Inhibitory G proteins play multiple roles to polarize sensory hair cell morphogenesis.

Inhibitory G alpha (GNAI or Gαi) proteins are critical for the polarized morphogenesis of sensory hair cells and for hearing. The extent and nature of their actual contributions remains unclear, however, as previous studies did not investigate all GNAI proteins and included non-physiological approaches. Pertussis toxin can downregulate functionally redundant GNAI1, GNAI2, GNAI3, and GNAO proteins, but may also induce unrelated defects. Here, we directly and systematically determine the role(s) of each individual GNAI protein in mouse auditory hair cells. GNAI2 and GNAI3 are similarly polarized at the hair cell apex with their binding partner G protein signaling modulator 2 (GPSM2), whereas GNAI1 and GNAO are not detected. In Gnai3 mutants, GNAI2 progressively fails to fully occupy the sub-cellular compartments where GNAI3 is missing. In contrast, GNAI3 can fully compensate for the loss of GNAI2 and is essential for hair bundle morphogenesis and auditory function. Simultaneous inactivation of Gnai2 and Gnai3 recapitulates for the first time two distinct types of defects only observed so far with pertussis toxin: (1) a delay or failure of the basal body to migrate off-center in prospective hair cells, and (2) a reversal in the orientation of some hair cell types. We conclude that GNAI proteins are critical for hair cells to break planar symmetry and to orient properly before GNAI2/3 regulate hair bundle morphogenesis with GPSM2.

G protein subunit alpha i2's pivotal role in angiogenesis.

Here we explored the potential role of Gαi2 (G protein subunit alpha i2) in endothelial cell function and angiogenesis. Methods: Genetic methodologies such as shRNA, CRISPR/Cas9, dominant negative mutation, and overexpression were utilized to modify Gαi2 expression or regulate its function. Their effects on endothelial cell functions were assessed in vitro. In vivo, the endothelial-specific Gαi2 shRNA adeno-associated virus (AAV) was utilized to silence Gαi2 expression. The impact of this suppression on retinal angiogenesis in control mice and streptozotocin (STZ)-induced diabetic retinopathy (DR) mice was analyzed. Results: Analysis of single-cell RNA sequencing data revealed Gαi2 (GNAI2) was predominantly expressed in retinal endothelial cells and expression was increased in retinal endothelial cells following oxygen-induced retinopathy (OIR) in mice. Moreover, transcriptome analysis linking Gαi2 to angiogenesis-related processes/pathways, supported by increased Gαi2 expression in experimental OIR mouse retinas, highlighted its possible role in angiogenesis. In various endothelial cell types, shRNA-induced silencing and CRISPR/Cas9-mediated knockout (KO) of Gαi2 resulted in substantial reductions in cell proliferation, migration, invasion, and capillary tube formation. Conversely, Gαi2 over-expression in endothelial cells induced pro-angiogenic activities, enhancing cell proliferation, migration, invasion, and capillary tube formation. Furthermore, our investigation revealed a crucial role of Gαi2 in NFAT (nuclear factor of activated T cells) activation, as evidenced by the down-regulation of NFAT-luciferase reporter activity and pro-angiogenesis NFAT-targeted genes (Egr3, CXCR7, and RND1) in Gαi2-silenced or -KO HUVECs, which were up-regulated in Gαi2-overexpressing endothelial cells. Expression of a dominant negative Gαi2 mutation (S48C) also down-regulated NFAT-targeted genes, slowing proliferation, migration, invasion, and capillary tube formation in HUVECs. Importantly, in vivo experiments revealed that endothelial Gαi2 knockdown inhibited retinal angiogenesis in mice, with a concomitant down-regulation of NFAT-targeted genes in mouse retinal tissue. In contrast, Gαi2 over-expression in endothelial cells enhanced retinal angiogenesis in mice. Single-cell RNA sequencing data confirmed increased levels of Gαi2 specifically in retinal endothelial cells of mice with streptozotocin (STZ)-induced diabetic retinopathy (DR). Importantly, endothelial Gαi2 silencing ameliorated retinal pathological angiogenesis in DR mice. Conclusion: Our study highlights a critical role for Gαi2 in NFAT activation, endothelial cell activation and angiogenesis, offering valuable insights into potential therapeutic strategies for modulating these processes.

GNAI2 Is a Risk Factor for Gastric Cancer: Study of Tumor Microenvironment (TME) and Establishment of Immune Risk Score (IRS).

Purpose: Although the G protein subunit α i2 (GNAI2) is upregulated in multiple cancers, its prognostic value and exact role in the development of gastric cancer (GC) remain largely unknown. Methods: This study evaluated the effect of GNAI2 on the tumor microenvironment (TME) in GC, constructed an immune risk score (IRS) model based on differentially-expressed immune genes, and systematically correlated GNAI2 and epigenetic factor expression patterns with TME and IRS. Also, RT-qPCR, flow cytometry, Western blotting (WB), and transwell assays were carried out to explore the regulatory mechanism of GNAI2 in GC. Results: High GNAI2 expression was associated with poor prognosis. Cytokine activation, an increase in tumor-infiltrating immune cells (TIIC), and the accumulation of regulatory T cells in the tumor immune cycle were all promoted by the TME, which was significantly associated with GNAI2 expression. Two different differentially expressed mRNA (DER) modification patterns were determined. These two DERs-clusters had significantly different TME cell infiltrations and were classified as either noninflamed or immune-inflamed phenotypes. The IRS model constructed using differentially expressed genes (DEGs) had great potential in predicting GC prognosis. The IRS model was also used in assessing clinicopathological features, such as microsatellite instability (MSI) status, epithelial-mesenchymal transition (EMT) status, clinical stages, tumor mutational burden (TMB), and tumor immune dysfunction and exclusion (TIDE) scores. Low IRS scores were associated with high immune checkpoint gene expression. Cell and animal studies confirmed that GNAI2 activated PI3K/AKT pathway and promoted the growth and migration of GC cells. Conclusion: The IRS model can be used for survival prediction and GNAI2 serves as a candidate therapeutic target for GC patients.

Melatonin reverses tumor necrosis factor-alpha-induced metabolic disturbance of human nucleus pulposus cells via MTNR1B/Gαi2/YAP signaling.

Background: Intervertebral disc degeneration (IDD), the main cause of low back pain, is closely related to the inflammatory microenvironment in the nucleus pulposus (NP). Tumor necrosis factor-α (TNF-α) plays an important role in inflammation-related metabolic disturbance of NP cells. Melatonin has been proven to regulate the metabolism of NP cells, but whether it can protect NP cells from TNF-α-induced damage is still unclear. Therefore, this study aims to investigate the role and specific mechanism of melatonin on regulating the metabolism of NP cells in the inflammatory microenvironment. Methods: Western blotting, RT-qPCR and immunohistochemistry were used to detect the expression of melatonin membrane receptors (MTNR1A/B) and TNF-α in human NP tissues. In vitro, human primary NP cells were treated with or without vehicle, TNF-α and melatonin. And the metabolic markers were also detected by western blotting and RT-qPCR. The activity of NF-κB signaling and Hippo/YAP signaling were assessed by western blotting and immunofluorescence. Membrane receptors inhibitors, pathway inhibitors, lentiviral infection, plasmids transfection and immunoprecipitation were used to explore the specific mechanism of melatonin. In vivo, the rat IDD model was constructed and melatonin was injected intraperitoneally to evaluate its therapeutical effect on IDD. Results: The upregulation of TNF-α and downregulation of melatonin membrane receptors (MTNR1A/B) were observed in degenerative NP tissues. Then we demonstrated that melatonin could alleviate the development of IDD in a rat model and reverse TNF-α-impaired metabolism of NP cells in vitro. Further investigation revealed that the protective effects of melatonin on NP cells mainly rely on MTNR1B, which subsequently activates Gαi2 protein. The activation of Gαi2 could upregulate the yes-associated protein (YAP) level, resulting in anabolic enhancement of NP cells. In addition, melatonin-mediated YAP upregulation increased the expression of IκBα and suppressed the TNF-α-induced activation of the NF-κB pathway, thereby inhibiting the catabolism of NP cells. Conclusions: Our results revealed that melatonin can reverse TNF-α-impaired metabolism of NP cells via the MTNR1B/Gαi2/YAP axis and suggested that melatonin can be used as a potential therapeutic drug in the treatment of IDD.

Gαi2-induced conductin/axin2 condensates inhibit Wnt/ß-catenin signaling and suppress cancer growth.

Conductin/axin2 is a scaffold protein negatively regulating the pro-proliferative Wnt/ß-catenin signaling pathway. Accumulation of scaffold proteins in condensates frequently increases their activity, but whether condensation contributes to Wnt pathway inhibition by conductin remains unclear. Here, we show that the Gαi2 subunit of trimeric G-proteins induces conductin condensation by targeting a polymerization-inhibiting aggregon in its RGS domain, thereby promoting conductin-mediated ß-catenin degradation. Consistently, transient Gαi2 expression inhibited, whereas knockdown activated Wnt signaling via conductin. Colorectal cancers appear to evade Gαi2-induced Wnt pathway suppression by decreased Gαi2 expression and inactivating mutations, associated with shorter patient survival. Notably, the Gαi2-activating drug guanabenz inhibited Wnt signaling via conductin, consequently reducing colorectal cancer growth in vitro and in mouse models. In summary, we demonstrate Wnt pathway inhibition via Gαi2-triggered conductin condensation, suggesting a tumor suppressor function for Gαi2 in colorectal cancer, and pointing to the FDA-approved drug guanabenz for targeted cancer therapy.

Positive allosteric mechanisms of adenosine A1 receptor-mediated analgesia.

The adenosine A1 receptor (A1R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain1,2. However, development of analgesic orthosteric A1R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects3. Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A1R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A1R co-bound to adenosine, MIPS521 and a Gi2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts.

GNAi2/gip2-Regulated Transcriptome and Its Therapeutic Significance in Ovarian Cancer.

Increased expression of GNAi2, which encodes the α-subunit of G-protein i2, has been correlated with the late-stage progression of ovarian cancer. GNAi2, also referred to as the proto-oncogene gip2, transduces signals from lysophosphatidic acid (LPA)-activated LPA-receptors to oncogenic cellular responses in ovarian cancer cells. To identify the oncogenic program activated by gip2, we carried out micro-array-based transcriptomic and bioinformatic analyses using the ovarian cancer cell-line SKOV3, in which the expression of GNAi2/gip2 was silenced by specific shRNA. A cut-off value of 5-fold change in gene expression (p < 0.05) indicated that a total of 264 genes were dependent upon gip2-expression with 136 genes coding for functional proteins. Functional annotation of the transcriptome indicated the hitherto unknown role of gip2 in stimulating the expression of oncogenic/growth-promoting genes such as KDR/VEGFR2, CCL20, and VIP. The array results were further validated in a panel of High-Grade Serous Ovarian Carcinoma (HGSOC) cell lines that included Kuramochi, OVCAR3, and OVCAR8 cells. Gene set enrichment analyses using DAVID, STRING, and Cytoscape applications indicated the potential role of the gip2-stimulated transcriptomic network involved in the upregulation of cell proliferation, adhesion, migration, cellular metabolism, and therapy resistance. The results unravel a multi-modular network in which the hub and bottleneck nodes are defined by ACKR3/CXCR7, IL6, VEGFA, CYCS, COX5B, UQCRC1, UQCRFS1, and FYN. The identification of these genes as the critical nodes in GNAi2/gip2 orchestrated onco-transcriptome establishes their role in ovarian cancer pathophysiology. In addition, these results also point to these nodes as potential targets for novel therapeutic strategies.

GNAI2 Promotes Proliferation and Decreases Apoptosis in Rabbit Melanocytes.

GNAI2 (G protein subunit alpha i2) is a signaling modulator or transducer, involved in several transmembrane signaling systems, that plays a vital role in the melanogenesis signaling pathway. However, whether GNAI2 regulates cell proliferation and apoptosis in rabbit melanocytes is not known. We found that GNAI2 was differentially expressed in rabbits with different coat colors using qRT-PCR and Wes assays. Furthermore, it was observed that the rabbits with black skin had the highest GNAI2 levels, and those with white skin had the lowest expression. The coding sequence of GNAI2 was successfully cloned and inserted into pcDNA3.1 and pcDNA3.1-Myc vectors. It was observed that the GNAI2 protein was mainly localized in the cytoplasm using the indirect immunofluorescence staining assay. Overexpression of GNAI2 significantly increased melanin content, promoted melanocyte proliferation, and inhibited melanocyte apoptosis. On the contrary, the knockdown of GNAI2 using siRNA had the opposite effect. In addition, GNAI2 significantly increased the mRNA expression levels of the melanin-related genes TYR, GPNMB, PMEL, and DCT in rabbit melanocytes. The results suggested that GNAI2 regulated melanocyte development by promoting melanocyte proliferation and inhibiting apoptosis.

Translational control by DHX36 binding to 5'UTR G-quadruplex is essential for muscle stem-cell regenerative functions.

Skeletal muscle has a remarkable ability to regenerate owing to its resident stem cells (also called satellite cells, SCs). SCs are normally quiescent; when stimulated by damage, they activate and expand to form new fibers. The mechanisms underlying SC proliferative progression remain poorly understood. Here we show that DHX36, a helicase that unwinds RNA G-quadruplex (rG4) structures, is essential for muscle regeneration by regulating SC expansion. DHX36 (initially named RHAU) is barely expressed at quiescence but is highly induced during SC activation and proliferation. Inducible deletion of Dhx36 in adult SCs causes defective proliferation and muscle regeneration after damage. System-wide mapping in proliferating SCs reveals DHX36 binding predominantly to rG4 structures at various regions of mRNAs, while integrated polysome profiling shows that DHX36 promotes mRNA translation via 5'-untranslated region (UTR) rG4 binding. Furthermore, we demonstrate that DHX36 specifically regulates the translation of Gnai2 mRNA by unwinding its 5' UTR rG4 structures and identify GNAI2 as a downstream effector of DHX36 for SC expansion. Altogether, our findings uncover DHX36 as an indispensable post-transcriptional regulator of SC function and muscle regeneration acting through binding and unwinding rG4 structures at 5' UTR of target mRNAs.

S-nitrosylation-mediated coupling of G-protein alpha-2 with CXCR5 induces Hippo/YAP-dependent diabetes-accelerated atherosclerosis.

Atherosclerosis-associated cardiovascular disease is one of the main causes of death and disability among patients with diabetes mellitus. However, little is known about the impact of S-nitrosylation in diabetes-accelerated atherosclerosis. Here, we show increased levels of S-nitrosylation of guanine nucleotide-binding protein G(i) subunit alpha-2 (SNO-GNAI2) at Cysteine 66 in coronary artery samples from diabetic patients with atherosclerosis, consistently with results from mice. Mechanistically, SNO-GNAI2 acted by coupling with CXCR5 to dephosphorylate the Hippo pathway kinase LATS1, thereby leading to nuclear translocation of YAP and promoting an inflammatory response in endothelial cells. Furthermore, Cys-mutant GNAI2 refractory to S-nitrosylation abrogated GNAI2-CXCR5 coupling, alleviated atherosclerosis in diabetic mice, restored Hippo activity, and reduced endothelial inflammation. In addition, we showed that melatonin treatment restored endothelial function and protected against diabetes-accelerated atherosclerosis by preventing GNAI2 S-nitrosylation. In conclusion, SNO-GNAI2 drives diabetes-accelerated atherosclerosis by coupling with CXCR5 and activating YAP-dependent endothelial inflammation, and reducing SNO-GNAI2 is an efficient strategy for alleviating diabetes-accelerated atherosclerosis.

Guanine Nucleotide-Binding Protein G(i) Subunit Alpha 2 Exacerbates NASH Progression by Regulating Peroxiredoxin 1-Related Inflammation and Lipophagy.

BACKGROUND AND AIMS: NASH is an advanced stage of liver disease accompanied by lipid accumulation, inflammation, and liver fibrosis. Guanine nucleotide-binding protein G(i) subunit alpha-2 (GNAI2) is a member of the "inhibitory" class of α-subunits, and recent studies showed that Gnai2 deficiency is known to cause reduced weight in mice. However, the role of GNAI2 in hepatocytes, particularly in the context of liver inflammation and lipid metabolism, remains to be elucidated. Herein, we aim to ascertain the function of GNAI2 in hepatocytes and its impact on the development of NASH. APPROACH AND RESULTS: Human liver tissues were obtained from NASH patients and healthy persons to evaluate the expression and clinical relevance of GNAI2. In addition, hepatocyte-specific Gnai2-deficient mice (Gnai2hep-/- ) were fed either a Western diet supplemented with fructose in drinking water (WDF) for 16 weeks or a methionine/choline-deficient diet (MCD) for 6 weeks to investigate the regulatory role and underlying mechanism of Gnai2 in NASH. GNAI2 was significantly up-regulated in liver tissues of patients with NASH. Following feeding with WDF or MCD diets, livers from Gnai2hep-/- mice had reduced steatohepatitis with suppression of markers of inflammation and an increase in lipophagy compared to Gnai2flox/flox mice. Toll-like receptor 4 signals through nuclear factor kappa B to trigger p65-dependent transcription of Gnai2. Intriguingly, immunoprecipitation, immunofluorescence, and mass spectrometry identified peroxiredoxin 1 (PRDX1) as a binding partner of GNAI2. Moreover, the function of PRDX1 in the suppression of TNF receptor-associated factor 6 ubiquitin-ligase activity and glycerophosphodiester phosphodiesterase domain-containing 5-related phosphatidylcholine metabolism was inhibited by GNAI2. Suppression of GNAI2 combined with overexpression of PRDX1 reversed the development of steatosis and fibrosis in vivo. CONCLUSIONS: GNAI2 is a major regulator that leads to the development of NASH. Thus, inhibition of GNAI2 could be an effective therapeutic target for the treatment of NASH.

Unrestrained Gαi2 Signaling Disrupts Neutrophil Trafficking, Aging, and Clearance.

Neutrophil trafficking, homeostatic and pathogen elicited, depends upon chemoattractant receptors triggering heterotrimeric G-protein Gαißγ signaling, whose magnitude and kinetics are governed by RGS protein/Gαi interactions. RGS proteins typically limit Gαi signaling by reducing the duration that Gαi subunits remain GTP bound and able to activate downstream effectors. Yet how in totality RGS proteins shape neutrophil chemoattractant receptor activated responses remains unclear. Here, we show that C57Bl/6 mouse neutrophils containing a genomic knock-in of a mutation that disables all RGS protein-Gαi2 interactions (G184S) cannot properly balance chemoattractant receptor signaling, nor appropriately respond to inflammatory insults. Mutant neutrophils accumulate in mouse bone marrow, spleen, lung, and liver; despite neutropenia and an intrinsic inability to properly mobilize from the bone marrow. In vitro they rapidly adhere to ICAM-1 coated plates, but in vivo they poorly adhere to blood vessel endothelium. Those few neutrophils that cross blood vessels and enter tissues migrate haphazardly. Following Concanavalin-A administration fragmented G184S neutrophils accumulate in liver sinusoids leading to thrombo-inflammation and perivasculitis. Thus, neutrophil Gαi2/RGS protein interactions both limit and facilitate Gαi2 signaling thereby promoting normal neutrophil trafficking, aging, and clearance.

Effects of Gαi2 and Gαz protein knockdown on alpha2A-adrenergic and cannabinoid CB1 receptor regulation of MEK-ERK and FADD pathways in mouse cerebral cortex.

BACKGROUND: Alpha2A-adrenergic (α2A-AR) and cannabinoid CB1 (CB1-R) receptors exert their functions modulating multiple signaling pathways, including MEK-ERK (extracellular signal-regulated kinases) and FADD (Fas-associated protein with death domain) cascades. These molecules are relevant in finding biased agonists with fewer side effects, but the mechanisms involving their modulations by α2A-AR- and CB1-R in vivo are unclear. This study investigated the roles of Gαi2 and Gαz proteins in mediating α2A-AR- and CB1-R-induced alterations of MEK-ERK and FADD phosphorylation (p-) in mouse brain cortex. METHODS: Gαi2 or Gαz protein knockdown was induced in mice with selective antisense oligodeoxinucleotides (ODNs; 3 nmol/day, 5 days) prior to UK-14,304 (UK or brimonidine; 1 mg/kg) or WIN55212-2 (WIN; 8 mg/kg) acute treatments. Inactivated (p-T286) MEK1, activated (p-S217/221) MEK1/2, activated (p-T202/Y204) ERK1/2, p-S191 FADD, and the corresponding total forms of these proteins were quantified by immunoblotting. RESULTS: Increased (+ 88%) p-T286 MEK1 cortical density, with a concomitant reduction (-43%) of activated ERK was observed in UK-treated mice. Both effects were attenuated by Gαi2 or Gαz antisense ODNs. Contrastingly, WIN induced Gαi2- and Gαz-independent upregulations of p-T286 MEK1 (+ 63%), p-S217/221 MEK1/2 (+ 86%), and activated ERK (+ 111%) in brain. Pro-apoptotic FADD was downregulated (- 34 to 39%) following UK and WIN administration, whereas the neuroprotective p-S191 FADD was increased (+ 74%) in WIN-treated mice only. None of these latter effects required from Gαi2 or Gαz protein integrity. CONCLUSION: The results indicate that α2A-AR (UK), but not CB1-R (WIN), agonists use Gαi2 and Gαz proteins to modulate MEK-ERK, but not FADD, pathway in mouse brain cortex.

Increased functional coupling of the mu opioid receptor in the anterior insula of depressed individuals.

The mu opioid receptor (MOR) is a G protein-coupled receptor that plays an essential role in reward and hedonic processes, and that has been implicated in disorders such as depression and addiction. Over the last decade, several brain imaging studies in depressed patients have consistently found that dysregulation of MOR function occurs in particular in the anterior insular cortex, an important brain site for the perception of internal states and emotional regulation. To investigate molecular mechanisms that may underlie these effects, here we assessed genetic polymorphisms, expression, and functional G-protein coupling of MOR in a large post-mortem cohort (N = 95) composed of depressed individuals who died by suicide, and healthy controls. Results indicated that depression, but not comorbid substance use disorder or acute opiate consumption, was associated with increased MOR activity. This effect was partly explained by a specific increase in expression of the inhibitory alpha G-protein subunit GNAI2. Consistent with previous neuroimaging studies, our findings support the notion that enhanced endogenous opioidergic tone in the anterior insula may buffer negative affective states in depressed individuals, a mechanism that could potentially contribute to the antidepressant efficacy of emerging opioid-based medications.

Neuroanatomical characterization of Gαi2-expressing neurons in the hypothalamic paraventricular nucleus of male and female Sprague-Dawley rats.

Hypertension is a global health burden. The hypothalamic paraventricular nucleus (PVN) is an essential component of the neuronal network that regulates sodium homeostasis and blood pressure (BP). Previously, we have shown PVN-specific G protein-coupled receptor-coupled Gαi2 subunit proteins are essential to counter the development of salt-sensitive hypertension by mediating the sympathoinhibitory and natriuretic responses to increased dietary sodium intake to maintain sodium homeostasis and normotension. However, the cellular localization and identity of PVN Gαi2-expressing neurons are currently unknown. In this study using in situ hybridization, we determined the neuroanatomical characterization of Gαi2-expressing PVN neurons in 3-mo-old male and female Sprague-Dawley rats. We observed that Gαi2-expressing neurons containing Gnai2 mRNA are highly localized in the parvocellular region of the hypothalamic PVN. At level 2 of the hypothalamic PVN, Gnai2 mRNA colocalized with ∼ 85% of GABA-expressing neurons and ∼28% of glutamatergic neurons. Additionally, within level 2 Gnai2 mRNA colocalized with ∼75% of corticotrophin-releasing hormone PVN neurons. Gnai2 neurons had lower colocalization with tyrosine hydroxylase (∼33%)-, oxytocin (∼6%)-, and arginine vasopressin (∼10%)-expressing parvocellular neurons in level 2 PVN. Colocalization was similar among male and female rats. The high colocalization of Gnai2 mRNA with GABAergic neurons, in conjunction with our previous findings that PVN Gαi2 proteins mediate sympathoinhibition, suggests that Gαi2 proteins potentially modulate GABAergic signaling to impact sympathetic outflow and BP.

Mice with an RGS-insensitive Gαi2 protein show growth hormone axis dysfunction.

Mice carrying an RGS-insensitive Gαi2 mutation display growth retardation early after birth. Although the growth hormone (GH)-axis is a key endocrine modulator of postnatal growth, its functional state in these mice has not been characterized. The present study was undertaken to address this issue. Results revealed that pituitary mRNA levels for GH, prolactin (PRL), somatostatin (SST), GH-releasing-hormone receptor (GHRH-R) and GH secretagogue receptor (GHS-R) were decreased in mutants compared to controls. These changes were reflected by a significant decrease in plasma levels of GH, IGF-1 and IGF-binding protein-3 (IGFBP-3). Mutants were also less responsive to GHRH and ghrelin (GhL) on GH stimulation of release from pituitary primary cell cultures. In contrast, they were more sensitive to the inhibitory effect of SST. These data provide the first evidence for an alteration of the functional state of the GH-axis in Gαi2G184S mice that likely contributes to their growth retardation.

Lack of Gαi2 proteins in adipocytes attenuates diet-induced obesity.

OBJECTIVES: Typically, obesity results from an inappropriate balance between energy uptake from nutrient consumption and burning of calories, which leads to a pathological increase in fat mass. Obesity is a major cause of insulin resistance and diabetes. Inhibitory G proteins (Gαi) form a subfamily that is involved in the regulation of adipose tissue function. Among the three Gαi members, i.e. Gαi1, Gαi2, Gαi3, the Gαi2, protein is predominantly expressed in adipose tissue. However, the functions of the Gαi2 isoform in adipose tissue and its impact on the development of obesity are poorly understood. METHODS: By using AdipoqCreERT2 mice, we generated adipocyte-specific Gnai2-deficient mice to study Gαi2 function, specifically in white and brown adipocytes. These mice were fed either a control diet (CD) or a high fat diet (HFD). Mice were examined for obesity development, insulin resistance and glucose intolerance. We examined adipocyte morphology and the development of inflammation in the white adipose tissue. Finally, intracellular cAMP levels as an indicator of Gαi signaling and glycerol release as an indicator of lipolysis rates were measured to verify the impact of Gαi2 on the signaling pathway in brown and white adipocytes. RESULTS: An adipocyte-specific deficiency of Gαi2 significantly reduced diet-induced obesity, leading to decreased fat masses, smaller adipocytes and decreased inflammation in the white adipose tissue relative to littermate controls. Concurrently, oxygen consumption of brown adipocytes and in vivo measured energy expenditure were significantly enhanced. In addition, glucose tolerance and insulin sensitivity of HFD-fed adipocyte-specific Gnai2-deficient mice were improved compared to the respective controls. In the absence of Gαi2, adrenergic stimulation of intracellular adipocyte cAMP levels was increased, which correlated with increased lipolysis and energy expenditure. CONCLUSION: We conclude that adipocyte Gαi2 is a major regulator of adipocyte lipid content in diet-induced obesity by inhibiting adipocyte lipolysis in a cAMP-dependent manner resulting in increased energy expenditure.

Hypothalamic Paraventricular Nucleus Gαi2 (Guanine Nucleotide-Binding Protein Alpha Inhibiting Activity Polypeptide 2) Protein-Mediated Neural Control of the Kidney and the Salt Sensitivity of Blood Pressure.

We have previously reported that in salt-resistant rat phenotypes brain, Gαi2 (guanine nucleotide-binding protein alpha inhibiting activity polypeptide 2) proteins are required to maintain blood pressure and sodium balance. However, the impact of hypothalamic paraventricular nucleus (PVN) Gαi2 proteins on the salt sensitivity of blood pressure is unknown. Here, by the bilateral PVN administration of a targeted Gαi2 oligodeoxynucleotide, we show that PVN-specific Gαi2 proteins are required to facilitate the full natriuretic response to an acute volume expansion (peak natriuresis [µeq/min] scrambled (SCR) oligodeoxynucleotide 41±3 versus Gαi2 oligodeoxynucleotide 18±4; P<0.05) via a renal nerve-dependent mechanism. Furthermore, in response to chronically elevated dietary sodium intake, PVN-specific Gαi2 proteins are essential to counter renal nerve-dependent salt-sensitive hypertension (mean arterial pressure [mm Hg] 8% NaCl; SCR oligodeoxynucleotide 128±2 versus Gαi2 oligodeoxynucleotide 147±3; P<0.05). This protective pathway involves activation of PVN Gαi2 signaling pathways, which mediate sympathoinhibition to the blood vessels and kidneys (renal norepinephrine [pg/mg] 8% NaCl; SCR oligodeoxynucleotide 375±39 versus Gαi2 oligodeoxynucleotide 850±27; P<0.05) and suppression of the activity of the sodium chloride cotransporter assessed as peak natriuresis to hydrochlorothiazide. Additionally, central oligodeoxynucleotide-mediated Gαi2 protein downregulation prevented PVN parvocellular neuron activation, assessed by FosB immunohistochemistry, in response to increased dietary salt intake. In our analysis of the UK BioBank data set, it was observed that 2 GNAI2 single nucleotide polymorphism (SNP) (rs2298952, P=0.041; rs4547694, P=0.017) significantly correlate with essential hypertension. Collectively, our data suggest that selective targeting and activation of PVN Gαi2 proteins is a novel therapeutic approach for the treatment of salt-sensitive hypertension.

Gαi2+ vomeronasal neurons govern the initial outcome of an acute social competition.

Pheromone detection by the vomeronasal organ (VNO) mediates important social behaviors across different species, including aggression and sexual behavior. However, the relationship between vomeronasal function and social hierarchy has not been analyzed reliably. We evaluated the role of pheromone detection by receptors expressed in the apical layer of the VNO such as vomeronasal type 1 receptors (V1R) in dominance behavior by using a conditional knockout mouse for G protein subunit Gαi2, which is essential for V1R signaling. We used the tube test as a model to analyze the within-a-cage hierarchy in male mice, but also as a paradigm of novel territorial competition in animals from different cages. In absence of prior social experience, Gαi2 deletion promotes winning a novel social competition with an unfamiliar control mouse but had no effect on an established hierarchy in cages with mixed genotypes, both Gαi2-/- and controls. To further dissect social behavior of Gαi2-/- mice, we performed a 3-chamber sociability assay and found that mutants had a slightly altered social investigation. Finally, gene expression analysis in the medial prefrontal cortex (mPFC) for a subset of genes previously linked to social status revealed no differences between group-housed Gαi2-/- and controls. Our results reveal a direct influence of pheromone detection on territorial dominance, indicating that olfactory communication involving apical VNO receptors like V1R is important for the outcome of an initial social competition between two unfamiliar male mice, whereas final social status acquired within a cage remains unaffected. These results support the idea that previous social context is relevant for the development of social hierarchy of a group. Overall, our data identify two context-dependent forms of dominance, acute and chronic, and that pheromone signaling through V1R receptors is involved in the first stages of a social competition but in the long term is not predictive for high social ranks on a hierarchy.

GPSM2-GNAI Specifies the Tallest Stereocilia and Defines Hair Bundle Row Identity.

The transduction compartment of inner ear hair cells, the hair bundle, is composed of stereocilia rows of graded height, a property essential for sensory function that remains poorly understood at the molecular level. We previously showed that GPSM2-GNAI is enriched at stereocilia distal tips and required for their postnatal elongation and bundle morphogenesis-two characteristics shared with MYO15A (short isoform), WHRN, and EPS8 proteins. Here we first performed a comprehensive genetic analysis of the mouse auditory epithelium to show that GPSM2, GNAI, MYO15A, and WHRN operate in series within the same pathway. To understand how these functionally disparate proteins act as an obligate complex, we then systematically analyzed their distribution in normal and mutant bundles over time. We discovered that WHRN-GPSM2-GNAI is an extra module recruited by and added to a pre-existing MYO15A-EPS8 stereocilia tip complex. This extended complex is only present in the first, tallest row, and is required to stabilize larger amounts of MYO15A-EPS8 than in shorter rows, which at tips harbor only MYO15A-EPS8. In the absence of GPSM2 or GNAI function, including in the epistatic Myo15a and Whrn mutants, bundles retain an embryonic-like organization that coincides with generic stereocilia at the molecular level. We propose that GPSM2-GNAI confers on the first row its unique tallest identity and participates in generating differential row identity across the hair bundle.