Nitric oxide regulates pulmonary vascular smooth muscle cell expression of the inducible cAMP early repressor gene

Abstract

Nitric oxide (NO) regulates vascular smooth muscle cell (VSMC) structure and function, in part by activating soluble guanylate cyclase (sGC) to synthesize cGMP. The objective of this study was to further characterize the signaling mechanisms by which NO regulates VSMC gene expression using transcription profiling. DNA microarrays were hybridized with RNA extracted from rat pulmonary artery smooth muscle cells (RPaSMC) exposed to the NO donor compound, S-nitroso-glutathione (GSNO). Many of the genes, whose expression was induced by GSNO, contain a cAMP-response element (CRE), of which one encoded the inducible cAMP early repressor (ICER). sGC and cAMP-dependent protein kinase, but not cGMP-dependent protein kinase, were required for NO-mediated phosphorylation of CRE-binding protein (CREB) and induction of ICER gene expression. Expression of a dominant-negative CREB in RPaSMC prevented the NO-mediated induction of CRE-dependent gene transcription and ICER gene expression. Pre-treatment of RPaSMC with the intracellular calcium (Ca(2+)) chelator, BAPTA-AM, blocked the induction of ICER gene expression by GSNO. The store-operated Ca(2+) channel inhibitors, 2-ABP, and SKF-96365, reduced the GSNO-mediated increase in ICER mRNA levels, while 2-ABP did not inhibit GSNO-induced CREB phosphorylation. Our results suggest that induction of ICER gene expression by NO requires both CREB phosphorylation and Ca(2+) signaling. Transcription profiling of RPaSMC exposed to GSNO revealed important roles for sGC, PKA, CREB, and Ca(2+) in the regulation of gene expression by NO. The induction of ICER in GSNO-treated RPaSMC highlights a novel cross-talk mechanism between cGMP and cAMP signaling pathways.

Fig. 1

Panel A: Incubation with S-nitroso-L-glutathione (GSNO) induces ICER gene expression in rat pulmonary artery smooth muscle cells (RPaSMC) in a time- dependent manner. RNA was extracted from untreated RPaSMC and RPaSMC treated with GSNO for 1, 2, 4, 16, and 24h. RNA blot hybridization was performed using radiolabeled DNA probes specifying ICER. Ethidium bromide staining of 28S rRNA in the gel was scanned and confirmed the equal loading of RNA samples. A representative blot of at least 3 experiments is shown. Panel B: Incubation with S-nitroso-L-glutathione (GSNO) induces ICER gene expression in RPaSMC in a dose-dependent manner. RNA was extracted from untreated RPaSMC and RPaSMC exposed to varying concentrations of GSNO for 2h. RNA blot were hybridized with DNA probes specifying ICER. Ethidium bromide staining of 28S rRNA confirmed the equal loading of RNA samples. A representative blot of at least 3 experiments is shown. Panel C: Proteins were extracted from RPaSMC treated without or with 0.1, 1, 10, 100 μmol/L of GSNO for 4h. Immunoreactive ICER was detected using CREM-1 antibodies, and GAPDH was used as a loading control.

Fig. 2

Panel A: Cytokines increase ICER gene expression in RPaSMC via NO- and cGMP-dependent mechanisms. RNA was extracted from untreated RPaSMC or RPaSMC incubated with IL-1β (20 ng/ml) and TNFα (100 ng/ml) for 4h. Similarly-treated RPaSMC were pretreated without or with the NOS2 inhibitor, L-NIL (0.5 mmol/L); the sGC inhibitor, ODQ (10 μmol/L); PKG antagonists, Rp-8-pCPT-cGMP (100 μmol/L) and Rp-8-Br-cGMP (100 μmol/L); or the PKA inhibitor, H89 (10 μmol/L). Membranes were hybridized with DNA probes specifying rat ICER and NOS2; the experiment shown is representative of 3 separate experiments. Panel B: Inhaled nitric oxide (NO) increases pulmonary ICER gene expression in vivo. RNA was extracted from lungs of wild-type mice that breathed air supplemented with NO gas (80 ppm) for 2, 4, 8, or 24h. Membranes were hybridized with DNA probes specifying ICER; the experiment shown is representative of 3 different experiments. Panel C: GSNO induces ICER in RPaSMC via PKA. RNA blots were prepared from RPaSMC pretreated with or without ODQ (10 μmol/L), PKG inhibitors, Rp-8-pCPT-cGMP (100 μmol/L) and KT5823 (1 μmol/L); H89 (10 μmol/L); or cGMP analogues, Sp-pCPT-cGMP (100 μmol/L) and Sp-8-Br-cGMP (100 μmol/L) for 30 min followed by incubation with and without GSNO (100 μmol/L) for 2h. Membranes were hybridized with DNA probes specifying ICER; the experiment shown is representative of 3 different experiments. Panel D: WT and sGCα1^-/-mice breathed air supplemented with or without NO gas (80 ppm) for 2h. Pulmonary ICER mRNA levels were induced in WT mice breathing air supplemented with NO (*p<0.0001 lungs of WT mice breathing air vs breathing air supplemented with NO). sGCα1^-/-mice showed a minor trend to induce ICER mRNA levels when breathing NO, that was significantly less than the induction of WT mice that breathed air supplemented with NO (†p<0.0001 sGCα1^-/-mice breathing NO vs WT mice breathing air with iNO. WT mice increased hin=5 mice in each group; mean (SD).

Fig. 3

Panel A: GSNO mediated cAMP response element (CRE)-dependent gene transcription is sGC-dependent. RPaSMC transfected with CRE-luciferase reporter plasmid were preincubated with ODQ (10 μmol/L), KT5823 (1 μmol/L), Rp-8-pCPT-cGMP (100 μmol/L), or Rp-8-Br-cGMP (100 μmol/L) for 30min and then stimulated with or without GSNO (100 μmol/L) for 4h. The results are expressed as firefly luciferase activity normalized to control renilla luciferase activity. *p<0.0005 GSNO treated cells vs control cells, either unstimulated, or pretreated with KT5823, Rp-8-pCPT-cGMP or RP-8-Br-cGMP; and †p<0.0001 ODQ- vs vehicle-pretreated treated RPaSMC stimulated with GSNO. Panel B: RPaSMCs incubated with Rolipram (Rol) plus GSNO induce CRE-dependent gene transcription more effectively than RPaSMC treated with GSNO alone RPaSMCtreated with GSNO plus milrinone (mil). RPaSMC transfected with CRE-luciferase reporter plasmid were pretreated with H89 (10 μmol/L), Mil (30 μmol/L) or Rol (10 μmol/L) for 30 min and stimulated with or without GSNO (100 μmol/L) for 4h. Firefly and renilla luciferase activities were measured. *p<0.001 versus control, # p<0.001 vs GSNO only. n≥3 for each condition; mean (SD).

Fig. 4

Panel A: GSNO mediated CREB phosphorylation is sGC- and PKA-dependent. RPaSMC preincubated without or with ODQ (10 μmol/L) or H-89 (10 μmol/L) were stimulated with GSNO or forskolin. Phosphorylated CREB (p-CREB) and total CREB were detected with immunoblotting. Panel B: Activation of CREB is required for induction of CRE luciferase activity in GSNO-stimulated RPaSMC. RPaSMC were transfected with a CRE-responsive luciferase plasmid without or with a plasmid specifying a dominant-negative CREB (CREBml) and stimulated with and without GSNO. Four hours later,firefly and renilla luciferase activities were measured. n=3; means (SD); *p<0.001 vs control, †p<0.001 vs GSNO control). Panel C: Activation of CREB is required for GSNO-mediated ICER induction. Uninfected cells and cells infected with Ad.CREB-ml (100 and 200 M.O.I.) or Ad.βgal (200 M.O.I.) were incubated with GSNO. Membranes were hybridized with DNA probes specifying ICER. Ethidium bromide staining of 28S rRNA confirmed the equal loading of RNA samples.

Fig. 5

Intracellular calcium is required for GSNO-mediated ICER induction. RPaSMC were pretreated with the intracellular Ca²⁺-chelator, BAPTA-AM (50 μmol/L; Panel A) store-operated Ca²⁺inhibitors, 2-ABP and SKF-96365 (Panel B); or the voltage dependent Ca²⁺-channel inhibitor, nifedipine (100 μmol/L) (Panel C), followed by incubation with or without GSNO (100 μmol/L). ICER mRNA levels were detected using qRT-PCR. n=3; mean (SD). (Panel A)*p=0.041 control vs GSNO-treated cells, †p=0.042 vs GSNO-treated cells without BAPTA-AM. (Panel B)Left panel: *p=0.037 control vs GSNO-treated cells, †p=0.017 RPaSMC pretreated with 2-ABP followed by GSNO vs. GSNO-treated cells. Right panel: *p=0.007 control vs GSNO-treated cells, †p=0.008 RPaSMC pretreated with SKF followed by GSNO vs. GSNO-treated cells. (Panel C)*p=0.02 control vs GSNO treated cells. Incubation with nifedipine did not alter ICER mRNA levels. (Panel D)GSNO stimulates CREB phosphorylation in presence of 2-ABP. Cells were preincubated with or without 2-ABP for 30 min and exposed to GSNO (100 μmol/L) for 4hrs. p-CREB and CREB phosphorylation were detected using immunoblot. Incubation of RPaSMC with GSNO induced CREB phosphorylation. CREB phosphorylation was not prevented by preincubation with the store-operated Ca^2*inhibitor 2-ABP. A representative result of 3 different experiments is shown.

Fig. 6

Panel A: ICER inhibits cAMP-dependent gene transcription. RPaSMC were transiently transfected with the CRE-luciferase reporter plasmid and a control plasmid (pcDNA3) or a plasmid encoding ICER (pcDNA3-ICER) for 24h. Transfected cells were treated with GSNO (100 μmol/L) or forskolin (FK; 10 μmol/L) for 4h. Firefly and renilla luciferase activities was measured. n=3, mean (SD); *p<0.001 RPaSMC transfected with pcDNA3 and stimulated with GSNO vs cells transfected with pcDNA3-ICER stimulated with GSNO, †p<0.001 RPaSMC transfected with pcDNA3 and stimulated with forskolin vs cells transfected with pcDNA3-ICER and stimulated with forskolin. Panel B: ICER inhibits cAMP-stimulated CRE-dependent gene expression. Uninfected RPaSMC and RPaSMC infected with Ad.ICER or Ad.βgal (100 M.O.I. each) were incubated with FK (10 μmol/L) for 2h. RNA blot hybridization was performed using radiolabeled DNA probes specifying hexokinase2 (HK2) and γ-glutamylcysteine synthetase heavy chain (GCS:hc) probes.

Fig. 7

Proposed pathway of NO-mediated ICER induction. NO induces ICER expression in RPaSMC via cGMP–mediated inhibition of phosphodiesterase3 (PDE3), thereby elevating cAMP concentrations (likely in a specific intracellular compartment), leading to activation of PKA and CREB phosphorylation. Phosphorylated CREB binds to the cAMP-response element (CRE) leading to activation of gene transcription. Ca²⁺is also required for ICER gene induction by GSNO. Once the ICER gene is induced, it inhibits CRE-dependent transcription and its own expression via a negative feedback loop.