Thrombin, a serine/threonine protease, besides its role in blood coagulation, plays a significant role as a mitogen and motogen to many cell types, in particular to vascular smooth muscle cells (VSMCs). Thrombin mediates its effects via protease-activated receptors (PARs), namely PAR-1, PAR-2, PAR-3 and PAR-4. Downstream to PARs, it requires the need for the participation of G proteins, notably Gq/11 or G12/13 in the regulation of cell migration and proliferation. In addition, thrombin possesses the capacity to transactivate receptor tyrosine kinases (RTKs), among which epidermal growth factor receptor (EGFR) gained more attention. While protease-dependent shedding of heparin-binding growth factors, in this case, heparin-binding epidermal growth factor (HB-EGF), appears to be accountable for thrombin transactivation of EGFR, there appears to be a gap in our understanding of how this EGFR transactivation influences thrombin-induced VSMC mitogenesis and motogenesis. The major question is, is transactivation of EGFR sufficient in the stimulation of mitogenic and/or motogenic signal flows downstream to the receptor? Towards elucidating these signal flows, we discovered that thrombin activates GRB2-associated binding protein 1 (Gab1) and Src homology 2- containing protein tyrosine phosphatase (Shp2), whose stimulation is otherwise expected in response to EGFR activation by its true ligand, EGF, and the stimulation of this multifunctional signaling complex requires EGFR tyrosine kinase activity. What is more exciting is that Gab1-Shp2 activation is required for thrombin-induced Rho GTPase stimulation and F-actin stress fiber formation. Based on these novel observations, we propose to test the following specific aims with a goal to elucidate the G protein-coupled receptor (GPCR) signal flows that are upstream and downstream to EGFR transactivation in human aortic smooth muscle cells (HASMCs) and test their strength in the mediation of F-actin stress fiber formation, migration and proliferation of these cells in response to thrombin in vitro and vascular wall remodeling after angioplasty in vivo.
The specific aims that will be addressed in this research proposal are as follows: 1. Thrombin-induced HASMC F-actin stress fiber formation, migration, proliferation and neointima formation require Gab1 activation. 2. Thrombin activates Rho GTPases via recruitment of RhoGEFs by Gab1 and RhoGEF-dependent RhoA, Rac1 and Cdc42 activation mediate HASMC F-actin stress fiber formation, migration, proliferation and neointima formation. 3. Gab1 targets PAK1 in mediating thrombin-induced HASMC F-actin stress fiber formation, migration, proliferation and neointima formation. Briefly, the results of the proposed experiments will fill the gap in our understanding of how GPCR signaling via crosstalk with RTK signaling and targeting a scaffold adaptor molecule, Gab1, leading to RhoGEF-mediated RhoA-Rac1/Cdc42-PAK1 activation plays a role in vascular wall remodeling following injury. Such comprehensive knowledge on the pathobiology of vascular wall diseases could become a valuable tool in the development of drugs for the control of these vascular lesions.

Public Health Relevance

Vascular smooth muscle cell (VSMC) migration and proliferation play an important role in peripheral vascular diseases such as atherosclerosis and restenosis following angioplasty or vein grafting. Elucidating the molecular mechanisms underlying VSMC migration and proliferation is crucial in the development of therapeutic agents to control the disease process of these vascular lesions. In this regard, the present grant proposal seeks to study the mechanisms by which thrombin, a G protein-coupled receptor agonist and a clotting factor that is produced at the site of vascular injury could transactivate receptor tyrosine kinase signaling in the stimulation of both VSMC migration and proliferation and ignite the disease process.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL103575-03
Application #
8444697
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Gao, Yunling
Project Start
2011-04-01
Project End
2016-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
3
Fiscal Year
2013
Total Cost
$440,954
Indirect Cost
$143,012
Name
University of Tennessee Health Science Center
Department
Physiology
Type
Schools of Medicine
DUNS #
941884009
City
Memphis
State
TN
Country
United States
Zip Code
38163
Janjanam, Jagadeesh; Rao, Gadiparthi N (2016) Novel role of cortactin in G protein-coupled receptor agonist-induced nuclear export and degradation of p21Cip1. Sci Rep 6:28687
Janjanam, Jagadeesh; Chandaka, Giri Kumar; Kotla, Sivareddy et al. (2015) PLCβ3 mediates cortactin interaction with WAVE2 in MCP1-induced actin polymerization and cell migration. Mol Biol Cell 26:4589-606
Singh, Nikhlesh K; Kotla, Sivareddy; Dyukova, Elena et al. (2015) Disruption of p21-activated kinase 1 gene diminishes atherosclerosis in apolipoprotein E-deficient mice. Nat Commun 6:7450
Gadepalli, Ravisekhar; Kotla, Sivareddy; Heckle, Mark R et al. (2013) Novel role for p21-activated kinase 2 in thrombin-induced monocyte migration. J Biol Chem 288:30815-31
Kundumani-Sridharan, Venkatesh; Singh, Nikhlesh K; Kumar, Sanjay et al. (2013) Nuclear factor of activated T cells c1 mediates p21-activated kinase 1 activation in the modulation of chemokine-induced human aortic smooth muscle cell F-actin stress fiber formation, migration, and proliferation and injury-induced vascular wall remodeli J Biol Chem 288:22150-62
Singh, Nikhlesh K; Kundumani-Sridharan, Venkatesh; Kumar, Sanjay et al. (2012) Protein kinase N1 is a novel substrate of NFATc1-mediated cyclin D1-CDK6 activity and modulates vascular smooth muscle cell division and migration leading to inward blood vessel wall remodeling. J Biol Chem 287:36291-304
Gadepalli, Ravisekhar; Singh, Nikhlesh K; Kundumani-Sridharan, Venkatesh et al. (2012) Novel role of proline-rich nonreceptor tyrosine kinase 2 in vascular wall remodeling after balloon injury. Arterioscler Thromb Vasc Biol 32:2652-61
Keilani, Serene; Chandwani, Samira; Dolios, Georgia et al. (2012) Egr-1 induces DARPP-32 expression in striatal medium spiny neurons via a conserved intragenic element. J Neurosci 32:6808-18