Regulation of protein expression by transforming growth factor beta and other growth modulators

The main goal of this project was to understand the signal transduction pathways through which growth modulators such as TGF-[beta], PMA and retinoic acid mediate protein expression and DNA synthesis. Transforming growth factor [beta] (TGF-[beta]) induces synthesis and secretion of a 48,000 M r protein that we have found to be type I plasminogen activator inhibitor (PAI-1). TGF-[beta] induces PAI-1 in every cell line that we have studied including near term mink lung epithelial CCL64 cells, mouse embryo fibroblast AKR-2B 84A cells, African green monkey kidney epithelial BSC-1 cells, and normal rat kidney fibroblast NRK 49F cells. PAI-1 is induced synergistically by TGF-[beta] and EGF in CCL64 cells. Phorbol 12-myristate 13-acetate induces PAI-1 in CCL64, BSC-1 and NRK cells but not in AKR-2B cells. TGF-[beta] and retinoic acid both induce synthesis and secretion of a 73,000 M r protein by CCL64 cells that we call inhibitor-induced protein, 73 kDa (IIP73). Retinoic acid does not significantly regulate PAI-1 expression. PMA does not induce IIP73. To test whether cAMP was a regulator of the growth modulator effects, we treated cells with agents which increase intracellular cAMP (forskolin, cholera toxin, and dibutyryl cAMP) and measured the effects on expression of PAI-1 and IIP73 and on DNA synthesis. We found that agents that increase intracellular cAMP lowered the basal and induced levels of PAI-1 coordinately and lowered DNA synthesis rates in AKR-2B, BSC-1, CCL64 and NRK cells. However, these same factors had little or no effect on the ability of TGF-[beta] or retinoic acid to induce IIP73. To test for the necessity of activated protein kinase C (PKC) in the induction of PAI-1 and IIP73 expression and to test the role of PKC in stimulation of DNA synthesis, we down-regulated PKC in CCL64 cells by a 48 h incubation with a high concentration of PMA. While PMA could no longer induce PAI-1, there was no effect on the ability of TGF-[beta] to induce PAI-1 and no effect on the ability of TGF-[beta] and retinoic acid to induce IIP73. However, down-regulation of PKC prevented retinoic acid stimulation of DNA synthesis in CCL64 cells. This shows that induction of PAI-1 by TGF-[beta] does not depend on activation of PKC and that retinoic acid may induce proliferation in CCL64 cells through a PKC-dependent mechanism