The Smooth Muscle Cell-restricted KCNMB1 Ion Channel Subunit Is a Direct Transcriptional Target of Serum Response Factor and Myocardin

Abstract

Large conductance calcium-activated potassium (MaxiK) channels play a pivotal role in maintaining normal arterial tone by regulating the excitation-contraction coupling process. MaxiK channels comprise ␣ and ␤ subunits encoded by Kcnma and the cell-restricted Kcnmb genes, respectively. Although the functionality of MaxiK channel subunits has been well studied, the molecular regulation of their transcription and modulation in smooth muscle cells (SMCs) is incomplete. Using several model systems, we demonstrate down-regulation of Kcnmb1 mRNA upon SMC phenotypic modulation in vitro and in vivo. As part of a broad effort to define all functional CArG elements in the genome (i.e. the CArGome), we discovered two conserved CArG boxes located in the proximal promoter and first intron of the human KCNMB1 gene. Gel shift and chromatin immunoprecipitation assays confirmed serum response factor (SRF) binding to both CArG elements. A luciferase assay showed myocardin (MYOCD)-mediated transactivation of the KCNMB1 promoter in a CArG element-dependent manner. In vivo analysis of the human KCNMB1 promoter disclosed activity in embryonic heart and aortic SMCs; mutation of both conserved CArG elements completely abolished in vivo promoter activity. Forced expression of MYOCD increased Kcnmb1 expression in a variety of rodent and human non-SMC lines with no effect on expression of the Kcnma1 subunit. Conversely, knockdown of Srf resulted in decreases of endogenous Kcnmb1. Functional studies demonstrated MYOCD-induced, iberiotoxin-sensitive potassium currents in porcine coronary SMCs. These results reveal the first ion channel subunit as a direct target of SRF-MYOCD transactivation, providing further insight into the role of MYOCD as a master regulator of the SMC contractile phenotype. Smooth muscle cells (SMCs)2 are of crucial importance in maintaining normal structural and contractile integrity of various organs and tissues throughout the vertebrate body. Unlike skeletal and cardiac muscle cells, which largely undergo irreversible terminal differentiation, SMCs have the inherent ability to alter their differentiated state in response to diverse stimulatory cues. This process of SMC phenotypic modulation is a hallmark of several human pathological conditions, including vascular occlusive disease, asthma, intestinal and bladder obstruction, and Alzheimer disease. SMC phenotypic modulation is often defined in terms of unique molecular signatures of gene expression involved with contraction and cytoskeletal architecture as well as extracellular matrix remodeling (1, 2). For example, vascular SMCs display reduced levels of contractile filaments following injury to the vessel wall, adopting the so-called synthetic phenotype characterized by an overabundance of rough endoplasmic reticulum (3). On the other hand, recent studies have shown an exaggerated SMC contractile phenotype thought to contribute to disease progression (4, 5).The majority of SMC-restricted genes contain one or more conserved CArG elements that bind the widely express...

Transcriptional profiling reveals ductus arteriosus-specific genes that regulate vascular tone

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Targeted next-generation sequencing detects point mutations, insertions, deletions and balanced chromosomal rearrangements as well as identifies novel leukemia-specific fusion genes in a single procedure

“…This gene, a potassium large conductance calcium-activated channel family member on chromosome 10q22.3, has been reported to be fundamental to the control of smooth muscle tone and neuronal excitability. [31][32][33] In our patient the RUNX1-KCNMA1 fusion, as confirmed by RT-PCR and Sanger sequencing, led to the disruption of the 'Runt homology domain' (RHD) of RUNX1 (Figure 4b). The break point in RUNX1 was detected in intron 3-4 (ENST00000300305), whereas in t(8;21) the genomic break point is located in intron 5, after the RHD domain.…”

Transforming Growth Factor-β1 (TGF-β1) Utilizes Distinct Pathways for the Transcriptional Activation of MicroRNA 143/145 in Human Coronary Artery Smooth Muscle Cells

“…Western Blotting-Cells were rinsed with phosphate-buffered saline (PBS) twice, and protein was extracted in cold lysis buffer containing 1% protease inhibitor mixture (Sigma) as described (43). Protein concentration was determined by a detergent-compatible protein assay (Bio-Rad).…”

“…Ϫ⌬⌬Ct method for normalization of raw data as described (43). The primers used for different target mRNA and miRs are listed in supplemental Table 1.…”