Involvement of mitogen-activated protein kinase pathways in expression of the water channel protein aquaporin-4 after ischemia in rat cortical astrocytes

Abstract

Brain edema after ischemic brain injury is a key determinant of morbidity and mortality. Aquaporin-4 (AQP4) plays an important role in water transport in the central nervous system and is highly expressed in brain astrocytes. However, the AQP4 regulatory mechanisms are poorly understood. In this study, we investigated whether mitogen-activated protein kinases (MAPKs), which are involved in changes in osmolality, might mediate AQP4 expression in models of rat cortical astrocytes after ischemia. Increased levels of AQP4 in primary cultured astrocytes subjected to oxygen-glucose deprivation (OGD) and 2 h of reoxygenation were observed, after which they immediately decreased at 0 h of reoxygenation. Astrocytes exposed to OGD injury had significantly increased phosphorylation of three kinds of MAPKs. Treatment with SB203580, a selective p38 MAPK inhibitor, or SP600125, a selective c-Jun N-terminal kinase inhibitor, significantly attenuated the return of AQP4 to its normal level, and SB203580, but not SP600125, significantly decreased cell death. In an in vivo study, AQP4 expression was upregulated 1-3 days after reperfusion, which was consistent with the time course of p38 phosphorylation and activation, and decreased by the p38 inhibition after transient middle cerebral artery occlusion (MCAO). These results suggest that p38 MAPK may regulate AQP4 expression in cortical astrocytes after ischemic injury.

FIG. 1.

Western blot analysis of the water channel protein in rat cortical astrocytes that underwent OGD/reoxygenation. Expression of AQP4 was significantly decreased by OGD injury, but gradually recovered after reoxygenation. AQP4 was significantly upregulated at 16 h. Data are expressed as mean±standard deviation of three independent experiments (*p<0.01 compared with control [cont]; ODG, oxygen-glucose deprivation; AQP4, aquaporin-4).

FIG. 2.

Changes in the levels of phospho-ERK1/2 (p-ERK) and ERK1/2 (A) or phospho-JNK (p-JNK) and JNK (B) in rat cortical astrocytes during reoxygenation after 6 h of OGD. Changes in the levels of p-p38 and p38 MAPK (C) in rat cortical astrocytes during reoxygenation after 6 h of OGD. ERK1/2 activity (A) was also examined after reoxygenation. Tissue homogenates were subjected to kinase assays using GST-Elk-1 as the substrate (cont, control; GST, glutathione S-transferase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; OGD, oxygen-glucose deprivation; MAPK, mitogen-activated protein kinase).

FIG. 3.

(A) p38 MAPK activity was examined after reoxygenation. Tissue homogenates were subjected to kinase assays using GST-ATF-2 as the substrate. Activation of p38 was gradually increased after reoxygenation and the peak level of expression occurred at 16 h. (B) Quantitative analysis showed that activation of p38 increased significantly 16 h after reoxygenation compared with the control (cont). Data are expressed as mean±standard deviation of four independent experiments (*p<0.05 compared with control; GST, glutathione S-transferase; ATF-2, activating transcription factor-2; MAPK, mitogen-activated protein kinase).

FIG. 4.

The cells were treated with 10 μM or 1 μM of PD98059, SP600125, or SB203580, before OGD. Sixteen hours after reoxygenation, the AQP4 level was determined by Western blot analysis. Ten micromoles of SB203580 or SP600125 significantly suppressed expression of AQP4 to the basal level 16 h after reoxygenation. Data are expressed as mean±standard deviation of four independent experiments (*p<0.05 compared with the OGD group; cont, control; ODG, oxygen-glucose deprivation; AQP4, aquaporin-4; PD10, 10 μM PD98059; PD1, 1 μM PD98059; SP10, 10 μM SP600125; SP1, 1 μM SP600125; SB10, 10 μM SB203580; SB1, 1 μM SB203580).

FIG. 5.

LDH release after OGD injury in rat cortical astrocytes. Cells were subjected to 6 h of OGD, followed by 24 h of reoxygenation. Cell death was assessed by LDH release. SB203580 (10 μM) significantly reduced OGD-induced cell death. Data are expressed as mean±standard deviation of three independent experiments performed in triplicate (#p<0.05 compared with the OGD group; cont, control; PD10, 10 μM PD98059; PD1, 1 μM PD98059; SP10, 10 μM SP600125; SP1, 1 μM SP600125; SB10, 10 μM SB203580; SB1, 1 μM SB203580; LDH, lactate dehydrogenase; OGD, oxygen-glucose deprivation).

FIG. 6.

(A) Western blot analysis of p-p38 and p38 in the rat cortex 1, 3, and 7 days after focal ischemia. After 90 min of MCAO, expression of p-p38 was increased from day 1 to day 3. (B) p38 MAPK activity (GST-ATF-2) was examined 1, 3, and 7 days after 90 min of MCAO. Activation of p38 was increased maximally on day 1. (C) Western blot analysis of AQP4 in the rat cortex 1, 3, and 7 days after focal ischemia. After 90 min of MCAO, AQP4 protein levels continued to increase significantly until 3 days. AQP4 expression was significantly decreased at 7 days. Data are expressed as mean±standard deviation (*p<0.05, **p<0.001, #p<0.05 compared with the sham group; sh, sham; GST, glutathione S-transferase; MCAO, middle cerebral artery occlusion; AQP4, aquaporin-4; MAPK, mitogen-activated protein kinase; ATF-2, activating transcription factor-2).

FIG. 7.

(A) Suppression of p38 MAPK activity by administration of SB203580. p38 MAPK activity was examined 1 and 3 days after reperfusion with or without SB203580 administration. SB203580 was administered 30 min before ischemic insult, and phosphorylation of ATF-2, a downstream substrate, was examined by Western blot analysis. (B) Suppression of AQP4 expression in SB203580-treated animals. Expression of AQP4 with or without SB203580 administration was examined by Western blot analysis. Values are mean±standard deviation (n=4 in each group; *p<0.05 for the SB203580-treated groups versus the vehicle-treated groups; GST, glutathione S-transferase; cont, control; ATF-2, activating transcription factor-2; MAPK, mitogen-activated protein kinase; AQP4, aquaporin-4).

FIG. 8.

Infarct volume and edema volume 3 days after transient focal cerebral ischemia in vehicle-treated and SB203580-treated rats (n=6 each; *p<0.05, **p<0.01 compared with ischemic controls; V, vehicle-treated rats; SB, SB203580-treated rats).