Pulsatile equibiaxial stretch inhibits thrombin-induced RhoA and NF-kappaB activation.

This study investigated interactions between the effects of mechanical stretch and thrombin on RhoA activation in rat aortic smooth muscle cells (RASMC). Equibiaxial, pulsatile stretch, or thrombin produced a significant increase in RhoA activation. Surprisingly, in combination, 30 min of stretch inhibited the ability of thrombin to activate RhoA. NO donors and 8-bromo-cGMP significantly inhibited thrombin-induced RhoA activation. Interestingly, the nitric oxide synthase (NOS) inhibitor L-NAME increased basal RhoA activity, suggesting that NOS activity exerts a tonic inhibition on RhoA. Stretching RASMC increases nitrite production, consistent with the idea that NO contributes to the inhibitory effects of stretch. Thrombin stimulates MAP kinase and NF-kappaB pathways through Rho and these responses were blocked by 8-bromo-cGMP or stretch and restored by L-NAME. These data suggest that stretch, acting through NO and cGMP, can prevent the ability of thrombin to stimulate Rho signaling pathways that contribute to pathophysiological proliferative and inflammatory responses.

Tumor necrosis factor-alpha-stimulated cell proliferation is mediated through sphingosine kinase-dependent Akt activation and cyclin D expression.

Tumor necrosis factor-alpha (TNF-alpha) has been shown to activate sphingosine kinase (SphK) in a variety of cell types. The extent to which SphK signaling mediates the pleiotropic effects of TNF-alpha is not entirely clear. The current study examined the role of SphK activity in TNF-alpha-stimulated cell proliferation in 1321N1 glioblastoma cells. We first demonstrated that pharmacological inhibitors of SphK markedly decrease TNF-alpha-stimulated DNA synthesis. Signaling mechanisms through which SphK mediated the effect of TNF-alpha on DNA synthesis were then examined. Inhibition of Rho proteins with C3 exoenzyme or of Rho kinase with Y27632 attenuated TNF-alpha-stimulated DNA synthesis. However, RhoA activation by TNF-alpha was not blocked by SphK inhibition. ERK activation was also required for TNF-alpha-stimulated DNA synthesis but likewise TNF-alpha-induced ERK activation was not blocked by inhibition of SphK. Thus, neither RhoA nor ERK activation are the SphK-dependent transducers of TNF-alpha-induced proliferation. In contrast, TNF-alpha-stimulated Akt phosphorylation, which was also required for DNA synthesis, was attenuated by SphK inhibition or SphK1 knockdown by small interfering RNA. Furthermore, cyclin D expression was increased by TNF-alpha in a SphK- and Akt-dependent manner. Additional studies demonstrated that TNF-alpha effects on DNA synthesis, ERK, and Akt phosphorylation are not mediated through cell surface Gi -coupled S1P receptors, because none of these responses were inhibited by pertussis toxin. We conclude that SphK-dependent Akt activation plays a significant role in TNF-alpha-induced cyclin D expression and cell proliferation.

Rho kinase polymorphism influences blood pressure and systemic vascular resistance in human twins: role of heredity.

The Rho/Rho kinase (ROCK) pathway is implicated in experimental hypertension. We, therefore, explored the role of ROCK2 genetic variation in human blood pressure (BP) regulation, exploiting the advantages of a human twin sample to probe heritability. The focus of this work is the common nonsynonymous variant at ROCK2: Thr431Asn. Cardiovascular and autonomic traits displayed substantial heritability (from approximately 33% to 71%; P<0.05). The Asn/Asn genotype (compared with Asn/Thr or Thr/Thr) was associated with greater resting systolic (P<0.001), diastolic (P<0.0001), and mean BP (P<0.0001); allelic variation at ROCK2 accounted for up to approximately 5% of BP variation (P<0.0001). Systemic vascular resistance was higher in Asn/Asn individuals (P=0.049), whereas cardiac output, large artery compliance, and vasoactive hormone secretion were not different. Coupling of the renin-angiotensin system to systemic resistance and BP was diminished in Asn/Asn homozygotes, suggesting genetic pleiotropy of Thr431Asn, confirmed by bivariate genetic analyses. The Asn/Asn genotype also predicted higher BP after environmental (cold) stress. The rise in heart rate after cold was less pronounced in Asn/Asn individuals, consistent with intact baroreceptor function, and baroreceptor slope was not influenced by genotype. Common genetic variation (Thr431Asn) at ROCK2 predicts increased BP, systemic vascular resistance (although not large artery compliance), and resistance in response to the endogenous renin-angiotensin system, indicating a resistance vessel-based effect on elevated BP. The results suggest that common variation in ROCK2 exerts systemic resistance-mediated changes in BP, documenting a novel mechanism for human circulatory control, and suggesting new possibilities for diagnostic profiling and treatment of subjects at risk of developing hypertension.

Enhanced activation of RhoA by angiotensin II in SHR preglomerular microvascular smooth muscle cells.

Angiotensin II causes a greater renal vasoconstriction in spontaneously hypertensive rats (SHR) than in normotensive Wistar Kyoto rats (WKY), and alpha2-adrenoceptor agonists potentiate angiotensin II-induced renal vasoconstriction more in SHR. Because angiotensin II activates RhoA, and RhoA contributes to vasoconstriction, we tested the hypothesis that the ability of angiotensin II to stimulate RhoA in preglomerular vascular smooth muscle cells and the ability of alpha2-adrenoceptor activation to potentiate this response are augmented in cells from SHR. In SHR preglomerular vascular smooth muscle cells, angiotensin II (1 micromol/L) greatly stimulated RhoA activity, and this effect was markedly potentiated by UK 14,304 (1 micromol/L; alpha2-adrenoceptor agonist) (fold increase from vehicle-treated cells: 9.0 +/- 2, 0.8 +/- 0.2, and 13.6 +/- 3.2 in cells treated with angiotensin II, UK 14,304, and angiotensin II + UK 14,304, respectively). In contrast, in WKY cells, angiotensin II only mildly activated RhoA (2.0 +/- 0.50), and this response was not enhanced by UK 14,304. The expression of Galpha12 and Galpha13, G-proteins thought to link G-protein-coupled receptors to RhoA, was not increased in SHR cells. We conclude that angiotensin II-induced activation of RhoA is much more robust in the preglomerular microcirculation of SHR compared with WKY and that this may contribute to the etiology of genetic hypertension.

RHO SIGNALING in vascular diseases.

Rho signaling pathways in vascular smooth muscle cells are highly activated in hypertension, a condition associated with a variety of vascular diseases, including restenosis injury and atherosclerosis. In this review we suggest that inflammatory cytokines and agonists of G protein-coupled receptors that activate Rho are effective triggers of vascular disease. Accordingly, Rho kinase inhibitors and statins may have therapeutic potential for preventing vascular disease characterized by Rho-mediated cell proliferation and gene expression.

Rho-mediated cytoskeletal rearrangement in response to LPA is functionally antagonized by Rac1 and PIP2.

G-protein-coupled receptors signal through Rho to induce actin cytoskeletal rearrangement. We previously demonstrated that thrombin stimulates Rho-dependent process retraction and rounding of 1321N1 astrocytoma cells. Surprisingly, while lysophosphatidic acid (LPA) activated RhoA in 1321N1 cells, it failed to produce cell rounding. Thrombin, unlike LPA, decreased Rac1 activity, and activated (GTPase-deficient) Rac1 inhibited thrombin-stimulated cell rounding, while expression of dominant-negative Rac1 promoted LPA-induced rounding. LPA and thrombin receptors appear to differ in coupling to Gi, as LPA but not thrombin-stimulated 1321N1 cell proliferation was pertussis toxin-sensitive. Blocking Gi with pertussis toxin enabled LPA to induce cell rounding and to decrease activated Rac1. These data support the hypothesis that Rac1 and Gi activation antagonize cell rounding. Thrombin and LPA receptors also differentially activated Gq pathways as thrombin but not LPA increased InsP3 formation and reduced phosphatidylinositol 4,5-bisphosphate (PIP2) levels. Microinjection of the plekstrin homology domain of phospholipase C (PLC)delta1, which binds PIP2, enabled LPA to elicit cell rounding, consistent with a requirement for PIP2 reduction. We suggest that Rho-mediated cytoskeletal responses are enhanced by concomitant reductions in cellular levels of PIP2 and Rac1 activation and thus effected only by G-protein-coupled receptors with appropriate subsets of G protein activation.

G protein mediated signaling pathways in lysophospholipid induced cell proliferation and survival.

Agonist activation of a subset of G protein coupled receptors (GPCRs) stimulates cell proliferation, mimicking the better known effects of tyrosine kinase growth factors. Cell survival or apoptosis is also regulated via pathways initiated by stimulation of these same GPCRs. This review focuses on aspects of signaling by the lysophospholipid mediators, lysophosphatidic acid (LPA), and sphingosine 1 phosphate (S1P), which make these agonists uniquely capable of modulating cell growth and survival. The general features of GPCR coupling to specific G proteins, downstream effectors and signaling cascades are first reviewed. GPCR coupling to G(i) and Ras/MAPK or to G(q) and phospholipase generated second messengers are insufficient to regulate cell proliferation while G(12/13)/Rho engagement provides additional complementary signals required for cell proliferation. Survival is best predicted by coupling to G(i) pathways that regulate PI3K and Akt, but other signals generated through different G protein pathways are also implicated. The unique ability of LPA and S1P to concomitantly stimulate G(i), G(q), and G(12/13) pathways, given the proper complement of expressed LPA or S1P receptors, allows these receptors to support cell survival and proliferation. In pathophysiological situations, e.g., vascular disease, cancer, brain injury, and inflammation, components of the signaling cascade downstream of lysophospholipid receptors, in particular those involving Ras or Rho, may be altered. In addition, up or downregulation of LPA or S1P receptor subtypes, altering their ratio, and increased availability of the lysophospholipid ligands at sites of injury or inflammation, likely contribute to disease and may be important targets for therapeutic intervention.

RGS16 inhibits signalling through the G alpha 13-Rho axis.

G alpha 13 stimulates the guanine nucleotide exchange factors (GEFs) for Rho, such as p115Rho-GEF. Activated Rho induces numerous cellular responses, including actin polymerization, serum response element (SRE)-dependent gene transcription and transformation. p115Rho-GEF contains a Regulator of G protein Signalling domain (RGS box) that confers GTPase activating protein (GAP) activity towards G alpha 12 and G alpha 13 (ref. 3). In contrast, classical RGS proteins (such as RGS16 and RGS4) exhibit RGS domain-dependent GAP activity on G alpha i and G alpha q, but not G alpha 12 or G alpha 13 (ref 4). Here, we show that RGS16 inhibits G alpha 13-mediated, RhoA-dependent reversal of stellation and SRE activation. The RGS16 amino terminus binds G alpha 13 directly, resulting in translocation of G alpha 13 to detergent-resistant membranes (DRMs) and reduced p115Rho-GEF binding. RGS4 does not bind G alpha 13 or attenuate G alpha 13-dependent responses, and neither RGS16 nor RGS4 affects G alpha 12-mediated signalling. These results elucidate a new mechanism whereby a classical RGS protein regulates G alpha 13-mediated signal transduction independently of the RGS box.

Localization of adenylyl cyclase isoforms and G protein-coupled receptors in vascular smooth muscle cells: expression in caveolin-rich and noncaveolin domains.

A number of different agonists activate G protein-coupled receptors to stimulate adenylyl cyclase (AC), increase cAMP formation, and promote relaxation in vascular smooth muscle. To more fully understand this stimulation of AC, we assessed the expression, regulation, and compartmentation of AC isoforms in rat aortic smooth muscle cells (RASMC). Reverse transcription-polymerase chain reaction detected expression of AC3, AC5, and AC6 mRNA, whereas immunoblot analysis indicated expression of AC3 and AC5/6 protein primarily in caveolin-rich membrane (cav) fractions relative to noncaveolin (noncav) fractions. Beta(1)-adrenergic receptors (AR), beta(2)AR, and G(s) were detected in both cav and noncav fractions, whereas the prostanoid receptors EP(2)R and EP(4)R were excluded from cav fractions. We used an adenoviral construct to increase AC6 expression. Overexpressed AC6 localized only in noncav fractions. Two-fold overexpression of AC6 caused enhancement of forskolin-, isoproterenol- and prostaglandin E(2)-stimulated cAMP formation but no changes in basal levels of cAMP. At higher levels of AC6 overexpression, basal and adenosine receptor-stimulated cAMP levels were increased. Stimulation of cAMP levels by agents that increase Ca(2+) in native cells was consistent with the expression of AC3, but overexpression of AC6, which is inhibited by Ca(2+), blunted the Ca(2+)-stimulable cAMP response. These data indicate that: 1) RASMC express multiple AC isoforms that localize in both caveolin-rich and noncaveolin domains, 2) expression of AC6 in non-caveolin-rich membranes can increase basal levels of cAMP and response to several stimulatory agonists, and 3) Ca(2+)-mediated regulation of cAMP formation depends upon expression of different AC isoforms in RASMC. Compartmentation of GPCRs and AC is different in cardiomyocytes than in RASMC, indicating that targeting of these components to caveolin-rich membranes can be cell-specific. Moreover, our results imply that the colocalization of GPCRs and the AC isoforms they activate need not occur in caveolin-rich fractions.

cGMP-dependent protein kinase inhibits serum-response element-dependent transcription by inhibiting rho activation and functions.

RhoA, in its active GTP-bound form, stimulates transcription through activation of the serum-response factor (SRF). We found that cGMP inhibited serum-induced Rho.GTP loading and transcriptional activation of SRF-dependent reporter genes in smooth muscle and glial cells in a cGMP-dependent protein kinase (G-kinase)-dependent fashion. Serum stimulation of the SRF target gene vinculin was also blocked by cGMP/G-kinase. G-kinase activation inhibited SRF-dependent transcription induced by upstream RhoA activators including Galpha(13) and p115RhoGEF, with Galpha(13)-induced Rho.GTP loading inhibited by G-kinase. G-kinase had no effect on the high activation levels of RhoA(63L) or the double mutant RhoA(63L,188A) but inhibited transcriptional activation by these two RhoA mutants to a similar extent, suggesting an effect downstream of RhoA and independent of RhoA Ser(188) phosphorylation. Constitutively active forms of the Rho effectors Rho kinase (ROK), PKN, and PRK-2 induced SRF-dependent transcription in a cell type-specific fashion with ROK being the most efficient; G-kinase inhibited transcription induced by all three effectors without affecting ROK catalytic activity. G-kinase had no effect on RhoA(63L)-induced morphological changes in glial cells, suggesting distinct transcriptional and cytoskeletal effectors of RhoA. We conclude that G-kinase inhibits SRF-dependent transcription by interfering with RhoA signaling; G-kinase acts both upstream of RhoA, inhibiting serum- or Galpha(13)-induced Rho activation, and downstream of RhoA, inhibiting steps distal to the Rho targets ROK, PKN, and PRK-2.