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AT₂ receptor activation regulates myocardial eNOS expression via the calcineurin–NF-AT pathway¹

OLIVER RITTER², KAI SCHUH², MARC BREDE^*, NICOLA RÖTHLEIN, NATALIE BURKARD, LUTZ HEIN^* and LUDWIG NEYSES³

Department of Medicine, University of Wuerzburg, Germany;
^* Department of Pharmacology and Toxicology, University of Wuerzburg, Germany; and
University Department of Medicine, Manchester Royal Infirmary, Manchester, UK

³Correspondence: University Department of Medicine, Manchester Heart Center, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, UK. E-mail: ludwig.neyses{at}mhc.cmht.nwest.nhs.uk

SPECIFIC AIMS

We tested the hypothesis that eNOS is up-regulated in cardiomyocytes on angiotensin II (AngII) stimulation, that this up-regulation is mediated via the AT₂ receptor, and that it involves the calcineurin/NF-AT pathway.

PRINCIPAL FINDINGS

1. AngII increased eNOS protein expression
eNOS protein expression was increased (332±34%, n=8; P<0.001) after 48 h stimulation with 100 nM AngII. Cyclosporin A (CsA; 0.5 mg/mL) completely blocked eNOS protein expression induced by AngII (n=8; P<0.01).

Incubation of cells with the specific AT₂ receptor agonist CPG 42112 (10 µM) caused a 2.4-fold increase (238±18%, n=8; P<0.001) in eNOS protein expression. In AngII-treated cells, eNOS protein expression could be fully blocked (n=8; P<0.01) with PD 123319 (10 µM), a specific AT₂ receptor antagonist. To ensure the in vivo relevance of AT₂ receptor-mediated eNOS expression, mice with targeted deletion of the AT₂ receptor and WT mice were investigated for eNOS expression in the myocardium. eNOS protein expression was significantly reduced in AT₂ receptor KO (75±8%) vs. WT mice (100±9%) (mean±SE, n=6; P<0.05).

2. AngII-stimulated eNOS promoter activity
To determine whether the promoter region of the eNOS gene was responsive to the stimulatory effect of AngII, a 1.6 kb fragment of the 5'-regulatory region of the human eNOS gene was cloned into a plasmid containing a luciferase reporter gene (NP3 luci) and transiently transfected into neonatal rat cardiomyocytes. A 333 ± 24% increase (n=8; P<0.001) of reporter activity transcribed from NP3 luci was detected after 48 h stimulation with 10 nM AngII compared with unstimulated cells (Fig. 1A ). CsA (0.5 µg/mL) blocked AngII-induced eNOS promoter activity by 77% (n=8; P<0.001) (Fig. 1B ). To delineate which angiotensin receptor is responsible for the induction, specific AT₁ and AT₂ receptor agonists and antagonists were used. Incubation of transfected cells with the AT₂ receptor agonist CPG 42112 caused a 462 ± 56% increase (n=8; P<0.001) in eNOS promoter activity. In AngII-treated transfected cells, the increase in eNOS promoter activity could be blocked 73%; (n=8; P<0.001) by using PD 123319. Addition of Losartan did not reduce eNOS promoter activity in cardiomyocytes treated with AngII (535±47%; n=8; P<0.001). (Fig. 1 A–C).

View larger version (11K): [in this window] [in a new window]   Figure 1. A) Effect of AngII on a 1.6 kb fragment of the human eNOS promoter. Cardiomyocytes were transfected with "NP1 luci" (eNOS promoter in non-sense orientation, followed by luciferase gene) or "NP3 luci" (eNOS promoter gene in sense orientation followed by luciferase). eNOS promoter ("NP3 luci") was activated only weakly in unstimulated cells but increased on stimulation with angiotensin II. AngII concentrations were 10, 100, and 500 nM. B) Blockade of the calcineurin/NF-AT pathway by CsA (0.5 µg/mL) prevented the AngII-induced increase in eNOS promoter activity. C) AT1 and AT2 receptor-mediated effect on eNOS promoter activity. AngII concentration was 100 nM unless stated otherwise. Asterisks denote values significantly different from control (n.s.=not significant; **P<0.01; ***P<0.001).

3. Two of four putative NF-AT sites in the human eNOS promoter showed NF-AT binding; different NF-AT factors were present in neonatal cardiac myocytes
EMSAs were used to test the binding of NF-AT factors to bona fide NF-AT consensus sites in the human eNOS promoter. Labeled double-strand oligonucleotides of the eNOS promoter were used as probes. All sites contain typical NF-AT core binding sequences (AGGA^C/_A^G/_A^T/_A) and the distal -1535 NF-AT site a typical composite NF-AT/AP-1 binding site, including an AP-1 binding motif (TGAGTCA). Induction of cells with AngII lead to formation of a DNA binding complex with the -1535 NF-AT site (Fig. 2 A) containing predominantly NF-AT2 (=NF-ATc1) (Fig. 2A ). Antibodies to other NF-AT isoforms generated no supershift complex. 50 ng of unlabeled probe was sufficient to abolish formation of the NF-AT/DNA complex; the same amount of mutated control oligonucleotide was insufficient (Fig. 2A ). No NF-AT binding was observed using the mutated double-strand -1535 oligonucleotide as a probe, demonstrating the necessity of an NF-AT consensus site for NF-AT binding. The second putative NF-AT site (-650 NF-AT site) of the human eNOS promoter showed weak binding of NF-AT to this site; the others showed no binding of NF-AT factors.

View larger version (58K): [in this window] [in a new window]   Figure 2. A) EMSA demonstrating NF-AT factor binding to the upstream -1535 NF-AT consensus site. Stimulation of cardiomyocytes with AngII increased DNA binding capacity, resulting in a DNA binding complex that could be attenuated by costimulation with CsA (0.5 µg/mL, A, lanes 1–3). Competition with 50 ng wild-type probe (wt NF-AT) resulted in an impairment of DNA binding (A, lane 4), whereas competition with 50 ng mutated (mut NF-AT) unlabeled oligonucleotide did not inhibit DNA binding activity (A, lane 5). Supershift assays with NF-AT-specific antibodies revealed participation of NF-AT2 in the DNA binding complex, other antibodies showed no obvious supershift (A, lanes 6–10). Using the mutated oligonucleotide as labeled probe resulted in a strong decrease in AngII-induced DNA binding (lane 11). Lanes 12–16: antibody controls without nuclear extracts demonstrating an absence of unspecific antibody/DNA complexes. B) EMSA using nuclear extracts from P/I-(PMA/ionomycin)-stimulated Jurkat T cells, demonstrating ability of antibodies to supershift NF-AT control complexes. C) Western blot showing presence of different NF-AT factors in nuclear extracts of cardiomyocytes; stimulated Jurkat T cells serving as positive controls.

In nuclear extracts of neonatal cardiomyocytes, NF-AT1 (NF-ATc2, NFATp), -2 (-c1) and -5 (-c5) were clearly detectable (Fig. 2B ). Only a weak signal was detectable for NF-AT4 (NF-ATc3) and no signal for NF-AT3 (NF-ATc4) vs. signals detected in nuclear extracts of PMA/ionomycin-stimulated Jurkat T cells (Fig. 2C ).

4. Effect of AngII stimulation on different NF-AT consensus sites in the eNOS promoter
To confirm the relevance of the NF-AT sites 1 (-1535) and 2 (-650) in an in situ promoter context, we transfected a series of plasmids carrying mutations destroying the core binding site in the -535 (pM1), the -650 site (pM2), or both sites (pM1+M2). Stimulation of cardiomyocytes transfected with pM1 did not result in a significant increase of eNOS promoter activity (55±7% in unstimulated cells vs. 84±10% in stimulated cells; n=8; n.s.). However, stimulation of cells transfected with pM2 increased eNOS promoter activity (120±11% vs. 220±28%; n=8; P<0.001). Transfection and stimulation of pM1 +2 did not increase eNOS promoter activity (117±11% vs. 141±39%; n=8; n.s.). These results demonstrate that the -1535 NF-AT consensus site is important for activation of the eNOS promoter by the calcineurin-NF-AT pathway, whereas the -650 NF-AT site may participate only to a minor extent in the activation of the eNOS promoter.

CONCLUSIONS

We aimed at defining new NF-AT targets and their upstream activating receptors in cardiomyocytes. Our results suggest that stimulation of cardiomyocytes in vivo by AngII is accompanied by increased expression of eNOS, which is mediated by a pathway encompassing the AT₂ receptor and calcineurin/NF-AT. This induction is (at least in part) transcriptionally regulated through an NF-AT element in the eNOS promoter.

Angiotensin stimulation of the myocardium has been shown to activate key signal transduction pathways. The simple hypothesis that AT₁ and AT₂ activation are counter-regulatory mechanisms has been challenged by results from AT₂-deficient mice showing that deletion of the AT₂ gene is conducive to a blunted hypertrophic response, in contrast to the increase expected if AT₂ were counter-regulatory to the AT₁-mediated hypertrophic response.

To test whether the effect on eNOS is mediated via the AT₁ or AT₂ receptor, specific AT₁ and AT₂ receptor agonists and antagonists were used. We found that activation of the calcineurin/NF-AT pathway and increased expression of eNOS in cardiomyocytes are mediated by the AT₂ receptor. These results could be confirmed in AT₂ receptor knockout mice.

Our results support the idea that the distal -1535 NF-AT site of the eNOS promoter is most likely responsible for induction of the eNOS gene via the calcineurin pathway and that NF-AT2 affects eNOS expression in the myocardium.

The primary goal of the present work was to identify eNOS as a new downstream target of AT₂ receptor stimulation and define calcineurin activation as a mediating step. This is likely to contribute to solving the conundrum of the AT₂ receptor action in the myocardium, but much more work is needed to delineate the alterations in gene program induced by the complex array of AT₂ actions.

We conclude, 1) AngII stimulation of neonatal rat cardiomyocytes is accompanied by increased expression of eNOS; 2) this effect is mediated by the calcineurin pathway and induced by the AT₂ receptor; 3) these results define a calcineurin/NF-AT/eNOS pathway as downstream effector of AT₂ receptor activation in the myocardium.

View larger version (45K): [in this window] [in a new window]   Figure 3. Diagram displaying principal findings. Stimulation of cardiomyocytes with AngII resulted in activation of phosphatase calcineurin consisting of catalytic subunit A (CnA) and regulatory subunit B (CnB). Specificity of calcineurin activation was demonstrated by blockade of pathway by cyclosporin A (CsA). NF-AT dephosphorylated by active calcineurin entered the nucleus and participated in induction of NOS-III promotor, resulting in elevated NOS-III protein expression in angiotensin-stimulated cardiomyocytes. Use of different antagonists and agonists (Losartan, CPG 42112, and PD 123319) of AT receptors revealed mainly the participation of AT2 receptor subtype in this pathway.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0321fje; to cite this article, use FASEB J. (December 17, 2002) 10.1096/fj.02-0321fje

² Both authors contributed equally to this work.

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