Expression of Semaphorin-3A and its receptors in endochondral ossification: potential role in skeletal development and innervation.

Bone tissue is densely innervated, and there is increasing evidence for a neural control of bone metabolism. Semaphorin-3A is a very important regulator of neuronal targeting in the peripheral nervous system as well as in angiogenesis, and knockout of the Semaphorin-3A gene induces abnormal bone and cartilage development. We analyzed the spatial and temporal expression patterns of Semaphorin-3A signaling molecules during endochondral ossification, in parallel with the establishment of innervation. We show that osteoblasts and chondrocytes differentiated in vitro express most members of the Semaphorin-3A signaling system (Semaphorin-3A, Neuropilin-1, and Plexins-A1 and -A2). In vitro, osteoclasts express most receptor chains but not the ligand. In situ, these molecules are all expressed in the periosteum and by resting, prehypertrophic and hypertrophic chondrocytes in ossification centers before the onset of neurovascular invasion. They are detected later in osteoblasts and also osteoclasts, with differences in intensity and regional distribution. Semaphorin-3A and Neuropilin-1 are also expressed in the bone marrow. Plexin-A3 is not expressed by bone cell lineages in vitro. It is detected early in the periosteum and hypertrophic chondrocytes. After the onset of ossification, this chain is restricted to a network of cell processes in close vicinity to the cells lining the trabeculae, similar to the pattern observed for neural markers at the same stages. After birth, while the density of innervation decreases, Plexin-A3 is strongly expressed by blood vessels on the ossification front. In conclusion, Semaphorin-3A signaling is present in bone and seems to precede or coincide at the temporal but also spatial level with the invasion of bone by blood vessels and nerve fibers. Expression patterns suggest Plexin-A3/Neuropilin-1 as a candidate receptor in target cells for the regulation of bone innervation by Semaphorin-3A

Improvement of the chondrocyte-specific phenotype upon equine bone marrow mesenchymal stem cell differentiation. Influence of TGF-ß1 or TGF-ß3, associated with BMP-2 and type I collagen siRNAs

International audienceArticular cartilage is a tissue characterized by its poor intrinsic capacity for self-repair. This tissue is frequently altered upon trauma or in osteoarthritis (OA), a degenerative disease that is currently incurable. Consequently, cartilage markers, such as type II collagen, are degraded whereas atypic molecules, such as type I collagen, are newly synthetized. Another essential phenomenon occurring in OA is the upregulation of HtrA1, a serine protease targeting upstream receptors of signalling pathways involved in the synthesis of articular cartilage markers. OA incurs considerable economic loss for the equine sector. In the view to develop new therapies for humans and horses, significant progress in tissue engineering has led to the emergence of new generations of cartilage therapy. Matrix-associated autologous chondrocyte implantation is an advanced 3D cell-based therapy that holds promise for cartilage repair. The aim of this study is to improve the autologous chondrocyte implantation strategy by enhancing the chondrogenic differentiation of mesenchymal stem cells (MSCs) in order to increase the type II collagen/ type I collagen ratio

ZAK beta is activated by cellular compression and mediates contraction-induced MAP kinase signaling in skeletal muscle

Mechanical inputs give rise to p38 and JNK activation, which mediate adaptive physiological responses in various tissues. In skeletal muscle, contraction-induced p38 and JNK signaling ensure adaptation to exercise, muscle repair, and hypertrophy. However, the mechanisms by which muscle fibers sense mechanical load to activate this signaling have remained elusive. Here, we show that the upstream MAP3K ZAK beta is activated by cellular compression induced by osmotic shock and cyclic compression in vitro, and muscle contraction in vivo. This function relies on ZAKO's ability to recognize stress fibers in cells and Z-discs in muscle fibers when mechanically perturbed. Consequently, ZAK-deficient mice present with skeletal muscle defects characterized by fibers with centralized nuclei and progressive adaptation towards a slower myosin profile. Our results highlight how cells in general respond to mechanical compressive load and how mechanical forces generated during muscle contraction are translated into MAP kinase signaling.Peer reviewe

Hypoxia potentiates the BMP-2 driven COL2A1 stimulation in human articular chondrocytes via p38 MAPK

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Localization of the expression of type I, II and III collagen genes in human normal and hypochondrogenesis cartilage canals.

International audienceThe expression of type I, II and III collagens genes was examined in human normal and hypochondrogenesis cartilage canals employing electrophoretic analysis, immunohistochemistry and in situ hybridization techniques. In normal cartilage, collagens type I and III were present in perichondrium, in the connective tissue surrounding the vessels of cartilage canals and in the dense fibrous tissue. However, types I and III procollagen mRNAs were detected only in fibroblasts of the perichondrium and of the canals, but not in the polymorphic cells. Type II collagen was present in the cartilage matrix and in the dense fibrous tissue, in good accordance with the localization of type II procollagen mRNAs detected in the chondrocytes and in the polymorphic cells. These data suggest that there are no transitional cells expressing type I, II and III collagen genes and that polymorphic cells are of chondrocytic origin. In the case of hypochondrogenesis, type II collagen was less abundant than in normal cartilage, whereas the corresponding mRNA level was equivalent. That suggests that a postranscriptional regulation of this protein is involved in the decrease of type II collagen production. Type I collagen, unexpectedly detected in the cartilage matrix, was synthesized by chondrocytes and polymorphic cells, suggesting a replacement of type II by type I collagen. The canal hypertrophy observed in this pathological case could thus be due to a modification in the regulation of the growth of cartilage canals caused by a defective cartilage matrix.The expression of type I, II and III collagens genes was examined in human normal and hypochondrogenesis cartilage canals employing electrophoretic analysis, immunohistochemistry and in situ hybridization techniques. In normal cartilage, collagens type I and III were present in perichondrium, in the connective tissue surrounding the vessels of cartilage canals and in the dense fibrous tissue. However, types I and III procollagen mRNAs were detected only in fibroblasts of the perichondrium and of the canals, but not in the polymorphic cells. Type II collagen was present in the cartilage matrix and in the dense fibrous tissue, in good accordance with the localization of type II procollagen mRNAs detected in the chondrocytes and in the polymorphic cells. These data suggest that there are no transitional cells expressing type I, II and III collagen genes and that polymorphic cells are of chondrocytic origin. In the case of hypochondrogenesis, type II collagen was less abundant than in normal cartilage, whereas the corresponding mRNA level was equivalent. That suggests that a postranscriptional regulation of this protein is involved in the decrease of type II collagen production. Type I collagen, unexpectedly detected in the cartilage matrix, was synthesized by chondrocytes and polymorphic cells, suggesting a replacement of type II by type I collagen. The canal hypertrophy observed in this pathological case could thus be due to a modification in the regulation of the growth of cartilage canals caused by a defective cartilage matrix

Different effects of bone morphogenetic proteins 2, 4, 12, and 13 on the expression of cartilage and bone markers in the MC615 chondrocyte cell line.

International audienceIn order to study the lineage leading to chondrocyte and osteoblast phenotype in vertebrate development, we examined the effect of recombinant human bone morphogenetic protein (BMP)-2, BMP-4, BMP-12 [or growth and differentiation factor (GDF)-7], and BMP-13 (or GDF-6) on the phenotypic expression of the mouse chondrocyte cell line MC615, grown for 1 or 2 weeks in monolayer. Protein synthesis rates were monitored after incubation with [(14)C]proline. BMP-2 and BMP-4 increased protein synthesis, in agreement with our observation by phase-contrast microscopy of a highly refractile matrix around MC615 cells treated with BMP-2 and -4. Markers of the chondrocytic and osteoblastic differentiation were analyzed at mRNA level. Expression of the type II collagen gene, a marker of the cartilage phenotype, was up-regulated in the presence of low concentration of BMP-2 or -4 (50 ng/ml) and down-regulated at higher concentrations (100-400 ng/ml). In parallel, this expression was stable in the presence of BMP-12 or -13 in the dose range tested (50-400 ng/ml). Expression of the matrix Gla protein (MGP) gene, another marker of cartilage, was also reduced in the presence of 100 ng/ml BMP-2 or -4, while it remained stable in the presence of BMP-12 or -13 at the same concentration. In contrast, expression of the bone Gla protein (BGP) gene, or osteocalcin, a marker of the bone phenotype, was induced when the cells were treated with BMP-2 or -4 but was not detected when the cells were treated with BMP-12 or -13. At the same time, BMP-2 or -4 markedly up-regulated expression of type X collagen mRNA, indicating that MC615 cells possess the ability to express traits associated with endochondral ossification, when exposed to specific BMPs. Furthermore, detailed analysis of type II collagen expression showed that the alternatively spliced transcript collagen IIB, specific for cartilage, is expressed concomitantly with BGP. Therefore, MC615 chondrocytes can simultaneously express chondrocytic and osteoblastic markers, in response to BMP-2 or -4, but show minimal response to BMP-12 (or GDF-7) or to BMP-13 (or GDF-6). These results raise the possibility that chondrocytes in vivo can express osteoblastic properties, provided they are induced by BMP-2 or -4.In order to study the lineage leading to chondrocyte and osteoblast phenotype in vertebrate development, we examined the effect of recombinant human bone morphogenetic protein (BMP)-2, BMP-4, BMP-12 [or growth and differentiation factor (GDF)-7], and BMP-13 (or GDF-6) on the phenotypic expression of the mouse chondrocyte cell line MC615, grown for 1 or 2 weeks in monolayer. Protein synthesis rates were monitored after incubation with [(14)C]proline. BMP-2 and BMP-4 increased protein synthesis, in agreement with our observation by phase-contrast microscopy of a highly refractile matrix around MC615 cells treated with BMP-2 and -4. Markers of the chondrocytic and osteoblastic differentiation were analyzed at mRNA level. Expression of the type II collagen gene, a marker of the cartilage phenotype, was up-regulated in the presence of low concentration of BMP-2 or -4 (50 ng/ml) and down-regulated at higher concentrations (100-400 ng/ml). In parallel, this expression was stable in the presence of BMP-12 or -13 in the dose range tested (50-400 ng/ml). Expression of the matrix Gla protein (MGP) gene, another marker of cartilage, was also reduced in the presence of 100 ng/ml BMP-2 or -4, while it remained stable in the presence of BMP-12 or -13 at the same concentration. In contrast, expression of the bone Gla protein (BGP) gene, or osteocalcin, a marker of the bone phenotype, was induced when the cells were treated with BMP-2 or -4 but was not detected when the cells were treated with BMP-12 or -13. At the same time, BMP-2 or -4 markedly up-regulated expression of type X collagen mRNA, indicating that MC615 cells possess the ability to express traits associated with endochondral ossification, when exposed to specific BMPs. Furthermore, detailed analysis of type II collagen expression showed that the alternatively spliced transcript collagen IIB, specific for cartilage, is expressed concomitantly with BGP. Therefore, MC615 chondrocytes can simultaneously express chondrocytic and osteoblastic markers, in response to BMP-2 or -4, but show minimal response to BMP-12 (or GDF-7) or to BMP-13 (or GDF-6). These results raise the possibility that chondrocytes in vivo can express osteoblastic properties, provided they are induced by BMP-2 or -4

Keratinocyte motility induced by TGF-beta1 is accompanied by dramatic changes in cellular interactions with laminin 5.

International audienceTransforming growth factor-beta1 (TGF-beta1) has the ability to induce epithelial cell migration while stopping proliferation. In this study, we show that, concomitant to promoting migration of normal human keratinocytes in vitro, TGF-beta1 induced a marked decrease in their adhesion capacity to processed alpha3-containing laminin 5-coated surfaces. Indeed, the expression levels of alpha3 and alpha6 integrin subunit mRNA and protein, as well as the cell surface alpha3beta1 and alpha6beta4 integrins, were down-regulated. Recent studies showed that keratinocytes over express and deposit laminin 5 during migration and we have shown that laminin 5 found in the matrix of TGF-beta1 induced migrating keratinocytes is present in its unprocessed form [Decline and Rousselle, 2001: J. Cell Sci. 114:811-823]. We show here that TGF-beta1 treatment of the cells promoted a significant increase in their adhesion to the alpha3 chain carboxy-terminal LG4/5 subdomain and that this interaction is likely to be mediated by a heparan sulfate proteoglycan type of receptor. Our results indicate that alpha6beta4 and alpha3beta1 integrin interactions with laminin 5 are diminished during migration while a specific interaction occurs between an additional cellular receptor and the alpha3 LG4/5 module present on unprocessed laminin 5.Transforming growth factor-beta1 (TGF-beta1) has the ability to induce epithelial cell migration while stopping proliferation. In this study, we show that, concomitant to promoting migration of normal human keratinocytes in vitro, TGF-beta1 induced a marked decrease in their adhesion capacity to processed alpha3-containing laminin 5-coated surfaces. Indeed, the expression levels of alpha3 and alpha6 integrin subunit mRNA and protein, as well as the cell surface alpha3beta1 and alpha6beta4 integrins, were down-regulated. Recent studies showed that keratinocytes over express and deposit laminin 5 during migration and we have shown that laminin 5 found in the matrix of TGF-beta1 induced migrating keratinocytes is present in its unprocessed form [Decline and Rousselle, 2001: J. Cell Sci. 114:811-823]. We show here that TGF-beta1 treatment of the cells promoted a significant increase in their adhesion to the alpha3 chain carboxy-terminal LG4/5 subdomain and that this interaction is likely to be mediated by a heparan sulfate proteoglycan type of receptor. Our results indicate that alpha6beta4 and alpha3beta1 integrin interactions with laminin 5 are diminished during migration while a specific interaction occurs between an additional cellular receptor and the alpha3 LG4/5 module present on unprocessed laminin 5

Type X collagen in rabbit and human meniscus.

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Differential gene expression analysis in a rabbit model of osteoarthritis induced by anterior cruciate ligament (ACL) section.

International audienceOsteoarthritis (OA) is the most common of all joint diseases to affect mankind and is characterized by the degradation of articular cartilage. The low availability of normal and pathologic human cartilage and the inability to study the early stages of the disease in humans has led to the development of numerous animal models of OA. The aim of our study was to establish gene expression profiles during the progression of a rabbit model of OA induced by anterior cruciate ligament (ACL) section. Semiquantitative RT-PCR was used to follow expression of several relevant molecules (type II and X collagens, aggrecan, osteonectin, betaig-h3, BiP, TIMP-1, MMP-1, -3, -13, aggrecanase-1, -2) during development of OA in articular cartilage. In parallel, we monitored the activities of collagenase, caseinase, phospholipase A2 and glycosyltransferases (xylosyl-, galactosyl-, glucuronyl- and N-acetyl-galactosaminyl-transferase). Novel cDNA clones for rabbit type X collagen, aggrecanase-1 and -2, osteonectin and BiP were constructed to obtain species-specific primers. Ours result show that MMP-13 (collagenase-3) gene expression increased dramatically early after ACL surgery and remained high thereafter. An increase in MMP-1 (collagenase-1) and MMP-3 expression was also noted with an absence of variation for TIMP-1 expression. In addition, the global MMPs activities paralleled the MMP gene expression. These data together characterize at the molecular level the evolution of OA in this rabbit model. Furthermore, we have undertaken a search for identifying differentially expressed genes in normal and OA cartilage in this model, by differential display RT-PCR. We present here preliminary results with the determination of the best technical conditions to obtain reproducible electrophoresis patterns of differential display RT-PCR.Osteoarthritis (OA) is the most common of all joint diseases to affect mankind and is characterized by the degradation of articular cartilage. The low availability of normal and pathologic human cartilage and the inability to study the early stages of the disease in humans has led to the development of numerous animal models of OA. The aim of our study was to establish gene expression profiles during the progression of a rabbit model of OA induced by anterior cruciate ligament (ACL) section. Semiquantitative RT-PCR was used to follow expression of several relevant molecules (type II and X collagens, aggrecan, osteonectin, betaig-h3, BiP, TIMP-1, MMP-1, -3, -13, aggrecanase-1, -2) during development of OA in articular cartilage. In parallel, we monitored the activities of collagenase, caseinase, phospholipase A2 and glycosyltransferases (xylosyl-, galactosyl-, glucuronyl- and N-acetyl-galactosaminyl-transferase). Novel cDNA clones for rabbit type X collagen, aggrecanase-1 and -2, osteonectin and BiP were constructed to obtain species-specific primers. Ours result show that MMP-13 (collagenase-3) gene expression increased dramatically early after ACL surgery and remained high thereafter. An increase in MMP-1 (collagenase-1) and MMP-3 expression was also noted with an absence of variation for TIMP-1 expression. In addition, the global MMPs activities paralleled the MMP gene expression. These data together characterize at the molecular level the evolution of OA in this rabbit model. Furthermore, we have undertaken a search for identifying differentially expressed genes in normal and OA cartilage in this model, by differential display RT-PCR. We present here preliminary results with the determination of the best technical conditions to obtain reproducible electrophoresis patterns of differential display RT-PCR