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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

PKD inhibitors CID755673 and CRT0066101 decrease necrosis and enhance apoptosis in pancreatic acini induced by supramaximal dose CCK. Rat pancreatic acini were pre-incubated for 1 h with CID755673 (CID, 25 μM) or CRT0066101 (CRT, 10 μM), followed by incubation with 100 nM CCK for 4 h. (A) Acinar cell necrosis was measured by the percentage of total cellular LDH released into the medium, as described in Section “Experimental Procedures.”(B) Apoptosis was measured by the percentage of TUNEL staining positive cells. For each condition, at least 3000 cells were counted. (C) Caspase activities were measured as described in Section “Experimental Procedures” in the lysates of isolated pancreatic acini. Graphs (A–C) show mean ± SE (n = 3–5) for each condition. **P < 0.01 comparing the control versus CCK alone without inhibitor pretreatments as indicated with dash lines; *P < 0.05 comparing CID + CCK or CRT + CCK versus CCK alone without inhibitor.
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Figure 2: PKD inhibitors CID755673 and CRT0066101 decrease necrosis and enhance apoptosis in pancreatic acini induced by supramaximal dose CCK. Rat pancreatic acini were pre-incubated for 1 h with CID755673 (CID, 25 μM) or CRT0066101 (CRT, 10 μM), followed by incubation with 100 nM CCK for 4 h. (A) Acinar cell necrosis was measured by the percentage of total cellular LDH released into the medium, as described in Section “Experimental Procedures.”(B) Apoptosis was measured by the percentage of TUNEL staining positive cells. For each condition, at least 3000 cells were counted. (C) Caspase activities were measured as described in Section “Experimental Procedures” in the lysates of isolated pancreatic acini. Graphs (A–C) show mean ± SE (n = 3–5) for each condition. **P < 0.01 comparing the control versus CCK alone without inhibitor pretreatments as indicated with dash lines; *P < 0.05 comparing CID + CCK or CRT + CCK versus CCK alone without inhibitor.

Mentions: We next investigated the role of PKD/PKD1 in death pathways by examining the effect of the two PKD inhibitors on cell necrosis and apoptosis death pathways in pancreatic acinar cells stimulated with a supramaximal dose of CCK, which serves as an in vitro experimental pancreatitis model (Figure 2). Isolated pancreatic acini were pretreated with CID755673 or CRT0066101 for 1 h and then further incubated with 100 nM CCK for 4 h.


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)

PKD inhibitors CID755673 and CRT0066101 decrease necrosis and enhance apoptosis in pancreatic acini induced by supramaximal dose CCK. Rat pancreatic acini were pre-incubated for 1 h with CID755673 (CID, 25 μM) or CRT0066101 (CRT, 10 μM), followed by incubation with 100 nM CCK for 4 h. (A) Acinar cell necrosis was measured by the percentage of total cellular LDH released into the medium, as described in Section “Experimental Procedures.”(B) Apoptosis was measured by the percentage of TUNEL staining positive cells. For each condition, at least 3000 cells were counted. (C) Caspase activities were measured as described in Section “Experimental Procedures” in the lysates of isolated pancreatic acini. Graphs (A–C) show mean ± SE (n = 3–5) for each condition. **P < 0.01 comparing the control versus CCK alone without inhibitor pretreatments as indicated with dash lines; *P < 0.05 comparing CID + CCK or CRT + CCK versus CCK alone without inhibitor.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: PKD inhibitors CID755673 and CRT0066101 decrease necrosis and enhance apoptosis in pancreatic acini induced by supramaximal dose CCK. Rat pancreatic acini were pre-incubated for 1 h with CID755673 (CID, 25 μM) or CRT0066101 (CRT, 10 μM), followed by incubation with 100 nM CCK for 4 h. (A) Acinar cell necrosis was measured by the percentage of total cellular LDH released into the medium, as described in Section “Experimental Procedures.”(B) Apoptosis was measured by the percentage of TUNEL staining positive cells. For each condition, at least 3000 cells were counted. (C) Caspase activities were measured as described in Section “Experimental Procedures” in the lysates of isolated pancreatic acini. Graphs (A–C) show mean ± SE (n = 3–5) for each condition. **P < 0.01 comparing the control versus CCK alone without inhibitor pretreatments as indicated with dash lines; *P < 0.05 comparing CID + CCK or CRT + CCK versus CCK alone without inhibitor.
Mentions: We next investigated the role of PKD/PKD1 in death pathways by examining the effect of the two PKD inhibitors on cell necrosis and apoptosis death pathways in pancreatic acinar cells stimulated with a supramaximal dose of CCK, which serves as an in vitro experimental pancreatitis model (Figure 2). Isolated pancreatic acini were pretreated with CID755673 or CRT0066101 for 1 h and then further incubated with 100 nM CCK for 4 h.

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