Deoxycholic acid activates protein kinase C and phospholipase C 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]-PLCδ-GFP), and translocation of protein kinase C (PKC) and a major PKC substrate, myristolated alanine-rich C-kinase substrate (MARCKS). Results DCA (50-250 μmol/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-PLCδ-GFP, and conventional, Ca2+/diacylglycerol (DAG)-dependent isoforms of PKC (PKC-βI and PKC-α), and MARCKS-GFP, but only in Ca2+-containing solutions. DCA had no effect on localization of a novel (PKCδ) or an atypical (PKCζ) 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.