[Show abstract][Hide abstract] ABSTRACT: Vascular smooth muscle cells (VSMCs) of the arterial wall play a critical role in the development of occlusive vascular diseases. Cysteine-rich protein 2 (CRP2) is a VSMC-expressed LIM-only protein, which functionally limits VSMC migration and protects against pathological vascular remodeling. The multifunctional cytokine TGFbeta has been implicated to play a role in the pathogenesis of atherosclerosis through numerous downstream signaling pathways. We showed previously that TGFbeta upregulates CRP2 expression; however, the detailed signaling mechanisms remain unclear.
TGFbeta treatment of VSMCs activated both Smad2/3 and ATF2 phosphorylation. Individually knocking down Smad2/3 or ATF2 pathways with siRNA impaired the TGFbeta induction of CRP2, indicating that both contribute to CRP2 expression. Inhibiting TbetaRI kinase activity by SB431542 or TbetaRI knockdown abolished Smad2/3 phosphorylation but did not alter ATF2 phosphorylation, indicating while Smad2/3 phosphorylation was TbetaRI-dependent ATF2 phosphorylation was independent of TbetaRI. Inhibiting Src kinase activity by SU6656 suppressed TGFbeta-induced RhoA and ATF2 activation but not Smad2 phosphorylation. Blocking ROCK activity, the major downstream target of RhoA, abolished ATF2 phosphorylation and CRP2 induction but not Smad2 phosphorylation. Furthermore, JNK inhibition with SP600125 reduced TGFbeta-induced ATF2 (but not Smad2) phosphorylation and CRP2 protein expression while ROCK inhibition blocked JNK activation. These results indicate that downstream of TbetaRII, Src family kinase-RhoA-ROCK-JNK signaling pathway mediates TbetaRI-independent ATF2 activation. Promoter analysis revealed that the TGFbeta induction of CRP2 was mediated through the CRE and SBE promoter elements that were located in close proximity.
Our results demonstrate that two signaling pathways downstream of TGFbeta converge on the CRE and SBE sites of the Csrp2 promoter to cooperatively control CRP2 induction in VSMCs, which represents a previously unrecognized mechanism of VSMC gene induction by TGFbeta.
Cell Communication and Signaling 03/2014; 12(1):22. DOI:10.1186/1478-811X-12-22 · 4.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cysteine-rich protein (CRP) 2, a member of the LIM-only CRP family that contains two LIM domains, is expressed in vascular smooth muscle cells (VSMCs) of blood vessels and functions to repress VSMC migration and vascular remodeling. The goal of this study was to define the molecular mechanisms by which CRP2 regulates VSMC migration.
Transfection of VSMCs with CRP2-EGFP constructs revealed that CRP2 associated with the actin cytoskeleton. In response to chemoattractant stimulation, Csrp2 (mouse CRP2 gene symbol)-deficient (Csrp2(-/-)) VSMCs exhibited increased lamellipodia formation. Re-introduction of CRP2 abrogated the enhanced lamellipodia formation and migration of Csrp2(-/-) VSMCs following chemoattractant stimulation. Mammalian 2-hybrid and coimmunoprecipitation assays demonstrated that CRP2 interacts with p130Cas, a scaffold protein important for lamellipodia formation and cell motility. Immunofluorescence staining showed that CRP2 colocalized with phospho-p130Cas at focal adhesions (FAs)/terminal ends of stress fibers in non-migrating cells. Interestingly, in migrating cells phospho-p130Cas localized to the leading edge of lamellipodia and FAs while CRP2 was restricted to FAs and stress fibers. Furthermore, we demonstrated that p130Cas expression and phosphorylation promote neointima formation following arterial injury.
These studies demonstrate that CRP2 sequesters p130Cas at FAs, thereby reducing lamellipodia formation and blunting VSMC migration.
Cardiovascular Research 08/2013; 100(3). DOI:10.1093/cvr/cvt207 · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cardiovascular disease accounts for 1 of every 2.9 deaths in the United States, thus the burden of the disease remains high. Given the high mortality and escalating healthcare cost for the disease, it is of urgent need to treat cardiovascular disease effectively. Heme oxygenase-1 (HO-1) catalyzes the oxidation of heme to generate carbon monoxide, biliverdin, and iron. These reaction products of HO-1 have potent anti-inflammatory and anti-oxidative functions. Although HO-1 is expressed at low levels in most tissues under normal basal conditions, it is highly inducible in response to various pathophysiological stresses. Numerous studies have indicated that HO-1 induction is an adaptive defense mechanism to protect cells and tissues against injury in many disease settings. This review highlights the role of HO-1 in inflammation and several cardiovascular diseases-atherosclerosis, myocardial infarction, graft survival after heart transplantation, and abdominal aortic aneurysm. Given that inflammation and oxidative stress are associated with development of cardiovascular disease and that HO-1 has anti-inflammatory and anti-oxidative properties, HO-1 is emerging as a great potential therapeutic target for treating cardiovascular disease.
[Show abstract][Hide abstract] ABSTRACT: The intrinsic defense mechanisms of the body are critical in protecting tissues from injury in response to pathological stress. Heme oxygenase-1 (HO-1), a stress response protein, is induced in response to various pathological stimuli to serve a cytoprotective function. By degrading the oxidant heme and generating the antioxidant bilirubin and anti-inflammatory molecule carbon monoxide, HO-1 may protect cell from injury due to oxidative and pathological stress. Oxidative stress in the heart caused by ischemia and reperfusion leads to cardiomyocyte death and subsequent myocardial infarction. Vascular diseases including atherosclerosis, graft failure, and restenosis are all associated with reactive oxygen species-induced injury and inflammation. Given that cardiovascular disease is the leading cause of death worldwide, there is considerable interest in developing new strategies for preventing and treating cardiovascular disease. Since HO-1 is induced in the heart and blood vessels in response to various stresses, a role of HO-1 has been implicated in cardiovascular homeostasis. Numerous studies using pharmacological method or genetic approach have since demonstrated the cardiovascular protective function of HO-1. Importantly, a number of studies have associated human HO-1 gene promoter polymorphisms with risk for vascular diseases. Taken together, HO-1 has a great therapeutic potential for cardiovascular disease.