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.
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.
|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|