Volume 129, Issue 2, Pages 639-651 (August 2005) Tumor Necrosis Factor-α Mediates Pancreatitis Responses in Acinar Cells via Protein Kinase C and Proline-Rich Tyrosine Kinase 2  Akihiko Satoh, Anna S. Gukovskaya, Mouad Edderkaoui, Melissa S. Daghighian, Joseph R. Reeve, Tooru Shimosegawa, Stephen J. Pandol  Gastroenterology  Volume 129, Issue 2, Pages 639-651 (August 2005) DOI: 10.1053/j.gastro.2005.05.005 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 TNF-α induces tyrosine phosphorylation of Pyk2 in the pancreatic acinar cell. Isolated rat pancreatic acini were stimulated with 100 ng/mL TNF-α for the indicated times (A) or with the indicated concentrations of TNF-α for 5 minutes (B). Whole-cell lysates were immunoprecipitated (IP) with anti-phosphotyrosine monoclonal antibody (pTyr) and examined by Western blot analysis (WB) by using anti-Pyk2/CAKβ monoclonal antibody (Pyk2). The upper panels show representative Western blots. The intensity of the band was quantified by densitometry, and the values were normalized to Pyk2 basal levels in unstimulated acini (graphs). Values are expressed as means ± SE (n = 4–5). *P < .05 compared with basal levels in unstimulated acini. Gastroenterology 2005 129, 639-651DOI: (10.1053/j.gastro.2005.05.005) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Site-specific phosphorylation of Pyk2 induced by TNF-α. (A) Pancreatic acini were stimulated with 100 ng/mL TNF-α for the indicated times. Whole-cell lysates were examined by Western blot analysis by using Pyk2 phosphorylation site-specific antibodies against pY402, pY580, and pY881 and the anti-Pyk2/CAKβ monoclonal antibody (Pyk2). The upper panels show representative Western blots. The graphs show the kinetics of site-specific phosphorylation of Pyk2. Values are normalized to the basal phosphorylation levels at each site in unstimulated acini and are expressed as means ± SE (n = 5). *P < .05 compared with each basal level in unstimulated acini. (B) AR42J cells were stimulated with 100 ng/mL TNF-α for 5 minutes, and whole-cell lysates were examined by Western blot analysis. Shown are representative blots from 3 independent experiments. Gastroenterology 2005 129, 639-651DOI: (10.1053/j.gastro.2005.05.005) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 PKC and Src kinase inhibitors block TNF-α-induced Pyk2 phosphorylation at specific sites. Pancreatic acini were preincubated for 3 hours with the broad-spectrum PKC inhibitor GF109203X (10 μmol/L) or the Src kinase inhibitor PP2 (20 μmol/L) and then stimulated with 100 ng/mL TNF-α. (A) After 5 minutes of stimulation, whole-cell lysates were immunoprecipitated with anti-phosphotyrosine monoclonal antibody (pTyr) and examined by Western blot analysis by using anti-Pyk2/CAKβ monoclonal antibody (Pyk2). The left panel shows a representative Western blot. Graphs on the right show densitometric quantification of the band intensity for tyrosine-phosphorylated Pyk2. Values are expressed as means ± SE (n = 3). The response to TNF-α without the inhibitors was considered as 100%. *P < .05 compared with TNF-α alone. (B) Pancreatic acini were pretreated with GF109203X or PP2 and then stimulated with TNF-α for the indicated times. Whole-cell lysates were examined by Western blot analysis for Pyk2 phosphorylation by using site-specific antibodies against pY402, pY580, and pY881 and the anti-Pyk2/CAKβ monoclonal antibody (Pyk2). Left panels show representative Western blots. Graphs on the right show the densitometric quantification of Pyk2 phosphorylation at each site. Values are normalized to the basal phosphorylation level at each site in unstimulated acini and are expressed as means ± SE (n = 5). (C) The graphs show the increase in phosphorylation of each residue at the maximal response to TNF-α. The responses to TNF-α without inhibitors were considered as 100%. *P < .01 compared with TNF-α alone. Gastroenterology 2005 129, 639-651DOI: (10.1053/j.gastro.2005.05.005) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Effects of isoform-specific PKC inhibitors on Pyk2 phosphorylation at Y402, Y580, and Y881. Pancreatic acini were preincubated for 3 hours with 10 μmol/L each of PKC-δ translocation inhibitor (δV1-1), PKC-ϵ translocation inhibitor (ϵV1-2), or PKC-ζ inhibitor (ζ pseudosubstrate) and then stimulated with 100 ng/mL TNF-α. Whole-cell lysates were examined by Western blot analysis by using Pyk2 phosphorylation site-specific antibodies against pY402, pY580, and pY881 and the anti-Pyk2/CAKβ monoclonal antibody (Pyk2). (A) Representative Western blots. (B) Densitometric quantification of Pyk2 phosphorylation at Y402, Y580, and pY881. The responses to TNF-α without inhibitors were considered as 100%. *P < .01 compared with TNF-α alone. Gastroenterology 2005 129, 639-651DOI: (10.1053/j.gastro.2005.05.005) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 PKC-δ and -ϵ antisense oligonucleotides block Pyk2 phosphorylation at Y402 and Y580. AR42J cells were transfected with 10 μmol/L each of antisense (AS) or scrambled (SC) oligonucleotide specific for PKC-δ or PKC-ϵ. (A) Protein levels of PKC-δ and PKC-ϵ were examined in whole-cell lysates by Western blot analysis. (B) Transfected cells were stimulated with 100 ng/mL TNF-α for 5 minutes, and whole-cell lysates were examined by Western blot analysis with Pyk2 phosphorylation site-specific antibodies against pY402 and pY580 and the anti-Pyk2/CAKβ monoclonal antibody (Pyk2). Shown are representative blots from 3 independent experiments. Gastroenterology 2005 129, 639-651DOI: (10.1053/j.gastro.2005.05.005) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 TNF-α causes disorganization of the actin cytoskeleton in pancreatic acini. Pancreatic acini were incubated for 30 minutes with vehicle (dimethyl sulfoxide) (A–D), the broad-spectrum PKC inhibitor GF109203X (10 μmol/L) (E), or the Src kinase inhibitor PP2 (20 μmol/L) (F) and stimulated with 100 ng/mL TNF-α for the indicated times. Cells were fixed and stained for actin filaments with rhodamine-phalloidin (original magnification, 100×). Arrows show the apical lumen, and arrowheads show the basal membranes. Shown are representative images from 3 independent experiments. (G) An area of the apical and basal membrane was delineated, and the mean fluorescence intensity was determined. Sixty cells from 3 independent experiments were analyzed per condition. Values represent the intensity in the basolateral area vs the apical area and are expressed as means ± SE. *P < .05 compared with the value in unstimulated acini; #P < .05 compared with TNF-α alone for 3 hours. Gastroenterology 2005 129, 639-651DOI: (10.1053/j.gastro.2005.05.005) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 Pyk2 mediates TNF-α-induced disorganization of the actin cytoskeleton in AR42J cells. AR42J cells were transfected with Pyk2 antisense (C and D) or scrambled (E and F) oligonucleotides. Cells were stimulated (B, D, and F) or not (A, C, and E) for 3 hours with 100 ng/mL TNF-α and were then fixed and stained for actin filaments with rhodamine-phalloidin (original magnification, 100×). Shown are representative images from 3 independent experiments. Gastroenterology 2005 129, 639-651DOI: (10.1053/j.gastro.2005.05.005) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 TNF-α-induced apoptosis in pancreatic acinar cells is mediated by caspases and PKC, but not Src kinases. Pancreatic acini were incubated for 30 minutes with vehicle (dimethyl sulfoxide), the broad-spectrum PKC inhibitor GF109203X (10 μmol/L), the Src kinase inhibitor PP2 (20 μmol/L), or the broad-spectrum caspase inhibitor zVAD-fmk (100 μmol/L) and stimulated with 100 ng/mL TNF-α for an additional 6 hours. (A) The morphology of apoptosis was evaluated by Hoechst 33258 staining. Shown is a representative image (original magnification, 100×). Arrows show a typical apoptotic cell with nuclei containing condensed and fragmented chromatin. (B) For each condition, at least 2000 cells from 3 independent experiments were counted. Values are expressed as means ± SE. *P < .01 compared with the value in unstimulated acini; #P < .05 compared with TNF-α alone for 3 hours. Gastroenterology 2005 129, 639-651DOI: (10.1053/j.gastro.2005.05.005) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 9 Caspases do not mediate TNF-α-induced actin cytoskeletal change. Pancreatic acini were incubated for 30 minutes with the broad-spectrum caspase inhibitor zVAD-fmk (100 μmol/L) and stimulated with and without 100 ng/mL TNF-α for 3 hours. Cells were fixed and stained for actin filaments with rhodamine-phalloidin. (A) Shown are representative images from 3 independent experiments (original magnification, 100×). (B) An area of the apical and basal membrane was delineated, and the mean fluorescence intensity was determined. Sixty cells from 3 independent experiments were analyzed per condition. Values represent the intensity in the basolateral area vs the apical area and are expressed as means ± SE. *P < .05 compared with the value in unstimulated acini. Gastroenterology 2005 129, 639-651DOI: (10.1053/j.gastro.2005.05.005) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 10 PKC-δ and -ϵ, but not Pyk2, mediate NF-κB activation induced by TNF-α in AR42J cells. AR42J cells were transfected with antisense (AS) or scrambled (SC) oligodeoxynucleotides to PKC-δ, PKC-ϵ, or Pyk2. (A) Cells were stimulated with 100 ng/mL TNF-α for 30 minutes, and NF-κB binding activity was measured in nuclear extracts by EMSA. Shown are representative examples of 3 independent experiments. (B) The total NF-κB band intensities were quantified in the PhosphorImager and normalized to NF-κB band intensity in untransfected cells and each transfection. Values are expressed as means ± SE. *P < .05 compared with untransfected cells. (C) Protein levels of Pyk2 were examined in whole-cell lysates by Western blot analysis by using anti-Pyk2/CAKβ monoclonal antibody (Pyk2). Numbers on the bottom show the intensity of the Pyk2 band quantified by densitometry and normalized on that in untransfected cells. Gastroenterology 2005 129, 639-651DOI: (10.1053/j.gastro.2005.05.005) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 11 TNF-α-induced signaling pathways involved in pathologic responses of the pancreatic acinar cell. Binding of TNF-α to its receptor in the pancreatic acinar cell activates PKC-δ and -ϵ. These PKC isoforms mediate the activation of both Pyk2 and NF-κB, the 2 diverging pathways that play key roles in, respectively, the cytoskeletal changes and the inflammatory responses of pancreatitis. PKC also involves the induction of apoptosis, probably by affecting caspase activation. Although activation of Pyk2 is also regulated by Src kinases, this pathway is not involved in the TNF-α-induced NF-κB activation and apoptosis in the pancreatic acinar cell. Gastroenterology 2005 129, 639-651DOI: (10.1053/j.gastro.2005.05.005) Copyright © 2005 American Gastroenterological Association Terms and Conditions