Gα13 and Gα12, encoded by the GNA13 and GNA12 genes, respectively, are members of the G12 family of Gα proteins that, along with their associated Gβγ subunits, mediate signaling from specific G protein-coupled receptors (GPCRs). Advanced prostate cancers have increased expression of GPCRs such as CXC Motif Chemokine Receptor 4 (CXCR4), lysophosphatidic acid receptor (LPAR), and protease activated receptor 1 (PAR-1). These GPCRs signal through either the G12 family, or through Gα13 exclusively, often in addition to other G proteins. The effect of Gα13 can be distinct from that of Gα12, and the role of Gα13 in prostate cancer initiation and progression is largely unexplored. The oncogenic effect of Gα13 on cell migration and invasion in prostate cancer has been characterized, but little is known about other biological processes such as mitochondrial function and oxidative stress. Current knowledge on the link between Gα13 and oxidative stress is based on animal studies in which GPCR-Gα13 signaling decreased superoxide levels, and the overexpression of constitutively active Gα13 promoted antioxidant gene activation. In human samples, mitochondrial superoxide dismutase 2 (SOD2) correlates with prostate cancer risk and prognostic Gleason grade. However, overexpression of SOD2 in prostate cancer cells yielded conflicting results on cell growth and survival under basal versus oxidative stress conditions. Hence, it is necessary to explore the effect of Gα13 on prostate cancer tumorigenesis, as well as the effect of Gα13 on SOD2 in prostate cancer cell growth under oxidative stress conditions.

Previous studies have shown the essential roles of O-GlcNAc transferase (Ogt) and O-GlcNAcylation in neuronal development, function and neurologic diseases. However, the function of Ogt and O-GlcNAcylation in the adult cerebellum has not been well elucidated. Here, we have found that cerebellum has the highest level of O-GlcNAcylation relative to cortex and hippocampus of adult male mice. Specific deletion of Ogt in granule neuron precursors (GNPs) induces abnormal morphology and decreased size of the cerebellum in adult male Ogt deficient [conditional knock-out (cKO)] mice. Adult male cKO mice show the reduced density and aberrant distribution of cerebellar granule cells (CGCs), the disrupted arrangement of Bergman glia (BG) and Purkinje cells. In addition, adult male cKO mice exhibit aberrant synaptic connection, impaired motor coordination, and learning and memory abilities. Mechanistically, we have identified G-protein subunit α12 (Gα12) is modified by Ogt-mediated O-GlcNAcylation. O-GlcNAcylation of Gα12 facilitates its binding to Rho guanine nucleotide exchange factor 12 (Arhgef12) and consequently activates RhoA/ROCK signaling. RhoA/ROCK pathway activator LPA can rescue the developmental deficits of Ogt deficient CGCs. Therefore, our study has revealed the critical function and related mechanisms of Ogt and O-GlcNAcylation in the cerebellum of adult male mice.SIGNIFICANCE STATEMENT Cerebellar function are regulated by diverse mechanisms. To unveil novel mechanisms is critical for understanding the cerebellar function and the clinical therapy of cerebellum-related diseases. In the present study, we have shown that O-GlcNAc transferase gene (Ogt) deletion induces abnormal cerebellar morphology, synaptic connection, and behavioral deficits of adult male mice. Mechanistically, Ogt catalyzes O-GlcNAcylation of Gα12, which promotes the binding to Arhgef12, and regulates RhoA/ROCK signaling pathway. Our study has uncovered the important roles of Ogt and O-GlcNAcylation in regulating cerebellar function and cerebellum-related behavior. Our results suggest that Ogt and O-GlcNAcylation could be potential targets for some cerebellum-related diseases.

G protein-coupled receptors (GPCRs), as the largest family of receptors in the human body, are involved in the pathological mechanisms of many diseases. Heterotrimeric G proteins represent the main molecular switch and receive cell surface signals from activated GPCRs. Growing evidence suggests that Gα12 subfamily (Gα12/13)-mediated signaling plays a crucial role in cellular function and various pathological processes. The current research on the physiological and pathological function of Gα12/13 is constantly expanding, Changes in the expression levels of Gα12/13 have been found in a wide range of human diseases. However, the mechanistic research on Gα12/13 is scattered. This review briefly describes the structural sequences of the Gα12/13 isoforms and introduces the coupling of GPCRs and non-GPCRs to Gα12/13. The effects of Gα12/13 on RhoA and other signaling pathways and their roles in cell proliferation, migration, and immune cell function, are discussed. Finally, we focus on the pathological impacts of Gα12/13 in cancer, inflammation, metabolic diseases, fibrotic diseases, and circulatory disorders are brought to focus.

Rationale: Liver injury must be further characterized to identify novel therapeutic approaches. Endoplasmic reticulum (ER) stress may cause hepatocyte death. Gα12 affects cell viability and its expression varies depending on physiological conditions. This study investigated whether hepatocyte-specific Gα12 overexpression affects acute liver injury, and if so, what the underlying mechanisms and treatment strategies are. Methods: All experiments were performed using human liver, hepatocytes, and toxicant injury models with Gna12 KO and/or hepatocyte-specific Gα12 overexpression. RNA-sequencing, immunoblotting, immunohistochemistry, reporter assays, and mutation assays were conducted. Results: Hepatic Gα12 was overexpressed in mice challenged with acetaminophen or other ER stress inducers or in patients with acute liver injury or fibrosis/cirrhosis. Several Gα12 and ER-associated pathways were identified using transcriptomic analysis. Acetaminophen intoxication was characterized by lipid peroxide-induced ferroptosis and was less severe in Gα12-deficient animals and cells. Conversely, Gα12 overexpression in wild-type or Gna12 KO hepatocytes increased hepatotoxicity, promoting lipid peroxidation, inflammation, and ferroptosis. IRE1α-dependent Xbp1 transactivated Gna12. Moreover, Gα12 overexpression enhanced the ability of acetaminophen to induce ALOX12, while downregulating GPX4. The level of miR-15a, herein identified as an ALOX12 inhibitor, was decreased. siRNA knockdown or pharmacological inhibition of ROCK1 prevented dysregulation of ALOX12 and GPX4, rescuing animals from toxicant-induced ferroptosis. These changes or correlations among the targets were confirmed in human liver specimens and datasets of livers exposed to other injurious medications. Conclusions: Gα12 overexpression by ER stress facilitates hepatocyte ferroptosis through ROCK1-mediated dysregulation of ALOX12, and miR-15a, supporting the concept that inhibition of Gα12 overexpression and/or ROCK1 axis may constitute a promising strategy for acute liver injury.

The G12/13 subfamily of heterotrimeric guanine nucleotide binding proteins comprises the α subunits Gα12 and Gα13, which transduce signals for cell growth, cytoskeletal rearrangements, and oncogenic transformation. In an increasing range of cancers, overexpressed Gα12 or Gα13 are implicated in aberrant cell proliferation and/or metastatic invasion. Although Gα12 and Gα13 bind non-redundant sets of effector proteins and participate in unique signalling pathways, the structural features responsible for functional differences between these α subunits are largely unknown. Invertebrates encode a single G12/13 homolog that participates in cytoskeletal changes yet appears to lack signalling to SRF (serum response factor), a transcriptional activator stimulated by mammalian Gα12 and Gα13 to promote growth and tumorigenesis. Our previous studies identified an evolutionarily divergent region in Gα12 for which replacement by homologous sequence from Drosophila melanogaster abolished SRF signalling, whereas the same invertebrate substitution was fully tolerated in Gα13 [Montgomery et al. (2014) Mol. Pharmacol. 85: 586]. These findings prompted our current approach of evolution-guided mutagenesis to identify fine structural features of Gα12 and Gα13 that underlie their respective SRF activation mechanisms. Our results identified two motifs flanking the α4 helix that play a key role in Gα12 signalling to SRF. We found the region encompassing these motifs to provide an interacting surface for multiple Gα12-specific target proteins that fail to bind Gα13. Adjacent to this divergent region, a highly-conserved domain was vital for SRF activation by both Gα12 and Gα13. However, dissection of this domain using invertebrate substitutions revealed different signalling mechanisms in these α subunits and identified Gα13-specific determinants of binding Rho-specific guanine nucleotide exchange factors. Furthermore, invertebrate substitutions in the C-terminal, α5 helical region were selectively disruptive to Gα12 signalling. Taken together, our results identify key structural features near the C-terminus that evolved after the divergence of Gα12 and Gα13, and should aid the development of agents to selectively manipulate signalling by individual α subunits of the G12/13 subfamily.

Angiotensin II (Ang II) activates central Angiotensin II type 1 receptors to increase blood pressure via multiple pathways. However, whether central Gα proteins contribute to Ang II-induced hypertension remains unknown. We hypothesized that Angiotensin II type 1 receptors couple with Gα12 and/or Gαq to produce sympatho-excitation and increase blood pressure and downregulation of these Gα-subunit proteins will attenuate Ang II-dependent hypertension.
After chronic infusion of Ang II (s.c. 350 ng/kg/min) or vehicle for 2 weeks, Ang II evoked an increase in Gα12 expression, but not Gαq in the rostral ventrolateral medulla of Sprague-Dawley rats. In other studies, rats that received Ang II or vehicle infusion s.c. were simultaneously infused i.c.v. with a scrambled (SCR) or Gα12 oligodeoxynucleotide (ODN; 50 µg/day). Central Gα12 ODN infusion lowered mean blood pressure in Ang II infused rats compared with SCR ODN infusion (14-day peak; 133 ± 12 vs. 176 ± 11 mm Hg). Compared to the SCR ODN group, Ang II infused rats that received i.c.v. Gα12 ODN showed a greater increase in heart rate to atropine, an attenuated reduction in blood pressure to chlorisondamine, and an improved baroreflex sensitivity. In addition, central Gα12 and Gαq ODN pretreatment blunted the pressor response to an acute i.c.v. injection of Ang II (i.c.v., 200 ng).
These findings suggest that central Gα12 protein signaling pathways play an important role in the development of chronic Ang II-dependent hypertension in rats.

The myogenic response (MR) and myogenic tone (MT) in resistance vessels is crucial for maintaining peripheral vascular resistance and blood flow autoregulation. Development of MT involves G protein-coupled receptors, and may be affected by aging.
(1) to estimate the mesenteric blood flow in myogenically active small mesenteric arteries; (2) to investigate the signaling from Gαq/11 and/or Gα12 activation to MT development; (3) to investigate the role of Rho-kinase 2 and aging on MT in mesenteric resistance arteries.
we used pressure myography, quantitative real-time PCR, and immunolocalization to study small (<200 μm) mesenteric arteries (SMA) from young, mature adult, and middle aged mice.
Poiseuille flow calculations indicated autoregulation of blood flow at 60-120 mm Hg arterial pressure. Gαq/11 and Gα12 were abundantly expressed at the mRNA and protein levels in SMA. The Gαq/11 inhibitor YM-254890 suppressed MT development, and the Phosholipase C inhibitors U73122 and ET-18-OCH3 robustly inhibited it. We found an age-dependent increase in ROCK2 mRNA expression, and in basal MT. The specific ROCK2 inhibitor KD025 robustly inhibited MT in SMAs in all mice with an age-dependent variation in KD025 sensitivity. The inhibitory effect of KD025 was not prevented by the L-type Ca2+ channel activator BayK 8644. KD025 reversibly inhibited MT and endothelin-1 vasoconstriction in small pial arteries from Göttingen minipigs.
MT development in SMAs occurs through a Gαq/11 /PLC/Ca2+ -dependent pathway, and is maintained via ROCK2-mediated Ca2+ sensitization. Increased MT at mature adulthood can be explained by increased ROCK2 expression/activity.

Kidney injury molecule-1 (KIM-1) is a receptor for the \"eat me\" signal, phosphatidylserine, on apoptotic cells. The specific upregulation of KIM-1 by injured tubular epithelial cells (TECs) enables them to clear apoptotic cells (also known as efferocytosis), thereby protecting from acute kidney injury. Recently, we uncovered that KIM-1 binds directly to the α-subunit of heterotrimeric G12 protein (Gα12) and inhibits its activation by reactive oxygen species during renal ischemia-reperfusion injury (Ismail OZ, Zhang X, Wei J, Haig A, Denker BM, Suri RS, Sener A, Gunaratnam L. Am J Pathol 185: 1207-1215, 2015). Here, we investigated the role that Gα12 plays in KIM-1-mediated efferocytosis by TECs. We showed that KIM-1 remains bound to Gα12 and suppresses its activity during phagocytosis. When we silenced Gα12 expression using small interefering RNA, KIM-1-mediated engulfment of apoptotic cells was increased significantly; in contrast overexpression of constitutively active Gα12 (QLGα12) resulted in inhibition of efferocytosis. Inhibition of RhoA, a key effector of Gα12, using a chemical inhibitor or expression of dominant-negative RhoA, had the same effect as inhibition of Gα12 on efferocytosis. Consistent with this, silencing Gα12 suppressed active RhoA in KIM-1-expressing cells. Finally, using primary TECs from Kim-1+/+ and Kim-1-/- mice, we confirmed that engulfment of apoptotic cells requires KIM-1 expression and that silencing Gα12 enhanced efferocytosis by primary TECs. Our data reveal a previously unknown role for Gα12 in regulating efferocytosis and that renal TECs require KIM-1 to mediate this process. These results may have therapeutic implications given the known harmful role of Gα12 in acute kidney injury.

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Recent studies have shown that the gip2 and gep oncogenes defined by the α-subunits of Gi2 and G12 family of G proteins, namely Gαi2 and Gα12/13, stimulate oncogenic signaling pathways in cancer cells including those derived from ovarian cancer. However, the critical α-subunit involved in ovarian cancer growth and progression in vivo remains to be identified. Using SKOV3 cells in which the expressions of individual Gα-subunits were silenced, we demonstrate that the silencing of Gα12 and Gα13 drastically attenuated serum- or lysophosphatidic acid-stimulated proliferation. In contrast, the invasive migration of these cells were reduced only by the silencing of Gαi2 or Gα13. Analyses of the xenograft tumors derived from these Gα-silenced cells indicated that only the silencing of Gα13 drastically reduced xenograft tumor growth and prolonged the survival of the mice. Similar, but albeit reduced, effect was seen with the silencing of Gα12. On the contrary, the silencing of Gαi2 or Gαq failed to exert such effect. Thus, our studies establish for the first time that Gα12/13, the putative gep oncogenes, are the determinant α-subunits involved in ovarian cancer growth in vivo and their increased oncogenicity can be correlated with its ability to stimulate both proliferation and invasive migration.