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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

Effects of genetic down- or up-regulation of PKD/PKD1 expression on protein levels of IAPs and RIP1. AR42J cells were transfected with pooled four PKD1 siRNA duplexes or pcDNA3-PKD1 vector. Control cells received non-target (NT) siRNAs or empty vector (pcDNA3). Three days after transfection, the cells were incubated in serum-free F-12K medium for 1 h and then incubated for 5 h with or without 100 nM CCK. (A,B) Western blot analysis of the transfected AR42J cell lysates using antibodies against PKD1 C-20, XIAP, survivin, and FLIP to show effects of down- or up-regulation of PKD/PKD1 expression on the protein levels of these IAPs. Blots were re-probed for GAPDH to confirm equal protein loading. Shown are representative blots from at least three independent experiments. (C,D) Western blot analysis of the cell lysates using RIP1 antibody to detect the effect of up-regulation or knockdown of PKD/PKD1 expression on RIP1 expression and cleavage. The membranes were reblotted with GAPDH antibody to verify equal loading. Shown are representative blots from three independent experiments.
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Figure 8: Effects of genetic down- or up-regulation of PKD/PKD1 expression on protein levels of IAPs and RIP1. AR42J cells were transfected with pooled four PKD1 siRNA duplexes or pcDNA3-PKD1 vector. Control cells received non-target (NT) siRNAs or empty vector (pcDNA3). Three days after transfection, the cells were incubated in serum-free F-12K medium for 1 h and then incubated for 5 h with or without 100 nM CCK. (A,B) Western blot analysis of the transfected AR42J cell lysates using antibodies against PKD1 C-20, XIAP, survivin, and FLIP to show effects of down- or up-regulation of PKD/PKD1 expression on the protein levels of these IAPs. Blots were re-probed for GAPDH to confirm equal protein loading. Shown are representative blots from at least three independent experiments. (C,D) Western blot analysis of the cell lysates using RIP1 antibody to detect the effect of up-regulation or knockdown of PKD/PKD1 expression on RIP1 expression and cleavage. The membranes were reblotted with GAPDH antibody to verify equal loading. Shown are representative blots from three independent experiments.

Mentions: To provide insights into the mechanism of effects of PKD/PKD1 on IAPs and RIP1, we examined whether genetic regulation of PKD/PKD1 expression alters protein levels of IAPs and RIP1 in pancreatic acinar cells. We found that cells transfected with PKD1 siRNAs markedly down-regulated PKD/PKD1 expression, which resulted in dramatically decreased protein levels of XIAP, survivin, and FILPs (Figure 8A). Conversely, overexpression of PKD/PKD1 enhanced the levels of these IAPs (Figure 8B). Furthermore, up-regulation of PKD1 expression promoted RIP1 production and inhibited CCK-induced RIP1 cleavage/inactivation (Figure 8C) whereas knockdown PKD1 promoted RIP1 cleavage/inactivation (Figure 8D). These findings confirmed that PKD/PKD1 regulates IAPs and RIP1 protein levels, as observed in cerulein-induced 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)

Effects of genetic down- or up-regulation of PKD/PKD1 expression on protein levels of IAPs and RIP1. AR42J cells were transfected with pooled four PKD1 siRNA duplexes or pcDNA3-PKD1 vector. Control cells received non-target (NT) siRNAs or empty vector (pcDNA3). Three days after transfection, the cells were incubated in serum-free F-12K medium for 1 h and then incubated for 5 h with or without 100 nM CCK. (A,B) Western blot analysis of the transfected AR42J cell lysates using antibodies against PKD1 C-20, XIAP, survivin, and FLIP to show effects of down- or up-regulation of PKD/PKD1 expression on the protein levels of these IAPs. Blots were re-probed for GAPDH to confirm equal protein loading. Shown are representative blots from at least three independent experiments. (C,D) Western blot analysis of the cell lysates using RIP1 antibody to detect the effect of up-regulation or knockdown of PKD/PKD1 expression on RIP1 expression and cleavage. The membranes were reblotted with GAPDH antibody to verify equal loading. Shown are representative blots from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Effects of genetic down- or up-regulation of PKD/PKD1 expression on protein levels of IAPs and RIP1. AR42J cells were transfected with pooled four PKD1 siRNA duplexes or pcDNA3-PKD1 vector. Control cells received non-target (NT) siRNAs or empty vector (pcDNA3). Three days after transfection, the cells were incubated in serum-free F-12K medium for 1 h and then incubated for 5 h with or without 100 nM CCK. (A,B) Western blot analysis of the transfected AR42J cell lysates using antibodies against PKD1 C-20, XIAP, survivin, and FLIP to show effects of down- or up-regulation of PKD/PKD1 expression on the protein levels of these IAPs. Blots were re-probed for GAPDH to confirm equal protein loading. Shown are representative blots from at least three independent experiments. (C,D) Western blot analysis of the cell lysates using RIP1 antibody to detect the effect of up-regulation or knockdown of PKD/PKD1 expression on RIP1 expression and cleavage. The membranes were reblotted with GAPDH antibody to verify equal loading. Shown are representative blots from three independent experiments.
Mentions: To provide insights into the mechanism of effects of PKD/PKD1 on IAPs and RIP1, we examined whether genetic regulation of PKD/PKD1 expression alters protein levels of IAPs and RIP1 in pancreatic acinar cells. We found that cells transfected with PKD1 siRNAs markedly down-regulated PKD/PKD1 expression, which resulted in dramatically decreased protein levels of XIAP, survivin, and FILPs (Figure 8A). Conversely, overexpression of PKD/PKD1 enhanced the levels of these IAPs (Figure 8B). Furthermore, up-regulation of PKD1 expression promoted RIP1 production and inhibited CCK-induced RIP1 cleavage/inactivation (Figure 8C) whereas knockdown PKD1 promoted RIP1 cleavage/inactivation (Figure 8D). These findings confirmed that PKD/PKD1 regulates IAPs and RIP1 protein levels, as observed in cerulein-induced 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