SIGNAL-TRANSDUCTION PATHWAY OF ACYLATION STIMULATING PROTEIN - INVOLVEMENT OF PROTEIN-KINASE-C

Acylation Stimulating Protein (ASP) was recently purified to homogenei ty from human plasma and shown to be identical to C3adesArg. ASP stimu lates triglyceride synthesis in human skin fibroblasts and primary hum an adipocytes. In vitro differentiation of human preadipocytes to matu re fat cells results in increased expression and accumulation of ASP i n the medium. These differentiated human adipocytes are also much more responsive to ASP than preadipocytes. The object of this study was to investigate the signal transduction pathway by which ASP causes trigl yceride synthesis (TGS) to increase in human cultured fibroblasts and adipocytes. No evidence was found for a protein kinase A-mediated resp onse. ASP action was consistent with a protein kinase C (PKC)-mediated pathway in that: 1) the effect of ASP on TGS was mimicked by 1-10 nM phorbol 12-myristate 13-acetate (PMA), a potent activator of PKC; (202 % ASP vs. 178% PMA stimulation); 2) the effect of PMA and ASP were non -additive with respect to TGS; 3) staurosporine (50 nM) and GF109203X (bisindolylmaleimide) at 1 mu M, both competitive inhibitors of the AT P-binding site on PKC, inhibited both ASP and PMA stimulation of TGS ( -59% and -65% for ASP and -84% and -99% for PMA, respectively); 4) Cal phostin C (0.8 mu M) which interacts with the regulatory domain of PKC also inhibited the ASP- and PMA-mediated stimulation of PKC (-76% +/- 11% inhibition for ASP and -99% +/- 20% inhibition for PMA), although in all cases the inhibition of PMA-stimulated triglyceride synthesis was greater; 5) ASP caused a time-dependent increase in intracellular diacylglycerol accumulation; and finally 6) stimulation by ASP caused an increase in PKC activity and a time-dependent translocation of PKC (maximal effect at 30 min) from the soluble intracellular compartment to a membrane-bound fraction (basal activity 22% in the membrane-bound fraction, ASP 54%, P<0.05 and PMA 69% P<0.0025). Taken together, the data are consistent with the conclusion that ASP acts to stimulate tri glyceride synthesis via activation of the protein kinase C pathway.