Dominant Negative p38 Mitogen-Activated Protein Kinase Expression Inhibits NF-κB Activation in AR42J Cells

Abstract

Background: The role of the p38 mitogen-activated protein (MAP) kinase in acute pancreatitis pathogenesis is controversial. We hypothesize that p38 plays a role in regulating NF-ĸB activation in exocrine pancreatic cells. Methods: AR42J cells incorporating an NF-ĸB-responsive luciferase reporter, with and without adenoviral transduction of DNp38, were stimulated with cholecystokinin (CCK) or tumor necrosis factor-α (TNF-α) prior to measuring NF-ĸB activation. Results: CCK- or TNF-α-stimulated NF-ĸB-dependent gene transcription (luciferase assay) was substantially subdued by DNp38 expression. These findings were confirmed by electrophoretic mobility shift assay. Nuclear translocation of the p65 NF-ĸB subunit following agonist stimulation was evident (supershift). Characterization studies showed excellent adenoviral infection efficiency and cell viability in our AR42J cell model. Agonist-stimulated dose- and time-dependent p38 activation, with inhibition by DNp38 expression, was also confirmed. Conclusion: The p38 MAP kinase regulates NF-ĸB pathway activation in exocrine pancreatic cells, and thus potentially plays a role in the mechanism of acute pancreatitis pathogenesis.

Deciphering fact from artifact when using reporter assays to investigate the roles of host factors on L1 retrotransposition

“…Thus, a pool of excess, unactivated p38δ-WT could perturb this regulatory system, or may simply compete with the population of endogenous activated p38, resulting in inhibition of L1. Consistent with this possibility are several studies that showed expression of a nonfunctional p38 has a dominant negative effect on endogenous p38 activity [42–46]. In addition, during some of our own preliminary experiments, we found on rare occasion that exogenous p38δ-WT slightly increased rather than decreased the number of G418-resistant colonies (unpublished data), further suggesting that the effect of exogenous p38δ-WT could depend on cellular conditions that affect the p38 pathway.…”

Taurine Attenuates Liver Injury by Downregulating Phosphorylated p38 MAPK of Kupffer Cells in Rats with Severe Acute Pancreatitis

“…EMSA electrophoretic mobility shift assay, NF-κB nuclear factor κB, SO sham operation, SAP severe acute pancreatitis, Taur taurine. Studies showed that p38 MAPK plays an important role in the pathogenesis of SAP [3,30]. The expression of phosphorylated p38 MAPK in the pancreatic and lung tissue was increased rapidly in the SAP rat model [3].…”

Systemic Inflammation with Multiorgan Dysfunction Is the Cause of Death in Murine Ligation-Induced Acute Pancreatitis

“…4,5,[20][21][22][23][24][25][26] We have previously shown that ERK, NF-κB, and AP-1 are activated in the mouse pancreas within 1 h of pancreatic duct ligation, 1 while here we have shown that pancreatic ERK activation is sustained and also progresses until the premortal state in acute pancreatitis; also, pulmonary ERK activation peaks on day 3. As we have observed that ERK inhibition reverses agoniststimulated increases in NF-κB-dependent gene transcription in an exocrine pancreatic malignant cell line (AR42J cells) 5 while p38 inhibition only partially reduces it, 6 we are currently focusing on the role of ERK in disease pathogenesis in ligation-induced acute pancreatitis in mice.…”