Yuan Jingzhen, Lugea Aurelia, Zheng Ling, Gukovsky Ilya, Edderkaoui Mouad, Rozengurt Enrique, Pandol Stephen J
Veterans Affairs Greater Los Angeles Healthcare System, West Los Angeles VA Healthcare Center, Los Angeles, CA 90073, USA.
Am J Physiol Gastrointest Liver Physiol. 2008 Dec;295(6):G1190-201. doi: 10.1152/ajpgi.90452.2008. Epub 2008 Oct 9.
The transcription factor NF-kappaB 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 PKCdelta and epsilon are key regulators of NF-kappaB activation induced by cholecystokinin-8 (CCK-8), tumor necrosis factor-alpha, and ethanol. However, the downstream participants in regulating NF-kappaB activation in exocrine pancreas remain poorly understood. Here, we demonstrate that protein kinase D1 (PKD1) is a key downstream target of PKCdelta and PKCepsilon in pancreatic acinar cells stimulated by two major secretagogues, CCK-8 and the cholinergic agonist carbachol (CCh), and that PKD1 is necessary for NF-kappaB 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-kappaB activity stimulated by CCK-8 or CCh, as measured by NF-kappaB DNA binding. Either inhibition of PKCdelta or epsilon by isoform-specific inhibitory peptides, genetic deletion of PKCdelta 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-kappaB activation stimulated by CCK-8 or CCh. Conversely, overexpression of PKD1 resulted in augmentation of CCK-8- and CCh-stimulated NF-kappaB activation. Finally, the kinetics of PKD1 and NF-kappaB activation during cerulein-induced rat pancreatitis showed that both PKD1 and NF-kappaB 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 PKCdelta and epsilon in the signaling pathways mediating NF-kappaB activation in pancreatitis.
转录因子NF-κB在急性胰腺炎的炎症和细胞死亡反应中起关键作用。我们实验室之前的研究表明，蛋白激酶C（PKC）同工型PKCδ和ε是由胆囊收缩素-8（CCK-8）、肿瘤坏死因子-α和乙醇诱导的NF-κB激活的关键调节因子。然而，外分泌胰腺中调节NF-κB激活的下游参与者仍知之甚少。在此，我们证明蛋白激酶D1（PKD1）是PKCδ和PKCε在由两种主要促分泌剂CCK-8和胆碱能激动剂卡巴胆碱（CCh）刺激的胰腺腺泡细胞中的关键下游靶点，并且PKD1是CCK-8和CCh诱导的NF-κB激活所必需的。通过体外激酶测定以及PKD1激活环（Ser744/748）或自磷酸化位点（Ser916）的磷酸化检测发现，CCK-8和CCh均剂量依赖性地诱导大鼠胰腺腺泡细胞中PKD1的快速且显著激活。PKD1的磷酸化和激活与CCK-8或CCh刺激的NF-κB活性相关，通过NF-κB DNA结合来测定。通过同工型特异性抑制肽抑制PKCδ或ε、胰腺腺泡细胞中PKCδ和ε的基因缺失，或在AR42J细胞中使用小干扰RNA敲低PKD1，均导致CCK-8或CCh刺激的PKD1和NF-κB激活显著降低。相反，PKD1的过表达导致CCK-8和CCh刺激的NF-κB激活增强。最后，在雨蛙肽诱导的大鼠胰腺炎期间PKD1和NF-κB激活的动力学表明，PKD1和NF-κB激活都是急性胰腺炎期间的早期事件，并且它们的反应时间进程相似。我们的结果确定PKD1是PKCδ和ε在介导胰腺炎中NF-κB激活的信号通路中的一个新的早期汇聚点。
