GTP cyclohydrolase I expression is regulated by nitric oxide: role of cyclic AMP

Abstract

Our previous studies have demonstrated that nitric oxide (NO) leads to nitric oxide synthase (NOS) uncoupling and an increase in NOS-derived superoxide. However, the cause of this uncoupling has not been adequately resolved. The pteridine cofactor tetrahydrobiopterin (BH(4)) is a critical determinant of endothelial NOS (eNOS) activity and coupling, and GTP cyclohydrolase I (GCH1) is the rate-limiting enzyme in its generation. Thus the initial purpose of this study was to determine whether decreases in BH(4) could underlie, at least in part, the NO-mediated uncoupling of eNOS we have observed both in vitro and in vivo. Initially we evaluated the effect of inhaled NO levels on GCH1 expression and BH(4) levels in the intact lamb. Contrary to our hypothesis, we found that there was a significant increase in both plasma BH4 levels and peripheral lung GCH1 protein levels. Furthermore, in vitro, we found that exposure to the NO donor spermine NONOate (SPNONO) led to an increase in GCH1 protein and BH(4) levels in both COS-7 and pulmonary arterial endothelial cells. However, SPNONO treatment also caused a significant increase in phospho-cAMP response element binding protein (CREB) levels, as detected by Western blot analysis, and significantly increased cAMP levels, as detected by enzyme immunoassay. Furthermore, utilizing GCH1 promoter fragments fused to a luciferase reporter gene, we found that GCH1 promoter activity was enhanced by SPNONO in a CREB-dependent manner, and electromobility shift assays revealed an NO-dependent increase in the nuclear binding of CREB. These data suggest that NO increases BH(4) levels through a cAMP/CREB-mediated increase in GCH1 transcription and that the eNOS uncoupling associated with exogenous NO does not involved reduced BH(4) levels.

Figures

Fig. 1.

Tetrahydriobiopterin (BH4) and GTP cyclohydrolase I (GCH1) protein levels in lambs exposed to inhaled nitric oxide (NO). A: 4-wk-old lambs were exposed to inhaled NO (40 ppm, 0–24 h). Plasma BH4 levels were measured before (Pre) and after (Post) NO inhalation using HPLC. There was an increase in plasma BH4 levels post-inhaled NO. Values are means ± SE; n = 4. *P < 0.05 vs. pre-inhaled NO. B: protein extracts (50 μg) prepared from peripheral lung pre- and post-inhaled NO were analyzed using a specific antisera raised against GCH1. GCH1 expression was also normalized for loading using β-actin. A representative blot is shown. C: there is a significant increase in densitometric value for normalized GCH1 protein in peripheral lung tissue post-inhaled NO. Values are means ± SE with pre-inhaled NO values normalized to 1.0; n = 4. *P < 0.05 vs. pre-inhaled NO.

Fig. 2.

Effect of exogenous NO on GCH1 protein and BH4 levels in COS-7 cells. A: COS-7 cells were treated with increasing concentration of the NO donor sperminine NONOate (SPNONO; 0–100 μM, 4 h), and then whole cell extracts (10 μg) were subjected to Western blot analysis using an antibody raised against GCH1. GCH1 expression was also normalized for loading using β-actin. A representative blot is shown. B: the bar graph shows the relative change in GCH1 protein levels with SPNONO treatment. There was a dose-dependent increase in GCH1 protein levels. Values are means ± SE; n = 3. *P < 0.05 vs. untreated cells. †P < 0.05 vs. 20 μM SPNONO. C: COS-7 were treated with SPNONO (50 μM, 24 h), and BH4 levels were then measured using HPLC. There was a significant increase in BH4 levels with exogenous NO treatment. Values are means ± SE; n = 6. *P < 0.05 vs. untreated cells.

Fig. 3.

Exogenous NO stimulates cellular cAMP levels and phosphorylated cAMP response element binding protein (pCREB) levels in COS-7 cells. A: COS-7 cells were treated with SPNONO (0–100 μM, 4 h), and cAMP levels were determined by EIA. Exogenous NO increased cellular levels of cAMP in COS-7 cells. Values are means ± SE; n = 3. *P < 0.05 vs. untreated cells. B: COS-7 cells were treated with increasing concentrations of SPNONO (0–100 μM, 4 h), and then whole cell extracts (10 μg) were subjected to Western blot analysis using an antibody raised against pCREB. Relative pCREB levels were obtained by reprobing the membranes with an antibody specific to CREB. A representative blot is shown. C: although total CREB levels were unchanged, exogenous NO caused a dose-dependent increase in pCREB levels. Values are means ± SE; n = 3. *P < 0.05 vs. untreated cells. †P < 0.05 vs. 20 μM SPNONO.

Fig. 4.

Exogenous NO stimulates GCH1 promoter activity in COS-7 cells via CREB. A: the promoter constructs used contain 446 bp (613 GTPCH) or 146 bp (313 GTPCH) of the human GCH1 promoter sequence upstream of the transcription start site linked to a luciferase reporter gene. 313mutGTPCH is identical to the 313 GTPCH construct except for a 4-bp mutation in the CRE consensus sequence at −89. B: COS-7 cells were transfected with either the 613 GTPCH or the 313 GTPCH construct and then treated with increasing concentrations of SPNONO (0–50 μM, 16 h), and the luciferase activity was determined. NO significantly increased both 613 GTPCH and 313 GTPCH promoter activities. Values are means ± SE; n = 6. *P < 0.05 vs. untreated cells. C: COS-7 cells were transfected with the 313mutGTPCH construct and then treated with increasing concentrations of SPNONO (0–50 μM, 16 h), and the luciferase activity was determined. The mutation of the CRE attenuated the NO-mediated increase in GCH1 promoter activity. Values are means ± SE; n = 6. *P < 0.05 vs. untreated cells.

Fig. 5.

Modulation of cAMP-PKA signaling alters the NO-mediated stimulation of CREB phopshorylation and GCH1 promoter activity in COS-7 cells. A: COS-7 cells were treated with SPNONO (50 μM, 4 h) in the presence or absence of the PKA inhibitors H-89 (10 μM) or Rp-cAMPS (200 μM). Whole cell extracts (10 μg) were then subjected to SDS-PAGE, and immunoblots were performed using an antibody raised against pCREB. Loading was normalized by reprobing with an antibody specific to CREB. Values are means ± SE; n = 4. *P < 0.05 vs. untreated cells. B and C: COS-7 cells were transfected with the 313 GTPCH and 313mutGTPCH promoter constructs and treated with forskolin (30 μM, 16 h) in the presence or absence of the PKA inhibitors H-89 (10 μM) or Rp-cAMPS (200 μM). The luciferase activities were then determined. Forskolin significantly increased 313 GTPCH promoter activity, and this increase was attenuated in the presence of H-89 and Rp-cAMPS. Mutation of the CRE abolished the effect of both forskolin and PKA inhibition on GCH1 promoter activity. Values are means ± SE; n = 6. *P < 0.05 vs. untreated cells. †P < 0.05 vs. SPNONO alone.

Fig. 6.

Effect of exogenous NO on CREB binding to the CRE of the GCH1 promoter. A: COS-7 cells were treated with increasing concentration of SPNONO (0–50μM, 4 h) then nuclear extracts (10 μg) were exposed to biotinylated CRE oligonucleotide and analyzed using electrophoretic mobility shift assays. Arrow indicates the position of the CREB complex. Lane marked Free is biotinylated oligonucleotide without extract. A representative image is shown. B. Band intensities were calculated by densitometeric analysis. Binding is expressed as fold changes relative to untreated control. The data indicate that exogenous NO increases CREB binding to the GCH1 cAMP-response element (CRE). The values expressed are means ± SEM, n = 6. *P < 0.05 vs. untreated. C. To verify the specificity of CREB binding, a competition assay was performed using 50 × and 100 × excess of unlabeled CREB oligonucleotide. There was a decrease in the binding of CREB with 50 × and 100 × excess of CREB oligonucleotide. A representative image is shown from an n = 4. D. Supershift assays were also conducted by incubating nuclear extracts (NE) with polyclonal antibodies (4 μg) specific to either CREB or phosphorylated-CREB. Both the CREB and phosphorylated CREB antibodies retard the migration of the shifted complex confirming the presence of phosphorylated CREB in the complex. The supershifted complex is shown with an arrow. Panel is representative of three independent supershift experiments.

Fig. 7.

The effects of exogenous NO on GTP cyclohydrolase I are recapitulated in pulmonary arterial endothelial cells. A: PAECs were treated with SPNONO (0–50 μM, 4h) and cAMP levels determined by EIA. Exogenous NO increases cellular levels of cAMP in PAECs. The values expressed are means ± SEM, n = 4, *P < 0.05 vs. untreated cells. B: PAECs were transfected with the 313 GTPCH promoter construct then treated with SPNONO (0–50 μM, 16h) and the luciferase activities determined. SPNONO significantly increased 313 GTPCH promoter activity. The values expressed are means ± SEM, n = 6; *P < 0.05 vs. untreated. C: PAECs were transfected with the 313mut GTPCH promoter construct then treated with SPNONO (0–50 μM, 16h) and the luciferase activities determined. Mutation of the CRE attenuated the NO-mediated increase in GCH1 promoter activity. The values expressed are means ± SEM, n = 6. D: PAECs cells were treated with increasing concentration of SPNONO (0–50 μM, 4h) then whole cell extracts (10μg) were subjected to Western blot analysis using an antibody raised against GCH1. GCH1 expression was also normalized for loading using β-actin. Figure is a representative blot. E: The bar graph shows the relative change in GCH1 expression with SPNONO treatment. Exogenous NO induces a dose-dependent increase in GCH1 protein levels. Values expressed are means ± SEM, n = 3, *P < 0.05 vs. untreated. F: PAEC were treated with SPNONO (50 μM, 24h) and BH4 levels were then measured using high-performance liquid chromatography. There is a significant increase in BH4 levels with exogenous NO treatment. Values expressed are means ± SEM, n = 6, *P < 0.05 vs. untreated cells.