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Protein kinase d regulates cell death pathways in experimental pancreatitis.

Yuan J, Liu Y, Tan T, Guha S, Gukovsky I, Gukovskaya A, Pandol SJ - Front Physiol (2012)

Bottom Line: 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.Conversely, up-regulation of PKD expression in pancreatic acinar cells increased necrosis and decreased apoptosis.PKD/PKD1 inhibition by CID755673 significantly ameliorated necrosis and severity of pancreatitis in an in vivo experimental model of acute pancreatitis.

View Article: PubMed Central - PubMed

Affiliation: South California Research Center for Alcoholic Liver and Pancreatic Diseases, Veterans Affairs Greater Los Angeles Healthcare System, University of California at Los Angeles Los Angeles, CA, USA.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Effect of CID755673 on anti-apoptotic proteins levels and degradation of RIP1 in cerulein-induced pancreatitis. Western blot analysis of pancreas tissue lysate using antibodies against XIAP (A), cFLIP (B), survivin (C), and RIP1 (D) to measure the protein levels of these proteins at early (30 min) and late (4 h) stages of cerulein (CR)-induced pancreatitis. Blots were re-probed for GAPDH to confirm equal protein loading. The bar figures are the quantification of these protein band intensities that were normalized on the band intensity in control animals injected with saline (S). Graphs show means ± SE (n = 4) for each condition. *P < 0.05 versus cerulein alone (DMSO + CR) in first injection and fourth injection (#).
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Figure 7: Effect of CID755673 on anti-apoptotic proteins levels and degradation of RIP1 in cerulein-induced pancreatitis. Western blot analysis of pancreas tissue lysate using antibodies against XIAP (A), cFLIP (B), survivin (C), and RIP1 (D) to measure the protein levels of these proteins at early (30 min) and late (4 h) stages of cerulein (CR)-induced pancreatitis. Blots were re-probed for GAPDH to confirm equal protein loading. The bar figures are the quantification of these protein band intensities that were normalized on the band intensity in control animals injected with saline (S). Graphs show means ± SE (n = 4) for each condition. *P < 0.05 versus cerulein alone (DMSO + CR) in first injection and fourth injection (#).

Mentions: We first determined whether PKD/PKD1 regulates XIAP, the most potent among the eight mammalian IAPs (Stehlik et al., 1998; Zou et al., 2004) and an endogenous inhibitor of caspase-9 and -3, in in vivo experimental pancreatitis. Our previous report (Mareninova et al., 2006) showed that rate of XIAP degradation during cerulein-induced pancreatitis is a key determinant of the type of cell death and that pharmacological inhibition of XIAP ameliorated necrosis and severity of cerulein-induced pancreatitis. Here we found that PKD1 inhibition by CID755673 altered protein levels of XIAP in cerulein-induced pancreatitis (Figure 7A). Although both non-inhibitor-treated and inhibitor-treated groups exhibited XIAP degradation in pancreatitis after four hourly cerulein injections, of importance, XIAP in CID755673-treated rats was degraded earlier (in 30 min after first cerulein injection) and to a much greater extent than in non-inhibitor-treated animals, indicating that the PKD/PKD1 inhibition promotes degradation of XIAP in pancreatitis.


Protein kinase d regulates cell death pathways in experimental pancreatitis.

Yuan J, Liu Y, Tan T, Guha S, Gukovsky I, Gukovskaya A, Pandol SJ - Front Physiol (2012)

Effect of CID755673 on anti-apoptotic proteins levels and degradation of RIP1 in cerulein-induced pancreatitis. Western blot analysis of pancreas tissue lysate using antibodies against XIAP (A), cFLIP (B), survivin (C), and RIP1 (D) to measure the protein levels of these proteins at early (30 min) and late (4 h) stages of cerulein (CR)-induced pancreatitis. Blots were re-probed for GAPDH to confirm equal protein loading. The bar figures are the quantification of these protein band intensities that were normalized on the band intensity in control animals injected with saline (S). Graphs show means ± SE (n = 4) for each condition. *P < 0.05 versus cerulein alone (DMSO + CR) in first injection and fourth injection (#).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3313474&req=5

Figure 7: Effect of CID755673 on anti-apoptotic proteins levels and degradation of RIP1 in cerulein-induced pancreatitis. Western blot analysis of pancreas tissue lysate using antibodies against XIAP (A), cFLIP (B), survivin (C), and RIP1 (D) to measure the protein levels of these proteins at early (30 min) and late (4 h) stages of cerulein (CR)-induced pancreatitis. Blots were re-probed for GAPDH to confirm equal protein loading. The bar figures are the quantification of these protein band intensities that were normalized on the band intensity in control animals injected with saline (S). Graphs show means ± SE (n = 4) for each condition. *P < 0.05 versus cerulein alone (DMSO + CR) in first injection and fourth injection (#).
Mentions: We first determined whether PKD/PKD1 regulates XIAP, the most potent among the eight mammalian IAPs (Stehlik et al., 1998; Zou et al., 2004) and an endogenous inhibitor of caspase-9 and -3, in in vivo experimental pancreatitis. Our previous report (Mareninova et al., 2006) showed that rate of XIAP degradation during cerulein-induced pancreatitis is a key determinant of the type of cell death and that pharmacological inhibition of XIAP ameliorated necrosis and severity of cerulein-induced pancreatitis. Here we found that PKD1 inhibition by CID755673 altered protein levels of XIAP in cerulein-induced pancreatitis (Figure 7A). Although both non-inhibitor-treated and inhibitor-treated groups exhibited XIAP degradation in pancreatitis after four hourly cerulein injections, of importance, XIAP in CID755673-treated rats was degraded earlier (in 30 min after first cerulein injection) and to a much greater extent than in non-inhibitor-treated animals, indicating that the PKD/PKD1 inhibition promotes degradation of XIAP in pancreatitis.

Bottom Line: 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.Conversely, up-regulation of PKD expression in pancreatic acinar cells increased necrosis and decreased apoptosis.PKD/PKD1 inhibition by CID755673 significantly ameliorated necrosis and severity of pancreatitis in an in vivo experimental model of acute pancreatitis.

View Article: PubMed Central - PubMed

Affiliation: South California Research Center for Alcoholic Liver and Pancreatic Diseases, Veterans Affairs Greater Los Angeles Healthcare System, University of California at Los Angeles Los Angeles, CA, USA.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus