Deoxycholic acid activates protein kinase C (PKC) and phospholipase C (PLC) via increased Ca2+ entry at plasma membrane

Background & Aims: Secondary bile acids like deoxycholic acid (DCA) are well-established tumor promoters that may exert their pathologic actions by interfering with intracellular signaling cascades. Methods: We evaluated the effects of DCA on Ca2 signaling in BHK-21 fibroblasts using fura-2 and mag-fura-2 to measure cytoplasmic and intraluminal internal stores [Ca2], respectively. Furthermore, green fluorescent protein (GFP)-based probes were used to monitor time courses of phospholipase C (PLC) activation (pleckstrin-homology [PH]-PLC- GFP), and translocation of protein kinase C (PKC) and a major PKC substrate, myristolated alanine–rich C-kinase substrate (MARCKS). Results: DCA (50–250 mol/L) caused profound Ca2 release from intracellular stores of intact or permeabilized cells. Correspondingly, DCA increased cytoplasmic Ca2 to levels that were 120% of those stimulated by Ca2-mobilizing agonists in the presence of external Ca2, and 60% of control in Ca2-free solutions. DCA also caused dramatic translocation of PH-PLC-GFP, and conventional, Ca2/diacylglycerol (DAG)-dependent isoforms of PKC (PKC-I and PKC-), and MARCKS-GFP, but only in Ca2-containing solutions. DCA had no effect on localization of a novel (PKC) or an atypical (PKC) PKC isoform. Conclusions: Data are consistent with a model in which DCA directly induces both Ca2 release from internal stores and persistent Ca2 entry at the plasma membrane. The resulting microdomains of high Ca2 levels beneath the plasma membrane appear to directly activate PLC, resulting in modest InsP3 and DAG production. Furthermore, the increased Ca2 entry stimulates vigorous recruitment of conventional PKC isoforms to the plasma membrane.