Gα12 and endoplasmic reticulum stress-mediated pyroptosis in a single cycle of dextran sulfate-induced mouse colitis.

Inflammatory bowel disease (IBD) pathogenesis involves complex inflammatory events and cell death. Although IBD involves mainly necrosis in the digestive tract, pyroptosis has also been recognized. Nonetheless, the underlying basis is elusive. Gα12/13 overexpression may affect endoplasmic reticulum (ER) stress. This study examined how Gα12/13 and ER stress affect pyroptosis using dextran sulfate sodium (DSS)-induced colitis models. Gα12/13 levels were increased in the distal and proximal colons of mice exposed to a single cycle of DSS, as accompanied by increases of IRE1α, ATF6, and p-PERK. Moreover, Il-6, Il-1ß, Ym1, and Arg1 mRNA levels were increased with caspase-1 and IL-1ß activation, supportive of pyroptosis. In the distal colon, RIPK1/3 levels were enhanced to a greater degree, confirming necroptosis. By contrast, the mice subjected to three cycles of DSS treatments showed decreases of Gα12/13, as accompanied by IRE1α and ATF6 suppression, but increases of RIPK1/3 and c-Cas3. AZ2 treatment, which inhibited Gα12, has an anti-pyroptotic effect against a single cycle of colitis. These results show that a single cycle of DSS-induced colitis may cause ER stress-induced pyroptosis as mediated by Gα12 overexpression in addition to necroptosis, but three cycles model induces only necroptosis, and that AZ2 may have an anti-pyroptotic effect.

Alternative splicing of latrophilin-3 controls synapse formation.

The assembly and specification of synapses in the brain is incompletely understood1-3. Latrophilin-3 (encoded by Adgrl3, also known as Lphn3)-a postsynaptic adhesion G-protein-coupled receptor-mediates synapse formation in the hippocampus4 but the mechanisms involved remain unclear. Here we show in mice that LPHN3 organizes synapses through a convergent dual-pathway mechanism: activation of Gαs signalling and recruitment of phase-separated postsynaptic protein scaffolds. We found that cell-type-specific alternative splicing of Lphn3 controls the LPHN3 G-protein-coupling mode, resulting in LPHN3 variants that predominantly signal through Gαs or Gα12/13. CRISPR-mediated manipulation of Lphn3 alternative splicing that shifts LPHN3 from a Gαs- to a Gα12/13-coupled mode impaired synaptic connectivity as severely as the overall deletion of Lphn3, suggesting that Gαs signalling by LPHN3 splice variants mediates synapse formation. Notably, Gαs-coupled, but not Gα12/13-coupled, splice variants of LPHN3 also recruit phase-transitioned postsynaptic protein scaffold condensates, such that these condensates are clustered by binding of presynaptic teneurin and FLRT ligands to LPHN3. Moreover, neuronal activity promotes alternative splicing of the synaptogenic Gαs-coupled variant of LPHN3. Together, these data suggest that activity-dependent alternative splicing of a key synaptic adhesion molecule controls synapse formation by parallel activation of two convergent pathways: Gαs signalling and clustered phase separation of postsynaptic protein scaffolds.

Gα12 signaling regulates transcriptional and phenotypic responses that promote glioblastoma tumor invasion.

In silico interrogation of glioblastoma (GBM) in The Cancer Genome Atlas (TCGA) revealed upregulation of GNA12 (Gα12), encoding the alpha subunit of the heterotrimeric G-protein G12, concomitant with overexpression of multiple G-protein coupled receptors (GPCRs) that signal through Gα12. Glioma stem cell lines from patient-derived xenografts also showed elevated levels of Gα12. Knockdown (KD) of Gα12 was carried out in two different human GBM stem cell (GSC) lines. Tumors generated in vivo by orthotopic injection of Gα12KD GSC cells showed reduced invasiveness, without apparent changes in tumor size or survival relative to control GSC tumor-bearing mice. Transcriptional profiling of GSC-23 cell tumors revealed significant differences between WT and Gα12KD tumors including reduced expression of genes associated with the extracellular matrix, as well as decreased expression of stem cell genes and increased expression of several proneural genes. Thrombospondin-1 (THBS1), one of the genes most repressed by Gα12 knockdown, was shown to be required for Gα12-mediated cell migration in vitro and for in vivo tumor invasion. Chemogenetic activation of GSC-23 cells harboring a Gα12-coupled DREADD also increased THBS1 expression and in vitro invasion. Collectively, our findings implicate Gα12 signaling in regulation of transcriptional reprogramming that promotes invasiveness, highlighting this as a potential signaling node for therapeutic intervention.

Ogt Deficiency Induces Abnormal Cerebellar Function and Behavioral Deficits of Adult Mice through Modulating RhoA/ROCK Signaling.

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.

Adagrasib (Krasati®) en cáncer colorectal metastásico / Adagrasib (Krasati®) in metastatic colorectal cancer

INTRODUCCIÓN: El cáncer colorrectal (CCR) representa la segunda causa de mortalidad por tumores en la Argentina. Según la Agencia Internacional de Investigación sobre el Cáncer la incidencia en nuestro país durante el año 2020 se calculó en aproximadamente 15.600 casos, registrándose en el mismo período de tiempo unas 8.600 muertes. que otros pacientes diagnosticados en estadios iniciales progresarán en el transcurso de la enfermedad requiriendo quimioterapia sistémica. Sin tratamiento, el promedio de sobrevida de estos pacientes es de unos 6 meses. Los esquemas de quimioterapia utilizados frecuentemente están basados en la combinación de fluoropirimidinas con oxaliplatino o irinotecan asociados con anticuerpos monoclonales contra el factor de crecimiento endotelial vascular (bevacizumab) o el receptor del factor de crecimiento epidérmico (cetuximab, panitunumab) en tumores sin mutaciones en la vía RAS (wildtipe o "salvajes"). En aquellos tumores con deficiencia del sistema de reparación de apareamientos erróneos o alta inestabilidad de microsatélites (por sus siglas en inglés, dMMR o MSI-H, respectivamente) se listan dentro de las opciones terapéuticas el uso de inmunoterapia. En aquellos pacientes en los cuales la enfermedad progresa a pesar de los esquemas mencionados se pueden utilizar regorafenib o trifluridina/tipiracilo y en ciertos subtipos moleculares encorafenib (mutación V600E en el gen BRAF); trastuzumab +/-pertuzumab/lapatinib/tucatinib (HER2 amplificado y sin mutaciones en los genes RAS y BRAF), entre otras. Se debe tener en cuenta que alguna de estas opciones no cuentan con aprobación o no se encuentran disponibles aún en nuestro país. En este documento se plantea evaluar la eficacia y seguridad del uso de adagrasib en pacientes con carcinoma colorectal metastásico portadores de la mutación G12C en el gen KRAS. TECNOLOGÍA: Adagrasib (Krasati®) es un inhibidor irreversible y selectivo de la proteína mutante KRAS G12C (homólogo del oncogén vírico de sarcoma de rata Kirsten). La proteína pertenece a la subfamilia de proteínas RAS (KRAS, HRAS y NRAS) que actúan como GTPasas y se desempeñan como reguladores moleculares, controlando un amplio espectro de actividades celulares, como la proliferación y la sobrevida celular. Su inactivación por parte de adagrasib bloquea la transmisión de señales inhibiendo el crecimiento celular y favoreciendo la apoptosis de manera selectiva en tumores portadores de la mutación KRAS G12C. OBJETIVO: El objetivo del presente informe es evaluar rápidamente los parámetros de eficacia, seguridad, costos y recomendaciones disponibles acerca del empleo de adagrasib en pacientes con diagnóstico de carcinoma colorectal metastásico. MÉTODOS Se realizó una búsqueda bibliográfica en las principales bases de datos tales como PUBMED, LILACS, BRISA, COCHRANE, SCIELO, EMBASE, TRIPDATABASE como así también en sociedades científicas, agencias reguladoras, financiadores de salud y agencias de evaluación de tecnologías sanitarias. Se priorizó la inclusión de revisiones sistemáticas, ensayos clínicos controlados aleatorizados, evaluación de tecnología sanitaria y guías de práctica clínica de alta calidad metodológica. La fecha de búsqueda de información fue hasta el 19 de mayo de 2023. Para la búsqueda en Pubmed se utilizó la siguiente estrategia de búsqueda: (adagrasib [Supplementary Concept] OR adagrasib [tiab] OR MRTX849 [tiab]) AND ("Colonic Neoplasms"[Mesh] OR ¨Colorectal Cancer¨ [tiab]). CONCLUSIONES: La evidencia sobre el uso de adagrasib para el tratamiento de pacientes con diagnóstico de carcinoma colorectal metastásico progresados a múltiples líneas de tratamiento se basa en un único ensayo clínico no aleatorizado de fase I-II. Este estudio mostró que aquellos pacientes, progresados generalmente a tres líneas de tratamiento y con tumores que presentan mutaciones G12C en el gen KRAS, que utilizaron adagrasib con cetuximab reportaron una mediana de sobrevida global de 13,4 meses y libre de progresión de 6,9 meses, y una tasa de respuesta del 46%. Los eventos adversos severos fueron reportados en el 16% de los pacientes. Actualmente se encuentra en curso un ensayo clínico aleatorizado de fase III que cuenta con centros en Argentina y tratará de establecer la seguridad y eficacia de la adición de adagrasib a un esquema de poliquimioterapia como segunda línea de tratamiento La Administración de Alimentos y Medicamentos de Estados Unidos y la Agencia Europea de Medicamentos aún no ha autorizado la comercialización del medicamento en la indicación antes mencionada. En referencias a las recomendaciones relevadas, no se hallaron guías que mencionen los esquemas de adagrasib combinado con cetuximab o en monoterapia como una opción para el tratamiento del carcinoma colorectal avanzado. Utilizando precios de referencia internacionales, el costo de adquisición para un ciclo de tratamiento fue estimado en aproximadamente 4,8 millones de pesos argentinos.

Gα13-Mediated Signaling Cascade Is Related to the Tau Pathology Caused by Anesthesia and Surgery in 5XFAD Transgenic Mice.

BACKGROUND: Our previous studies indicated that anesthesia and surgery could aggravate cognitive impairment of 5XFAD transgenic (Tg) mice, and this aggravation was associated with tau hyperphosphorylation. We previously identified that GNA13 (the gene encoding Gα13) was a hub gene with tau hyperphosphorylation. OBJECTIVE: This study aims to further investigate the mechanism that whether the Gα13-mediated signaling pathway acts as an instigator to regulate cofilin activation and autophagy impairment in this process. METHODS: 5XFAD Tg mice and their littermate (LM) mice were randomly allocated into four groups: LM Control group, LM Anesthesia/Surgery group, AD Control group, and AD Anesthesia/Surgery group. For mice in the Anesthesia/Surgery groups, abdominal surgery was performed under 1.4% isoflurane anesthesia followed by sustaining anesthetic inhalation for up to 2 h. RESULTS: Compared with the AD Control group, protein levels of Gα13, ROCK2, LPAR5, and p-tau/tau46 ratio were increased, while p-cofilin/cofilin protein expression ratio was decreased in the AD Anesthesia/Surgery group. However, the differences in these protein levels were not significant among LM groups. CONCLUSION: This study demonstrated that anesthesia and surgery might exacerbate p-tau accumulation in 5XFAD Tg mice but not in LM mice. And this might be closely related to cofilin activation via Gα13-mediated signaling cascade.

Detrimental Role of PDZ-RhoGEF in Pathological Cardiac Hypertrophy.

BACKGROUND: Postsynaptic density 95/disk-large/ZO-1 Rho guanine nucleotide exchange factor (PDZ-RhoGEF, PRG) functions as a RhoGEF for activated Gα13 and transmits activation signals to downstream signaling pathways in various pathological processes. Although the prohypertrophic effect of activated Gα13 (guanine nucleotide binding protein alpha 13; a heterotrimeric G protein) is well-established, the role of PDZ-RhoGEF in pathological cardiac hypertrophy is still obscure. METHODS: Genetically engineered mice and neonatal rat ventricular myocytes were generated to investigate the function of PRG in pathological myocardial hypertrophy. The prohypertrophic stimuli-induced alternations in the morphology and intracellular signaling were measured in myocardium and neonatal rat ventricular myocytes. Furthermore, multiple molecular methodologies were used to identify the precise molecular mechanisms underlying PDZ-RhoGEF function. RESULTS: Increased PDZ-RhoGEF expression was documented in both hypertrophied hearts and neonatal rat ventricular myocytes. Upon prohypertrophic stimuli, the PDZ-RhoGEF-deficient hearts displayed alleviated cardiomyocyte enlargement and attenuated collagen deposition with improved cardiac function, whereas the adverse hypertrophic responses in hearts and neonatal rat ventricular myocytes were markedly exaggerated by PDZ-RhoGEF overexpression. Mechanistically, RhoA (ras homolog family member A)-dependent signaling pathways may function as the downstream effectors of PDZ-RhoGEF in hypertrophic remodeling, as confirmed by rescue experiments using a RhoA inhibitor and dominant-negative RhoA. Furthermore, PDZ-RhoGEF is associated with activated Gα13 and contributes to Gα13-mediated activation of RhoA-dependent signaling. CONCLUSIONS: Our data provide the first evidence that PDZ-RhoGEF promotes pathological cardiac hypertrophy by linking activated Gα13 to RhoA-dependent signaling pathways. Therefore, PDZ-RhoGEF has the potential to be a diagnostic marker or therapeutic target for pathological cardiac hypertrophy.

Overexpressed Gα13 activates serum response factor through stoichiometric imbalance with Gßγ and mislocalization to the cytoplasm.

Gα13, a heterotrimeric G protein α subunit of the G12/13 subfamily, is an oncogenic driver in multiple cancer types. Unlike other G protein subfamilies that contribute to cancer progression via amino acid substitutions that abolish their deactivating, intrinsic GTPase activity, Gα13 rarely harbors such mutations in tumors and instead appears to stimulate aberrant cell growth via overexpression as a wildtype form. It is not known why this effect is exclusive to the G12/13 subfamily, nor has a mechanism been elucidated for overexpressed Gα13 promoting tumor progression. Using a reporter gene assay for serum response factor (SRF)-mediated transcription in HEK293 cells, we found that transiently expressed, wildtype Gα13 generates a robust SRF signal, approximately half the amplitude observed for GTPase-defective Gα13. When epitope-tagged, wildtype Gα13 was titrated upward in cells, a sharp increase in SRF stimulation was observed coincident with a "spillover" of Gα13 from membrane-associated to a soluble fraction. Overexpressing G protein ß and γ subunits caused both a decrease in this signal and a shift of wildtype Gα13 back to the membranous fraction, suggesting that stoichiometric imbalance in the αßγ heterotrimer results in aberrant subcellular localization and signalling by overexpressed Gα13. We also examined the acylation requirements of wildtype Gα13 for signalling to SRF. Similar to GTPase-defective Gα13, S-palmitoylation of the wildtype α subunit was necessary for SRF activation but could be replaced functionally by an engineered site for N-terminal myristoylation. However, a key difference was observed between wildtype and GTPase-defective Gα13: whereas the latter protein lacking palmitoylation sites was rescued in its SRF signalling by either an engineered polybasic sequence or a C-terminal isoprenylation site, these motifs failed to restore signalling by wildtype, non-palmitoylated Gα13. These findings illuminate several components of the mechanism in which overexpressed, wildtype Gα13 contributes to growth and tumorigenic signalling, and reveal greater stringency in its requirements for post-translational modification in comparison to GTPase-defective Gα13.

Lysophosphatidic Acid Induced Apoptosis, DNA Damage, and Oxidative Stress in Spinal Cord Neurons by Upregulating LPA4/LPA6 Receptors.

Lysophosphatidic acid (LPA) has disruptive effects on lumbar spinal stenosis (LSS). Recently, LPA has been reported to be involved in spinal cord neuronal injury and toxicity, promoting the pathogenesis of LSS. However, the exact effects of LPA on spinal cord neurons remain unknown. The purpose of this study is to investigate the effects of LPA (18 : 1) on spinal cord neuronal cytotoxicity, apoptosis, DNA damage, and oxidative stress. After clinical detection of LPA secretion, spinal cord neurons were treated with LPA (18 : 1); cell viability was analyzed by MTT assay, and LDH leakage was detected by LDH kit; cell apoptosis was detected by flow cytometry; ROS production was measured by DCFDA staining and MitoSOX Red Staining; the activation of the Gα12/Gα13 signaling pathway was detected by serum response factor response element (SRF-RE) luciferase reporter gene; the relationship among LPA, LPA4/6, and ROCK was examined by western blotting. In spinal cord neurons treated with LPA (18 : 1), cellular activity decreased and LDH release increased. The Rho kinase inhibitor (Y-27632) can attenuate LPA-induced apoptosis, DNA damage, and oxidative stress in spinal cord neurons. Moreover mechanistic investigation indicated that LPA (18 : 1) activates Gα12/13-Rho-ROCK2-induced apoptosis, DNA damage, and oxidative stress in spinal cord neurons by upregulating LPA4/LPA6 receptors. Further, the Rho kinase inhibitor Y-27632 attenuates the effects of LPA by downregulating LPA4/LPA6 receptors. Taken together, the possible mechanism by which LPA secretion in LSS patients aggravates patient injury was further elucidated using an LPA-induced spinal cord neuronal injury cell model in vitro.

Neonatal Platelets: Lower G12/13 Expression Contributes to Reduced Secretion of Dense Granules.

Despite fully functional primary hemostasis, platelets of healthy neonates exhibit hypoaggregability and secretion defects, which may be adaptations to specific requirements in this developmental stage. The etiologies for reduced signal transduction vary with the type of agonist. The discovered peculiarities are lower receptor densities, reduced calcium mobilization, and functional impairments of G proteins. Reduced secretion of dense granules has been attributed to lower numbers of granules. Signaling studies with adult platelets have shown a regulating effect of the G12/13 signaling pathway on dense granule secretion via RhoA. We comparatively analyzed secretion profiles using flow cytometry and expression levels of Gq, Gi, and G12/13 using Western blot analysis in platelets from cord blood and adults. Furthermore, we evaluated Rho activation after in vitro platelet stimulation with thrombin using a pulldown assay. We observed a markedly reduced expression of the dense granule marker CD63 on neonatal platelets after thrombin stimulation. Gα12/13 expression was significantly decreased in neonatal platelets and correlated with lower Rho activation after thrombin stimulation. We conclude that lower expression of G12/13 in neonatal platelets results in attenuated activation of Rho and may contribute to reduced secretion of dense granules after exposure to thrombin.

Prostaglandin F2α and angiotensin II type 1 receptors exhibit differential cognate G protein coupling regulation.

Promiscuous G protein-coupled receptors (GPCRs) engage multiple Gα subtypes with different efficacies to propagate signals in cells. A mechanistic understanding of Gα selectivity by GPCRs is critical for therapeutic design, since signaling can be restrained by ligand-receptor complexes to preferentially engage specific G proteins. However, details of GPCR selectivity are unresolved. Here, we investigated cognate G protein selectivity using the prototypical promiscuous Gαq/11 and Gα12/13 coupling receptors, angiotensin II type I receptor (AT1R) and prostaglandin F2α receptor (FP), bioluminescence resonance energy transfer-based G protein and pathway-selective sensors, and G protein knockout cells. We determined that competition between G proteins for receptor binding occurred in a receptor- and G protein-specific manner for AT1R and FP but not for other receptors tested. In addition, we show that while Gα12/13 competes with Gαq/11 for AT1R coupling, the opposite occurs for FP, and Gαq-mediated signaling regulated G protein coupling only at AT1R. In cells, the functional modulation of biased ligands at FP and AT1R was contingent upon cognate Gα availability. The efficacy of AT1R-biased ligands, which poorly signal through Gαq/11, increased in the absence of Gα12/13. Finally, we show that a positive allosteric modulator of Gαq/11 signaling that also allosterically decreases FP-Gα12/13 coupling, lost its negative modulation in the absence of Gαq/11 coupling to FP. Together, our findings suggest that despite preferential binding of similar subsets of G proteins, GPCRs follow distinct selectivity rules, which may contribute to the regulation of ligand-mediated G protein bias of AT1R and FP.

Thromboxane A2 receptor activation via Gα13-RhoA/C-ROCK-LIMK2-dependent signal transduction inhibits angiogenic sprouting of human endothelial cells.

We could previously show that thromboxane A2 receptor (TP) activation inhibits the angiogenic capacity of human endothelial cells, but the underlying mechanisms remained unclear. Therefore, the aim of this study was to elucidate TP signal transduction pathways relevant to angiogenic sprouting of human endothelial cells. To clarify this matter, we used RNAi-mediated gene silencing as well as pharmacological inhibition of potential TP downstream targets in human umbilical vein endothelial cells (HUVEC) and VEGF-induced angiogenic sprouting of HUVEC spheroids in vitro as a functional read-out. In this experimental set-up, the TP agonist U-46619 completely blocked VEGF-induced angiogenic sprouting of HUVEC spheroids. Moreover, in live-cell analyses TP activation induced endothelial cell contraction, sprout retraction as well as endothelial cell tension and focal adhesion dysregulation of HUVEC. These effects were reversed by pharmacological TP inhibition or TP knockdown. Moreover, we identified a TP-Gα13-RhoA/C-ROCK-LIMK2-dependent signal transduction pathway to be relevant for U-46619-induced inhibition of VEGF-mediated HUVEC sprouting. In line with these results, U-46619-mediated TP activation potently induced RhoA and RhoC activity in live HUVEC as measured by FRET biosensors. Interestingly, pharmacological inhibition of ROCK and LIMK2 also normalized U-46619-induced endothelial cell tension and focal adhesion dysregulation of HUVEC. In summary, our work reveals mechanisms by which the TP may disturb angiogenic endothelial function in disease states associated with sustained endothelial TP activation.

Convergent selective signaling impairment exposes the pathogenicity of latrophilin-3 missense variants linked to inheritable ADHD susceptibility.

Latrophilin-3 (Lphn3; also known as ADGRL3) is a member of the adhesion G Protein Coupled Receptor subfamily, which participates in the stabilization and maintenance of neuronal networks by mediating intercellular adhesion through heterophilic interactions with transmembrane ligands. Polymorphisms modifying the Lphn3 gene are associated with attention-deficit/hyperactivity disorder (ADHD) in children and its persistence into adulthood. How these genetic alterations affect receptor function remains unknown. Here, we conducted the functional validation of distinct ADHD-related Lphn3 variants bearing mutations in the receptor's adhesion motif-containing extracellular region. We found that all variants tested disrupted the ability of Lphn3 to stabilize intercellular adhesion in a manner that was distinct between ligands classes, but which did not depend on ligand-receptor interaction parameters, thus pointing to altered intrinsic receptor signaling properties. Using G protein signaling biosensors, we determined that Lphn3 couples to Gαi1, Gαi2, Gαs, Gαq, and Gα13. However, all ADHD-related receptor variants consistently lacked intrinsic as well as ligand-dependent Gα13 coupling efficiency while maintaining unaltered coupling to Gαi, Gαs, and Gαq. Consistent with these alterations, actin remodeling functions as well as actin-relevant RhoA signaling normally displayed by the constitutively active Lphn3 receptor were impeded by select receptor variants, thus supporting additional signaling defects. Taken together, our data point to Gα13 selective signaling impairments as representing a disease-relevant pathogenicity pathway that can be inherited through Lphn3 gene polymorphisms. This study highlights the intricate interplay between Lphn3 GPCR functions and the actin cytoskeleton in modulating neurodevelopmental cues related to ADHD etiology.

GNA13 promotes the proliferation and migration of lung squamous cell carcinoma cells through regulating the PI3K/AKT signaling pathway.

The purpose of this study aimed to figure out the role of GNA13 in lung squamous cell carcinoma (LUSC) and the underlying mechanism. Male BALB/c mice were used to construct LUSC mouse model. Cell growth was examined using MTT and colony formation assay. Cell migration and invasion was determined using wound healing and transwell assay. The expression and phosphorylation of protein was detected by Western blotting assay. Immunohistochemistry staining was used to observe the tumor growth and metastasis. GNA13 was overexpressed in both LUSC tissues and LUSC cell lines. Knockdown of GNA13 in LUSC cells reduced cell viability and inhibited the formation of colonies in the SK-MES-1 and NCI-H520 cells. Cell migration and invasion was also prevented by inhibition of GNA13 in the LUSC cells. Phosphorylation of PI3K and AKT was downregulated by silencing GNA13 and upregulated by overexpression of GNA13 in the LUSC cells. In LUSC mouse model, tumor size and tumor weight were significantly decreased in si-GNA13 mice compared to control group. The expression of GNA13, Ki67, MMP2 and phosphorylation of AKT were significantly inhibited in si-GNA13 mice compared to control group. This study has demonstrated that knockdown of GNA13 could inhibit cell survival, migration and metastasis in LUSC.

Gα13 loss in Kras/Tp53 mouse model of pancreatic tumorigenesis promotes tumors susceptible to rapamycin.

Gα13 transduces signals from G-protein-coupled receptors. While Gα13 functions as a tumor suppressor in lymphomas, it is not known whether Gα13 is pro-tumorigenic or tumor suppressive in genetically engineered mouse (GEM) models of epithelial cancers. Here, we show that loss of Gα13 in the Kras/Tp53 (KPC) GEM model promotes well-differentiated tumors and reduces survival. Mechanistically, tumors developing in KPC mice with Gα13 loss exhibit increased E-cadherin expression and mTOR signaling. Importantly, human pancreatic ductal adenocarcinoma (PDAC) tumors with low Gα13 expression also exhibit increased E-cadherin expression and mTOR signaling. Treatment with the mTOR inhibitor rapamycin decreases the growth of syngeneic KPC tumors with Gα13 loss by promoting cell death. This work establishes a tumor-suppressive role of Gα13 in pancreatic tumorigenesis in the KPC GEM model and suggests targeting mTOR in human PDAC tumors with Gα13 loss.

CD97 promotes spleen dendritic cell homeostasis through the mechanosensing of red blood cells.

Dendritic cells (DCs) are crucial for initiating adaptive immune responses. However, the factors that control DC positioning and homeostasis are incompletely understood. We found that type-2 conventional DCs (cDC2s) in the spleen depend on Gα13 and adhesion G protein-coupled receptor family member-E5 (Adgre5, or CD97) for positioning in blood-exposed locations. CD97 function required its autoproteolytic cleavage. CD55 is a CD97 ligand, and cDC2 interaction with CD55-expressing red blood cells (RBCs) under shear stress conditions caused extraction of the regulatory CD97 N-terminal fragment. Deficiency in CD55-CD97 signaling led to loss of splenic cDC2s into the circulation and defective lymphocyte responses to blood-borne antigens. Thus, CD97 mechanosensing of RBCs establishes a migration and gene expression program that optimizes the antigen capture and presentation functions of splenic cDC2s.

ERα inhibits mesenchymal and amoeboidal movement of liver cancer cell via Gα12.

Hepatocellular carcinoma (HCC) is the second most common cancer worldwide, demonstrating aggressiveness and mortality more frequently in men than in women. Despite reports regarding the inhibitory ability of estrogen receptor alpha (ERα, ESR1) in certain cancer progression, targets and the basis of underlying gender disparity in HCC worsening remain elusive. Here, we report the ability of ERα to transcriptionally inhibit G protein subunit alpha 12 (Gα12) responsible for HCC worsening. First, using human samples and public database, the expression of ERα and Gα12 in HCC was examined. Then, quantitative real-time PCR, chromatin immunoprecipitation-assay, luciferase assay and immunoblottings of liver cancer cell lines confirmed the inhibitory ability of ERα on Gα12 and HCC progression. Gα12 promoted mesenchymal characteristics and amoeboidal movement, which was antagonized by ERα overexpression. Additionally, we found microRNA-141 and microRNA-200a as downstream targets of the Gα12 signaling axis for cancer malignancy regulation under the control of ERα. As for in-depth mechanism, PTP4A1 was found to be directly inhibited by microRNA-141 and microRNA-200a. Moreover, we found the inhibitory effect of ERα on amoeboidal movement by analyzing the morphology and blebbing of liver cancer cells and the active form of MLC levels. The identified targets and ESR1 levels are inversely correlated with human specimens, as well as with sex-biased survival rates of HCC patients. Collectively, ERα-dependent repression of Gα12 and consequent changes in the Gα12 signaling may explain the gender disparity in HCC, providing pharmacological clues for the control of metastatic HCC.

Size-Reduced Macrocyclic Analogues of [Pyr1]-apelin-13 Showing Negative Gα12 Bias Still Produce Prolonged Cardiac Effects.

We previously reported a series of macrocyclic analogues of [Pyr1]-apelin-13 (Ape13) with increased plasma stability and potent APJ agonist properties. Based on the most promising compound in this series, we synthesized and then evaluated novel macrocyclic compounds of Ape13 to identify agonists with specific pharmacological profiles. These efforts led to the development of analogues 39 and 40, which possess reduced molecular weight (MW 1020 Da vs Ape13, 1534 Da). Interestingly, compound 39 (Ki 0.6 nM), which does not activate the Gα12 signaling pathway while maintaining potency and efficacy similar to Ape13 to activate Gαi1 (EC50 0.8 nM) and ß-arrestin2 recruitment (EC50 31 nM), still exerts cardiac actions. In addition, analogue 40 (Ki 5.6 nM), exhibiting a favorable Gα12-biased signaling and an increased in vivo half-life (t1/2 3.7 h vs <1 min of Ape13), produces a sustained cardiac response up to 6 h after a single subcutaneous bolus injection.

The emerging roles of Gα12/13 proteins on the hallmarks of cancer in solid tumors.

G12 proteins comprise a subfamily of G-alpha subunits of heterotrimeric GTP-binding proteins (G proteins) that link specific cell surface G protein-coupled receptors (GPCRs) to downstream signaling molecules and play important roles in human physiology. The G12 subfamily contains two family members: Gα12 and Gα13 (encoded by the GNA12 and GNA13 genes, respectively) and, as with all G proteins, their activity is regulated by their ability to bind to guanine nucleotides. Increased expression of both Gα12 and Gα13, and their enhanced signaling, has been associated with tumorigenesis and tumor progression of multiple cancer types over the past decade. Despite these strong associations, Gα12/13 proteins are underappreciated in the field of cancer. As our understanding of G protein involvement in oncogenic signaling has evolved, it has become clear that Gα12/13 signaling is pleotropic and activates specific downstream effectors in different tumor types. Further, the expression of Gα12/13 proteins is regulated through a series of transcriptional and post-transcriptional mechanisms, several of which are frequently deregulated in cancer. With the ever-increasing understanding of tumorigenic processes driven by Gα12/13 proteins, it is becoming clear that targeting Gα12/13 signaling in a context-specific manner could provide a new strategy to improve therapeutic outcomes in a number of solid tumors. In this review, we detail how Gα12/13 proteins, which were first discovered as proto-oncogenes, are now known to drive several "classical" hallmarks, and also play important roles in the "emerging" hallmarks, of cancer.

High GNG13 expression is associated with poor survival in epithelial ovarian cancer and breast cancer.

Recently, change in the GNG13 expression has been shown to result in multiple congenital malformations and sexual reversal, and it was also found in the brain. The aim of this study was to measure the expression levels in epithelial ovarian cancer (EOC) and breast cancer (BC) and assess their value as a potential prognostic marker. The correlation of GNG13 protein expression was detected by immunohistochemistry (IHC) in 119 EOC and 125 BC tissues. Assessment of the associations between GNG13 levels and various clinicopathological features was identified, the relationship between GNG13 and prognosis in BC and EOC patients was analyzed using online resources of Oncomine and Kaplan-Meier plotter. Protein expression levels of GNG13 were both significantly lower in BC and EOC compared with normal tissues (p<0.0001 and p<0.001, respectively). Among the clinicopathological characteristics of BC, tumor grade (p=0.001) and TNM stage (p=0.001) were significantly associated with low expression of GNG13. While in EOC, low expression of GNG13 was significantly related to FIGO stage (p=0.001), presence of metastasis (p=0.001), and CA125 (p=0.001). Our data suggest that GNG13 expression maybe as a new inhibitor, which can strongly inhibit metastasis and partially attenuates tumor growth in EOC and BC.