ya Tan

Protein Kinase D Regulates Cell Death Pathways in Experimental Pancreatitis

Inflammation and acinar cell necrosis are two major pathological responses of acute pancreatitis, a serious disorder with no current therapies directed to its molecular pathogenesis. Serine/threonine protein kinase D family, which includes PKD/PKD1, PKD2, and PKD3, has been increasingly implicated in the regulation of multiple physiological and pathophysiological effects. We recently reported that PKD/PKD1, the predominant PKD isoform expressed in rat pancreatic acinar cells, mediates early events of pancreatitis including NF-κB activation and inappropriate intracellular digestive enzyme activation. In current studies, we investigated the role and mechanisms of PKD/PKD1 in the regulation of necrosis in pancreatic acinar cells by using two novel small molecule PKD inhibitors CID755673 and CRT0066101 and molecular approaches in in vitro and in vivo experimental models of acute pancreatitis. Our results demonstrated that both CID755673 and CRT0066101 are PKD-specific inhibitors and that PKD/PKD1 inhibition by either the chemical inhibitors or specific PKD/PKD1 siRNAs attenuated necrosis while promoting apoptosis induced by pathological doses of cholecystokinin-octapeptide (CCK) in pancreatic acinar cells. Conversely, up-regulation of PKD expression in pancreatic acinar cells increased necrosis and decreased apoptosis. We further showed that PKD/PKD1 regulated several key cell death signals including inhibitors of apoptotic proteins, caspases, receptor-interacting protein kinase 1 to promote necrosis. PKD/PKD1 inhibition by CID755673 significantly ameliorated necrosis and severity of pancreatitis in an in vivo experimental model of acute pancreatitis. Thus, our studies indicate that PKD/PKD1 is a key mediator of necrosis in acute pancreatitis and that PKD/PKD1 may represent a potential therapeutic target in acute pancreatitis.

Genetic inhibition of protein kinase Cε attenuates necrosis in experimental pancreatitis

Understanding the regulation of death pathways, necrosis and apoptosis, in pancreatitis is important for developing therapies directed to the molecular pathogenesis of the disease. Protein kinase Cε (PKCε) has been previously shown to regulate inflammatory responses and zymogen activation in pancreatitis. Furthermore, we demonstrated that ethanol specifically activated PKCε in pancreatic acinar cells and that PKCε mediated the sensitizing effects of ethanol on inflammatory response in pancreatitis. Here we investigated the role of PKCε in the regulation of death pathways in pancreatitis. We found that genetic deletion of PKCε resulted in decreased necrosis and severity in the in vivo cerulein-induced pancreatitis and that inhibition of PKCε protected the acinar cells from CCK-8 hyperstimulation-induced necrosis and ATP reduction. These findings were associated with upregulation of mitochondrial Bak and Bcl-2/Bcl-xL, proapoptotic and prosurvival members in the Bcl-2 family, respectively, as well as increased mitochondrial cytochrome c release, caspase activation, and apoptosis in pancreatitis in PKCε knockout mice. We further confirmed that cerulein pancreatitis induced a dramatic mitochondrial translocation of PKCε, suggesting that PKCε regulated necrosis in pancreatitis via mechanisms involving mitochondria. Finally, we showed that PKCε deletion downregulated inhibitors of apoptosis proteins, c-IAP2, survivin, and c-FLIPs while promoting cleavage/inactivation of receptor-interacting protein kinase (RIP). Taken together, our findings provide evidence that PKCε activation during pancreatitis promotes necrosis through mechanisms involving mitochondrial proapoptotic and prosurvival Bcl-2 family proteins and upregulation of nonmitochondrial pathways that inhibit caspase activation and RIP cleavage/inactivation. Thus PKCε is a potential target for prevention and/or treatment of acute pancreatitis.

Novel Small Molecule Inhibitors of Protein Kinase D Suppress NF-kappaB Activation and Attenuate the Severity of Rat Cerulein Pancreatitis

Nuclear factor-kappa B (NF-κB) activation is a key early signal regulating inflammatory and cell death responses in acute pancreatitis. Our previous in vitro studies with molecular approaches on AR42J cell showed that protein kinase D (PKD/PKD1) activation was required in NF-κB activation induced by cholecystokinin 8 (CCK) or carbachol (CCh) in pancreatic acinar cells. Recently developed small molecule PKD inhibitors, CID755673 and CRT0066101, provide potentially important pharmacological approaches to further investigate the effect of PKD in pancreatitis therapy. The aim of this study was to explore whether CID755673 and CRT0066101 block NF-κB activation with in vitro and in vivo models of experimental pancreatitis and whether the small molecule PKD inhibitors have therapeutic effects when given before or after the initiation of experimental pancreatitis. Freshly prepared pancreatic acini were incubated with CID755673 or CRT006101, followed by hyperstimulation with CCK or CCh. For in vivo experimental pancreatitis, rats were treated with intraperitoneal injection of CID755673 or CRT0066101 prior to or after administering cerulein or saline. PKD activation and NF-κB-DNA binding activity in nuclear extracts from pancreatic acini and tissue were measured. The effects of PKD inhibitors on pancreatitis responses were evaluated. Our results showed that both CID755673 or CRT0066101 selectively and specifically inhibited PKD without effects on related protein kinase Cs. Inhibition of PKD resulted in significantly attenuation of NF-κB activation in both in vitro and in vivo models of experimental pancreatitis. NF-κB inhibition by CID755673 was associated with decreased inflammatory responses and attenuated severity of the disease, which were indicated by less inflammatory cell infiltration, reduced pancreatic interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), decreased intrapancreatic trypsin activation, and alleviation in pancreatic necrosis, edema and vacuolization. Furthermore, PKD inhibitor CID755673, given after the initiation of pancreatitis in experimental rat model, significantly attenuated the severity of acute pancreatitis. Therapies for acute pancreatitis are limited. Our results indicate that small chemical PKD inhibitors have significant potential as therapeutic interventions by suppressing NF-κB activation.

Su1892 Protein Kinase C Epsilon Promotes Necrosis During Experimental Pancreatitis by Upregulation of Expression of Inhibitors of Apoptosis Proteins and Stabilization of Receptor-Interacting Protein Kinase

infiltration, and interstitial edema were all reduced in cerulein-treated PKD3Δpanc mice compared to controls. Conclusion: PKD3Δpanc mice displayed significant attenuation in inflammation, acinar cell necrosis, and severity of pancreatitis. These novel findings indicate that this animal model can be used to demonstrate the role of PKD in the pathobiologic responses in the pancreatic acinar cell during pancreatitis.

S1819 PKCɛ: A Critical Mediator of Mitochondria-Dependent Cell Death in Acute Pancreatitis

properties of colon specific DRG neurons. Results: We found that the visceromoter response to CRD in 6-to-8 week old female rats was significantly greater than that in age-matched male Sprague-Dawley rats (p<0.05 at 30, 40 and 50 mmHg). Patch clamp recordings showed significant differences between the colon-specific DRG neurons from female and male rats. The membrane input resistance in female DRG neurons was significantly lower than that in male DRG neurons (195±27 vs 424±54 Mohm, p<0.05); action potential duration and action potential threshold of DRG neurons were significantly greater in females than those in males (6.0±0.7 vs 3.6±0.5 msec, and -15.8±1.3 vs -22.3±1.4 mV, respectively, p<0.05). We obtained colon-specific DRG neurons 24-hours after daily treatment of male rats with i.m. progesterone (10mg/kg) for seven-days. Progesterone treatment significantly depolarized the resting membrane potential (-59±1 vs -51±1mV, p<0.05), increased input resistance (424±54 vs 872±88Mohm, p<0.05), decreased rheobase (0.42±0.05 vs 0.09±0.01 nA, p<0.05), increased action potential duration (3.6±0.5 vs 5.2±0.3msec, p<0.05) and decreased action potential threshold (-22.3±1.4 vs -26.6±1.0 mV, p<0.05). All these changes in the active and passivemembrane properties of DRGneurons indicate an increase in the excitability of DRG neurons. In support of this, we found that the visceromoter response to CRD was significantly greater in male rats 24-hours after 7-days of daily treatment with PG, when compared with baseline (p<0.05). However, the visceromoter response returned to normal range 7-days after the end of progesterone treatment. Conclusions: We conclude that the baseline visceromoter response to colorectal distension in female rats is greater than that in age-matched male rats. Daily treatment of male rats with exogenous PG sensitizes the DRG neurons and enhances the visceromoter response. The female sex hormone progesterone may be one of the factors that promote visceral hypersensitivity in females predisposing them to a higher incidence of visceral pain.