Laminin mediates tethering and spreading of colon cancer cells in physiological shear flow.
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Under the physiological shear condition, cultured colon cancer cells bound to laminin (LM), but not to fibronectin or vitronectin. Most of the tethered cells did not roll, but arrested immediately and spread within 10-30 min on LM under the continuous presence of shear flow. The tethering of Colo201 was partially inhibited by monoclonal antibodies (mAbs) to alpha6 integrin and a combination of mAbs to beta1 and beta4 integrins, but not by mAb to 67KD laminin receptor. Some Colo201 cells still tethered at 4 degrees C. This suggests that alpha6beta1 and alpha6beta4 integrins participate in Colo201 tethering on LM, although other non-integrin molecules play roles. In contrast, the spread of Colo201 was effectively inhibited by the mAbs to integrin alpha2, alpha6 and beta1 chains. The effect of anti-alpha2 plus anti-alpha6 mAbs was almost equal to anti-beta1, suggesting that Colo201 cells mainly use alpha2beta1 and alpha6beta1 integrins for spreading on LM. When the cells were perfused on subconfluent endothelial cells (HUVEC) cultured on LM, they did not tether on HUVEC but did on coated LM exposed at intercellular gap area. Immunohistochemistry revealed that LM abundantly existed in the cytosol of human portal and hepatic vein endothelial cells. These data suggest that LM can mediate from tethering to spreading of colon cancer cells under the blood flow and plays an essential role in haematogeneous metastasis. (+info)
Inhibition of matrix metalloproteinase-2 expression and bladder carcinoma metastasis by halofuginone.
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Matrix metalloproteinase-2 (MMP-2) plays a critical role in tumor cell invasion and metastasis. Inhibitors of this enzyme effectively suppress tumor metastasis in experimental animals and are currently being tested in clinical trials. MMP-2 transcriptional regulation is a part of a delicate balance between the expression of various extracellular matrix (ECM) constituents and ECM degrading enzymes. Halofuginone, a low-molecular-weight quinazolinone alkaloid, is a potent inhibitor of collagen type alpha1 (I) gene expression and ECM deposition. We now report that expression of the MMP-2 gene by murine (MBT2-t50) and human (5637) bladder carcinoma cells is highly susceptible to inhibition by halofuginone. Fifty percent inhibition was obtained in the presence of as little as 50 ng/ml halofuginone. This inhibition is due to an effect of halofuginone on the activity of the MMP-2 promoter, as indicated by a pronounced suppression of chloramphenicol acetyltransferase activity driven by the MMP-2 promoter in transfected MBT2 cells. There was no effect on chloramphenicol acetyltransferase activity driven by SV40 promoter in these cells. Halofuginone-treated cells failed to invade through reconstituted basement-membrane (Matrigel) coated filters, in accordance with the inhibition of MMP-2 gene expression. A marked reduction (80-90%) in the lung colonization of MBT2 bladder carcinoma cells was obtained after the i.v. inoculation of halofuginone-treated cells as compared with the high metastatic activity exhibited by control untreated cells. Under the same conditions, there was almost no effect of halofuginone on the rate of MBT2 cell proliferation. These results indicate that the potent antimetastatic activity of halofuginone is due primarily to a transcriptional suppression of the MMP-2 gene, which results in a decreased enzymatic activity, matrix degradation, and tumor cell extravasation. This is the first description, to our knowledge, of a drug that inhibits experimental metastasis through the inhibition of MMP-2 at the transcriptional level. Combined with its known inhibitory effect on collagen synthesis and ECM deposition, halofuginone is expected to exert a profound anticancerous effect by inhibiting both the primary tumor stromal support and metastatic spread. (+info)
Temporal expression of the matrix metalloproteinase MMP-2 correlates with fibronectin immunoreactivity during the development of the vascular system in the chick embryo chorioallantoic membrane.
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In this study we have examined in the chick embryo chorioallantoic membrane (ChAM) the expression of the matrix metalloproteinase 2 (MMP-2) and have correlated this parameter with the expansion of the ChAM vasculature and with the expression of 3 extracellular matrix components (fibronectin, laminin and type IV collagen), which differentially modulate angiogenesis. In the early phases of ChAM development, between d 6 and d 8 of incubation, when the increase of the ChAM vasculature is maximal, higher values of MMP-2 and, respectively, of fibronectin immunoreactive area, are detectable. These results indicate that MMP-2 activity and fibronectin expression are 2 strictly related components of angiogenesis occurring in vivo. (+info)
Biochemical characterization of the epithelial dystroglycan complex.
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Dystroglycan is a widely expressed extracellular matrix receptor that plays a critical role in basement membrane formation, epithelial development, and synaptogenesis. Dystroglycan was originally characterized in skeletal muscle as an integral component of the dystrophin glycoprotein complex, which is critical for muscle cell viability. Although the dystroglycan complex has been well characterized in skeletal muscle, there is little information on the structural composition of the dystroglycan complex outside skeletal muscle. Here we have biochemically characterized the dystroglycan complex in lung and kidney. We demonstrate that the presence of sarcoglycans and sarcospan in lung reflects association with dystroglycan in the smooth muscle. The smooth muscle dystroglycan complex in lung, composed of dystroglycan, dystrophin/utrophin, beta-, delta-, epsilon-sarcoglycan, and sarcospan, can be biochemically separated from epithelial dystroglycan, which is not associated with any of the known sarcoglycans or sarcospan. Similarly, dystroglycan in kidney epithelial cells is not associated with any of the sarcoglycans or sarcospan. Thus, our data demonstrate that there are distinct dystroglycan complexes in non-skeletal muscle organs as follows: one from smooth muscle, which is associated with sarcoglycans forming a similar complex as in skeletal muscle, and one from epithelial cells. (+info)
Division of labor among the alpha6beta4 integrin, beta1 integrins, and an E3 laminin receptor to signal morphogenesis and beta-casein expression in mammary epithelial cells.
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Contact of cultured mammary epithelial cells with the basement membrane protein laminin induces multiple responses, including cell shape changes, growth arrest, and, in the presence of prolactin, transcription of the milk protein beta-casein. We sought to identify the specific laminin receptor(s) mediating the multiple cell responses to laminin. Using assays with clonal mammary epithelial cells, we reveal distinct functions for the alpha6beta4 integrin, beta1 integrins, and an E3 laminin receptor. Signals from laminin for beta-casein expression were inhibited in the presence of function-blocking antibodies against both the alpha6 and beta1 integrin subunits and by the laminin E3 fragment. The alpha6-blocking antibody perturbed signals mediated by the alpha6beta4 integrin, and the beta1-blocking antibody perturbed signals mediated by another integrin, the alpha subunit(s) of which remains to be determined. Neither alpha6- nor beta1-blocking antibodies perturbed the cell shape changes resulting from cell exposure to laminin. However, the E3 laminin fragment and heparin both inhibited cell shape changes induced by laminin, thereby implicating an E3 laminin receptor in this function. These results elucidate the multiplicity of cell-extracellular matrix interactions required to integrate cell structure and signaling and ultimately permit normal cell function. (+info)
Stimulation of beta1-integrin function by epidermal growth factor and heregulin-beta has distinct requirements for erbB2 but a similar dependence on phosphoinositide 3-OH kinase.
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Integrins and growth factor receptors are important participants in cellular adhesion and migration. The EGF receptor (EGFR) family of tyrosine kinases and the beta1-integrin adhesion receptors are of particular interest, given the implication for their involvement in the initiation and progression of tumorigenesis. We used adhesion and chemotaxis assays to further elucidate the relationship between these two families of transmembrane signaling molecules. Specifically, we examined integrin-mediated adhesive and migratory characteristics of the metastatic breast carcinoma cell line MDA-MB-435 in response to stimulation with growth factors that bind to and activate the EGFR or erbB3 in these cells. Although ligand engagement of the EGFR stimulated modest beta1-dependent increases in cell adhesion and motility, heregulin-beta (HRGbeta) binding to the erbB3 receptor initiated rapid and potent induction of breast carcinoma cell adhesion and migration and required dimerization of erbB3 with erbB2. Pharmacologic inhibitors of phosphoinositide 3-OH kinase (PI 3-K) or transient expression of dominant negative forms of PI 3-K inhibited both EGF- and HRGbeta-mediated adhesion and potently blocked HRGbeta- and EGF-induced cell motility. Our results illustrate the critical role of PI 3-K activity in signaling pathways initiated by the EGFR or erbB3 to up-regulate beta1-integrin function. (+info)
Regulation of renal laminin in mice with type II diabetes.
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This study examines the regulation of renal laminin in the db/db mouse, a model of type II diabetes characterized by extensive remodeling of extracellular matrix. Immunohistochemistry demonstrated an increase in the contents of laminin chains including beta1 chain in the mesangium and tubular basement membranes at 1, 2, 3, and 4 mo of diabetes. Immunofluorescence with an antibody against the recently discovered laminin alpha5 chain showed that in the normal mouse, the protein had a restricted distribution to the glomerular and tubular basement membranes with scant expression in the mesangium of older mice. In the diabetic mouse, the laminin alpha5 chain content of the glomerular and tubular basement membranes was increased, with marked expression in the mesangium. Northern analysis revealed a significant decrease in the renal cortical contents of alpha5, beta1, and gamma1 chain mRNA in the diabetic mice compared to control, at each of the time points. In situ hybridization showed decreased abundance of alpha5 transcripts in the glomeruli of diabetic mice compared to nondiabetic controls. Analysis of mRNA changes by Northern and in situ hybridization studies demonstrated that the reduction in laminin transcripts involved both glomerular and tubular elements. These observations demonstrate that laminin accumulation in the db/db mice with type II diabetes is due to nontranscriptional mechanisms. Because previous investigations in rodents with type I diabetes have shown that the increase in renal laminin content was associated with a corresponding increment in laminin chain transcript levels, it appears that the mechanisms underlying augmentation in renal matrix laminin content may be distinct in the two types of diabetes. (+info)
The nonintegrin laminin binding protein (p67 LBP) is expressed on a subset of activated human T lymphocytes and, together with the integrin very late activation antigen-6, mediates avid cellular adherence to laminin.
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A search for genes expressed in activated T cells revealed that the nonintegrin, 67-kDa laminin binding protein (p67 LBP) is expressed on the surface of a subset (10-15%) of activated peripheral blood T cells. Surface p67 LBP expression is detectable by FACS using the anti-p67 LBP mAb, MLuC5, within 6 h of T cell activation with phorbol dibutyrate and ionomycin, peaks 18-36 h postactivation, and persists for 7-10 days. The subset of T cells expressing p67 LBP is composed of mature, single-positive cells (85% CD4+8-, 15% CD4-8+) of memory cell phenotype (100% CD45 RO+/CD45 RA-). The p67 LBP+ T cells also express the integrin alpha6 chain (CD49f), which is known to associate with p67 LBP on tumor cells. In addition, the p67 LBP+ T cells express the integrin beta1, which associates with alpha6 in the laminin-specific integrin receptor very late activation Ag (VLA)-6 (alpha6beta1). Expression of an exogenous cDNA encoding the 37-kDa LBP precursor (p37 LBPP) confers p67 LBP surface expression on a p67 LBP-negative Jurkat T cell line (B2.7). Expression of p67 LBP induces B2.7 transfectants to adhere to laminin, but avid laminin binding depends on coexpression of VLA-6. Taken together, these data indicate that p67 LBP is an activation-induced surface structure on memory T cells that, together with VLA-6, mediates cellular adherence to laminin. (+info)