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. "