Activation of protein kinase C alpha enhances human growth hormone-binding protein release

The effect of phorbol ester on human growth hormone-binding protein (hGH-BP) release was investigated. The hGH-BP release from human IM-9 cells measured by immunoblotting was dose-dependently enhanced by a phorbol ester, phorbol 12, 13-dibutyrate (PDBu), and reached plateau at 100 nM. The increased hGH-BP release was shown after 10 min incubation with PDBu and reached a plateau at 60 min after stimulation. Similarly, a diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol, enhanced hGH-BP release. The enhancement was not inhibited by cycloheximide pretreatment, suggesting that the enhanced hGH-BP release does not require de novo protein synthesis. The PDBu-enhanced hGH-BP release was strongly inhibited by extracellular EDTA, and was dose-dependently inhibited by protein kinase C (PKC)-specific inhibitor, Ro 31-8220. These results suggest that activation of PKC mediates the PDBu-enhanced hGH-BP release. Of the 11 known PKC isoforms in human cells, PKCalpha, delta, mu and iota were detected in IM-9 cells by immunoblotting. Of these isoforms, PKCalpha, delta and mu were present in the membrane fraction, which is a known activation marker of PKC. Furthermore, when several PKC-specific inhibitors (Gö 6976, GF 109203X or bisindolylmaleimide III) with different specificities for each isoform were used, there was a good correlation between inhibition of the enhancement of hGH-BP release and inhibition of the phosphorylation of PKC isoforms, another activation marker of PKC, in PKCalpha but not in PKCdelta and mu. These results suggest that activation of PKCalpha is involved in PDBu-enhanced hGH-BP release.