Overexpression of CHIP inhibits myocardin-induced SMC differentiation through downregulation of myocardin protein. (A) SMCs were transfected with CHIP or the empty vector for 24 h and then cultured in 2% horse serum for 48 h. The transcripts of SM genes, including those for SM22α, SM α-actin, SM-MHC, smoothelin, myocardin, and SRF, were examined by RT-PCR. (B) Quantitative analysis of the mRNA levels in panel A (n = 3, means ± SEM). *, P < 0.01 versus the vector. (C) SMCs were transfected with myocardin, WT CHIP, and U-box deletion mutant plasmids. Cells were cultured, and the mRNA levels of SM genes were analyzed as in panel A. (D) Quantitative analysis of mRNA transcripts in panel C (n = 3, means ± SEM). *, P < 0.01 versus myocardin alone. (E) SMCs were transfected with CHIP or vector plasmids and cultured in 2% horse serum for 0, 12, 24, 36, 48, or 60 h. The cell extracts were examined with antimyocardin, anti-CHIP, or β-actin antibody. (F) SMCs were transfected with increasing amounts of CHIP plasmid and cultured in 2% horse serum for 48 h. The cell extracts were examined as in panel E. (G, H) 293 cells were transfected with myocardin, SM22α, or ANF reporters and increasing amounts of WT CHIP or CHIP ΔU-box (0.25 and 0.5 μg). Luciferase (Luc) activity was measured (n = 3, means ± SEM). *, P < 0.01 versus myocardin alone. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Wt, wild type.

CHIP interacts with myocardin. (A) SMCs were cultured in 2% horse serum for 48 h, lysates were immunoprecipitated with anti-CHIP antibody or control IgG, and the immunoprecipitates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted (IB) with antimyocardin (top) or anti-CHIP (bottom) antibody. (B) SMCs were cultured in 2% horse serum for 48 h. Cell lysates were subjected to coimmunoprecipitation (IP) with antimyocardin or anti-CHIP antibody in the absence or presence of ATP (10 mM) and rabbit IgG as a control. The presence of CHIP, Hsp70, Hsp90, or myocardin in the precipitated complexes was determined by immunoblotting as indicated. (C) CHIP interacts with myocardin in GST pull-down assays. GST fusion proteins were analyzed by immunoblotting with anti-GST antibody (top). The ability of Flag-myocardin (middle) or HA-SRF (bottom) expressed in 293 cells to be retained by GST or GST-CHIP fusion protein was analyzed by immunoblotting after binding reactions. (D) 293 cells were transfected with the indicated plasmids. Equal amounts of protein lysates were immunoprecipitated with the anti-Myc or control IgG serum and analyzed by immunoblotting with anti-Flag or anti-Myc antibody. (E) SMCs were cotransfected with Myc-CHIP and GFP-myocardin and immunostained with anti-Myc antibody. For quantitation, 150 cells per coverslip were counted and each treatment was performed in triplicate. A representative image is shown for each condition.

CHIP as an E3 ligase targets myocardin for proteasomal degradation. (A) 293 cells were transfected with a Flag-myocardin, Myc-CHIP, or vector plasmid and treated with cycloheximide (CHX, 10 μg/ml) at the indicated time points. The half-life of myocardin was determined by immunoblotting with anti-Flag antibody and quantified in the presence of a CHIP or vector plasmid (n = 3, means ± SEM). (B) The effect of CHIP on steady-state levels of myocardin was examined by transfection with a Myc-CHIP or vector plasmid in the presence of MG132 for 4 h. (C) The ubiquitination of endogenous myocardin in SMCs was tested by transfection with a Myc-CHIP or vector plasmid. Cell extracts were immunoprecipitated (IP) with antimyocardin antibody and immunoblotted (IB) with an anti-Ub (top) or antimyocardin (middle) antibody. The expression of myocardin and CHIP in the cell extracts was determined with antimyocardin or anti-Myc antibody (bottom). (D) 293 cells were transfected with a His-Ub, Myc-CHIP, or CHIP ΔU-box mutant plasmid in the presence of Flag-myocardin WT or mutant protein 1-713 and incubated with MG132 for 4 h before harvesting. Ub-conjugated proteins were purified by nickel chromatography and analyzed by immunoblotting with anti-Flag (top) or anti-His (middle) antibody. The expression of myocardin and CHIP in the cell extracts was determined with anti-Flag or anti-Myc antibody (bottom). (E) In vitro ubiquitylation reactions were performed with purified Ub, E1, E2-Ubc5b, GST alone, GST-CHIP, and GST-CHIP ΔU-box mutant protein in the presence of Flag-myocardin WT or 1-713 mutant protein. Reaction products were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting with anti-Ub (top) or anti-Flag antibody (bottom).

CHIP gene transfer downregulates myocardin protein level and SM contractile gene expression in rat aortic rings. (A) Expression of GFP in aortic rings 2 days after transduction of Ad-GFP and Ad-CHIP was confirmed with a fluorescence microscope (magnification, ×40). (B) Myocardin and CHIP levels were determined by Western blot analysis. The blots are typical representatives of two different rings per group. (C) The expression of SM contractile gene mRNAs was detected by RT-PCR analysis. (D) Quantitative analysis of mRNA levels in panel C. Means ± SEM of four rings per group are shown. *, P < 0.01 versus Ad-GFP.

CHIP siRNA upregulates the expression of myocardin-induced SM contractile genes. (A) SMCs were transfected with control or CHIP siRNA and cultured in 2% horse serum for 48 h. CHIP and myocardin mRNA and protein levels were analyzed by RT-PCR and immunoblotting as indicated. (B) SMCs were transfected and cultured as in panel A. The transcripts of SMC genes, including those for SM22α, SM α-actin, SM-MHC, and myocardin, were examined by RT-PCR. (C) Quantitative analysis of the mRNA transcripts in panel B (n = 3, means ± SEM). *, P < 0.05 versus control siRNA. (D) SMCs were transfected with SM22α reporter, control siRNA, or CHIP siRNA for 48 h. Luciferase (luc) activity was measured (n = 3, means ± SEM). *, P < 0.01 versus control siRNA. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Mapping of the interactive domain of CHIP and myocardin. (A) The domain of CHIP involved in binding to myocardin. GST fusion proteins were analyzed by immunoblotting with anti-GST antibody (top). The ability of the truncated CHIP fusion proteins to bind to Flag-myocardin expressed in 293 cells was analyzed by immunoblotting with anti-Flag antibody (middle). Ten percent of the input is shown in the bottom panel. (B) Schematic representation of the CHIP mutant proteins used in panel A. (C) The domain of myocardin involved in binding to CHIP. GST-CHIP fusion proteins were analyzed by immunoblotting with GST antibody (top). The ability of the truncated myocardin proteins expressed in 293 cells to bind to GST-CHIP was analyzed by immunoblotting with anti-Flag antibody (middle). Ten percent of the input is shown in the bottom panel. (D) Schematic representation of deletion constructs of myocardin used in panel C. TPR, tetratricopeptide repeat; Q, a stretch of glutamine residues; SAP, SAF-A/B-acinus-PIAS domain; LZ, leucine zipper.

CHIP promotes the ubiquitination of phosphorylated myocardin. (A) 293 cells were transfected with His-Ub, Flag-myocardin, and Myc-CHIP and left untreated or treated with AR-A014418 (10 mM) for 12 h in the presence of MG132 for 4 h before harvesting. Cell extracts were immunoprecipitated (IP) with the anti-Flag antibody and immunoblotted (IB) with the anti-His (top) or -Flag (middle) antibody. Cell extracts were subjected to immunoblotting with anti-Flag or anti-Myc antibody (bottom). (B) 293 cells were transfected with His-Ub, Flag-myocardin, and Myc-CHIP plasmids and treated with increasing amounts of AR-A014418 (0, 5, and 10 mM) for 12 h in the presence of MG132 for 4 h before harvesting. Cell extracts were immunoprecipitated and immunoblotted as in panel A. (C) 293 cells were cotransfected with vectors expressing His-Ub, Flag-myocardin, Myc-CHIP, GSK-3β S9A, or Dn-GSK in the presence of MG132 for 4 h before harvesting. Ub-conjugated proteins were purified by nickel chromatography and analyzed by immunoblotting with anti-Flag (top) and anti-His (middle) antibodies. The expression of myocardin, CHIP, and GSK-3β in the cell extracts was determined with anti-Flag, anti-Myc, or anti-HA antibody (bottom). (D) In vitro ubiquitination reactions were performed with purified Ub, E1, E2-UbcH5b, GST alone, or GST-CHIP. Flag-myocardin was precipitated from transfected 293 cells treated with AR-A014418 or left untreated for 12 h. Reaction products were analyzed as described in the legend to Fig. 5E.

CHIP overexpression regulates the contractile phenotype in rat aortic rings. Shown are concentration-response curves for phenylephrine (A), acetylcholine (B), and SNP (C) in rat aortic rings transduced for 2 days with Ad-GFP (n = 4) or Ad-CHIP (n = 4). Means ± SEM from four rings per group are shown. *, P < 0.01 versus Ad-GFP. (D) Schematic model depicting the role of CHIP in SMC phenotype modulation via promoting myocardin degradation.