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PubMed1992

Glomerular matrix accumulation is linked to inhibition of the plasmin protease system.

TGF-beta plays a pivotal role in the pathological accumulation of extracellular matrix in experimental glomerulonephritis. Increased TGF-beta expression leads to increased synthesis and deposition of extracellular matrix components while administration of anti-serum to TGF-beta suppresses the major manifestations of the disease. We hypothesized that TGF-beta might also enhance matrix accumulation by decreasing matrix turnover via effects on protease/protease inhibitor balance. Plasmin is a potent protease capable of degrading a variety of matrix molecules. Plasmin generation from plasminogen is regulated by plasminogen activator(s) (PA) and plasminogen activator inhibitor(s) (PAI). In this study PA activity was markedly reduced and PAI-1 synthesis dramatically increased when TGF-beta was added to normal glomeruli. Diseased glomeruli also showed decreased PA activity, increased PAI-1 synthesis and increased PAI-1 deposition into matrix. Administration of anti-TGF-beta serum to glomerulonephritic rats blocked the expected increase in glomerular PAI-1 deposition. Thus changes in the PA/PAI balance favoring accumulation of matrix are induced by TGF-beta in normal glomeruli and are present in nephritic glomeruli when endogenous TGF-beta production is high. Our findings implicate the plasmin protease system in tissue repair following acute glomerular injury and suggest another mechanism by which TGF-beta enhances the matrix accumulation characteristic of many glomerular diseases.

Animals↗
PubMed2002

Mechanisms balancing skeletal matrix synthesis and degradation.

Bone is regulated by evolutionarily conserved signals that balance continuous differentiation of bone matrix-producing cells against apoptosis and matrix removal. This is continued from embryogenesis, where the skeleton differentiates as a solid mass and is shaped into separate bones by cell death and proteolysis. The two major tissues of the skeleton are avascular cartilage, with an extracellular matrix based on type II collagen and hydrophilic proteoglycans, and bone, a stronger and lighter material based on oriented type I collagen and hydroxyapatite. Both differentiate from the same mesenchymal stem cells. This differentiation is regulated by a family of related signals centred on bone morphogenic proteins. Fibroblast growth factors, Indian hedgehog and parathyroid hormone-related protein are important in determining the type of matrix and the relation of skeletal and non-skeletal structures. Removal of mineralized matrix involves apoptosis of matrix cells and differentiation of acid-secreting cells (osteoclasts) from macrophage precursors. Key regulators of matrix removal are signals in the tumour-necrosis-factor family. Osteoclasts dissolve bone by isolating a region of the matrix and secreting HCl and proteinases at that site. Successive cycles of removal and replacement allow growth, repair and remodelling. The signals for bone turnover are predominantly cell-membrane-associated, allowing very specific spatial regulation. In addition to its support function, bone is a reservoir of Ca2+, PO3-(4) and OH-. Secondary modulation of mineral secretion and bone degradation are mediated by humoral signals, including parathyroid hormone and vitamin D, as well as the cytokines that also regulate the underlying cell differentiation.

Animals↗
PubMed2002

Tissue-dependent expression of matrix proteins in human endothelin-1 transgenic mice.

Endothelin-1 (ET-1) is a potent pro-fibrotic growth factor. However, little is known about its specific effects on the synthesis of matrix proteins in vivo. We used male 12-month-old ET-1 transgenic mice characterized by transgene expression in the kidney and (to a lesser extent) in the heart. Global cardiac and renal matrix protein synthesis was analysed after Sirius Red and periodate-Schiff staining. Specific expression of collagen types I, III and IV, laminin and fibronectin was examined using immunohistochemistry followed by computer-aided image analysis. Analysis of blood pressure revealed that mean arterial blood pressure was similar in ET-1 transgenic mice and controls. The total cardiac matrix protein content was increased in the myocardium of ET-1 transgenic mice. Analysis of specific cardiac matrix proteins showed increased cardiac expression of collagen type III (+211%; P<0.001) and laminin (+128%; P<0.01) in transgenic mice. The expression of collagen types I and IV and fibronectin was not altered. Global analysis of renal matrix proteins confirmed earlier studies showing pronounced interstitial fibrosis and glomerulosclerosis. Laminin expression was markedly increased in the glomerula (+152%; P<0.01) and even more so in the interstitium (+211%; P<0.001), whereas expression of collagen type III was reduced in glomerula (-48%; P<0.01) and interstitial tissue (-55%; P<0.01) of ET-1 transgenic mice. In conclusion, a primary overexpression of ET-1 does not cause uniformly enhanced synthesis of matrix proteins. In contrast, the effects of ET-1 on the matrix protein pattern is tissue-specific. The major renal and cardiac alterations in matrix proteins induced by ET-1 is a marked enhancement of laminin expression.

Animals↗
PubMed1997

Isolation and characterization of an endodermally derived, proteoglycan-like extracellular matrix molecule that may be involved in larval starfish digestive tract morphogenesis.

A monoclonal antibody, anti-Pisaster matrix-1 (anti-PM1) has been developed against an extracellular matrix antigen, Pisaster matrix-1 (PM1) found in embryos and larvae of the starfish Pisaster ochraceus. Pisaster matrix-1 was first observed in endodermal cells of the early gastrula, and shortly thereafter it was secreted into the blastocoel where it accumulated steadily during gastrulation. During the late gastrula stage it also appeared in the extracellular matrix (ECM) of the gut lumen. Immunogold electron microscopy with anti-PM1 revealed that PM1 was found in condensations of ECM associated with blastocoel matrix fibers, in the trans Golgi network, in Golgi-associated vesicles in endoderm and mesenchyme cells and throughout the ECM lining the digestive tract of late gastrula and bipinnaria larvae. When blastula or early gastrula stage embryos were grown in the presence of the PM1 antibody, archenteron elongation, bending and mouth formation failed to occur. Pisaster matrix-1 stained with alcian blue and its assembly could be disrupted with the common inhibitor of O-linked glycosaminoglycan assembly, beta-xyloside but not by tunicamycin. It was not sensitive to enzymes that degrade vertebrate proteoglycans. Pisaster matrix-1 is a large (600 kDa) proteoglycan-like glycosaminoglycan, secreted exclusively by endodermal and/or endodermally derived cells that may be necessary for morphogenesis of the mouth and digestive tract of Pisaster ochraceus embryos/larvae.

Animals↗
PubMed2000

Matrix metalloproteinases in human melanoma.

Cutaneous melanoma is a highly invasive and metastatic tumor. Degradation of basement membranes and extracellular matrix is an essential step in melanoma cell migration, invasion, and metastasis formation. Matrix metalloproteinases and their tissue inhibitors play a crucial role in these complex multistep processes. Melanoma cells may express a number of matrix metalloproteinase family members (MMP-1, MMP-2, MMP-9, MMP-13, and MT1-MMP) as well as their tissue inhibitors (TIMP-1, TIMP-2, and TIMP-3). Numerous studies have examined matrix metalloproteinases, their tissue inhibitors, and the molecules that regulate their expression and/or activation in melanoma cell lines in vitro and in vivo, and in human melanocytic lesions. Recent results have indicated that adhesion molecules such as CD44 and integrin alphavbeta3 are involved in positioning activated matrix metalloproteinase molecules on the cell surface of invasive tumor cells. In this review we evaluate these novel aspects of the role of matrix metalloproteinases and their tissue inhibitors in melanoma progression. We conclude that the balance between levels of activated matrix metalloproteinases and expression levels of their tissue inhibitors, and the coexpression of activated matrix metalloproteinases and adhesion molecules are important factors in determining melanoma cell invasion, tumor growth, and metastasis formation.

Animals↗
PubMed2000

Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin.

Damage to human skin due to ultraviolet light from the sun (photoaging) and damage occurring as a consequence of the passage of time (chronologic or natural aging) are considered to be distinct entities. Photoaging is caused in part by damage to skin connective tissue by increased elaboration of collagen-degrading matrix metalloproteinases, and by reduced collagen synthesis. As matrix metalloproteinase levels are known to rise in fibroblasts as a function of age, and as oxidant stress is believed to underlie changes associated with both photoaging and natural aging, we determined whether natural skin aging, like photoaging, gives rise to increased matrix metalloproteinases and reduced collagen synthesis. In addition, we determined whether topical vitamin A (retinol) could stimulate new collagen deposition in sun-protected aged skin, as it does in photoaged skin. Sun-protected skin samples were obtained from 72 individuals in four age groups: 18-29 y, 30-59 y, 60-79 y, and 80+ y. Histologic and cellular markers of connective tissue abnormalities were significantly elevated in the 60-79 y and 80+ y groups, compared with the two younger age groups. Increased matrix metalloproteinase levels and decreased collagen synthesis/expression were associated with this connective tissue damage. In a separate group of 53 individuals (80+ y of age), topical application of 1% vitamin A for 7 d increased fibroblast growth and collagen synthesis, and concomitantly reduced the levels of matrix-degrading matrix metalloproteinases. Our findings indicate that naturally aged, sun-protected skin and photoaged skin share important molecular features including connective tissue damage, elevated matrix metalloproteinase levels, and reduced collagen production. In addition, vitamin A treatment reduces matrix metalloproteinase expression and stimulates collagen synthesis in naturally aged, sun-protected skin, as it does in photoaged skin.

Adolescent↗
PubMed1998

Regulation of survival and death of mesangial cells by extracellular matrix.

BACKGROUND: Cell-matrix interactions exert major effects on such phenotypic features as cell growth and differentiation. Apoptosis is an active form of cell death that is crucial for maintaining the appropriate number of cells as well as the organization of tissue. Recently, it has been suggested that apoptosis of the mesangial cells (MC) is important in glomerular remodeling after injury. The MC are surrounded by an extracellular matrix (ECM) in vivo. Since in disease conditions the mesangial matrix is altered quantitatively and qualitatively, it is of interest to determine whether cell-matrix interactions may influence apoptosis of the MC. METHODS: We first investigated the differences in the susceptibility to apoptotic stimuli of the MC cultured on various ECM components (type I collagen, fibronectin, basement membrane matrix). We then determined whether the inhibition of MC-matrix interactions would affect apoptosis. Finally, interactions between MC and matrix were disrupted by the inhibition of beta1-integrin expression with antisense oligonucleotides (ODN). RESULTS: When MC were cultured on type I collagen or fibronectin and deprived of serum for eight hours, the extracted DNA from the MC demonstrated an internucleosomal ladder pattern on gel electrophoresis that constituted the biochemical characteristic of apoptosis. However, no ladder pattern was apparent when MC were cultured on basement membrane matrix. The attachment of cells was completely inhibited when the MC were cultured on agarose-coated dishes for 24 hours. Gel electrophoresis of DNA extracted from these cells showed a ladder pattern. However, the MC attached to the substratum did not show any apoptosis. MC showed an increase in apoptotic cell death after treatment with antisense ODN against beta1-integrin molecule. CONCLUSIONS: These results indicate that normal ECM may prevent the MC from undergoing apoptosis and serve as a survival factor for MC. Signals from ECM that prevent apoptosis may be mediated by beta1-integrin molecules.

Apoptosis↗
PubMed2001

An elevated amniotic fluid matrix metalloproteinase-8 level at the time of mid-trimester genetic amniocentesis is a risk factor for spontaneous preterm delivery.

OBJECTIVE: Strong evidence implicates chronic intra-amniotic inflammation in the etiology of mid-trimester abortion and spontaneous preterm delivery. The purpose of this study was to determine if concentrations of amniotic fluid matrix metalloproteinase-8, and cytokines such as interleukin-6 and angiogenin can identify patients at risk for spontaneous preterm delivery in patients undergoing mid-trimester amniocentesis. STUDY DESIGN: A case-control study was conducted to compare mid-trimester concentrations of amniotic fluid matrix metalloproteinase-8, interleukin-6, and angiogenin in patients who delivered at term and in those who delivered before term. The study included 19 cases with spontaneous preterm delivery and 95 matched controls with normal outcomes. Patients with abnormal fetal karyotypes or major anomalies were excluded. Matrix metalloproteinase-8, interleukin-6, and angiogenin were measured by using specific immunoassays. Mann-Whitney U tests, Fisher exact tests, and receiver-operating characteristic curves were used for statistical analysis. RESULTS: The median amniotic fluid matrix metalloproteinase-8, interleukin-6, and angiogenin concentrations of patients with spontaneous preterm delivery were significantly higher than those of control cases (matrix metalloproteinase-8: median, 3.1 ng/mL [range, 0.3-1954.9 ng/mL] vs median, 1.3 ng/mL [range, <0.3-45.2 ng/mL], P <.01; interleukin-6: median, 0.32 ng/mL [range, 0.04-2.52 ng/mL] vs median, 0.18 ng/mL [range, 0.01-1.81 ng/mL], P <.01; angiogenin: median, 11.1 ng/mL [range, 4.5-30.7 ng/mL] vs median, 6.7 ng/mL [range, 1.3-21.9 ng/mL], P <.001). Amniotic fluid matrix metalloproteinase-8 concentrations higher than 23 ng/mL had the highest specificity and odds ratio (sensitivity, 42% [8/19]; specificity, 99% [94/95]; OR, 68.4 [95% CI, 7.8-599.1]) in the identification of the patients with preterm delivery after genetic amniocentesis. CONCLUSIONS: Elevated mid-trimester concentrations of amniotic fluid matrix metalloproteinase-8, interleukin-6, and angiogenin are a risk factor for early spontaneous preterm delivery (<32 weeks). An elevated matrix metalloproteinase-8 level of >23 ng/mL is a powerful predictor of spontaneous preterm delivery (<32 weeks) with an odds ratio of 68.4. Amniotic fluid studies can be used to improve the risk assessment for preterm delivery in women who undergo mid-trimester amniocentesis for genetic indications.

Adult↗
PubMed2001

Amniotic fluid matrix metalloproteinase-8 in preterm labor with intact membranes.

OBJECTIVE: Intra-amniotic inflammation is a major determinant of maternal and neonatal outcome in patients with preterm labor. Matrix metalloproteinase-8 is a sensitive marker of inflammation in body fluids. This study was conducted to examine the value of amniotic fluid matrix metalloproteinase-8 determinations in patients with preterm labor and intact membranes. STUDY DESIGN: Amniotic fluid was obtained by transabdominal amniocentesis from 371 patients with preterm labor. Fluid was cultured for aerobic and anaerobic bacteria and Mycoplasmas. Amniotic fluid analysis included Gram stain examination, white blood cell count, and matrix metalloproteinase-8 (enzyme-linked immunosorbent assay) determination. Nonparametric statistics were used for analysis. RESULTS: The rate of preterm delivery was 54% (200/371) and that of intra-amniotic infection was 9.2% (34/371). The median amniotic fluid matrix metalloproteinase-8 concentration was more than 50-fold higher in patients with intra-amniotic infection than in patients with no intra-amniotic infection (median, 605.6 ng/mL; range, 0.65-15,000 ng/mL vs median, 10.6 ng/mL; range, <0.06-16,600 ng/mL, respectively; P <.0001). The matrix metalloproteinase-8 amniotic fluid concentrations were significantly higher in patients who delivered preterm than in patients who delivered at term (median, 19.5 ng/mL; range, <0.06-16,600 ng/mL vs median, 2.1 ng/mL; range, <0.06-500 ng/mL, respectively; P <.001). After exclusion of patients with intra-amniotic infection, patients who delivered preterm had a significantly higher median amniotic fluid matrix metalloproteinase-8 than patients who delivered at term (P <.05). An amniotic fluid matrix metalloproteinase-8 level of >30 ng/mL was an independent predictor for the occurrence of neonatal morbidity (odds ratio, 3.4; 95% CI, 1.9-5.8; P <.01). CONCLUSION: Increased amniotic fluid matrix metalloproteinase-8 concentrations identify patients at risk for intra-amniotic infection, impending preterm delivery, and adverse neonatal outcome.

Amniocentesis↗
PubMed2001

Amniotic fluid matrix metalloproteinase-8 indicates intra-amniotic infection.

OBJECTIVE: This study was undertaken to determine whether matrix metalloproteinase-8, which is produced by neutrophils, is a useful marker for the detection of intra-amniotic infection. STUDY DESIGN: We performed a case-control study using enzyme-linked immunosorbent assays to detect matrix metalloproteinase-8 in 77 amniotic fluid specimens that were obtained by amniocentesis from women with preterm contractions or preterm labor and intact fetal membranes (n = 66) and from women with preterm premature rupture of membranes (n = 11). RESULTS: Thirty women had culture-proven intra-amniotic infection (cases), 21 of whom had intact membranes. After constructing receiver operating characteristic curves to establish the optimal threshold concentration of matrix metalloproteinase-8 for a positive test result, we detected matrix metalloproteinase-8 in 27 of 30 women with intra-amniotic infection; only 10 of 47 control specimens contained matrix metalloproteinase-8 (P <.001; odds ratio, 33.3; 95% CI, 8.4, 132.7). Matrix metalloproteinase-8 was present in 20 of 21 women with intact membranes and intra-amniotic infection and in only 10 of 45 control subjects (P <.001; odds ratio, 70.0; 95% CI, 8.3, 587.6). Among women with intact membranes, the sensitivity of the assay was 0.95 and the specificity was 0.78. CONCLUSION: Our results indicate that matrix metalloproteinase-8 is highly correlated with intra-amniotic infection and that enzyme-linked immunosorbent assay for matrix metalloproteinase-8 may be a clinically useful test for the diagnosis of intra-amniotic infection in women with preterm contractions and preterm labor.

Amnion↗
PubMed1998

Intranuclear targeting of AML/CBFalpha regulatory factors to nuclear matrix-associated transcriptional domains.

The AML/CBFalpha runt transcription factors are key regulators of hematopoietic and bone tissue-specific gene expression. These factors contain a 31-amino acid nuclear matrix targeting signal that supports association with the nuclear matrix. We determined that the AML/CBFalpha factors must bind to the nuclear matrix to exert control of transcription. Fusing the nuclear matrix targeting signal to the GAL4 DNA binding domain transactivates a genomically integrated GAL4 responsive reporter gene. These data suggest that AML/CBFalpha must associate with the nuclear matrix to effect transcription. We used fluorescence labeling of epitope-tagged AML-1B (CBFA2) to show it colocalizes with a subset of hyperphosphorylated RNA polymerase II molecules concentrated in foci and linked to the nuclear matrix. This association of AML-1B with RNA polymerase II requires active transcription and a functional DNA binding domain. The nuclear matrix domains that contain AML-1B are distinct from SC35 RNA processing domains. Our results suggest two of the requirements for AML-dependent transcription initiation by RNA polymerase II are association of AML-1B with the nuclear matrix together with specific binding of AML to gene promoters.

Cell Compartmentation↗
PubMed1995

Matrix nonenzymatic glycosylation leads to altered cellular phenotype and intracellular tyrosine phosphorylation.

The effect of matrix nonenzymatic glycosylation on signal transduction and the cellular phenotype was examined. Human microvascular endothelial cells were plated on control or glycated basement membrane-like matrix. Cells exhibited a decrease in their ability to adhere and spread on modified matrix. The pattern of intracellular tyrosine phosphorylation was examined by Western Immunoblotting; a band with 65 kDa mobility exhibited a marked reduction of tyrosine phosphorylation in cells adherent to modified matrix. Immunoprecipitation experiments provided evidence that this band is paxillin, a member of focal adhesion proteins. Immunoprecipitation with antibodies against focal adhesion kinase (pp125FAK), the enzyme that is thought to regulate paxillin tyrosine phosphorylation, also demonstrated a reduction in tyrosine phosphorylation of pp125FAK. To confirm these biochemical data, adherent cells were examined for the distribution of paxillin, using immunofluorescence microscopy; paxillin was seen in focal points peripherally located in cells on normal matrix, but lacked this pattern in cells on modified matrix. Actin filaments were also disorganized in cells plated on modified matrix. These data suggest that matrix nonenzymatic glycosylation can interfere with and potentially alter cellular phenotype and intracellular signaling.

Animals↗
PubMed1996

Formation of sodium dodecyl sulfate-stable fibronectin multimers. Failure to detect products of thiol-disulfide exchange in cyanogen bromide or limited acid digests of stabilized matrix fibronectin.

Fibronectin exists in a soluble form in body fluids and as a fibrillar component of the extracellular matrix. Matrix fibronectin associates as large complexes in SDS unless a reducing reagent is also present. This observation suggests that complex formation is due to interprotomeric disulfides that form by thiol-disulfide exchange. To localize the presumptive new disulfides, we labeled protomeric fibronectin by the chloramine-T method or with 125I-Bolton-Hunter reagent, incorporated 125I-fibronectin into the matrix of cultured fibroblasts, and subjected matrix fibronectin to acid or cyanogen bromide digestion. When cyanogen bromide digests of matrix 125I-fibronectin and protomeric 125I-fibronectin labeled with Bolton-Hunter reagent were analyzed by two-dimensional polyacrylamide gel electrophoresis in SDS, with the first dimension being nonreducing and the second reducing, we were not able to identify any fragments of matrix fibronectin that migrated as high molecular weight complexes in the first dimension. Limited acid digestion of matrix 125I-fibronectin also dissociated the majority of the high molecular weight complexes. Since we could account for all of the parts of fibronectin that contain cysteine or cystine, we conclude that matrix fibronectin is not stabilized by interprotomeric disulfides. We propose, instead, that stabilization is mediated by noncovalent protein-protein interactions that are sensitive to reduction, cyanogen bromide digestion, or limited acid digestion.

Acids↗
PubMed2002

The newly identified human nuclear protein NXP-2 possesses three distinct domains, the nuclear matrix-binding, RNA-binding, and coiled-coil domains.

Using a monoclonal antibody that recognizes a nuclear matrix protein, we selected a cDNA clone from a lambdagt11 human placenta cDNA library. This cDNA encoded a 939-amino acid protein designated nuclear matrix protein NXP-2. Northern blot analysis indicated that NXP-2 was expressed in various tissues at different levels. Forcibly expressed green fluorescent protein-tagged NXP-2 as well as endogenous NXP-2 was localized in the nucleus and distributed to the nuclear matrix. NXP-2 was released from the nuclear matrix when RNase A was included in the buffer for nuclear matrix preparation. Mapping of functional domains was carried out using green fluorescent protein-tagged truncated mutants of NXP-2. The region of amino acids 326-353 was responsible for nuclear matrix binding and contained a cluster of hydrophobic amino acids that was similar to the nuclear matrix targeting signal of acute myeloleukemia protein. The central region (amino acids 500-591) was demonstrated to be required for RNA binding by Northwestern analysis, although NXP-2 lacked a known RNA binding motif. The region of amino acid residues 682-876 was predicted to have a coiled-coil structure. The RNA-binding, nuclear matrix-binding, and coiled-coil domains are structurally separated, suggesting that NXP-2 plays important roles in diverse nuclear functions, including RNA metabolism and maintenance of nuclear architecture.

Amino Acid Sequence↗
PubMed1987

Rat hepatocytes in serum-free primary culture elaborate an extensive extracellular matrix containing fibrin and fibronectin.

Adult rat hepatocytes cultured on type IV collagen, fibronectin, or laminin and maintained in serum-free medium were examined by indirect immunofluorescence using polyclonal antibodies against extracellular matrix proteins. An extensive fibrillar matrix containing fibronectin and fibrin was detected in all hepatocyte cultures irrespective of the exogenous matrix substratum used to support cell adhesion. Fibrils radiated from the cell periphery and covered the entire culture substratum. In addition, thicker fibers or bundles of fibers were localized on top of hepatocytes. This matrix did not contain laminin or the major types of collagen found in the liver biomatrix (types I, III, and IV). Isolation of the fibrillar matrix and analysis on polyacrylamide gels under reducing conditions demonstrated a major 58-kD polypeptide, derived from beta-fibrinogen as indicated by immunoblotting and two-dimensional peptide mapping. Plasmin rapidly dissolved the matrix. Deposition of the fibrin matrix in hepatocyte cultures was arrested by hirudin, by specific heparin oligosaccharides that potentiate thrombin inhibition by antithrombin III, and by dermatan sulfate, an activator of heparin cofactor II-mediated inhibition of thrombin. The results indicate that hepatocytes in culture synthesize and activate coagulation zymogens. In the absence of inhibitory and fibrinolytic mechanisms, a fibrin clot is formed by the action of thrombin on fibrinogen. Fibronectin attaches to this fibrin clot but fails to elaborate a fibrillar matrix on its own in the presence of coagulation inhibitors.

Animals↗
PubMed1994

Latent transforming growth factor-beta 1 associates to fibroblast extracellular matrix via latent TGF-beta binding protein.

The role of latent transforming growth factor-beta (TGF-beta) binding protein (LTBP) in the association of TGF-beta 1 to the extracellular matrix of cultured fibroblasts and HT-1080 fibrosarcoma cells was studied by immunochemical methods. The matrices were isolated from the cells, and the levels of LTBP and TGF-beta 1 were estimated by immunoblotting and immunoprecipitation. LTBP, TGF-beta 1, and its propeptide (latency-associated peptide, LAP) were found to associate to the extracellular matrix. Immunoblotting analysis indicated that treatment of the cells with plasmin resulted in a concomitant time and dose dependent release of both LTBP and TGF-beta 1 from the extracellular matrix to the supernatant. Comparison of molecular weights suggested that plasmin treatment resulted in the cleavage of LTBP from the high molecular weight fibroblast form to a form resembling the low molecular weight LTBP found in platelets. Pulse-chase and immunoprecipitation analysis indicated that both the free form of LTBP and LTBP complexed to latent TGF-beta were efficiently incorporated in the extracellular matrix, from where both complexes were slowly released to the culture medium. Addition of plasmin to the chase solution resulted, however, in a rapid release of LTBP from the matrix. Fibroblast derived LTBP was found to associate to the matrix of HT-1080 cells in a plasmin sensitive manner as shown by immunoprecipitation analysis. These results suggest that the latent form of TGF-beta 1 associates with the extracellular matrix via LTBP, and that the release of latent TGF-beta 1 from the matrix is a consequence of proteolytic cleavage(s) of LTBP.

Carrier Proteins↗
PubMed1995

The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/rac GTPases.

Interaction of cells with extracellular matrix via integrin adhesion receptors plays an important role in a wide range of cellular: functions, for example cell growth, movement, and differentiation. Upon interaction with substrate, integrins cluster and associate with a variety of cytoplasmic proteins to form focal complexes and with the actin cytoskeleton. Although the intracellular signals induced by integrins are at present undefined, it is thought that they are mediated by proteins recruited to the focal complexes. It has been suggested, for example, that after recruitment to focal adhesions p125FAK can activate the ERK1/2 MAP kinase cascade. We have previously reported that members of the rho family of small GTPases can trigger the assembly of focal complexes when activated in cells. Using microinjection techniques, we have now examined the role of the extracellular matrix and of the two GTP-binding proteins, rac and rho, in the assembly of integrin complexes in both mouse and human fibroblasts. We find that the interaction of integrins with extracellular matrix alone is not sufficient to induce integrin clustering and focal complex formation. Similarly, activation of rho or rac by extracellular growth factors does not lead to focal complex formation in the absence of matrix. Focal complexes are only assembled in the presence of both matrix and functionally active members of the rho family. In agreement with this, the interaction of integrins with matrix in the absence of rho/rac activity is unable to activate the ERK1/2 kinases in Swiss 3T3 cells. In fact, ERK1/2 can be activated fully by growth factors in the absence of matrix and it seems unlikely, therefore, that the adhesion dependence of fibroblast growth is mediated through the ras/MAP kinase pathway. We conclude that extracellular matrix is not sufficient to trigger focal complex assembly and subsequent integrin-dependent signal transduction in the absence of functionally active members of the rho family of GTPases.

3T3 Cells↗
PubMed2006

Extracellular matrix of the human cyclic corpus luteum.

Extracellular matrix regulates many cellular processes likely to be important for development and regression of corpora lutea. Therefore, we identified the types and components of the extracellular matrix of the human corpus luteum at different stages of the menstrual cycle. Two different types of extracellular matrix were identified by electron microscopy; subendothelial basal laminas and an interstitial matrix located as aggregates at irregular intervals between the non-vascular cells. No basal laminas were associated with luteal cells. At all stages, collagen type IV alpha1 and laminins alpha5, beta2 and gamma1 were localized by immunohistochemistry to subendothelial basal laminas, and collagen type IV alpha1 and laminins alpha2, alpha5, beta1 and beta2 localized in the interstitial matrix. Laminin alpha4 and beta1 chains occurred in the subendothelial basal lamina from mid-luteal stage to regression; at earlier stages, a punctate pattern of staining was observed. Therefore, human luteal subendothelial basal laminas potentially contain laminin 11 during early luteal development and, additionally, laminins 8, 9 and 10 at the mid-luteal phase. Laminin alpha1 and alpha3 chains were not detected in corpora lutea. Versican localized to the connective tissue extremities of the corpus luteum. Thus, during the formation of the human corpus luteum, remodelling of extracellular matrix does not result in basal laminas as present in the adrenal cortex or ovarian follicle. Instead, novel aggregates of interstitial matrix of collagen and laminin are deposited within the luteal parenchyma, and it remains to be seen whether this matrix is important for maintaining the luteal cell phenotype.

Collagen Type IV↗