Bone morphogenetic protein-induced MSX1 and MSX2 inhibit myocardin-dependent smooth muscle gene transcription

Abstract

During the onset and progression of atherosclerosis, the vascular smooth muscle cell (VSMC) phenotype changes from differentiated to dedifferentiated, and in some cases, this change is accompanied by osteogenic transition, resulting in vascular calcification. One characteristic of dedifferentiated VSMCs is the down-regulation of smooth muscle cell (SMC) marker gene expression. Bone morphogenetic proteins (BMPs), which are involved in the induction of osteogenic gene expression, are detected in calcified vasculature. In this study, we found that the BMP2-, BMP4-, and BMP6-induced expression of Msx transcription factors (Msx1 and Msx2) preceded the down-regulation of SMC marker expression in cultured differentiated VSMCs. Either Msx1 or Msx2 markedly reduced the myocardin-dependent promoter activities of SMC marker genes (SM22alpha and caldesmon). We further investigated interactions between Msx1 and myocardin/serum response factor (SRF)/CArG-box motif (cis element for SRF) using coimmunoprecipitation, gel-shift, and chromatin immunoprecipitation assays. Our results showed that Msx1 or Msx2 formed a ternary complex with SRF and myocardin and inhibited the binding of SRF or SRF/myocardin to the CArG-box motif, resulting in inhibition of their transcription.

FIG. 1.

BMP-induced expression of Msx transcription factors and down-regulation of SMC marker expression in VSMCs. Differentiated VSMCs were stimulated with 10 ng/ml (a) or 100 ng/ml (b) of the indicated BMP family members (B2, BMP2; B4, BMP4; and B6, BMP6) or 2 ng/ml IGF-I (I) for 1 to 3 days. (A) RT-PCR analysis of the expression of Msx1, Msx2, and the indicated SMC marker mRNAs. RT-PCR was performed using RNAs isolated from the indicated VSMC cultures, as described in Materials and Methods. Cycle numbers of the PCRs and the sizes of PCR products are as follows: Msx1, 32, 446 bp; Msx2, 32, 420 bp; SM22α, 26, 461 bp; h- and l-CaD, 34, 537 bp; calponin, 32, 459 bp; MHC-SM2, 34, 498 bp; and GAPDH, 24, 575 bp. The mRNAs of the caldesmon isoforms, h- and l-CaD, are transcribed from the same promoter and are generated by SMC phenotype-dependent alternative splicing; h-CaD is specifically distributed in differentiated SMCs, whereas l-CaD is in dedifferentiated SMCs and nonmuscle cells. Expression levels of Msx transcription factor and SMC marker mRNAs were normalized to GAPDH mRNA and were quantified (B, C, D, and E). (B) Open and closed bars indicate Msx1 and Msx2 mRNAs, respectively. (E) Open bars, calponin mRNA; closed bars, MHC-SM2 mRNA. (F) The expression profiles of Msx1, Msx2, SM22a, and h- and l-CaD were confirmed by real-time PCR. Relative abundance of transcripts was presented based on those in VSMCs cultured under IGF-I-stimulated conditions for 1 day, which were set as 100. Each value represents the mean ± standard deviations of results from three independent experiments. (G) Whole-cell lysates from the indicated VSMC cultures were probed with anti-Msx1 and anti-Msx2 antibodies by immunoblot analysis. These results are taken from one representative experiment (of at least three).

FIG. 2.

Effects of Msx1 or Msx2 on the Mycd-dependent activation of the SM22α and CaD promoters. SM22P-Luc (wt, dCArGmut, pCArGmut, or dpCArGmut) (400 ng) (A and C), CaD GP2-Luc (wt or CArGmut) (400 ng) (B and D), pGL3 control (E) (400 ng), pSVβ-gal (200 ng), and empty plasmid were cotransfected into 10T1/2 cells with or without the indicated expression plasmids (25 or 50 ng) (total, 1.0 μg plasmids/well of a 12-well culture plate). The culture conditions of the 10T1/2 cells and the assay procedures are described in Materials and Methods. Relative luciferase activities normalized to the β-galactosidase activity are shown. Each value represents the mean ± standard deviation of results from three independent experiments.

FIG. 3.

Inhibition of the Mycd-induced expression of endogenous SMC markers by the coexpression of Msx transcription factors. 10T1/2 cells were transfected with Flag-Mycd (1.0 μg) and/or HA-Msx1 (0.5 μg) or HA-Msx2 (0.5 μg) expression plasmids with (+) or without (−) empty plasmid (total 2 μg plasmids/well of a six-well culture plate) and were cultured as described in Materials and Methods. Expression of the indicated SMC markers was analyzed by RT-PCR (A), immunoblotting (B), and immunocytochemistry (C and D). (A) Cycle numbers of the PCRs and the sizes of PCR products are as follows: SM22α, 28, 329 bp; CaD, 24, 380 bp; calponin, 30, 400 bp; MHC-SM2, 28, 323 bp; GAPDH, 22, 984 bp. (B) Whole-cell lysates of 10T1/2 cells from the indicated cultures were probed with anti-SM22α, anti-CaD, and anti-HA (Msx1 or Msx2) antibodies by immunoblot analysis. In this analysis, 10T1/2 cells were transfected with the indicated expression plasmids as described above except for HA-Msx1 or HA-Msx2 expression plasmid; + and ++ indicate 0.25 μg and 0.5 μg of the respective expression plasmid. (C) Tagged Mycd (red) and Msx transcription factors (green) were stained with anti-Flag and anti-HA antibodies, respectively, and endogenous SM22α and CaD (blue) were stained using their respective antibodies.

FIG. 4.

Interactions between Msx1, SRF, and Mycd. Cos7 cells were transfected with the indicated expression plasmids and were cultured in the presence of 2% HS for 20 h. (A) Tagged SRF (red), Mycd (red), and Msx1 (green) were localized by staining with antibodies against the indicated tags, and the nuclei (blue) were stained with Hoechst 33258. (B) Cos7 cells were cotransfected with expression plasmids for SRF-Flag and HA-Msx1 (lanes 1 and 3) or Flag-Mycd and HA-Msx1 (lanes 2 and 4). The cell extracts from respective transfectants were incubated with anti-Flag antibody (lanes 1 and 2) or control IgG (lanes 3 and 4), and the extracts (right panels) and immunoprecipitates (IP) (left panels) were probed with an anti-Flag antibody (SRF or Mycd) or anti-HA antibody (Msx1) by immunoblot (IB) analysis. (C) Cos7 cells were cotransfected with expression plasmids for Flag-Mycd and HA-Msx1 (lane 1), Flag-Mycd and SRF-HA (lane 2), or Flag-Mycd, SRF-HA, and HA-Msx1 (lane 3). The cell extracts were incubated with anti-Flag antibody (Ab), and the extracts (right panel) and immunoprecipitates (left panels) were probed with an anti-Flag antibody (Mycd) or anti-HA antibody (SRF and/or Msx1) by immunoblot analysis. (D) Endogenous Msx1, SRF, and Mycd formed a ternary complex in VSMCs cultured under BMP2-stimulated conditions. VSMCs were cultured in the presence of IGF-I (2 ng/ml) (lane 1) or BMP2 (10 ng/ml) (lane 2) for 24 h. The cell extracts were incubated with an anti-SRF antibody, and the extracts and immunoprecipitates were probed with the indicated antibodies by immunoblot analysis.

FIG. 5.

Domain mapping of Mycd's interaction with Msx1. (A) In vitro-translated Flag-Mycd derivatives, as indicated, were incubated with HA-Msx1, and their interactions were analyzed by immunoprecipitation (IP) using an anti-Flag antibody (Ab) (for the Mycd derivatives) followed by immunoblotting (IB) using the indicated antibodies. Input HA-Msx1 protein is also shown (lane 1). (B) The results from panel A are presented schematically. The binding of the Mycd derivatives to Msx1 are presented as follows: ++, strong binding; +, moderate binding; −, no binding.

FIG. 6.

Domain mapping of SRF's interaction with Msx1. (A) The localization of the SRF derivatives is shown. Cos7 cells were transfected with the indicated expression plasmids for SRF-Flag derivatives [wt, SRFwt; Pm, SRF Pm143-146; PRGI, SRF PRGI-In206; ΔMADS, SRFΔMADS (133-266); MADS, MADS (133-266)] and cultured as described in the legend to Fig. 4. The SRF derivatives (red) and the nuclei (blue) were stained with an anti-Flag antibody and Hoechst 33258, respectively. (B) Identification of the Msx1-interacting domain of SRF. Cos7 cells were cotransfected with each of the indicated expression plasmids for the SRF-Flag derivatives and the HA-Msx1 expression plasmid. Interactions between SRF derivatives and Msx1 were analyzed as described in the legend to Fig. 4B. (C) The results of panel B are presented schematically. The ability of SRF derivatives to bind Msx1 is presented as follows: ++, strong binding; ±, faint binding; −, no binding. IP, immunoprecipitation; IB, immunoblotting; Ab, antibody.

FIG. 7.

Domain mapping of Msx1's interaction with SRF or Mycd. (A) In vitro-translated SRF-Flag or Flag-Mycd was incubated with in vitro-translated HA-Msx1 or HA-Msx1ΔH as follows: lane 1, input HA-Msx1ΔH; lane 2, SRF-Flag plus HA-Msx1; lane 3, SRF-Flag plus HA-Msx1ΔH; lane 4, input HA-Msx1; lane 5, Flag-Mycd plus HA-Msx1; lane 6, Flag-Mycd plus HA-Msx1ΔH. Their interactions were analyzed by immunoprecipitation (IP) followed by immunoblotting (IB) as described in the legend to Fig. 5A. Ab, antibody. (B) The results of panel A are presented schematically. The ability of Msx1 derivatives to bind Mycd or SRF is presented as follows: ++, strong binding; +, moderate binding; −, no binding.

FIG. 8.

Msx1 inhibits the binding of SRF or the SRF/Mycd complex to the CArG-box motif. (A and B) The ³²P-labeled pCArG-box motif of the SM22α promoter was incubated with the indicated in vitro-translated proteins, antibodies, and/or double-stranded oligonucleotides, and their interactions were analyzed by 5% polyacrylamide gel electrophoresis. (A) Constant amounts of SRF (5 μl of in vitro-translated aliquot) (lanes 1 to 8) or Msx1 (4 μl of in vitro-translated aliquot) (lane 9) were used. One hundred-fold excess amounts of indicated cold competitors were added (lanes 2 and 3). Increasing amounts of Msx1 (2 μl, 4 μl or 8 μl) (lanes 6 and 7) were mixed with SRF (5 μl) (lanes 6 and 7). (B) Constant amounts of Mycd (8 μl of in vitro-translated aliquot) and SRF (4 μl) were used. Increasing amounts of Msx1 (2 μl, 4 μl, or 8 μl) (lanes 4 to 6) were mixed with Mycd/SRF. Arrows and asterisks show the indicated pCArG-box motif/protein complexes and supershifted complexes, respectively. +, present; −, absent. (C) ChIP assays were performed using endogenous proteins associated with extracted chromatin fragments prepared from VSMCs by BMP2 stimulation and differentiated VSMCs cultured in the presence of IGF-I (left panels) or 10T1/2 cells expressing Mycd or Mycd and Msx1 (right panels). The extracted chromatin fragments were immunoprecipitated with control IgG (lane 2) or antibody against SRF (lane 3), and precipitated genomic DNA was analyzed by PCR using primers for the SM22α and CaD promoter regions containing the CArG-box motif. The sizes of PCR products are as follows: rat SM22α promoter, 195 bp; rat CaD promoter, 141 bp; mouse SM22α promoter, 196 bp; mouse CaD promoter, 141 bp. PCR amplification was also performed prior to immunoprecipitation for the input control (lane 1). −, transfection of control vector.

FIG. 9.

Model for the inhibition of SRF/Mycd-dependent SMC marker gene transcription by Msx transcription factors.