Chondrocyte survival and differentiation in situ are integrin mediated

Chondrocytes in specific areas of the chick sternum have different developmental fates. Cephalic chondrocytes become hypertrophic and secrete type X collagen into the extracellular matrix prior to bone deposition. Middle and caudal chondrocytes remain cartilaginous throughout development and continue to secrete collagen types II, IX, and XI. The interaction of integrin receptors with extracellular matrix molecules has been shown to affect cytoskeleton organization, proliferation, differentiation, and gene expression in other cell types. We hypothesized that chondrocyte survival and differentiation including the deposition into interstitial matrix of type X collagen may be integrin receptor mediated. To test this hypothesis, a serum‐free organ culture sternal model that recapitulates normal development and maintains the three‐dimensional relationships of the tissue was developed. We examined chondrocyte differentiation by five parameters: type X collagen deposition into interstitial matrix, sternal growth, actin distribution, cell shape, and cell diameter changes. Additional sterna were analyzed for apoptosis using a fragmented DNA assay. Sterna were organ cultured with blocking antibodies specific for integrin subunits (α2, α3, or β1). In the presence of anti‐β1 integrin (25 μg/ml, clone W1B10), type X collagen deposition into interstitial matrix and sternal growth were significantly inhibited. In addition, all chondrocytes were significantly smaller, the actin was disrupted, and there was a significant increase in apoptosis throughout the specimens. Addition of anti‐α2 (10 μg/ml, clone P1E6) or anti‐ α3 (10 μg/ml, clone P1B5) integrin partially inhibited type X collagen deposition into interstitial matrix; however, sternal growth and cell size were significantly decreased. These data are the first obtained from intact tissue and demonstrate that the interaction of chondrocytes with extracellular matrix is required for chondrocyte survival and differentiation. Dev. Dyn. 1997;210:249–263. © 1997 Wiley‐Liss, Inc.

[1]  S. Albelda,et al.  Integrins and other cell adhesion molecules , 1990, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[2]  V. Trinkaus-Randall,et al.  Distribution of F-actin, vinculin and integrin subunits (alpha 6 and beta 4) in response to corneal substrata. , 1995, Experimental eye research.

[3]  T. Schmid,et al.  Immunoelectron microscopy of type X collagen: supramolecular forms within embryonic chick cartilage. , 1990, Developmental biology.

[4]  D. Bonen,et al.  Elevated extracellular calcium concentrations induce type X collagen synthesis in chondrocyte cultures , 1991, The Journal of cell biology.

[5]  F. Re,et al.  Inhibition of anchorage-dependent cell spreading triggers apoptosis in cultured human endothelial cells , 1994, The Journal of cell biology.

[6]  V. Trinkaus-Randall,et al.  The intracellular distribution of vinculin and alpha 2 integrin in epithelial cells and chondrocytes. , 1994, Scanning.

[7]  A. Ridley,et al.  Signal transduction pathways regulating Rho‐mediated stress fibre formation: requirement for a tyrosine kinase. , 1994, The EMBO journal.

[8]  Z. Werb,et al.  Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression , 1989, The Journal of cell biology.

[9]  D. Boettiger,et al.  Beta 1 integrins mediate chondrocyte interaction with type I collagen, type II collagen, and fibronectin. , 1993, Experimental cell research.

[10]  B. Lanske,et al.  PTH/PTHrP Receptor in Early Development and Indian Hedgehog--Regulated Bone Growth , 1996, Science.

[11]  T. Schmid,et al.  Immunohistochemical localization of short chain cartilage collagen (type X) in avian tissues , 1985, The Journal of cell biology.

[12]  M. Hirsch,et al.  Confocal microscopy of whole mount embryonic cartilage: Intracellular localization of F-actin, chick prolyl hydroxylase and type II collagen mRNA , 1993 .

[13]  D. Bonen,et al.  Late events in chondrocyte differentiation: hypertrophy, type X collagen synthesis and matrix calcification. , 1991, In vivo.

[14]  J. Gamble,et al.  Regulation of in vitro capillary tube formation by anti-integrin antibodies , 1993, The Journal of cell biology.

[15]  T. Aigner,et al.  In situ hybridization studies on the expression of type X collagen in fetal human cartilage. , 1991, Developmental biology.

[16]  M. Pacifici,et al.  Ascorbic acid induces alkaline phosphatase, type X collagen, and calcium deposition in cultured chick chondrocytes. , 1989, The Journal of biological chemistry.

[17]  C. Farnum,et al.  Morphologic stages of the terminal hypertrophic chondrocyte of growth plate cartilage , 1987, The Anatomical record.

[18]  D. Boettiger,et al.  Occupation of the extracellular matrix receptor, integrin, is a control point for myogenic differentiation , 1987, Cell.

[19]  M J Bissell,et al.  Cellular growth and survival are mediated by beta 1 integrins in normal human breast epithelium but not in breast carcinoma. , 1995, Journal of cell science.

[20]  Clifford J. Tabin,et al.  Regulation of Rate of Cartilage Differentiation by Indian Hedgehog and PTH-Related Protein , 1996, Science.

[21]  S. Santoro,et al.  Widespread histologic distribution of the alpha 2 beta 1 integrin cell-surface collagen receptor. , 1990, The American journal of pathology.

[22]  R. Cancedda,et al.  Thyroid hormone, insulin, and glucocorticoids are sufficient to support chondrocyte differentiation to hypertrophy: a serum-free analysis , 1992, The Journal of cell biology.

[23]  Joseph Haydn,et al.  A Theme and Variations , 1956 .

[24]  A. Ridley,et al.  Rho: theme and variations , 1996, Current Biology.

[25]  E. Hay,et al.  Cell Biology of Extracellular Matrix , 1988, Springer US.

[26]  B. Olsen,et al.  Transcriptional regulation of type X collagen during chondrocyte maturation. , 1989, Developmental biology.

[27]  T. Krieg,et al.  Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins , 1995, The Journal of cell biology.

[28]  H. Lebovitz,et al.  Triiodothyronine stimulation of in vitro growth and maturation of embryonic chick cartilage. , 1982, Endocrinology.

[29]  K. Daniels,et al.  Type X collagen is transcriptionally activated and specifically localized during sternal cartilage maturation. , 1992, Matrix.

[30]  R. Loeser.  Integrin-mediated attachment of articular chondrocytes to extracellular matrix proteins. , 1993, Arthritis and rheumatism.

[31]  R. Eavey,et al.  Intrinsic and extrinsic controls of the hypertrophic program of chondrocytes in the avian columella. , 1988, Developmental biology.

[32]  K. Burridge,et al.  Rho-stimulated contractility drives the formation of stress fibers and focal adhesions , 1996, The Journal of cell biology.

[33]  Z. Werb,et al.  Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix , 1995, Science.

[34]  R. Killiany,et al.  Increased cell diameter precedes chondrocyte terminal differentiation, whereas cell‐matrix attachment complex proteins appear constant , 1996, The Anatomical record.

[35]  M. Schwartz,et al.  The extracellular matrix as a cell survival factor. , 1993, Molecular biology of the cell.

[36]  D. Boettiger,et al.  Expression and Function of Chicken Integrin / 31 Subunit and Its Cytoplasmic Domain Mutants in Mouse NIH 313 Cells , 1990 .

[37]  C. Nobes,et al.  Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia , 1995, Cell.

[38]  S. Goodman,et al.  Localization of beta 1-integrins in human cartilage and their role in chondrocyte adhesion to collagen and fibronectin. , 1993, Experimental cell research.

[39]  R. Mayne,et al.  Collagen types IX and X in the developing chick tibiotarsus: analyses of mRNAs and proteins. , 1991, Development.

[40]  I. Shapiro,et al.  Regulated Production of Mineralization-competent Matrix Vesicles in Hypertrophic Chondrocytes , 1997, The Journal of cell biology.

[41]  H. Lebovitz,et al.  Triiodothyronine stimulates maturation of porcine growth-plate cartilage in vitro. , 1982, The Journal of clinical investigation.

[42]  Michelle S. Hirsch,et al.  Chondrocytes provide a model for in-situ confocal microscopy and 3D reconstructions , 1994, Electronic Imaging.

[43]  Richard O. Hynes,et al.  Integrins: Versatility, modulation, and signaling in cell adhesion , 1992, Cell.

[44]  J. Parsons,et al.  Integrin-mediated signalling: regulation by protein tyrosine kinases and small GTP-binding proteins. , 1996, Current opinion in cell biology.

[45]  D. Boettiger,et al.  Expression and function of chicken integrin beta 1 subunit and its cytoplasmic domain mutants in mouse NIH 3T3 cells , 1990, The Journal of cell biology.

[46]  R. Loeser,et al.  Expression of beta 1 integrins by cultured articular chondrocytes and in osteoarthritic cartilage. , 1995, Experimental cell research.

[47]  A. Horwitz,et al.  Integrin cytoplasmic domains: mediators of cytoskeletal linkages and extra- and intracellular initiated transmembrane signaling. , 1993, Current opinion in cell biology.

[48]  S. Frisch,et al.  Disruption of epithelial cell-matrix interactions induces apoptosis , 1994, The Journal of cell biology.

[49]  S. Ben‐Sasson,et al.  Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation , 1992, The Journal of cell biology.

[50]  Y. Takada,et al.  Identification of a regulatory region of integrin beta 1 subunit using activating and inhibiting antibodies. , 1993, The Journal of biological chemistry.

[51]  T. Tschan,et al.  Induction of proliferation or hypertrophy of chondrocytes in serum-free culture: the role of insulin-like growth factor-I, insulin, or thyroxine , 1992, The Journal of cell biology.

[52]  John Calvin Reed,et al.  Anchorage dependence, integrins, and apoptosis , 1994, Cell.

[53]  M. Hirsch,et al.  β1 Integrin Antibodies Inhibit Chondrocyte Terminal Differentiation in Whole Sterna a , 1996 .