Regulation of Kupffer Cell Cytokine Gene Expression during Experimental Acute Pancreatitis: The Central Role of P38-MAPK, ERK1/2 and JNK
Abstracts
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Background: We have demonstrated that the pancreatic elastase-induced, and Kupffer cell-derived cytokine TNF, mediates liver injury during acute pancreatitis. Aim: To determine the role of P38 MAPK, ERK1/2 and JNK in the regulation of TNF gene expression within Kupffer cells. Methods: Rat livers (n=5) were perfused with elastase in situ for 3hr to mimic acute pancreatitis. TNF was measured in the effluent (ELISA) and TNF mRNA in liver tissue (RT-PCR). Rat Kupffer cells (KC) were cultured and treated with elastase (1U/ml) ? Gadolinium (0.5mg/ml) to inhibit KC cytokine production, SB203580 (1uM/ml) to inhibit P38 MAPK, or UO126 (1mM/ml) to inhibit ERK1/2. TNF protein, TNF mRNA, NF-kB activation (EMSA) and phosphorylated P38 MAPK, JNK and ERK1/2 (western blot) were determined at 0, 7, 15, 30, 60 and 120 min. Results: In vivo, elastase increased TNF (data not shown, p<0.03 vs sham) and upregulated TNF mRNA (TNF/BMG: 0.22?0.03 vs 0.06?0.03, p<0.01 vs sham). In vitro, elastase induced TNF production (1447?71 vs 30?2pg/ml, p<0.001 vs control), and upregulation of TNF mRNA within KC (TNF/BMG: 0.9?0.01 vs 0.02?0.01, p<0.001 vs control). Elastase-induced activation of NF-kB and phosphorylation of JNK, ERK1/2 and P38 MAPK were detected as early as 7 min. Activation of NF-kB peaked at 15 min and returned to baseline by 60 min. Phosphorylated JNK peaked at 15 min, followed by P38 MAPK at 15-30 min and ERK1/2 at 30 min; all three kinases remained activated at 60 min. TNF mRNA was upregulated by 15 min but peaked at 60 min; whereas, TNF protein production peaked at 120 min. Gadolinium inhibited elastase-induced upregulation of TNF mRNA (TNF/BMG: 0.04 ? 0.02 vs. 0.3 ? 0.03, p<0.001 vs elastase), TNF protein production (42?8 vs 1447?71pg/ml, p<0.001 vs elastase) and significantly reduced phosphorylated JNK and ERK1/2 but not P38 MAPK. UO126 inhibited both elastase-induced TNF production (212?34 vs 695?22pg/ml p<0.001, vs elastase) and activation of NF-kB; whereas, SB203580 inhibited TNF production (195?11 vs 695?22pg/ml, p<0.001 vs elastsae) but not NF-kB activation. Conclusion: Intrahepatic TNF gene expression within Kupffer cells follows an orderly and temporally related activation of cell signaling pathways. Inhibition of P38 MAPK and ERK1/2 attenuated elastase-induced upregulation of TNF gene expression through different pathways that do not converge on NF-kB. Nevertheless, the limited duration of NF-kB activation may be an important step in "switching off" the cytokine cascade. |
