Stimulation of Peritoneal Mesothelial Cells to Secrete Matrix Metalloproteinase-9 (MMP-9) by TNF-α: A Role in the Invasion of Gastric Carcinoma Cells.

It has recently been recognized that inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), upregulate the secretion of matrix metalloproteinase-9 (MMP-9) from cancer cells and thereby promote peritoneal dissemination. In this study, we found that TNF-α also stimulated peritoneal mesothelial cells to secrete MMP-9 as assessed by zymography. MMP-9 gene expression in mesothelial cells induced by TNF-α was confirmed by quantitative RT-PCR analysis. We then utilized the reconstituted artificial mesothelium, which was composed of a monolayer of mesothelial cells cultured on a Matrigel layer in a Boyden chamber system, to examine the effects of TNF-α on carcinoma cell invasion. The transmigration of MKN1 human gastric carcinoma cells through the reconstituted mesothelium was promoted by TNF-α in a dose-dependent manner. The increased MKN1 cell migration was partially inhibited by the anti-α3 integrin antibody, indicating that the invasion process involves an integrin-dependent mechanism. Finally, we observed that the invasion of MMP-9-knockdown MKN1 cells into Matrigel membranes was potentiated by the exogenous addition of purified proMMP-9. These results suggest that TNF-α-induced MMP-9 secretion from mesothelial cells plays an important role in the metastatic dissemination of gastric cancer.

Potentiation of cell invasion and matrix metalloproteinase production by alpha3beta1 integrin-mediated adhesion of gastric carcinoma cells to laminin-5.

We previously reported that the adhesion of gastric carcinoma cells to the peritoneum mediated by the alpha3beta1 integrin-laminin interaction is a key step in the initial process of peritoneal metastatic dissemination. Carcinoma cells subsequently invade through the intercellular gaps of mesothelial linings. In this study, we examined the role of the interaction of carcinoma cells with laminin-5, which is a major component of submesothelial basement membranes and serves as a high-affinity ligand for alpha3beta1 integrin, in carcinoma cell invasion. Human gastric carcinoma cell lines (MKN1, GT3TKB, and NUGC-4) adhered in an alpha3beta1 integrin-dependent manner to the extracellular matrix deposited by peritoneal mesothelial cells. An in vitro invasion assay using the Boyden chamber system revealed that MKN1 cell migration through the membranes increased when the membranes were coated with matrices produced by mesothelial cells or with laminin-5-containing Matrigel as compared to Matrigel alone. The cell migration promoted by laminin-5-containing Matrigel was inhibited by the presence of anti-alpha3 integrin antibody. When MKN1 cells were cultured in a laminin-5-coated plate, these cells were promoted to produce matrix metalloproteinase (MMP)-9, as assessed by gelatin zymography, enzyme-linked immunosorbent assay, and reverse transcription-polymerase chain reaction. These results suggest that the production of MMP-9 by MKN1 cells was potentiated by the alpha3beta1 integrin-laminin-5 interaction, which facilitated their invasion via degradation of the matrix.

Changes in adhesive and migratory characteristics of hepatocellular carcinoma (HCC) cells induced by expression of alpha3beta1 integrin.

The invasive and metastatic potentials of hepatocellular carcinoma are positively correlated with the expression level of alpha3beta1 integrin, a high-affinity adhesion receptor for laminin isoforms including laminin-5. In this study, we investigated changes in the adhesive and invasive behaviors of human HCC HepG2 cells after transfection with cDNA for alpha3 integrin in order to elucidate the direct involvement of this integrin in these cellular processes. We introduced cDNA for splice variants of alpha3 integrin (alpha3A and alpha3B) into the cells, and selected two transfectant clones (HepG2-3A and HepG2-3B), which express the alpha3A and alpha3B integrins, respectively. Both transfectant cells adhered almost equally to laminin-5-coated plates in an alpha3 integrin-dependent manner, indicating that transfected alpha3Abeta1 and alpha3Bbeta1 integrins were functionally active in these cells. The migratory and invasive potentials of the transfectant cells were assessed by scratch wound assay and in vitro chemoinvasion assay. The results demonstrated that the migration of HepG2-3A and HepG2-3B cells but not of mock transfectant (HepG2-M) cells was stimulated on the plates coated with laminin-5. Furthermore, HepG2-3A and HepG2-3B cells were found to be more invasive into laminin-5-containing matrices than were HepG2-M cells. These results strongly suggest that enhanced expression of alpha3beta1 integrin on HCC cells is directly involved in their malignant phenotypes such as invasion and metastasis.

Characterization of the promoter for the alpha3 integrin gene in various tumor cell lines: roles of the Ets- and Sp-family of transcription factors.

The alpha3beta1 integrin is an adhesion receptor for extracellular matrix proteins, including laminin isoforms, and plays crucial roles in the organization of epithelial and endothelial tissues. The aberrant expression of this adhesion molecule on tumor cells is associated with their invasive and metastatic potentials. In the present study, we analyzed the elements essential for alpha3 integrin gene expression in various tumor cell lines with different tissue origins by luciferase assay. An approximately 0.3 kb fragment of the 5'-flanking region of the mouse alpha3 integrin gene (-260/+84, relative to the major transcription start site) showed strong promoter activity in all six examined tumor cell lines. However, we found that these cell lines could be divided into two groups according to the level of dependency on the putative Ets-transcription factor binding motif located at -133. This motif was previously shown to be crucial for alpha3 integrin expression in MKN1 gastric carcinoma cells. The gene expression in one group of cell lines was upregulated mainly by the Ets motif, whereas that in the other group was less dependent on the Ets motif. We then postulated that additional regulatory elements were responsible for the expression of alpha3 integrin, and found that a GC-rich motif at -69 was another important element. An electrophoretic mobility shift assay using specific antibodies and a Western blot analysis of nuclear proteins revealed that the Sp3-transcription factor bound to this GC-rich motif. These results suggest that the Sp3 and Ets transcription factors cooperatively regulate alpha3 integrin gene expression and that the contribution of each element depends on the type of tumor cells.

Transforming growth factor-beta1 upregulates transcription of alpha3 integrin gene in hepatocellular carcinoma cells via Ets-transcription factor-binding motif in the promoter region.

The invasive and metastatic potentials of hepatocellular carcinoma (HCC) are positively correlated with the expression level of alpha3beta1 integrin, a high-affinity adhesion receptor for laminin isoforms. Transforming growth factor (TGF)-beta1 stimulates non-invasive HCC cells to acquire invasive phenotypes in association with the enhanced expression of alpha3 integrin. In this study, we investigated the molecular mechanism underlying the upregulation of alpha3beta1 integrin by TGF-beta1 in non-invasive HepG2 HCC cells. The treatment of HepG2 cells with TGF-beta1 induced the expression of alpha3 integrin and potentiated these cells to adhere to laminin-5 and to migrate through laminin-5-coated membranes. The promoter activity was measured by luciferase assay with a series of deletion constructs of the 5'-flanking region of the mouse alpha3 integrin gene, and the results showed that the -260/-119 region (relative to the major transcription start site) contained elements responsive to TGF-beta1 stimulation. The introduction of mutations into the putative consensus binding sequence for the Ets-family of transcription factors located at -133 greatly decreased the promoter activity responding to TGF-beta1 stimulation. The nuclear proteins extracted from TGF-beta1-stimulated HepG2 cells yielded a larger amount of DNA-nuclear protein complexes than did those extracted from unstimulated cells, as determined by an electrophoretic mobility shift assay using an oligonucleotide containing the Ets-site as a probe. These results suggest that TGF-beta1 stimulates HepG2 cells to express a higher level of alpha3 integrin by transcriptional upregulation via Ets transcription factors and to exhibit a more invasive phenotype.

Adhesion of gastric carcinoma cells to peritoneum mediated by alpha3beta1 integrin (VLA-3).

The interaction between gastric carcinoma cells and the peritoneal lining is a key step in peritoneal dissemination. In this study, we examined the roles of the beta1 family of integrin receptors in the adhesion of such cells to the peritoneum. The adhesion of several gastric carcinoma cell lines to peritonea excised from mice was inhibited most by an anti-alpha3 integrin antibody and to a lesser extent by an anti-alpha2 integrin antibody. In the peritoneal implantation of NUGC-4 human gastric carcinoma cells in athymic mice, treatment of the cells with anti-alpha2 or anti-alpha3 integrin antibody reduced the number of disseminated nodules; suppression by the anti-alpha3 integrin antibody was stronger than that by the anti-alpha2 integrin antibody. The cDNAs to human alpha2 and alpha3 integrins were introduced into K562 leukemic cells, which were positive for the integrin beta1 subunit but negative for the alpha2 or alpha3 subunit. The alpha3 integrin-transfected cells adhered to excised peritoneum and to a monolayer of peritoneal mesothelial cells more firmly than did the alpha2 integrin-transfected cells or the mock transfectant. Reverse transcription-PCR was used to analyze the expression of laminin-5 and laminin-10/11, which have been reported to serve as high-affinity ligands for alpha3beta1 integrin. mRNA for these laminin isoforms was found in mesothelial cells from the diaphragm and parietal peritoneum. These results strongly suggest that alpha3beta1 integrin plays an essential role in mediating the initial attachment of cancer cells to the peritoneum, leading to the formation of peritoneal metastasis.