Regulation of the Muscarinic M3 Receptor by Myocardin-Related Transcription Factors

Abstract

Myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MRTFA, and MRTF-B/MRTFB) are co-factors of serum response factor (SRF) that activate the smooth muscle cell (SMC) gene program and that play roles in cardiovascular development and mechanobiology. Gain and loss of function experiments have defined the SMC gene program under control of MRTFs, yet full understanding of their impact is lacking. In the present study, we tested the hypothesis that the muscarinic M3 receptor (CHRM3) is regulated by MRTFs together with SRF. Forced expression of MYOCD (8d) in human coronary artery (SMC) followed by RNA-sequencing showed increased levels of M2, M3, and M5 receptors (CHRM2: 2-fold, CHRM3: 16-fold, and CHRM5: 2-fold). The effect of MYOCD on M3 was confirmed by RT-qPCR using both coronary artery and urinary bladder SMCs, and correlation analyses using human transcriptomic datasets suggested that M3 may also be regulated by MRTF-B. Head-to-head comparisons of MYOCD, MRTF-A and MRTF-B, argued that while all MRTFs are effective, MRTF-B is the most powerful transactivator of CHRM3, causing a 600-fold increase at 120h. Accordingly, MRTF-B conferred responsiveness to the muscarinic agonist carbachol in Ca2+ imaging experiments. M3 was suppressed on treatment with the MRTF-SRF inhibitor CCG-1423 using SMCs transduced with either MRTF-A or MRTF-B and using intact mouse esophagus in culture (by 92±2%). Moreover, silencing of SRF with a short hairpin reduced CHRM3 (by >60%) in parallel with α-actin (ACTA2). Tamoxifen inducible knockout of Srf in smooth muscle reduced Srf (by 54±4%) and Chrm3 (by 41±6%) in the urinary bladder at 10days, but Srf was much less reduced or unchanged in aorta, ileum, colon, trachea, and esophagus. Longer induction (21d) further accentuated the reduction of Chrm3 in the bladder and ileum, but no change was seen in the aorta. Single cell RNA-sequencing revealed that Mrtfb dominates in ECs, while Myocd dominates in SMCs, raising the possibility that Chrm3 may be driven by Mrtfb-Srf in the endothelium and by Myocd-Srf in SMCs. These findings define a novel transcriptional control mechanism for muscarinic M3 receptors in human cells, and in mice, that could be targeted for therapy.

Cell Type Dependent Suppression of Inflammatory Mediators by Myocardin Related Transcription Factors

“…Upon first reflection, this contradiction makes little sense, but it is possible that the different MRTFs are expressed in entirely different cell types with different roles in vascular inflammation. Indeed, in parallel work ( Liu et al, 2021 ) we find that MYOCD is enriched in SMCs as expected, and that MRTF-B is enriched in endothelial cells, while MRTF-A primarily resides in fibroblasts. Additional explanations for the discrepant effects of MRTF-A and MYOCD in atherosclerosis include coactivator-specific effects.…”

“…Here, we show that overexpression of MRTFs downregulate several pro-inflammatory transcripts and proteins relevant for vascular pathophysiology and development of vascular disease. However, the RNA-seq experiment providing impetus for the present work ( Liu et al, 2021 ) revealed additional pro-inflammatory genes, such as SGPP2 (sphingosine 1-phosphate phosphatase 2), IL32 (interleukin 32), and TSLP (thymic stromal lymphopoietin), as being repressed. Notably, downregulation of SGPP2 gene activity and sphingosine 1-phosphate phosphatase 2 protein levels reduces TNFα-induced IL1B and CXCL8 production in endothelial cells, and moreover, knockdown of NF-κB/RelA shows that SGPP2 is an NF-κB-regulated gene ( Mechtcheriakova et al, 2007 ).…”

“…In a parallel study (submitted to the same thematic issue of this journal, Liu et al, 2021 ), we generated an RNA-sequencing (RNA-seq) dataset after viral overexpression of myocardin (MYOCD) in human coronary artery SMCs (hCASMCs) for 8 days. Thousands of transcripts were differentially expressed, and among them, numerous inflammatory mediators stood out as being robustly reduced ( Figure 1A , adjusted P < 0.0001 throughout).…”

“…Transcript read counts (in TPM, transcripts per million) for SRF , MYOCD , MRTFA , and MRTFB were correlated with 13 inflammatory transcripts identified in an RNA-seq experiment where MYOCD was overexpressed. The study describing this RNA-seq experiment was submitted in parallel to the Frontiers’ theme in Cardiovascular mechanobiology ( Liu et al, 2021 ) and has been deposited with the temporary submission ID SUB9688745, and release date 2022-06-01 (or with the release of linked data). Correlation matrices for MRTFs and the inflammatory transcripts were generated using the Pearson method using GraphPad Prism in all three arteries represented in the GTExPortal.…”

“…Inducible and SMC-specific knockout of Srf in mice was accomplished as described (accompanying paper submitted to Frontier’s theme on mechanobiology, Liu et al, 2021 ). Floxed Cre-negative mice treated with tamoxifen (TC) and Floxed Cre-positive mice treated with vehicle (VC) were used as controls.…”