Changes in secreted and cell associated proteoglycan synthesis during conversion of myoblasts to osteoblasts in response to bone morphogenetic protein-2: role of decorin in cell response to BMP-2

Proteoglycans have been identified within the extracellular matrices (ECM) of bone and are known to play a role in ECM assembly, mineralization, and bone formation. Bone morphogenetic protein-2 (BMP-2) specifically converts the differentiation pathway of C2C12 myoblasts into that of osteoblast lineage cells. Microarray analyses of the mouse myoblast cell line C2C12 and its differentiation into osteoblastic cells in response to BMP-2 have suggested the up-regulation of several proteoglycan species, although there is a lack of biochemical evidence for this response. In this study we have biochemically analyzed and characterized the proteoglycan populations that are induced in C2C12 cells upon osteoblastic differentiation produced by BMP-2. An important and specific increase in the synthesis of secreted decorin was observed in BMP-2-treated cells, as compared to untreated myoblasts and myoblasts induced to differentiate into myotubes. Decorin was seen to contain larger glycosaminoglycan (GAG) chains in induced than in non-induced cells. BMP-2 also produced an augment in the synthesis of different heparan sulfate proteoglycans such syndecan-2, - 3, glypican, and perlecan in detergent-soluble and non-soluble cellular fractions. We also examined whether the evident changes induced by BMP-2 in secreted decorin could have a functional role. BMP-2 signaling dependent as well as induction of alkaline phosphatase (ALP) activity was diminished in decorin null myoblasts compared to wild type myoblats although cell surface level of BPM-2 receptors was unchanged. These results are the first biochemical evidence and analysis for the effect of BMP-2 on the synthesis of proteoglycan during osteogenic conversion of myoblasts and suggest a role for decorin in cell response to BMP-2.     

Effect of p-nitrophenyl-beta-D-xyloside on proteoglycan synthesis and extracellular matrix formation by bovine corneal endothelial cell cultures

The effect of p-nitrophenyl-beta-D-xyloside on proteoglycan synthesis and extracellular matrix (ECM) formation by cultured bovine corneal endothelial (BCE) cells was investigated. BCE cells actively proliferating on plastic dishes produced in the absence of xyloside an ECM containing various proteoglycans. Heparan sulfate was the main 35S-labeled glycosaminoglycan component (83%). Dermatan sulfate (14%) and chondroitin sulfate (3%) were also present. Exposure of actively proliferating BCE cells to xyloside totally inhibited synthesis of proteoglycans containing dermatan sulfate or chondroitin sulfate and caused an 86% inhibition of heparan sulfate proteoglycan synthesis. The heparan sulfate proteoglycans that were extracted from the ECM produced by BCE cells exposed to xyloside had a smaller size and a reduced charge density compared to their counterparts extracted from the ECM of cultures not exposed to xyloside. In contrast to the inhibitory effect of the xyloside on proteoglycan synthesis, exposure of actively proliferating BCE cells to xyloside stimulated synthesis of free chondroitin sulfate and heparan sulfate chains. All of the xyloside-initiated glycosaminoglycan chains were secreted into the culture medium. The proteoglycan-depleted matrices produced by BCE cells exposed to xyloside were used to study the effect of these matrices on proteoglycan synthesis by BCE cells. BCE cells growing on proteoglycan-depleted ECM showed a considerable increase in the rate of proteoglycan synthesis compared to BCE cells growing on normal ECM. Moreover, the pattern of glycosaminoglycan synthesis by BCE cells growing on proteoglycan-depleted ECM was changed to one which resembled that of BCE cells actively proliferating on plastic dishes. It is postulated that BCE cells are able to recognize when an ECM is depleted of proteoglycan and to respond to it by increasing their rate of proteoglycan synthesis and incorporation into the ECM.     

Domains in proteins and proteoglycans of the extracellular matrix with functions in assembly and cellular activities

Most proteins of the extracellular matrix (ECM), such as the glycoproteins, collagens and proteoglycans, consist of many structurally autonomous domains that are often functionally distinct. Consequently these proteins are designated as mosaic proteins. Related domains are often found in several different ECM proteins. Domains which are of importance for assembly have been identified by fragmentation and other approaches. Triple-stranded coiled-coil domains in laminin and probably also in tenascin and thrombospondin are responsible for chain selection, a process which may be important for the formation of tissue specific isoforms. Globular domains at the C-terminus of collagenous domains are essential for the registration of the three chains and triple-helix formation. Fibrillar assemblies of these triple helices with constituent globular domains serve important assembly functions in many collagens including collagens IV and VI. Many other domains with more specialized functions in assembly have been identified in laminin, fibronectin and other ECM proteins. Cys-rich domains with either distant or close homology with epidermal growth factor are repeated manifold in rod-like regions of a number of ECM proteins including laminin, tenascin and thrombospondin. They may serve as spacer elements but as suggested for laminin some domains of this type may also function as signals for cellular growth and differentiation. Another important cellular function common to many ECM proteins is cell attachment. Several cell attachment sites have been localized in structurally unrelated domains of the same or of different ECM proteins.     

Fibronectin fibril alignment is established upon initiation of extracellular matrix assembly

The physical structure of the extracellular matrix (ECM) is tissue-specific and fundamental to normal tissue function. Proper alignment of ECM fibers is essential for the functioning of a variety of tissues. While matrix assembly in general has been intensively investigated, little is known about the mechanisms required for formation of aligned ECM fibrils. We investigated the initiation of fibronectin (FN) matrix assembly using fibroblasts that assemble parallel ECM fibrils and found that matrix assembly sites, where FN fibrillogenesis is initiated, were oriented in parallel at the cell poles. We show that these polarized matrix assembly sites progress into fibrillar adhesions and ultimately into aligned FN fibrils. Cells that assemble an unaligned meshwork matrix form matrix assembly sites around the cell periphery, but the distribution of matrix assembly sites in these cells could be modulated through micropatterning or mechanical stretch. While an elongated cell shape corresponds with a polarized matrix assembly site distribution, these two features are not absolutely linked, since we discovered that transforming growth factor beta (TGF-β1) enhances matrix assembly site polarity and assembly of aligned fibrils independent of cell elongation. We conclude that the ultimate orientation of FN fibrils is determined by the alignment and distribution of matrix assembly sites that form during the initial stages of cell–FN interactions.     

Extracellular matrix is required for skeletal muscle differentiation but not myogenin expression

Skeletal muscle cells are a useful model for studying cell differentiation. Muscle cell differentiation is marked by myoblast proliferation followed by progressive fusion to form large multinucleated myotubes that synthesize muscle-specific proteins and contract spontaneously. The molecular analysis of myogenesis has advanced with the identification of several myogenic regulatory factors, including myod1, myd, and myogenin. These factors regulate each other's expression and that of muscle-specific proteins such as the acetylcholine receptor and acetylcholinesterase (AChE). In order to investigate the role of extracellular matrix (ECM) in myogenesis we have cultured myoblasts (C₂C₁₂) in the presence or absence of an exogenous ECM (Matrigel). In addition, we have induced differentiation of myoblasts in the presence or absence of Matrigel and/or chlorate, a specific inhibitor of proteoglycan sulfation. Our results indicated that the formation of fused myotubes and expression of AChE was stimulated by Matrigel. Treatment of myoblasts induced to differentiate with chlorate resulted in an inhibition of cell fusion and AChE activity. Chlorate treatment was also found to inhibit the deposition and assembly of ECM components such fibronectin and laminin. The expression of myogenin mRNA was observed when myoblasts were induced to differentiate, but was unaffected by the presence of Matrigel or by culture of the cells in the presence of chlorate. These results suggest that the expression of myogenin is independent of the presence of ECM, but that the presence of ECM is essential for the formation of myotubes and the expression of later muscle-specific gene products. © 1996 Wiley-Liss, Inc.     

Biosynthesis and deposition of a noncovalent laminin-heparan sulfate proteoglycan complex and other basal lamina components by a human malignant cell line

The basal lamina components laminin, heparan sulfate proteoglycan (HSPG), and type IV collagen were synthesized and codeposited in the extracellular matrix (ECM) by a cultured human cell line from gestational choriocarcinoma (JAR). Laminin and HSPG formed a noncovalent complex detected by the coimmunoprecipitation of HSPG with laminin from cell lysates and culture media. The complex was stable in the cell lysis buffer that contained detergents (1% Triton X-100, 0.5% deoxycholate, and 0.1% sodium dodecyl sulfate) and sodium chloride (from 0.15 to 1.0 M), but was dissociated by adding 8 M urea to the detergent lysates. Even though JAR cells produced roughly equal amounts of HSPG and chondroitin sulfate proteoglycan, only HSPG complexed with laminin, suggesting a specific interaction between these basal lamina components. The laminin-HSPG complex was deposited and retained in the ECM. This was shown biochemically by isolating an enriched fraction of ECM from JAR cells cultured on native type I collagen gels. At steady state, more than half (52%) of the laminin-HSPG in the culture was recovered in the ECM fraction, in contrast to 16% of the total laminin and 29% of the total type IV collagen, which were secreted to a greater extent than laminin-HSPG into the culture medium. The retention of the laminin-HSPG complex in the ECM suggests that it may participate in the assembly of the basal lamina-like extracellular matrix deposited by JAR cultures. Omission of ascorbate from the culture medium abolished the ECM deposition of type IV collagen but had little effect on the deposition of laminin or laminin-HSPG. This demonstrates that the stable deposition of laminin-HSPG and laminin in the collagen-based choriocarcinoma cultures is not dependent on an assembled network of type IV collagen.     

Biochemical and functional characterization of proteoglycans produced by Sl/Sld murine bone marrow stromal cell lines

The Steel anemia of mice results from an inherited defect in the hematopoietic microenvironment. Proteoglycans synthesized by bone marrow stromal cells are an important functional component of the hematopoietic microenvironment in normal animals. It is thus possible that Steel anemia results from a molecular abnormality involving bone marrow stromal proteoglycans. To investigate this possibility, we studied proteoglycan synthesis in three stromal cell lines from Steel anemic (Sl/Sld) animals and two control stromal cell lines, one (+/+2.4) from a non-anemic littermate, and one (GBl/6) from a normal mouse. Proteoglycans were precursor labelled with 35S sulfate and separated by ion exchange HPLC, CsCl density gradient centrifugation, and molecular sieve HPLC. Glycosaminoglycan (GAG) moieties were characterized by molecular sieve HPLC and enzyme sensitivity. There were no consistent differences in total proteoglycan synthesis, proteoglycan heterogeneity, GAG hydrodynamic size, or enzyme sensitivity among the cell lines studied. Growth factor binding to stromal extracellular matrix (ECM) was studied by co-culture of an IL-3-dependent cell line (FDC-P1) with cell-free ECM preparations from an Sl/Sld and a control (GBl/6) stromal cell line, with and without pre-incubation with IL-3. Cell-free ECM preparations from Sl/Sld and control cell lines supported FDC-P1 growth to an approximately equal extent after pre-incubation with IL-3. FDC-P1 growth support by ECM preparations from both cell lines was also observed without IL-3 pre-incubation, although to a lesser extent, suggesting ECM binding of endogenous growth factors synthesized by the stromal cells.     

Retention of the native chondrocyte pericellular matrix results in significantly improved matrix production.

The interaction of the cell with its surrounding extracellular matrix (ECM) has a major effect on cell metabolism. We have previously shown that chondrons, chondrocytes with their in vivo-formed pericellular matrix, can be enzymatically isolated from articular cartilage. To study the effect of the native chondrocyte pericellular matrix on ECM production and assembly, chondrons were compared with chondrocytes isolated without any pericellular matrix. Immediately after isolation from human cartilage, chondrons and chondrocytes were centrifuged into pellets and cultured. Chondron pellets had a greater increase in weight over 8 weeks, were more hyaline appearing, and had more type II collagen deposition and assembly than chondrocyte pellets. Minimal type I procollagen immunofluorescence was detected for both chondron and chondrocyte pellets. Chondron pellets had a 10-fold increase in proteoglycan content compared with a six-fold increase for chondrocyte pellets over 8 weeks (P<0.0001). There was no significant cell division for either chondron or chondrocyte pellets. The majority of cells within both chondron and chondrocyte pellets maintained their polygonal or rounded shape except for a thin, superficial edging of flattened cells. This edging was similar to a perichondrium with abundant type I collagen and fibronectin, and decreased type II collagen and proteoglycan content compared with the remainder of the pellet. This study demonstrates that the native pericellular matrix promotes matrix production and assembly in vitro. Further, the continued matrix production and assembly throughout the 8-week culture period make chondron pellet cultures valuable as a hyaline-like cartilage model in vitro.     

Post-translational addition of chondroitin sulfate glycosaminoglycans. Role of N-linked oligosaccharide addition, trimming, and processing

A melanoma proteoglycan model system has been used to examine the role of core protein asparagine-linked (N-linked) oligosaccharides in the transport and assembly of proteoglycan molecules. The use of agents which block discrete steps in the trimming and processing of core oligosaccharides (castanospermine, 1-deoxynojirimycin, N-methyldeoxynojirimycin, 1-deoxymannojirimycin, and swainsonine) demonstrates that removal of glucose residues from the N-linked oligosaccharides is required for the cell surface expression of a melanoma proteoglycan core protein and for the conversion of the core protein to a chondroitin sulfate proteoglycan. However, complete maturation of the oligosaccharides to a "complex" form is not required for these events. Treatment of M21 human melanoma cells with the glucosidase inhibitors castanospermine, 1-deoxynojirimycin, or N-methyldeoxynojirimycin results in a dose-dependent inhibition of glycosaminoglycan (GAG) addition to the melanoma antigen recognized by monoclonal antibody 9.2.27. In contrast, treatment with the mannosidase inhibitors 1-deoxymannojirimycin and swainsonine does not effect GAG addition. Identical results are obtained when the major histocompatibility complex class II antigen gamma chain proteoglycan is examined in inhibitor-treated melanoma and B-lymphoblastoid cells. These data, in conjunction with the known effects of the glucosidase and mannosidase inhibitors on the transport and secretion of other glycoproteins support the hypothesis that the addition, trimming, and processing of N-linked oligosaccharides is involved in the transport of certain proteoglycan core proteins to the site of GAG addition and to the cell surface.     

Structural and compositional divergencies in the extracellular matrix encountered by neural crest cells in the white mutant axolotl embryo

The skin of the white mutant axolotl larva is pigmented differently from that of the normal dark due to a local inability of the extracellular matrix (ECM) to support subepidermal migration of neural crest-derived pigment cell precursors. In the present study, we have compared the ECM of neural crest migratory pathways of normal dark and white mutant embryos ultrastructurally, immunohistochemically and biochemically to disclose differences in their structure/composition that could be responsible for the restriction of subepidermal neural crest cell migration in the white mutant axolotl. When examined by electron microscopy, in conjunction with computerized image analysis, the structural assembly of interstitial and basement membrane ECMs of the two embryos was found to be largely comparable. At stages of initial neural crest cell migration, however, fixation of the subepidermal ECM in situ with either Karnovsky-ruthenium red or with periodate-lysine-paraformaldehyde followed by ruthenium red-containing fixatives, revealed that fibrils of the dark matrix were significantly more abundant in associated electron-dense granules. This ultrastructural discrepancy of the white axolotl ECM was specific for the subepidermal region and suggested an abnormal proteoglycan distribution. Dark and white matrices of the medioventral migratory route of neural crest cells had a comparable appearance but differed from the corresponding subepidermal ECMs. Immunohistochemistry revealed only minor differences in the distribution of fibronectin, laminin, collagen types I, and IV, whereas collagen type III appeared differentially distributed in the two embryos. Chondroitin- and chondroitin-6-sulfate-rich proteoglycans were more prevalent in the white mutant embryo than in the dark, especially in the subepidermal space. Membrane microcarriers were utilized to explant site-specifically native ECM for biochemical analysis. Two-dimensional gel electrophoresis of these regional matrices revealed a number of differences in their protein content, principally in constituents of apparent molecular masses of 30-90,000. Taken together our observations suggest that local divergences in the concentration/assembly of low and high molecular mass proteins and proteoglycans of the ECM encountered by the moving neural crest cells account for their disparate migratory behavior in the white mutant axolotl.     

Angiogenesis

Extracellular matrix (ECM) is essential for all stages of angiogenesis. In the adult, angiogenesis begins with endothelial cell (EC) activation, degradation of vascular basement membrane, and vascular sprouting within interstitial matrix. During this sprouting phase, ECM binding to integrins provides critical signaling support for EC proliferation, survival, and migration. ECM also signals the EC cytoskeleton to initiate blood vessel morphogenesis. Dynamic remodeling of ECM, particularly by membrane-type matrix metalloproteases (MT-MMPs), coordinates formation of vascular tubes with lumens and provides guidance tunnels for pericytes that assist ECs in the assembly of vascular basement membrane. ECM also provides a binding scaffold for a variety of cytokines that exert essential signaling functions during angiogenesis. In the embryo, ECM is equally critical for angiogenesis and vessel stabilization, although there are likely important distinctions from the adult because of differences in composition and abundance of specific ECM components.     

Diabetic Nephropathy and Extracellular Matrix

Diabetic nephropathy (DN) is a serious complication in diabetes. Major typical morphological changes are the result of changes in the extracellular matrix (ECM). Thus, basement membranes are thickened and the glomerular mesangial matrix and the tubulointerstitial space are expanded, due to increased amounts of ECM. One important ECM component, the proteoglycans (PGs), shows a more complex pattern of changes in DN. PGs in basement membranes are decreased but increased in the mesangium and the tubulointerstitial space. The amounts and structures of heparan sulfate chains are changed, and such changes affect levels of growth factors regulating cell proliferation and ECM synthesis, with cell attachment affecting endothelial cells and podocytes. Enzymes modulating heparan sulfate structures, such as heparanase and sulfatases, are implicated in DN. Other enzyme classes also modulate ECM proteins and PGs, such as matrix metalloproteinases (MMPs) and serine proteases, such as plasminogen activator, as well as their corresponding inhibitors. The levels of these enzymes and inhibitors are changed in plasma and in the kidneys in DN. Several growth factors, signaling pathways, and hyperglycemia per se affect ECM synthesis and turnover in DN. Whether ECM components can be used as markers for early kidney changes is an important research topic, whereas at present, the clinical use remains to be established.     

Identification of the precursor protein to basement membrane heparan sulfate proteoglycans

The precursor protein of a basement membrane specific heparan sulfate proteoglycan has been identified as a 400,000 Mr polypeptide. Antibodies against large and small forms of this proteoglycan, isolated from a basement membrane (Engelbreth-Holm-Swarm, EHS) tumor, immunoprecipitated the same 400,000 protein from pulse-labeled EHS cells. The proteoglycan precursor protein was not recognized by antibodies against other basement membrane components or by antibodies to the cartilage proteoglycan. Furthermore, heparan sulfate proteoglycan purified from the EHS tumor blocked the immunoprecipitation of the precursor protein. Pulse-chase studies with [35S]methionine showed the precursor protein was converted to a proteoglycan. Pulse-chase studies with 35SO4 showed the large, low density proteoglycan appeared first and was degraded to a smaller, high density proteoglycan. We propose that the precursor protein is used after very little or no modification in the assembly of a large, low density heparan sulfate proteoglycan and that a portion of the population of these macromolecules are subsequently degraded to a smaller form.     

Coordinate role for cell surface chondroitin sulfate proteoglycan and alpha 4 beta 1 integrin in mediating melanoma cell adhesion to fibronectin

Cellular recognition and adhesion to the extracellular matrix (ECM) has a complex molecular basis, involving both integrins and cell surface proteoglycans (PG). The current studies have used specific inhibitors of chondroitin sulfate proteoglycan (CSPG) synthesis along with anti-alpha 4 integrin subunit monoclonal antibodies to demonstrate that human melanoma cell adhesion to an A-chain derived, 33-kD carboxyl-terminal heparin binding fragment of human plasma fibronectin (FN) involves both cell surface CSPG and alpha 4 beta 1 integrin. A direct role for cell surface CSPG in mediating melanoma cell adhesion to this FN fragment was demonstrated by the identification of a cationic synthetic peptide, termed FN-C/H-III, within the fragment. FN-C/H-III is located close to the amino terminal end of the fragment, representing residues #1721-1736 of intact FN. FN-C/H-III binds CSPG directly, can inhibit CSPG binding to the fragment, and promotes melanoma cell adhesion by a CSPG-dependent, alpha 4 beta 1 integrin-independent mechanism. A scrambled version of FN-C/H-III does not inhibit CSPG binding or cell adhesion to the fragment or to FN-C/H-III, indicating that the primary sequence of FN-C/H-III is important for its biological properties. Previous studies have identified three other synthetic peptides from within this 33-kD FN fragment that promote cell adhesion by an arginyl-glycyl-aspartic acid (RGD) independent mechanism. Two of these synthetic peptides (FN-C/H-I and FN-C/H-II) bind heparin and promote cell adhesion, implicating cell surface PG in mediating cellular recognition of these two peptides. Additionally, a third synthetic peptide, CS1, is located in close proximity to FN-C/H-I and FN-C/H-II and it promotes cell adhesion by an alpha 4 beta 1 integrin-dependent mechanism. In contrast to FN-C/H-III, cellular recognition of these three peptides involved contributions from both CSPG and alpha 4 integrin subunits. Of particular importance are observations demonstrating that CS1-mediated melanoma cell adhesion could be inhibited by interfering with CSPG synthesis or expression. Since CS1 does not bind CSPG, the results suggest that CSPG may modify the function and/or activity of alpha 4 beta 1 integrin on the surface of human melanoma cells. Together, these results support a model in which the PG and integrin binding sites within the 33-kD fragment may act in concert to focus these two cell adhesion receptors into close proximity on the cell surface, thereby influencing initial cellular recognition events that contribute to melanoma cell adhesion on this fragment.     

Versican and the regulation of cell phenotype in disease

Background

Versican is an extracellular matrix (ECM) proteoglycan that is present in the pericellular environment of most tissues and increases in many different diseases. Versican interacts with cells to influence the ability of cells to proliferate, migrate, adhere and assemble an ECM.

Scope of review

The structure of the versican molecule is briefly reviewed and studies highlighting those factors that promote versican synthesis and degradation and their impact on cell phenotype in disease are discussed. Particular attention is given to vascular disease, but other diseases where versican is important are covered as well, most notably different forms of cancers. Attention is given to mechanisms(s) by which versican influences cell behaviors through either direct or indirect processes. Versican produced by either stromal cells or myeloid cells can have a major impact influencing immunity and inflammation. Finally, studies controlling versican accumulation that either delay or inhibit the progression of disease will be highlighted.

Major conclusions

Versican is one component of the ECM that can influence the ability of cells to proliferate, migrate, adhere, and remodel the ECM. Targeting versican as a way to control cell phenotype offers a novel approach in the treatment of disease.

Significance

ECM molecules such as versican contribute to the structural integrity of tissues and interact with cells through direct and indirect means to regulate, in part, cellular events that form the basis of disease. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

The role of TIMPs in regulation of extracellular matrix proteolysis

Tissue inhibitors of metalloproteinases (TIMPs), which inhibit matrix metalloproteinases (MMPs) as well as the closely related, a disintegrin and metalloproteinases (ADAMs) and ADAMs with thrombospondin motifs (ADAMTSs), were traditionally thought to control extracellular matrix (ECM) proteolysis through direct inhibition of MMP-dependent ECM proteolysis. This classical role for TIMPs suggests that increased TIMP levels results in ECM accumulation (or fibrosis), whereas loss of TIMPs leads to enhanced matrix proteolysis. Mice lacking TIMP family members have provided support for such a role; however, studies with these TIMP deficient mice have also demonstrated that loss of TIMPs can often be associated with an accumulation of ECM. Collectively, these studies suggest that the divergent roles of TIMPs in matrix accumulation and proteolysis, which together can be referred to as ECM turnover, are dependent on the TIMP, specific tissue, and local tissue environment (i.e. health vs. injury/disease). Ultimately, these combined factors dictate the specific metalloproteinases being regulated by a given TIMP, and it is likely the diversity of metalloproteinases and their physiological substrates that determines whether TIMPs inhibit matrix proteolysis or accumulation. In this review, we discuss the evidence for the dichotomous roles of TIMPs in ECM turnover highlighting some of the common findings between different TIMP family members. Importantly, while we now have a better understanding of the role of TIMPs in regulating ECM turnover, much remains to be determined. Data on the specific metalloproteinases inhibited by different TIMPs in vivo remains limited and must be the focus of future studies.     

CD44-Anchored Hyaluronan-Rich Pericellular Matrices: An Ultrastructural and Biochemical Analysis

The chondrocyte pericellular matrix is an essential zone for cartilage matrix assembly and turnover. Electron micrographs of native endogenous and composition-defined exogenous pericellular matrices, both preserved via ruthenium hexaminetrichloride fixation procedures, depict strikingly similar networks of hyaluronan and proteoglycan extending out from the cell surface. Biochemical and morphological analyses of matrix regrowth show that monoclonal antibodies directed against the hyaluronan receptor CD44 blocked chondrocyte pericellular matrix assembly. Immunoperoxidase electron microscopy was used to display regular repeating spacing patterns of hyaluronan/proteoglycan assembly at the cell surface. These patterns compared well with the ultrastructural immunolocalization of CD44 at the cell surface. All of these data suggest that the hyaluronan receptor CD44 retains and participates in the assembly of the chondrocyte pericellular matrix.     

A comparison of fibronectin, laminin, and galactosyltransferase adhesion mechanisms during embryonic cardiac mesenchymal cell migration in vitro

Embryonic hearts contain a homogeneous population of mesenchymal cells which migrate through an extensive extracellular matrix (ECM) to become the earliest progenitors of the cardiac valves. Since these cells normally migrate through an ECM containing several adhesion substrates, this study was undertaken to examine and compare three ECM binding mechanisms for mesenchymal cell migration in an in vitro model. Receptor mechanisms for the ECM glycoproteins fibronectin (FN) and laminin (LM) and the cell surface receptor galactosyltransferase (GalTase), which binds an uncharacterized ECM substrate, were compared. Primary cardiac explants from stage 17 chick embryos were cultured on three-dimensional collagen gels. Mesenchymal cell outgrowth was recorded every 24 hr and is reported as a percentage of control. Migration was perturbed using specific inhibitors for each of the three receptor mechanisms. These included the hexapeptide GRGDSP (300-1000 micrograms/ml), which mimics a cell binding domain of FN, the pentapeptide YIGSR (300-1000 micrograms/ml), which mimics a binding domain of LM, and alpha-lactalbumin (1-10 mg/ml), a protein modifier of GalTase activity. The functional role of these adhesion mechanisms was further tested using antibodies to avian integrin (JG22) and avian GalTase. While the FN-related peptide had no significant effect on cell migration it did produce a rounded cellular morphology. The LN-related peptide inhibited mesenchymal migration 70% and alpha-lactalbumin inhibited cell migration 50%. Antibodies against integrin and GalTase inhibited mesenchymal cell migration by 80 and 50%, respectively. The substrate for GalTase was demonstrated to be a single high molecular weight substrate which was not LM or FN. Control peptides, proteins and antibodies demonstrated the specificity of these effects. These data demonstrate that multiple adhesion mechanisms, including cell surface GalTase, are potentially functional during cardiac mesenchymal cell migration. The sensitivity of cell migration to the various inhibitors suggests that occupancy of specific ECM receptors can modulate the activity of other, unrelated, ECM adhesion mechanisms utilized by these cells.     

力学环境对软骨基质代谢的影响

正常关节软骨所受压力是由动态压力与静态压力交替完成。压力引起软骨一系列生理变化包括细胞及细胞外基质成分变形、组织内液体流动、水流电位和生理生化变化。这些变化直接调控细胞外基质代谢。体外构建有良好功能的组织工程化软骨是目前软骨病变、缺损理想的修复方法。研究力学环境对软骨基质代谢的影响,对构建组织工程化软骨有深远意义。