Tuesday, August 7, 2012


The critical role of the epidermal growth factor receptor in endochondral ossification.

"Loss of epidermal growth factor receptor (EGFR) {LSJL upregulates EGFR 2-fold} activity in mice alters growth plate development, impairs endochondral ossification, and retards growth. Administration of an EGFR-specific small-molecule inhibitor, gefitinib, into 1-month-old rats for 7 days produced profound defects in long bone growth plate cartilage characterized by epiphyseal growth plate thickening and massive accumulation of hypertrophic chondrocytes. Growth plate chondrocytes express EGFR, but endothelial cells and osteoclasts show little to no expression{so increase in fold factor of EGFR may be a sign of increased chondrogenic differentiation}. Gefitinib did not alter chondrocyte proliferation or differentiation and vascular invasion into the hypertrophic cartilage.  osteoclast recruitment and differentiation at the chondro-osseous junction were attenuated owing to decreased RANKL expression in the growth plateGefitinib treatment inhibited the expression of matrix metalloproteinases (MMP-9, -13, and -14){MMP14 increased 2-fold due to LSJL}, increased the amount of collagen fibrils, and decreased degraded extracellular matrix products in the growth plate. In vitro, the EGFR ligand transforming growth factor α (TGF-α) strongly stimulated RANKL and MMPs expression and suppressed osteoprotegerin (OPG) expression in primary chondrocytes. A mouse model of cartilage-specific EGFR inactivation exhibited a similar phenotype of hypertrophic cartilage enlargement. EGFR signaling supports osteoclastogenesis at the chondro-osseous junction and promotes chondrogenic expression of MMPs in the growth plate."

"The epidermal growth factor receptor (EGFR) belongs to a tyrosine kinase receptor family that also contains ErbB2, ErbB3 {upregulated by LSJL}, and ErbB4. Its ligands include EGF, amphiregulin{Areg an amphiregulin pre-protein is upregulated by LSJL}, and transforming growth factor α (TGF-α), which only bind to EGFR, and heparin-binding EGF (HB-EGF){HBEGF is upregulated by LSJL}, betacellulin, and epiregulin, which bind to both EGFR and ErbB4"

"Upon ligand binding, EGFR either homodimerizes or heterodimerizes with other ErbBs. The dimerized receptors then undergo auto- or transphosphorylation on tyrosine residues in the intracellular domain, thus activating several important intracellular signal-transduction pathways, including Ras-Raf-MAP-kinase and PI-3-kinase-Akt. ErbB2 is the preferred partner for EGFR, and the signals mediated by EGFR/ErbB2 are thought to account for most of EGFR's biologic activities."

"Overexpression of herstatin, a soluble ErbB2 receptor that acts in a dominant-negative fashion to inhibit EGFR signaling, under the limb-specific Prx1 promoter in mice resulted in shortened limbs and an expanded hypertrophic zone"

"This change in the growth plate was reversible because the length of the growth plate returned to normal 1 week after withdrawal of the EGFR inhibitor"

"osteoblasts express EGFR but that osteoclast progenitors and mature osteoclasts do not bind EGF ligand and do not possess functional EGFR"<-Thus osteoblasts could account for some increase in EGFR expression.

"RNA expression of Vegfa, along with another angiogenesis factor, transferrin, was increased about 5.3- and 4.8-fold, respectively, in the growth plate after gefitinib treatment, which is consistent with expansion of the hypertrophic zone."

"knocking down specific MMP genes, such as Mmp9, Mmp13,2 and Mmp14, diminishes matrix degradation and expands the growth plate"

"protein levels of MMP-9, -13, and -14 in the growth plates from gefitinib-treated rats were decreased to 7.9% ± 2.1%, 14.0% ± 12.6%, and 39.2% ± 10.5%"<-this is inconsistent in the fact that MMP14 was the only one of these three MMP's that increased above threshold.  According to this data, with an EGFR increase you'd expect MMP9 and MMP13 to increase more.

"To form new bone, hypertrophic chondrocytes and osteoclasts need to partially degrade the growth plate ECM at the COJ, generating space for osteoblasts to deposit bone matrix. MMPs, in particular MMP-9, -13, and -14, are key enzymes involved in this process. MMP-13, also named collagenase 3, is the major protease that degrades native type 2 collagen. MMP-9 and -13 together are responsible for MMP-dependent aggrecan cleavage."

"MMP9, -13, and -14 are expressed by cells in and surrounding the growth plate. MMP-13 is expressed mainly in hypertrophic chondrocytes and osteoblasts. Cells with high MMP-9 expression are osteoclasts and TRAP− cells of unknown lineage beneath the hypertrophic cartilage. MMP-9 expression was identified in the hypertrophic chondrocytes of mouse cartilage and throughout the growth plate in 4-week-old rats. MMP-14 is expressed mainly in the resting and proliferative zones, whereas the hypertrophic zone showed little expression. MMP-14 is abundant in osteoclasts and exhibits intense staining in the region beneath the COJ."<-The elevated levels of MMP-14 over MMP9 and MMP13 is consistent with that LSJL induces chondrogenic differentiation and osteoclast remodeling which would be needed for new growth plate formation.

"Activation of EGFR signaling strongly stimulates chondrocyte proliferation, inhibits chondrogenesis, and suppresses the expression of chondrocyte matrix genes."

"During limb development, epithelial cells in the apical ectodermal ridge keep the underlying mesenchymal cells in a proliferative state preventing differentiation by secreting signaling molecules such as epidermal growth factor (EGF). Micromass culture-induced chondrogenesis of chick limb bud mesenchymal cells [was the] model system. Expression and tyrosine phosphorylation of the EGF receptor was increased transiently during chondrogenesis. Exogenous EGF inhibited chondrogenic differentiation of mesenchymal cells, and this effect was reversed by the EGF receptor inhibitor AG1478. EGF treatment also inhibited the expression and activation of protein kinase C-alpha, whereas it activated Erk-1 and inhibited p38 mitogen-activated protein kinase{Both ERK and p38 pathways are involved in LSJL but not clear how}, all of which appeared to be involved in the EGF-induced inhibition of chondrogenesis{we can't know about the activation of Erk-1 and p38 MAPK as LSJL does not present phosphorylation data}. Stimulation of the EGF receptor blocked precartilage condensation and altered the expression of cell adhesion molecules such as N-cadherin and integrins alpha(5){altered expression by LSJL is predicted} and beta(1). All these EGF effects were reversible by AG1478. EGF negatively regulate chondrogenesis of chick limb bud mesenchymal cells by inhibiting precartilage condensation and by modulating signaling pathways including those of protein kinase C-alpha, Erk-1, and p38 mitogen-activated protein kinase."

"Precartilage condensations differentiate into cartilage nodules where differentiated chondrocytes are located"<-this would be critical to form new growth plates.

"Limb mesenchymal cells also undergo spontaneous differentiation to chondrocytes in vitro when cultured at a high seeding density such as in micromass culture. During in vitro chondrogenesis, mesenchymal cells proliferate increasing the number of chondrogenic competent cells that undergo extensive cell to cell and cell to matrix interactions."<-LSJL by compressing the bone may alter the density of MSCs facilitating chondrocyte differentiation.

PRKCDP is upregulated by LSJL a PKC binding protein.  Protein Kinase C gamma is downregulated by LSJL.  "protein kinase C (PKC) positively regulates chondrogenesis of mesenchymal cells"

"Among the PKC isoforms expressed in differentiating mesenchymal cells, such as α, ε, ζ[delta], and λ/ι[gamma], the expression of both PKCα and less dramatically PKCε increased as chondrogenesis proceeded, whereas the expression of PKCζ and λ/ι did not vary significantly during chondrogenesis"

"The increase in the expression of PKCα was completely inhibited when the cells were treated with EGF, and the EGF effect could be blocked by inhibition of EGFR with AG1478. In contrast to PKCα, the amounts and expression patterns of the other PKC isoforms were essentially the same between cells cultured either in the absence or presence of EGF"

"[condensation] takes place during days 1–2 and chondrogenesis happens during days 3–5, the altered expression of adhesion molecules by EGF signaling appears to be associated with the formation of cartilage nodules rather than precartilage condensation."<-LSJL gene expression was measured on day 3 so maybe there wasn't EGF expression between days 1-2 to inhibit chondrogenesis and only appeared on day 3.

Disruption of actin cytoskeleton induces chondrogenesis of mesenchymal cells by activating protein kinase C-alpha signaling.

"Disruption of actin cytoskeleton with cytochalasin D [can] induce chondrogenic differentiation of chick embryo limb bud mesenchymal cells.  Protein kinase C (PKC) and extracellular signal-regulated protein kinase (Erk-1) [is involved] in chondrogenesis of mesenchymal cells induced by disruption of actin cytoskeleton. Disruption of actin cytoskeleton with cytochalasin D or latrunculin B induced chondrogenesis of mesenchymal cells cultured at subconfluent cell density, as determined by type II collagen expression. Among the expressed PKC isoforms, cytochalasin D dramatically increased expression and activation of PKCalpha in a dose-dependent manner, and inhibition or downregulation of PKCalpha blocked cytochalasin D-induced chondrogenesis. Cytochalasin D downregulated Erk-1 phosphorylation that is associated with chondrogenesis. Disruption of actin cytoskeleton induces chondrogenesis of mesenchymal cells by activating PKCalpha and by inhibiting Erk-1 signaling."

"Although the expression of Erk-1 was not changed, its phosphorylation decreased during cytochalasin D-induced chondrogenesis"

"inhibition of Erk-1 phosphorylation by PD98059 is not sufficient to enhance chondrogenesis by cytochalasin D."

Immunosuppressant rapamycin inhibits protein kinase C alpha and p38 mitogen-activated protein kinase leading to the inhibition of chondrogenesis.

"we examined the effects of the immunosuppressant rapamycin on the chondrogenesis of mesenchymal cells and on the cell signaling that is required for chondrogenesis, such as protein kinase C, extracellular signal-regulated kinase-1 (ERK-1), and p38 mitogen-activated protein (MAP) kinase pathways. Rapamycin inhibited the expression of type II collagen and the accumulation of sulfate glycosaminoglycan, indicating inhibition of the chondrogenesis of mesenchymal cells. Rapamycin treatment did not affect precartilage condensation, but it prevented cartilage nodule formation. Exposure of chondrifying mesenchymal cells to rapamycin blocked activation of the protein kinase C alpha and p38 MAP kinase, but had no discernible effect on ERK-1 signaling. Selective inhibition of PKCalpha or p38 MAP kinase activity, which is dramatically increased during chondrogenesis, with specific inhibitors in the absence of rapamycin blocked the chondrogenic differentiation of mesenchymal cells."

"In comparison to a density of 2×107 cells/ml, in cells cultured at a density of 4×107 cells/ml there was 218% and 263% increase in chondrogenesis and cell number, respectively"

"Phosphorylation of p70 S6 kinase was completely abolished when mesenchymal cells were treated with 1 nM rapamycin. However, the phosphorylation state of p70 S6 kinase was not affected by the inhibition of protein kinase C with Go6976, ERK-1 with PD98059, or p38 MAP kinase with SB203580"

Transient anabolic effects accompany epidermal growth factor receptor signal activation in articular cartilage in vivo.

"Transgenic mice with conditional, limb-targeted deletion of the endogenous intracellular EGFR inhibitor Mig-6 were generated using CreLoxP (Mig-6-flox;Prx1Cre) recombination.
The articular cartilage of Mig-6-conditional knockout (Mig-6-cko) mice was dramatically and significantly thicker than normal articular cartilage at 6 and 12 weeks of age{so stimulating EGFR by lowering levels of the inhibitor Mig-6 increases articular cartilage thickness, would this have an effect on growth plate cartilage and does this increase in thickness increase height by increasing knee height?}. Mig-6-cko articular cartilage contained a population of chondrocytes in which EGFR signaling was activated, and which were 3-4 times more proliferative than normal Mig-6-flox articular chondrocytes. These cells expressed high levels of the master chondrogenic regulatory factor Sox9, as well as high levels of putative progenitor cell markers including superficial zone protein (SZP), growth and differentiation factor-5 (GDF-5), and Notch1. Expression levels were also high for activated beta-catenin and the transforming growth factor beta (TGF-beta) mediators phospho-Smad2/3 (pSmad2/3). Anabolic effects of EGFR activation in articular cartilage were followed by catabolic events including matrix degradation as determined by accumulation of aggrecan cleavage fragments, and onset of hypertrophy as determined by type X collagen expression{maybe enhancement of EGFR causes progression towards endochondral ossification?}. By 16 weeks of age, the articular cartilage of Mig-6-cko knees was no longer thickened, and was degenerating."

"EGFR signals typically suppress chondrogenic differentiation and/or homeostasis. For example, in vitro studies show that EGFR signals suppress initial chondrogenic differentiation by limb mesenchymal cells"

"The articular cartilage of the femoral surfaces of Mig-6-cko joints was also increased (283 ±19um thick for Mig-6-cko, compared to 132 ±16um thick for control Mig-6-flox" <-this could be an increase of up to double knee height.

"Mig-6-cko knee joints also contained thickened lateral and central ligaments which stained
intensely with Safranin-O, abundant connective tissue, and enlarged menisci"<-so stimulating EGFR stimulated other things that could increase knee height.

"At 6 weeks of age EGFR signaling in normal tibial articular cartilage was limited to the
superficial zone. In the normal knee at 12 weeks of age, few superficial chondrocytes were EGFR-positive, but EGFR-positive chondrocytes were relatively abundant in the calcified zone adjacent to the chondro-osseous junction, as well as in the subchondral bone itself. In Mig-6-cko knee articular cartilage, EGFR signaling was dramatically enhanced in these regions compared to controls."

"The knee joints of 16 week old Mig-6-cko mice{at which point articular cartilage thickness was similar to controls} contained fused and highly chondrified central ligaments; thickened and fibrogenic menisci; reduced subchondral bone area; and prominent central and lateral osteophytes"

"Mig-6 can directly bind to and inhibit signal transduction by the EGFR-related receptor, ErbB2"<-Mig-6 can also inhibit the ERK pathway.

The scientists do not believe that egfr causes dedifferentiation of articular cartilage chondrocytes which would increase proliferation due to the fact that the stimulation of articular cartilage thickness occurs so early in development.

"at 6 weeks of age, which is when the Mig-6-cko articular cartilage is thickest, and proliferation is greatest, hypertrophic chondrocytes are not yet detected. Anabolic effects of EGFR signal activation precede catabolic ones, and are not necessarily coincident."

Or transient activation of EGFR has anabolic effects but too much results in negative feedback.

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