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Supplemental Digital Content is available in the text., Objective: Despite the importance of reactive oxygen species (ROS) and NOX (nicotinamide adenine dinucleotide phosphate [NADPH] oxidase) 2 in platelet activation and in vivo thrombosis, it is unclear how ROS and NOX2 play a role in platelet activation and why NOX2 deficiencies in humans and mice do not affect hemostasis. Outside-in signaling of integrin αIIbβ3 mediates platelet response to shear stress, secondary platelet activation, and thrombus expansion and is critical to thrombosis but dispensable for hemostasis. We studied the mechanisms of platelet ROS generation, ROS-mediated platelet response, and the role of ROS in integrin αIIbβ3 outside-in signaling. Approach and Results: ROS generation in activated platelets was low and slow without shear but was robust under shear. Shear-enhanced ROS generation and activation of p47phox, an important regulatory subunit of NOX2, were diminished by the integrin antagonist integrilin or β3 knockout, and by Gα13 knockout or blocking the Gα13-β3 interaction. Resting platelets spreading on integrin ligand fibrinogen also Gα13-dependently stimulated ROS generation and p47phox activation. Hence, Gα13-mediated outside-in signaling induces NOX2 activation and ROS generation which is greatly enhanced by shear. Outside-in NOX2 activation requires Src, phosphoinositide 3-kinase and Akt downstream of Gα13. Importantly, NOX2-knockout platelets showed defective ROS generation, reduced platelet spreading without shear, and reduced platelet adhesion and thrombus volume on collagen and VWF (von Willibrand factor) under shear, whereas ROS inhibition diminished activation of tyrosine kinase Syk. Conclusions: Outside-in signaling activates the mainly NOX2-mediated ROS generation, which mediates Syk-dependent secondary platelet activation, adhesion, and thrombosis with minimal effect on hemostasis.

BACKGROUND: Diabetic nephropathy (dNP), now the leading cause of ESKD, lacks efficient therapies. Coagulation protease–dependent signaling modulates dNP, in part via the G protein–coupled, protease-activated receptors (PARs). Specifically, the cytoprotective protease-activated protein C (aPC) protects from dNP, but the mechanisms are not clear. METHODS: A combination of in vitro approaches and mouse models evaluated the role of aPC-integrin interaction and related signaling in dNP. RESULTS: The zymogen protein C and aPC bind to podocyte integrin-β(3), a subunit of integrin-α(v)β(3). Deficiency of this integrin impairs thrombin-mediated generation of aPC on podocytes. The interaction of aPC with integrin-α(v)β(3) induces transient binding of integrin-β(3) with G(α13) and controls PAR-dependent RhoA signaling in podocytes. Binding of aPC to integrin-β(3) via its RGD sequence is required for the temporal restriction of RhoA signaling in podocytes. In podocytes lacking integrin-β(3), aPC induces sustained RhoA activation, mimicking the effect of thrombin. In vivo, overexpression of wild-type aPC suppresses pathologic renal RhoA activation and protects against dNP. Disrupting the aPC–integrin-β(3) interaction by specifically deleting podocyte integrin-β(3) or by abolishing aPC’s integrin-binding RGD sequence enhances RhoA signaling in mice with high aPC levels and abolishes aPC’s nephroprotective effect. Pharmacologic inhibition of PAR1, the pivotal thrombin receptor, restricts RhoA activation and nephroprotects RGE-aPC(high) and wild-type mice. Conclusions aPC–integrin-α(v)β(3) acts as a rheostat, controlling PAR1-dependent RhoA activation in podocytes in diabetic nephropathy. These results identify integrin-α(v)β(3) as an essential coreceptor for aPC that is required for nephroprotective aPC-PAR signaling in dNP.

Supplemental Digital Content is available in the text., GNA13 is a G protein involved in modulating tumor proliferative capacity, infiltration, metastasis, and migration. Genomic alteration of GNA13 was frequently observed in follicular lymphoma (FL). In this study, we examined 167 cases of FL by immunostaining of GNA13 using tissue microarray to evaluate the clinical significance. There were 26 GNA13-positive cases (15.6%) and 141 GNA13-negative cases (84.4%). GNA13-positive cases had a higher incidence of early progression of disease for which disease progression was recognized within 2 years compared with GNA13-negative cases (P=0.03). There were no significant differences in other clinicopathologic factors including histological grade, BCL2-IGH translocation, immunohistochemical phenotype, and Follicular Lymphoma International Prognostic Index. In addition, overall survival and progression-free survival were poorer in GNA13-positive cases than in GNA13-negative cases (P=0.009 and 0.005, respectively). In multivariate analysis, GNA13 positivity was found to be a poor prognostic factor for overall survival and progression-free survival. Thus, GNA13 protein expression was an independent prognostic factor and may affect disease progression in FL.

Background: Chronic heart failure (HF) is associated with altered signal transduction via β-adrenoceptors and G proteins and with reduced cAMP formation. Nucleoside diphosphate kinases (NDPKs) are enriched at the plasma membrane of patients with end-stage HF, but the functional consequences of this are largely unknown, particularly for NDPK-C. Here, we investigated the potential role of NDPK-C in cardiac cAMP formation and contractility. Methods: Real-time polymerase chain reaction, (far) Western blot, immunoprecipitation, and immunocytochemistry were used to study the expression, interaction with G proteins, and localization of NDPKs. cAMP levels were determined with immunoassays or fluorescent resonance energy transfer, and contractility was determined in cardiomyocytes (cell shortening) and in vivo (fractional shortening). Results: NDPK-C was essential for the formation of an NDPK-B/G protein complex. Protein and mRNA levels of NDPK-C were upregulated in end-stage human HF, in rats after long-term isoprenaline stimulation through osmotic minipumps, and after incubation of rat neonatal cardiomyocytes with isoprenaline. Isoprenaline also promoted translocation of NDPK-C to the plasma membrane. Overexpression of NDPK-C in cardiomyocytes increased cAMP levels and sensitized cardiomyocytes to isoprenaline-induced augmentation of contractility, whereas NDPK-C knockdown decreased cAMP levels. In vivo, depletion of NDPK-C in zebrafish embryos caused cardiac edema and ventricular dysfunction. NDPK-B knockout mice had unaltered NDPK-C expression but showed contractile dysfunction and exacerbated cardiac remodeling during long-term isoprenaline stimulation. In human end-stage HF, the complex formation between NDPK-C and Gα i2 was increased whereas the NDPK-C/Gα s interaction was decreased, producing a switch that may contribute to an NDPK-C–dependent cAMP reduction in HF. Conclusions: Our findings identify NDPK-C as an essential requirement for both the interaction between NDPK isoforms and between NDPK isoforms and G proteins. NDPK-C is a novel critical regulator of β-adrenoceptor/cAMP signaling and cardiac contractility. By switching from Gα s to Gα i2 activation, NDPK-C may contribute to lower cAMP levels and the related contractile dysfunction in HF.

The rich diversity of lipids and the specific signalling pathways they recruit provides tremendous scope for modulation of biological functions. Lysophosphatidylinositol (LPI) is emerging as a key modulator of cell proliferation, migration, and function, and holds important pathophysiological implications due to its high levels in diseased tissues, such as in cancer. Here we report a novel role for LPI in sensitization of peripheral sensory neurons, which was evident as exaggerated sensitivity to painful and innocuous pressure. Histopathological analyses indicated lack of involvement of myelin pathology and immune cell recruitment by LPI. Using pharmacological and conditional genetic tools in mice, we delineated receptor-mediated from non-receptor-mediated effects of LPI and we observed that GPR55, which functions as an LPI receptor when heterologously expressed in mammalian cells, only partially mediates LPI-induced actions in the context of pain sensitization in vivo; we demonstrate that, in vivo, LPI functions by activating Gα(13) as well as Gα(q/11) arms of G-protein signalling in sensory neurons. This study thus reports a novel pathophysiological function for LPI and elucidates underlying molecular mechanisms.

Tumor microenvironment, defined by a variety of growth factors including lysophosphatidic acid (LPA), whose levels are increased in pancreatic cancer patients, plays a major role in the genesis and progression of pancreatic cancer. Because the gep proto-oncogenes, Gα12 and Gα13, are implicated in LPA-stimulated oncogenic signaling, this study is focused on evaluating the role of these proto-oncogenes in LPA-stimulated invasive migration of pancreatic cancer cells.Effect of LPA on the migration and proliferation of pancreatic cancer cells was assessed using BxPC3, Dan-G, MDAPanc-28, Panc-1, and PaCa-2 cell lines. The role of Gα13 in the migration of pancreatic cancer cells was interrogated by disrupting lysophosphatidic acid receptor-Gα13 interaction using CT13, a dominant negative mutant of Gα13, and by silencing the expression of Gα13.Results indicate that LPA stimulates the migration of pancreatic cancer cells and such LPA-stimulated migratory response is mediated by Gα13. Furthermore, the results establish that the silencing of Gα13, but not Gα12, abrogates LPA-stimulated invasive migration of pancreatic cancer cells.These results report for the first time a critical role for Gα13 in LPA-stimulated invasive migration of pancreatic cancer cells. These findings identify LPA-lysophosphatidic acid receptor-Gα13 signaling node as a novel therapeutic target for pancreatic cancer treatment and control.

Rationale: The peptide ligand apelin and its receptor APJ constitute a signaling pathway with numerous effects on the cardiovascular system, including cardiovascular development in model organisms such as xenopus and zebrafish. Objective: This study aimed to characterize the embryonic lethal phenotype of the Apj −/− mice and to define the involved downstream signaling targets. Methods and Results: We report the first characterization of the embryonic lethality of the Apj −/− mice. More than half of the expected Apj −/− embryos died in utero because of cardiovascular developmental defects. Those succumbing to early embryonic death had markedly deformed vasculature of the yolk sac and the embryo, as well as poorly looped hearts with aberrantly formed right ventricles and defective atrioventricular cushion formation. Apj −/− embryos surviving to later stages demonstrated incomplete vascular maturation because of a deficiency of vascular smooth muscle cells and impaired myocardial trabeculation and ventricular wall development. The molecular mechanism implicates a novel, noncanonical signaling pathway downstream of apelin-APJ involving Gα13, which induces histone deacetylase (HDAC) 4 and HDAC5 phosphorylation and cytoplasmic translocation, resulting in activation of myocyte enhancer factor 2. Apj −/− mice have greater endocardial Hdac4 and Hdac5 nuclear localization and reduced expression of the myocyte enhancer factor 2 (MEF2) transcriptional target Krüppel-like factor 2 . We identify a number of commonly shared transcriptional targets among apelin-APJ, Gα13, and MEF2 in endothelial cells, which are significantly decreased in the Apj −/− embryos and endothelial cells. Conclusions: Our results demonstrate a novel role for apelin-APJ signaling as a potent regulator of endothelial MEF2 function in the developing cardiovascular system.

Background— Cardiac remodeling in response to pressure or volume overload plays an important role in the pathogenesis of heart failure. Various mechanisms have been suggested to translate mechanical stress into structural changes, one of them being the release of humoral factors such as angiotensin II and endothelin-1, which in turn promote cardiac hypertrophy and fibrosis. A large body of evidence suggests that the prohypertrophic effects of these factors are mediated by receptors coupled to the G q/11 family of heterotrimeric G proteins. Most G q/11 -coupled receptors, however, can also activate G proteins of the G 12/13 family, but the role of G 12/13 in cardiac remodeling is not understood. Methods and Results— We use siRNA-mediated knockdown in vitro and conditional gene inactivation in vivo to study the role of the G 12/13 family in pressure overload–induced cardiac remodeling. We show in detail that inducible cardiomyocyte-specific inactivation of the α subunit of G 13 , Gα 13 , does not affect basal heart function but protects mice from pressure overload–induced hypertrophy and fibrosis as efficiently as inactivation of Gα q/11 . Furthermore, inactivation of Gα 13 prevents the development of heart failure up to 1 year after overloading. On the molecular level, we show that Gα 13 , but not Gα q/11 , controls agonist-induced expression of hypertrophy-specific genes through activation of the small GTPase RhoA and consecutive activation of myocardin-related transcription factors. Conclusion— Our data show that the G 12/13 family of heterotrimeric G proteins is centrally involved in pressure overload–induced cardiac remodeling and plays a central role in the transition to heart failure.

Objective— Gα 12/13 play a role in oncogenic transformation and tumor growth. Cysteine-rich protein 61 (CYR61) is a growth-factor-inducible angiogenic factor. In view of potential overlapping functions between Gα 12/13 and CYR61, this study investigated the role of these G proteins in CYR61 induction in association with hyperplastic vascular abnormality. Methods and Results— Overexpression of activated Gα 12 or Gα 13 induced CYR61 expression in vascular smooth muscle cells (VSMCs). Gene knockdown and knockout experiments revealed that sphingosine-1-phosphate (S1P) treatment induced CYR61 via Gα 12/13 . JunD/activator protein-1 (AP-1) was identified as a transcription factor required for CYR61 transactivation by S1P. Deficiencies in Gα 12/13 abrogated AP-1 activation and AP-1-mediated CYR61 induction. c-Jun N-terminal kinase was responsible for CYR61 induction. Moreover, deficiencies of Gα 12/13 abolished c-Jun N-terminal kinase–dependent CYR61 induction by S1P. N -acetyl- l -cysteine or NADPH oxidase inhibitor treatment reversed CYR61 induction by S1P, indicating that reactive oxygen species are responsible for this process. The levels of Gα 12/13 were increased within thickened intimas and medias in wire-injured mouse femoral arteries, which was accompanied by simultaneous CYR61 induction. Moreover, Gα 12/13 and CYR61 were costained in the arteriosclerotic lesions immediately adjacent to human tumor tissues. Conclusion— Gα 12/13 regulate AP-1-dependent CYR61 induction in VSMCs and promote VSMC migration, and they are upregulated with CYR61 in arteriosclerotic lesions.

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 barrier dysfunction caused by inflammatory agonists is a frequent underlying cause of vascular leak and edema. Novel strategies to preserve barrier integrity could have profound clinical impact. Adenosine triphosphate (ATP) released from endothelial cells by shear stress and injury has been shown to protect the endothelial barrier in some settings. We have demonstrated that ATP and its nonhydrolyzed analogues enhanced barrier properties of cultured endothelial cell monolayers and caused remodeling of cell–cell junctions. Increases in cytosolic Ca 2+ and Erk activation caused by ATP were irrelevant to barrier enhancement. Experiments using biochemical inhibitors or siRNA indicated that G proteins (specifically G αq and G αi2 ), protein kinase A (PKA), and the PKA substrate vasodilator-stimulated phosphoprotein were involved in ATP-induced barrier enhancement. ATP treatment decreased phosphorylation of myosin light chain and specifically activated myosin-associated phosphatase. Depletion of G αq with siRNA prevented ATP-induced activation of myosin phosphatase. We conclude that the mechanisms of ATP-induced barrier enhancement are independent of intracellular Ca 2+ , but involve activation of myosin phosphatase via a novel G-protein–coupled mechanism and PKA.

The roles of the G-protein α-subunits, G s , G i , and G q/11 , in the signal transduction of the D 1 -like dopamine receptors, D 1 and D 5 , have been deciphered. Gα 12 and Gα 13 , members of the 4th family of G protein subunits, are not linked with D 1 receptors, and their linkage to D 5 receptors is not known. Therefore, we studied the expression of Gα 12 and Gα 13 and interaction with D 5 dopamine receptors in the kidney from normotensive Wistar-Kyoto (WKY) rats and D 5 receptor-transfected HEK293 cells. Gα 12 and Gα 13 were found in the proximal tubule, distal convoluted tubule, and artery and vein in the WKY rat kidney. Whereas Gα 12 was expressed in the ascending limb of Henle, Gα 13 was expressed in the collecting duct and juxtaglomerular cells. In renal proximal tubules, Gα 12 and Gα 13 , as with D 5 receptors, were expressed in brush border membranes. Laser confocal microscopy revealed the colocalization of D 5 receptors with Gα 12 and Gα 13 in rat renal brush border membranes, immortalized rat renal proximal tubule cells, and D 5 receptor-transfected HEK293 cells. In these cells, a D 1 -like agonist, fenoldopam, increased the association of Gα 12 and Gα 13 with D 5 receptors, results that were corroborated by immunoprecipitation experiments. We conclude that although both D 1 and D 5 receptors are linked to Gαs, they are differentially linked to Gα 12 and Gα 13 . The consequences of the differential G-protein subunit linkage on D 1 - and D 5 -mediated sodium transport remains to be determined.

Abstract —Receptor-induced vascular smooth muscle cell contraction is mediated by dual regulation of myosin light chain (MLC 20 ) phosphorylation through Ca 2+ -dependent stimulation of myosin light chain kinase and Rho/Rho-kinase–mediated inhibition of myosin phosphatase. Although myosin light chain kinase regulation is initiated by the coupling of receptors to G proteins of the G q family, G q and G 11 , it is not known how receptors regulate the Rho/Rho-kinase–mediated pathway. In vascular smooth muscle cells, receptor-mediated MLC 20 phosphorylation and cell contraction was blocked by inhibitors of each of the pathways. Receptors of various vasocontractors were found to couple to G q /G 11 and G 12 /G 13 , and constitutively active forms of Gα 12 and Gα 13 induced a pronounced contraction of vascular smooth muscle cells that could be blocked by C3 exoenzyme, by inhibition of Rho-kinase, and by stable analogues of cGMP and cAMP. Receptor-mediated smooth muscle cell contraction was strongly inhibited by dominant-negative forms of Gα 12 and Gα 13 . These data indicate that a G 12 /G 13 -mediated Rho/Rho-kinase–dependent pathway operates in smooth muscle cells and that dual regulation of MLC 20 phosphorylation by vasocontractors is initiated by the dual coupling of their receptors to G proteins of the G q and G 12 families.