PHORBOL ESTER STIMULATES CHOLINE UPTAKE IN SWISS 3T3 FIBROBLASTS FOLLOWING INTRODUCTION OF THE GENE ENCODING PROTEIN-KINASE C-ALPHA

Phorbol 12-myristate 13-acetate (PMA) stimulated radiolabelled choline uptake and incorporation into phosphatidylcholine (PtdCho) in a time- and concentration-dependent manner in wild-type NIH 3T3 fibroblasts. The accumulation of labelled choline induced by PMA was paralled by an increase in choline mass. The results implicate protein kinase C (PKC ) in the regulation of choline uptake. In order to address the PKC-sub type specificity of this response, a study was undertaken in Swiss 3T3 fibroblast cells, which normally express very low levels of PKC alpha . A retroviral expression system was used to introduce the genes for P KC alpha and neomycin resistance (used for selection) into the cells. Two resulting lines expressed PKC alpha at levels that were 20-fold hi gher than those found in the control (neomycin-resistant) line, or in the wild-type cells. In control Swiss 3T3 fibroblasts, 1 mu M PMA elev ated choline levels by only 30%, whereas, in Swiss 3T3 cell lines that stably over-expressed PKC alpha, PMA caused a 5-fold enhancement in [ C-14]choline accumulation. This concentration of PMA significantly inc reased [C-14]PtdCho levels in both control and PKC alpha-over-expressi ng lines, although the effect in the latter was significantly greater. The effects of PMA. were inhibited by the PKC antagonist sphingosine. These results implicate PKC alpha in the regulation of choline accumu lation and phospholipid synthesis in fibroblasts. Although additional PKC subtypes appear to participate in the control of PtdCho synthesis in these cells, PMA-stimulated choline uptake in Swiss 3T3 fibroblasts is almost entirely dependent on the presence of PKC alpha.