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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

CID755673 and CRT0066101 selectively inhibit supramaximal dose CCK-induced PKD/PKD1 activation in pancreatic acinar cells. Isolated pancreatic acini were pre-incubated for 3 h with CID755673 (CID), CRT0066101 (CRT), or GF 109203X (GF1), followed by incubation with 100 nM CCK for 30 min. The acini were collected and lysed. (A) PKD/PKD1 autophosphorylation at its Ser916 (pS916 PKD1) was determined by Western blot analysis of the acinar lysates using anti-phospho-Ser916 PKD/PKD1 antibody, as described in Section “Experimental Procedures.” Blots were stripped and re-probed with PKD1 C-20 antibody for loading control. Shown are representative blots from at least three independent experiments. (B) PKD/PKD1 was immunoprecipitated from the acini lysate with PKD1 C-20 antibody and the PKD/PKD1 catalytic activity was measured with in vitro kinase assay as described in Section “Experimental Procedures.” Activity values were normalized by the control without CCK stimulation. Results are means ± SE (n = 3) for each condition. **P < 0.01 comparing the control versus CCK alone without inhibitor pretreatments, and comparing CID + CCK or CRT + CCK versus CCK alone, as indicated with dash lines. (C) Western blot analyses of the acini lysates with anti-phospho-Ser744/748 PKD or anti-phospho-Ser152/156-MARCKS antibodies. Blots were stripped and re-probed with GAPDH antibody for loading controls. (D) Western blot analyses of the acinar lysates with anti-phospho-Ser82 Hsp27 antibodies. Blots were stripped and re-probed with Hsp27 antibody for loading controls.
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Figure 1: CID755673 and CRT0066101 selectively inhibit supramaximal dose CCK-induced PKD/PKD1 activation in pancreatic acinar cells. Isolated pancreatic acini were pre-incubated for 3 h with CID755673 (CID), CRT0066101 (CRT), or GF 109203X (GF1), followed by incubation with 100 nM CCK for 30 min. The acini were collected and lysed. (A) PKD/PKD1 autophosphorylation at its Ser916 (pS916 PKD1) was determined by Western blot analysis of the acinar lysates using anti-phospho-Ser916 PKD/PKD1 antibody, as described in Section “Experimental Procedures.” Blots were stripped and re-probed with PKD1 C-20 antibody for loading control. Shown are representative blots from at least three independent experiments. (B) PKD/PKD1 was immunoprecipitated from the acini lysate with PKD1 C-20 antibody and the PKD/PKD1 catalytic activity was measured with in vitro kinase assay as described in Section “Experimental Procedures.” Activity values were normalized by the control without CCK stimulation. Results are means ± SE (n = 3) for each condition. **P < 0.01 comparing the control versus CCK alone without inhibitor pretreatments, and comparing CID + CCK or CRT + CCK versus CCK alone, as indicated with dash lines. (C) Western blot analyses of the acini lysates with anti-phospho-Ser744/748 PKD or anti-phospho-Ser152/156-MARCKS antibodies. Blots were stripped and re-probed with GAPDH antibody for loading controls. (D) Western blot analyses of the acinar lysates with anti-phospho-Ser82 Hsp27 antibodies. Blots were stripped and re-probed with Hsp27 antibody for loading controls.

Mentions: To demonstrate effects of these two PKD inhibitors in pancreas, we first examined their specificity for PKD inhibition in an in vitro experimental pancreatitis model of isolated pancreatic acini (Figure 1). Isolated pancreatic acini were pretreated with CID755673 or CRT0066101 and then stimulated with a supramaximal dose (100 nM) of the pancreatic secretagogue CCK which is known to cause cell pathologies of pancreatitis in vitro (Gukovskaya and Pandol, 2004). We applied 10 μM CRT0066101 and 25 μM CID755673 for maximal inhibition of PKD activation based on previous reports including our own measurements (Sharlow et al., 2008; Harikumar et al., 2010; Thrower et al., 2011).


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)

CID755673 and CRT0066101 selectively inhibit supramaximal dose CCK-induced PKD/PKD1 activation in pancreatic acinar cells. Isolated pancreatic acini were pre-incubated for 3 h with CID755673 (CID), CRT0066101 (CRT), or GF 109203X (GF1), followed by incubation with 100 nM CCK for 30 min. The acini were collected and lysed. (A) PKD/PKD1 autophosphorylation at its Ser916 (pS916 PKD1) was determined by Western blot analysis of the acinar lysates using anti-phospho-Ser916 PKD/PKD1 antibody, as described in Section “Experimental Procedures.” Blots were stripped and re-probed with PKD1 C-20 antibody for loading control. Shown are representative blots from at least three independent experiments. (B) PKD/PKD1 was immunoprecipitated from the acini lysate with PKD1 C-20 antibody and the PKD/PKD1 catalytic activity was measured with in vitro kinase assay as described in Section “Experimental Procedures.” Activity values were normalized by the control without CCK stimulation. Results are means ± SE (n = 3) for each condition. **P < 0.01 comparing the control versus CCK alone without inhibitor pretreatments, and comparing CID + CCK or CRT + CCK versus CCK alone, as indicated with dash lines. (C) Western blot analyses of the acini lysates with anti-phospho-Ser744/748 PKD or anti-phospho-Ser152/156-MARCKS antibodies. Blots were stripped and re-probed with GAPDH antibody for loading controls. (D) Western blot analyses of the acinar lysates with anti-phospho-Ser82 Hsp27 antibodies. Blots were stripped and re-probed with Hsp27 antibody for loading controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: CID755673 and CRT0066101 selectively inhibit supramaximal dose CCK-induced PKD/PKD1 activation in pancreatic acinar cells. Isolated pancreatic acini were pre-incubated for 3 h with CID755673 (CID), CRT0066101 (CRT), or GF 109203X (GF1), followed by incubation with 100 nM CCK for 30 min. The acini were collected and lysed. (A) PKD/PKD1 autophosphorylation at its Ser916 (pS916 PKD1) was determined by Western blot analysis of the acinar lysates using anti-phospho-Ser916 PKD/PKD1 antibody, as described in Section “Experimental Procedures.” Blots were stripped and re-probed with PKD1 C-20 antibody for loading control. Shown are representative blots from at least three independent experiments. (B) PKD/PKD1 was immunoprecipitated from the acini lysate with PKD1 C-20 antibody and the PKD/PKD1 catalytic activity was measured with in vitro kinase assay as described in Section “Experimental Procedures.” Activity values were normalized by the control without CCK stimulation. Results are means ± SE (n = 3) for each condition. **P < 0.01 comparing the control versus CCK alone without inhibitor pretreatments, and comparing CID + CCK or CRT + CCK versus CCK alone, as indicated with dash lines. (C) Western blot analyses of the acini lysates with anti-phospho-Ser744/748 PKD or anti-phospho-Ser152/156-MARCKS antibodies. Blots were stripped and re-probed with GAPDH antibody for loading controls. (D) Western blot analyses of the acinar lysates with anti-phospho-Ser82 Hsp27 antibodies. Blots were stripped and re-probed with Hsp27 antibody for loading controls.
Mentions: To demonstrate effects of these two PKD inhibitors in pancreas, we first examined their specificity for PKD inhibition in an in vitro experimental pancreatitis model of isolated pancreatic acini (Figure 1). Isolated pancreatic acini were pretreated with CID755673 or CRT0066101 and then stimulated with a supramaximal dose (100 nM) of the pancreatic secretagogue CCK which is known to cause cell pathologies of pancreatitis in vitro (Gukovskaya and Pandol, 2004). We applied 10 μM CRT0066101 and 25 μM CID755673 for maximal inhibition of PKD activation based on previous reports including our own measurements (Sharlow et al., 2008; Harikumar et al., 2010; Thrower et al., 2011).

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