Oncostatin M Regulation of the Tissue Inhibitor of Matrix Metalloproteinases-1 Promoter

Abstract

<p>The progression of an inflammatory response is largely dictated by soluble<br />factors termed cytokines reknown for their redundant and pleiotropic nature in<br />modulation of both immune and stromal cells. Individual members of the<br />interleukin-6 (IL-6)-type cytokine family possess both unique and shared biological<br />activities. These cytokines may participate in tissue remodelling by promoting<br />reconstruction ofextracellular matrix (ECM) following nonspecific tissue damage by<br />inflammatory cells. Consistent with this view, these cytokines upregulate expression<br />of an ECM protease inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1) and<br />thus may alter net enzymatic degradation of ECM. The overall goal of this thesis<br />is to examine mechanisms by which TIMP-1 is regulated by IL-6-type cytokines,<br />especially by the cytokine oncostatin M (OSM). The promoter of TIMP-1 has<br />therefore been studied in detail to address the mechanisms by which OSM (and IL-6<br />type cytokines) regulate the transcription of the TIMP-1 gene. The approaches<br />undertaken have included deletion analysis of the TIMP-1 gene nucleotide<br />sequences proximal to the start of transcription to define DNA sequences<br />necessary/sufficient for cytokine-induced TIMP-1 promoter activity. In addition, the<br />binding of nuclear factors to these DNA elements and cytokine-response elements, their expression and involvement in the regulation of TIMP-1 transcription have been explored.</p> <p>We have identified sequences proximal to the start of TIMP-1 transcription<br />that are necessary for maximal responsiveness to OSM and IL-6. Deletion analysis<br />of the proximal TIMP-1 promoter (-95 to +47 TIMP-1 sequences) has identified a<br />nucleotide sequence within -59 to -53 ofthe murine TIMP-1 promoter that harbours<br />an AP-1 consensus DNA binding element. This element is necessary for maximal<br />OSM or IL-6 induced promoter activity of TIMP1-CAT reporter gene constructs<br />transfected into human hepatoma HepG2 cells. OSM is the most potent stimulus<br />(approx. 11-fold for OSM, and 4-fold for IL-6) of this response and additional<br />sequences 3-prime to +1 ofthe TIMP-1 gene are also necessary for maximal OSM<br />responsiveness.</p> <p>Electrophoretic mobility shift assays demonstrated two gel-shifted complexes<br />which bind the TIMP-1 AP-1 site. An AP-1 gel-shifted complex is present in the<br />absence of cytokine stimulation ("complex 1"), while OSM and not other IL-6-type<br />cytokines, stimulated the formation of a second AP-1 gel shifted complex.<br />Nuclear factors binding to TIMP-1 AP-1 complex 1 include junB, junD and fosrelated<br />antigens. However, unlike complex1, c-fos is present and necessary for the<br />formation of the OSM-induced TIMP-1 AP-1 complex2. Consistent with this, OSM<br />is a potent inducerofc-fos protein expression among IL-6-type cytokines. Both the<br />formation of complex2 and c-fos expression require new protein synthesis. JunB<br />and junO are constitutively expressed, while the expression of fos-related antigens are induced in response to OSM. In addition, although PMA was also a potent<br />inducer of c-fos expression, induction of TIMP-1 promoter activity by the<br />combination of PMA and IL-6 was comparable to IL-6 alone and did not equal the<br />significantly higher induction by OSM. Within the same cells, OSM and IL-6 equally<br />induced STATDNA-binding activity. An Ets-consensus site (nucleotides -45 to -40)<br />flanking the 3-prime end of the AP-1 site is a weak binding site for Ets-related<br />nuclear factors, and an SP-1 site near +1 (-11 to -6) is a strong binding siteforSP1<br />nuclear factors and related SP-1 site binding proteins. No STAT nuclear factor<br />binding to the proximal TIMP-1 promoterwas detected. Taken together, the TIMP1<br />AP-1 site and c-fos represent a unique target of OSM signalling and activation of<br />AP-1 complexes (possibly containing c-fos) by OSM as well as sequences<br />downstream of TIMP-1 +1 contribute to maximal responsiveness of the promoter<br />to this cytokine among IL-6 family members.</p> <p>The contribution of c-fos to OSM-induced TIMP-1 expression was further<br />explored in murine cells. OSM stimulates the expression of c-fos and activates<br />STATs 1, 3 and 5 DNA-binding activity in murine fibroblasts. As observed in<br />human cells, OSM but not other IL-6-type cytokines upregulated c-fos expression<br />which participated in complexes binding the TIMP-1 AP-1 site. OSM was also<br />unique among IL-6 family members in activating STAT5 DNA-binding activity in<br />murine fibroblasts. However, in contrast to observations in human HepG2 cells,<br />deletion analysis of the TIMP-1 promoter showed that the AP-1 site (-59/-53) was<br />v not necessary for OSM-mediated upregulation of the TIMP-1 proximal promoter<br />activity over basal levels in murine NIH3T3 fibroblasts and co-transfection of a<br />dominant-negative of AP-1 had no effect. However, transfection of dominant-negative<br />STATs1, 3 or5 (especially STAT3) could diminish cytokine-induced TIMP1<br />promoter activity. In addition, c-Fos was dispensable for OSM-mediated<br />upregulation ofTIMP-1 mRNA levels as TIMP-1 expression was detected in wildtype<br />and c-fos knockout murine lung fibroblasts. Consistent with deletion analysis<br />ofthe TIMP-1 promoter in human cells, dominant-negative AP-1 expression vectors<br />abrogated OSM-mediated TIMP-1 promoter activity, while Stat-dominant negative<br />expression vectors did not. Taken together, the examination of murine and human<br />systems suggests that AP-1 and STAT nuclear factors can contribute to the<br />regulation of the TIMP-1 promoter.</p>