of compression provided about a 15-g force. The direction of the force was assumed to be parallel to the Frankfort plane."<-The force used in the LSJL studies was 0.5N which is much less force.

"The lateral force loaded on the mandible was 15 g; at that level, the rats could move the mandible back to its normal position by themselves when awake. Force was loaded for 24 hours a day."<-LSJL loading is intermittent.

At 2 weeks the absolute mandibular length increased from 17 to 18.  That is huge.  There was no change in the control group.

B and D are loaded side and A and C are non-loaded.  It's hard to tell if there's any evidence of new mesenchymal chondrogenesis or articular cartilage endochondral ossification which would be what increases height.  However, the control group did not grow at all indicating that this is not merely an increase in growth rate.

So lateral loading of the mandible increases it's length and perhaps intermittent loading could provide this increase without the deformity.  You could also load both sides equally.  Growth can be stimulated in the mandible by lateral loading.  No clamp is likely needed and you can just use your hands.  Ultrasound has been tested on the condyle as well.

Grow Taller by manipulating ERK1/2?

ERK related proteins have been mentioned in several loading modalities and supplements mentioned on this site.  Therefore, it is important to understand ERK to better understand those methods.  Vitamin C activates ERK.  LIPUS activates ERK.  FGF21 antagonizes GH via ERK.  Acteoside inhibits ERK phosphorylation. Mechanical strain causes cell proliferation by ERK activation.  Huperzine A increases ERK1/2 phosphorylation.  IGF-1 activates Twist1 via ERK1/2.  Myostatin operates through ERK.  c-Fos is upregulated by LSJL which is a target of ERK1/2 so LSJL likely operates via the ERK1/2 pathway.

Extracellular signal-regulated kinase (ERK) dictates osteogenic and/or chondrogenic lineage commitment of mesenchymal stem cells under dynamic compression.

"Bone marrow mesenchymal stem cells (MSC) were encapsulated in fibrin gel scaffolds and subjected to a dynamic mechanical compression stimulus to induce chondrogenic differentiation of the cells with and without the addition of PD98059, a selective inhibitor for the ERK1/2 pathway. Dynamic compression induces the chondrogenic differentiation of the cells and inhibition of the ERK1/2 pathway completely abolishes this chondrogenic response. Inhibition of ERK1/2 under dynamic compression augments the osteogenic response of the cells and significantly increases their expression of alkaline phosphatase (ALP), collagen type I (COLI) and osteocalcin (OCN). Dynamically compressed samples show staining for sulfated glycosaminoglycans (sGAG) while the inhibited and compressed samples show no sGAG but present positive staining for microcalcifications. The activation of ERK1/2 can determine the ultimate cell fate between the chondrogenic and osteogenic programs in cells stimulated under dynamic unconfined mechanical compression."

So you want to upregulate ERK 1/2 to increase the early stages of chondrogenic differentiation.  Likely the mesenchymal condensation stage.

The Bone Marrow MSCs were CD73, CD90, CD105-positive.

"Dynamically compressed samples were stimulated by a 10% sinusoidal (1 Hz) strain with an initial 5% strain (yielding a 15% max strain) for a period of 6 h."

"When subjected to microenvironmental cues within a type-1 collagen scaffold, the inhibition of the ERK1/2 pathway actually resulted in an augmented osteogenic response from the cells and an inhibited chondrogenic differentiation concurrently."  ERK1/2 inhibition in the loaded samples reduced Sox9 and Aggrecan expression.

"The activation of ERK1/2 [is] at least partially non-critical for the hydrostatic pressure-induced osteogenesis of bone marrow mesenchymal stem cells"<-this is interesting as we believed hydrostatic pressure to always be pro-chondrogenic.

"Dynamic compression led to significant increase in the expression of both TGF-β 1 and II receptors and potential involvement of p42/44 MAPK due to the rapid upregulation in the expression of c-Fos, which is a known downstream target of p42/44 MAPK. Cyclic unconfined mechanical compression activated the p42/44 pathway but not the p38 or JNK pathways in the stem cells and the inhibition of the p42/44 pathway did not result in the activation of any of the other two MAPK cascades and completely abolished the chondrogenic response seen in the cells"

"Osteogenesis was suppressed in the presence of U0126, a potent selective inhibitor of p42/44 MAPK, in both compressed and non-compressed cellular constructs."

"Dynamic compression led to significant increase in the expression of both TGF-β 1 and II receptors and potential involvement of p42/44 MAPK due to the rapid upregulation in the expression of c-Fos"

Note that chondroinducers like TGF-Beta and BMP-2 were not present.  Although dynamic compression likely results in ERK phosphorylation.

Activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) is needed for the TGFβ-induced chondrogenic and osteogenic differentiation of mesenchymal stem cells

"Bone marrow-derived MSC were cultured in three-dimensional fibrin gel scaffolds and stimulated down the chondrogenic and osteogenic programs by addition of TGF-β3 to and osteogenic buffer media. Cells were cultured under control conditions (no cytokine supplementation), treated with TGF-β3 or treated with PD98059+TGF-β3 for 7 days. Addition of TGF-β3 significantly upregulates the phosphorylation of ERK1/2 and induces the cells down the chondrogenic and osteogenic pathways (as demonstrated by the significant upregulation of aggrecan, sox9, collagen types 1 & 2 gene expressions). Inhibition of ERK1/2 phosphorylation with PD98059 led to the abolishment of the upregulation of chondrogenic and osteogenic-specific gene expressions. ERK1/2 is needed for the chondrogenic and osteogenic differentiation of MSC as induced by TGF-β3 supplementation."

"The activation of ERK1/2 is necessary for the induction of the chondrogenic gene SOX-9"<-Since Sox9 is more helpful during the early stages whereas CNP is more helpful in the later stages this could be why ERK1/2 is only helpful in some cases which is before Sox9 is induced and before CNP takes over.

"ERK1/2 phosphorylation decreased during chondrogenic differentiation of the chick mesenchyme"<-which would make sense as ERK1/2 plays a role during the early stages of the chondrogenic lineage.

"[In another chick mesenchyme model], inhibition of chondrogenesis increased phosphorylation of ERK1/2"

TGF-Beta3 is highly pro-chondrogenic maybe TGF-Beta3 phosphorylates ERK1/2 more than other high proteins thus making increasing ERK 1/2 phosphorylation a potential way to increase chondrogenesis.

The study mentions though that ERK1/2 phosphorylation has had mixed effects on chondrogenesis and osteogenesis.

In this study ERK1/2 phosphorylation only increased COL2A1 levels but decreased osteogenesis markers ALP and OCN. This study may disagree that ERK 1/2 inhibition increases osteogenesis but it confirms that ERK 1/2 phosphorylation encourages chondrogenesis.  This study also mentions a connection to Akt phosphorylation since LSJL phosphorylates Akt maybe Akt phosphorylation is needed to make ERK1/2 anabolic.  TGFB3 can phosphorylate AKT.

The role of the ERK1/2 pathway as an alternative to the aging-diminished cyclic AMP pathway in calcitonin-mediated chondrogenesis in human nucleus pulposus.

"Human disc degeneration initiated by aging in the central nucleus pulposus (hNP) is an irreversible process. the related mechanisms of calcitonin on the regeneration of hNP and the effects of calcitonin on the age-related alterations were examined. The harvested hNP population was designated as YhNP (from young donor, age <50) and OhNP (from old donor, age >50). Primary OhNP cells showed more hypertrophic phenotypes than YhNP. Calcitonin (10(-8)-10(-6) M) was able to induce the same chondrogenesis in both YhNP and OhNP by elevating chondrogenic specific-mRNA and protein expressions. Their cell viabilities were increased with calcitonin treatment. No significant differences of calcitonin receptor (CTR) were expressed between YhNP and OhNP cells. In calcitonin-induced pathways for chondrogenesis, highly increased cyclic AMP (cAMP) was detected in YhNP but was strongly diminished by aging in OhNP after calcitonin treatment. To maintain the chondrogenesis, calcitonin-induced an alterative phosphorylated ERK1/2 (p-ERK) in both cells. After inhibiting ERK1/2 by PD98059, calcitonin-induced chondrogenesis in OhNP was almost restrained while YhNP cells were not affected. The regeneration of calcitonin on hNP was maintained with aging which was satisfied by an alternative signaling pathway. Calcitonin shows great potential for clinical therapy for disc regeneration without aging considerations."

The proteins that initiate chondrogenesis in the discs may be different than those in other areas.

"Platelet-rich plasma (PRP) comprised of various growth factors promotes chondrogenic re-differentiation in degenerated-NP cells via TGF-β1-induced Smad signaling"

"The calcitonin receptor (CTR) is a metabotropic or seven transmembrane (7TM) receptor and also belongs to the G-coupled proteins receptor (GCPR) on cell membrane. The binding of calcitonin to the CTR has been demonstrated to activate intracellular signaling, including the cyclic AMP (cAMP)/protein kinase C (PKC) pathway and the mitogen-activated protein kinase (MAPK) pathway"

Calitonin increased Sox9, COL2A1, Agn, and p-Sox9.

"The cAMP level in YhNP cells was dose-dependently and highly increased with calcitonin. In contrast, the cAMP in OhNP was very low and no dose-dependent expression was seen even after Forskolin activation"

"For chondrogenesis, the cAMP could increase the transcriptional activity of SOX9 during mesenchymal condensation in early embryogenesis. However, cAMP was strongly induced only in YhNP but not OhNP"

Huperzine A's increase in ERK 1/2 was like via TGF-Beta1. LSJL and LIPUS also increase TGF-Beta1. So the best way to increase ERK 1/2 phosphorylation as of now is to do either LSJL or LIPUS.

So in addition to manipulating calcium secretions another potential mechanism for LSJL action on height is that LSJL increases TGF-Beta which increases ERK 1/2 phosphorylation which allows for chondrogenic differentiation. The entirety of the TGF-Beta3 upregulation of chondrogenic genes was via ERK1/2. There ERK1/2 phosphorylation likely induces the upregulation of chondrogenic genes.

In the LSJL gene expression study,  C-fos was upregulated.  In the elbow loading study, c-Fos was upregulated by 2.9 in the distal humerus and 4.5 in the proximal ulna.  The cutoff of displayed genes in the LSJL gene expression study was 2.8.  Since the authors didn't observe any length increase in the LSJL gene expression study (although they weren't looking for it) and c-Fos is downstream of ERK 1/2 the reason that the mice grew longer limbs in the elbow loading study could be a result of ERK 1/2 phosphorylation increase.  

In the gene expression study the mice were loaded at 0.5 N at 5 H/z for 3 min/day for 3 days.  For elbow loading this duration was 5 min/day for 10 days.  We need RT-PCR tests using phospho-specific ERK1/2 to see if they were phosphorylated.


Estrogen stimulates leptin receptor expression in ATDC5 cells via the estrogen receptor and extracellular signal-regulated kinase pathways.

"The effects of estrogen are mediated not only through the direct activity of estrogen receptors (ERs) but also through cross talk with other signaling systems implicated in chondrogenesis. The receptors of both estrogen and leptin (OBR (LEPR)) are detectable in growth plate chondrocytes of all zones. The expression of mRNA and protein of OBR in chondrogenic ATDC5 cells and the effect of 17β-estradiol (E(2)) stimulation were assessed.  The mRNA of Obr was dynamically expressed during the differentiation of ATDC5 cells over 21 days. Application of E(2) (10(-7) M) at day 14 for 48 h significantly upregulated OBR mRNA and protein levels. The upregulation of Obr mRNA by E(2) was shown to take place in a concentration-dependent manner, with a concentration of 10(-7) M E(2) having the greatest effect. E(2) affected the phosphorylation of ERK1/2 (MAPK1/MAPK3) {ERK1-p may be chondroinhibitory} in a time-dependent manner where a maximal fourfold change was observed at 10 min following application of E(2). Pretreatment of the cells with either U0126 (ERK1/2 inhibitor) or ICI 182 780 (ER antagonist) blocked the upregulation of OBR by E(2) and prevented the E(2)-induced phosphorylation of ERK."

"Gender- and region-specific differences in ERα and ERβ gene expression in the limb and spine growth plate during development in both male and female rats."

"Activation of OBR by leptin leads to the activation of many signaling pathways including the JAK1/STAT1 pathway and the phosphatidylinositol 3-kinase (PI3K (PIK3R1))/MAPK1 pathway,"

"At low concentrations of estrogen, ERβ is able to inhibit ERα-activated transcription from estrogen response elements. At high concentrations of a ligand, ERβ does not inhibit ERα action; moreover, it induces its own transcription"

Leptin regulates estrogen receptor gene expression in ATDC5 cells through the extracellular signal regulated kinase signaling pathway.

"Both ERα and ERβ were dynamically expressed during the ATDC5 cell differentiation for 21 days. Leptin (50 ng/ml) significantly upregulated ERα and ERβ mRNA and protein levels 48 h after leptin stimulation at day 14. The up-regulation of ERα and ERβ{upregulated by LSJL as esr2} mRNA by leptin was shown in a dose-dependent manner, but the most effective dose of leptin was different (100 and 1,000 ng/ml, respectively) {Leptin can be used to manipulate the ERalpha and ERBeta ratio}. leptin augmented the phosphorylation of ERK1/2 in a time-dependent manner. A maximum eightfold change was observed at 15 min. a specific ERK1/2 inhibitor, UO126, blocked leptin-induced ERs regulation in ATDC5 cells, indicating that ERK1/2 mediates, partly, the effects of leptin on ERs."

"ERα and ERβ exert opposite effects in the regulation of longitudinal bone growth: the former accelerates bone growth and contributes to growth spurt in puberty, while the latter inhibits growth"

"ERα strongly increased leptin-induced STAT3 transactivation"

"ERα exerted stimulatory effects on leptin production in adipocytes, whereas ERβ produced inhibitory effects"

"In humans, higher doses of estrogen during puberty can lead to growth plate fusion, which are primarily mediated through ERα"

"ERα [may] not [be] required for skeletal growth during early sexual maturation."<-although some ERalpha KO studies have found height decrease.

Gas6, a new regulator of chondrogenic differentiation from mesenchymal cells.

"Growth arrest-specific 6 (Gas6) [is a] gene that was clearly downregulated by this [instance] of chondrogenic differentiation. Blockage of Gas6 mRNA expression by siRNA remarkably enhanced the chondrogenic differentiation {Gas6 inhibitors could help you grow taller}, while stimulation with recombinant Gas6 inhibited the mRNA expressions of type II collagen (Col2a1) and aggrecan. Gas6 signaling activated the phosphorylation of ERK1/2, SAPK/JNK, and Akt, but not p38 MAPK. Gas6 negatively regulates chondrogenic differentiation, at least through the MAPK pathway."

"Gas6 [has] mitogenic and anti-apoptotic effects through MAPK and Akt pathways in multiple systems. rGas6 induced transient phosphorylation of ERK1/2 that peaked at 5 min and slight phosphorylation of SAPK/JNK at 5 min. p38 MAPK phosphorylation was not affected by the stimulation until 60 min, while phosphorylation of Akt increased at 5 min and continued until 60 min"

"Gas6 inhibits chondrogenic differentiation of mesenchymal cells."


Cartducin stimulates mesenchymal chondroprogenitor cell proliferation through both extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt pathways.

"Cartducin[LSJL upregulates carducin as C1qtnf3 by 6 fold], a paralog of Acrp30/adiponectin, is a secretory protein produced by both chondrogenic precursors and proliferating chondrocytes, and belongs to [the] C1q family of proteins. Cartducin promotes the growth of both mesenchymal chondroprogenitor cells and chondrosarcoma-derived chondrocytic cells in vitro. [Does] cartducin exist in serum and [what] intracellular signaling pathways [are] stimulated by cartducin in mesenchymal chondroprogenitor cells? Unlike Acrp30/adiponectin, cartducin was undetectable in mouse serum. mesenchymal chondroprogenitor N1511 cells were stimulated with cartducin, and three major groups of mitogen-activated protein kinase (MAPK) pathways and the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway were examined. Cartducin activated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt, but not c-jun N-terminal kinase (JNK) nor p38 MAPK. The MEK1/2 inhibitor, U0126, blocked cartducin-stimulated ERK1/2 phosphorylation and suppressed the DNA synthesis induced by cartducin in N1511 cells. The PI3K inhibitor, LY294002, blocked cartducin-stimulated Akt phosphorylation and a decrease in cartducin-induced DNA synthesis in N1511 cells was also observed."

A high mitogenic activity of mesenchymal chondroprogenitor cells is initially required to produce enough cells for the process of chondrogenesis"

"Both ERK1 and ERK2 have been shown to be activated by their upstream activators, MEK1 and MEK2."

This study suggests that ERK1-p is pro chondrogenic.  Note that ERK1 is pro chondrogenic when both it and Akt are phosphorylated.

"mitogenic response to cartducin by mesenchymal chondroprogenitor cells requires the activation of both the ERK1/2 and PI3K/Akt pathways."<-We know that LSJL induces p-Akt.


Cartducin, a paralog of Acrp30/adiponectin, is induced during chondrogenic differentiation and promotes proliferation of chondrogenic precursors and chondrocytes. states that Cartducin is a mesenchymal chondrogenic marker providing further evidence that LSJL induces chondrogenesis.  It also increases chondrocyte proliferation and chondrocyte precursor differentiation.  Cartducin is also induced by TGFB1.

Progression of chondrogenesis in C3H10T1/2 cells is associated with prolonged and tight regulation of ERK1/2.

"Close contact of mesenchymal cells in vivo and also in super dense micromass cultures in vitro results in cellular condensation and alteration of existing cellular signaling required for initiation and progression of chondrogenesis. To investigate chondrogenesis related changes in the activity of ubiquitous cell signaling mediated by mitogen-activated protein kinases (MAP kinase), we have compared the effect of cell seeding of pluripotent C3H10T1/2 mesenchymal cells as monolayers (non-chondrogenic culture) or high density micromass cultures (chondrogenic) on the regulation and phosphorylation state of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and also on regulation of ERK1/2 nuclear targets, namely, activation protein-1 (AP-1) and serum response factor (SRF). Increasing cell density resulted in reduced DNA binding as well as activity of AP-1. SRF activity was up-regulated in confluent monolayer cultures but like AP-1 was inhibited in micromass cultures. Low levels of PD 98059 (5 microM), a specific inhibitor of ERK1/2, resulted in delayed induction of AP-1 and SRF activity whereas higher concentrations of this inhibitor (10-50 microM) conferred an opposite effect. Increasing concentrations of the PD 98059 inhibitor in long term monolayer or micromass cultures (2.5 day) resulted in differential regulation of c-Fos and c-Jun protein levels as well as total expression and phosphorylation levels of ERK1/2. PD 98059 treatment of C3H10T1/2 micromass cultures also resulted in up-regulation of type IIB collagen and Sox9 gene expression. While high expression of aggrecan and type IIB collagen genes were dependent on BMP-2 signaling, ERK inhibition of BMP-2 treated micromass cultures resulted in reduced activity of both genes. ERK1/2 in chondrogenic cultures of C3H10T1/2 cells is tightly controlled and can cross interact with other signaling activities mediated by BMP-2[BMP2 can induce Akt phosphorylation] to positively regulate chondrogensis."

"Inhibition of p38 results in reduced chondrogenesis whereas inhibition of MEK/ERK results in increased chondrogenesis. Over expression of c-Fos, a target of the MAP kinase signaling pathway (MEK/ERK), inhibits chondrogenic activity of a mouse carcinoma derived chondrogenic cell line (ATDC5) in culture"

"The long term maintenance of low AP-1 activity [plays] a necessary role in the overall molecular context of prechondrogenic cells which [favors] the initiation and maintenance of chondrogenic gene expression."

"Synthesis of c-Fos in C3H10T1/2 cells is dependent on cellular density; although cells which establish intercellular contacts up-regulate c-Fos, this activity peaks in super dense micromass cultures"<-So LSJL upregulation of c-Fos is consistent with the fact that LSJL increases cellular density.

"Expression of type IIB collagen, a spliced form of type II collagen associated with differentiating chondrocytes, is up-regulated in cells making increasing contact or when C3H10T1/2 cells are cultured in micromass densities"

"Although there was a large increase in levels of c-Fos in micromass cultures of C3H10T1/2 cells, the binding and activity of AP-1 was maintained low"

EGFR may be one of the targets of AP-1. Also Ptn which was upregulated in LSJL. LSJL upregulates Cxcl1 over six fold which is a target of AP-1.

Stretch-induced modulation of matrix metalloproteinases in mineralizing osteoblasts via extracellular signal-regulated kinase-1/2.

"Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) produced by osteoblasts play an essential role in bone remodeling. We examined the effect of stretch on MMP-1, -2, -3, -8, -9, -13, and -14, as well as TIMP-1 and -2 gene expression in differentiating, mineralizing, and nonmineralizing human SV-40 immortalized preosteoblast cells. In the mineralizing osteoblast culture, but not in the nonmineralizing cultures, cyclic stretch for only 15 min resulted in an increase of MMP-1 (fourfold) and -3 {upregulated by LSJL 3.677 fold in other data upregulated 5.3 fold} (depending on differentiation stage up to 25-fold) transcript abundance. The increase of MMP-1 and -3 was confirmed on the protein level. Stretching experiments performed in the presence of a specific inhibitor of extracellular signal-regulated kinase (ERK) showed a strong suppression of the stretch-induced increase in MMP-1 and -3. MMP-1 and MMP-3 are mechanosensitive genes in mineralizing the human osteoblast, and that the mechano-induction of these genes is mediated via the ERK pathway.  With the ability to generate MMPs at highly stretched sites, osteoblasts can potantially direct osteoclasts to specific bone surface areas prepared for resorption."

"MMPs are synthesized in a latent form, and become activated extracellularly by proteolytic cleavage requiring plasmin"

"At the onset of mineralization (day 14), this induction was reduced to five fold, while on day 21 MMP-3 expression was not affected."  MMP3 levels in LSJL are most consistent with onset of mineralization.

"When the cells were not induced to mineralize, stretch weakly induced MMP-1, and did not change MMP-3 expression"

"An increase of phosphorylated ERK levels [exists] in mineralizing osteoblast cultures after mechanical stimulation"

"the very potent induction of MMP-3 by stretch already after 6 h on day 7 was strongly inhibited by blocking ERK1/2 phosphorylation"

"MMP-3 can activate the inactive procollagenases MMP-1, -8, and -13."

"Stretch is able to induce ERK phosphorylation at all stages of differentiation and mineralization,"

Mechanical impact induces cartilage degradation via mitogen activated protein kinases.

"The phosphorylation of MAP kinases was examined. The effects of MAP kinase inhibitors on impaction-induced chondrocyte death and proteoglycan (PG) loss were determined. The expression of catabolic genes at mRNA levels was examined.

Early p38{mentioned as being involved in LSJL by Hiroki Yokota} activation was detected at 20 min and 1h post-impaction. At 24h, enhanced phosphorylation of p38 and extracellular signal-regulated protein kinase (ERK)1/2 was visualized in chondrocytes from in and around impact sites. The phosphorylation of p38 was increased by 3.0-fold in impact sites and 3.3-fold in adjacent cartilage. The phosphorylation of ERK-1 was increased by 5.8-fold in impact zone and 5.4-fold in adjacent cartilage; the phosphorylation of ERK-2 increased by 4.0-fold in impacted zone and 3.6-fold in adjacent cartilage. Furthermore, the blocking of p38 pathway did not inhibit impaction-induced ERK activation. The inhibition of p38 or ERK pathway significantly reduced injury-related chondrocyte death and PG losses. Blunt impaction significantly up-regulated matrix metalloproteinase (MMP)-13, Tumor necrosis factor (TNF)-α, and ADAMTS-5 expression."

"MEK1/2, the upstream kinase of ERK1/2, and p38 MAP kinase was demonstrated to be responsible for the up-regulation of iNOS and COX-2{up in LSJL as PTGS2} which mediate chondrocyte apoptosis"

"Elevated phosphorylation of JNK-1 (p46) and JNK-2 (p54) [was present] in both the impact and annulus cartilage at 20 min post-impaction,. The enhanced phosphorylation declined to baseline at 1 hr post-impaction."  LSJL gene expression was taken at one hour so this may be why signs of JNK phosphorylation were not detected.

"acute impact-induced cell death was substantially inhibited by immediate post-impact treatment (within 4 hrs) with n-acetyl cysteine (NAC), a free radical scavenger"

Gene disruption of Spred-2 causes dwarfism.

"Overexpression of fibroblast growth factors (FGFs), several gain-of-function mutations in the FGFR3, and constitutive activation of mitogen-activated protein kinase (MAPK) kinase (MEK1) in chondrocytes have been shown to cause dwarfism in mice by activation of the MAPK signaling pathway. To investigate the inhibitory role of Spred in the FGFR3/MAPK pathway, we generated mice with a trapped Spred-2 gene.  Lack of functional Spred-2 protein in mice caused a dwarf phenotype, similar to achondroplasia, the most common form of human dwarfism. Spred-2(-/-) mice showed reduced growth and body weight, they had a shorter tibia length, and showed narrower growth plates as compared with wild-type mice. We detected promoter activity and protein expression of Spred-2 in chondrocytes. Stimulation of chondrocytes with different FGF concentrations showed earlier and augmented ERK phosphorylation in Spred-2(-/-) chondrocytes in comparison to Spred-2(+/+) chondrocytes."

So ERK-p reduces height when it occurs in chondrocytes(not necessarily stem cells).

"Sprouty from Drosophila was identified as a negative regulator of growth factor-induced ERK activation"

ERK1/2-p is likely good at the earlier stage of chondrocyte growth when Sox9 is good but bad later when CNP takes over as the primary determinant of height growth.

Complementary antagonistic actions between C-type natriuretic peptide and the MAPK pathway through FGFR-3 in ATDC5 cells.

"[In] ATDC5 cells, a mouse chondrogenic cell line, FGF2 and FGF18 markedly reduced CNP-dependent intracellular cGMP production, and these effects were attenuated by MAPK inhibitors. The level of GC-B, a particulate guanylyl cyclase specific for CNP, was not changed by treatment with FGFs. CNP and 8-bromo-cGMP strongly and dose-dependently inhibited the induction of ERK phosphorylation by FGF2 and FGF18 without changing the level of FGFR-3, although they did not affect the phosphorylation of STAT-1. In the organ-cultured fetal mouse tibias, CNP and FGF18 counteracted on the longitudinal bone growth, and both the size and number of hypertrophic chondrocytes. The FGF/FGFR-3 pathway is known as the negative regulator of endochondral ossification. FGFs inhibited CNP-stimulated cGMP production by disrupting the signaling pathway through GC-B while CNP antagonized the activation of the MAPK cascade by FGFs."

"ATDC5 cells contain particularly high activity levels for GC-B and also appear to contain low levels of GC-A and the soluble form of guanylyl cyclase, which is responsive to nitric oxide"

"the hypertrophic chondrocyte layer increased after treatment with 10−7 M CNP compared with treatment with the vehicle. The mean size of hypertrophic chondrocytes was markedly increased (vehicle treated: 497.85 ± 19.2 vs. CNP treated: 1071.42 ± 53.5 μm2), and the number of cells in the hypertrophic chondrocyte layer was reduced (vehicle treated: 152.67 ± 4.1 vs. CNP treated: 118 ± 3.61 cells)"

"Not only the cell size (vehicle treated: 497.85 ± 19.2 vs. FGF18 treated: 314.01 ± 23.67 μm2), but also the number of hypertrophic chondrocytes was reduced by the treatment with 10 ng/ml FGF18 (vehicle treated: 152.67 ± 4.1 vs. FGF18 treated: 89.3 ± 1.45 cells)"

"ATDC5 cells express GC-B, FGF2 and FGF18 reduce CNP-dependent cGMP production in a dose-dependent manner without changing the amount of GC-B, MAPK inhibitors attenuate the FGF inhibition of CNP-dependent cGMP production, both CNP and cGMP inhibit the MAPK pathway but not the STAT-1 pathway of FGFR-3 activation without changing the amount of FGFR-3"

More on the sometimes inhibitory/stimulatory role of ERK:

MEK-ERK signaling plays diverse roles in the regulation of facial chondrogenesis.

"We employed the micromass culture system to define the roles of MEK-ERK signaling in the chondrogenic differentiation of neural crest-derived ectomesenchyme cells of the embryonic chick facial primordia. In cultures of frontonasal mesenchyme isolated from stage 24/25 embryos, treatment with the MEK inhibitor U0126 increased type II collagen and glycosaminoglycan deposition into cartilage matrix, elevated mRNA levels for three chondrogenic marker genes (col2a1, aggrecan, and sox9), and increased expression of a Sox9-responsive collagen II enhancer-luciferase reporter gene. Transfection of frontonasal mesenchyme cells with dominant negative ERK increased collagen II enhancer activation, whereas transfection of constitutively active MEK decreased its activity. MEK-ERK signaling inhibits chondrogenesis in stage 24/25 frontonasal mesenchyme. MEK-ERK signaling enhanced chondrogenic differentiation in mesenchyme of the stage 24/25 mandibular arch. In mandibular mesenchyme cultures, pharmacological MEK inhibition decreased cartilage matrix deposition, cartilage-specific RNA levels, and collagen II enhancer activity. Expression of constitutively active MEK increased collagen II enhancer activation in mandibular mesenchyme, while dominant negative ERK had the opposite effect. MEK-ERK modulation had no significant effects on cultures of maxillary or hyoid process mesenchyme cells. [There was] a striking shift in the response of frontonasal mesenchyme to MEK-ERK modulation by stage 28/29 of development."

The frontonasal cells are not within bone whereas the mandibular arch is within bone which may be the reason for the difference.

"the ERK pathway is triggered by a set of adaptor proteins, like Shc, GRB2, and Pax, that link the receptor to a guanine nucleotide exchange factor, like Sos, C3G, or EPAC. These GDP/GTP exchange factors subsequently activate small GTP binding proteins, like Ras, Rap1, and Rac, which, in turn, activate the Raf family of serine threonine kinases (A-Raf, B-Raf, and c-Raf1) at the top of the triple kinase phospho-relay unit of the cascade. The Raf kinases activate MEK1 and MEK2. MEK1 and 2 activate ERK1 and ERK2, their only characterized downstream substrates, by dual phosphorylation at a conserved TEY motif. ERK activation results in dissociation from its cytoplasmic anchor MEK and activation of various nuclear, cytosolic, and cytoskeletal substrates such as Elk1, Rsk, and Tau, respectively"

"expression of constitutively active MEK inhibited chondrogenesis in stage 24/25 frontonasal mesenchyme, but enhanced chondrogenesis in stage 28/29 frontonasal mesenchyme"

"MEK inhibition enhanced the initiation of sox9 and col2a1 expression in pluripotent C3H10T1/2 mesenchymal stem cells, but suppressed cartilage-specific gene expression after chondrogenesis was induced in the presence of exogenous BMP."<-LSJL upregulates BMP2 but not after two weeks so this may be way LSJL results are reduced.

This study provides evidence that ERK-p is pro-chondrogenic.

Pharmacological modulation of human mesenchymal stem cell chondrogenesis by a chemically oversulfated polysaccharide of marine origin: potential application to cartilage regenerative medicine.

"Human adipose tissue-derived MSCs (hATSCs) were cultured in pellets with transforming growth factor (TGF)-β1-supplemented chondrogenic medium containing either the polysaccharide GY785 DR or its oversulfated isoform GY785 DRS.  Pellet volume, total collagens, and GAG production [was increased] with GY785 DRS and chondrogenic medium. The enhanced chondrogenic differentiation of hATSC was demonstrated by increased expression of several chondrogenic markers. TGF-β1 bound GY785 DRS with higher affinity compared to GY785 DR. In association with TGF-β1, GY785 DRS was found to upregulate the phosphorylation of extracellular signal-regulated kinase 1/2, indicating that oversulfated polysaccharide affects the mitogen activated protein kinase signaling activity.  TGF-β1-dependent stem cell chondrogenesis [is upregulated] by a chemically oversulfated marine polysaccharide. This polysaccharide of marine origin is easily producible."

"Alteromonas infernus[a deep sea bacteria] has been shown to produce a branched high-molecular weight polysaccharide: GY785 (∼106 g/mol)"

"volume estimation revealed that pellets exposed to GY785 DRS in combination with the chondrogenic medium underwent a massive increase by nearly eightfold"

"ERK1/2 phosphorylation was barely stimulated by TGF-β1 or GY785 DRS alone, the concomitant treatment of cells with TGF-β1 and GY785 DRS induced a marked upregulation of the phosphorylation of ERK1/2 as early as 4 hours. This stimulation was maintained up to 24 hours. Analysis of the phosphorylation of the other MAPK showed no detectable phosphorylation of either JNK or p38 in response to TGF-β1 and GY785 DRS treatment alone or combined. GY785 DR alone failed to affect any tested signaling pathway"

"Sulfated GAGs were able to bind and regulate a number of proteins such as cytokines, chemokines, growth factors, morphogens, enzymes, and adhesion molecules. Some growth factors such as VEGF and FGF have been extensively described as being stored, stabilized, and protected from degradation in the matrix through interactions with GAG. Under the action of a stimulus, these growth factors can then be released and exert their biological functions. These observations suggest that not only the binding affinity of a ligand to its receptor but also the stability of the ligand-receptor complex on the cell surface is one of the key factors that control the biological activity of the ligand in the targeted cells. "

MAP kinases in chondrocyte differentiation

"JNK phosphorylation is not affected during chondrogenesis"

"During chondrogenesis of chick mesenchymal cells, p38 phosphorylation is increased and ERK phosphorylation is decreased"

"MEK/ERK signaling [is required] for the induction of the chondrogenic master gene Sox9 by FGF signaling "

"CTGF/Hcs24-induced proliferation of chondrocytes is mediated through the ERK signaling pathway, while CTGF/Hcs24-induced differentiation of chondrocytes is mediated through p38 MAPK."

"The c-Raf kinase, which is a MAP3K for ERK1/2, has been shown to be upregulated during chondrocyte differentiation in vivo. Recent evidence from our laboratory also demonstrated that c-Raf, MEK1/2, and ERK1/2 are required for the normal expression of the collagen X and p21WAF1/CIP1 genes, which are markers for hypertrophic chondrocytes"<-LSJL downregulates c-Raf as Raf1.

"Generation of a hypomorphic allele of c-Raf in mice, where c-Raf activity is reduced to approximately 10% of wild type activity, causes a severe delay in growth and endochondral ossification"<-Further evidence that LSJL stimulates the early stages of growth and not the late stages.

"p38, in contrast to ERK and JNK, is activated by retinoic acid in chondrocytes and is necessary for the induction of collagenase 3, Cbfa1, and osteocalcin expression by retinoic acid, which might represent transdifferentiation of chondrocytes to osteoblast-like cells"

"[A] target of ERK proteins, the ribosomal S6 kinase 2 (RSK2), is mutated in Coffin-Lowry syndrome, a genetic disease characterized by reduced growth and skeletal and craniofacial malformations. RSK2 (and its upstream regulators) [has a role] in human cartilage development, which is in agreement with the roles of the RSK2 targets c-Fos and CREB in chondrocytes."

Signaling responses of osteoblast cells to hydroxyapatite: the activation of ERK and SOX9.

"ERK signaling molecule is activated in response to HA[Bone mineral hydroxyapatite]. Eleven genes, including those involved in calcium regulation and bone matrix formation, showed a greater than 2.0-fold change in expression level in response to HA. Among those genes upregulated by HA was the gene encoding SOX9 with a 5.7-fold increase in expression."

"Interactions of bone cells with HA surfaces are mediated by adhesion receptors belonging to the integrin superfamily that recognize binding domains within proteins of the extracellular matrix (ECM). Integrin-mediated adhesion to extracellular proteins activates multiple cytoskeletalassociated

and intracellular signaling proteins, such as focal adhesion kinase (FAK). FAK associates with Shc protein activating Ras, which leads to the stimulation of the extracellular regulated kinases (ERK) signaling cascade"

Upregulated genes also upregulated in LSJL:

Hapln2

Sox9

RRAGA{down}

Mechanical signals control SOX-9, VEGF, and c-Myc expression and cell proliferation during inflammation via integrin-linked kinase, B-Raf, and ERK1/2-dependent signaling in articular chondrocytes.

"ACs isolated from articular cartilage were exposed to low/physiologic levels of dynamic strain in the presence of IL-1beta. The cell extracts were probed for differential activation/inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling cascade.

Mechanoactivation, but not IL-1beta treatment, of ACs initiated integrin-linked kinase activation. Mechanical signals induced activation and subsequent C-Raf-mediated activation of MAP kinases (MEK1/2). However, IL-1beta activated B-Raf kinase activity. Dynamic strain did not induce B-Raf activation but instead inhibited IL-1beta-induced B-Raf activation. Both mechanical signals and IL-1beta induced ERK1/2 phosphorylation but discrete gene expression. ERK1/2 activation by mechanical forces induced SRY-related protein-9 (SOX-9), vascular endothelial cell growth factor (VEGF), and c-Myc mRNA expression and AC proliferation. IL-1beta did not induce SOX-9, VEGF, and c-Myc gene expression and inhibited AC cell proliferation. SOX-9, VEGF, and Myc gene transcription and AC proliferation induced by mechanical signals were sustained in the presence of IL-1beta."

"Phosphorylated Rafs activate mitogen-activated protein kinase (MAPK) kinase (MEK1/2) by phosphorylation of Ser217/Ser221. Subsequently, MEK1/2 activates extracellular receptor kinase 1/2 (ERK1/2) by phosphorylating Thr202/Tyr204. ERK1/2 activation is associated with growth signals. However, cytokines like interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α) also phosphorylate ERK1/2 to regulate certain proinflammatory genes. Following activation, ERK1/2 translocates to the nucleus and activates transcription factors that are specific to the signals perceived by cells"

"Cells exposed to IL-1β lose their ability to express SRY-related protein-9 (SOX-9) and vascular endothelial cell growth factor (VEGF)"

"10% compressive forces as well as 6% tensile forces suppress proinflammatory gene induction, upregulate total proteoglycan contents, and aggrecan, collagen type II, and SOX-9 mRNA induction in ACs"

"Mechanical signals trigger c-Raf kinase activity by phosphorylating Ser338 residues. However, IL-1β induces Ser445-B-Raf phosphorylation. B-Raf was not activated by mechanical signals."

Extracellular Signal-Regulated Kinase 1 (ERK1) and ERK2 Play Essential Roles in Osteoblast Differentiation and in Supporting Osteoclastogenesis

"Osteoblasts and chondrocytes arise from common osteo-chondroprogenitor cells. Inactivation of ERK1 and ERK2 in osteo-chondroprogenitor cells causes a block in osteoblast differentiation and leads to ectopic chondrogenic differentiation in the bone-forming region in the perichondrium. Furthermore, increased mitogen-activated protein kinase signaling in mesenchymal cells enhances osteoblast differentiation and inhibits chondrocyte differentiation. The inactivation of ERK1 and ERK2 resulted in reduced beta-catenin expression[and Beta-catenin inhibits chondrocyte differentiation].  Inactivation of ERK1 and ERK2 significantly reduced RANKL expression, accounting for a delay in osteoclast formation."

"Expression of a constitutively active mutant of MEK1 in chondrocytes caused a dwarf phenotype and inhibited hypertrophic chondrocyte differentiation"

"Chondroclast/osteoclast formation is supported by receptor activator of nuclear factor-kappa B ligand (RANKL) secreted from osteoblasts and bone marrow stromal cells"

"Inactivation of ERK1 and ERK2 significantly reduced RANKL expression, accounting for a delay in osteoclast formation."

"Inactivation of ERK1 and ERK2 did not abolish Runx2 mRNA [and] did not affect Osterix, ATF4, JunB{up} and RSK2 protein expression"  However osteocalcin{up}, Fra1, Fra2, c-Fos{up}, and Bsp{up} levels were reduced.

"Ectopic cartilage formation [was observed] in the perichondria of ERK1−/−; ERK2flox/flox; Prx1-Cre mice. [This occurred] as early as E13.5 in the mutant humerus"  The ectopic chondrocytes expressed Ihh, Pth, Pthr, Col10a1, and MMP13.

"Cells in the ectopic cartilage expressed a master transcription factor for chondrocyte differentiation, Sox9, and a cartilage-specific marker, Col2a1, indicating chondrogenic differentiation. These observations suggest that osteo-chondroprogenitor cells in the perichondrium were blocked in their differentiation into osteoblasts and instead differentiated into chondrocytes. Interestingly, the cells in the ectopic cartilage also expressed markers for prehypertrophic chondrocytes (Indian hedgehog [Ihh] and Parathyroid hormone/Parathyroid hormone-related peptide receptor) as well as markers for hypertrophic chondrocytes (Col10a1 and Mmp13)"

"Although the matrix showed intense staining with anti-type X collagen antibody, these cells expressed Col10a1 at a reduced level and instead expressed markers for terminally differentiated hypertrophic chondrocytes (Vegf, Mmp13, and Osteopontin)."

"Loss of ERK1 and ERK2 may result in low β-catenin protein levels through intracellular cross talk between ERK and canonical Wnt signaling. "

"Loss of ERK1 and ERK2 caused severe disorganization of the epiphyseal cartilage and significant reduction in chondrocyte proliferation."

"Prx1-MEK1 transgenic mice showed a dramatic increase in cortical bone formation, fusion of long bones"

"MAPK signaling recruits and directs osteo-chondral progenitor cells toward the osteoblastic lineage"<-Thus ERK may accelerate fusion with enhanced bone formation.

Sotos Syndrome Is Associated with Deregulation of the MAPK/ERK-Signaling Pathway.

"Sotos syndrome (SoS), characterized by tall stature, is caused by haploinsufficiency of the NSD1 gene. [We] identify downstream effectors of NSD1 and map these effectors in signaling pathways associated with growth. Genome-wide expression studies were performed on dermal fibroblasts from SoS patients with a confirmed NSD1 abnormality. Phosphorylation, siRNA and transfection experiments were performed. A significant association was demonstrated with the Mitogen-Activated Protein Kinase (MAPK) pathway. Members of the fibroblast growth factor family such as FGF4 and FGF13{down} contributed strongly to the differential expression in this pathway. a diminished activity state of the MAPK/ERK pathway was demonstrated in SoS. Ras Interacting Protein 1 (RASIP1) [is] upregulated in SoS. RASIP1 dose-dependently potentiated [enhanced] bFGF induced expression of the MAPK responsive SBE reporter providing further support for a link between NSD1 and the MAPK/ERK signaling pathway. NSD1 expression [occurs] in the terminally differentiated hypertrophic chondrocytes of normal human epiphyseal growth plates. In short stature syndromes such as hypochondroplasia and Noonan syndrome, the activation level of the FGF-MAPK/ERK-pathway in epiphyseal growth plates is a determining factor for statural growth. Deregulation of the MAPK/ERK pathway in SoS results in altered hypertrophic differentiation of NSD1 expressing chondrocytes and may be a determining factor in statural overgrowth and accelerated skeletal maturation in SoS."

ERK1/2 may be chondroinductive but it may be inhibitory to the hypertrophic stage which is the predominant cause of height growth.  Thus ERK1/2 may be good for inducing new growth plates with LSJL but bad for existing growth plates.  This property can be observed in proteins like Twist1, Beta-Catenin, Sox9(in the inverse), etc.

"Nsd1 interacted with a number of nuclear hormone receptors, such as the estrogen receptor, retinoic acid and thyroid hormone receptors"

"[An NSD1 domain] specifically methylates lysine 36 at histone H3 (H3-K36), lysine 20 at histone H4 (H4-K20) and other non-histone substrates (4-7), resulting overall in the regulation of chromatin transcription."

NSD1 regulates NF-kB and reduces BMP4 expression.

"Heterozygous inactivation of NSD1 results in loss of repression of growth promoting genes"

"heterozygous knock out mice of Nsd1 do not show a SoS phenotype"

"[SoS patients showed] increased plasma levels of IGFBP-2 and IGFBP-6 and reduced levels of IGF-I, IGF-II, IGFBP-3 and IGFBP-4 were detected"

"NSD1 was expressed in [human] terminally differentiated hypertrophic chondrocytes"

"Activity of the MAPK/ERK pathway [is decreased] in SoS."

"Constitutive active mutations in more downstream genes such as KRAS and BRAF result in an increased activation of the MAPK/ERK pathway and hence in short stature syndromes"

"Fibroblast Growth Factor 13 (FGF13) [is one] of the most down regulated gene in SoS"

Regulation of cartilage formation and maturation by mitogen-activated protein kinase signaling.

"Prechondrogenic mesenchyme cells migrate to the site of the prospective skeletal element. Once there, the mesenchymal cells assemble into compact cellular aggregates, or condensations, a process that is mediated by cell adhesion molecules such as neural cadherin (N-cadherin) and neural cell adhesion molecule (N-CAM), as well as extracellular matrix (ECM) components such as fibronectin and syndecan. These prechondrogenic condensations serve to establish the size and position of each nascent cartilage anlage. Next, the close proximity of the aggregated mesenchymal cells permits critical cell–cell surface interactions and signaling events that initiate intracellular changes culminating in the activation of overt chondrocyte differentiation. In this stage, the chondrogenic progenitor cells exchange their stellate, fibroblastic-like phenotype for the spherical morphology of chondrocytes and commence synthesis of cartilage-specific ECM molecules such as collagen types II, IX, and XI and the highly sulfated proteoglycan, aggrecan"

"transcription factors implicated in early-stage chondrogenic differentiation and skeletal patterning include Msx1 and 2, β-catenin, lymphocyte enhancer-binding factor 1 (Lef1), AP-1 , AP-2, and Runx2/Cbfa1"

"The ERK1/2 pathway is triggered by a set of adaptor proteins, such as Shc, growth factor receptor-bound protein 2 (GRB2), and paired box (Pax), that link the receptor to a guanine nucleotide exchange factor, such as son of sevenless (Sos), CRK SH3-binding guanine nucleotide releasing factor (C3G), or exchange protein directly activated by cAMP (EPAC). These GDP/GTP exchange factors subsequently activate small GTP binding proteins, such as Ras, Rac, and repressor activator protein 1 (Rap1), which, in turn, activate the Raf family of serine threonine kinases (A-Raf, B-Raf, and C-Raf) at the top of the triple kinase phosphorelay unit. The Raf kinases activate MEK1 and MEK2. MEK1 and 2 activate ERK1 and ERK2, their only characterized downstream substrates, by dual phosphorylation at a conserved TEY motif "

"ERK1/2 activation results in dissociation from its cytoplasmic anchor MEK1/2 (Adachi et al.,1999), and phosphorylation of various nuclear, cytosolic, and cytoskeletal substrates such as Ets-like protein-1 (Elk-1), ribosomal S6 kinase (Rsk), and Tau, respectively"

"Common substrates of p38 include other kinases: p38-regulated/activated protein kinase (PRAK), mitogen- and stress-activated kinase 1 (MSK1), and MAPK-interacting kinases 1 and 2 (MNK1/2); as well as transcription factors, including myocyte enhancer factor 2 (MEF2), activating transcription factor 2 (ATF2), and C/EBP homologous protein (CHOP)"

"JNK substrates include c-jun, Elk-1, ATF2, and serum response factor accessory protein 1a (Sap1a)"

"incubation of whole chick or mouse embryos with SU5402, a pharmacological inhibitor of FGF receptor tyrosine kinase (FGFR) activity, strongly depresses ERK1/2 phosphorylation at most locations"

"The prechondrogenic mesenchyme of the embryonic limbs is derived from somatopleural mesoderm of the lateral body wall. At the time of limb bud initiation, the mesodermal cells of the prospective limb-forming region secrete FGF10, which signals to the overlying surface ectoderm, leading to the formation of a thickened apical ectodermal ridge (AER). The AER at the distal tip of the limb bud, in turn, secretes FGF8 and other growth factors that stimulate proliferation of the underlying limb mesenchyme and inhibit precocious differentiation of the mesodermal cells that reside in the region immediately subjacent to the AER. Chondrogenic differentiation is initiated as the proximodistal outgrowth of the limb bud allows some mesodermal cells to emerge from the region of AER influence (known as the progress or subridge zone) and begin forming prechondrogenic condensations. Thus, chondrogenesis is initiated in the proximal limb region, but as limb outgrowth continues, the mesenchymal condensations corresponding to more distal cartilage elements are progressively established, culminating in formation of the phalangeal primordia. Importantly, the condensation process initiates expression of the Sox9 transcription factor, which then activates the genes for the principle cartilage matrix components (collagen type II and aggrecan) and triggers overt differentiation of limb mesenchyme cells into hyaline chondrocytes."

"Treatment of limb mesenchyme micromass cultures with MEK1/2 inhibitor PD98059 accelerated the decline in expression of cell adhesion molecules (N-cadherin, fibronectin, and α5β1 integrin) involved in the prechondrogenic condensation process"

ERK1/2 may be stimulatory towards forming growth plates but once they have been established may be inhibitory towards much like FGFR.

"the ERK1/2 cascade may relay signals that selectively inhibit hypertrophic chondrocyte differentiation within the growth plate, while a different signal transduction pathway mediates the inhibitory effects of FGFR3 on chondrocyte proliferation. This additional relay system appears to involve signal transducer and activator of transcription 1 (Stat1) activation, as ablation of Stat1 function restored normal rates of chondrocyte proliferation in mice that express the achondroplasia-type FGFR3 mutation in cartilage tissues"

"Stat1 deletion only minimally restored bone growth, therefore, elevated ERK signaling may be predominantly responsible for the dwarfism phenotype in mice that express activating FGFR3 mutations."

"prolongation of Sox9 expression induced by upregulated ERK1/2 signaling might account for the delay in hypertrophic maturation of cartilages expressing the ca-MEK transgene, because chondrocytes undergoing hypertrophy normally cease Sox9 expression"<-although phosphorylation of Sox9 may be an alternative to ceasing Sox9 expression.

"the Sox9 transcription factor normally acts to inhibit the maturation of hyaline chondrocytes into hypertrophic chondrocytes."

"The levels of ERK activity in growth plate chondrocytes [may need to] be tightly controlled to allow proper hypertrophic maturation, and that either insufficient or excessive ERK pathway signaling interferes with hypertrophy and subsequent mineralization. Expression of either constitutively active C-Raf or dominant negative C-Raf decreased activation of the collagen type X promoter in the MCT chondrogenic cell line."

"Treatments with any one of several pharmacological inhibitors of p38 (e.g., SB203580, PD169316, SB202190, or SB220025) have consistently been shown to suppress cartilage matrix production in micromass cultures of embryonic chick or mouse limb mesenchyme cells, and also to inhibit expression of mRNAs for the chondrocyte marker genes, sox9, col2a1, and aggrecan"

"Transfection of prechondrogenic limb mesenchyme with constitutively active MKK6, a MAPKK that activates p38, was shown to markedly increase expression of a Sox9-responsive reporter gene"

"p38 and MEK inhibition have opposite effects on the expression of cell adhesion molecules involved in mesenchymal condensation. Treatment with the p38 inhibitor SB203580 causes increased expression of N-cadherin, fibronectin, and α5β1 integrin in limb mesenchyme cultures, apparently by reducing expression of matrix metalloproteinase 2 (MMP2), which degrades fibronectin and other ECM components. Thus, p38 inhibition may suppress chondrogenesis by delaying progression from mesenchymal condensation to overt chondrocyte differentiation."

So ERK1/2 is important for mesenchymal condensation whereas p38 is important for the cells in the condensation to express chondrogenic genes.

"In micromass cultures, BMP2 treatment triggers p38 activation in limb mesenchyme cells and stimulates increased chondrogenesis by downregulating Wnt7a/β-catenin signals that promote Sox9 protein ubiquitination and degradation. In cultures of embryonic chick limb bud mesenchyme, TGF-β3 treatment was shown to enhance both phosphorylation of ATF2 and production of Alcian blue-positive ECM. Treatment with PD169316 was able to block these TGF-β3-induced increases in ATF2 phosphorylation and chondrogenesis"

"The stimulatory actions of Wnt5a on chondrogenesis in micromass cultures are dependent on the activation of both p38 and PKC signaling. In contrast, the inhibitory actions of retinoid compounds on chondrocyte differentiation involve suppression of p38 signaling in cultured limb mesenchyme. Activation of the p38 pathway was able to rescue the inhibitory effects of retinoic acid receptor (RAR)-mediated signaling on Sox9 protein levels in prechondrogenic limb mesenchyme, while inhibition of RAR signaling increased activation of the ATF2 transcription factor, a nuclear target of p38"

"Once the cartilage precursors of the long bones are established, signaling through the p38 pathway functions to suppress the maturation of hyaline chondrocytes into hypertrophic chondrocytes, thereby retarding any subsequent endochondral ossification events"

"The level of p38 signaling in chondrocytes [may have to] be held within normative limits to enable the progression of immature chondrocytes to hypertrophy, such that overly high or low p38 activity interferes with hypertrophy and endochondral ossification. This could reconcile the apparent paradox that upregulation of p38 signaling in ca-MKK6 transgenic mice inhibits chondrocyte hypertrophy and endochondral ossification, while inhibition of p38 activity in various in vitro models induces similar effects."

"treatment with JNK inhibitor SP600125 has no impact on cartilage matrix formation when applied to micromass cultures preparedfrom embryonic chick wing bud, frontonasal, or mandibular mesenchyme cells"

"cyclin-dependent kinase 6 (Cdk6), a promoter of the G1-S cell-cycle transition, is regulated by p38 during chondrogenesis. It is hypothesized that cell-cycle stoppage at G1-S is required for differentiation. the expression of Cdk6 decreases along the ATDC5 cell chondrogenic timeline and that Cdk6 overexpression represses ATDC5 cell chondrogenic differentiation as indicated by reduced collagen type II and X mRNA levels. the p38 inhibitor SB203580 blocked the depression in Cdk6 levels induced by incubation in chondrogenic differentiation medium, indicating that p38 is responsible for promoting chondrogenesis by decreasing Cdk6 levels. MEK inhibition via PD98059 had no effect on Cdk6 levels."

"FGFs 2 and 18 were able to stimulate ERK phosphorylation. pretreatment of the cells with CNP attenuated the effect of the FGFs on ERK phosphorylation"

"T3 enhanced both FGF2- and FGF18-induced ERK phosphorylation, while blocking the activation of Stat1 stimulated by these FGFs. T3 inhibited both EGF- and PDGF-induced activation of the ERK signaling pathway."

"in C3H10T1/2 cells induced to differentiate via BMP2 treatment, [the] siRNA knockdown of Gas6, a γ-carboxylated glutamic acid protein, enhanced chondrogenesis whereas addition of recombinant Gas6 inhibited chondrogenic differentiation"

"TGF-β1-induced chondrogenesis of trabecular bone-derived MSCs is positively transduced by ERK, p38, and JNK; and these three MAPKs promote chondrogenesis by enhancing cell adhesion through elevated N-cadherin levels and repressing β-catenin-transduced canonical Wnt signaling."

Genetic inactivation of ERK1 and ERK2 in chondrocytes promotes bone growth and enlarges the spinal canal.

"Activating mutations in FGFR3 cause the most common forms of human dwarfism: achondroplasia and thanatophoric dysplasia. In mouse models of achondroplasia, the ERK MAPK pathway, a pathway activated by FGFR3, [creates] reduced bone growth. Increased Fgfr3 and ERK MAPK signaling in chondrocytes{although ERK signaling in MSCs may be anabolic and encourage MSC differentiation into chondrocytes} also causes premature synchondrosis closure in the cranial base and vertebrae, accounting for the sometimes fatal stenosis of the foramen magnum and spinal canal in achondroplasia. Conversely, whether the decrease--or inactivation--of ERK1 and ERK2 promotes bone growth and delays synchondrosis closure remains to be investigated. In this study, we inactivated ERK2 in the chondrocytes of ERK1-null mice using the Col2a1-Cre and Col2a1-CreER transgenes{since this is specific to Col2a1 it would not be the activation of ERK in MSCs which is where ERK seems to be chondroinductive}.  Genetic inactivation of ERK1 and ERK2 in chondrocytes enhances the growth of cartilaginous skeletal elements. Postnatal inactivation of ERK1 and ERK2 in chondrocytes delays synchondrosis closure and enlarges the spinal canal."

"The inactivation of all four alleles of ERK1 and ERK2 resulted in longer and wider epiphyses of the long bones and larger vertebral bodies. "

"ERK1/2/Col2a1Cre embryos show a remarkable expansion of the zone of hypertrophic chondrocytes in the long bones, while chondrocyte proliferation is strongly inhibited. It has been shown that chondrocyte hypertrophy is a major determinant of longitudinal bone growth. Chondrocyte hypertrophy is also likely to account for the growth in the width in addition to periosteal apposition. Since ERK1/2/Col2a1Cre embryos do not show an obvious periosteal phenotype, periosteal apposition may not be significantly affected in these embryos. The increase in the size of the vertebral body is also associated with the predominant presence of hypertrophic chondrocytes "

P38 mitogen-activated protein kinase promotes dedifferentiation of primary articular chondrocytes in monolayer culture.

"Mitogen-activated protein kinase (MAPK) signalling is crucial for chondrocyte metabolism and matrix production, and changes in MAPK signals can affect the phenotype of cultured cells. We investigated the effects of inhibition of MAPK signalling on chondrocyte dedifferentiation during monolayer culture. Blockade of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signalling caused a significant increase in cartilage gene expression, however, also caused up-regulation of fibrotic gene expression. Inhibition of p38 MAPK (p38) caused a significant up-regulation of collagen type II while suppressing collagen type I expression. P38 inhibition also resulted in consistently more organized secretion of collagen type II protein deposits on cell culture surfaces. Follow-on pellet culture of treated cells revealed that MAPK inhibition reduced cell migration from the pellet. ERK and JNK inhibition caused more collagen type I accumulation in pellets versus controls while p38 inhibition strongly promoted collagen type II accumulation with no effect on collagen type I. Blockade of all three MAPKs caused increased GAG content in pellets."

The fibrogenic marker was Col1a2.  Chonrogenic markers were Col2a1, Acan, Sox9, and COMP.

"inhibition of JNK caused a significant decrease in viability which strongly correlated with a significant increase in cells stained positive for the active apoptosis marker cleaved caspase-3"

"ERK and JNK blockade caused up-regulation of the fibrotic marker collagen type I (Col1a2)"

The Ras-GTPase activity of neurofibromin restrains ERK-dependent FGFR signaling during endochondral bone formation.

"The severe defects in growth plate development caused by chondrocyte ERK1/2 gain or loss-of-function suggest that tight spatial and temporal regulation of MAPK signaling is necessary to achieve harmonious growth plate elongation and structure.  Neurofibromin, via its Ras GTPase-activating activity, controls ERK1/2-dependent FGFR signaling in chondrocytes. We show first that neurofibromin is expressed in FGFR-positive prehypertrophic and hypertrophic chondrocytes during growth plate endochondral ossification. Using mice lacking Nf1 in type II collagen-expressing cells, (Nf1col2-/- mutant mice), we then show that lack of neurofibromin in post-mitotic chondrocytes triggers a number of phenotypes reminiscent of the ones observed in mice characterized by FGFR gain-of-function mutations. Those include dwarfism, constitutive ERK1/2 activation, strongly reduced Ihh expression and decreased chondrocyte proliferation and maturation, increased chondrocytic expression of Rankl, Mmp9 and Mmp13 and enhanced growth plate osteoclastogenesis, as well as increased sensitivity to caspase-9 mediated apoptosis. Using wildtype (WT) and Nf1-/- chondrocyte cultures in vitro, we show that FGF2 pulse-stimulation triggers rapid ERK1/2 phosphorylation in both genotypes, but that returns to basal level is delayed in Nf1-/- chondrocytes. Importantly, in vivo ERK1/2 inhibition by daily injection of a recombinant form of C-type natriuretic peptide (CNP) to post-natal pups for 18 days was able to correct the short stature of Nf1col2-/- mice. Together, these results underscore the requirement of neurofibromin and ERK1/2 for normal endochondral bone formation and support the notion that neurofibromin, by restraining RAS-ERK1/2 signaling, is a negative regulator of FGFR signaling in differentiating chondrocytes."

"lack of Spred2, an inhibitor of FGF-induced MAPK signaling that binds to Ras and inhibits phosphorylation of Raf-1, causes an achondroplasia-like dwarfism phenotype. The product of the NF1 gene, neurofibromin, is a large cytoplasmic protein with a small central region that shares homology with GTPase-activating family proteins (GAP).  Through its GAP domain, it negatively regulates p21-Ras in multiple cell types"

"Nf1 mRNA expression increased during chondrocyte differentiation, with a pattern similar to the one of Fgfr1 expression"

NF1 inhibition did not affect the expression of the receptor for CNP(Npr-B).