Angiotensin II represses Npr1 expression and receptor function by recruitment of transcription factors CREB and HSF-4a and activation of HDACs

Figures

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  Figure 1. Effect of ANG II on luciferase activity of Npr1 promoter deletion constructs in a time- and dose-dependent manner. (A) Different deletional constructs of the 5′-flanking region of mouse Npr1 promoter, inserted upstream of the firefly luciferase gene. The numbers next to the schematics of Npr1 promoter indicate the nucleotide positions for the 5′ and 3′ ends of the constructs, respectively. MMCs were transiently transfected with 1 µg of the deletion constructs and 0.3 µg of pRL-TK and after 24 h, cells were treated with ANG II (10 nM) for 16 h. (B) MMCs were transiently transfected with 1 µg of Npr1 promoter construct −1182/+55 bp and 0.3 µg of pRL-TK and were treated with varying concentrations of ANG II (0.01 nM to 1000 nM) for 16 h. (C) Cells were transfected with Npr1 promoter construct −1182/+55 bp and treated for varying time periods of 0, 8, 16, 24, and 36 h with ANG II (10 nM). Normalized luciferase activity is shown as a percentage of the activity of pNPRA-luc1. (D) Schematic representation of the putative cis-acting elements presents in the region (−1982 to +55 bp) of Npr1 promoter. Bars represent the mean ± S.E. of 6–8 independent experiments in triplicates. *p < 0.05;…

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  Figure 2. Effect of ANG II on the expression of Npr1 mRNA and protein levels of NPRA in MMCs. (A) Effect of increasing concentration of ANG II with varying time periods (0, 8, 16, 24, and 36 h) of treatments on the Npr1 promoter activity. (B) Effect of ANG II (10 nM) on Npr1 mRNA expression as determined by quantitative real-time RT-PCR. (C) Western blot and densitometry analyses of NPRA protein (135 kDa) levels in MMCs treated with ANG II and β-actin is shown as loading control (full-length image: Supplementary Fig. 1). (D) Intracellular accumulation of cGMP in MMCs-treated cells with ANG II (10 nM) and ANP (100 nM). Bar represents the mean ± SE of 6 independent experiments in triplicates. The time-course are indicated as 8 h (Blue), 16 h (Pink), 24 h (Green), and 36 h (Red) **p < 0.01; ***p < 0.001.

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  Figure 3. Effect of ANG II Type 1 and Type 2 receptor antagonists on ANG II-mediated transcriptional repression of Npr1 gene promoter in the ∆R1 and ∆R5 constructs. (A) Schematic representation of the Npr1 promoter constructs ∆R1 (−1182 to −1127 bp) and ∆R5 (−984 to −914 bp). MMCs were transiently transfected with 1 µg of either ∆R1 construct (B,C) or ∆R5 construct (D,E) and 0.3 µg of pRL-TK. After 24 h, the cells were treated with either 100 nM PD 123319 (B,D) or 100 nM candesartan (C,E), followed by incubation with 10 nM ANG II for 16 h. Normalized luciferase activity is shown as a percentage of the activity of an untreated group. The results are expressed as mean + SE from 7–8 independent experiments. **p < 0.01.

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  Figure 4. Effect of protein kinase inhibitors on ANG II-mediated transcriptional repression of Npr1 gene promoter in ∆R1 and ∆R5 constructs. MMCs were transiently transfected with 1 µg of ∆R1 or ∆R5 construct and 0.3 µg of pRL-TK. After 24 h, cells were treated with 100 nM H-89 dihydrochloride (A,D), 100 nM wortmannin (B,E), or 100 nM genistein (C,F) and were incubated further with 10 nM ANG II for 16 h. Normalized luciferase activity is shown as a percentage of the activity of untreated groups. (G) Schematic representation of the effect of protein kinase inhibitors on Npr1 gene transcription. The results are expressed as mean + SE from 7–8 independent experiments. **p < 0.01.

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  Figure 5. Effect of mutation in ∆R1 and ∆R5 constructs and overexpression of HSF-4a and CREB transcription factors in ANG II-mediated transcriptional repression of Npr1 promoter. (A) Schematic diagram showing the sequence of the wild-type and mutated HSF-4a binding site in the Npr1 promoter. Underlined nucleotides show the mutated sequence. (B) MMCs were transiently transfected with wild-type ∆R1 or mutant ∆R1 constructs, treated with ANG II for 16 h, and the promoter activity was measured. (C) MMCs were cotransfected with HSF-4a expression plasmid and ∆R1 promoter construct, treated with 10 nM ANG II for 16 h, and the luciferase activity was measured. Normalized luciferase activity is shown as a percentage of the activity of untreated control groups. (D) Schematic diagram showing the sequence of the wild-type and mutated CREB binding site in the Npr1 promoter. (E) MMCs were transiently transfected with wild-type ∆R5 or mutant ∆R5 construct, treated with ANG II for 16 h, and luciferase promoter activity was measured. (F) MMCs were cotransfected with CREB expression plasmid and ∆R5 promoter construct treated with ANG II for 16 h, and the luciferase activity was measured. (G) Western blot…

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  Figure 6. Gel electrophoretic mobility shift assay and UV crosslinking analysis of HSF-4a and CREB: Double-stranded oligonucleotides containing the consensus binding site for HSF-4a and CREB were end-labeled with [γ-p32]ATP. The DNA-protein complex was resolved from the free-labeled DNA by electrophoresis in 4% native polyacrylamide gel. (A,B) Representative autoradiographs of HSF-4a and CREB binding activity in nuclear extract from ANG II treated cells. Lane 1 shows the labeled probe without nuclear extract. Line 2 indicates the nuclear protein from untreated MMCs, lane 3 indicates nuclear protein complex binding in 10 nM ANG II-treated cells. Lane 4 shows the replacement of specific binding with 100 x excess concentrations of cold probe. Lane 5 indicates the absence of binding pattern with mutant probe, and lane 6 shows the mutant probe in the presence of nuclear extract. (C) UV crosslinking analysis in ANG II-treated MMCs nuclear extract. Lane 1 shows ∆R1a probe alone and lane 2 indicates binding of nuclear protein from ANG II-treated nuclear extract. (D) UV cross linking analysis of ANG II-treated MMCs nuclear extract. Lane 1 shows only ∆R5a probe alone and lane 2 indicates binding of…

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  Figure 7. Effect of ANG II on HDAC activity and protein levels of class I HDACs in MMCs. (A) Effect of increasing concentrations of ANG II on total HDAC activity. (B) Western blot and densitometry analyses of HDAC 1, 2, and 3 protein expression in ANG II-treated cells. H3 was used as loading control (full-length image: Supplementary Fig. 4). (C) Effect of MGCD0103 on Npr1 mRNA expression and (D) NPRA protein (135 kDa) levels in ANG II-pretreated MMCs (full-length image: Supplementary Fig. 5). (E) Western blot and densitometry analyses of acetylated histones H3-K9/14 and H4-K8 protein expression in ANG II-treated cells (full-length image: Supplementary Fig. 6). The results are expressed as mean ± SE from 6–8 independent experiments. WB, Western blot; *p < 0.05; **p < 0.01; **p < 0.001.

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  Figure 8. Effect of ANG II treatment on Npr1 gene expression and ANP-induced vasorelaxation mouse of aortic rings of male mice ex vivo. (A) Npr1 mRNA expression by real time RT-PCR and (B) Western blot analysis of NPRA protein (135 kDa) expression in ANG II-induced aortic rings. β-actin expression was used as loading controls (full-length image: Supplementary Fig. 7). (C) Vasorelaxation of aortic rings in the presence of ANP with or without ANG II treatments. (D) Overall, schematic model showing the antagonistic effect of ANG II on Npr1 gene transcription and functional expression using cultured MMCs and aortic rings containing VSMCs. The model depicts that ANG II represses the Npr1 gene transcription and expression via its AT1 receptor that enhances the PI-3K and TK signaling and recruitment of HSF-4a and CREB to the Npr1 promoter. Furthermore, ANG II induces the HDAC activity and represses the acetylation of histones H3-K9/14 and H4-K8. The upward arrow indicates an increased HDAC activity and enhanced binding of HSF-4a and CREB to the Npr1 promoter. The downward arrow indicates decreased Npr1 gene transcription and reduced NPRA and cGMP levels, thereby the decreased renal and vascular…