The goal of this project is to elucidate the novel regulatory mechanism by which the Rac1 pathway regulates smooth muscle contraction. The smooth muscle contraction is a critical component for the regulation of constriction of hollow organs such as airway and arteries, thus controlling airflow and blood pressure, therefore, the proposed study will provide a novel insight into vascular and airway diseases. Smooth muscle contraction is primarily regulated by myosin light chain (MLC) phosphorylation, however, recent studies have suggested that actin cytoskeletal rearrangement may be in part responsible for the change in contraction. In this proposal, we hypothesize that the Rac signaling pathway concertedly controls smooth muscle contraction by changing MLC phosphorylation and cytoskeletal rearrangement. MLC phosphorylation is regulated by both Ca2+ dependent and Ca2+ independent pathways, and MLC phosphatase (MLCP) plays a key role in the latter mechanism. MLCP activity is regulated by the phosphorylation of MYPT1, a myosin binding regulatory subunit of MLCP, and CPI-17, a MLCP specific inhibitor. The research in the past has focused on the kinases responsible for MYPT1 and CPI-17 phosphorylation, such as Rho kinase and PKC, but nothing is known about the protein phosphatases that dephosphorylate MYPT1 and CPI-17. Based upon our findings, we hypothesize that the Rac pathway regulates MYPT1/CPI-17 phosphatases during agonist stimulation, which regulates MLCP and is in part responsible for the Rac dependent contractile regulation. Since smooth muscle undergoes rapid mechanical plasticity involving actin cytoskeletal change, we hypothesize that agonist stimulation induces Rac translocation to the membrane, where it activates its down-stream targets such as WAVE and PAK to recruit adhesion junction proteins, which strengthen the connections between the membrane adhesion junctions and actomyosin filaments to transmit force. We will first determine if Rac1 is activated after agonist stimulation. To evaluate the role of Rac1 in contraction, we will use pharmacological specific Rac inhibitors and molecular biological tools and gene silencing. Furthermore, we will clarify the mechanism by which Rac activation regulates the contraction. The change in MYPT1 phosphatase and/or CPI-17 phosphatase activities will be determined along with the Rac activity change using biochemical means. We will also examine if Rac1 activation stimulates the actin cytoskeletal change via WAVE and ARP2/3 translocation to the cell periphery. We will measure actin polymerization, and the binding of Rac and its down stream proteins. Translocation of Rac1 and its down-stream targets will also be studied with arterial tissues and single cells using a two-photon digital microscope, 3D digital confocal microscope, and a total internal reflection fluorescence (TIRF) microscope with super resolution analysis. The Rac1 dependent ultrastructural change will be achieved by electron microscopy using tomography technique to obtain 3D structural images.

Public Health Relevance

Smooth muscle plays an important role in the constriction/dilation of hollow organs such as airway and blood vessels and controls airway flow and blood pressure. Therefore, its malfunction causes various diseases such as high blood pressure and asthma. The proposed project will clarify the role of the novel regulatory pathway, i.e., the Rac signaling pathway, linking the external stimuli and the contraction of smooth muscle, and thus will improve our understanding of vascular and airway diseases.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL111696-04
Application #
8829892
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Olive, Michelle
Project Start
2012-07-05
Project End
2017-03-31
Budget Start
2016-04-01
Budget End
2017-03-31
Support Year
4
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of Texas Health Center at Tyler
Department
Type
Overall Medical
DUNS #
800772337
City
Tyler
State
TX
Country
United States
Zip Code
75708
Komatsu, Satoshi; Kitazawa, Toshio; Ikebe, Mitsuo (2018) Visualization of stimulus-specific heterogeneous activation of individual vascular smooth muscle cells in aortic tissues. J Cell Physiol 233:434-446
Lee, Kyoung Hwan; Sulbarán, Guidenn; Yang, Shixin et al. (2018) Interacting-heads motif has been conserved as a mechanism of myosin II inhibition since before the origin of animals. Proc Natl Acad Sci U S A 115:E1991-E2000
Philley, Julie V; Kannan, Anbarasu; Qin, Wenyi et al. (2016) Complex-I Alteration and Enhanced Mitochondrial Fusion Are Associated With Prostate Cancer Progression. J Cell Physiol 231:1364-74
Tiwari, Nivedita; Marudamuthu, Amarnath S; Tsukasaki, Yoshikazu et al. (2016) p53- and PAI-1-mediated induction of C-X-C chemokines and CXCR2: importance in pulmonary inflammation due to cigarette smoke exposure. Am J Physiol Lung Cell Mol Physiol 310:L496-506
Owens, Shuzi; Jeffers, Ann; Boren, Jake et al. (2015) Mesomesenchymal transition of pleural mesothelial cells is PI3K and NF-?B dependent. Am J Physiol Lung Cell Mol Physiol 308:L1265-73
Hosoba, Kosuke; Komatsu, Satoshi; Ikebe, Mitsuo et al. (2015) Phosphorylation of myosin II regulatory light chain by ZIP kinase is responsible for cleavage furrow ingression during cell division in mammalian cultured cells. Biochem Biophys Res Commun 459:686-91
Marudamuthu, Amarnath S; Shetty, Shwetha K; Bhandary, Yashodhar P et al. (2015) Plasminogen activator inhibitor-1 suppresses profibrotic responses in fibroblasts from fibrotic lungs. J Biol Chem 290:9428-41
Shibata, Keita; Sakai, Hiroyasu; Huang, Qian et al. (2015) Rac1 regulates myosin II phosphorylation through regulation of myosin light chain phosphatase. J Cell Physiol 230:1352-64
Komatsu, Satoshi; Ikebe, Mitsuo (2014) ZIPK is critical for the motility and contractility of VSMCs through the regulation of nonmuscle myosin II isoforms. Am J Physiol Heart Circ Physiol 306:H1275-86
Zhang, Cheng-Hai; Lifshitz, Lawrence M; Uy, Karl F et al. (2013) The cellular and molecular basis of bitter tastant-induced bronchodilation. PLoS Biol 11:e1001501