Genomes and Genes
SMOOTH MUSCLE MYOSIN PHOSPHATASE SUBUNIT ISOFORMS
Principal Investigator: Steven A Fisher
Abstract: DESCRIPTION (provided by applicant): Myosin phosphatase (MP) is the primary effector of smooth muscle relaxation and a key target of signaling pathways that regulate vessel tone. MP is a hetero-trimer composed of catalytic (PP1c), targeting/regulatory (MYPT1) and 21 kD (M21) subunits. Our long term goals are to understand the regulated expression of MP/MYPT1 isoforms in relation to vascular function in development and disease. We have shown that isoforms of MYPT1 generated by alternative splicing of a 31 nt exon (E23) are tissue-specific, developmentally regulated, evolutionarily conserved and modulate in disease. E23 is spliced in smooth muscle tissues with fast (phasic) or intermediate contractile properties, e.g. portal vein and mesenteric resistance arteries, and skipped in the slow (tonic) smooth muscle of the large arteries and veins. A switch from E23 skipping to splicing occurs in the perinatal period in tissues that acquire a fast phenotype. In disease models of altered blood flow/pressure, the PV and mesenteric resistance arteries shift to E23 skipping as part of a generalized shift towards the slow phenotype. E23 skipping codes for a MYPT1 C-terminus leucine zipper (LZ) motif required for cGMP-dependent protein kinase (cGK1) dimerization and activation of MP, resulting in calcium de-sensitization of force production. Inclusion of the 31 nt exon codes for the MYPT1 LZ- isoform. We have shown in a number of models that tissues that express the E23-included/LZ- isoform are less sensitive to NO/cGMP-mediated relaxation, suggesting that the regulated splicing of E23 serves as a way for vascular smooth muscle to fine tune its sensitivity to NO/cGMP signaling. This renewal focuses on the regulation of MYPT1 E23 splicing. We propose to test the hypothesis that Transformer2b is a novel regulator of E23 splicing/vascular smooth muscle phenotypic specification in development and modulation in disease, and the corollary that Tra2b can be used as a novel nodal point in this line of investigation. The Transformer splicing factors were originally identified as master regulators of sexually dimorphic traits in the fly. The role of the vertebrate homologues in phenotypic specification is unknown. In the previous funding period we showed 1) a strong and evolutionarily conserved correlation between Tra2b expression and E23 splicing in developmental and disease models 2) Tra2b binds E23 and trans-activates its splicing from a mini-gene construct. The following aims are proposed: Aim 1: Define the role of Tra2b cis-element in the regulation of MYPT1 E23 splicing through the generation of mutation/deletion/chimeric constructs, measurements of binding affinity, and assay for binding in vivo. Aim 2: Test the role of Tra2b in vivo with a) LacZ targeting the Tra2b locus as a reporter for the expression of Tra2b in different vascular beds throughout development b) Cre-lox mediated conditional inactivation of TRa2b in VSM (loss-of-function) c) forced expression of Tra2b in the mesenteric arteries in the disease models (gain-of-function/rescue). Aim 3: Tra2b as a novel nodal point for the dissection of VSM diversification. An ~500 nt ultra-conserved sequence (UCS) in the first intron of Tra2b will be tested for its ability to drive tissue-specific transcription in fast smooth muscle in a transgenic assay. The UCS will be dissected through bio-informatics, deletion/mutation, and candidate factor (T3,NFAT) approaches. These studies will provide novel insights regarding the role of Tra2b, and novel links between transcriptional and splicing controls, in the generation and modulation of vascular smooth muscle contractile phenotypic diversity.
Funding Period: 2001-02-01 - 2014-04-30
more information: NIH RePORT
- Myosin phosphatase isoform switching in vascular smooth muscle developmentMichael C Payne
Department of Medicine, 422 BRB, 2109 Adelbert Road, Case Western Reserve School of Medicine, Cleveland, OH 44106 4958, USA
J Mol Cell Cardiol 40:274-82. 2006..We propose that MP isoform switching during neonatal vascular smooth muscle phenotypic specification may determine changing vascular responses to NO/cGMP signaling in the transition from the fetal to the adult circulation...
- Smooth muscle contractile diversity in the control of regional circulationsJohn J Reho
Division of Cardiology, School of Medicine, University of Maryland, Baltimore, Maryland
Am J Physiol Heart Circ Physiol 306:H163-72. 2014..We consider how these unique properties may allow for selective drug targeting of regional circulations for therapeutic benefit and point out gaps in our knowledge and areas in need of further investigation. ..
- Notch transcriptional control of vascular smooth muscle regulatory gene expression and functionSanchita Basu
Department of Medicine, Case Cardiovascular Research Institute, Case Western Reserve University, Cleveland, Ohio 44106, USA
J Biol Chem 288:11191-202. 2013..Genetic or pharmacological manipulation of Notch signaling is a potential strategy for modulating arterial function in human disease...
- Tra2β protein is required for tissue-specific splicing of a smooth muscle myosin phosphatase targeting subunit alternative exonKang Fu
Department of Medicine Cardiology, Case Western Reserve University, Cleveland, Ohio 44106, USA
J Biol Chem 287:16575-85. 2012..Tra2β, by regulating the splicing of Mypt1 E23, sets the sensitivity of smooth muscle to cGMP-mediated relaxation...
- Regulation of basal LC20 phosphorylation by MYPT1 and CPI-17 in murine gastric antrum, gastric fundus, and proximal colon smooth musclesB P Bhetwal
Department of Physiology and Cell Biology, Center of Biomedical Research Excellence, University of Nevada School of Medicine, 1664 N Virginia St, Reno, NV 89557, USA
Neurogastroenterol Motil 23:e425-36. 2011..Here, we investigate the role of ROK in regulating LC20 phosphorylation and spontaneous contractions of gastric fundus, gastric antrum, and proximal colon smooth muscles...
- Vascular smooth muscle phenotypic diversity and functionSteven A Fisher
Department of Medicine, and Cardiovascular Research Institute, Case Western Reserve University, Cleveland, Ohio 44106 7290, USA
Physiol Genomics 42:169-87. 2010..This review focuses on smooth muscle contractile phenotypic diversity in the vascular system, how it is generated, and how it may determine vascular function in developmental and disease contexts...
- Induction of PDE5 and de-sensitization to endogenous NO signaling in a systemic resistance artery under altered blood flowHaiying Zhang
Department of Medicine Cardiology, Case Western Reserve School of Medicine, 4 533 Wolstein Research Bldg, 2103 Cornell Rd, Cleveland, OH 44106, USA
J Mol Cell Cardiol 47:57-65. 2009..Treatment with PDE5 antagonists, in contrast to NO donors, may more specifically and effectively increase blood flow to chronically hypo-perfused tissues...
- Tra2beta as a novel mediator of vascular smooth muscle diversificationSupriya Shukla
Departments of Medicine Cardiology, Case Western Reserve School of Medicine, Cleveland, Ohio 44106, USA
Circ Res 103:485-92. 2008....
- Uterine artery myosin phosphatase isoform switching and increased sensitivity to SNP in a rat L-NAME model of hypertension of pregnancyYuan Lu
Department of Medicine, Case Western Reserve School of Medicine, Cleveland, OH 44106 7290, USA
Am J Physiol Cell Physiol 294:C564-71. 2008..We propose that MYPT1 isoform switching is an adaptive response to reduce vascular resistance and maintain uterine blood flow in the setting of hypertension-triggered inward remodeling of the UAs in hypertension of pregnancy...
- Conditioning effect of blood flow on resistance artery smooth muscle myosin phosphataseHaiying Zhang
Department of Medicine Cardiology, Case Western Reserve School of Medicine, Cleveland, Ohio 44106, USA
Circ Res 100:730-7. 2007..The loss of this conditioning effect significantly increases the sensitivity to vasodilator signals in the setting of chronically reduced blood flow...
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