Peroxynitrite-induced p38 MAPK pro-apoptotic signaling in enterocytes

Abstract

Enterocyte apoptosis in necrotizing enterocolitis is partly due to the elaboration of toxic intermediates of nitric oxide (NO), such as peroxynitrite (PN). Because p38 mitogen-activated protein kinase (MAPK) and serine-threonine kinase (AKT) are well-characterized pro- and anti-apoptotic mediators, respectively, we hypothesized that PN could induce enterocyte apoptosis via activation of p38 and deactivation of AKT. To test this hypothesis, the rat intestinal cell line, IEC-6, was treated with PN. PN caused phosphorylation of p38, its upstream activator, MKK3/6, and downstream effector, transcription factor ATF-2. PN-induced apoptosis was inhibited by the p38 inhibitor, SB202190, and by p38 siRNA. PN decreased AKT phosphorylation; this effect was abrogated by pre-treatment with SB202190 or p38 siRNA. PN exposure also increased the activity of the protein phosphatase 2A (PP2A). These data demonstrate that PN-mediated apoptosis depends on the p38 pathway and that p38 mediates deactivation of AKT survival pathways possibly by the involvement of PP2A.

Figure 1. PN increases phosphorylation of p38, MKK3/6 and ATF-2

IEC-6 cells were exposed to 50 M PN and DPN for the indicated times. Naïve (untreated cells) are indicated by 0min. Levels of phospho-p38, phospho-MKK3/6, phospho-ATF-2, total p38, total MMK3 and total ATF-2 were determined by Western blotting. UV, cells exposed to 50mJ/cm² UVC and allowed to recover in growth medium for 1h (positive control for p38 activation). Data shown are representative of at least 3 independent experiments.

Figure 2. PN-induced apoptosis is prevented by p38 inhibition

(A) IEC-6 cells were transfected with p38 siRNA, or control (Mock) siRNA as indicated. 36h post transfection, cells were exposed to PBS, or 50 M PN, or equivalent concentration of DPN for 60min as indicated, and levels of phospho-p38, total p38, and -actin were determined. UV, cells exposed to 50 mJ/cm² UVC and allowed to recover in growth medium for 1h (positive control for p38 activation). (B) IEC-6 cells were treated with PN or equivalent concentration of DPN for 1h, and allowed to recover for 6h. Percentage of apoptotic (sub-G1 DNA content) cells was determined by flow cytometry of 7-AAD stained cells were determined by flow cytometry. Cells were pre-treated with 10 M SB202190 (SB), or transfected with p38 siRNA or control siRNA 30 min or 48h prior to PN or DPN treatment, respectively, as indicated. GT, cells treated with 2.5 mol/L of gliotoxin for 6h. (*) p<0.001, gliotoxin treated cells vs. naïve DPN-treated, SB+PN-treated or p38 siRNA-transfected cells. (†) p<0.001, PN-treated vs. DPN-treated, SB+PN-treated and p38 siRNA-transfected PN-treated cells. (‡) p<0.001 control-siRNA-transfected vs.p38-siRNA-transfectedPN-treated cells. Data are depicted as the mean S.E. (n=6).

Figure 3. AKT phosphorylation in IEC-6 cells treated with PN

(A) Cells were treated with PN, or equivalent concentration of DPN for indicated time. Levels of phospho-AKT and total AKT were determined by Western blotting. (B) Levels of phospho-AKT and total AKT in cells untreated naive, or treated with PN or DPN for 1h, with or without pre-treatment with 10 M SB202190 (SB) as indicated. Data are representative of 3 independent experiments.

Figure 3. AKT phosphorylation in IEC-6 cells treated with PN

(A) Cells were treated with PN, or equivalent concentration of DPN for indicated time. Levels of phospho-AKT and total AKT were determined by Western blotting. (B) Levels of phospho-AKT and total AKT in cells untreated naive, or treated with PN or DPN for 1h, with or without pre-treatment with 10 M SB202190 (SB) as indicated. Data are representative of 3 independent experiments.

Figure 4. PN increases PP2A activity

IEC-6 cells were treated with PN (50 M) or equivalent concentration of DPN for 1 h. Cells were pre-treated with 10 M SB202190 (SB), 10 M okadaic acid (OA) or 100nM I₁^PP2A (PP2A INH) 30m prior to PN or DPN exposure. PP2A activity in cell lysates were measured. (*) PN exposure significantly elevated PP2A activity compared to all other variables (p<0.001, n=5). Data are shown as pmoles phosphate produced per mg protein in the form of mean S.E.