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 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.
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.
"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.
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"
"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"
"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.
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.
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.
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
"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.
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."
"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)."
"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.
"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"
"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.
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).
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).
Hi Tyler what do you think about height loss that occurs with age? You are in your late 20s so in your height peak, aren't you concerned with height loss that usually begins in yout 30s? What parts of the body shrink , disks ,vertebras or other bones?
ReplyDelete