Cartilage matrix protein forms a type II collagen-independent filamentous network: analysis in primary cell cultures with a retrovirus expression system.

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136

Matrilin-3 is a recently identified member of the superfamily of proteins containing von Willebrand factor A-like domains and is able to form hetero-oligomers with matrilin-1 (cartilage matrix protein) via a C-terminal coiled-coil domain. Full-length matrilin-3 and a fragment lacking the assembly domain were expressed in 293-EBNA cells, purified, and subjected to biochemical characterization. Recombinantly expressed full-length matrilin-3 occurs as monomers, dimers, trimers, and tetramers, as detected by electron microscopy and SDSpolyacrylamide gel electrophoresis, whereas matrilin-3, purified from fetal calf cartilage, forms homotetramers as well as hetero-oligomers of variable stoichiometry with matrilin-1. In the matrix formed by cultured chondrosarcoma cells, matrilin-3 is found in a filamentous, collagen-dependent network connecting cells and in a collagen-independent pericellular network. Affinity-purified antibodies detect matrilin-3 expression in a variety of mouse cartilaginous tissues, such as sternum, articular, and epiphyseal cartilage, and in the cartilage anlage of developing bones. It is found both inside the lacunae and in the interterritorial matrix of the resting, proliferating, hypertrophic, and calcified cartilage zones, whereas the expression is lower in the superficial articular cartilage. In trachea and in costal cartilage of adult mice, an expression was seen in the perichondrium. Furthermore, matrilin-3 is found in bone, and its expression is, therefore, not restricted to chondroblasts and chondrocytes.The matrilins constitute a recently discovered family of noncollagenous proteins (1) belonging to the von Willebrand factor A (vWFA) 1 -like domain superfamily. To date, there are four matrilins known. Matrilin-2 (2, 3) and matrilin-4 (4, 5) have a broad tissue distribution, whereas the expression of matrilin-1 (also known as cartilage matrix protein) (6 -8) and matrilin-3 (9 -11) is more restricted to skeletal tissues. The division of the family into two subgroups can also be concluded from evolutionary studies (1). The descent from a common ancestor and the divergence through duplication of whole domains indicates the possibility of the different family members providing similar functions in different tissues. The at least partially coordinated expression of matrilin-1 and -3 gains further functional significance through the recent discovery of hetero-oligomers formed by matrilin-1 and -3 in epiphyseal cartilage of fetal calf femur (12).Matrilin-3 has most features of the modular structure typical for matrilins and consists of an N-terminal vWFA-like domain, four EGF-like domains, and a C-terminal ␣-helical coiled-coil oligomerization domain (9 -11), but it lacks the second vWFAlike domain that is present in all the other matrilins. Similarly, a unique mouse matrilin-4 splice variant lacking the N-terminal vWFA-like domain was recently identified (4). In addition, matrilin-3 possesses a domain with a high content of positively charged amino acids between the N-terminal vWFA-like domain ...

Section: Matrilin-3 Is Able To Form Homo-andsupporting

confidence: 72%

“…These extended fibrils could not be detected in the absence of ascorbate (Fig. 5B), conditions known to inhibit collagen secretion (21). Under those conditions, matrilin-3 was seen on the surface of individual cells or in network structures connecting adjacent cells (Fig.…”

Section: Recombinant Expression Of Matrilin-3mentioning

confidence: 90%

Molecular Structure and Tissue Distribution of Matrilin-3, a Filament-forming Extracellular Matrix Protein Expressed during Skeletal Development

Klatt

Nitsche

Kobbe

et al. 2000

116

136

“…CMP forms two types of filamentous networks in the pericellular matrix: one that contains type II collagen and another that does not contain type II collagen (30). The collagen-independent CMP filaments may also serve as a scaffold for cell adhesion in vivo because CMP at high concentrations (10 -20 g/ml) enhanced cell spreading in the absence of type II collagen in vitro.…”

Section: Discussionmentioning

confidence: 99%

Enhancement of Cell Adhesion and Spreading by a Cartilage-specific Noncollagenous Protein, Cartilage Matrix Protein (CMP/Matrilin-1), via Integrin α1β1

Makihira

Yan

Ohno³

et al. 1999

Cartilage matrix protein (CMP; also known as matrilin-1), one of the major noncollagenous proteins in most cartilages, binds to aggrecan and type II collagen. We examined the effect of CMP on the adhesion of chondrocytes and fibroblasts using CMP-coated dishes. The CMP coating at 10 -20 g/ml enhanced the adhesion and spreading of rabbit growth plate, resting and articular chondrocytes, and fibroblasts and human epiphyseal chondrocytes and MRC5 fibroblasts. The effect of CMP on the spreading of chondrocytes was synergistically increased by native, but not heated, type II collagen (gelatin). The monoclonal antibody to integrin ␣ 1 or ␤ 1 abolished CMP-induced cell adhesion and spreading, whereas the antibody to integrin ␣ 2 , ␣ 3 , ␣ 5 , ␤ 2 , ␣ 5 ␤ 1 , or ␣ V ␤ 5 had little effect on cell adhesion or spreading. The antibody to integrin ␣ 1 , but not to other subunits, coprecipitated 125 I-CMP that was added to MRC5 cell lysates, indicating the association of CMP with the integrin ␣ 1 subunit. Unlabeled CMP competed for the binding to integrin ␣ 1 with 125 I-CMP. These findings suggest that CMP is a potent adhesion factor for chondrocytes, particularly in the presence of type II collagen, and that integrin ␣ 1 ␤ 1 is involved in CMP-mediated cell adhesion and spreading. Since CMP is expressed almost exclusively in cartilage, this adhesion factor, unlike fibronectin or laminin, may play a special role in the development and remodeling of cartilage.Cartilage matrix protein (CMP 1 /matrilin-1) was originally isolated as a protein that binds to aggrecan and thereafter was shown to bind to type II collagen (1-3). CMP is synthesized in a cartilage-specific manner, except that eye tissues, notochord, and tendon express CMP at low levels (4 -7). The role of CMP is unknown, but it may have a structural role, modulating physical properties of cartilage, or may be involved in matrixcell interactions.CMP exists in vivo as a homotrimer of 148 kDa, as measured by sedimentation equilibrium centrifugation, although estimates of the molecular mass of this protein by SDS-polyacrylamide gel electrophoresis (PAGE) yield a higher value. CMP migrates at positions corresponding to 215 and 60 kDa under nonreducing and reducing conditions, respectively, during SDS-PAGE (2, 8). The subunits of CMP are connected in the C-terminal region by disulfide bonding and the presence of the coiled-coil ␣-helical assembly domain (9, 10). The monomer has two type A-like (von Willebrand factor-like or I) domains connected with an epidermal growth factor-like domain in addition to the short C-terminal domain (9,11,12). A type A-like domain is present in several proteins such as complement factors B and C2; type VI, XII, XIV, and XVI collagens; and ␣ subunits of several integrins (13). Some members of this protein family are involved in cell adhesion. However, whether CMP is an adhesion factor remains unknown.Numerous adhesion proteins are recognized by integrins, which are heterodimeric proteins with two (␣ and ␤) membrane-spanning subunits. The extracel...

“…Anti-p38 MAPK, anti-JNK1, anti-BCL-2, and protein A/G were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA). Monoclonal antibodies including anti-CMP and anti-link protein were prepared as described previously (16 Cell Culture and Preparation of Cell Lysates-Primary chondrocyte cultures were established as described previously (1). Briefly, hypertrophic chondrocytes were obtained from the cephalic part of sterna cartilage from 17-day-old embryonic chickens, and prehypertrophic chondrocytes were obtained from the caudal part of the cartilage.…”

mentioning

confidence: 99%

Mitogen-activated Protein Kinase p38 Mediates Regulation of Chondrocyte Differentiation by Parathyroid Hormone

Zhen¹,

Wei²,

Wu³

et al. 2001

Parathyroid hormone (PTH) and its related peptide regulate endochondral ossification by inhibiting chondrocyte differentiation toward hypertrophy. However, the intracellular pathway for transducing PTH/PTH-related peptide signals in chondrocytes remains unclear. Here, we show that this pathway is mediated by mitogen-activated protein kinase (MAPK) p38. Incubation of hypertrophic chondrocytes with PTH (1-34) induces an inhibition of p38 kinase activity in a time-and dose-dependent manner. Inhibition of protein kinase C prevents PTH-induced p38 MAPK inhibition, whereas inhibition of protein kinase A has no effect. Thus, protein kinase C, but not protein kinase A, is required for the inhibition of p38 MAPK by PTH. Treatment of hypertrophic chondrocytes by PTH or by p38 MAPK inhibitor SB203580 up-regulates Bcl-2, suggesting that Bcl-2 lies downstream of p38 MAPK in the PTH signaling pathway. Inhibition of p38 MAPK in hypertrophic chondrocytes by either PTH, SB303580, or both together leads to a decrease of hypertrophic marker type X collagen mRNA and an increase of the expression of prehypertrophic marker cartilage matrix protein. Therefore, inhibition of p38 converts a hypertrophic cell phenotype to a prehypertrophic one, thereby preventing precocious chondrocyte hypertrophy. Taken together, these data suggest a major role for p38 MAPK in transmitting PTH signals to regulate chondrocyte differentiation.During endochondral ossification, chondrocytes undergo a differentiation process including proliferation, maturation, hypertrophy, and apoptosis (1). Cells from these different stages of differentiation synthesize specific molecules, for example, type X collagen is synthesized specifically by hypertrophic chondrocytes (2, 3), whereas cartilage matrix protein (CMP) 1 / matrilin-1, is a marker of prehypertrophic chondrocytes (1). Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) are major regulators of the chondrocyte differentiation process. PTH and PTHrP both bind to PTH receptors, which belong to G protein-coupled receptors (4). In a growth plate, PTH receptors are expressed at specific stages during chondrocyte differentiation, with the highest level at the prehypertrophic to hypertrophic transition (5). This suggests that PTH receptors may be important for the regulation of chondrocyte differentiation from the prehypertrophic state to the hypertrophic state. Indeed, overexpression of PTHrP or its constitutively active receptor in growth plates of transgenic mice delays endochondral bone formation (6), whereas mice with ablation of the PTH/PTHrP receptor gene develop skeletal dysplasia because of accelerated chondrocyte hypertrophy (7-9). Thus, the PTH/PTHrP receptor plays a fundamental role in the control of endochondral bone formation by transducing signals inhibiting chondrocyte hypertrophy. However, the intracellular signaling mechanism for PTH/PTHrP to regulate chondrocyte differentiation remains unknown.The PTH/PTHrP receptor may exert its biological action via at least two signaling p...