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. 2015 Jan 5:746:106-14.
doi: 10.1016/j.ejphar.2014.10.042. Epub 2014 Oct 30.

Angiotensin II limits NO production by upregulating arginase through a p38 MAPK-ATF-2 pathway

Alia Shatanawi  1 , Tahira Lemtalsi  2 , Lin Yao  3 , Chintan Patel  2 , Ruth B Caldwell  4 , R William Caldwell  3
Affiliations

Angiotensin II limits NO production by upregulating arginase through a p38 MAPK-ATF-2 pathway

Alia Shatanawi et al. Eur J Pharmacol. .

Abstract

Enhanced vascular arginase activity can impair endothelium-dependent vasorelaxation by decreasing l-arginine availability to endothelial nitric oxide (NO) synthase, thereby reducing NO production and uncoupling NOS function. Elevated angiotensin II (Ang II) is a key component of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated arginase activity. In this study we explored the signaling pathway leading to increased arginase expression/activity in response to Ang II in bovine aortic endothelial cells (BAEC). Our previous studies indicate involvement of p38 mitogen activated protein kinase (MAPK) in Ang II-induced arginase upregulation and reduced NO production. In this study, we further investigated the Ang II-transcriptional regulation of arginase 1 in endothelial cells. Our results indicate the involvement of ATF-2 transcription factor of the AP1 family in arginase 1 upregulation and in limiting NO production. Using small interfering RNA (siRNA) targeting ATF-2, we showed that this transcription factor is required for Ang II-induced arginase 1 gene upregulation and increased arginase 1 expression and activity, leading to reduced NO production. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay further confirmed the involvement of ATF-2. Moreover, our data indicate that p38 MAPK phosphorylates ATF-2 in response to Ang II. Collectively, our results indicate that Ang II increases endothelial arginase activity/expression through a p38 MAPK/ATF-2 pathway leading to reduced endothelial NO production. These signaling steps might be therapeutic targets for preventing vascular endothelial dysfunction associated with elevated arginase activity/expression.

Keywords: ATF-2; Angiotensin II; Arginase; Nitric oxide (NO); Transcription factors; p38 MAPK.

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Figures

Fig 1
Fig 1. Arginase 1 promoter luciferase constructs and luciferase activity in Ang II treated BAECs
(A) Scheme representing a series of luciferase constructs driven by different lengths of the murine arginase 1 promoter and illustrating several putative transcription factor binding sequences in the 4.8 kb promoter region upstream of arginase 1 gene. (B) BAECs were co-transfected with the full length luciferase construct- −4.78kb arginase 1 promoter-Luc for 48 h and Renilla luciferase gene plasmid to normalize for transfection efficiency. Cells were then treated with Ang II (0.1 µM, 24 h). Luciferase activity shows a 50% increase with Ang II treatment versus transfected untreated cells (control). n=4 times each performed in triplicates; *P<0.05 vs. control.
Fig 2
Fig 2. Analysis of the arginase 1 promoter in response to Ang II
BAECs were co-transfected with one of three different deletion arginase 1 promoter-luciferase (Luc) constructs and Renilla luciferase gene plasmid to normalize for transfection efficiency. Luciferase activity induction showed that the Ang II-responsive element was located between −3.29 kb to −2.78 kb in the arginase 1 promoter as arrows indicate. n=3 each performed in triplicates; *P<0.05 vs. corresponding length of promoter (no treatment), #P<0.05 vs Ang II.
Fig 3
Fig 3. Role of ATF-2 and c-Jun in Ang II-induced arginase 1 luciferase activity in BAECs
BAECs were co-transfected with the full length luciferase construct- −4.78kb arginase 1 promoter-Luc for 48 h and Renilla luciferase gene plasmid to normalize for transfection efficiency. In addition co-transfection of BAECs with non-targeting siRNA (SC siRNA) or siRNA targeting either ATF-2 or c-Jun was performed. Cells were then treated with Ang II (0.1 µM, 24 h). (A) Representation of Western blot probed with ATF-2 antibody shows the efficiency of ATF-2 siRNA transfection. Band intensities from multiple experiments were quantified and normalized to α-actin. n=3 (B) Representation of Western blot probed with c-Jun antibody shows the efficiency of c-Jun siRNA transfection. Band intensities from multiple experiments were quantified and normalized to α-actin. n=3 (C) Luciferase activity was measured in cell lysate, normalized to Renilla luciferase and expressed as percent of scrambled (SC) siRNA transfected controls. n=3 each performed in triplicates; *P<0.05 vs. SC control, #P<0.05 vs. SC Ang II.
Fig 4
Fig 4. Arginase Activity and Arginase 1 Expression in BAECs
BAECs were transfected with either siRNA for ATF-2, c-Jun or scrambled (SC) siRNA. BAECs were then treated with Ang II (0.1 µM, 24h) (A) Elevation of arginase activity by exposure of BAECs to Ang II was prevented by transfection of cells with ATF-2 siRNA and partially prevented by c-Jun transfection. Transfection with sc-siRNA did not prevent response to Ang II. n= 3 *P<0.05 vs. control, #P<0.05 vs. SC siRNA for Ang II. (B) Elevation of arginase 1 expression by exposure of BAECs to Ang II (0.1 µM, 24 h) was prevented by transfection of cells with ATF-2 siRNA. Transfection with sc-siRNA did not prevent response to Ang II. Values are expressed as means of percent of control ± S.E.M..n= 3. *P<0.05 vs. control, #P<0.05 vs. SC siRNA for Ang II.
Fig 5
Fig 5. Activation of ATF-2 by Ang II
(A) BAECs exposed to Ang II (0.1 µM) for 5 to 60 mins show a time-dependent increase in phospho-ATF-2 expression first evident at 10 mins and peaking at 30 mins. (B) Pretreatment of BAEC with SB-202190 (2 µM, 2 h) blocked the phosphorylation of ATF-2 caused by Ang II (0.1 µM, 30 min). Representative autographs are shown. Values are expressed fold change over control ± S.E.M. of phospho-ATF-2/total-ATF-2 ratio. n=3 of independent experiments carried out in duplicate. *P<0.05 vs. control. #P<0.05 vs. Ang II.
Fig 6
Fig 6. Electrophoretic mobility shift assay in Ang II treated cells
Electrophoretic mobility shift assay (EMSA) analysis was performed with nuclear extracts from control BAECs or Ang II treated BAECs (0.1 µM, 30 mins). Ang II treatment enhanced formation of binding complex between nuclear extracts with oligonucleotide containing the wild-type AP-1 site compared to untreated BAECs (Left panel). No specific complex formation was observed for either nuclear extracts with the oligonucleotide containing the mutant AP-1 sequence (Right panel). Result shown is representative of three independent experiments. n=3.
Fig 7
Fig 7. Identification of activating transcription factor 2 (ATF-2) binding to AP-1 elements within the arginase 1 promoter using supershift assay
Supershift assay was performed with nuclear extracts from BAECs treated with Ang II (0.1 µM, 30 mins) in the absence and presence ATF-2 specific antibody (1:100) before performing the EMSA with oligonucleotide containing the wild-type AP-1 site. Results shown are representative of three independent experiments. n=3.
Fig 8
Fig 8. ChIP assay determines ATF-2 association with the arginase 1 promoter
ChIP assays were performed in BAECs treated with Ang II versus untreated control. anti-ATF-2 antibody and arginase 1 PCR primer were used. IgG (5 µg/IP) was used as negative IP control. qPCR was performed with primers for the 18S promoter and for bovine arginase 1 gene. qPCR analysis was performed using delta-delta circle time (Ct). Fig. shows percentage of DNA input (relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis normalized to the internal control 18S). IP; Immunoprecipation, α-ATF-2; anti-ATF-2. n=3 of independent experiments. *P<0.05 vs. control, #P<0.05 vs. Ang II IP: α-ATF-2.
Fig 9
Fig 9. Role of ATF-2 in limiting NO production in endothelial cells in response to Ang II
lonomycin stimulated NO production was determined in non-transfected, scrambled (SC) or ATF-2 siRNA transfected cell lysate with or without exposure to Ang II (0.1 µM, 24 h) as nitrites released in cell media of the treated cells. Basal control NO production was 60.5 pmol/ml/hr. Ang II decreased NO production. This effect was prevented with ATF-2 siRNA transfection. Data represent mean ± S.E.M. n=4 independent experiments carried out in triplicates. *P<0.05 vs. control. #P<0.05 vs. Ang II.
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