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PubMed1989

Matrix-associated heparan sulfate proteoglycan: core protein-specific monoclonal antibodies decorate the pericellular matrix of connective tissue cells and the stromal side of basement membranes.

Cultured human lung fibroblasts produce a large, nonhydrophobic heparan sulfate proteoglycan that accumulates in the extracellular matrix of the monolayer (Heremans, A., J. J. Cassiman, H. Van den Berghe, and G. David. 1988. J. Biol. Chem. 263: 4731-4739). A panel of four monoclonal antibodies, specific for four distinct epitopes on the 400-kD core protein of this extracellular matrix heparan sulfate proteoglycan, detects similar proteoglycans in human epithelial cell cultures. Immunohistochemistry of human tissues with the monoclonal antibodies reveals that these proteoglycans are concentrated at cell-matrix interfaces. Immunogold labeling of ultracryosections of human skin indicates that the proteoglycan epitopes are nonhomogeneously distributed over the width of the basement membrane. Immunochemical investigations and amino acid sequence analysis indicate that the proteoglycan from the fibroblast matrix shares several structural features with the large, low density heparan sulfate proteoglycan isolated from the Engelbreth-Holm-Swarm sarcoma. Thus, both epithelial cell sheets and individual mesenchymal cells accumulate a large heparan sulfate proteoglycan(s) at the interface with the interstitial matrix, where the proteoglycan may adopt a specific topological orientation with respect to this matrix.

Amino Acid Sequence↗

PubMed1991

Association between proteoglycans and matrix vesicles in the extracellular matrix of growth plate cartilage.

Matrix vesicles (MV) are microstructures localized to the extracellular matrix of developing hard tissues that induce mineral formation. MV proteins are not well characterized, and little is known of how they interact with the surrounding matrix. However, recent electron microscopic studies indicate that MV interact with matrix proteins in growth plate cartilage. In the studies now reported, procedures developed for dissecting various components from isolated MV led to the discovery that two major vesicle proteins (38 and 46 kDa) are readily released from MV by low ionic strength solutions. These low ionic strength-soluble proteins (LISSP) were shown to be major fragments of the link protein (LP) and hyaluronic acid-binding region (HABR) of matrix proteoglycans: they react immunologically with highly specific monoclonal antibodies to LP and HABR, and the NH2-terminal sequence of the 38-kDa LISSP is essentially identical to residues 40-78 of chicken cartilage LP and that the 46-kDa LISSP represents HABR. Release of both LISSP is enhanced by hyaluronidase treatment, indicating anchorage by a hyaluronate-mediated mechanism. Both LP and HABR are firmly attached to MV in either isotonic or hypertonic solutions. In contrast, our other studies show that dissociation of type II collagen from MV occurs only with hypertonic salts which do not release the LISSP. Thus, strong interactions occur under physiological conditions between MV and both the proteoglycans and collagens, but these take place by different mechanisms.

Amino Acid Sequence↗

PubMed1991

The matrix of endochondral bone differs from the matrix of intramembranous bone.

Osseous tissue develops via two distinctly different processes: endochondral (EC) ossification and intramembranous (IM) ossification. The present study tests the hypothesis that each type of osseous tissue contains unique inducing factors for the promotion of cartilage and bone development. Previous work suggests that subcutaneous implants of demineralized EC and IM bone matrices both induce endochondral ossification. Thus, it concludes that the bone growth promotion properties of the respective matrices are very similar. As it was unclear to us why EC and IM bone powders should possess identical osteoinductive properties, we attempted to reproduce these results. We implanted EC (femoral) demineralized bone matrix (DBM), IM (frontal) DBM, or a mixture of the two into the ventral thoracic subcutaneous tissue of 12 to 15-week-old male Sprague Dawley rats. Morphological and radiolabeling techniques in this study demonstrated that implants of EC bone matrix induce bone formation via EC ossification in contrast to implants of IM bone matrix which do not induce EC ossification. Our findings suggest that the matrix of EC bone differs qualitatively from the matrix of IM bone due to their respective abilities to induce cartilage and/or bone formation. These observations differ from those previously reported possibly because our IM DBM preparations were not contaminated with tissues of endochondral origin. In current clinical practice, EC DBM allografts are often used to induce new bone formation in defects involving both IM and EC bone. We conclude that there may be clinical settings in which it would be more appropriate to replace bone originally formed via IM ossification with IM DBM rather than EC DBM.

Animals↗

PubMed1990

Regulation of extracellular matrix degradation by cell-extracellular matrix interactions.

An appropriate balance of extracellular matrix synthesis and degradation is required for normal morphogenesis and maintenance of tissue architecture. Extracellular matrix molecules and their receptors, as well as proteinases and their inhibitors, are all involved in matrix remodeling. In this report we show that signal transduction through extracellular matrix receptors regulates matrix remodeling.

Animals↗

PubMed1991

Collagen-binding proteins in collagenase-released matrix vesicles from cartilage. Interaction between matrix vesicle proteins and different types of collagen.

Recent evidence indicates that matrix vesicles (MV) interact with cartilage-specific collagens and other matrix proteins. Both type II and X collagens bind to and cosediment with MV. Our companion study shows that MV also are tightly coupled to proteoglycan link proteins (LP) and hyaluronic acid-binding region (HABR) in cartilage matrix. Here we sought to identify proteins responsible for the nexus between MV and matrix collagens using affinity chromatography with types I, II, and X collagen-Sepharose columns. Elution with NaCl step-gradients in the presence of nonionic detergent was used to assess the affinity between the MV proteins and the covalently attached collagens. Several MV proteins were found to bind to native type I, II, and X collagens but none bound to denatured type I collagen. Alkaline phosphatase, proteoglycan LP and HABR, and the 33- and 67-kDa annexins, bound with varying affinities to the native type I, II and X columns. In particular, LP and HABR, the 67-kDa annexin, and alkaline phosphatase bound with high affinity to the cartilage-specific collagens, although LP, HABR, and a 37-kDa protein also bound less tightly to native type I collagen. Thus, several MV proteins bind specifically to native type II and X collagens and should promote interaction between MV and the extracellular matrix. Such interactions may be important in MV formation, or in MV-mediated mineralization.

Animals↗

PubMed1991

[Cell membrane binding components for constituents of the extracellular matrix: structural mediator of the epithelial matrix interaction in the gastrointestinal tract].

Growth, migration, differentiation and metabolic functions of the epithelium in the gastrointestinal tract are regulated by the extracellular matrix. Different cell membrane binding components including the integrins for constituents of the extracellular matrix are expressed in the epithelial cells. These cell membrane binding components may be structural mediators of cell-matrix interaction in the gastrointestinal tract. A characterization of this interaction is of great importance, not only to understand physiological processes, such as epithelial migration and differentiation, but also for the pathogenesis of healing processes (ulcer healing), immunologically mediated processes (adhesion of immunocompetent cells), and especially for tumor pathology (invasion and metastasis). Gastrointestinal diseases are characterized by alterations in the expression of cell membrane binding components for different constituents of the extracellular matrix. In chronic inflammatory bowel disease, a changed expression can be identified on epithelial cells and, in especially malignant transformation of epithelial cells resulted in a pathologic expression of cell adhesion molecules. Oncogenes may modify the expression and function of these cell membrane binding components in the course of malignant transformation. In animal models, it was possible to reduce the frequency of tumor invasion and metastasis and to achieve longer survival times by blocking the cell membrane binding components on malignant cells. An increasing understanding of the role of cell membrane binding components in the epithelium-matrix interaction will certainly also be translated in the future into new therapeutic concepts.

Cell Adhesion↗

PubMed1988

The build-up of the thrombospondin extracellular matrix. An apparent dependence on synthesis and on preformed fibrillar matrix.

Thrombospondin (TSP) is a multifunctional protein synthesized by several cell types in culture, among them endothelial cells, and incorporated into the extracellular matrix (ECM) of these cells. In vitro it has been detected in most interstitial spaces, and its presence has been suggested to be developmentally controlled. With the aim of elucidating the role of TSP in the extracellular matrix, we studied the build-up of this protein in the matrix as a function of cell type density and age in culture. The development of the TSP matrix was compared to that of fibronectin (FN) and of von Willebrand Factor (VWF) in the same cultures using our monoclonal anti-TSP antibody B7-3 and monospecific anti-FN or anti-VWF antibodies. In cultures of bovine aortic endothelial cells (BAEC) we observed that both the pattern and distribution of TSP in the matrix changed with cell density and time in culture; it started as a completely amorphous dense layer of protein when the cells were sparse and changed to well organized fibrils when the cells had been confluent for a while. The first appearance of the fibrillar arrays of TSP coincides perfectly with that of the FN fibers; extracellular VWF also was first detected at this point. From this time in culture, there was an increasing coincidence of the TSP with the FN and the VWF matrices suggesting the formation of a complex multicomponent structure of the fibrillar network.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1990

Maintenance of embedded pig pancreatic pseudo-islets in a collagen gel matrix: study of the effect of hydrocortisone, a collagenase inhibitor, and nicotinamide on collagenolysis and the morphogenesis of pancreatic islet-cells in collagen gel matrix.

We describe a method for maintaining neonatal pig pancreatic isletlike cell clusters (as pseudo-islets) embedded in a collagen gel matrix for long periods. The pseudo-islets were formed from single cells of pig pancreas maintained in a suspension culture and then embedded in pepsin-solubilized type I collagen. When the pseudo-islets were cultured in the collagen matrix, the amount of collagen in the culture decreased gradually during the culture period as soluble hydroxyproline-containing material accumulated in the medium. A low concentration of collagen (0.16%) degraded the collagen gels more rapidly than did high concentrations of collagen (0.64%). The degradation of collagen depended both on the number of pseudo-islets embedded in the gel matrix and on the culture conditions used to maintain them. With added nicotinamide, the accumulation of hydroxyproline decreased in the medium and the structure of the gel matrix was well maintained. Hydrocortisone or a specific inhibitor of collagenase did not decrease the solubilization of embedded pseudo-islet cultures and did not help to maintain their structure. These observations indicate the possible utility of long-term maintenance of pseudo-islets in collagen gel matrix in the presence of nicotinamide.

Animals↗

PubMed1989

Inhibition of mitochondrial-matrix inorganic pyrophosphatase by physiological [Ca2+], and its role in the hormonal regulation of mitochondrial matrix volume.

1. The pyrophosphatase activity in cytosolic and mitochondrial fractions of rat liver was 1.7 and 0.26 units/mg of protein respectively when assayed at 37 degrees C in the presence of physiological [Mg2+] (0.3 mM). 2. Approx. 80% of the mitochondrial pyrophosphatase was inaccessible to extramitochondrial PPi, of which 40% represented soluble matrix enzyme (0.38 unit/mg of matrix protein). 3. Ca2+ inhibited the soluble matrix enzyme; the effective K0.5 for inhibition increased as [Mg2+], an essential cofactor of the enzyme, increased. Measured values were 0.39, 1.15, 3.7, 8.3 and 12.5 microM at 0.04 mM-, 0.1 mM-, 0.3 mM-, 0.6 mM- and 1 mM-Mg2+ respectively. 4. The data were analysed by a kinetic model similar to that for yeast pyrophosphatase, which assumes the substrate to be MgPPi (Km 5 microM) with Mg2+ also activating at an additional site (K0.5 23 microM). Ca2+ inhibits through the formation of CaPPi, a strong competitive inhibitor (Ki 0.067 microM). 5. Heart mitochondria also contain a soluble matrix pyrophosphatase of similar activity to that of liver mitochondria and with the same sensitivity to [Ca2+]. 6. The data provide an explanation for the increase in mitochondrial PPi, mediated by Ca2+, which is responsible for the increase in matrix volume induced by gluconeogenic hormones [Davidson & Halestrap (1988) Biochem. J. 254, 379-384].

Animals↗

PubMed1989

Role of extracellular matrix in the action of basic fibroblast growth factor: matrix as a source of growth factor for long-term stimulation of plasminogen activator production and DNA synthesis.

When bovine capillary endothelial (BCE) cells were treated with 10 ng/ml of basic fibroblast growth factor (bFGF) for 10 or 30 minutes at 37 degrees C, washed extensively with phosphate-buffered saline (PBS) and incubated in bFGF-free medium, plasminogen activator (PA) production was stimulated to the same extent as in cells exposed continuously to bFGF. Three methods of removing bFGF from heparin-like binding sites in the extracellular matrix, but not from bFGF receptors, abolished this long-term effect of a brief exposure to bFGF. First, BCE cells exposed to bFGF for 30 minutes were washed with 2M NaCl and incubated in bFGF-free medium. Second, BCE cells were incubated with bFGF for 10 minutes in the presence of heparin, and cells were washed with PBS and incubated in bFGF-free medium. Third, BCE cell cultures were treated with heparinase and exposed to bFGF. Each of these treatments abolished the long-term (24-48 hours) stimulation of PA production normally observed after brief exposure to bFGF. In each of these experiments, incubation of cells in bFGF-containing medium after the treatments resulted in normal stimulation of PA production, demonstrating that the treatments did not harm the cells. Stimulation of DNA synthesis was observed when cells were exposed to bFGF for 2 hours at 4 degrees C, incubated in bFGF-free medium for 24 hours at 37 degrees C, and assayed for 3H-thymidine incorporation. However, no stimulation was observed if the 2 hours incubation at 4 degrees C was carried out in the presence of heparin. Thus, long-term stimulation of PA activity and DNA synthesis after a brief exposure to bFGF seems to be a consequence of bFGF binding to the extracellular matrix. The extracellular matrix may act as a physiologic buffer, binding bFGF when concentrations are high and releasing it later for interaction with its receptor. This interaction with matrix may be required for the in vivo action of bFGF.

Animals↗

PubMedDate not supplied

Nuclear matrix bound terminal deoxynucleotidyl transferase in rat thymus nuclei. II. Effect of ATP on free and matrix bound TdT.

Endogenous nuclease digestion of thymus nuclei from 3-4 week old rats followed by a step wise extraction with low salt, 0.5 M salt and 1 M salt removed approximately 70-85% of total nuclear terminal deoxynucleotidyl transferase (TdT) whereas approximately 15-30% of the enzyme remained tightly bound to the residual nuclear matrix. The cytoplasmic TdT as well as the bulk of nuclear TdT extracted in low salt and 0.5 M salt was found to be strongly inhibited at low concentration of ATP whereas matrix bound TdT and a significant portion of the enzyme in 1 M salt extract was completely insensitive to this nucleotide. The ATP resistant enzyme in the 1 M salt extract was unstable and slowly converted to ATP sensitive form upon prolonged preincubation on ice whereas under similar conditions it remained unaffected in the matrix bound form. These observations lead us to suggest that ATP resistant matrix bound TdT being capable of discriminating unnatural rNTPs against the natural dNTP substrates, may be the functionally organized form of the enzyme and that free TdT having lost the capability to distinguish between dNTP and rNTP may be the nonfunctional form of the enzyme in the thymus gland.

Adenosine Triphosphate↗

PubMed1991

Extracellular matrix contraction by cultured mesangial cells: an assay system for mesangial cell-matrix interaction.

To investigate the interaction between glomerular mesangial cells (MCs) and extracellular matrix (ECM), we have developed a quantitative assay using a floating culture of MC-populated collagen gel matrix (MCGM). Since MCs can contract such a matrix consisting of type I collagen, MCGM exhibited marked contraction. The apparent cause was tensional interaction between MCs and collagen fibers, because: (1) MCs in the gel attached tightly to surrounding collagen fibers; (2) collagen fibrils surrounding MCs were ordered in a radial array; (3) collagen fibers aggregated around MCs in the contracted gel, but not in an uncontracted gel; (4) cytochalasin B, an actin polymerization blocker, inhibited gel contraction in a dose-dependent manner. We found that this interaction was modulated by some factors. Serum in the medium stimulated the contraction of MCGM. The degree of MCGM contraction was proportional to the number of MCs embedded above 2.3 x 10(4) cells/ml of gel. In MCGM containing a basement membrane-type gel matrix (BGM), gel contraction was increasingly inhibited as the content of BGM rose. Our method is useful for elucidating physiological and pathological interactions between MCs and ECM.

Angiotensin II↗

PubMed1990

Polarized fibronectin secretion and localized matrix assembly sites correlate with subendothelial matrix formation.

Endothelial cells in vivo form the interface between the vascular and interstitial compartments and are strategically located to mediate vascular permeability and hemostasis. One mechanism endothelial cells use to maintain a nonthrombogenic surface is to polarize basement membrane constituents to the basolateral cell surface. In the present study, we began characterization of the mechanisms used by endothelial cells in the assembly of a subcellular fibronectin matrix. Immunofluorescence microscopy was used to localize extracellular matrix fibronectin in endothelial cell cultures. In contrast to preconfluent and newly confluent cultures, post-confluent cultures assembled a fibronectin matrix that was restricted to the basolateral cell surface. To determine if endothelial cells polarize fibronectin secretion, Millicell culture inserts were used to distinguish proteins secreted from apical and basal surfaces. Preconfluent and newly confluent cultures secreted fibronectin equally into apical and basal media. In contrast, post-confluent endothelial cells secreted fibronectin preferentially into the basal chamber. The degree to which fibronectin secretion was polarized varied with time in culture and with the ability of the monolayers to act as a barrier to the movement of 125I-fibronectin from the apical to basal chamber. In addition, high affinity binding sites for exogenous 125I-fibronectin were found to be present on the basolateral, but not apical, surface of post-confluent endothelial monolayers. These results indicate that subendothelial matrix assembly correlates with polarized fibronectin secretion, culture confluence, and expression of high affinity binding sites for fibronectin on the basolateral cell surface.

Animals↗

PubMed1988

Two mitochondrial matrix proteases act sequentially in the processing of mammalian matrix enzymes.

The imported precursors of the mammalian matrix enzymes malate dehydrogenase [(S)-malate:NAD+ oxidoreductase, EC 1.1.1.37] and ornithine transcarbamylase (carbamoyl-phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) are cleaved to their mature subunits in two steps, each catalyzed by matrix-localized processing proteases. The number and properties of these proteases are the subjects of this report. We have identified and characterized two distinct protease activities in a crude matrix fraction from rat liver: processing protease I, which cleaves these precursors to the corresponding intermediate form; and processing protease II, which cleaves the intermediate forms to mature subunits. Protease I is insensitive to chelation by EDTA and to inactivation with N-ethylmaleimide; protease II is inhibited by 5 mM EDTA and is inactivated by treatment with N-ethylmaleimide. We have prepared from mitochondrial matrix an 800-fold-enriched protease I fraction free of protease II activity by using the following steps: ion exchange, hydroxyapatite, molecular sieving, and hydrophobic chromatography. Using similar procedures, we also have prepared an approximately 2000-fold-enriched protease II fraction, which has a trace amount of contaminating protease I. This enriched protease II fraction has little or no cleavage activity toward mitochondrial precursors but rapidly and efficiently converts intermediate forms to mature size. Finally, we show that protease I alone is sufficient to cleave the precursor of a third nuclear-encoded mitochondrial protein subunit--the beta subunit of propionyl-CoA carboxylase [propanoyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.3]--to its mature size.

Animals↗

PubMed1992

Matrix metalloproteinase 3 (stromelysin) activates the precursor for the human matrix metalloproteinase 9.

Matrix metalloproteinase 9 (MMP-9), also known as 92-kDa gelatinase/type IV collagenase, is secreted from neutrophils, macrophages, and a number of transformed cells in zymogen form. Here we report that matrix metalloproteinase 3 (MMP-3/stromelysin) is an activator of the precursor of matrix metalloproteinase 9 (proMMP-9). MMP-3 initially cleaves proMMP-9 at the Glu40-Met41 bond located in the middle of the propeptide to generate an 86-kDa intermediate. Cleavage of this bond triggers a change in proMMP-9 that renders the Arg87-Phe88 bond susceptible to the second cleavage by MMP-3, resulting in conversion to an 82-kDa form. alpha 2-Macroglobulin binding studies of partially activated MMP-9 demonstrate that the 82-kDa species is proteolytically active, but not the initial intermediate of 86 kDa. This stepwise activation mechanism of proMMP-9 is analogous to those of other members of the MMP family, but the action of MMP-3 on proMMP-9 is the first example of zymogen activation that can be triggered by another member of the MMP family. The results imply that MMP-3 may be an effective activator of proMMP-9 in vivo.

Amino Acid Sequence↗

PubMed1988

Lung, heart, and kidney express high levels of mRNA for the vitamin K-dependent matrix Gla protein. Implications for the possible functions of matrix Gla protein and for the tissue distribution of the gamma-carboxylase.

We have used cDNA probes for two small vitamin K-dependent bone matrix proteins, bone Gla protein (BGP) and matrix Gla protein (MGP), to evaluate the possibility that either of these proteins might be synthesized by the various soft tissues previously shown to have gamma-carboxylase activity. BGP mRNA was found in bone but not in any of the soft tissues tested, a result which reinforces the view that plasma BGP is a specific marker for bone metabolism. In contrast, MGP mRNA was found in all rat tissues examined. Lung and heart have 10-fold higher levels of MGP mRNA than bone, and kidney has a 5-fold higher level. Despite the high levels of MGP mRNA in heart and kidney, these tissues contain 40-500-fold lower concentrations of MGP protein than bone. Immunofluorescence was used to identify cells that contain MGP in kidney, lung, heart, and spleen. In each tissue, MGP was found in discrete tissue-specific cell types. In most of the soft tissues tested, MGP is the first well characterized substrate for the vitamin K-dependent carboxylase found to be synthesized. The exceptionally broad tissue distribution for MGP synthesis demonstrates that the function of MGP is not specific to connective tissues, and the low levels of MGP antigen in soft tissues with high MGP mRNA levels indicate that MGP is unlikely to act solely by virtue of its accumulation in an extracellular matrix.

Animals↗

PubMedDate not supplied

Synovial extracellular matrix II. Specific incorporation of immunoglobulin into the cell-free matrix of pannus.

To determine whether immune complex-like material is incorporated into the extracellular matrix (ECM) of proliferated RA synovium, cell-free matrices were isolated from pannus removed at joint replacement surgery, and were subjected to differential extraction. When the IgG and albumin concentrations in the ECM extracts were compared to those in simultaneously obtained synovial fluids, the IgG was found to be enriched 8.8-fold. Approximately 95% of the IgG was extractable with 6M Guanidine-HCl and 8 M Urea-B-ME. Further extraction with collagenase and low-pH buffers did not result in any additional recovery of IgG. Matrix-associated IgG demonstrated a restricted mobility on IEF with a pI of 4.8. The extracellular matrix of RA pannus is enriched in an acidic IgG species. Incorporation of IgG appears to be secondary to non-covalent interactions and may represent an additional reservoir of immune complex material in the rheumatoid joint.

Albumins↗

PubMed1989

DNA fragments which specifically bind to isolated nuclear matrix in vitro interact with matrix-associated DNA topoisomerase II.

A matrix-associated region (MAR)-containing fragment has been selected from the library of cloned chicken nuclear matrix-associated DNA fragments. Factors, which determine the specific binding of DNA fragments have been studied. Using topoisomerase II-specific inhibitor VM 26 we established that nuclear matrix-associated topoisomerase II interacted with the MAR-containing DNA fragment producing specific cleavage sites on DNA of the fragment.

Animals↗