Phorbol ester and interleukin-1 induce interleukin-6 gene expression in vascular smooth muscle cells via independent pathways

Previous studies using bioassays suggest that interleukin 6 (IL-6) is a major secretory product of vascular smooth muscle cells (VSMC), which is induced by proinflammatory cytokines. This study investigated whether activation of the protein kinase C (PKC) pathway induces IL-6 gene expression and release in VSMC, by using both bioassay and specific immunoassay methods to measure IL-6 release. Activation of PKC with a phorbol ester, PMA (phorbol myristate acetate), induced a rapid and transient (1-4 h) increase in the levels of both 1.2- and 2.4-kb IL-6 transcripts in rat aortic SMCs (RASMC), as determined by Northern analysis, which was followed by increased release of bioactive IL-6, as determined by a B9 cell-proliferation assay. IL-1, a physiological activator of PKC, induced a rapid increase in IL-6 messenger RNA (mRNA) levels, which was sustained at 24 h. PMA-induced IL-6 mRNA levels in RASMC were markedly attenuated after downregulation of PKC with PMA and by the selective PKC inhibitor, bisindolylmaleimide. In contrast, IL-1-induced increases in IL-6 mRNA were not affected by either PKC downregulation or bisindolylmaleimide. Angiotensin II (Ang II), also known to activate PKC, likewise induced a rapid increase in IL-6 mRNA levels and IL-6 release in RASMC, but the effect was not blocked by PKC downregulation. VSMC derived from human saphenous vein (HSVSMC) released substantial amounts of immunoreactive IL-6 in the absence of stimulation by exogenous growth factors, and both PMA and IL-1 markedly increased IL-6 release. Furthermore, downregulation of PKC and bisindolylmaleimide blocked the effect of PMA but not that of IL-1 in HSVSMC. These results suggest that activation of phorbol ester-responsive PKC induces IL-6 gene expression in both rat and human VSMC. In contrast, IL-1 and Ang II activate IL-6 gene expression by a pathway distinct from that of phorbol ester-responsive PKC.