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PubMed2002

The interaction between the human beta-globin locus control region and nuclear matrix.

Our previous study showed that hydroxyurea (Hu) could induce HEL cells to express human beta-globin gene. However the molecular mechanisms by which the expression of beta-globin gene is activated and regulated are poorly understood. Here we show that the binding patterns between the core DNA sequences (HS2 core sequence -10681 approximately -10971 bp, HS3 core sequence -14991 approximately -14716 bp and HS4 core sequence -18586 approximately -18306 bp) of DNase I hypersensitive sites in the human beta-globin LCR and nuclear matrix proteins isolated from Hu induced and uninduced HEL cells are quite different. Results demonstrated that nuclear matrix proteins might play important roles in regulating the expression of human beta-like globin genes through their interaction with HSs (HS2, HS3 and HS4 core sequences) in the LCR. Moreover, the results obtained from the in vitro DNA-matrix binding assay showed that the core DNA sequences of DNase I hypersensitive sites (HS2, HS3 and HS4) were unable to bind to the nuclear matrix isolated from uninduced HEL cells; in addition, HS2 core DNA sequence was capable of binding to the nuclear matrix prepared from Hu-induced HEL cells, while both HS3 and HS4 core DNA sequences could not do so. Results indicated that the HS2 core DNA sequence may be a functional MAR (matrix attachment region). We suggest that the HS2 core DNA sequence binding to the nuclear matrix in Hu-induced HEL cells may open the structure of chromatin to make the LCR accessible to the promoter of beta-globin gene and to promote its transcription.

Binding Sites↗
PubMed2006

Role of cathepsin K in the turnover of the dermal extracellular matrix during scar formation.

Cathepsins are a group of cysteine proteinases that are involved in various aspects of extracellular matrix turnover. The collagenolytic activity of cathepsin K plays a pivotal role in bone resorption and lung matrix homeostasis, but so far has not been described in skin. To study the role of cathepsin K in the turnover of the cutaneous extracellular matrix, we studied the expression of cathepsin K in human skin and in cultured primary neonatal skin fibroblasts. Normal skin exhibited only low levels or no expression of cathepsin K. In contrast, dermal fibroblasts in surgical scars showed strong cytoplasmic cathepsin K expression. Cathepsin K expression was most prominent in young scars and declined with time. Cultured neonatal primary fibroblasts showed strong cathepsin K staining in the perinuclear endosomal compartment, consistent with intracellular degradation of internalized collagen in lysosomes. Cathepsin K was also found to be strongly expressed in keloids and dermatofibromas, but not in sclerotic areas of morphea. Our data suggest that cathepsin K may play an important role in the homeostasis of the dermal extracellular matrix and the dynamic equilibrium between matrix synthesis and proteolytic degradation, by counteracting deposition of matrix proteins during scar formation with its matrix-degrading activity.

Cathepsin K↗
PubMed2001

Blocking sp1 transcription factor broadly inhibits extracellular matrix gene expression in vitro and in vivo: implications for the treatment of tissue fibrosis.

Fibrosis is a consequence of injury characterized by accumulation of excess collagen and other extracellular matrix components, resulting in the destruction of normal tissue architecture and loss of function. Sp1 was originally described as a ubiquitous transcription factor. It is involved in the basal expression of extracellular matrix genes and may, therefore, be important in fibrotic processes. To evaluate the effect of Sp1 blockade on the expression of extracellular matrix genes, clones of NIH 3T3 fibroblasts stably transfected with an anti-sense Sp1 expression vector. Simultaneously reduced expression of several extracellular matrix genes as compared with mock-transfected clones was noted using differential hybridization of cDNA microarrays, without significant alteration in cell growth. Transfection of human dermal fibroblasts with several extracellular matrix gene (COL1A1, COL1A2, COL3A1, COL5A2, COL7A1, TIMP-1, and decorin) promoter/reporter constructs demonstrated that anti-sense Sp1-induced reduction of extracellular matrix gene mRNA steady-state levels results from transcriptional repression, consistent with the role of Sp1 as a transcription factor. Decoy Sp1 binding oligonucleotides inhibited COL1A2 promoter activity both in cultured fibroblasts and in vivo, in the skin of transgenic mice, which have integrated a mouse COL1A2 promoter/luciferase reporter gene construct. These results indicate that targeting Sp1 efficiently blocks extracellular matrix gene expression, and suggest that such an approach may represent an interesting therapeutic alternative toward the treatment of fibrotic disorders.

3T3 Cells↗
PubMed2001

Acid polysaccharides in the skeletal matrix and calicoblastic epithelium of the stony coral Mycetophyllia reesi.

Like many corals the skeletal organic matrix and associated epithelium of Mycetophyllia reesi is physico-chemically unstable to preparative procedures for electron microscopy. Ethanol cryofracture of mineralized and demineralized material is accompanied by delamination of tissue and skeleton. Filamentous algae occur in the interface and account for some but not all of the separation artifact. Transmission microscopy accompanied by decalcification requires embedment in glycerol jelly to preserve the skeletal organic matrix. Even then, the matrix is not fixed and is not retained within the gel using standard double fixation with or without tannic acid as an additive. Ruthenium red, in combination with osmium, prevents the matrix from physical disruption, although positional artifacts relative to the calicoblastic epithelium are still evident. Inclusion of other glycan precipitating agents in the fixative sequence (Alcian blue, iron diamine or the detergent cetylpyridinium chloride) are more useful in preserving an acid polysaccharide-rich, fibrillar, extracellular matrix after demineralization. This material is not observed in SEM preparations. The calicoblast cells appear to be the source of this extracellular material that also appears to contribute to the composition of the mineralizing matrix. Moreover, a hyaluronan-like substance appears to play a significant role in matrix structure as suggested by its degradation by hyaluronidase.

Animals↗
PubMed2004

Concentration of insulin-like growth factor (IGF)-I in iliac crest bone matrix in premenopausal women with idiopathic osteoporosis.

Previous studies have shown a link between low serum insulin-like growth factor-I (IGF-I) and decreased bone mass of patients with osteoporosis. However, whether serum levels are representative for the growth factor concentration or activity available in human bone tissue is controversial. In the present study, IGF-I was assessed in serum and bone matrix extracts from the iliac crest in 19 eugonadal women with idiopathic osteoporosis and in 38 age-matched controls. In addition, the relationship between the skeletal levels of IGF-I and bone mineral density (BMD) or the susceptibility to osteoporotic fractures in women with osteoporosis was examined. Bone matrix extraction was performed based on a guanidine-HCL/ethylendiamine-tetraacetic acid (EDTA) method. No significant difference in both serum and bone matrix IGF-I levels between groups was observed. Serum IGF-I concentrations failed to be associated with bone matrix IGF-I levels in osteoporotic patients. However, in premenopausal women with idiopathic osteoporosis, skeletal IGF-I positively correlated with BMD at the lumbar spine (r = + 0.58, p = 0.01). In contrast, neither femoral neck BMD nor Ward's triangle BMD was associated with bone matrix IGF-I concentrations. A tendency towards lower levels of bone matrix IGF-I in subjects with vertebral fractures as compared to those without fractures was observed in age-adjusted analyses, however the difference failed to remain statistically significant after adjustment for bone mineral density. These data provide no clear evidence for low bone matrix IGF-I as a determinant factor of age-unrelated osteoporosis. However, low skeletal IGF-I concentrations may aggravate osteoporosis in these women.

Absorptiometry, Photon↗
PubMed2001

Amniotic fluid concentrations of matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 during pregnancy and labor.

OBJECTIVE: This study was undertaken to determine whether obstetric factors affect the concentrations of matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 in the amniotic fluid. STUDY DESIGN: We prospectively collected amniotic fluid samples from 109 women at various stages of pregnancy and labor and determined matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 concentrations by means of enzyme-linked immunosorbent sandwich assay systems. With multiple regression analysis we evaluated relationships between amniotic fluid matrix metalloproteinase 9 concentration and tissue inhibitor of metalloproteinase 1 concentration and the following factors: gestational age, presence of labor, cervical dilatation, membrane status, presence of clinical chorioamnionitis, and microbial colonization of the amniotic fluid. RESULTS: The detectable presence of amniotic fluid matrix metalloproteinase 9 was independently associated with intra-amniotic infection, labor, cervical dilatation, and spontaneous rupture of membranes. Chorioamnionitis and amniotic fluid matrix metalloproteinase 9 concentrations were correlated with tissue inhibitor of metalloproteinase 1 levels. CONCLUSIONS: Intra-amniotic infection, advanced labor, and rupture of membranes before the onset of labor were independently associated with the presence of matrix metalloproteinase 9 in the amniotic fluid. Both pathologic and physiologic processes appear to produce shifts in the balance between degradation and synthesis of the extracellular matrix.

Amniotic Fluid↗
PubMed2001

The relationship between amniotic fluid matrix metalloproteinase-8 and funisitis.

OBJECTIVE: The fetal inflammatory response syndrome is a multisystem disorder associated with impending preterm delivery and adverse neonatal outcome. Inflammation of the umbilical cord--funisitis--is the histologic counterpart of fetal inflammatory response syndrome and has been associated with an increased risk for the development of cerebral palsy. Neutrophils found in the amniotic cavity are of fetal origin. Therefore, neutrophil secretory products may be an index of the fetal inflammatory response syndrome. To test this hypothesis, we examined the relationship between levels of amniotic fluid matrix metalloproteinase-8 and funisitis. STUDY DESIGN: The relationship between the presence of funisitis and concentrations of amniotic fluid matrix metalloproteinase-8 was examined in 255 consecutive patients who delivered preterm singleton neonates (gestational age, <36 weeks) within 72 hours of amniocentesis. Amniotic fluid was cultured for aerobic and anaerobic bacteria and for mycoplasmas. Funisitis was diagnosed in the presence of neutrophil infiltration into the umbilical vessel walls or Wharton jelly. Matrix metalloproteinase-8 was measured by use of a specific immunoassay. Nonparametric statistics were used for analysis. RESULTS: Funisitis was present in 23% (59/255) of cases. Patients with funisitis had a significantly higher median concentration of amniotic fluid matrix metalloproteinase-8 than those without funisitis (median, 433.7 ng/mL [range, 1.5-3836.8 ng/mL] vs median, 1.9 ng/mL [range, <0.3-4202.7 ng/mL]; P <.001). The diagnostic indices of matrix metalloproteinase-8 (cutoff, 23 ng/mL) in the identification of funisitis were: sensitivity of 90% (53/59), specificity of 78% (153/196), positive predictive value of 55% (53/96), and negative predictive value of 96% (153/159). CONCLUSIONS: There is a strong association between increased levels of amniotic fluid matrix metalloproteinase-8 and funisitis. We propose that determination of amniotic fluid matrix metalloproteinase-8 concentrations may assist the assessment of the fetal inflammatory status, thereby eliminating the need for fetal blood sampling.

Adult↗
PubMed1990

Plasminogen-activator inhibitor type 1 is a potent natural inhibitor of extracellular matrix degradation by fibrosarcoma and colon carcinoma cells.

We have analyzed the role of plasminogen-activator inhibitor type 1 (PAI-1) in the regulation of tumor cell-mediated extracellular matrix degradation. Immunocytochemical analysis revealed PAI-1 associated with microgranular and fibrillar material of the extracellular matrix and demonstrated the presence of PAI-1 as a cell surface-associated antigen. Transforming growth factor beta significantly reduced matrix degradation mediated by HT-1080 human fibrosarcoma cells. This inhibition was correlated with an increase in PAI-1 antigen expression, whereas urinary-type plasminogen activator (u-PA) secretion was unaffected. In this experimental system, PAI-1 regulated extracellular matrix breakdown, as added PAI-1 inhibited matrix solubilization, whereas monoclonal antibodies to PAI-1 increased it. A cell line (LPAI) producing high levels of biologically active PAI-1 was established by transfection of a human PAI-1 cDNA clone into mouse L cells. Coculture experiments demonstrated that LPAI cells prevented matrix degradation by Lu-PA cells (L cells expressing high levels of u-PA) or Co-115 human colon carcinoma cells (expressing tissue-type plasminogen activator). These results indicate that PAI-1 may play a critical role in the regulation of extracellular matrix degradation during tumor cell invasion.

Animals↗
PubMed1992

Sequence-specific DNA-binding proteins are components of a nuclear matrix-attachment site.

We have identified a nuclear matrix-attachment region within an upstream element of a human H4 histone gene promoter. Nuclear matrix proteins, isolated and solubilized from HeLa S3 cells, were found to interact with sequence specificity at this matrix-attachment region. Several types of assays for protein-DNA interaction showed that the minimal sequence for the nuclear matrix protein-DNA interaction was 5'-TGACGTCCATG-3'; the underlined region corresponds to the core consensus sequence for ATF transcription factor binding. Two proteins with molecular masses of 43 and 54 kDa were identified by UV-crosslinking analysis as integral components of this protein-DNA complex. The molecular masses of these proteins and the ATF-binding site consensus sequence suggest that these proteins are members of the ATF family. Our results provide direct evidence for nuclear matrix localization of sequence-specific DNA-binding factors for an actively transcribed gene. The proximity of a strong positive transcriptional regulatory element to the matrix-attachment region of this gene suggests that the nuclear matrix may serve to localize and concentrate trans-acting factors that facilitate regulation of gene expression.

Base Sequence↗
PubMed1993

Transmembrane neural cell-adhesion molecule (NCAM), but not glycosyl-phosphatidylinositol-anchored NCAM, down-regulates secretion of matrix metalloproteinases.

During embryogenesis interactions between cells and extracellular matrix play a central role in the modulation of cell motility, growth, and differentiation. Modulation of matrix structure is therefore crucial during development; extracellular matrix ligands, their receptors, extracellular proteinases, and proteinase inhibitors all participate in the construction, maintenance, and remodeling of extracellular matrix by cells. The neural cell-adhesion molecule (NCAM)-negative rat glioma cell line BT4Cn secretes substantial amounts of metalloproteinases, as compared with its NCAM-positive mother cell line BT4C. We have transfected the BT4Cn cell line with cDNAs encoding the human NCAM-B and -C isoforms. We report here that the expression of transmembrane NCAM-B, but not of glycosyl-phosphatidylinositol-linked NCAM-C, induces a down-regulation of 92-kDa gelatinase (matrix metalloproteinase 9) and interstitial collagenase (matrix metalloproteinase 1), indicating that cellular expression of the recognition molecule NCAM regulates the metabolism of the surrounding matrix.

Animals↗
PubMed1996

Cellular response to transforming growth factor-beta1 and basic fibroblast growth factor depends on release kinetics and extracellular matrix interactions.

The extracellular matrix plays an important role in growth factor biology, serving as a potential platform for rapid growth factor mobilization or a sink for concentrated sequestration. We now demonstrate that when a growth factor binds reversibly to the matrix, its effects are augmented by this interaction, and when the factor is absorbed irreversibly to the extracellular matrix, it becomes sequestered. These findings call into question the notion that all growth factors are best presented to cells and tissues in a sustained and controlled fashion. In our studies, we examined basic fibroblast growth factor (bFGF) and transforming growth factor-beta1 (TGF-beta1) release kinetics from synthetically fabricated microsphere devices and naturally synthesized extracellular matrix. While the sustained release of bFGF was up to 3.0-fold more potent at increasing vascular endothelial and smooth muscle cell proliferation than bolus administration, the reverse was true for TGF-beta1. A bolus of TGF-beta1 inhibited vascular cells up to 3.8-fold more efficiently than the same amount of TGF-beta1 if control-released. Both growth factors bound to the extracellular matrix, but only bFGF was released in a controlled fashion (2.8%/day). Contact with the extracellular matrix and subsequent release enhanced bFGF activity such that it was 86% more effective at increasing smooth muscle cell numbers than equal amounts of growth factor diluted from frozen stock. TGF-beta1 remained tightly adherent. The small amount of TGF-beta1 released from the extracellular matrix was approximately 30% less effective than bolus administration at inhibiting vascular endothelial and smooth muscle cell growth. Sustained growth factor release may be the preferable mode of administration, but only when a similar mode of metabolism is utilized endogenously.

Animals↗
PubMed1997

Norrie disease protein (norrin) forms disulfide-linked oligomers associated with the extracellular matrix.

COS-7 cells transfected with a DNA construct encoding the 133 amino acids in norrin plus six histidine residues at its carboxyl terminus were pulse-labeled with [35S]cysteine, and the labeled norrin was examined in cell lysates, the medium, and the extracellular matrix. SDS-gel electrophoresis under reducing conditions showed that the norrin expressed had an apparent Mr = 14,000 and was present only in cell lysates and the extracellular matrix. Under nonreducing conditions, most of the norrin in the extracellular matrix was oligomers that contained up to approximately 20 monomers. One of the major extracellular species of norrin under reducing conditions after cross-linking of norrin oligomers with bis(sulfosuccinimidyl)suberate had an apparent Mr = 28,000, consistent with covalent cross-linked dimers. Thus the covalently cross-linked dimers are key structural components of norrin oligomers. By site-directed mutagenesis, the codon for half-cystine 95 in norrin was changed to one encoding alanine. The norrin C95A found in the extracellular matrix of cells transfected with this mutant was the size of dimers, indicating that half-cystine 95 is involved in oligomer formation. The corresponding half-cystine residue in human prepro-von Willebrand factor is also involved in interchain disulfide bond formation, which is consistent with the sequence identity of the half-cystine residues in norrin and part of the half-cystine residues in a disulfide-rich domain of von Willebrand factor. Replacement of valine at residue 60 in norrin by glutamic acid, a mutation found in humans with a severe type of Norrie disease, results in a considerable reduction (50%) in the amount of norrin in the extracellular matrix of transfected COS-7 cells. Replacement of arginine at residue 121 by glutamine, which is associated with a less severe type of Norrie disease, results in a reduction in the amount of norrin R121Q in the extracellular matrix (26%). These studies suggest that norrin is a secreted protein that forms disulfide-bonded oligomers that are associated with the extracellular matrix upon secretion from cells. Moreover, the disulfide-rich motif of norrin and prepro-von Willebrand factor promotes interchain disulfide bond formation among polypeptides in which it is found.

Animals↗
PubMed1998

Induction of matrix metalloproteinase-9 requires a polymerized actin cytoskeleton in human malignant glioma cells.

Alterations in cytoskeleton and subsequent cell shape changes exert specific effects on the expression of various genes. Our previous results suggested that malignant human gliomas express elevated levels of matrix metalloproteinases compared with normal brain tissue and low grade gliomas. To understand the role of cell shape changes on matrix metalloproteinase expression in human glioma cells, we treated SNB19 cells with cytochalasin-D, an inhibitor of actin polymerization, and colchicine-B, a tubulin inhibitor, in the presence of phorbol 12-myristate 13-acetate. Cytochalasin-D treatment of SNB19 cells resulted in the loss of phorbol 12-myristate 13-acetate-induced matrix metalloproteinase-9 (also known as gelatinase-B) expression and coincided with inhibition of actin polymerization, resulting in cell rounding. Moreover, compared with monolayers, cells grown as spheroids or cell aggregates failed to express matrix metalloproteinase-9 in the presence of phorbol 12-myristate 13-acetate. Matrix metalloproteinase-9 expression was also inhibited by calphostin-C, a protein kinase inhibitor, suggesting the involvement of protein kinase C in matrix metalloproteinase-9 expression. Phorbol 12-myristate 13-acetate-induced invasion of SNB19 cells through Matrigel was inhibited by cytochalasin-D and calphostin-C. These results suggest that the actin polymerization transduces signals that modulate the expression of matrix metalloproteinase-9 expression and the subsequent invasion of human glioma cells.

Actins↗
PubMed1998

Control of cell cycle progression by fibronectin matrix architecture.

Developmental patterning and differentiation, maintenance of parenchymal cell function, and the size, shape, and invasiveness of tumors are all orchestrated by cell interactions with the extracellular matrix. Here we show that the fibrillar structure of fibronectin (FN) matrix encodes essential regulatory cues and controls cell proliferation and signaling through changes in matrix architecture. A matrix assembled from native FN stimulated cell growth. In contrast, a mutant FN (FNDeltaIII1-7) that contains all known cell binding motifs but forms a structurally distinct matrix inhibited progression from G0/G1 into S phase. Furthermore, FNDeltaIII1-7 suppressed the stimulatory capacity of native FN and induced different levels of tyrosine phosphorylation of pp125(FAK). The differential effects on cell growth were ablated by blocking formation of matrix fibrils. Thus, modification of matrix architecture provides a novel approach to control cell proliferation.

Bromodeoxyuridine↗
PubMed1999

Inhibition of fibronectin matrix assembly by the heparin-binding domain of vitronectin.

The deposition of fibronectin into the extracellular matrix is an integrin-dependent, multistep process that is tightly regulated in order to ensure controlled matrix deposition. Reduced fibronectin deposition has been associated with altered embryonic development, tumor cell invasion, and abnormal wound repair. In one of the initial steps of fibronectin matrix assembly, the amino-terminal region of fibronectin binds to cell surface receptors, termed matrix assembly sites. The present study was undertaken to investigate the role of extracellular signals in the regulation of fibronectin deposition. Our data indicate that the interaction of cells with the extracellular glycoprotein, vitronectin, specifically inhibits matrix assembly site expression and fibronectin deposition. The region of vitronectin responsible for the inhibition of fibronectin deposition was localized to the heparin-binding domain. Vitronectin's heparin-binding domain inhibited both beta(1) and non-beta(1) integrin-dependent matrix assembly site expression and could be overcome by treatment of cells with lysophosphatidic acid, an agent that promotes actin polymerization. The interaction of cells with the heparin-binding domain of vitronectin resulted in changes in actin microfilament organization and the subcellular distribution of the actin-associated proteins alpha-actinin and talin. These data suggest a mechanism whereby the heparin-binding domain of vitronectin regulates the deposition of fibronectin into the extracellular matrix through alterations in the organization of the actin cytoskeleton.

Animals↗
PubMed2005

Nuclear matrix binding regulates SATB1-mediated transcriptional repression.

Special AT-rich binding protein 1 (SATB1) originally was identified as a protein that bound to the nuclear matrix attachment regions (MARs) of the immunoglobulin heavy chain intronic enhancer. Subsequently, SATB1 was shown to repress many genes expressed in the thymus, including interleukin-2 receptor alpha, c-myc, and those encoded by mouse mammary tumor virus (MMTV), a glucocorticoid-responsive retrovirus. SATB1 binds to MARs within the MMTV provirus to repress transcription. To address the role of the nuclear matrix in SATB1-mediated repression, a series of SATB1 deletion constructs was used to determine protein localization. Wild-type SATB1 localized to the soluble nuclear, chromatin, and nuclear matrix fractions. Mutants lacking amino acids 224-278 had a greatly diminished localization to the nuclear matrix, suggesting the presence of a nuclear matrix targeting sequence (NMTS). Transient transfection experiments showed that NMTS fusions to green fluorescent protein or LexA relocalized these proteins to the nuclear matrix. Difficulties with previous assay systems prompted us to develop retroviral vectors to assess effects of different SATB1 domains on expression of MMTV proviruses or integrated reporter genes. SATB1 overexpression repressed MMTV transcription in the presence and absence of functional glucocorticoid receptor. Repression was alleviated by deletion of the NMTS, which did not affect DNA binding, or by deletion of the MAR-binding domain. Our studies indicate that both nuclear matrix association and DNA binding are required for optimal SATB1-mediated repression of the integrated MMTV promoter and may allow insulation from cellular regulatory elements.

Animals↗
PubMed2006

Urokinase-type plasminogen activator receptor regulates a novel pathway of fibronectin matrix assembly requiring Src-dependent transactivation of epidermal growth factor receptor.

Previous studies have indicated that the urokinase-type plasminogen activator receptor (uPAR) can functionally interact with integrins thereby modulating integrin activity. We have previously demonstrated that treatment of fibroblasts with the uPAR ligand, P25, results in an increase in the activation of the beta1 integrin and a 35-fold increase in fibronectin matrix assembly (Monaghan, E., Gueorguiev, V., Wilkins-Port, C., and McKeown-Longo, P. J. (2004) J. Biol. Chem. 279, 1400-1407). Experiments were conducted to address the mechanism of uPAR regulation of matrix assembly. Treatment of fibroblasts with P25 led to an increase in the activation of the epidermal growth factor receptor (EGFR) and a colocalization of activated EGFR with beta1 integrins in cell matrix contacts. The effects of P25 on matrix assembly and beta1 integrin activation were inhibited by pretreatment with EGFR or Src kinase inhibitors, suggesting a role for both Src and EGFR in integrin activation by uPAR. Phosphorylation of EGFR in response to P25 occurred on Tyr-845, an Src-dependent phosphorylation site and was inhibited by PP2, the Src kinase inhibitor, consistent with Src kinase lying upstream of EGFR and integrin activation. Cells null for Src kinases also showed a loss of P25-induced matrix assembly, integrin activation, and EGFR phosphorylation. These P25-induced effects were restored following Src re-expression. The effects of P25 were specific for uPAR as enhanced matrix assembly by P25 was not seen in uPAR-/- cells, but was restored upon uPAR re-expression. These data provide evidence for a novel pathway of fibronectin matrix assembly through the uPAR-dependent sequential activation of Src kinase, EGFR, and beta1 integrin.

Cell Culture Techniques↗
PubMed2005

SPARC regulates extracellular matrix organization through its modulation of integrin-linked kinase activity.

SPARC, a 32-kDa matricellular glycoprotein, mediates interactions between cells and their extracellular matrix, and targeted deletion of Sparc results in compromised extracellular matrix in mice. Fibronectin matrix provides provisional tissue scaffolding during development and wound healing and is essential for the stabilization of mature extracellular matrix. Herein, we report that SPARC expression does not significantly affect fibronectin-induced cell spreading but enhances fibronectin-induced stress fiber formation and cell-mediated partial unfolding of fibronectin molecules, an essential process in fibronectin matrix assembly. By phage display, we identify integrin-linked kinase as a potential binding partner of SPARC and verify the interaction by co-immunoprecipitation and colocalization in vitro. Cells lacking SPARC exhibit diminished fibronectin-induced integrin-linked kinase activation and integrin-linked kinase-dependent cell-contractile signaling. Furthermore, induced expression of SPARC in SPARC-null fibroblasts restores fibronectin-induced integrin-linked kinase activation, downstream signaling, and fibronectin unfolding. These data further confirm the function of SPARC in extracellular matrix organization and identify a novel mechanism by which SPARC regulates extracellular matrix assembly.

Actins↗