Chondrocyte Aggregation in Suspension Culture Is GFOGER-GPP- and 1 Integrin-dependent (original) (raw)
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Matrix biology : journal of the International Society for Matrix Biology, 2017
Interactions of cells with supramolecular aggregates of the extracellular matrix (ECM) are mediated, in part, by cell surface receptors of the integrin family. These are important molecular components of cell surface-suprastructures regulating cellular activities in general. A subfamily of β1-integrins with von Willebrand-factor A-like domains (I-domains) in their α-chains can bind to collagen molecules and, therefore, are considered as important cellular mechano-receptors. Here we show that chondrocytes strongly bind to cartilage collagens in the form of individual triple helical molecules but very weakly to fibrils formed by the same molecules. We also find that chondrocyte integrins α1β1-, α2β1- and α10β1-integrins and their I-domains have the same characteristics. Nevertheless we find integrin binding to mechanically generated cartilage fibril fragments, which also comprise peripheral non-collagenous material. We conclude that cell adhesion results from binding of integrin-conta...
Chondrocyte survival and differentiation in situ are integrin mediated
Developmental Dynamics, 1997
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 (␣2, ␣3, or 1). In the presence of anti-1 integrin (25 g/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-␣2 (10 g/ml, clone P1E6) or anti-␣3 (10 g/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.
Chondrocytes and chondrons for tissue engineering of cartilage
OBJECTIVES. To investigate whether maintaining the chondrocyte's native pericellular matrix prevents collagen-induced up-regulation of collagenase-3 (MMP-13) and whether integrin α1 (ITGα1) and /or discoidin domain receptor 2 (DDR2) modulate MMP-13 expression and which signalling pathway plays a role in collagen-stimulated MMP-13 expression. METHODS. Goat articular chondrocytes and chondrons were cultured on collagen coatings. Small interfering RNA (siRNA) oligonucleotides targeted against Itgα1 and Ddr2 were transfected into primary chondrocytes. Chemical inhibitors for MEK1 (PD98059), FAK (FAK inhibitor 14), JNK (SP600125) and PKC (PKC412), and a calcium chelator (BAPTA-AM) were used in cell cultures. Real-time polymerase chain reaction was performed to examine gene expression levels of Mmp-13, Itgα1 and Ddr2 and collagenolytic activity was determined by measuring the amount of hydroxyproline released in the culture medium. RESULTS. Maintaining the chondrocyte's native pericellular matrix prevented Mmp-13 upregulation and collagenolytic activity when the cells were cultured on a collagen coating. Silencing of Itgα1 and Ddr2 reduced Mmp-13 gene expression and collagenolytic activity by primary chondrocytes cultured on collagen. Incubation with the PKC inhibitor strongly reduced Mmp-13 gene expression levels. Gene expression levels of Mmp-13 were also decreased by chondrocytes incubated with the MEK, FAK or JNK inhibitor. CONCLUSIONS. Maintaining the native pericellular matrix of chondrocytes prevents collagen-induced up-regulation of MMP-13. Both integrin α1 and discoidin domain receptor 2 modulate Mmp-13 expression after direct contact between chondrocytes and collagen. PKC, FAK, MEK and JNK are involved in collagen-stimulated expression of Mmp-13.
Cell and Tissue Research, 2004
Articular cartilage is subjected to cyclic compressive stresses during joint loading. There is increasing experimental evidence that this loading is essential for the chondrocytes to maintain the functionality of the cartilage extracellular matrix (ECM) and that members of the integrin family of transmembrane receptors may play an important role in signal mechanotransduction between the ECM and chondrocytes. Of particular interest are the integrin subunits α5 and β1, which are known to form the receptor for fibronectin, an important ECM protein, and to be involved in mechanotransduction as well as in the regulation of cytokine production. In this study, we measured the amounts of the integrin subunits α5 and β1 in chondrocytes from young (immature) and adult (mature) bovine articular cartilage explants which were subjected to a continuously applied cyclic compressive stress of 1 MPa for 6 and 24 h. The integrin content per chondrocyte was measured immediately after load cessation by flow cytometry following matrix digestion to release the cells. We found that a mechanical stress induced an increase in the number of integrin subunit α5 in immature and mature cartilage but not in the integrin subunit β1 content. The integrin contents were greatest after 6 h of loading and returned to control levels after 24 h of unloading. The results of this study supply further experimental evidence that chondrocytes respond to changes in their mechanical environment and that the integrin α5β1 may act as a mechanical signal transducer between the chondrocyte and the ECM for the modulation of cellular physiology.
Inhibition of Chondrogenesis by Integrin Antibodyin Vitro
Experimental Cell Research, 1998
sion during embryogenesis and development. Integrins Integrins mediate cell attachment to a variety of ex-bind extracellular matrix to the cytoskeleton and can tracellular matrix proteins. These interactions play an thereby transduce extracellular signals into the intraimportant role in morphogenesis and differentiation. cellular machinery controlling cell behavior [1-8]. In The mediating functions of integrins during chondrofact, the influence of collagen type II on the behavior genesis in vitro were investigated by using mesenchyof chondrocytes depends on integrins participating in mal cells from limb buds of day 12 mouse embryos. The chondrocyte-collagen type II interactions. Hence, these cells were treated with anti-b1,-a1, and-a5 integrin integrins mediate the interaction of chondrocytes and antibodies (a) from day 1 to day 3 and (b) from day 3 collagen types I and II and fibronectin [9-12]. to day 7 of cultivation. The total culture period was 7 Chondrogenesis involves the recruitment of mesendays. The presence of exogenous anti-b1, but not-a1 chymal cells to chondroblasts and their differentiation, and-a5 integrin antibodies, from day 1 to 3 completely e.g., the capability to synthesize a cartilage-specific exinhibited the differentiation of blastemal cells to chontracellular matrix. The organoid culture system allows droblasts and the formation of cartilage matrix. On the the differentiation of prechondrogenic mesenchymal other hand, the presence of exogenous anti-b1,-a1, cells from dissociated limb buds, as reported on by a and-a5 integrin antibodies from day 3 of cultivation number of publications. During cultivation the initially onwards had no effect. Immunoblotting and immunosmall cartilage nodules become larger, due to apposimorphological findings in the cultures treated with tional growth and matrix synthesis, which involves the anti-b1 antibody from day 1 to day 3 revealed a pattern of integrins and collagen composed of b1, a1, a5b1 inte-transition of perichondral cells to chondrocytes [13grins and collagen type I. The cartilage-specific chon-15]. In the developing organoid culture, prechondrodroitin sulfate proteoglycan (CSPG) could not be demgenic mesenchymal cells readily aggregate. They cononstrated in these cultures. The cultures treated later sist of densely packed cells with tight surface contacts. (day 3 to day 7) showed a pattern of b1, a3, a5b1, and This is of great importance for the triggering of differ-avb3 integrins, collagen types I and II, and CSPG idenentiation to cartilage [16-18]. It is known that Ca 2/ tical to that of the untreated controls. These findings [19], adhesion molecules such as the neural cell adheindicate that b1-integrins play a crucial role in early sion molecule (N-CAM), N-cadherin [20-22], and hyacartilage differentiation and point to a possible imluronan [23] influence and establish cell-cell contacts, portant cell-matrix interaction in the induction of chondrocyte aggregation, and chondrocyte differentiachondrogenesis. ᭧ 1998 Academic Press tion during cartilage formation. Blockage of N-CAM,
Changes in integrin expression during chondrogenesis in vitro: an immunomorphological study
Journal of Histochemistry & Cytochemistry, 1995
Integrins are receptors composed of ligand-specific alpha-chains and cell type-specific beta-chains which are involved in cell-cell and cell-matrix interactions. The distribution of alpha 1- and alpha 3-integrins as well as collagen Types I and II, was investigated by immunofluorescence and immunoelectron microscopy during chondrogenesis in organ culture after various culture periods. Mesenchymal cells from limb buds of Day 12 mouse embryos were grown at high density. Within the first 2 days of the culture period, only alpha 1-integrin could be detected. Formation of cartilage-specific matrix on Day 3 was accompanied by the occurrence of alpha 3-integrin. On Day 7, alpha 3 was present only in cartilage nodules, whereas alpha 1 was strongly expressed in the perichondrium and was more or less homogeneously distributed in the surrounding mesenchyme. On Day 14, alpha 1-integrin was again detectable in cartilage. We suggest that the change in collagen formation from Type I to Type II dur...