Role of TAB1 in nitric oxide-induced p38 activation in insulin-producing cells

Abstract

The aim of present study was to elucidate the role of TAB1 in nitric oxide-induced activation of p38 MAPK. For this purpose we over-expressed TAB1 in insulin-producing beta-TC6 cells. We observed in cells transiently over-expressing TAB1 that p38 activation was enhanced in response to DETA/NONOate. A lowering of TAB1 levels, using the siRNA technique, resulted in the opposite effect. The DETA/NONOate-induced cell death rate was increased in cells transiently overexpressing TAB1. In stable beta-TC6 cell clones with very high TAB1 levels p38 phosphorylation was enhanced also at basal conditions. DETA/NONOate increased also the phosphorylation of JNK and ERK in beta-TC6 cells, but these events were not affected by TAB1. Interestingly, the inhibitory effect of SB203580 on p38 phosphorylation was paralleled by a stimulatory effect on JNK phosphorylation and an inhibitory effect on ERK phosphorylation. In summary, we propose that TAB1 promotes nitric oxide-induced p38 autophosphorylation. In addition, nitric oxide-induced p38 activation seems to promote JNK inhibition and ERK activation, but this effect appears to not require TAB1. A better understanding of how the TAB1/p38 pathway promotes beta-cell death in response to nitric oxide might help in the development of novel pharmacological approaches in the treatment of diabetes.

Fig 1

(A) Transient overexpession of TAB1 affects cytokine- and DETA/NO–induced p38 phosphorylation. Two days after transfection and 1 day after FACS sorting, the GFP positive cells were pretreated with SB203580 (10 μM) for 30 min and then stimulated with IL-1β (50 U/ml), DETA/NONOate (2.5 mM) and sorbitol (0.4 M) for 30 min. Cells were lysed, separated by SDS gel electrophoresis and analysed by immunoblotting with p38 and phospho-specific antibodies. The immunoblot is representative for two separate experiments. (B) Effect of TAB1 overexpression on DETA/NO-induced cell death in transiently transfected cells. Two days after transfection and 1 day after cell sorting, GFP-positive β-TC6 cells were exposed to DETA/NONOate (2.5 mM) for 24 h. Cells were then stained with propidium iodide and cell viability was assessed using the FACSCalibur flow cytometer. Results are presented as percentage of dead cells of the total cell count. Bars are means±S.E.M for three independent experiments. * denotes P<0.05 using Student´s paired t-test. (C) Treatment with TAB1 siRNA decreases DETA/NON-induced p38 phosphorylation. Cells were transfected with GL3 lucifierase siRNA (control) or TAB1 siRNA. Two days later cells were incubated with DETA/NONOate (2.5 mM) for 30 min. Cells were lysed, separated by SDS gel electrophoresis and analysed by immunoblotting. The immunoblot is representative for two separate experiments.

Fig 2

TAB1 overexpression in stable cell clones. The β-TC6 cells were transfected for expression of TAB1 and neomycin resistance and selected by treatment with G418. After 10-14 days cells were lysed and analysed by immunoblot. The three clones A2, A3 and D3 were selected for further experimentation.

Fig 3

Effect of stable TAB1 overexpression on DETA/NO-stimulated p38, JNK and ERK phosphorylation. (A) The stable clones were pretreated with SB203580 (10 μM) and then stimulated with DETA/NO (2.5 mM) for 30 min. The cells were lysed, separated by SDS gel electrophoresis and analysed by immunoblotting with phospho-specific antibody recognizing phosphorylated p38 and total p38 (Panel A), phosphorylated JNK and total JNK (Panel B) or phosphorylated ERK and total ERK (Panel C). Values of phospho-protein bands were related to those of non-phospho-specific protein bands. Results shown are means±S.E.M. for 3-6 observations. * denotes P<0.05 using two-way ANOVA and Student´s t-test.