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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/PKD1 inhibition by CID755673 ameliorates necrosis and other pancreatitis parameters in cerulein-induced pancreatitis. Rats received intraperitoneal (IP) injection of CID755673 (CID, 15 mg/kg) or same volume of vehicle DMSO. At 60 min after the pretreatment, pancreatitis was induced by four hourly IP injections of cerulein (CR), as described in Section “Experimental Procedures.” Control rats received saline (S). The blood and pancreas were harvested in 4 h after the first injection of cerulein for the following measurements. (A) Serum amylase and lipase levels were measured. (B) H&E staining of pancreatic tissue sections from the experiments. Each image is representative of at least 6 rats with similar results at each condition. Bars represent 20 μm. (C) Necrosis was measured on H&E stained pancreatic tissue sections. Cells with swollen cytoplasm, loss of plasma membrane integrity, and leakage of organelles into interstitium were considered necrotic. For necrosis measurement and the quantifications of following other histological measurements, a total of at least 2000 acinar cells were counted on tissue sections from each animal and three to five animals each condition were counted. (D) Pancreatic edema grading was made from 1 to 3 on the H&E stained tissue sections with Schoenberg grading system as described in Section “Experimental Procedures.”(E,F) Number of vacuole and inflammatory cell infiltration were counted on these H&E stained pancreatic tissue sections and expressed as percentage of total acinar cells. Graphs (A,C–F) show means ± SE (n = 4) for each condition. **P < 0.01 or *P < 0.05 versus cerulein alone without inhibitor pretreatments as indicated.
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Figure 4: PKD/PKD1 inhibition by CID755673 ameliorates necrosis and other pancreatitis parameters in cerulein-induced pancreatitis. Rats received intraperitoneal (IP) injection of CID755673 (CID, 15 mg/kg) or same volume of vehicle DMSO. At 60 min after the pretreatment, pancreatitis was induced by four hourly IP injections of cerulein (CR), as described in Section “Experimental Procedures.” Control rats received saline (S). The blood and pancreas were harvested in 4 h after the first injection of cerulein for the following measurements. (A) Serum amylase and lipase levels were measured. (B) H&E staining of pancreatic tissue sections from the experiments. Each image is representative of at least 6 rats with similar results at each condition. Bars represent 20 μm. (C) Necrosis was measured on H&E stained pancreatic tissue sections. Cells with swollen cytoplasm, loss of plasma membrane integrity, and leakage of organelles into interstitium were considered necrotic. For necrosis measurement and the quantifications of following other histological measurements, a total of at least 2000 acinar cells were counted on tissue sections from each animal and three to five animals each condition were counted. (D) Pancreatic edema grading was made from 1 to 3 on the H&E stained tissue sections with Schoenberg grading system as described in Section “Experimental Procedures.”(E,F) Number of vacuole and inflammatory cell infiltration were counted on these H&E stained pancreatic tissue sections and expressed as percentage of total acinar cells. Graphs (A,C–F) show means ± SE (n = 4) for each condition. **P < 0.01 or *P < 0.05 versus cerulein alone without inhibitor pretreatments as indicated.

Mentions: Next, we evaluated the effect of PKD/PKD1 inhibition on the parameters of cerulein-induced pancreatitis. After 1 h pretreatment of CID755673 (15 mg/kg) or vehicle as described above, the rats received up to four hourly IP injections of cerulein or saline. The animals were sacrificed at 30 min and 4 h after the first cerulein injection. As illustrated in Figure 4A, cerulein caused striking increases of blood amylase and lipase after four hourly IP injections of cerulein, which were significantly attenuated in CID755673-treated rats. Furthermore, CID755673 treatment dramatically ameliorated the histological damage in cerulein-induced pancreatitis (Figure 4B). The salient feature of pancreas histological changes in CID755673-pretreated animals was that cerulein-induced severe acinar cell necrosis was strikingly attenuated and almost prevented (Figure 4C). Other histopathologic features of pancreatitis including interstitial edema, accumulation of cytoplasmic vacuoles, and inflammatory cell infiltration were also greatly attenuated by CID755673 treatment (Figures 4D–F). These results from the in vivo pancreatitis model demonstrated that PKD/PKD1 inhibition blocks necrosis and ameliorates severity of acute 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)

PKD/PKD1 inhibition by CID755673 ameliorates necrosis and other pancreatitis parameters in cerulein-induced pancreatitis. Rats received intraperitoneal (IP) injection of CID755673 (CID, 15 mg/kg) or same volume of vehicle DMSO. At 60 min after the pretreatment, pancreatitis was induced by four hourly IP injections of cerulein (CR), as described in Section “Experimental Procedures.” Control rats received saline (S). The blood and pancreas were harvested in 4 h after the first injection of cerulein for the following measurements. (A) Serum amylase and lipase levels were measured. (B) H&E staining of pancreatic tissue sections from the experiments. Each image is representative of at least 6 rats with similar results at each condition. Bars represent 20 μm. (C) Necrosis was measured on H&E stained pancreatic tissue sections. Cells with swollen cytoplasm, loss of plasma membrane integrity, and leakage of organelles into interstitium were considered necrotic. For necrosis measurement and the quantifications of following other histological measurements, a total of at least 2000 acinar cells were counted on tissue sections from each animal and three to five animals each condition were counted. (D) Pancreatic edema grading was made from 1 to 3 on the H&E stained tissue sections with Schoenberg grading system as described in Section “Experimental Procedures.”(E,F) Number of vacuole and inflammatory cell infiltration were counted on these H&E stained pancreatic tissue sections and expressed as percentage of total acinar cells. Graphs (A,C–F) show means ± SE (n = 4) for each condition. **P < 0.01 or *P < 0.05 versus cerulein alone without inhibitor pretreatments as indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: PKD/PKD1 inhibition by CID755673 ameliorates necrosis and other pancreatitis parameters in cerulein-induced pancreatitis. Rats received intraperitoneal (IP) injection of CID755673 (CID, 15 mg/kg) or same volume of vehicle DMSO. At 60 min after the pretreatment, pancreatitis was induced by four hourly IP injections of cerulein (CR), as described in Section “Experimental Procedures.” Control rats received saline (S). The blood and pancreas were harvested in 4 h after the first injection of cerulein for the following measurements. (A) Serum amylase and lipase levels were measured. (B) H&E staining of pancreatic tissue sections from the experiments. Each image is representative of at least 6 rats with similar results at each condition. Bars represent 20 μm. (C) Necrosis was measured on H&E stained pancreatic tissue sections. Cells with swollen cytoplasm, loss of plasma membrane integrity, and leakage of organelles into interstitium were considered necrotic. For necrosis measurement and the quantifications of following other histological measurements, a total of at least 2000 acinar cells were counted on tissue sections from each animal and three to five animals each condition were counted. (D) Pancreatic edema grading was made from 1 to 3 on the H&E stained tissue sections with Schoenberg grading system as described in Section “Experimental Procedures.”(E,F) Number of vacuole and inflammatory cell infiltration were counted on these H&E stained pancreatic tissue sections and expressed as percentage of total acinar cells. Graphs (A,C–F) show means ± SE (n = 4) for each condition. **P < 0.01 or *P < 0.05 versus cerulein alone without inhibitor pretreatments as indicated.
Mentions: Next, we evaluated the effect of PKD/PKD1 inhibition on the parameters of cerulein-induced pancreatitis. After 1 h pretreatment of CID755673 (15 mg/kg) or vehicle as described above, the rats received up to four hourly IP injections of cerulein or saline. The animals were sacrificed at 30 min and 4 h after the first cerulein injection. As illustrated in Figure 4A, cerulein caused striking increases of blood amylase and lipase after four hourly IP injections of cerulein, which were significantly attenuated in CID755673-treated rats. Furthermore, CID755673 treatment dramatically ameliorated the histological damage in cerulein-induced pancreatitis (Figure 4B). The salient feature of pancreas histological changes in CID755673-pretreated animals was that cerulein-induced severe acinar cell necrosis was strikingly attenuated and almost prevented (Figure 4C). Other histopathologic features of pancreatitis including interstitial edema, accumulation of cytoplasmic vacuoles, and inflammatory cell infiltration were also greatly attenuated by CID755673 treatment (Figures 4D–F). These results from the in vivo pancreatitis model demonstrated that PKD/PKD1 inhibition blocks necrosis and ameliorates severity of acute 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