Roles of the Ser146, Tyr159, and Lys163 residues in the catalytic action of 7alpha-hydroxysteroid dehydrogenase from Escherichia coli

Adhesion to collagens by most cell types is mediated by the integrins alpha1beta1 and alpha2beta1. Both integrin alpha subunits belong to a group which is characterized by the presence of an I domain in the N-terminal half of the molecule, and this domain has been implicated in the ligand recognition. Since purified alpha1beta1 and alpha2beta1 differ in their binding to collagens I and IV and recognize different sites within the major cell binding domain of collagen IV, we investigated the potential role of the alpha1 and alpha2 I domains in specific collagen adhesion. We find that introducing the alpha2 I domain into alpha1 results in surface expression of a functional collagen receptor. The adhesion mediated by this chimeric receptor (alpha1-2-1beta1) is similar to the adhesion profile conferred by alpha2beta1, not alpha1beta1. The presence of alpha2 or alpha1-2-1 results in preferential binding to collagen I, whereas alpha1 expressing cells bind better to collagen IV. In addition, alpha1 containing cells bind to low amounts of a tryptic fragment of collagen IV, whereas alpha2 or alpha1-2-1 bearing cells adhere only to high concentrations of this substrate. We also find that collagen adhesion of NIH-3T3 mediated by alpha2beta1 or alpha1-2-1beta1, but not by alpha1, requires the presence of Mn2+ ions. This ion requirement was not found in CHO cells, implicating the I domain in cell type-specific activation of integrins.     

Flow cytometric analysis of platelet activation by different collagen types present in the vessel wall

The interaction of platelets with collagens of the vessel wall is a critical event in primary haemostasis. Although numerous studies have examined the ability of various collagen types to support platelet adhesion, little is known concerning the relative ability of different collagens to elicit specific activation markers in platelets. In this report, flow cytometric analysis has been utilized to evaluate the ability of various native collagen types to elicit secondary activation events in human platelets. Collagen types I, III, V and VI induced alpha-granule secretion and up-regulation of cell surface glycoprotein (GP) IIb/IIIa. In contrast, collagen type IV did not elicit these responses in the concentration ranges examined. Dose-response curves for alpha-granule secretion induced by the various collagen types indicated that human type III and human type I collagens were less effective than human type V, human type VI and calf skin type I. In addition, the ability of these various collagens to activate GPIIb/IIIa to its ligand binding conformation was even more heterogenous with only human type VI and calf skin type I readily promoting this transition. These data demonstrate that flow cytometric analysis of collagen-induced platelet activation is feasible and that collagen-mediated alpha-granule secretion and membrane glycoprotein redistribution in human platelets are separate events from activation of GPIIb/IIIa.     

Adhesion of mammalian cells to polymer surfaces: from physical chemistry of surfaces to selective adhesion on defined patterns

The study of the adsorption of type I collagen from a solution containing Pluronic F68 has shown that the latter prevents collagen adsorption on polystyrene and does not prevent it on surface-oxidized polystyrene. This explains the control of mammalian cell adhesion by substrate surface hydrophobicity and composition of pre-conditioning solution. On that basis, selective adhesion of different types of mammalian cells (PC12 pheochromocytoma, MSC80 schwannoma, Hep G2 hepatoblastoma, rat hepatocytes) on patterned surfaces was achieved. Therefore tracks (width in the range of a few tens of microm) of reduced hydrophobicity were produced on polystyrene by photolithography and oxygen plasma treatment. After conditioning by a solution containing both Pluronic F68 and extracellular matrix protein (collagen, fibronectin), the latter adsorbed selectively on these paths thus allowing selective adhesion of the cells.     

Galectin-3, a beta-galactoside-binding animal lectin, binds to neural recognition molecules

In this study, we have investigated the ability of galectin-3, a beta-galactoside-binding animal lectin, to interact in vitro with different neural tissue-derived glycoproteins involved in cell-cell and cell-substrate adhesion. Galectin-3 interacted to varying degrees with the cell recognition molecules L1, the myelin-associated glycoprotein, and the neural cell adhesion molecule and the extracellular matrix molecules tenascin-C and tenascin-R but not with collagen type I. Binding of galectin-3 to the different glycoproteins tested was carbohydrate dependent and could be specifically inhibited by the addition of lactose and, to a lesser extent, galactose.     

Identification in collagen type I of an integrin alpha2 beta1-binding site containing an essential GER sequence

The collagen type I-derived fragment alpha1(I)CB3 is known to recognize the platelet collagen receptor integrin alpha2beta1 as effectively as the parent collagen, although it lacks platelet-aggregatory activity. We have synthesized the fragment as seven overlapping peptides that spontaneously assemble into triple helices. On the basis of their capacity to bind purified alpha2 beta1 and the recombinant alpha2 A-domain, and their ability to support alpha2 beta1-mediated cell adhesion, we identified two peptides, CB3(I)-5 and -6, which contain an alpha2 beta1 recognition site. Synthesis of the peptide CB3(I)-5/6, containing the overlap sequence between peptides 5 and 6, allowed us to locate the binding site within the 15-residue sequence, GFP*GERGVEGPP*GPA (where P* represents hydroxyproline), corresponding to residues 502-516 of the collagen type I alpha1 chain. The Glu and Arg residues in the GER triplet were found to be essential for recognition since substitution of either residue with Ala caused a loss of alpha2 A-domain binding. By contrast, substitution of the Glu in GVE did not reduce binding, but rather enhanced it slightly. We were unable to detect significant recognition of alpha2 beta1 by the peptide CB3(I)-2 containing the putative alpha2 beta1 recognition sequence DGEA. Peptides CB3(I)-1 to -6, together with peptide CB3(I)-5/6, exhibited good platelet-aggregatory activity, in some cases better than collagen. However, peptide CB3(I)-7 was inactive, suggesting the presence of an inhibitory element that might account for the lack of aggregatory activity of the parent alpha1(I)CB3 fragment.     

Platelets interact with soluble and insoluble collagens through characteristically different reactions

Platelet interaction with soluble and insoluble collagens was characterized through binding studies. In contrast to resting platelets, cells reacted with activators, TS2/16 (integrin alpha2 beta1-activating antibody), thrombin, collagen-related peptide, or ADP, exhibited specific soluble collagen binding that is Mg2+-dependent, but inhibited by prostaglandin I2, Ca2+, and Gi9 (anti-integrin alpha2 beta1 antibody). Each platelet has 1500-3500 soluble collagen binding sites, with a dissociation constant of 3. 5-9 x 10(-8) M. This is the first study to show the specific binding of soluble collagen to platelets; our data strongly suggest that the receptor is integrin alpha2 beta1 after it becomes activated upon platelet activation. These results suggest that activation of platelets transforms integrin alpha2 beta1 to a state with higher affinity binding sites for soluble collagen. The soluble collagen-platelet interaction was compared with the platelet interaction with fibrillar collagen, which has until now not been demonstrated to bind specifically to platelets. Here, we demonstrated specific, biphasic fibrillar collagen binding. One phase is rapid and metal ion-independent, and accounts for most of the binding. The other phase is slow and Mg2+-dependent. The characteristic differences in the specific bindings of soluble and fibrous collagens demonstrate the different contributions of two different collagen receptors.     

Homology modelling by distance geometry

The collateral ligaments can be clearly distinguished in the 25-day fetal rabbit knee joint. Type I and V collagens are present in the extracellular matrix between the cells of the lateral and medial collateral ligaments and this distribution persists until the rabbit is skeletally mature. From 8 months onwards type III collagen is also present, particularly around the cells. Type I collagen mRNA is expressed by the cells from the 25-day fetal to 8-month-old adult ligament. The ligament sheath is composed of types III and V collagens. The cruciate ligaments are present between the femur and tibia in the 20-day fetus. The matrix is composed of types I and V collagens from the 25-day fetus until at 12- to 14-weeks postnatal, type III collagen appears in the pericellular regions together with type V. At 8 months and 2 years the amount of type III collagen has increased. All the cells express the mRNA for type I collagen at 12- to 14-weeks, but only isolated cells express this mRNA at 8 months. Thus, both the collateral and cruciate ligaments undergo changes in their complement of collagens during postnatal development and ageing. The implications of these complex interactions of different types of collagen are discussed in relation to healing and the surgical replacement of torn ligaments by tendons.     

Involvement of integrins with adhesion-promoting, heparin-binding peptides of type IV collagen in cultured human corneal epithelial cells

PURPOSE: The aim of this work was to identify the integrin subunits present on the cell surface of human corneal epithelial cells. The authors determined to show whether type IV collagen, heparin-binding peptides of type IV collagen (Hep-I, Hep-II, and Hep-III), fibronectin, and GRGDSP promote cell adhesion of human corneal epithelial cells. Type IV collagen and heparin-binding peptides of type IV collagen may be important in corneal epithelial cell adhesion in normal and pathologic conditions and reepithelialization. The authors assess the role of cell surface integrins in mediating cell adhesion to these proteins and peptides. METHODS: Fluorescence-activated cell sorter (FACS) analysis was used to determine the integrin subunits expressed at the cell surface of the cultured human corneal epithelial cells. Cell adhesion was assessed with type IV collagen, heparin-binding peptides of type IV collagen, fibronectin, and GRGDSP: Antibodies to the integrin subunits were used to determine the potential role of integrins in cell adhesion to the above proteins and peptides. RESULTS: FACS analysis identified the beta 1, beta 4, alpha 2, alpha 3, alpha 5, alpha 6, and alpha v integrin subunits on human corneal epithelial cells grown as primary cultures. The anti-beta 1 antibody inhibited cell adhesion to heparin-binding peptides of type IV collagen, type IV collagen, fibronectin, and GRGDSP: Antibodies to the alpha 2 integrin subunit inhibited cell adhesion to the heparin-binding peptides of type IV collagen and slightly inhibited cell adhesion to intact type IV. Antibodies to the alpha 3 integrin subunit exhibited a somewhat lesser effect compared to the anti-alpha 2 integrin antibody. CONCLUSIONS: These data show that the alpha 2 beta 1 integrin of human corneal epithelial cells recognize heparin-binding peptide sequences derived from human type IV collagen. It seems likely that these sequences play an important role in integrin-mediated corneal epithelial cell adhesion. In addition, the alpha 3 beta 1 integrin may mediate similar events.     

Cell adhesion molecules--update

Cell adhesion molecules are glycoproteins expressed on the cell surface and play an important role in inflammatory as well as neoplastic diseases. There are four main groups: the integrin family, the immunoglobulin superfamily, selectins, and cadherins. The integrin family has eight subfamilies, designated as beta 1 through beta 8. The most widely studied subfamilies are beta 1 (CD29, very late activation [VLA] members), beta 2 (leukocyte integrins such as CD11a/CD18, CD11b/CD18, CD11c/CD18, and alpha d beta 2), beta 3 (CD61, cytoadhesions), and beta 7 (alpha 4 beta 7 and alpha E beta 7). The immunoglobulin superfamily includes leukocyte function antigen-2 (LFA-2 or CD2), leukocyte function antigen-3 (LFA-3 or CD58), intercellular adhesion molecules (ICAMs), vascular adhesion molecule-1 (VCAM-1), platelet-endothelial cell adhesion molecule-1 (PE-CAM-1), and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). The selectin family includes E-selectin (CD62E), P-selectin (CD62P), and L-selectin (CD62L). Cadherins are major cell-cell adhesion molecules and include epithelial (E), placental (P), and neural (N) subclasses. The binding sites (ligands/receptors) are different for each of these cell adhesion molecules (e.g., ICAM binds to CD11/CD18; VCAM-1 binds to VLA-4). The specific cell adhesion molecules and their ligands that may be involved in pathologic conditions and potential therapeutic strategies by modulating the expression of these molecules will be discussed.     

Extracellular matrix of opacified anterior capsule after endocapsular cataract surgery

BACKGROUND: We determined the types and distribution of glycosaminoglycans (GAGs) and collagens, in anterior capsular opacification after endocapsular phacoemulsification and aspiration (ECPEA) and intraocular lens implantation. METHODS: Opacified anterior capsules were removed from human eyes after ECPEA. Immunohistochemical staining was performed to determine GAGs with monoclonal antibodies to chondroitin, chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), dermatan sulfate (DS), and keratan sulfate (KS); collagens with monoclonal antibodies to types I, II, and III collagens; and cellular characteristics with monoclonal antibodies to vimentin, desmin, alpha-smooth muscle actin, and cytokeratin. Decorin mRNA and type I collagen mRNA were detected by in situ hybridization. RESULTS: In the capsules, the C6S, DS, KS, and types I and III collagens were similar to the chemical components found at the adhesion site of the anterior and posterior capsules after extracapsular cataract extraction, and cellular components contained vimentin, desmin, alpha-smooth muscle actin, cytokeratin, decorin mRNA, and type I collagen mRNA. CONCLUSIONS: The GAGs and collagens in opacified anterior capsule after ECPEA were similar to those found during wound healing, although KS is present in normal anterior segment tissue during development and only in the cornea postnatally. These chemical components may be produced by myofibroblast-like cells presumably transformed from lens epithelial cells.     

Differential effects of integrin alpha chain mutations on invasin and natural ligand interaction

To determine if recognition of the Yersinia pseudotuberculosis invasin protein and natural substrates requires identical integrin residues, a region of the human alpha3 integrin chain predicted to be involved in substrate adhesion was targeted for mutation. One point mutation located in a region of the third N-terminal repeat of the alpha3 chain, alpha3-W220A, failed to promote adhesion to the natural alpha3 beta1 substrate epiligrin but maintained near wild type levels of adhesion to invasin. A second nearby mutation, alpha3-Y218A, which showed no detectable adhesion to epiligrin, was only partially attenuated for invasin binding as well as invasin-mediated bacterial uptake. A third substitution, alpha3-D154A, predicted to be in the second N-terminal repeat not known to be implicated in cell adhesion, was competent for invasin-promoted adhesion events and appeared to encode a receptor of increased activity, as it had a higher efficiency than wild type receptor for adhesion to epiligrin. Cell lines expressing this derivative were not recognized by a function blocking anti-alpha3 antibody, indicating that the second and third repeats of the alpha3 chain are either closely linked in space or the second repeat can modulate activity of the third. Differential effects on substrate adhesion do not appear to be associated with all integrin alpha chain mutations, as alpha4 chain mutations affecting the divalent cation binding domains depressed adhesion to invasin to a significant extent.     

Identification of a domain on the integrin alpha5 subunit implicated in cell spreading and signaling

The alpha5 beta1 integrin is a cell surface receptor for fibronectin implicated in several cellular activities including cell proliferation, differentiation, and migration. The primary site at which the alpha5 beta1 integrin interacts with fibronectin is the RGD (Arg-Gly-Asp) amino acid sequence. In general, the sites on the integrin alpha subunits involved in ligand binding are not well characterized. Based on previous cross-linking studies, sequence alignment, predicted conformation, and intron-exon boundaries, we identified a 144-residue region (positions 223-367) on the alpha5 subunit as a putative binding region and divided it into four subdomains named domains I, II, III, and IV. Chimeric receptors were prepared in which sequences on the alpha5 subunit were exchanged with the corresponding sequences on the alpha6 subunit, which is specific for laminin and does not bind via an RGD sequence. The mutated human alpha5 integrin gene was transfected into CHO B2 cells, which are deficient in alpha5 expression. Only chimeras of domain III or IV express on the cell surface. Both of these chimeras decreased the adhesion, spreading, focal adhesion assembly, and migration on fibronectin. The adhesion of the chimeric receptors to fibronectin remained sensitive to the RGD peptide, and antibodies that inhibit interaction with the fibronectin synergy site and RGD loop remain inhibitory for the chimeras, indicating that our chimeras do not inhibit binding to either the RGD or synergy sites. Finally, the affinity of soluble fibronectin to cells via the alpha5 beta1 receptor decreased only about 3-fold. This decrease is substantially less than the observed effects on migration and spreading, which were not altered by changes in substrate concentration. Thus, the alteration in binding sites does not easily account for the changes in cell spreading and focal adhesion assembly. The tyrosine phosphorylation and focal adhesion assembly that are seen when cells expressing the wild type alpha5 receptor adhere to fibronectin were inhibited in cells expressing the chimeric receptors. Therefore, our results suggest that the chimeras of these domains likely interrupt alpha5-mediated conformational signaling.     

Alternative adhesion sites in human fibrinogen for vascular endothelial cells

Fibrinogen mediates endothelial cell adhesion, spreading, and angiogenesis through integrin alphavbeta3. Previous studies by several investigators have suggested that the Arg-Gly-Asp (RGD) site at position 572-574 on the alpha chain of human fibrinogen can bind to alphavbeta3. However, this RGD sequence is absent in fibrinogen from most other species, including bovine, hamster, monkey, mouse, pig, and rat fibrinogen. In these species, an RGD site exists at the equivalent of position alpha252-254, which has the sequence RGG in humans. In addition, the role of an integrin binding site on the gamma chain at position 400-411 has been an issue of controversy. In the present studies, recombinant fibrinogen molecules with mutations in the potential endothelial cell binding sites have been used to test the role of these sites directly. The results show that the RGD at alpha572-574 is the primary adhesion site, and that the gamma chain site plays no significant role. Human and bovine plasma fibrinogens were also assayed for their ability to support adhesion of human and bovine vascular endothelial cells. The results show that although the two types of fibrinogen have RGD sequences at widely divergent sites, there is no significant difference in their ability to support endothelial cell adhesion. Furthermore, a chimeric human fibrinogen molecule with an RGD sequence at the bovine site, position alpha252-254, also supported adhesion. These results indicate that an RGD site in human fibrinogen at either position alpha252-254 or position alpha572-574 can mediate endothelial cell adhesion.     

Ontogenic regulation of phospholipase C-gamma 1 activity and expression in the rat small intestine

The accumulation of low density lipoprotein (LDL) in the arterial intima is an important characteristic of atherosclerosis. We investigated the mechanisms by which LDL binds to different types of collagen. The binding activities of 125I-labeled human native LDL (nLDL) and copper-oxidized LDL (oxLDL) with different collagen gels prepared in type I collagen-based mixtures with types I, III, IV and V (I+I, I+III, I+IV and I+V, respectively) were examined. A concentration of 20 micrograms LDL protein/150 micrograms collagen/well was used. The diffusion of both nLDL and oxLDL into the collagen gels reached an equilibrium after 48 h. All of the collagen gels showed the same rates of diffusion with both LDLs. The binding activities of oxLDL were significantly greater than those of nLDL (P < 0.001%), while the binding activities for both LDLs followed the order I+I and I+III > I+V > I+IV. However, the increased binding rate of oxLDL compared to nLDL was 1.66 for I+IV, 1.50 for I+V, 1.33 for I+I and 1.19 for I+III. When a 10-fold higher dose of NaCl (1 M) was added to the oxLDL medium, the binding rate of oxLDL was reduced (rate of reduction: 52% (I+I), 48% (I+III), 35% (I+IV), 13% (I+V)). These results suggest that oxLDL binds more to type I and III collagens by negative charge-dependent mechanisms than to type IV and V collagens. Therefore, types I and III collagens may play an important role in trapping LDL, especially oxLDL. Therefore, oxidatively modified LDL may contribute to atherogenesis due to its longer retention in the arterial wall.     

Collagen type I modulates the platelet-derived growth factor (PDGF) regulation of the growth and expression of beta1 integrins by rat mesangial cells

Mesangial cell (MC) proliferation and the deposition of collagen type I (collagen I) are the major pathological features in many types of glomerulonephritis (GN). Recent work suggested that beta-integrins play a critical role in the cell proliferation and extracellular matrix (ECM) remodeling observed in tissue repair after injury. To examine the involvement of beta-integrins in MC proliferation in association with the interaction of MCs with pathological collagen I, we investigated the effect of a prominent mitogen, platelet-derived growth factor-BB (PDGF-BB) on the growth and expression of beta-integrins by MCs cultured on plastic or in a three-dimensional collagen I gel. Immunoprecipitation using 35S-metabolic labeling, flow cytometry and a 3H-thymidine-uptake analysis demonstrated that PDGF-BB stimulated the cell mitogenicity and the expression of alpha5beta1 integrin (a fibronectin receptor), but not alpha1beta1 integrin (a collagen and laminin receptor) of MCs on plastic, in a dose-dependent manner. In contrast, MCs in the collagen I gels showed no significant changes in mitogenicity or alpha1beta1 and alpha5beta1 integrin expression, but increased alpha1beta1 integrin-mediated gel contraction was observed after PDGF-BB stimulation. Thus, the parallel up-regulation of MC-mitogenicity and alpha5beta1 integrin expression by PDGF-BB suggested that alpha5beta1 integrin is an important ECM receptor involved in the proliferative phenotype of MC. A spatial interaction between MCs and pathological collagen I in GN may influence the PDGF regulation of the MC phenotype regarding the cell growth and the expression of beta1 integrins.     

Crystal structure of ICAM-2 reveals a distinctive integrin recognition surface

Recognition by integrin proteins on the cell surface regulates the adhesive interactions between cells and their surroundings. The structure of the 'I' domain that is found in some but not all integrins, has been determined. However, the only integrin ligands for which structures are known, namely fibronectin and VCAM-1, are recognized by integrins that lack I domains. The intercellular adhesion molecules ICAM-1, 2 and 3 are, like VCAM-1, members of the immunoglobulin superfamily (IgSF), but they are recognized by an I domain-containing integrin, lymphocyte-function-associated antigen 1 (LFA-1, or CD11a/CD18). Here we present the crystal structure of the extracellular region of ICAM-2. The glutamic acid residue at position 37 is critical for LFA-1 binding and is proposed to coordinate the Mg2+ ion in the I domain; this Glu 37 is surrounded by a relatively flat recognition surface and lies in a beta-strand, whereas the critical aspartic acid residue in VCAM-1 and fibronectin lie in protruding loops. This finding suggests that there are differences in the architecture of recognition sites between integrins that contain or lack I domains. A bend between domains 1 and 2 of ICAM-2 and a tripod-like arrangement of N-linked glycans in the membrane-proximal region of domain 2 may be important for presenting the recognition surface to LFA-1. A model of ICAM-1 based on the ICAM-2 structure provides a framework for understanding its recognition by pathogens.     

Integrin-mediated entry into S phase of human gastric adenocarcinoma cells

The integrins are a family of integral membrane receptors that participate in binding to various extracellular and cell surface proteins during adhesion, migration, and homing of normal and neoplastic cells. In this study, we characterized the involvement of integrins in mediating the growth of an adhesion-dependent gastric adenocarcinoma line, ST2. This line was distinguished and selected for study based on its inability to grow when suspended in soft agar or plated on poly(2-hydroxyethyl methacrylate)-coated dishes. ST2 cells arrested in G0/G1 of the cell cycle when deprived of adhesion to substrate. Using purified matrix components, collagen was found to be highly active in promoting beta 1 integrin-mediated cell attachment and spreading. Subsequent to spreading on collagen, the cells were released from G0/G1 block and progressed into S phase. Monoclonal antibodies to alpha 2 or beta 1 integrin blocked the reinduction of both cell spreading and entry into S phase. These studies suggest that during the metastatic process, integrin receptor interaction with the insoluble matrix may be an important step leading to proliferation of some tumors.     

Crystal structure and mapping by site-directed mutagenesis of the collagen-binding epitope of an activated form of BM-40/SPARC/osteonectin

The extracellular calcium-binding domain (positions 138-286) of the matrix protein BM-40 possesses a binding epitope of moderate affinity for several collagen types. This epitope was predicted to reside in helix alphaA and to be partially masked by helix alphaC. Here we show that deletion of helix alphaC produces a 10-fold increase in collagen affinity similar to that seen after proteolytic cleavage of this helix. The predicted removal of the steric constraint was clearly demonstrated by the crystal structure of the mutant at 2.8 A resolution. This constitutively activated mutant was used to map the collagen-binding site following alanine mutagenesis at 13 positions. Five residues were crucial for binding, R149 and N156 in helix alphaA, and L242, M245 and E246 in a loop region connecting the two EF hands of BM-40. These residues are spatially close and form a flat ring of 15 A diameter which matches the diameter of a triple-helical collagen domain. The mutations showed similar effects on binding to collagens I and IV, indicating nearly identical binding sites on both collagens. Selected mutations in the non-activated mutant DeltaI also reduced collagen binding, consistent with the same location of the epitope but in a more cryptic form in intact BM-40.     

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 (alpha2, alpha3, or beta1). In the presence of anti-beta1 integrin (25 microg/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-alpha2 (10 microg/ml, clone P1E6) or anti-alpha3 (10 microg/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.