Expression of Nur77 in vascular smooth muscle cells (VSMCs) in vivo and in vitro in response to angiotensin II (Ang II). A and B, Representative images of dual immunofluorescence staining of Nur77 (green) and SM-α-actin (red), and quantification of the ratio of Nur77-positive area to α-actin–positive area (n=6, *P<0.05). C, Nur77 mRNA expression measured by real-time polymerase chain reaction (PCR) in thoracic aorta sections after systemic Ang II infusion for 0, 7, and 14 days. Values are expressed as the fold change relative to the control (n=6, *P<0.05). D, Primary rat VSMCs were serum-starved and stimulated with Ang II (500 nmol/L) for the indicated time. The mRNA level of Nur77 was quantified by real-time PCR (n=3, *P<0.05 vs 0 h). E, Primary VSMCs were treated with increasing concentrations of Ang II for 3 h. Protein expression of Nur77 was examined by Western blotting (n=3, *P<0.05 vs 0 nmol/L). F, Time-dependent effect of Ang II (500 nmol/L) on protein expression of Nur77 in VSMCs, as determined by Western blotting (n=3, *P<0.05 vs 0 h).
Angiotensin II (Ang II) induces Nur77 expression in cultured vascular smooth muscle cells (VSMCs) via the MAPK/PKA-CREB pathway. A, Schematic representation of the Nur77 promoter. There are 4 CREs in the promoter region. B, Primary VSMCs were treated with Ang II (500 nmol/L) for the indicated time. The phosphorylation of CREB was examined by Western blotting (n=3, *P<0.05 vs 0 min). C and D, Primary VSMCs were pretreated either with vehicle or with (C) 20 μmol/L MAPK inhibitors (PD98059, ERK inhibitor; SB203580, P38 inhibitor; SP600125, JNK inhibitor) or (D) 10 μmol/L protein kinase A (PKA) inhibitor H-89 for 1 h before Ang II stimulation for another 5 min. The phosphorylation of CREB was examined by Western blotting (n=3; *P<0.05 vs vehicle/Ang II [−], #P<0.05 vs vehicle/Ang II [+]). E and F, Primary VSMCs were transfected with either control siRNA (si-CTL) or cAMP response element–binding protein (CREB)-specific siRNA (si-CREB). Seventy-two hours after transfection, VSMCs were stimulated with Ang II (500 nmol/L) for 1 h. E, Protein and (F) mRNA levels of CREB were assessed by Western blotting and real-time polymerase chain reaction, respectively (n=3, *P<0.05 vs si-CTL/Ang II [−], #P<0.05 vs si-CTL/Ang II [+]). G, Primary VSMCs were pretreated either with vehicle or with 20 μmol/L MAPK inhibitors (PD98059, ERK inhibitor; SB203580, P38 inhibitor; alone or both) or 10 μmol/L PKA inhibitor H-89 for 1 h before Ang II stimulation for another 1 h. Protein expression of Nur77 was examined by Western blotting (n=3, *P<0.05 vs vehicle/Ang II [−], #P<0.05 vs vehicle/Ang II [+]).
Nur77 inhibits angiotensin II (Ang II)–induced vascular smooth muscle cell (VSMC) proliferation, migration, and phenotypic switching in primary VSMCs in vitro. A–C, VSMCs isolated from wild-type (WT) and Nur77−/− mice were serum-starved for 24 h and stimulated with Ang II (500 nmol/L) for another 48 h. Quantification by (A) cell counting and (B) cell counting kit-8 assay. C, Protein expression of cyclin D1, PCNA, and P27 were examined by Western blotting. D, Monolayer confluent cells were serum-starved and scraped in the presence of Ang II (500 nmol/L) to stimulate VSMC migration toward the wound area. Representative images of the in vitro scratch-wound assay and quantification of migrated cells (number/field) are presented. E, Representative images of transwell migration assay and quantification of migrated cells (number per field). F and G, WT and Nur77−/− VSMCs were serum-starved for 24 h before stimulation with Ang II (500 nmol/L) for another 24 h. F, Protein and (G) mRNA levels of VSMC-specific genes were assessed by Western blotting and real-time polymerase chain reaction, respectively. A–G, n=3, *P<0.05.
Nur77 deletion exacerbates angiotensin II (Ang II)–induced vascular remodeling in vivo. A, Representative images of hematoxylin and eosin staining and quantification of medial area and luminal diameter results in wild-type (WT) and Nur77−/− mice (n=11–12, *P<0.05). B, Representative images of dual immunofluorescence staining of PCNA (green) and SM-α-actin (red), and quantification of percentage of PCNA-positive VSMCs in Ang II–induced remodeling aortas from WT and Nur77−/− mice (n=6, *P<0.05). C and D, Representative images of immunofluorescence staining of matrix metalloproteinase (MMP)-8, MMP-13, SM-22α, and α-actin, and quantification of their relative fluorescence intensity in Ang II–induced remodeling aortas from WT and Nur77−/− mice (n=6, *P<0.05).
Angiotensin II (Ang II) activates the β-catenin signaling pathway, which is attenuated by Nur77. A, Primary vascular smooth muscle cells (VSMCs) were treated with Ang II (500 nmol/L) for the indicated time. Protein expression of active β-catenin was examined by Western blotting (n=3, *P<0.05 vs 0 min). B, Time course of subcellular localization of β-catenin after Ang II stimulation. Western blotting of cytosolic and nuclear extracts (n=3, *P<0.05 vs 0 min). C, Representative confocal images of VSMCs treated with or without Ang II (500 nmol/L) for 30 min and stained for β-catenin (green) and nuclei (blue). D, Primary VSMCs were infected with an increasing dose of green fluorescent protein (GFP)–Nur77 adenovirus (Ad-GFP-Nur77) and GFP control adenovirus (Ad-GFP) for 48 h. Endogenous β-catenin protein level was determined by Western blotting (n=3, *P<0.05 vs Ad-GFP–infected group). E, Two hundred ninety-three T cells were cotransfected with 0.5 μg of HA-β-catenin plasmid and an increasing dose of Myc-Nur77 plasmid for 48 h. Exogenous HA-β-catenin protein level was determined by Western blotting (n=3, *P<0.05 vs myc-mock transfected group). F, Two hundred ninety-three T cells were transfected with HA-β-catenin and Flag-Nur77 plasmids for 48 h. The exogenous interaction of β-catenin and Nur77 was determined using co-immunoprecipitated (IP) assay. G, Two hundred ninety-three T cells were transfected with HA-β-catenin and Flag-Nur77 or Flag-pcDNA vector for 48 h and were treated with MG-132 (10 μmol/L) for the last 10 h. HA-β-catenin protein level was determined by Western blotting. H, Two hundred ninety-three T cells were transfected with TOPFlash and renilla luciferase reporter plasmid with or without β-catenin and Nur77 expression vectors. Renilla luciferase activities were measured using a dual-luciferase reporter assay (n=3, *P<0.05 vs TOPFlash alone group, #P<0.05 vs TOPFlash+HA-β-catenin group). I, Primary VSMCs were infected with Ad-GFP–Nur77 and Ad-GFP for 48 h. Protein levels of β-catenin and cyclin D1 were determined by Western blotting.
Effect of wild-type (WT) and Nur77-deficient genotype on β-catenin expression after systemic angiotensin II (Ang II) administration. Representative images of immunohistochemical staining of β-catenin, and quantification of β-catenin–positive area (% of intima-media area) and percentage of β-catenin–positive cells (n=6, *P<0.05).
Expression of Nur77 in vascular smooth muscle cells (VSMCs) in vivo and in vitro in response to angiotensin II (Ang II). A and B, Representative images of dual immunofluorescence staining of Nur77 (green) and SM-α-actin (red), and quantification of the ratio of Nur77-positive area to α-actin–positive area (n=6, *P<0.05). C, Nur77 mRNA expression measured by real-time polymerase chain reaction (PCR) in thoracic aorta sections after systemic Ang II infusion for 0, 7, and 14 days. Values are expressed as the fold change relative to the control (n=6, *P<0.05). D, Primary rat VSMCs were serum-starved and stimulated with Ang II (500 nmol/L) for the indicated time. The mRNA level of Nur77 was quantified by real-time PCR (n=3, *P<0.05 vs 0 h). E, Primary VSMCs were treated with increasing concentrations of Ang II for 3 h. Protein expression of Nur77 was examined by Western blotting (n=3, *P<0.05 vs 0 nmol/L). F, Time-dependent effect of Ang II (500 nmol/L) on protein expression of Nur77 in VSMCs, as determined by Western blotting (n=3, *P<0.05 vs 0 h).
Angiotensin II (Ang II) induces Nur77 expression in cultured vascular smooth muscle cells (VSMCs) via the MAPK/PKA-CREB pathway. A, Schematic representation of the Nur77 promoter. There are 4 CREs in the promoter region. B, Primary VSMCs were treated with Ang II (500 nmol/L) for the indicated time. The phosphorylation of CREB was examined by Western blotting (n=3, *P<0.05 vs 0 min). C and D, Primary VSMCs were pretreated either with vehicle or with (C) 20 μmol/L MAPK inhibitors (PD98059, ERK inhibitor; SB203580, P38 inhibitor; SP600125, JNK inhibitor) or (D) 10 μmol/L protein kinase A (PKA) inhibitor H-89 for 1 h before Ang II stimulation for another 5 min. The phosphorylation of CREB was examined by Western blotting (n=3; *P<0.05 vs vehicle/Ang II [−], #P<0.05 vs vehicle/Ang II [+]). E and F, Primary VSMCs were transfected with either control siRNA (si-CTL) or cAMP response element–binding protein (CREB)-specific siRNA (si-CREB). Seventy-two hours after transfection, VSMCs were stimulated with Ang II (500 nmol/L) for 1 h. E, Protein and (F) mRNA levels of CREB were assessed by Western blotting and real-time polymerase chain reaction, respectively (n=3, *P<0.05 vs si-CTL/Ang II [−], #P<0.05 vs si-CTL/Ang II [+]). G, Primary VSMCs were pretreated either with vehicle or with 20 μmol/L MAPK inhibitors (PD98059, ERK inhibitor; SB203580, P38 inhibitor; alone or both) or 10 μmol/L PKA inhibitor H-89 for 1 h before Ang II stimulation for another 1 h. Protein expression of Nur77 was examined by Western blotting (n=3, *P<0.05 vs vehicle/Ang II [−], #P<0.05 vs vehicle/Ang II [+]).
Nur77 inhibits angiotensin II (Ang II)–induced vascular smooth muscle cell (VSMC) proliferation, migration, and phenotypic switching in primary VSMCs in vitro. A–C, VSMCs isolated from wild-type (WT) and Nur77−/− mice were serum-starved for 24 h and stimulated with Ang II (500 nmol/L) for another 48 h. Quantification by (A) cell counting and (B) cell counting kit-8 assay. C, Protein expression of cyclin D1, PCNA, and P27 were examined by Western blotting. D, Monolayer confluent cells were serum-starved and scraped in the presence of Ang II (500 nmol/L) to stimulate VSMC migration toward the wound area. Representative images of the in vitro scratch-wound assay and quantification of migrated cells (number/field) are presented. E, Representative images of transwell migration assay and quantification of migrated cells (number per field). F and G, WT and Nur77−/− VSMCs were serum-starved for 24 h before stimulation with Ang II (500 nmol/L) for another 24 h. F, Protein and (G) mRNA levels of VSMC-specific genes were assessed by Western blotting and real-time polymerase chain reaction, respectively. A–G, n=3, *P<0.05.
