Stromelysin-2 (matrix metalloproteinase 10) is inducible in lymphoma cells and accelerates the growth of lymphoid tumors in vivo.

Matrix metalloproteinase (MMP) 10 (stromelysin-2) is known to degrade various components of the extracellular matrix; however, the signals that regulate its expression and its role in lymphoma growth remain unknown. In the present work, we report the up-regulated expression of MMP10 in T lymphoma cells following contact with endothelial cells. The induction of MMP10 was found to be dependent on the specific interaction between LFA-1 and ICAM-1, which play a central role in regulating the expression of genes involved in the rate-limiting steps of lymphoma development. MMP10, but not MMP3 (stromelysin-1), was also up-regulated in human B lymphoma cells following exposure to IL-4, IL-6, and IL-13, but not to IL-1. To gain further insight into the role of MMP10 in lymphoma development, we generated lymphoma cell lines constitutively expressing high levels of MMP10 and studied these cells for their ability to form thymic lymphoma in vivo. Mice injected with lymphoma cells constitutively expressing MMP10 developed thymic lymphoma more rapidly than those injected with control lymphoma cells. These results provide the first in vivo evidence that overexpression of MMP10 promotes tumor development, and indicate that MMP10 induction is an important pathway activated not only upon ICAM-1/LFA-1-mediated intercellular contact, but also following activation of tumor cells with inflammatory cytokines.

Stromelysin-1 (MMP-3) is inducible in T lymphoma cells and accelerates the growth of lymphoid tumors in vivo.

Matrix metalloproteinase (MMP)-3 (stromelysin-1) degrades various components of the extracellular matrix as well as several non-matrix components; it has notably been shown to activate other MMPs relevant to cancer and metastasis, including MMP-9. MMP-3 gene expression in the tumor microenvironment could therefore contribute to cancer progression. Transcriptional regulation of MMP genes was often described to occur upon intercellular interactions, leading to overexpression of these genes by cancer and/or stromal cells. In the present work, we report that expression of MMP-3 in T lymphoma cells is transiently induced during specific intercellular contact with endothelial cells (EC). Moreover, mice injected with lymphoma cells expressing MMP-3 constitutively developed thymic lymphoma more rapidly than those injected with control lymphoma cells. We also found that overexpression of MMP-3 in lymphoma transfectants significantly improved their ability to migrate through the matrix when compared to cells transfected with the control vector. These results provide the first in vivo evidence that local expression of MMP-3 promotes lymphoma progression and indicate that MMP-3 expression is tightly regulated upon lymphoma cell/stromal cell interaction.

ICAM-1 isoforms: specific activity and sensitivity to cleavage by leukocyte elastase and cathepsin G.

The extracellular moiety of ICAM-1 consists of five Ig-like domains, the first and third domains mediating adhesion to integrin ligands. The ICAM-1 gene, however, gives rise to the expression of five alternative splice variants containing two, three, or four Ig-like domains. In this work, we have investigated whether the rearrangement of the architecture of ICAM-1 affects its structural properties and function. We showed that, in contrast to the common form, all alternative isoforms of ICAM-1 were susceptible to cleavage by leukocyte elastase and cathepsin G. We found that the length of an isoform did not influence the susceptibility to proteolysis. The molecular diversity provided by the skipping of entire Ig domains and the level of expression on the APC, however, significantly influenced their ability to potentiate the proliferation of T cells. Finally, we found that the expression of minor ICAM-1 isoforms encoding the third Ig-like domains was sufficient to sustain neutrophil infiltration in the liver and confer exon-5-targeted ICAM-1-deficient mice susceptibility to LPS-induced septic shock. These findings not only demonstrate that ICAM-1 isoforms are fully functional, but support the concept that alternative RNA splicing in the Ig superfamily may fulfill distinct roles during the development of the immune response.

Evidence for the role of promoter methylation in the regulation of MMP-9 gene expression.

Several studies have reported that elevated MMP-9 expression in lymphoma tissues correlated with tumor stage, grade, or prognosis. Because the DNA methylation pattern is critical for gene expression, detailed methylation analysis using genomic bisulfite sequencing was performed on a series of lymphoma cell lines. We found an inverse correlation between level of methylation of the MMP-9 promoter and the level of MMP-9 expression. Treating lymphoma cells with a DNA methylation inhibitor decreased MMP-9 promoter methylation and increased MMP-9 messenger RNA and protein secretion. This increased expression was potentiated by PMA, a known stimulus of MMP-9 in lymphoma cells. Finally, experiments using in vitro methylated MMP-9 promoter constructs confirmed the fact that DNA methylation exerts suppression on transcriptional activity. The results thus indicate that methylation may contribute to the transcriptional activity of the MMP-9 promoter.

Induced expression of MMP-9 in C6 glioma cells is inhibited by PDGF via a PI 3-kinase-dependent pathway.

The involvement of phosphatidylinositol 3 (PI 3)-kinase in the signalling pathways leading to MMP-9 expression in glioma cells remains unclear. Here, we report that PI 3-kinase inhibits MMP-9 expression induced by either IL-1 or TNF-alpha in rat C6 glioma cells. Using zymography and semi-quantitative RT-PCR analysis, we showed that treatment of C6 cells with wortmannin, an inhibitor of PI 3-kinase activity, potentiated the expression of MMP-9 induced by both cytokines. In contrast, platelet-derived growth factor (PDGF), an inducer of PI 3-kinase activity in C6 cells, inhibited IL-1- or TNF-alpha-induced MMP-9 secretion. Accordingly, this inhibition by PDGF was prevented by wortmannin. Furthermore, stable C6 clones over-expressing the dominant-negative form the regulatory subunit of PI 3-kinase potentiated the expression of MMP-9 induced by IL-1 or TNF-alpha. Taken together, these results suggest that PI 3-kinase may act as a negative regulator of MMP-9 expression in C6 glioma cells.

Protein kinase C-zeta regulates transcription of the matrix metalloproteinase-9 gene induced by IL-1 and TNF-alpha in glioma cells via NF-kappa B.

The regulation of matrix metalloproteinase-9 (MMP-9) expression in glioma cells is one of the key processes in tumor invasion through the brain extracellular matrix. Although some studies have demonstrated the implication of classic protein kinase C (PKC) isoforms in the regulation of MMP-9 production by phorbol esters or lipopolysaccharide, the involvement of specific PKC isoforms in the signaling pathways leading to MMP-9 expression by inflammatory cytokines remains unclear. Here we report that the atypical PKC-zeta isoform participates in the induction of MMP-9 expression by interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) in rat C6 glioma cells. Indeed, zymography and semi-quantitative reverse transcriptase-PCR analysis showed that pretreatment of C6 cells with PKC-zeta pseudosubstrate abolished MMP-9 activity and gene expression induced by IL-1 or TNF-alpha. Accordingly, IL-1 and TNF-alpha were able to induce PKC-zeta activity, as demonstrated by in vitro kinase assay using immunoprecipitated PKC-zeta. Furthermore, stable C6 clones overexpressing PKC-zeta, but not PKC-epsilon, displayed an up-regulation of MMP-9 constitutive expression as well as an increase of mmp-9 promoter activity. These processes were inhibited by an NF-kappaB-blocking peptide and completely prevented by NF-kappaB-binding site mutation in the mmp-9 promoter. Taken together, these results indicate that PKC-zeta plays a key role in the regulation of MMP-9 expression in C6 glioma cells through NF-kappaB.