The signaling pathways mediating lysophosphatidic acid (LPA)-stimulated PKD2 activation and the potential contribution of PKD2 in regulating LPA-induced interleukin-8 (IL-8) secretion in nontransformed, human colonic epithelial NCM460 cells were examined. Treatment of serum-deprived NCM460 cells with LPA led to a rapid and striking activation of PKD2, as measured by in vitro kinase assay and phosphorylation at the activation loop (Ser706/710) and autophosphorylation site (Ser876). PKD2 activation induced by LPA was abrogated by preincubation with selective protein kinase C (PKC) inhibitors GF-I and Ro 31-8220 in a dose-dependent manner. These inhibitors did not have any direct inhibitory effect on PKD2 activity. LPA induced a striking increase in IL-8 production and stimulated nuclear factor (NF)-B activation as measured by NF-B-DNA binding, NF-B driven luciferase reporter activity, and IB- phosphorylation. PKD2 gene silencing utilizing siRNAs targeting distinct PKD2 sequences dramatically reduced LPA-stimulated NF-B promoter activity and IL-8 production. PKD2 activation is a novel early event in the biological action of LPA and mediates LPA-stimulated IL-8 secretion in NCM460 cells through a NF-B-dependent pathway. Our results demonstrate, for the first time, the involvement of a member of the PKD family in the production of IL-8, a potent pro-inflammatory chemokine, by epithelial cells.