SMOOTH MUSCLE MYOSIN PHOSPHATASE SUBUNIT ISOFORMS

Summary

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

Top Publications

  1. ncbi Myosin phosphatase isoform switching in vascular smooth muscle development
    Michael 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
  2. pmc Smooth muscle contractile diversity in the control of regional circulations
    John J Reho
    Division of Cardiology, School of Medicine, University of Maryland, Baltimore, Maryland
    Am J Physiol Heart Circ Physiol 306:H163-72. 2014
  3. pmc Notch transcriptional control of vascular smooth muscle regulatory gene expression and function
    Sanchita Basu
    Department of Medicine, Case Cardiovascular Research Institute, Case Western Reserve University, Cleveland, Ohio 44106, USA
    J Biol Chem 288:11191-202. 2013
  4. pmc Tra2β protein is required for tissue-specific splicing of a smooth muscle myosin phosphatase targeting subunit alternative exon
    Kang Fu
    Department of Medicine Cardiology, Case Western Reserve University, Cleveland, Ohio 44106, USA
    J Biol Chem 287:16575-85. 2012
  5. pmc Regulation of basal LC20 phosphorylation by MYPT1 and CPI-17 in murine gastric antrum, gastric fundus, and proximal colon smooth muscles
    B 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
  6. pmc Vascular smooth muscle phenotypic diversity and function
    Steven A Fisher
    Department of Medicine, and Cardiovascular Research Institute, Case Western Reserve University, Cleveland, Ohio 44106 7290, USA
    Physiol Genomics 42:169-87. 2010
  7. pmc Induction of PDE5 and de-sensitization to endogenous NO signaling in a systemic resistance artery under altered blood flow
    Haiying 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
  8. pmc Tra2beta as a novel mediator of vascular smooth muscle diversification
    Supriya Shukla
    Departments of Medicine Cardiology, Case Western Reserve School of Medicine, Cleveland, Ohio 44106, USA
    Circ Res 103:485-92. 2008
  9. ncbi Uterine artery myosin phosphatase isoform switching and increased sensitivity to SNP in a rat L-NAME model of hypertension of pregnancy
    Yuan Lu
    Department of Medicine, Case Western Reserve School of Medicine, Cleveland, OH 44106 7290, USA
    Am J Physiol Cell Physiol 294:C564-71. 2008
  10. ncbi Conditioning effect of blood flow on resistance artery smooth muscle myosin phosphatase
    Haiying Zhang
    Department of Medicine Cardiology, Case Western Reserve School of Medicine, Cleveland, Ohio 44106, USA
    Circ Res 100:730-7. 2007

Research Grants

  1. Excitation contraction coupling in bladder smooth muscle
    Robert S Moreland; Fiscal Year: 2013
  2. Physiological Regulation of MLCK in Intact Arteries
    Withrow Gil Wier; Fiscal Year: 2013
  3. MITOCHONDRIAL ENCEPHALOMYOPATHIES AND MENTAL RETARDATION
    Salvatore DiMauro; Fiscal Year: 2013
  4. HORMONAL REGULATION OF BLOOD PRESSURE
    Michal Laniado Schwartzman; Fiscal Year: 2013
  5. Molecular Analyses and Interventions for Biodefense and Emerging Pathogens
    Olaf Schneewind; Fiscal Year: 2013
  6. IPF Fibroblast Phenotype
    Craig A Henke; Fiscal Year: 2013
  7. Neurohumoral control of veins in hypertension
    Gregory D Fink; Fiscal Year: 2013
  8. MINNESOTA OBESITY CENTER
    Allen S Levine; Fiscal Year: 2013
  9. Interactive Signaling Modules in Vascular Inflammation
    Linda H Shapiro; Fiscal Year: 2013
  10. KCNQ Channels and Vasoconstrictor Signal Transduction
    Kenneth L Byron; Fiscal Year: 2013

Detail Information

Publications11

  1. ncbi Myosin phosphatase isoform switching in vascular smooth muscle development
    Michael 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...
  2. pmc Smooth muscle contractile diversity in the control of regional circulations
    John 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. ..
  3. pmc Notch transcriptional control of vascular smooth muscle regulatory gene expression and function
    Sanchita 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...
  4. pmc Tra2β protein is required for tissue-specific splicing of a smooth muscle myosin phosphatase targeting subunit alternative exon
    Kang 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...
  5. pmc Regulation of basal LC20 phosphorylation by MYPT1 and CPI-17 in murine gastric antrum, gastric fundus, and proximal colon smooth muscles
    B 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...
  6. pmc Vascular smooth muscle phenotypic diversity and function
    Steven 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...
  7. pmc Induction of PDE5 and de-sensitization to endogenous NO signaling in a systemic resistance artery under altered blood flow
    Haiying 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...
  8. pmc Tra2beta as a novel mediator of vascular smooth muscle diversification
    Supriya Shukla
    Departments of Medicine Cardiology, Case Western Reserve School of Medicine, Cleveland, Ohio 44106, USA
    Circ Res 103:485-92. 2008
    ....
  9. ncbi Uterine artery myosin phosphatase isoform switching and increased sensitivity to SNP in a rat L-NAME model of hypertension of pregnancy
    Yuan 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...
  10. ncbi Conditioning effect of blood flow on resistance artery smooth muscle myosin phosphatase
    Haiying 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...

Research Grants31

  1. Excitation contraction coupling in bladder smooth muscle
    Robert S Moreland; Fiscal Year: 2013
    ..The studies proposed in this application will provide this missing information. If we can learn how normal bladder is controlled, we can then design experiments to study and hopefully repair abnormal bladder ..
  2. Physiological Regulation of MLCK in Intact Arteries
    Withrow Gil Wier; Fiscal Year: 2013
    ..This research will utilize a mouse model of salt-induced hypertension to provide specific new information on the role of MLCK in high blood pressure. ..
  3. MITOCHONDRIAL ENCEPHALOMYOPATHIES AND MENTAL RETARDATION
    Salvatore DiMauro; Fiscal Year: 2013
    ....
  4. HORMONAL REGULATION OF BLOOD PRESSURE
    Michal Laniado Schwartzman; Fiscal Year: 2013
    ..ular tone, in the pathophysiology of hypertension and cardiovascular disease. ..
  5. Molecular Analyses and Interventions for Biodefense and Emerging Pathogens
    Olaf Schneewind; Fiscal Year: 2013
    ..Research and training at the GLRCE is governed by a mechanism involving ongoing review of scientific excellence and translational goals, inter-institutional advisory boards and external scientific advisory bodies. ..
  6. IPF Fibroblast Phenotype
    Craig A Henke; Fiscal Year: 2013
    ..A major objective of this Program Project is to inform decisions of the IPF Clinical Network by providing information that can be translated into novel therapeutic strategies for IPF. ..
  7. Neurohumoral control of veins in hypertension
    Gregory D Fink; Fiscal Year: 2013
    ..This project tests the idea that altered structure or function of veins also may cause hypertension, and that it may be possible to treat hypertension using drugs that affect veins. ..
  8. MINNESOTA OBESITY CENTER
    Allen S Levine; Fiscal Year: 2013
    ..Resources for oilot/feasibilitv proiects and an educational program will be established. ..
  9. Interactive Signaling Modules in Vascular Inflammation
    Linda H Shapiro; Fiscal Year: 2013
    ..abstract_text> ..
  10. KCNQ Channels and Vasoconstrictor Signal Transduction
    Kenneth L Byron; Fiscal Year: 2013
    ....