Tumor necrosis factor-alpha mediates pancreatitis responses in acinar cells via protein kinase C and proline-rich tyrosine kinase 2.
OBJECTIVE
Although tumor necrosis factor alpha is implicated as an important mediator of the inflammatory response in acute pancreatitis, its role in other pathologic features of the disease remains unknown. We investigated the role for tumor necrosis factor alpha in cytoskeletal responses and the underlying signaling mechanisms in pancreatic acinar cells.


METHODS
In isolated rat pancreatic acini and AR42J cells, we determined the effect of tumor necrosis factor alpha on the actin cytoskeleton by rhodamine-phalloidin. Using pharmacological and molecular approaches, we assessed the involvement of protein kinase C, Src kinases, and proline-rich tyrosine kinase 2 in the process. We also studied the involvement of these signaling pathways in tumor necrosis factor-alpha-induced nuclear factor-kappaB activation and apoptosis.


RESULTS
Tumor necrosis factor-alpha increased the tyrosine phosphorylation of proline-rich tyrosine kinase 2 in acinar cells. The broad-spectrum protein kinase C inhibitor and the Src kinase inhibitor both inhibited tumor necrosis factor-alpha-induced proline-rich tyrosine kinase 2 phosphorylation, but at different tyrosine residues. Using protein kinase C isoform-specific inhibitors and the antisense approach, we showed that protein kinase C delta and mediate proline-rich tyrosine kinase 2 tyrosine phosphorylation. Tumor necrosis factor-alpha caused disorganization of the actin cytoskeleton by a mechanism dependent on protein kinase C, Src kinases, and proline-rich tyrosine kinase 2. Inhibition of protein kinase C, but not Src kinases, decreased tumor necrosis factor-alpha-induced apoptosis. Furthermore, with antisense transfections, we showed that protein kinase C delta and , but not proline-rich tyrosine kinase 2, mediate tumor necrosis factor alpha-induced nuclear factor-kappaB activation.


CONCLUSIONS
Tumor necrosis factor-alpha activates proline-rich tyrosine kinase 2 to cause cytoskeletal disorganization and nuclear factor-kappaB to cause inflammatory response, and it triggers cell death signaling through divergent mechanisms mediated by protein kinase C. The results provide insights into the mechanisms in pancreatic acinar cells that link tumor necrosis factor alpha to critical processes in acute pancreatitis.
Satoh
Akihiko
A
Veterans Affairs Greater Los Angeles Health Care System and University of California, 90073, USA.
Gukovskaya
Anna S
AS
Edderkaoui
Mouad
M
Daghighian
Melissa S
MS
Reeve
Joseph R
JR
Shimosegawa
Tooru
T
Pandol
Stephen J
SJ
D000208:Acute Disease; D000704:Analysis of Variance; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D004195:Disease Models, Animal; D005260:Female; D051418:Focal Adhesion Kinase 2; D008297:Male; D008969:Molecular Sequence Data; D010179:Pancreas / Q000166:cytology*; D010195:Pancreatitis / Q000188:drug therapy* / Q000201:enzymology*; D016133:Polymerase Chain Reaction / Q000379:methods; D011336:Probability; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism*; D051745:Protein Kinase C-delta; D011505:Protein-Tyrosine Kinases / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D014409:Tumor Necrosis Factor-alpha; C497260:Prkcd protein, rat; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; C496054:Ptk2b protein, rat; D011493:Protein Kinase C; D051745:Protein Kinase C-delta