Caveolin-1 negatively regulates a metalloprotease-dependent epidermal growth factor receptor transactivation by angiotensin II.

A metalloprotease, ADAM17, mediates the generation of mature ligands for the epidermal growth factor receptor (EGFR). This is the key signaling step by which angiotensin II (AngII) induces EGFR transactivation leading to hypertrophy and migration of vascular smooth muscle cells (VSMCs). However, the regulatory mechanism of ADAM17 activity remains largely unclear. Here we hypothesized that caveolin-1 (Cav1), the major structural protein of a caveolae, a membrane microdomain, is involved in the regulation of ADAM17. In cultured VSMCs, infection of adenovirus encoding Cav1 markedly inhibited AngII-induced EGFR ligand shedding, EGFR transactivation, ERK activation, hypertrophy and migration, but not intracellular Ca(2+) elevation. Methyl-ß-cyclodextrin and filipin, reagents that disrupt raft structure, both stimulated an EGFR ligand shedding and EGFR transactivation in VSMCs. In addition, non-detergent sucrose gradient membrane fractionations revealed that ADAM17 cofractionated with Cav1 in lipid rafts. These results suggest that lipid rafts and perhaps caveolae provide a negative regulatory environment for EGFR transactivation linked to vascular remodeling induced by AngII. These novel findings may provide important information to target cardiovascular diseases under the enhanced renin angiotensin system.

Distinct roles of protease-activated receptors in signal transduction regulation of endothelial nitric oxide synthase.

Protease-activated receptors (PARs), such as PAR1 and PAR2, have been implicated in the regulation of endothelial NO production. We hypothesized that PAR1 and PAR2 distinctly regulate the activity of endothelial NO synthase through the selective phosphorylation of a positive regulatory site, Ser(1179), and a negative regulatory site, Thr(497), in bovine aortic endothelial cells. A selective PAR1 ligand, TFLLR, stimulated the phosphorylation of endothelial NO synthase at Thr(497). It had a minimal effect on Ser(1179) phosphorylation. In contrast, a selective PAR2 ligand, SLIGRL, stimulated the phosphorylation of Ser(1179) with no noticeable effect on Thr(497). Thrombin has been shown to transactivate PAR2 through PAR1. Thus, thrombin, as well as a peptide mimicking the PAR1 tethered ligand, TRAP, stimulated phosphorylation of both sites. Also, thrombin and SLIGRL, but not TFLLR, stimulated cGMP production. A G(q) inhibitor blocked thrombin- and SLIGRL-induced Ser(1179) phosphorylation, whereas it enhanced thrombin-induced Thr(497) phosphorylation. In contrast, a G(12/13) inhibitor blocked thrombin- and TFLLR-induced Thr(497) phosphorylation, whereas it enhanced the Ser(1179) phosphorylation. Although a Rho-kinase inhibitor, Y27632, blocked the Thr(497) phosphorylation, other inhibitors that targeted Rho-kinase failed to block TFLLR-induced Thr(497) phosphorylation. These data suggest that PAR1 and PAR2 distinctly regulate endothelial NO synthase phosphorylation and activity through G(12/13) and G(q), respectively, delineating the novel signaling pathways by which the proteases act on protease-activated receptors to potentially modulate endothelial functions.

Endothelial nitric oxide synthase inhibits G12/13 and rho-kinase activated by the angiotensin II type-1 receptor: implication in vascular migration.

BACKGROUND: Although, endothelial nitric oxide (NO) synthase (eNOS) is believed to antagonize vascular remodeling induced by the angiotensin II (AngII) type-1 receptor, the exact signaling mechanism remains unclear. METHODS AND RESULTS: By expressing eNOS to vascular smooth muscle cells (VSMCs) via adenovirus, we investigated a signal transduction mechanism of the eNOS gene transfer in preventing vascular remodeling induced by AngII. We found marked inhibition of AngII-induced Rho/Rho-kinase activation and subsequent VSMC migration by eNOS gene transfer whereas G(q)-dependent transactivation of the epidermal growth factor receptor by AngII remains intact. This could be explained by the specific inhibition of G(12/13) activation by eNOS-mediated G(12/13) phosphorylation. CONCLUSIONS: The eNOS/NO cascade specifically targets the Rho/Rho-kinase system via inhibition of G(12/13) to prevent vascular migration induced by AngII, representing a novel signal cross-talk in cardiovascular protection by NO.

Central role of Gq in the hypertrophic signal transduction of angiotensin II in vascular smooth muscle cells.

The angiotensin II (AngII) type 1 receptor (AT(1)) plays a critical role in hypertrophy of vascular smooth muscle cells (VSMCs). Although it is well known that G(q) is the major G protein activated by the AT(1) receptor, the requirement of G(q) for AngII-induced VSMC hypertrophy remains unclear. By using cultured VSMCs, this study examined the requirement of G(q) for the epidermal growth factor receptor (EGFR) pathway, the Rho-kinase (ROCK) pathway, and subsequent hypertrophy. AngII-induced intracellular Ca(2+) elevation was completely inhibited by a pharmacological G(q) inhibitor as well as by adenovirus encoding a G(q) inhibitory minigene. AngII (100nm)-induced EGFR transactivation was almost completely inhibited by these inhibitors, whereas these inhibitors only partially inhibited AngII (100nm)-induced phosphorylation of a ROCK substrate, myosin phosphatase target subunit-1. Stimulation of VSMCs with AngII resulted in an increase of cellular protein and cell volume but not in cell number. The G(q) inhibitors completely blocked these hypertrophic responses, whereas a G protein-independent AT(1) agonist did not stimulate these hypertrophic responses. In conclusion, G(q) appears to play a major role in the EGFR pathway, leading to vascular hypertrophy induced by AngII. Vascular G(q) seems to be a critical target of intervention against cardiovascular diseases associated with the enhanced renin-angiotensin system.

Novel role of protein kinase C-delta Tyr 311 phosphorylation in vascular smooth muscle cell hypertrophy by angiotensin II.

We have shown previously that activation of protein kinase C-delta (PKC delta) is required for angiotensin II (Ang II)-induced migration of vascular smooth muscle cells (VSMCs). Here, we have hypothesized that PKC delta phosphorylation at Tyr(311) plays a critical role in VSMC hypertrophy induced by Ang II. Immunoblotting was used to monitor PKC delta phosphorylation at Tyr(311), and cell size and protein measurements were used to detect hypertrophy in VSMCs. PKC delta was rapidly (0.5 to 10.0 minutes) phosphorylated at Tyr(311) by Ang II. This phosphorylation was markedly blocked by an Src family kinase inhibitor and dominant-negative Src but not by an epidermal growth factor receptor kinase inhibitor. Ang II-induced Akt phosphorylation and hypertrophic responses were significantly enhanced in VSMCs expressing PKC delta wild-type compared with VSMCs expressing control vector, whereas the enhancements were markedly diminished in VSMCs expressing a PKC delta Y311F mutant. Also, these responses were significantly inhibited in VSMCs expressing kinase-inactive PKC delta K376A compared with VSMCs expressing control vector. From these data, we conclude that not only PKC delta kinase activation but also the Src-dependent Tyr(311) phosphorylation contributes to Akt activation and subsequent VSMC hypertrophy induced by Ang II, thus signifying a novel molecular mechanism for enhancement of cardiovascular diseases induced by Ang II.

Angiotensin II signal transduction through the AT1 receptor: novel insights into mechanisms and pathophysiology.

The intracellular signal transduction of AngII (angiotensin II) has been implicated in cardiovascular diseases, such as hypertension, atherosclerosis and restenosis after injury. AT(1) receptor (AngII type-1 receptor), a G-protein-coupled receptor, mediates most of the physiological and pathophysiological actions of AngII, and this receptor is predominantly expressed in cardiovascular cells, such as VSMCs (vascular smooth muscle cells). AngII activates various signalling molecules, including G-protein-derived second messengers, protein kinases and small G-proteins (Ras, Rho, Rac etc), through the AT(1) receptor leading to vascular remodelling. Growth factor receptors, such as EGFR (epidermal growth factor receptor), have been demonstrated to be 'trans'-activated by the AT(1) receptor in VSMCs to mediate growth and migration. Rho and its effector Rho-kinase/ROCK are also implicated in the pathological cellular actions of AngII in VSMCs. Less is known about the endothelial AngII signalling; however, recent studies suggest the endothelial AngII signalling positively, as well as negatively, regulates the NO (nitric oxide) signalling pathway and, thereby, modulates endothelial dysfunction. Moreover, selective AT(1)-receptor-interacting proteins have recently been identified that potentially regulate AngII signal transduction and their pathogenic functions in the target organs. In this review, we focus our discussion on the recent findings and concepts that suggest the existence of the above-mentioned novel signalling mechanisms whereby AngII mediates the formation of cardiovascular diseases.

Small GTP-binding proteins and mitogen-activated protein kinases as promising therapeutic targets of vascular remodeling.

PURPOSE OF REVIEW: To summarize the most recent findings concerning the targeting of mitogen-activated protein kinases and small GTP-binding proteins toward vascular remodeling together with molecular mechanisms of their activations in vascular pathophysiology. RECENT FINDINGS: In addition to targeting the classical Ras/extracellular signal-regulated kinase cascade, Rho-kinase inhibitors, as well as the HMG-CoA reductase inhibitors, or 'statins', have pleiotropic efficacy for experimental cardiovascular diseases that involve inhibition of the signal transduction cascades originated by the small GTP-binding proteins such as Rho and Rac. Moreover, the underlying molecular mechanisms of the activation of these small GTP-binding proteins and downstream mitogen-activated protein kinases in cardiovascular tissue and cells have recently been better characterized. Additionally, gene-targeting studies in animal models are revealing select roles of the isoforms of these signaling proteins in the pathophysiology of cardiovascular disease. This is exemplified by the role of c-Jun NH(2)-terminal kinases in mediating atherosclerosis and diabetes. SUMMARY: Characterization of the function of small GTP-binding proteins, mitogen-activated protein kinases and their effectors in cardiovascular pathophysiology can be readily identified by using select inhibitors, dominant-negative gene transfer and the generation of select gene-targeted animals. These findings strongly support the notion that small GTP-binding proteins and mitogen-activated protein kinases are promising therapeutic targets toward cardiovascular diseases.