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.

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.

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.