A p38MAPK/MK2 signaling pathway leading to redox stress, cell death and ischemia/reperfusion injury

Abstract

Background: Many diseases and pathological conditions are characterized by transient or constitutive overproduction of reactive oxygen species (ROS). ROS are causal for ischemia/reperfusion (IR)-associated tissue injury (IRI), a major contributor to organ dysfunction or failure. Preventing IRI with antioxidants failed in the clinic, most likely due to the difficulty to timely and efficiently target them to the site of ROS production and action. IR is also characterized by changes in the activity of intracellular signaling molecules including the stress kinase p38MAPK. While ROS can cause the activation of p38MAPK, we recently obtained in vitro evidence that p38MAPK activation is responsible for elevated mitochondrial ROS levels, thus suggesting a role for p38MAPK upstream of ROS and their damaging effects.

Results: Here we identified p38MAPKα as the predominantly expressed isoform in HL-1 cardiomyocytes and siRNA-mediated knockdown demonstrated the pro-oxidant role of p38MAPKα signaling. Moreover, the knockout of the p38MAPK effector MAPKAP kinase 2 (MK2) reproduced the effect of inhibiting or knocking down p38MAPK. To translate these findings into a setting closer to the clinic a stringent kidney clamping model was used. p38MAPK activity increased upon reperfusion and p38MAPK inhibition by the inhibitor BIRB796 almost completely prevented severe functional impairment caused by IR. Histological and molecular analyses showed that protection resulted from decreased redox stress and apoptotic cell death.

Conclusions: These data highlight a novel and important mechanism for p38MAPK to cause IRI and suggest it as a potential therapeutic target for prevention of tissue injury.

Figure 1

Knockdown of p38MAPKα (p38α) decreases ROS levels following HR. (A) Quantitative RT-PCR analysis of p38MAPK isoform expression in HL-1 cells (n = 3). (B-D) Effect of p38MAPKα knockdown on downstream signaling and mitochondrial ROS production. 72 hours after transfection with p38MAPKα siRNAs (250 nM) or control siRNAs (250 nM), HL-1 cells were exposed to the following HR protocol: hypoxia (1 hour) and reoxygenation (15 min) and analyzed for the expression of p38MAPKα (B), phosphorylation of MK2 and ATF2 (C) and mitochondrial ROS levels (D) as described in Methods. Representative immunoblots and summary graphs are shown (B-D). The data are expressed as mean ± SEM (n = 3-4). **p < 0.01, ***p < 0.001 control siRNAs transfected cells vs. control siRNA transfected cells undergoing HR; §p < 0.01, #p < 0.001 control siRNA transfected HL-1 cells vs. p38MAPKα siRNA transfected cells, subjected to HR.

Figure 2

p38MAPK (p38) increases mitochondrial ROS levels via MK2. (A) WT and MK2-/- MEFs were pretreated with vehicle or BIRB796 (B-796) (50 nM) for 1 hour and then subjected to HR: hypoxia (6 hours) and reoxygenation (15 min). Expression of phosphorylated and non-phosphorylated p38MAPK, MK2 and HSP25 was determined. (B, C) For mitochondrial ROS measurements WT and MK2-/- MEFs were pretreated with either vehicle, BIRB796 (B-796) (50 nM) or N-acetyl cysteine (NAC) (7.5 mM) for 1 hour and exposed to the HR protocol followed by ROS measurement as described in Methods. (D-F) 72 hours after transfection with MK2 siRNAs (500 nM) or control siRNAs (500 nM), HL-1 cells were subjected to HR: hypoxia (6 hours) and reoxygenation (15 min) and analyzed for the effect of MK2 knockdown on p38MAPK/MK2 signaling (D), and mitochondrial ROS production (E, F) during HR as described in the Methods. Representative immunoblots (A, D), fluorescence images (B, E) and summary graphs (C, F) are shown. The data are expressed as mean ± SEM (n = 6-8). **p < 0.01, ***p < 0.001 vs. WT MEFs undergoing HR; #p < 0.001 vs. control siRNAs transfected HL-1 cells, subjected to HR.

Figure 3

Effect of p38MAPK (p38) inhibition on intracellular signaling following IR. Rats were pretreated with the carrier DMSO or BIRB796 (B-796) (5 mg/kg BW) for 1 hour and subjected to 1 hour of renal ischemia followed by different time points of reperfusion (15 min, 2 days, 7 days). Kidneys were harvested at given time points of reperfusion and total tissue lysates were used to determine activation pattern of MAPKs (p38MAPK, JNK, ERK) and the downstream target of p38MAPK (MK2) by phosphorylation specific antibodies. A representative immunoblot (A) and summary graphs (B, C) are shown. Results are given as mean ± SEM (n = 3). ** p < 0.01, ***p < 0.001 vs. sham-operated group; §p < 0.01, #p < 0.001 vs. IR-15 min group.

Figure 4

p38MAPK (p38) inhibition prevents ischemia/reperfusion-induced increase in the serum levels of kidney function markers and oxidative stress indicators. Serum levels of creatinine (A) and urea (B) were measured on indicated days (D0 to D7) following IR in rats pretreated with either BIRB796 (B-796) at two different doses (5 mg or 20 mg/kg BW) or vehicle (DMSO) only. Day 0 represents measurements before ischemia in every group. Likewise, serum cystatin C (C) and NGAL (D) levels were measured on indicated days of reperfusion in another set of experiments where rats were pretreated with either BIRB796 (B-796) (5 mg/kg BW) or DMSO only. Results are given as mean ± SEM (n = 4-7). *p < 0.05, **p < 0.01 ***p < 0.001, difference between DMSO- and BIRB796-treated groups at the given time points. (E-H) Rats were pretreated with BIRB796 (B-796) (5 mg/kg BW) for 1 hour and subjected to 1 hour of renal ischemia followed by different time points of reperfusion (15 min, 2 days, 7 days). Kidneys were harvested at given time points of reperfusion and total tissue lysates were used to determine the expression level of HSP70 (E), the abundance of 3-nitrotyrosine (3-Nitrotyr) (F) and 4-HNE modified proteins (G), and the phosphorylation of H2AX (H). Results are given as mean ± SEM (n = 3-4). $p < 0.01, Øp < 0.05 vs. sham-operated group, **p < 0.01 vs. IR-15 min group, §p < 0.01, #p < 0.05 vs. IR-2d group, *p < 0.05 vs. IR-7d group.

Figure 5

p38MAPK (p38) inhibition prevents ischemia/reperfusion-induced apoptosis of tubular cells. Rats were pretreated with the carrier DMSO or BIRB796 (B-796) (5 mg/kg BW) for 1 hour and subjected to 1 hour of renal ischemia followed by different time points of reperfusion (15 min, 2 days, 7 days). Kidneys were harvested at given time points of reperfusion and total tissue lysates were used to determine activation of caspase-3. A representative immunoblot is shown (A). IR-induced tubular cell death was assessed by TdT-mediated dUTP nick end labeling (TUNEL) staining at day 2 of reperfusion as described in Material and Methods. Representative images of the three regions of the kidney (cortex, corticomedullary junction and medulla) at 400x magnification and summary graph of the TUNEL positive cells are shown (B-C). Arrows point to the apoptotic cells with condensed nuclear material. Results are given as mean ± SEM (n = 4). *p < 0.05, ***p < 0.001 vs. vehicle-treated IR group.