The effect of the tumor promoter, 12-0-tetradeconoyl-phorbol-13 acetate (TPA)on the expression of 65kDa and 92kDa gelatinoloytic metalloproteinases (gelatinases) was studied in a variety of normal and malignant cell lines. Enzyme activity was assessed by gelatin zymography. At mRNA levels the 65kDa gelatinase was expressed equally by normal fibroblasts and tumor cells and was minimally affected by TPA treatment. In contrast, the 92kDa gelatinase which was constitutively expressed only by tumor cell lines was stimulated to a varying degree by TPA in all tumor cell lines. TPA-mediated stimulation of the 92kDa gelatinase was abolished by inhibitors of protein kinase C (PKC), as well as by inhibitors of protein synthesis and RNA transcription. Analysis of transcription binding proteins containing c-fos capable of binding to type I collagenase and stromelysin AP-1 consensus sequences in lysates from TPA treated cells was undertaken. No relationship was observed between AP-1 levels and 65kDa gelatinase expression. However, AP-1 binding activity did follow a similar pattern to the expression of 92kDa gelatinase. Interstitial collagenase mRNA expression, known to contain an AP-1 site in a positive regulatory element, appeared to follow the same pattern of expression as 92kDa gelatinase following TPA treatment. Two physiologically relevant cytokines, thought to act via the PKC pathway, also induced cell line specific selective stimulation of 92kDa gelatinase activity. These effects were blocked by inhibitors of protein synthesis, RNA transcription and PKC. The project has been extended to evaluate the effects of TPA on the RNA expression of two inhibitors of metalloproteinases, TIMP-1 and TIMP-2.
