"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.
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"
"[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"
"Constitutive active mutations in more downstream genes such as KRAS and BRAF result in an increased activation of the MAPK/ERK pathway and hence in short stature syndromes"
"Fibroblast Growth Factor 13 (FGF13) [is one] of the most down regulated gene in SoS"
Regulation of cartilage formation and maturation by mitogen-activated protein kinase signaling.
"Prechondrogenic mesenchyme cells migrate to the site of the prospective skeletal element. Once there, the mesenchymal cells assemble into compact cellular aggregates, or condensations, a process that is mediated by cell adhesion molecules such as neural cadherin (N-cadherin) and neural cell adhesion molecule (N-CAM), as well as extracellular matrix (ECM) components such as fibronectin and syndecan. These prechondrogenic condensations serve to establish the size and position of each nascent cartilage anlage. Next, the close proximity of the aggregated mesenchymal cells permits critical cell–cell surface interactions and signaling events that initiate intracellular changes culminating in the activation of overt chondrocyte differentiation. In this stage, the chondrogenic progenitor cells exchange their stellate, fibroblastic-like phenotype for the spherical morphology of chondrocytes and commence synthesis of cartilage-specific ECM molecules such as collagen types II, IX, and XI and the highly sulfated proteoglycan, aggrecan"
"transcription factors implicated in early-stage chondrogenic differentiation and skeletal patterning include Msx1 and 2, β-catenin, lymphocyte enhancer-binding factor 1 (Lef1), AP-1 , AP-2, and Runx2/Cbfa1"
"The ERK1/2 pathway is triggered by a set of adaptor proteins, such as Shc, growth factor receptor-bound protein 2 (GRB2), and paired box (Pax), that link the receptor to a guanine nucleotide exchange factor, such as son of sevenless (Sos), CRK SH3-binding guanine nucleotide releasing factor (C3G), or exchange protein directly activated by cAMP (EPAC). These GDP/GTP exchange factors subsequently activate small GTP binding proteins, such as Ras, Rac, and repressor activator protein 1 (Rap1), which, in turn, activate the Raf family of serine threonine kinases (A-Raf, B-Raf, and C-Raf) at the top of the triple kinase phosphorelay unit. The Raf kinases activate MEK1 and MEK2. MEK1 and 2 activate ERK1 and ERK2, their only characterized downstream substrates, by dual phosphorylation at a conserved TEY motif "
"ERK1/2 activation results in dissociation from its cytoplasmic anchor MEK1/2 (Adachi et al.,1999), and phosphorylation of various nuclear, cytosolic, and cytoskeletal substrates such as Ets-like protein-1 (Elk-1), ribosomal S6 kinase (Rsk), and Tau, respectively"
"Common substrates of p38 include other kinases: p38-regulated/activated protein kinase (PRAK), mitogen- and stress-activated kinase 1 (MSK1), and MAPK-interacting kinases 1 and 2 (MNK1/2); as well as transcription factors, including myocyte enhancer factor 2 (MEF2), activating transcription factor 2 (ATF2), and C/EBP homologous protein (CHOP)"
"JNK substrates include c-jun, Elk-1, ATF2, and serum response factor accessory protein 1a (Sap1a)"
"incubation of whole chick or mouse embryos with SU5402, a pharmacological inhibitor of FGF receptor tyrosine kinase (FGFR) activity, strongly depresses ERK1/2 phosphorylation at most locations"
"The prechondrogenic mesenchyme of the embryonic limbs is derived from somatopleural mesoderm of the lateral body wall. At the time of limb bud initiation, the mesodermal cells of the prospective limb-forming region secrete FGF10, which signals to the overlying surface ectoderm, leading to the formation of a thickened apical ectodermal ridge (AER). The AER at the distal tip of the limb bud, in turn, secretes FGF8 and other growth factors that stimulate proliferation of the underlying limb mesenchyme and inhibit precocious differentiation of the mesodermal cells that reside in the region immediately subjacent to the AER. Chondrogenic differentiation is initiated as the proximodistal outgrowth of the limb bud allows some mesodermal cells to emerge from the region of AER influence (known as the progress or subridge zone) and begin forming prechondrogenic condensations. Thus, chondrogenesis is initiated in the proximal limb region, but as limb outgrowth continues, the mesenchymal condensations corresponding to more distal cartilage elements are progressively established, culminating in formation of the phalangeal primordia. Importantly, the condensation process initiates expression of the Sox9 transcription factor, which then activates the genes for the principle cartilage matrix components (collagen type II and aggrecan) and triggers overt differentiation of limb mesenchyme cells into hyaline chondrocytes."
"Treatment of limb mesenchyme micromass cultures with MEK1/2 inhibitor PD98059 accelerated the decline in expression of cell adhesion molecules (N-cadherin, fibronectin, and α5β1 integrin) involved in the prechondrogenic condensation process"
ERK1/2 may be stimulatory towards forming growth plates but once they have been established may be inhibitory towards much like FGFR.
"the ERK1/2 cascade may relay signals that selectively inhibit hypertrophic chondrocyte differentiation within the growth plate, while a different signal transduction pathway mediates the inhibitory effects of FGFR3 on chondrocyte proliferation. This additional relay system appears to involve signal transducer and activator of transcription 1 (Stat1) activation, as ablation of Stat1 function restored normal rates of chondrocyte proliferation in mice that express the achondroplasia-type FGFR3 mutation in cartilage tissues"
"Stat1 deletion only minimally restored bone growth, therefore, elevated ERK signaling may be predominantly responsible for the dwarfism phenotype in mice that express activating FGFR3 mutations."
"prolongation of Sox9 expression induced by upregulated ERK1/2 signaling might account for the delay in hypertrophic maturation of cartilages expressing the ca-MEK transgene, because chondrocytes undergoing hypertrophy normally cease Sox9 expression"<-although phosphorylation of Sox9 may be an alternative to ceasing Sox9 expression.
"the Sox9 transcription factor normally acts to inhibit the maturation of hyaline chondrocytes into hypertrophic chondrocytes."
"The levels of ERK activity in growth plate chondrocytes [may need to] be tightly controlled to allow proper hypertrophic maturation, and that either insufficient or excessive ERK pathway signaling interferes with hypertrophy and subsequent mineralization. Expression of either constitutively active C-Raf or dominant negative C-Raf decreased activation of the collagen type X promoter in the MCT chondrogenic cell line."
"Treatments with any one of several pharmacological inhibitors of p38 (e.g., SB203580, PD169316, SB202190, or SB220025) have consistently been shown to suppress cartilage matrix production in micromass cultures of embryonic chick or mouse limb mesenchyme cells, and also to inhibit expression of mRNAs for the chondrocyte marker genes, sox9, col2a1, and aggrecan"
"Transfection of prechondrogenic limb mesenchyme with constitutively active MKK6, a MAPKK that activates p38, was shown to markedly increase expression of a Sox9-responsive reporter gene"
"p38 and MEK inhibition have opposite effects on the expression of cell adhesion molecules involved in mesenchymal condensation. Treatment with the p38 inhibitor SB203580 causes increased expression of N-cadherin, fibronectin, and α5β1 integrin in limb mesenchyme cultures, apparently by reducing expression of matrix metalloproteinase 2 (MMP2), which degrades fibronectin and other ECM components. Thus, p38 inhibition may suppress chondrogenesis by delaying progression from mesenchymal condensation to overt chondrocyte differentiation."
So ERK1/2 is important for mesenchymal condensation whereas p38 is important for the cells in the condensation to express chondrogenic genes.
"In micromass cultures, BMP2 treatment triggers p38 activation in limb mesenchyme cells and stimulates increased chondrogenesis by downregulating Wnt7a/β-catenin signals that promote Sox9 protein ubiquitination and degradation. In cultures of embryonic chick limb bud mesenchyme, TGF-β3 treatment was shown to enhance both phosphorylation of ATF2 and production of Alcian blue-positive ECM. Treatment with PD169316 was able to block these TGF-β3-induced increases in ATF2 phosphorylation and chondrogenesis"
"The stimulatory actions of Wnt5a on chondrogenesis in micromass cultures are dependent on the activation of both p38 and PKC signaling. In contrast, the inhibitory actions of retinoid compounds on chondrocyte differentiation involve suppression of p38 signaling in cultured limb mesenchyme. Activation of the p38 pathway was able to rescue the inhibitory effects of retinoic acid receptor (RAR)-mediated signaling on Sox9 protein levels in prechondrogenic limb mesenchyme, while inhibition of RAR signaling increased activation of the ATF2 transcription factor, a nuclear target of p38"
"Once the cartilage precursors of the long bones are established, signaling through the p38 pathway functions to suppress the maturation of hyaline chondrocytes into hypertrophic chondrocytes, thereby retarding any subsequent endochondral ossification events"
"The level of p38 signaling in chondrocytes [may have to] be held within normative limits to enable the progression of immature chondrocytes to hypertrophy, such that overly high or low p38 activity interferes with hypertrophy and endochondral ossification. This could reconcile the apparent paradox that upregulation of p38 signaling in ca-MKK6 transgenic mice inhibits chondrocyte hypertrophy and endochondral ossification, while inhibition of p38 activity in various in vitro models induces similar effects."
"treatment with JNK inhibitor SP600125 has no impact on cartilage matrix formation when applied to micromass cultures preparedfrom embryonic chick wing bud, frontonasal, or mandibular mesenchyme cells"
"cyclin-dependent kinase 6 (Cdk6), a promoter of the G1-S cell-cycle transition, is regulated by p38 during chondrogenesis. It is hypothesized that cell-cycle stoppage at G1-S is required for differentiation. the expression of Cdk6 decreases along the ATDC5 cell chondrogenic timeline and that Cdk6 overexpression represses ATDC5 cell chondrogenic differentiation as indicated by reduced collagen type II and X mRNA levels. the p38 inhibitor SB203580 blocked the depression in Cdk6 levels induced by incubation in chondrogenic differentiation medium, indicating that p38 is responsible for promoting chondrogenesis by decreasing Cdk6 levels. MEK inhibition via PD98059 had no effect on Cdk6 levels."
"FGFs 2 and 18 were able to stimulate ERK phosphorylation. pretreatment of the cells with CNP attenuated the effect of the FGFs on ERK phosphorylation"
"T3 enhanced both FGF2- and FGF18-induced ERK phosphorylation, while blocking the activation of Stat1 stimulated by these FGFs. T3 inhibited both EGF- and PDGF-induced activation of the ERK signaling pathway."
"in C3H10T1/2 cells induced to differentiate via BMP2 treatment, [the] siRNA knockdown of Gas6, a γ-carboxylated glutamic acid protein, enhanced chondrogenesis whereas addition of recombinant Gas6 inhibited chondrogenic differentiation"
"TGF-β1-induced chondrogenesis of trabecular bone-derived MSCs is positively transduced by ERK, p38, and JNK; and these three MAPKs promote chondrogenesis by enhancing cell adhesion through elevated N-cadherin levels and repressing β-catenin-transduced canonical Wnt signaling."
Genetic inactivation of ERK1 and ERK2 in chondrocytes promotes bone growth and enlarges the spinal canal.
"Activating mutations in FGFR3 cause the most common forms of human dwarfism: achondroplasia and thanatophoric dysplasia. In mouse models of achondroplasia, the ERK MAPK pathway, a pathway activated by FGFR3, [creates] reduced bone growth. Increased Fgfr3 and ERK MAPK signaling in chondrocytes{although ERK signaling in MSCs may be anabolic and encourage MSC differentiation into chondrocytes} also causes premature synchondrosis closure in the cranial base and vertebrae, accounting for the sometimes fatal stenosis of the foramen magnum and spinal canal in achondroplasia. Conversely, whether the decrease--or inactivation--of ERK1 and ERK2 promotes bone growth and delays synchondrosis closure remains to be investigated. In this study, we inactivated ERK2 in the chondrocytes of ERK1-null mice using the Col2a1-Cre and Col2a1-CreER transgenes{since this is specific to Col2a1 it would not be the activation of ERK in MSCs which is where ERK seems to be chondroinductive}. Genetic inactivation of ERK1 and ERK2 in chondrocytes enhances the growth of cartilaginous skeletal elements. Postnatal inactivation of ERK1 and ERK2 in chondrocytes delays synchondrosis closure and enlarges the spinal canal."
"The inactivation of all four alleles of ERK1 and ERK2 resulted in longer and wider epiphyses of the long bones and larger vertebral bodies. "
"ERK1/2/Col2a1Cre embryos show a remarkable expansion of the zone of hypertrophic chondrocytes in the long bones, while chondrocyte proliferation is strongly inhibited. It has been shown that chondrocyte hypertrophy is a major determinant of longitudinal bone growth. Chondrocyte hypertrophy is also likely to account for the growth in the width in addition to periosteal apposition. Since ERK1/2/Col2a1Cre embryos do not show an obvious periosteal phenotype, periosteal apposition may not be significantly affected in these embryos. The increase in the size of the vertebral body is also associated with the predominant presence of hypertrophic chondrocytes "
P38 mitogen-activated protein kinase promotes dedifferentiation of primary articular chondrocytes in monolayer culture.
"Mitogen-activated protein kinase (MAPK) signalling is crucial for chondrocyte metabolism and matrix production, and changes in MAPK signals can affect the phenotype of cultured cells. We investigated the effects of inhibition of MAPK signalling on chondrocyte dedifferentiation during monolayer culture. Blockade of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signalling caused a significant increase in cartilage gene expression, however, also caused up-regulation of fibrotic gene expression. Inhibition of p38 MAPK (p38) caused a significant up-regulation of collagen type II while suppressing collagen type I expression. P38 inhibition also resulted in consistently more organized secretion of collagen type II protein deposits on cell culture surfaces. Follow-on pellet culture of treated cells revealed that MAPK inhibition reduced cell migration from the pellet. ERK and JNK inhibition caused more collagen type I accumulation in pellets versus controls while p38 inhibition strongly promoted collagen type II accumulation with no effect on collagen type I. Blockade of all three MAPKs caused increased GAG content in pellets."
The fibrogenic marker was Col1a2. Chonrogenic markers were Col2a1, Acan, Sox9, and COMP.
"inhibition of JNK caused a significant decrease in viability which strongly correlated with a significant increase in cells stained positive for the active apoptosis marker cleaved caspase-3"
"ERK and JNK blockade caused up-regulation of the fibrotic marker collagen type I (Col1a2)"
The Ras-GTPase activity of neurofibromin restrains ERK-dependent FGFR signaling during endochondral bone formation.
"The severe defects in growth plate development caused by chondrocyte ERK1/2 gain or loss-of-function suggest that tight spatial and temporal regulation of MAPK signaling is necessary to achieve harmonious growth plate elongation and structure. Neurofibromin, via its Ras GTPase-activating activity, controls ERK1/2-dependent FGFR signaling in chondrocytes. We show first that neurofibromin is expressed in FGFR-positive prehypertrophic and hypertrophic chondrocytes during growth plate endochondral ossification. Using mice lacking Nf1 in type II collagen-expressing cells, (Nf1col2-/- mutant mice), we then show that lack of neurofibromin in post-mitotic chondrocytes triggers a number of phenotypes reminiscent of the ones observed in mice characterized by FGFR gain-of-function mutations. Those include dwarfism, constitutive ERK1/2 activation, strongly reduced Ihh expression and decreased chondrocyte proliferation and maturation, increased chondrocytic expression of Rankl, Mmp9 and Mmp13 and enhanced growth plate osteoclastogenesis, as well as increased sensitivity to caspase-9 mediated apoptosis. Using wildtype (WT) and Nf1-/- chondrocyte cultures in vitro, we show that FGF2 pulse-stimulation triggers rapid ERK1/2 phosphorylation in both genotypes, but that returns to basal level is delayed in Nf1-/- chondrocytes. Importantly, in vivo ERK1/2 inhibition by daily injection of a recombinant form of C-type natriuretic peptide (CNP) to post-natal pups for 18 days was able to correct the short stature of Nf1col2-/- mice. Together, these results underscore the requirement of neurofibromin and ERK1/2 for normal endochondral bone formation and support the notion that neurofibromin, by restraining RAS-ERK1/2 signaling, is a negative regulator of FGFR signaling in differentiating chondrocytes."
"lack of Spred2, an inhibitor of FGF-induced MAPK signaling that binds to Ras and inhibits phosphorylation of Raf-1, causes an achondroplasia-like dwarfism phenotype. The product of the NF1 gene, neurofibromin, is a large cytoplasmic protein with a small central region that shares homology with GTPase-activating family proteins (GAP). Through its GAP domain, it negatively regulates p21-Ras in multiple cell types"
"Nf1 mRNA expression increased during chondrocyte differentiation, with a pattern similar to the one of Fgfr1 expression"
NF1 inhibition did not affect the expression of the receptor for CNP(Npr-B).
"Ectopic cartilage induced: A histological time course of the effects of a single intramuscular injection of buffer or hBMP-2 protein into quadriceps muscles of C57B1/6J SCID females. A, C, E, G, and I correspond to muscles removed after 1, 3, 4, 7, and 14 days, respectively, following an injection of formulation buffer. B, D, F, H, and J correspond to muscles removed after 1, 3, 4, 7, and 14 days, respectively, following an injection of 50 μg of hBMP-2 protein. Muscles were fixed and stained with H&E as described under Methods. Abbreviations: M, normal skeletal muscle fiber; I, inflammatory cells; DM, degenerated skeletal muscle fiber; F, fibroplasia; Cb, chondroblast; C, chondrocyte; Ob, osteoblast; Oc, osteoclast; B, bone; BM, bone marrow. The bar in each panel = 0.1 mm."
