Activation of protein kinase-C differentially regulates insulin-like growth factor-I and basic fibroblast growth factor messenger RNA levels

Fibroblasts represent one of the in vivo sites of insulin-like growth factor-I (IGF-I) production. In this study rat dermal fibroblasts in culture were used as a model system to assess the effect of activation of protein kinase-C on the levels of the mRNAs encoding IGF-I and another growth factor, basic fibroblast growth factor (bFGF). IGF-I and bFGF mRNA levels were determined using a solution hybridization/ RNase protection assay. Treatment of cells in serum-free medium containing 0.25% BSA (MEM + BSA) with the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) decreased IGF-I and increased bFGF mRNA levels in a time- and dose-dependent fashion. The peak effect of 100 nM PMA on IGF-I mRNA levels occurred at 9 h, whereas the peak effect on bFGF mRNA levels occurred after 3 h of incubation. In dose-response studies, halfmaximal inhibition of IGF-I mRNA levels was achieved with approximately 0.08 nM PMA, while half-maximal stimulation of bFGF mRNA levels was achieved with approximately 3 nM PMA. Inhibition of protein synthesis with cycloheximide abrogated the effect of PMA on bFGF mRNA levels, but only partially inhibited the effect of PMA on IGF-I mRNA levels. Studies employing sphingosine or staurosporine to inhibit protein kinase-C or preincubation in high doses of PMA to down-regulate protein kinase-C suggested that the effect of PMA on IGF-I and bFGF mRNA levels was mediated by activation of protein kinase-C, although both staurosporine and sphingosine had independent effects on the levels of these mRNAs and down-regulation of protein kinase-C had a sustained effect on IGF-I mRNA levels. Ligands known to activate protein kinase-C were then tested. Treatment of cells with 100 μg/ml of the synthetic diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol decreased IGF-I mRNA levels to 25% and increased bFGF mRNA levels to 520% of the level present in cells maintained in MEM + BSA. Treatment of cells with thrombin or bradykinin also decreased IGF-I mRNA levels and increased bFGF mRNA levels, but whereas the effect of thrombin on IGF-I mRNA levels was marked, the effect of bradykinin was minimal, and whereas the effect of thrombin on bFGF mRNA levels was sustained, the effect of bradykinin was transient. Bradykinin and thrombin were equally active in the cells, as demonstrated by the ability of each to increase accumulation of inositol phosphates, stimulate translocation of protein kinase-C from the cytosol to the membrane, and activate S6 kinase. Consistent with their effects on IGF-I mRNA levels, treatment of cells for 48 h with 1 nM PMA or 2 U/ml thrombin decreased immunoreactive IGF-I levels in conditioned medium to 64% and 52% of the level present in medium conditioned by cells maintained in MEM + BSA. In summary, activation of protein kinase-C in fibroblasts has differential effects on IGF-I and bFGF mRNA levels. The difference in the effect of thrombin and PMA compared to that of bradykinin on bFGF and IGF-I mRNA levels suggests that cellular events activated by PMA and thrombin, but not bradykinin, are important in the regulation of IGF-I and bFGF mRNA levels by these ligands.