Protein kinase D1 mediates NF-kappa B activation induced by cholecystokinin and cholinergic signaling in pancreatic acinar cells

The transcription factor NF-kappa B plays a critical role in inflammatory and cell death responses during acute pancreatitis. Previous studies in our laboratory demonstrated that protein kinase C (PKC) isoforms PKC delta and epsilon are key regulators of NF-kappa B activation induced by cholecystokinin-8 (CCK-8), tumor necrosis factor-alpha, and ethanol. However, the downstream participants in regulating NF-kappa B activation in exocrine pancreas remain poorly understood. Here, we demonstrate that protein kinase D1 (PKD1) is a key downstream target of PKC delta and PKC epsilon in pancreatic acinar cells stimulated by two major secretagogues, CCK-8 and the cholinergic agonist carbachol (CCh), and that PKD1 is necessary for NF-kappa B activation induced by CCK-8 and CCh. Both CCK-8 and CCh dose dependently induced a rapid and striking activation of PKD1 in rat pancreatic acinar cells, as measured by in vitro kinase assay and by phosphorylation at PKD1 activation loop (Ser744/748) or autophosphorylation site (Ser916). The phosphorylation and activation of PKD1 correlated with NF-kappa B activity stimulated by CCK-8 or CCh, as measured by NF-kappa B DNA binding. Either inhibition of PKC delta or epsilon by isoform-specific inhibitory peptides, genetic deletion of PKC delta and epsilon in pancreatic acinar cells, or knockdown of PKD1 by using small interfering RNAs in AR42J cells resulted in a marked decrease in PKD1 and NF-kappa B activation stimulated by CCK-8 or CCh. Conversely, overexpression of PKD1 resulted in augmentation of CCK-8 and CCh-stimulated NF-kappa B activation. Finally, the kinetics of PKD1 and NF-kappa B activation during cerulein-induced rat pancreatitis showed that both PKD1 and NF-kappa B activation were early events during acute pancreatitis and that their time courses of response were similar. Our results identify PKD1 as a novel early convergent point for PKC delta and epsilon in the signaling pathways mediating NF-kappa B activation in pancreatitis.