Myogenic vasoconstriction requires G12/G13 and LARG to maintain local and systemic vascular resistance.

Myogenic vasoconstriction is an autoregulatory function of small arteries. Recently, G-protein-coupled receptors have been involved in myogenic vasoconstriction, but the downstream signalling mechanisms and the in-vivo-function of this myogenic autoregulation are poorly understood. Here, we show that small arteries from mice with smooth muscle-specific loss of G12/G13 or the Rho guanine nucleotide exchange factor ARHGEF12 have lost myogenic vasoconstriction. This defect was accompanied by loss of RhoA activation, while vessels showed normal increases in intracellular [Ca2+]. In the absence of myogenic vasoconstriction, perfusion of peripheral organs was increased, systemic vascular resistance was reduced and cardiac output and left ventricular mass were increased. In addition, animals with defective myogenic vasoconstriction showed aggravated hypotension in response to endotoxin. We conclude that G12/G13- and Rho-mediated signaling plays a key role in myogenic vasoconstriction and that myogenic tone is required to maintain local and systemic vascular resistance under physiological and pathological condition.

S1pr2/Gα13 signaling regulates the migration of endocardial precursors by controlling endoderm convergence.

Formation of the heart tube requires synchronized migration of endocardial and myocardial precursors. Our previous studies indicated that in S1pr2/Gα13-deficient embryos, impaired endoderm convergence disrupted the medial migration of myocardial precursors, resulting in the formation of two myocardial populations. Here we show that endoderm convergence also regulates endocardial migration. In embryos defective for S1pr2/Gα13 signaling, endocardial precursors failed to migrate towards the midline, and the presumptive endocardium surrounded the bilaterally-located myocardial cells rather than being encompassed by them. In vivo imaging of control embryos revealed that, like their myocardial counterparts, endocardial precursors migrated with the converging endoderm, though from a more anterior point, then moved from the dorsal to the ventral side of the endoderm (subduction), and finally migrated posteriorly towards myocardial precursors, ultimately forming the inner layer of the heart tube. In embryos defective for endoderm convergence due to an S1pr2/Gα13 deficiency, both the medial migration and the subduction of endocardial precursors were impaired, and their posterior migration towards the myocardial precursors was premature. This placed them medial to the myocardial populations, physically blocking the medial migration of the myocardial precursors. Furthermore, contact between the endocardial and myocardial precursor populations disrupted the epithelial architecture of the myocardial precursors, and thus their medial migration; in embryos depleted of endocardial cells, the myocardial migration defect was partially rescued. Our data indicate that endoderm convergence regulates the medial migration of endocardial precursors, and that premature association of the endocardial and myocardial populations contributes to myocardial migration defects observed in S1pr2/Gα13-deficient embryos. The demonstration that endoderm convergence regulates the synchronized migration of endocardial and myocardial precursors reveals a new role of the endoderm in heart development.

S1P2/G12/13 Signaling Negatively Regulates Macrophage Activation and Indirectly Shapes the Atheroprotective B1-Cell Population.

OBJECTIVES: Monocyte/macrophage recruitment and activation at vascular predilection sites plays a central role in the pathogenesis of atherosclerosis. Heterotrimeric G proteins of the G12/13 family have been implicated in the control of migration and inflammatory gene expression, but their function in myeloid cells, especially during atherogenesis, is unknown. APPROACH AND RESULTS: Mice with myeloid-specific deficiency for G12/13 show reduced atherosclerosis with a clear shift to anti-inflammatory gene expression in aortal macrophages. These changes are because of neither altered monocyte/macrophage migration nor reduced activation of inflammatory gene expression; on the contrary, G12/13-deficient macrophages show an increased nuclear factor-κB-dependent gene expression in the resting state. Chronically increased inflammatory gene expression in resident peritoneal macrophages results in myeloid-specific G12/13-deficient mice in an altered peritoneal micromilieu with secondary expansion of peritoneal B1 cells. Titers of B1-derived atheroprotective antibodies are increased, and adoptive transfer of peritoneal cells from mutant mice conveys atheroprotection to wild-type mice. With respect to the mechanism of G12/13-mediated transcriptional control, we identify an autocrine feedback loop that suppresses nuclear factor-κB-dependent gene expression through a signaling cascade involving sphingosine 1-phosphate receptor subtype 2, G12/13, and RhoA. CONCLUSIONS: Together, these data show that selective inhibition of G12/13 signaling in macrophages can augment atheroprotective B-cell populations and ameliorate atherosclerosis.

Evidence of a common mechanism of disassembly of adherens junctions through Gα13 targeting of VE-cadherin.

The heterotrimeric G protein Gα13 transduces signals from G protein-coupled receptors (GPCRs) to induce cell spreading, differentiation, migration, and cell polarity. Here, we describe a novel GPCR-independent function of Gα13 in regulating the stability of endothelial cell adherens junctions (AJs). We observed that the oxidant H2O2, which is released in response to multiple proinflammatory mediators, induced the interaction of Gα13 with VE-cadherin. Gα13 binding to VE-cadherin in turn induced Src activation and VE-cadherin phosphorylation at Tyr 658, the p120-catenin binding site thought to be responsible for VE-cadherin internalization. Inhibition of Gα13-VE-cadherin interaction using an interfering peptide derived from the Gα13 binding motif on VE-cadherin abrogated the disruption of AJs in response to inflammatory mediators. These studies identify a unique role of Gα13 binding to VE-cadherin in mediating VE-cadherin internalization and endothelial barrier disruption and inflammation.

PRR5L degradation promotes mTORC2-mediated PKC-δ phosphorylation and cell migration downstream of Gα12.

Mammalian target of rapamycin complex 2 (mTORC2) phosphorylates AGC protein kinases including protein kinase C (PKC) and regulates cellular functions such as cell migration. However, its regulation remains poorly understood. Here we show that lysophosphatidic acid (LPA) induces two phases of PKC-δ hydrophobic motif phosphorylation. The late phase is mediated by Gα(12), which specifically activates ARAF, leading to upregulation of the RFFL E3 ubiquitin ligase and subsequent ubiquitylation and degradation of the PRR5L subunit of mTORC2. Destabilization of PRR5L, a suppressor of mTORC2-mediated hydrophobic motif phosphorylation of PKC-δ, but not AKT, results in PKC-δ hydrophobic motif phosphorylation and activation. This Gα(12)-mediated signalling pathway for mTORC2 regulation is critically important for fibroblast migration and pulmonary fibrosis development.

G(alpha)12/13 induction of CYR61 in association with arteriosclerotic intimal hyperplasia: effect of sphingosine-1-phosphate.

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. CONCLUSIONS: Gα(12/13) regulate AP-1-dependent CYR61 induction in VSMCs and promote VSMC migration, and they are upregulated with CYR61 in arteriosclerotic lesions.

Suppression of tumor angiogenesis by Galpha(13) haploinsufficiency.

Heterotrimeric G proteins are critical transducers of cellular signaling. Of the four families of G proteins, the physiological function of Galpha(13) is less well understood. Galpha(13) gene-deleted mice die at embryonic day approximately 9.5. Here, we show that heterozygous Galpha(13)(+/-) mice display defects in adult angiogenesis. Female Galpha(13)(+/-) mice showed a higher number of immature follicles and a lower density of blood vessels in the mature corpus luteum compared with Galpha(13)(+/+) mice. Furthermore, implanted tumors grew slower in Galpha(13)(+/-) host mice. These tumor tissues had many fewer blood vessels compared with those from Galpha(13)(+/+) host mice. Moreover, bone marrow-derived progenitor cells from Galpha(13)(+/+) mice rescued the failed growth of allografted tumors when reconstituted into irradiated Galpha(13)(+/-) mice. Hence, Galpha(13) is haploinsufficient for adult angiogenesis in both the female reproductive system and tumor angiogenesis.

Galpha12/Galpha13 deficiency causes localized overmigration of neurons in the developing cerebral and cerebellar cortices.

The heterotrimeric G proteins G(12) and G(13) link G-protein-coupled receptors to the regulation of the actin cytoskeleton and the induction of actomyosin-based cellular contractility. Here we show that conditional ablation of the genes encoding the alpha-subunits of G(12) and G(13) in the nervous system results in neuronal ectopia of the cerebral and cerebellar cortices due to overmigration of cortical plate neurons and cerebellar Purkinje cells, respectively. The organization of the radial glia and the basal lamina was not disturbed, and the Cajal-Retzius cell layer had formed normally in mutant mice. Embryonic cortical neurons lacking G(12)/G(13) were unable to retract their neurites in response to lysophosphatidic acid and sphingosine-1-phosphate, indicating that they had lost the ability to respond to repulsive mediators acting via G-protein-coupled receptors. Our data indicate that G(12)/G(13)-coupled receptors mediate stop signals and are required for the proper positioning of migrating cortical plate neurons and Purkinje cells during development.

G proteins G12 and G13 control the dynamic turnover of growth factor-induced dorsal ruffles.

Growth factors induce massive actin cytoskeletal remodeling in cells. These reorganization events underlie various cellular responses such as cell migration and morphological changes. One major form of actin reorganization is the formation and disassembly of dorsal ruffles (also named waves, dorsal rings, or circular ruffles). Dorsal ruffles are involved in physiological functions including cell migration, invasion, macropinocytosis, plasma membrane recycling, and others. Growth factors initiate rapid formation (within 5 min) of circular membrane ruffles, and these ruffles move along the dorsal side of the cells, constrict, close, and eventually disassemble ( approximately 20 min). Considerable attention has been devoted to the mechanism by which growth factors induce the formation of dorsal ruffles. However, little is known of the mechanism by which these ruffles are disassembled. Here we have shown that G proteins G(12) and G(13) control the rate of disassembly of dorsal ruffles. In Galpha(12)(-/-)Galpha(13)(-/-) fibroblast cells, dorsal ruffles induced by growth factor treatment remain visible substantially longer ( approximately 60 min) than in wild-type cells, whereas the rate of formation of these ruffles was the same with or without Galpha(12) and Galpha(13). Thus, Galpha(12)/Galpha(13) critically regulate dorsal ruffle turnover.

G12/G13 family G proteins regulate marginal zone B cell maturation, migration, and polarization.

G protein-coupled receptors play an important role in the regulation of lymphocyte functions such as migration, adhesion, proliferation, and differentiation. Although the role of G(i) family G proteins has been intensively studied, no in vivo data exist with respect to G12/G13 family G proteins. We show in this study that mice that lack the G protein alpha-subunits G alpha12 and G alpha13 selectively in B cells show significantly reduced numbers of splenic marginal zone B (MZB) cells, resulting in a delay of Ab production in response to thymus-independent Ags. Basal and chemokine-induced adhesion to ICAM-1 and VCAM-1, two adhesion molecules critically involved in MZB localization, is normal in mutant B cells, and the same is true for chemokine-induced migration. However, migration in response to serum and sphingosine 1-phosphate is strongly increased in mutant MZB cells, but not in mutant follicular B cells. Live-cell imaging studies revealed that G alpha12/G alpha13-deficient MZB cells assumed more frequently an ameboid form than wild-type cells, and pseudopod formation was enhanced. In addition to their regulatory role in serum- and sphingosine 1-phosphate-induced migration, G12/G13 family G proteins seem to be involved in peripheral MZB cell maturation, because also splenic MZB cell precursors are reduced in mutant mice, although less prominently than mature MZB cells. These data suggest that G12/G13 family G proteins contribute to the formation of the mature MZB cell compartment both by controlling MZB cell migration and by regulating MZB cell precursor maturation.

The G protein G alpha(13) is required for growth factor-induced cell migration.

Heterotrimeric G proteins are critical cellular signal transducers. They are known to directly relay signals from seven-transmembrane G protein-coupled receptors (GPCRs) to downstream effectors. On the other hand, receptor tyrosine kinases (RTKs), a different family of membrane receptors, signal through docking sites in their carboxy-terminal tails created by autophosphorylated tyrosine residues. Here we show that a heterotrimeric G protein, G alpha(13), is essential for RTK-induced migration of mouse fibroblast and endothelial cells. G alpha(13) activity in cell migration is retained in a C-terminal mutant that is defective in GPCR coupling, suggesting that the migration function is independent of GPCR signaling. Thus, G alpha(13) appears to be a critical signal transducer for RTKs as well as GPCRs. This broader role of G alpha(13) in cell migration initiated by two types of receptors could provide a molecular basis for the vascular system defects exhibited by G alpha(13) knockout mice.

Relative contribution of G-protein-coupled pathways to protease-activated receptor-mediated Akt phosphorylation in platelets.

Protease-activated receptors (PARs) activate Gq and G(12/13) pathways, as well as Akt (protein kinase B [PKB/Akt]) in platelets. However, the relative contribution of different G-protein pathways to Akt phosphorylation has not been elucidated. We investigated the contribution of Gq and G(12/13) to Gi/Gz-mediated Akt phosphorylation downstream of PAR activation. Selective G(12/13) activation failed to cause Akt phosphorylation in human and Galpha q-deficient mouse platelets. However, supplementing Gi/Gz signaling to G(12/13) caused significant increase in Akt phosphorylation, confirming that G(12/13) potentiates Akt phosphorylation. Inhibition of PAR-mediated Akt phosphorylation in the presence of the Gq-selective inhibitor YM-254890 was restored to the normal extent achieved by PAR agonists if supplemented with Gi signaling, indicating that Gq does not have any direct effect on Akt phosphorylation. Selective G(12/13) activation resulted in Src kinase activation, and Akt phosphorylation induced by costimulation of G(12/13) and Gi/Gz was inhibited by a Src kinase inhibitor but not by a Rho kinase inhibitor. These data demonstrate that G(12/13), but not Gq, is essential for thrombin-induced Akt phosphorylation in platelets, whereas Gq indirectly contributes to Akt phosphorylation through Gi stimulation by secreted ADP. G(12/13) activation might mediate its potentiating effect through Src activation, and Src kinases play an important role in thrombin-mediated Akt phosphorylation.

Characteristic defects in neural crest cell-specific Galphaq/Galpha11- and Galpha12/Galpha13-deficient mice.

The endothelin/endothelin receptor system plays a critical role in the differentiation and terminal migration of particular neural crest cell subpopulations. Targeted deletion of the G-protein-coupled endothelin receptors ET(A) and ET(B) was shown to result in characteristic developmental defects of derivatives of cephalic and cardiac neural crest and of neural crest-derived melanocytes and enteric neurons, respectively. Since both endothelin receptors are coupled to G-proteins of the G(q)/G(11)- and G(12)/G(13)-families, we generated mouse lines lacking Galpha(q)/Galpha(11) or Galpha(12)/Galpha(13) in neural crest cells to study their roles in neural crest development. Mice lacking Galpha(q)/Galpha(11) in a neural crest cell-specific manner had craniofacial defects similar to those observed in mice lacking the ET(A) receptor or endothelin-1 (ET-1). However, in contrast to ET-1/ET(A) mutant animals, cardiac outflow tract morphology was intact. Surprisingly, neither Galpha(q)/Galpha(11)- nor Galpha(12)/Galpha(13)-deficient mice showed developmental defects seen in animals lacking either the ET(B) receptor or its ligand endothelin-3 (ET-3). Interestingly, Galpha(12)/Galpha(13) deficiency in neural crest cell-derived cardiac cells resulted in characteristic cardiac malformations. Our data show that G(q)/G(11)- but not G(12)/G(13)-mediated signaling processes mediate ET-1/ET(A)-dependent development of the cephalic neural crest. In contrast, ET-3/ET(B)-mediated development of neural crest-derived melanocytes and enteric neurons appears to involve G-proteins different from G(q)/G(11)/G(12)/G(13).

Essential role for Galpha13 in endothelial cells during embryonic development.

Toward identifying the roles of protease-activated receptor-1 (PAR1) and other G protein-coupled receptors important for vascular development, we investigated the role of Galpha13 in endothelial cells in the mouse embryo. LacZ inserted into Galpha13 exon 1 was highly expressed in endothelial cells at midgestation. Endothelial-specific Galpha13 knockout embryos died at embryonic days 9.5-11.5 and resembled the PAR1 knockout. Restoration of Galpha13 expression in endothelial cells by use of a Tie2 promoter-driven Galpha13 transgene rescued development of endothelial-specific Galpha13 knockout embryos as well the embryonic day 9.5 vascular phenotype in Galpha13 conventional knockouts; transgene-positive Galpha13-/- embryos developed for several days beyond their transgene-negative Galpha13-/- littermates and then manifested a previously uncharacterized phenotype that included intracranial bleeding and exencephaly. Taken together, our results suggest a critical role for Galpha13 in endothelial cells during vascular development, place Galpha13 as a candidate mediator of PAR1 signaling in this process, and reveal roles for Galpha13 in other cell types in the mammalian embryo.

Anti-glycoprotein VI treatment severely compromises hemostasis in mice with reduced alpha2beta1 levels or concomitant aspirin therapy.

BACKGROUND: Platelet inhibition is a major strategy to prevent arterial thrombosis, but it is frequently associated with increased bleeding because of impaired primary hemostasis. The activating platelet collagen receptor, glycoprotein VI (GP VI), may serve as a powerful antithrombotic target because its inhibition or absence results in profound protection against arterial thrombosis but no major bleeding in mice. METHODS AND RESULTS: Mice lacking (-/-) or expressing half-levels (+/-) of the other major platelet collagen receptor, integrin alpha2beta1, were injected with the anti-GP VI antibody JAQ1 and analyzed on day 5. Anti-GP VI treatment resulted in a marked hemostatic defect in alpha2-/- or alpha2+/- mice, as shown by dramatically prolonged tail bleeding times. Platelet adhesion to collagen was studied in an ex vivo whole-blood perfusion system under high shear conditions. Weak integrin activation by thromboxane A2 (TxA2) receptor stimulation restored defective adhesion of anti-GP VI-treated wild-type but not alpha2-/- or alpha2+/- platelets to collagen. This process required the simultaneous activation of the G(q) and G13 signaling pathways, as demonstrated by use of the respective knockout strains. Conversely, inhibition of TxA2 production by aspirin severely compromised hemostasis in anti-GP VI-treated or GP VI/Fc receptor gamma-chain-deficient but not control mice. CONCLUSIONS: Anti-GP VI therapy may result in defective hemostasis in patients with reduced alpha2beta1 levels or concomitant aspirin therapy. These observations may have important implications for a potential use of anti-GP VI-based therapeutics in the prevention of cardiovascular disease.