Fig 1: SRF SUMOylation and the SRF-ELK complex regulate VSMC proliferation, migration and phenotypic switching.A–F. MOVAS-1 cells were stably transfected with empty vector, Flag-SRF or Flag-SRFK143R and pooled clones were expanded for the designed experiments. (A). Cells were untreated or treated with PDGF-BB (10 ng/ml) for 0–24 h followed by western blot for VSMC proliferation, migration and phenotypic markers. Relative protein levels are presented by taking untreated SRF-WT as 1.0 (n=3). (B). Cells were untreated or treated with PDGF-BB (10 ng/ml) for 24 h and were subjected to immunofluorescence co-staining with VSMC contractile/synthetic markers as indicated (n=3). (C–F). Cells were serum-starved for 24 hours followed by treatment with PDGF-BB (10 ng/ml) for 24 h. (C) Cells were used for Edu incorporation assay or Ki67 staining with α-SMA. Representative images are presented. (D) % Ki67+ and Edu+ VMSC were quantified (n=8 per group). (E) Cells were subjected to scratch and transwell assays in the absence or presence of PDGF-BB for 24 h before fixation and imaging. Representative images are presented. (F) % wound closure and # of invaded VMSCs were quantified (n = 8 per group). G–K. Primary aortic VSMCs from WT and Senp1SMCKO mice were treated with PDGF-BB (10 ng/ml) for 0–24 h in the absence or presence of AZD6244 (1 μmol/L). (G) Cell lysates were subjected to western blot for VSMC proliferation, migration and phenotypic markers. Relative protein levels are presented by taking untreated WT as 1.0 (n=3). (H). Cells were subjected to immunofluorescence co-staining with VSMC contractile/synthetic markers as indicated (n=3). (I–K). Cells were serum-starved for 24 hours and subjected to Edu incorporation, scratch and transwell assays. Representative images are presented in (I). % Ki67+ or Edu+ VMSC (J), % wound closure and # of invaded VMSCs (K) were quantified (n = 8 per group). Data are mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, no significance, using one-way ANOVA followed by Tukey’s multiple comparisons test. Scale bars, 10 μm (B, H); 50 μm (C, E, I).
Fig 2: SRF SUMOylation regulates the SRF-ELK1 complex formation.A–C. VSMCs were isolated from un-injured WT and Senp1SMCKO mice. (A). Cells were treated with PDGF-BB (10 ng/ml) for 24 h and subjected to immunofluorescence co-staining with SRF and LAMP2. High magnification of merged images is shown at the bottom of each panel. Lysosomal and nuclear SRF are indicated by arrows and arrowheads, respectively. (B). Cells were treated with PDGF-BB (10 ng/ml) for 0–24. Cytoplasm and nuclear fractions were isolated by cellular fractionation followed by western blot for SRF, lysosomal marker LAMP2 and nuclear marker lamin A. Relative protein levels are presented by taking untreated WT as 1.0 (n=3 per group). (C). VSMCs were treated with CHX (100 ng/ml) for 0–24 h followed by western blot for SRF and GAPDH. Relative protein levels are presented by taking untreated WT as 1.0 (n=3). D. A diagram for the structural domains of mouse SRF protein, containing the DNA-binding MADS domain and the transactivation domain. The putative SUMOylation K residues are indicated. E–I. MOVAS-1 cells were transiently transfected with Flag-tagged SRF-WT, SRFK131R, SRFK161R or SRFK143R without (E, F, H) or with (G, I) HA-SUMO1. (E) Cell lysates were subjected to western blot with the anti-Flag antibody. GAPDH as the loading control (n = 3 per group). (F) Total RNAs were used for qRT-PCR for Srf mRNA levels. (G) Cells lysates were subjected to coimmunoprecipitation assays under denaturing condition with anti-Flag followed by western blot with anti-HA (SUMO1). The original input lysates were also analyzed with GAPDH as a loading control. Relative protein levels are presented by taking untreated WT as 1.0 (n=3). (H) Flag-SRF and Flag-SRFK143R -expressing cells were subjected to immunofluorescence co-staining of Flag (green) and anti-LAMP2 (red) with DAPI counterstaining for nucleus (blue). (I) Flag-SRF and Flag-SRFK143R-expressing cells were untreated or treated with PDGF-BB for 24. Cells lysates were subjected to co-immunoprecipitation assays under non-denaturing condition with anti-Flag or IgG control followed by western blot with anti-HA (SUMO1), anti-ELK1 or antimyocardin antibodies. The original input lysates were also analyzed with GAPDH as a loading control. Relative protein levels are presented by taking untreated WT as 1.0 (n=3). Data are mean ± SEM. ** P < 0.01; ns, no significance. One-way ANOVA with Bonferroni post hoc analysis. Scale bars, 20 μm (A, I).
Fig 3: A model for the SRF SUMOylation and the SRF-ELK1 complex mediated vascular remodeling.A. Under normal condition, SRF is present in a deSUMOylation state and retains its binding with MYCD to maintain the VSMC contractile phenotype. B. In response to vascular injury, activated ELK1 and increased SUMOylated SRF form a complex and induce VSMC synthetic phenotypic switch, VSMC proliferation and migration, leading to neointimal formation. C. SENP1 deficiency in VSMCs dramatically increases SUMO1mediated SRF SUMOylation at lysine 143 and the SRF-ELK1 complex, which augments VSMC phenotypic switch and neointimal formation. D. Pharmacological inhibition of phosphor-ELK1 by AZD6244 prevents the SRF-ELK complex and/or restores the SRF-MYCD complex, attenuating the excessive proliferation, migration, and neointimal formation. AZD: AZD6244; CArG: CA(A/T)6G box; MYCD: myocardin; TCE: ternary complex element; Su: SUMO1.
Fig 4: SENP1 deficiency in VSMCs augments the SRF-ELK complex during vascular remodeling.Carotid arteries from WT and Senp1SMCKO mice on day 0–28 post-injury. A. Western blots for proteins in the MEK-ERK1/2-ELK1 pathway. Relative protein levels are presented by taking non-injured WT as 1.0 (n=3 per group). B–C. Immunofluorescence staining for p-ELK in carotid arteries from WT and Senp1SMCKO mice on day 28 post-injury. Four color images are presented with p-ELK (red), α-SMA (green), CD31 (APC-conjugated; pseudo-colored by blue) and DAPI (blue; pseud-colored by white). (C) MFI of p-ELK within the neointimal areas or ECs were quantified (n =10 per group). D–E. Immunofluorescence staining for SRF in carotid arteries from WT and Senp1SMCKO mice on day 28 post-injury. (D) Four color images are presented with SRF (red), α-SMA (green), CD31 (APC; pseudo-colored by blue) and DAPI (blue; pseud-colored by white). (E) MFI of SRF within the neointimal areas or ECs were quantified (n =10 per group). F. Western blots for SRF and myocardin. Relative protein levels are presented by taking non-injured WT as 1.0 (n=3 per group). G. qRTPCR for Srf mRNAs in carotid arteries from WT and Senp1SMCKO mice. Gapdh served as a control. Relative mRNA levels are presented by taking non-injured WT as 1.0 (n=3 per group). H. Detection of SRF SUMOylation in day 7 post-injury carotid arteries from WT and Senp1SMCKO mice. Artery lysates were subjected to co-immunoprecipitation assays with anti-SRF or IgG followed by western blot with SUMO1 or SUMO2/3 as indicated. Relative protein levels in the input and co-immunoprecipitates are presented by taking non-injured carotid artery from WT mice as 1.0 (n=3 per group). I. Detection of SRF-SUMO1, SRF-ELK and SRF-myocardin complexes. VSMCs from WT and Senp1SMCKO mice were untreated or treated with PDGFBB (10 ng/ml for 60 min). VSMCs lysates were subjected to co-immunoprecipitation assays with anti-SRF or IgG followed by western blot with SUMO1, ELK1, and myocardin as indicated. Relative protein levels in the input and co-immunoprecipitates are presented by taking untreated VSMCs from WT mice as 1.0 (n=3 per group). J. Effects of AZD6244 on the SRF complexes in PDGF-treated VSMCs from WT and Senp1SMCKO mice. VSMCs were treated with PGDF-BB in the absence or presence of AZD6244 (0.5 μmol/L) for 60 min. Cell lysates were subjected to co-immunoprecipitation assays with anti-SRF or IgG followed by Western blot with SUMO1, ELK or myocardin as indicated. Relative protein levels in the input and co-immunoprecipitates are presented by taking untreated VSMCs from WT mice as 1.0 (n=3 per group). Data are mean ± SEM. ns, no significance, compared with WT mice (two-tailed Student’s t-test) (C, E). # P < 0.05; #### P < 0.0001, compared with un-injured or untreated groups (One-way ANOVA with Bonferroni post hoc analysis) (G). Scale bars, 50 μm (B, D).
Fig 5: Blocking shift from SRF-myocardin to SRF-ELK complex by AZD6244 inhibits injury-induced neointimal formation.A. A diagram for AZD6244 injection protocol. 12-week-old WT and Senp1SMCKO mice were received injection of AZD6244 (25 mg/kg) or vehicle (PBS) once daily from 4 days before carotid artery injury to 3 days after injury (D-4 to D3). Artery tissues were harvested on D3, D7, D14 and D28. B. Carotid artery tissues were subjected to western blot for the MEK-ERK-ELK1 signaling. Relative protein levels are presented by taking untreated WT as 1.0 (n=3). C. Carotid artery tissues were subjected to co-immunoprecipitation assays with anti-SRF followed by western blot with anti-ELK1 and anti-myocardin. Relative protein levels are presented by taking untreated WT as 1.0 (n=3). D. Carotid artery tissues were subjected to western blot for the VSMC proliferation, migration and phenotypic markers. Relative protein levels are presented by taking untreated WT as 1.0 (n=3). E. Representative photomicrographs of EVG staining and HE staining of carotid arteries harvested on day 28 post-injury from WT and Senp1SMCKO mice. F. Circumference of EEL, neointimal area, media area, and neointima/media ratio in carotid arteries were measured (n=12 per group). G–H. Carotid artery tissues were subjected to immunofluorescence staining with VSMC contractile and synthetic markers. (G) Representative images are presented. (H) MFI of each marker within the neointimal areas were quantified (n = 10 per group). I–J. Carotid artery tissues were subjected to immunofluorescence co-staining of α-SMA (green) with Ki67, MMP2 or MMP9 (red) with DAPI counterstaining for nuclei (blue). (I) Representative images are presented. (J) Ki67+α-SMA+ VSMCs, MFI of MMP2 and MMP9 within the neointimal areas were quantified (n = 10 per group). Data are mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.001; ns, no significance. One-way ANOVA with Bonferroni post hoc analysis. Scale bars, 50 μm (E, G, I). MFI: mean fluorescence intensity.
Supplier Page from OriGene Technologies for Srf Mouse shRNA Plasmid (Locus ID 20807)