Fig. 7 Localization of the element(s) responsible for the transcriptional suppression by PPAR-γ. A, Rat VSMCs were transfected with either −1969/+104-luc, −987/+104-luc, −331/+104-luc, or− 58/+104-luc. Bar graphs represent mean ± se (%) of luciferase activity (line 1 as 100%) (n = 6). *, P < 0.01, as compared with line 1; **, P < 0.01, as compared with line 5;*** , P < 0.01, as compared with line 9;**** , P < 0.01, as compared with line 13. B, Rat VSMCs were transfected either with −58/+104-luc, −58/-1-luc,− 34/-1-luc, −1/-34-luc, or mutated −58/-1-luc. Bar graphs represent mean ± se (%) of luciferase activity (line 1 as 100%) (n = 6). Arrows indicate the orientation of the promoter. TATA, TATA box; GC, GC-box-related sequence. *, P < 0.01, as compared with line 1;** , P < 0.01, as compared with line 5. C, Rat VSMCs were transfected with either −1969/+104-luc or mutated− 1969/+104-luc. Bar graphs represent mean ± se (%) of luciferase activity (line 1 as 100%) (n = 6). *, P < 0.01, as compared with line 1. The shaded area in A, B, and C is 5′-UTR. The cells were treated with indicated concentrations of 15dPGJ₂ or troglitazone for 12 h. Transcriptional Suppression of Type 1 Angiotensin II Receptor Gene Expression by Peroxisome Proliferator-Activated Receptor-γ in Vascular Smooth Muscle Cells* Endocrinology. 2001;142(7):3125-3134. doi:10.1210/endo.142.7.8272 Endocrinology | Copyright © 2001 by The Endocrine Society

Fig. 10 Effect of Sp1 and PPAR-γ overexpression on the AT1R gene promoter. Rat VSMCs were transfected with expression plasmid of Sp1 alone (lines 2, 7, and 10) or Sp1 plus PPAR-γ1 (lines 3–5, 8, and 11) with either −58/−1-luc (lines 1–5), −34/−1-luc (lines 6–8), or mutated −58/−1-luc (lines 9–11). In some experiments, the cells were treated with 2.5 μm 15dPGJ₂ (line 4) or 10 μm troglitazone (line 5). Bar graphs represent mean ± se (%) of luciferase activity (line 1 as 100%) (n = 6). TATA, TATA box; GC, GC-box-related sequence. *, P < 0.01, as compared with line 1; **, P < 0.05, as compared with line 2; ***, P < 0.01, as compared with line 2. Transcriptional Suppression of Type 1 Angiotensin II Receptor Gene Expression by Peroxisome Proliferator-Activated Receptor-γ in Vascular Smooth Muscle Cells* Endocrinology. 2001;142(7):3125-3134. doi:10.1210/endo.142.7.8272 Endocrinology | Copyright © 2001 by The Endocrine Society

Fig. 6 Effect of PPAR-γ overexpression on the AT1R gene promoter Fig. 6 Effect of PPAR-γ overexpression on the AT1R gene promoter. Rat VSMCs were transfected with expression plasmid of PPAR-γ1 (lines 2, 7, 11, and 15), PPAR-α (line 5), RXR-α (lines 3, 8, 12, and 16), or both (lines 4, 9, 13, and 17) as well as− 1969/+104-luc. The transfected cells were treated with 2.5μ m 15dPGJ₂ (lines 6–9), 10 μm troglitazone (lines 10–13), or 50 μm troglitazone (lines 14–17) for 12 h. Bar graphs represent mean ± se (%) of luciferase activity (line 1 as 100%) (n = 6). *, P < 0.01, as compared with line 1; **, P < 0.01, as compared with line 6; ***, P < 0.01, as compared with line 10; ****, P < 0.05, as compared with line 14. Transcriptional Suppression of Type 1 Angiotensin II Receptor Gene Expression by Peroxisome Proliferator-Activated Receptor-γ in Vascular Smooth Muscle Cells* Endocrinology. 2001;142(7):3125-3134. doi:10.1210/endo.142.7.8272 Endocrinology | Copyright © 2001 by The Endocrine Society

Fig. 5 Expression of PPAR-γ mRNA in VSMCs Fig. 5 Expression of PPAR-γ mRNA in VSMCs. Total RNAs extracted from rat VSMCs were subjected to semiquantitative RT-PCR using specific primers for rat PPAR-γ and rat GAPDH. The lower band, indicated by an arrow, is PCR products from rat PPAR-γ mRNA (250 bp), and the upper band indicated by an arrow is PCR products from rat GAPDH mRNA (308 bp). Lane 1, size marker; lane 2, PCR products of rat VSMCs total RNA in the absence of RT (note that no PCR products are observed); lane 3, RT-PCR products of rat VSMCs total RNA. Transcriptional Suppression of Type 1 Angiotensin II Receptor Gene Expression by Peroxisome Proliferator-Activated Receptor-γ in Vascular Smooth Muscle Cells* Endocrinology. 2001;142(7):3125-3134. doi:10.1210/endo.142.7.8272 Endocrinology | Copyright © 2001 by The Endocrine Society

Fig. 4 Effect of PPAR-γ ligands on AT1R gene promoter activity Fig. 4 Effect of PPAR-γ ligands on AT1R gene promoter activity. Rat VSMCs transfected with −1969/+104-luc were treated with indicated concentrations of 15dPGJ₂, troglitazone, rosiglitazone, LTB₄, or 9cRA for 12 h and harvested for measuring luciferase activities. Line 1, no ligands. Bar graphs represent mean ± se (%) of luciferase activity (line 1 as 100%) (n = 6). *, P < 0.01 as compared with line 1. **, P < 0.05 as compared with line 1. Transcriptional Suppression of Type 1 Angiotensin II Receptor Gene Expression by Peroxisome Proliferator-Activated Receptor-γ in Vascular Smooth Muscle Cells* Endocrinology. 2001;142(7):3125-3134. doi:10.1210/endo.142.7.8272 Endocrinology | Copyright © 2001 by The Endocrine Society

Fig. 3 Effect of PPAR-γ ligands on AII induced-³H-thymidine incorporation. Rat VSMCs incubated either without (line 1) or with 1 μm AII (line 2), AII plus 1 μm candesartan (line 3), AII plus 1μ m 15dPGJ₂ (line 4), or AII plus 10μ m troglitazone (line 5) for 12 h with 1 μCi/ml ³H-thymidine were harvested, and their ³H-thymidine incorporation was measured. The bar graph represents the mean ± sd of the percentage of ³H-thymidine incorporation compared with control value (line 1 as 100%) (n = 6). *, P< 0.01, as compared with line 2. Transcriptional Suppression of Type 1 Angiotensin II Receptor Gene Expression by Peroxisome Proliferator-Activated Receptor-γ in Vascular Smooth Muscle Cells* Endocrinology. 2001;142(7):3125-3134. doi:10.1210/endo.142.7.8272 Endocrinology | Copyright © 2001 by The Endocrine Society

Fig. 2 Effect of PPAR-γ ligands on AT1R mRNA stability Fig. 2 Effect of PPAR-γ ligands on AT1R mRNA stability. Rat VSMCs were treated in the absence (□) or presence of 1μ m 15dPGJ₂ () or 10 μm troglitazone (▪) for 12 h. Actinomycin D (5μ g/ml) was then added and incubated for an additional 4 or 8 h. Extracted RNAs were then processed to Northern blot analysis. The horizontal line represents mean ± se (%) of AT1R mRNA expression levels normalized by densities of 28S rRNA (time 0 as 100%) (n = 4). Transcriptional Suppression of Type 1 Angiotensin II Receptor Gene Expression by Peroxisome Proliferator-Activated Receptor-γ in Vascular Smooth Muscle Cells* Endocrinology. 2001;142(7):3125-3134. doi:10.1210/endo.142.7.8272 Endocrinology | Copyright © 2001 by The Endocrine Society

Fig. 1 Effect of PPAR-γ ligands on AT1R expression Fig. 1 Effect of PPAR-γ ligands on AT1R expression. A, Effect of PPAR-γ ligands on AT1R mRNA expression. The top panel shows a representative result. The upper bands represent AT1R mRNA expression (3.5-kb, indicated by an arrow) in rat VSMC total RNA demonstrated by Northern blot analysis. The lower bands represent ethidium bromide staining of 28S rRNA (indicated by an arrow) of the corresponding samples. Bar graphs in the bottom panel represent mean± se (%) of AT1R mRNA expression levels normalized by densities of 28S rRNA (incubated in the absence of ligands as 100%) (n = 4). *, P < 0.01, as compared with the control value. B, Effect of PPAR-γ ligands on AT1R protein expression. The bottom panel shows a representative blot. Anti-AT1R antibody detected AT1R protein at the expected molecular mass (41-kDa, indicated by an arrow) in rat VSMC membrane proteins (lanes 1–4). Bar graphs represent mean ± se (%) of AT1R protein expression levels (lane 1 as 100%) (n = 4). *, P < 0.01, as compared with lane 1 (control). Transcriptional Suppression of Type 1 Angiotensin II Receptor Gene Expression by Peroxisome Proliferator-Activated Receptor-γ in Vascular Smooth Muscle Cells* Endocrinology. 2001;142(7):3125-3134. doi:10.1210/endo.142.7.8272 Endocrinology | Copyright © 2001 by The Endocrine Society

Fig. 8 Interaction between PPAR-γ/Sp1 and the AT1R gene promoter Fig. 8 Interaction between PPAR-γ/Sp1 and the AT1R gene promoter. A, Interaction between PPAR-γ and the AT1R gene promoter. ³²P-labeled oligonucleotides containing HMG-CoA synthase gene PPRE (HMGS-PPRE; lanes 1–4) or the −58/−34 region of the AT1R gene promoter (−58/−34; lanes 5–8) were incubated with 2 μl in vitro translated PPAR-γ1 (PPAR-γ; lanes 4 and 8), RXR-α (RXR-α; lanes 2 and 6), or both factors (lanes 3 and 7). Total amounts of RL were adjusted to 4 μl by adding 4 μl (lanes 1 and 5) or 2 μl (lanes 2, 4, 6, and 8) unprogrammed RL to each lane. PPAR-γ/RXR-α heterodimer is indicated by an arrow. B, Interaction between Sp1 and the AT1R gene promoter. ³²P-labeled oligonucleotides containing the −58/−34 region of the AT1R gene promoter (−58/−34; lanes 1–3) were incubated with 2 μl unprogrammed RL (RL; lane 2) or 2 μl in vitro translated Sp1 (lane 3). ³²P-labeled oligonucleotides containing the mutated GC-box-related sequence in the− 58/−34 region (GC Mut) were also incubated with 2 μl in vitro translated Sp1 (lane 4). Sp1-DNA complex is indicated by an arrow (Sp1). The asterisk represents nonspecific binding formed with unprogrammed RL. Transcriptional Suppression of Type 1 Angiotensin II Receptor Gene Expression by Peroxisome Proliferator-Activated Receptor-γ in Vascular Smooth Muscle Cells* Endocrinology. 2001;142(7):3125-3134. doi:10.1210/endo.142.7.8272 Endocrinology | Copyright © 2001 by The Endocrine Society

Fig. 9 Interaction between VSMC nuclear extracts and the AT1R gene promoter. ³²P-labeled oligonucleotides containing the −58/−34 region of the AT1R gene promoter (−58/−34; lanes 1–7) were incubated with 2 μg VSMC nuclear extracts (VSMC NE) and were sequentially incubated with 1 μl anti-Sp1 antibody (lane 2), anti-Sp2 antibody (lane 3), anti-Sp3 antibody (lane 4), or anti-Sp4 antibody (lane 5). Protein-DNA complexes are indicated by arrows (Protein/DNA Complexes). Supershifted bands by anti-Sp1 antibody (lane 2) (Supershifted Sp1), anti-Sp2 antibody (lane 3), or by anti-Sp3 antibody (lane 4) (Supershifted Sp2/Sp3) are also indicated by arrows. ³²P-labeled oligonucleotides containing the mutated GC-box-related sequence in the −58/−34 region (GC Mut) were also used (lane 8). Unlabeled oligonucleotides (100-fold excess) containing the −58/−34 region (−58/−34; lane 6) or SV40 early promoter Sp1 site (Sp1; lane 7) were used for competition. Transcriptional Suppression of Type 1 Angiotensin II Receptor Gene Expression by Peroxisome Proliferator-Activated Receptor-γ in Vascular Smooth Muscle Cells* Endocrinology. 2001;142(7):3125-3134. doi:10.1210/endo.142.7.8272 Endocrinology | Copyright © 2001 by The Endocrine Society

Fig. 11 Effect of PPAR-γ on Sp1 binding to the AT1R gene promoter Fig. 11 Effect of PPAR-γ on Sp1 binding to the AT1R gene promoter. ³²P-labeled oligonucleotides containing the− 58/−34 region of the AT1R gene promoter (−58/−34; lanes 1–4) were incubated with 2 μg VSMC nuclear extracts (VSMC NE) in the absence (lane 1) or presence of 2 μl in vitro translated PPAR-γ1 (PPAR-γ; lane 2), RXR-α (RXR-α; lane 3), or both factors (lane 4). Total amounts of RL were adjusted to 4 μl by adding 4 μl (lane 1) or 2 μl (lanes 2 and 3) unprogrammed RL to each lane. Protein-DNA complexes are indicated by arrows (Protein/DNA Complexes). Transcriptional Suppression of Type 1 Angiotensin II Receptor Gene Expression by Peroxisome Proliferator-Activated Receptor-γ in Vascular Smooth Muscle Cells* Endocrinology. 2001;142(7):3125-3134. doi:10.1210/endo.142.7.8272 Endocrinology | Copyright © 2001 by The Endocrine Society

Fig. 12 Protein-protein interaction between PPAR-γ and Sp1 Fig. 12 Protein-protein interaction between PPAR-γ and Sp1. GST pull-down assays using GST alone (lane 2) or GST-PPAR-γ fusion protein (lanes 3 and 4) and in vitro translated ³⁵S-labeled Sp1 (5 μl) were performed. An arrow indicates Sp1. In lane 4, the sample was incubated in the presence of 50 μm troglitazone. Lane 1 represents the 50% volume of in vitro translated ³⁵S-labeled Sp1 used in the assays. Transcriptional Suppression of Type 1 Angiotensin II Receptor Gene Expression by Peroxisome Proliferator-Activated Receptor-γ in Vascular Smooth Muscle Cells* Endocrinology. 2001;142(7):3125-3134. doi:10.1210/endo.142.7.8272 Endocrinology | Copyright © 2001 by The Endocrine Society