Microenvironmental changes during differentiation of mesenchymal stem cells towards chondrocytes.
"the induction of MSC differentiation towards chondrocytes might be induced and/or influenced by molecules from the microenvironment[through diet, supplementation, and loading we can influence the microenvironment]. We identified genes that are regulated during MSC differentiation towards chondrocytes [and the chondrogenic microenvironment]. Chondrogenesis of bone-marrow-derived human MSCs was induced by culture in micropellet for various periods of time. Total RNA was extracted. We identified molecules already known to be involved in attachment and cell migration, including syndecans, glypicans, gelsolin, decorin, fibronectin, and type II, IX and XI collagens. Importantly, we detected the expression of molecules that were not previously associated with MSCs or chondrocytes, namely metalloproteases (MMP-7 and MMP-28), molecules of the connective tissue growth factor (CTGF); cef10/cyr61{up in LSJL} and nov (CCN) family (CCN3 {up in LSJL} and CCN4), chemokines and their receptors chemokine CXC motif ligand (CXCL1), Fms-related tyrosine kinase 3 ligand (FlT3L), chemokine CC motif receptor (CCR3 and CCR4), molecules with A Disintegrin And Metalloproteinase domain (ADAM8{down in LSJL}, ADAM9, ADAM19, ADAM23, A Disintegrin And Metalloproteinase with thrombospondin type 1 motif ADAMTS-4{up} and ADAMTS-5), cadherins (4 and 13{up in LSJL}) and integrins (alpha4{down}, alpha7 and beta5). Our data suggest that crosstalk between ECM components of the microenvironment and MSCs within the cartilage is responsible for the differentiation of MSCs into chondrocytes"
"mesenchymal stem cells (MSCs) have been recently identified in articular cartilage and are thought to represent up to 3.5% of the constituent cells"<-If this many cells are in the AC how many would you think are in the epiphyseal bone marrow?
"normal chondrocytes adhere to various amounts of type I and IV collagens, thrombospondin, vitronectin, fibronectin, laminin and fibrinogen through the RGD (Arg-Gly-Asp) sequence and integrin-mediated interactions"
"MMP-28 [was expressed] and MMP-7 [was absent] in MSCs. During chondrogenesis, all the MMPs tested [were upregulated], except for MMP-9[MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-12 and MMP-28]"<-So probably the scientist is planning to use MMP-7 as an off switch to turn chondrogenesis off in the scaffold.
"Of the cell-surface components, syndecan-4{up} is more abundant than syndecan-2{down}, whereas syndecan-3 is briefly expressed during the early stages of chondrogenesis"
"MMP-9 was shown to be involved in regulating pericellular proteolysis for correct endochondral bone formation during the in vitro differentiation pathway and in vivo cartilage repair process"
"CX3CR1 is the only receptor that showed total inhibition of its mRNA during chondrogenesis (from day 2)"
"we report the expression of ADAM8, ADAM9, ADAM19, ADAM23, A Disintegrin-like And Metalloproteinase with thrombospondin type 1 motif (ADAMTS)-4 and ADAM-TS5 in MSCs and their upregulation at the late stage of chondrogenesis, in particular ADAMTS-4 and ADAMTS-5"
"chondrocytes express only ALCAM and VCAM1{down}"
Genes that were not expressed at day 0 but were expressed at later stages of differentiation(2-21) that were also upregulated during LSJL(thus which genes correlate with beginning of chondrogenesis):
Slc38a4
Kdr
Ccr1
"Of the cell-surface components, syndecan-4{up} is more abundant than syndecan-2{down}, whereas syndecan-3 is briefly expressed during the early stages of chondrogenesis"
"MMP-9 was shown to be involved in regulating pericellular proteolysis for correct endochondral bone formation during the in vitro differentiation pathway and in vivo cartilage repair process"
"CX3CR1 is the only receptor that showed total inhibition of its mRNA during chondrogenesis (from day 2)"
"we report the expression of ADAM8, ADAM9, ADAM19, ADAM23, A Disintegrin-like And Metalloproteinase with thrombospondin type 1 motif (ADAMTS)-4 and ADAM-TS5 in MSCs and their upregulation at the late stage of chondrogenesis, in particular ADAMTS-4 and ADAMTS-5"
"chondrocytes express only ALCAM and VCAM1{down}"
Genes that were not expressed at day 0 but were expressed at later stages of differentiation(2-21) that were also upregulated during LSJL(thus which genes correlate with beginning of chondrogenesis):
Slc38a4
Kdr
Ccr1
Col2a1{not expressed until day 21}
ENPP3{down}{also not expressed on day 2}
Genes that were expressed at day 0 but were not expressed at one or more of the later stages of differentiation 2, 7, 21 that were also upregulated during LSJL:
IL6{not expressed at days 2 and 21}
Ogn{down}{not expressed on day 7}
Wnt2{not expressed at days 7 and 21}
ENPP3{down}{also not expressed on day 2}
Genes that were expressed at day 0 but were not expressed at one or more of the later stages of differentiation 2, 7, 21 that were also upregulated during LSJL:
IL6{not expressed at days 2 and 21}
Ogn{down}{not expressed on day 7}
Wnt2{not expressed at days 7 and 21}
"Continuous Wnt/beta-catenin signaling in nascent cartilaginous skeletal elements blocks chondrocyte hypertrophy and endochondral ossification, whereas signaling starting at later stages stimulates hypertrophy and ossification, indicating that Wnt/beta-catenin roles are developmentally regulated. We created transgenic mice expressing a fusion mutant protein of beta-catenin and LEF (CA-LEF) in nascent chondrocytes. Transgenic mice had severe skeletal defects, particularly in limbs. Growth plates were totally disorganized, lacked maturing chondrocytes expressing Indian hedgehog and collagen X, and failed to undergo endochondral ossification. Interestingly, the transgenic cartilaginous elements were ill defined, intermingled with surrounding connective and vascular tissues, and even displayed abnormal joints. However, when activated beta-catenin mutant (delta-beta-catenin) was expressed in chondrocytes already engaged in maturation such as those present in chick limbs, chondrocyte maturation and bone formation were greatly enhanced. Differential responses to Wnt/beta-catenin signaling were confirmed in cultured chondrocytes. Activation in immature cells blocked maturation and actually de-stabilized their phenotype, as revealed by reduced expression of chondrocyte markers, abnormal cytoarchitecture, and loss of proteoglycan matrix. Activation in mature cells instead stimulated hypertrophy, matrix mineralization, and expression of terminal markers such as metalloprotease (MMP)-13 and vascular endothelial growth factor[so you don't want beta catenin in immature chondrocytes and you might want Beta Catenin in mature chondrocytes]. Because proteoglycans are crucial for cartilage function, we tested possible mechanisms for matrix loss. Delta-beta-catenin expression markedly increased expression of MMP-2, MMP-3, MMP-7, MMP-9, MT3-MMP, and ADAMTS5[maybe it's MMP-7 that de-stabilizes the phenotype]. In conclusion, Wnt/beta-catenin signaling regulates chondrocyte phenotype, maturation, and function in a developmentally regulated manner, and regulated action by this pathway is critical for growth plate organization, cartilage boundary definition, and endochondral ossification."
"continuous expression of an activated β-catenin form (Δ-β-catenin) in nascent chondrocytes using collagen II promoter sequences blocks further chondrocyte development and also ossification, as indicated by low expression of chondrocyte master regulator Sox9 and bone regulator Runx2."
"Transgenic mice expressing CA-LEF[constitutively active form of Beta-Catenin] died at birth of respiratory failure and displayed severe dwarfism with shortened limbs and a flattened skull"
Overexpression of GDF5 was observed in Beta-Catenin overexpression mice and abnormal expression of GAS1 and ERG was observed.
"when Δ-β-catenin is overexpressed in the developing chick embryo limb long bone anlagen, endochondral ossification and bone deposition are enhanced in diaphyseal regions, whereas immature but disorganized cartilage persists at the epiphyseal regions"
Beta-Catenin, Wnt14 transgenes decreased expression of Col9 and Aggrecan. These same transgenic mice overexpress MMP13 and VEGF and underexpressed Col10 in mature chondrocytes.
This study linked MMP-7 to growth plate disruption:
Expression of genes encoding extracellular matrix macromolecules and metalloproteinases in avian tibial dyschondroplasia.
"Avian tibial dyschondroplasia (TD) is a skeletal disease characterized by disruption of endochondral bone formation. The aim of this study was to determine the expression of extracellular matrix (ECM) macromolecules and ECM-degrading enzymes [matrix metalloproteinases (MMPs)] in the growth plates of normal and TD-affected 3-week-old broiler chicks (Cobb strain). Protein levels were analyzed by immunoblotting and gelatin zymography and gene expression by polymerase chain reaction. Expression of genes encoding the ECM macromolecules (collagen types II, IX, X and XI; and aggrecan) was not altered in dyschondroplasia; however, there was down-regulation of genes encoding MMP-9, MMP-13, MMP-10 and MMP-11 in addition to reduced amounts of MMP-2 and MMP-13 proteins. In contrast, there was up-regulation of genes encoding MMP-7 and the vascular endothelial growth factor. These findings suggest that the accumulation of cartilage associated with the disease may be the result of decreased proteolysis due to the down-regulation of MMPs and not to an increased production of ECM macromolecules."
"TD is characterized by the formation of a lesion composed of non-vascularized and non-mineralized cartilage that can extend from the epiphyseal growth plate into the metaphysis"<-So with upregulated MMP-7 chondrocytes still manage to form they just don't undergo endochondral ossification.
"The matrix metalloproteinases MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13 are highly expressed in the tumor-bone microenvironment, and, of these, MMP-7 and MMP-9 were found to be localized to bone-resorbing osteoclasts in human breast-to-bone metastases. In a bid to define the roles of host-derived MMP-7 and MMP-9 in the tumor-bone microenvironment, the tibias of MMP-7 and MMP-9 null mice were injected with osteolytic luciferase-tagged mammary tumor cell lines. Our data show that osteoclast-derived MMP-7 significantly contributes to tumor growth and tumor-induced osteolysis whereas osteoclast-derived MMP-9 had no effect on these processes. MMP-7 is capable of processing a number of nonmatrix molecules to soluble active forms that have profound effects on cell-cell communication, such as RANKL, a crucial mediator of osteoclast precursor recruitment and maturation. Therefore, the ability of osteoclast-derived MMP-7 to promote RANKL solubilization in the tumor-bone microenvironment was explored. Results revealed that levels of soluble RANKL were significantly lower in the MMP-7 null mice compared with wild-type (WT) controls. In keeping with this observation, MMP-7 null mice had significantly fewer osteoclast numbers at the tumor-bone interface compared with the WT controls. In summary, we propose that the solubilization of RANKL by MMP-7 is a potential mechanism through which MMP-7 mediates mammary tumor-induced osteolysis. Our studies indicate that the selective inhibition of MMP-7 in the tumor-bone microenvironment may be of benefit for the treatment of lytic breast-to-bone metastases."
"Osteoclasts express a variety of proteases including the cysteine protease, cathepsin-K and matrix metalloproteinases (MMPs)"
" lower levels of total RANKL (membrane bound and soluble) in the tumor injected tibias of MMP-7 null mice compared to wild type control mice"<-Note no mention is made of TRAP which is also associated with osteoclasts. TRAP is essential for proper growth plate development.
"An osteoclast derived protease, MMP-7, can promote osteoclast activation in the tumor-bone microenvironment by generating an active soluble form of the osteoclastogenic factor, RANKL"
MMP-7 can also be expressed by macrophages and other cells so inhibiting osteoclasts especially given the importance of TRAP may not be an option.
According to Matrilysin (MMP-7) inhibition of BMP-7 induced renal tubular branching morphogenesis suggests a role in the pathogenesis of human renal dysplasia, MMP-7 inhibits BMP-7. BMP-7 has been shown to be able to induce chondrogenesis.
Matrix metalloproteinases that associate with and cleave bone morphogenetic protein-2 in vitro are elevated in hypertrophic fracture nonunion tissue.
Matrix metalloproteinases that associate with and cleave bone morphogenetic protein-2 in vitro are elevated in hypertrophic fracture nonunion tissue.
"Both MMP-7 and MMP-12 mRNAs were significantly elevated in nonunion tissue when compared with local mineralized callus from the same site; the elevated protein levels of interest were visualized through immunoblotting and immunohistochemistry. These two MMPs were found to directly bind to and degrade bone morphogenetic protein-2 in vitro."
So decreasing MMP-7 levels may be a way to increase height. No mention was made if MMP-7 null mice suffered any adverse height growth effects. The good news is that MMP-7 is involved in cancer which means that potential therapies to inhibit it are likely in development.
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