-
[show abstract]
[hide abstract]
ABSTRACT: Overactive bladder (OAB) is a pervasive clinical problem involving alterations in both neurogenic and myogenic activity. While there has been some progress in understanding neurogenic inputs to OAB, the mechanisms controlling myogenic bladder activity are unclear. We report the involvement of myocardin (MYOCD) and microRNA-1 (miR-1) in the regulation of connexin 43 (GJA1), a major gap junction in bladder smooth muscle, and the collective role of these molecules during post-natal bladder development. Wild type mouse bladders showed normal development from early postnatal to adult including increases in bladder capacity and maintenance of normal sensitivity to cholinergic agents concurrent with down-regulation of MYOCD and several smooth muscle cell (SMC) contractile genes. Myocardin heterozygous-knockout mice exhibited reduced expression of Myocd mRNA and several SMC contractile genes concurrent with bladder SMC hypersensitivity that was mediated by gap junctions. In both cultured rat bladder SMC and in vivo bladders, MYOCD down-regulated GJA1 expression through miR-1 up-regulation. Interestingly, adult myocardin heterozygous-knockout mice showed normal increases in bladder and body weight but lower bladder capacity compared to wild type mice. These results suggest that MYOCD down-regulates GJA1 expression via miR-1 up-regulation, thereby contributing to maintenance of normal sensitivity and development of bladder capacity. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
Journal of Cellular Physiology 01/2013; · 3.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Smooth muscle calponin (CNN1) contains multiple conserved intronic CArG elements that bind serum response factor and display enhancer activity in vitro. The objectives here were to evaluate these CArG elements for activity in transgenic mice and determine the effect of human CNN1 on injury-induced vascular remodeling.
Mice carrying a lacZ reporter under control of intronic CArG elements in the human CNN1 gene failed to show smooth muscle cell (SMC)-restricted activity. However, deletion of the orthologous sequences in mice abolished endogenous Cnn1 promoter activity, suggesting their necessity for in vivo Cnn1 expression. Mice carrying a 38-kb bacterial artificial chromosome (BAC) harboring the human CNN1 gene displayed SMC- restricted expression of the corresponding CNN1 protein, as measured by immunohistochemistry and Western blotting. Extensive BAC recombineering studies revealed the absolute necessity of a single intronic CArG element for correct SMC-restricted expression of human CNN1. Overexpressing human CNN1 suppressed neointimal formation following arterial injury. Mice with an identical BAC carrying mutations in CArG elements that inhibit human CNN1 expression showed outward remodeling and neointimal formation.
A single intronic CArG element is necessary but insufficient for proper CNN1 expression in vivo. CNN1 overexpression antagonizes arterial injury-induced neointimal formation.
Arteriosclerosis Thrombosis and Vascular Biology 08/2011; 31(10):2172-80. · 6.37 Impact Factor
-
Jeffrey W Streb,
Xiaochun Long,
Ting-Hein Lee,
Qiang Sun,
Chad M Kitchen,
Mary A Georger, Orazio J Slivano,
William S Blaner,
Daniel W Carr,
Irwin H Gelman,
Joseph M Miano
[show abstract]
[hide abstract]
ABSTRACT: Retinoids are used clinically to treat a number of hyper-proliferative disorders and have been shown in experimental animals to attenuate vascular occlusive diseases, presumably through nuclear receptors bound to retinoic acid response elements (RARE) located in target genes. Here, we show that natural or synthetic retinoids rapidly induce mRNA and protein expression of a specific isoform of A-Kinase Anchoring Protein 12 (AKAP12β) in cultured smooth muscle cells (SMC) as well as the intact vessel wall. Expression kinetics and actinomycin D studies indicate Akap12β is a retinoid-induced, immediate-early gene. Akap12β promoter analyses reveal a conserved RARE mildly induced with atRA in a region that exhibits hyper-acetylation. Immunofluorescence microscopy and protein kinase A (PKA) regulatory subunit overlay assays in SMC suggest a physical association between AKAP12β and PKA following retinoid treatment. Consistent with its designation as a tumor suppressor, inducible expression of AKAP12β attenuates SMC growth in vitro. Further, immunohistochemistry studies establish marked decreases in AKAP12 expression in experimentally-injured vessels of mice as well as atheromatous lesions in humans. Collectively, these results demonstrate a novel role for retinoids in the induction of an AKAP tumor suppressor that blocks vascular SMC growth thus providing new molecular insight into how retiniods may exert their anti-proliferative effects in the injured vessel wall.
PLoS ONE 01/2011; 6(4):e18538. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
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 alpha and beta 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.
Journal of Biological Chemistry 10/2009; 284(48):33671-82. · 4.77 Impact Factor
