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.

Public Health Relevance

The smooth muscle of the small resistance arteries by contracting and relaxing is the primary regulator of blood flow to the tissues. The proposed experiments will test the role of Transformer proteins, master determinants of sexual features in flies, in regulating gene expression and function of the small arteries in development and disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL066171-12
Application #
8469330
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Olive, Michelle
Project Start
2001-02-01
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
12
Fiscal Year
2013
Total Cost
$362,950
Indirect Cost
$127,330
Name
University of Maryland Baltimore
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Reho, John J; Fisher, Steven A (2015) The stress of maternal separation causes misprogramming in the postnatal maturation of rat resistance arteries. Am J Physiol Heart Circ Physiol 309:H1468-78
Zheng, Xiaoxu; Reho, John J; Wirth, Brunhilde et al. (2015) TRA2β controls Mypt1 exon 24 splicing in the developmental maturation of mouse mesenteric artery smooth muscle. Am J Physiol Cell Physiol 308:C289-96
Reho, John J; Zheng, Xiaoxu; Asico, Laureano D et al. (2015) Redox signaling and splicing dependent change in myosin phosphatase underlie early versus late changes in NO vasodilator reserve in a mouse LPS model of sepsis. Am J Physiol Heart Circ Physiol 308:H1039-50
Zheng, Xiaoxu; Heaps, Cristine L; Fisher, Steven A (2015) Myosin phosphatase isoforms and related transcripts in the pig coronary circulation and effects of exercise and chronic occlusion. Microvasc Res 98:166-71
Dippold, Rachael P; Fisher, Steven A (2014) Myosin phosphatase isoforms as determinants of smooth muscle contractile function and calcium sensitivity of force production. Microcirculation 21:239-48
Reho, John J; Zheng, Xiaoxu; Fisher, Steven A (2014) Smooth muscle contractile diversity in the control of regional circulations. Am J Physiol Heart Circ Physiol 306:H163-72
Dippold, Rachael P; Fisher, Steven A (2014) A bioinformatic and computational study of myosin phosphatase subunit diversity. Am J Physiol Regul Integr Comp Physiol 307:R256-70
Basu, Sanchita; Srinivasan, Dinesh Kumar; Yang, Ke et al. (2013) Notch transcriptional control of vascular smooth muscle regulatory gene expression and function. J Biol Chem 288:11191-202
Fu, Kang; Mende, Ylva; Bhetwal, Bhupal P et al. (2012) Tra2β protein is required for tissue-specific splicing of a smooth muscle myosin phosphatase targeting subunit alternative exon. J Biol Chem 287:16575-85
Bhetwal, B P; An, C L; Fisher, S A et al. (2011) Regulation of basal LC20 phosphorylation by MYPT1 and CPI-17 in murine gastric antrum, gastric fundus, and proximal colon smooth muscles. Neurogastroenterol Motil 23:e425-36

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