G-protein-coupled Receptor 31 Research Articles

12-HETE/GPR31 induces endothelial dysfunction in diabetic retinopathy.

12-hydroxyeicosatetraenoic acid (12-HETE), a major metabolite of arachidonic acid, is converted by 12/15-lipoxygenase and implicated in diabetic retinopathy (DR). Our previous study demonstrated a positive correlation between 12-HETE and the prevalence of DR. However, reasons for the increased production of 12-HETE are unclear, and the underlying mechanisms through which 12-HETE promotes DR are unknown. This study aimed to elucidate the correlation between 12-HETE and DR onset, investigate potential mechanisms through which 12-HETE promotes DR, and seek explanations for the increased production of 12-HETE in diabetes. We conducted a prospective cohort study, which revealed that higher serum 12-HETE levels could induce DR. Additionally, G protein-coupled receptor 31 (GPR31), a high-affinity receptor for 12-HETE, was expressed in human retinal microvascular endothelial cells (HRMECs). 12-HETE/GPR31-mediated HRMEC inflammation occurred via the p38 MAPK pathway. 12-HETE levels were significantly higher in the retina of mice with high-fat diet (HFD)- and streptozotocin (STZ)-induced diabetes than in those with only STZ-induced diabetes and healthy controls. They were positively correlated with the levels of inflammatory cytokines in the retina, indicating that HFD could induce increased 12-HETE synthesis in patients with diabetes in addition to hyperglycemia. Conclusively, 12-HETE is a potential risk factor for DR. The 12-HETE/GPR31 axis plays a crucial role in HRMEC dysfunction and could be a novel target for DR prevention and control. Nevertheless, further research is warranted to provide comprehensive insights into the complex underlying mechanisms of 12-HETE in DR.

SAT070 The G Protein Coupled Receptor GPR31 Promotes Islet Inflammation And Diabetes

Abstract Disclosure: C. Checkcinco: None. S.A. Tersey: None. R.G. Mirmira: None. In type 1 diabetes a characteristic immune response targets insulin secreting beta cells in pancreatic islets, resulting in their destruction. This pathogenesis features inflammatory signaling cascades in both beta cells and infiltrating immune cell macrophages. 12-Lipoxygenase (12-LOX) contributes to inflammation in β cells and macrophages by producing the eicosanoid 12-HETE, which induces oxidative and ER stress resulting in cellular dysfunction. G-protein coupled receptor 31 (GPR31) was identified as a high affinity 12-HETE receptor and is increased in human islets after cytokine induced inflammation, but it remains unknown if GPR31 might mediate the downstream effects of 12-LOX. We found increased expression of GPR31 in C57Bl/6J islets, over 7 times higher relative to macrophages. To investigate the role of GPR31 during diabetic inflammation, whole body GPR31 knockout mice were generated on the C57Bl/6J background and studied for glucose homeostasis in the setting of diabetic inflammation. Islets and macrophages from these mice were studied following manipulations in vitro. Compared to wildtype (WT) littermates, Gpr31b-/- mice maintained similar body weight, beta-cell mass, and glucose homeostasis as mice aged. To mimic the setting of islet inflammation as seen in type 1 diabetes, WT and Gpr31b-/- mice were treated with the pro-diabetic toxin streptozotocin (which induces beta cell death and macrophage influx). Compared to WT, Gpr31b-/- mice showed protection from development of hyperglycemia and glucose tolerance tests showed >30% protection reduction in area-under-the-curve, suggesting that GPR31 promotes islet dysfunction and hyperglycemia. To interrogate the role of GPR31 in the beta cell, we isolated islets from WT and Gpr31b-/- mice and treated them with proinflammatory cytokines followed by RNA sequencing to interrogate gene expression pathways. Differentially expressed genes in Gpr31b-/- islets included those pertaining to oxidative stress, MAPK signaling, and cellular homeostasis by Wnt/β-catenin, a finding reminiscent of pathways that are also altered following 12-LOX inhibition. To assess how GPR31 might impact macrophage function, we induced proinflammatory M1 polarization by lipopolysaccharide and interferon gamma in bone marrow-derived macrophages. We saw no significant difference in the extent of M1 polarization between GPR31b-/- and WT macrophages by flow cytometry, as assessed by the production of iNOS. We then asked if GPR31 was involved in the mobility of macrophages using a transwell assay with chemoattractant. We saw a ∼50% reduction in migrating bone marrow-derived macrophages in GPR31b-/- compared to WT. Taken together, these data are consistent with our previously published data on the 12-LOX knockout mice, suggesting that GPR31 may be involved in mediating the proinflammatory responses of 12-HETE in both β cells and macrophages. Presentation: Saturday, June 17, 2023

Lipid Receptor GPR31 (G-Protein-Coupled Receptor 31) Regulates Platelet Reactivity and Thrombosis Without Affecting Hemostasis.

12-LOX (12-lipoxygenase) produces a number of bioactive lipids including 12(S)-HETE that are involved in inflammation and platelet reactivity. The GPR31 (G-protein-coupled receptor 31) is the proposed receptor of 12(S)-HETE; however, it is not known whether the 12(S)-HETE-GPR31 signaling axis serves to enhance or inhibit platelet activity. Approach and Results: Using pepducin technology and biochemical approaches, we provide evidence that 12(S)-HETE-GPR31 signals through Gi to enhance PAR (protease-activated receptor)-4-mediated platelet activation and arterial thrombosis using both human platelets and mouse carotid artery injury models. 12(S)-HETE suppressed AC (adenylyl cyclase) activity through GPR31 and resulted in Rap1 (Ras-related protein 1) and p38 activation and low but detectable calcium flux but did not induce platelet aggregation. A GPR31 third intracellular (i3) loop-derived pepducin, GPR310 (G-protein-coupled receptor 310), significantly inhibited platelet aggregation in response to thrombin, collagen, and PAR4 agonist, AYPGKF, in human and mouse platelets but relative sparing of PAR1 agonist SFLLRN in human platelets. GPR310 treatment gave a highly significant 80% protection (P=0.0018) against ferric chloride-induced carotid artery injury in mice by extending occlusion time, without any effect on tail bleeding. PAR4-mediated dense granule secretion and calcium flux were both attenuated by GPR310. Consistent with these results, GPR310 inhibited 12(S)-HETE-mediated and PAR4-mediated Rap1-GTP and RASA3 translocation to the plasma membrane and attenuated PAR4-Akt and ERK activation. GPR310 caused a right shift in thrombin-mediated human platelet aggregation, comparable to the effects of inhibition of the Gi-coupled P2Y12 receptor. Co-immunoprecipitation studies revealed that GPR31 and PAR4 form a heterodimeric complex in recombinant systems. The 12-LOX product 12(S)-HETE stimulates GPR31-Gi-signaling pathways, which enhance thrombin-PAR4 platelet activation and arterial thrombosis in human platelets and mouse models. Suppression of this bioactive lipid pathway, as exemplified by a GPR31 pepducin antagonist, may provide beneficial protective effects against platelet aggregation and arterial thrombosis with minimal effect on hemostasis.

2111-P: Role of G-Protein Coupled Receptor 31 (GPR31) in ß-Cell Health and Disease

Islet β-cell inflammation, dysfunction, and death are characteristic of diabetes. 12/15-Lipoxygenase (12/15-LOX) produces 12-HETE and is found activated under conditions of insulin resistance and systemic inflammation. 12-HETE acts as an inflammatory mediator that promotes β-cell dysfunction and death through oxidative stress. Since 12-HETE has been shown to bind the G-protein coupled receptor 31 (GPR31), we hypothesized that this binding activates pathways leading to β-cell dysfunction. To test our hypothesis, we screened primary islets and β-cell lines for the presence of GPR31 and studied islets and glucose homeostasis in Gpr31b-/- mice. We observed that GPR31 is present in human and mouse islets, and the mouse β cell-derived cell line (βTC3). Consistent with a role in inflammation, GPR31 expression was increased upon stimulation with a cocktail of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β). We also created a germline knockout of the gene encoding GPR31 (Gpr31b) in mice. Similar to mice with deletion of the gene for 12/15-LOX (Alox15-/-), Gpr31b-/- mice were viable and healthy, with body weight and glucose tolerance indistinguishable from control littermates. To mimic conditions of insulin resistance in vitro, we treated isolated islets Gpr31-/- and control littermates with 10 µM S961, an insulin receptor inhibitor. Treated control islets showed an expected decrease in β-cell identity genes (Ins1/2, Pdx1), genes encoding antioxidant enzymes (Gpx1), and an increase in genes associated with ER stress (Chop, sXbp1). In contrast, Gpr31b-/- islets expression of these genes was resistant to S961. Compared to control littermates, Gpr31-/- mice were resistant to glucose intolerance induced by the β-cell inflammatory toxin streptozotocin. Together these data support the hypothesis that the major product of 12/15-LOX, 12-HETE, acts via GPR31 in promoting β-cell dysfunction in the setting of insulin resistance and inflammation. Disclosure M. Hernandez-Perez: None. I. Haider: None. R.M. Anderson: None. S.A. Tersey: None. R. Mirmira: Advisory Panel; Self; Hibercell, Sigilon Therapeutics, Veralox Therapeutics. Employee; Spouse/Partner; Eli Lilly and Company. Funding JDRF (3-PDF-2019-750-A-N); National Institutes of Health (T32DK064466, R01DK105588, (P30DK097512)

Blockade of Lipid Receptor GPR31 Suppresses Platelet Reactivity and Thrombosis with Minimal Effect on Hemostasis

Objective: Platelet agonist-activated 12-lipoxygenase (12-LOX)/12-Hydroxyeicosatetraenoic acid (12-HETE)/G protein-coupled receptor 31 (GPR31) signaling has been proposed to regulate platelet reactivity. While inhibition or genetic ablation of 12-LOX supports an important role of 12-HETE in response to platelet agonists thrombin and collagen, the participation of GPR31 in platelet lipid signaling has not been examined. We developed a potent pepducin inhibitor, GPR-310, to test the downstream involvement of GPR31 in thrombin and collagen mediated platelet activation and thrombosis. Approach and Results: Treatment of mice with GPR-310 reversibly inhibited ex vivo platelet aggregation in response to thrombin and the PAR4 agonist, AYPGKF. There was significant protection (P<0.002) against FeCl3-induced carotid artery injury in mice by extending occlusion time from 100% occlusion at 27 min in the vehicle cohort to 20% occluded at 45 min in the GPR-310 cohort. GPR-310 treatment did not affect tail bleeding time. In human platelets, GPR-310 significantly (P<0.001) inhibited PAR4 agonist and collagen-mediated platelet aggregation and PAR4 calcium release. GPR-310 inhibited 12(S)-HETE- and PAR4-mediated RAP1 activation, with no effect on the PAR1-RAP1 signal. Accordingly, PAR1-mediated aggregation of human platelets was not affected by either GPR-310 or the 12-LOX inhibitor, ML355. GPR-310 caused a 5-fold shift in thrombin-mediated human platelet aggregation, comparable to a direct P2Y12 inhibitor, AZD1283. Dual GPR31 and P2Y12 inhibition showed synergy and protected against thrombin-mediated human platelet aggregation with a 19-fold shift. Blockade of GPR31 was more effective than the P2Y12 inhibitor in a thrombin-mediated clot retraction assay. Co-immunoprecipitation studies revealed that GPR31 and PAR4 form a heterodimeric complex in recombinant systems. Conclusions: GPR31 may serve as a new therapeutic target in platelet-dependent arterial thrombosis and aggregation in humans. Disclosures No relevant conflicts of interest to declare.

An ALOX12-12-HETE-GPR31 signaling axis is a key mediator of hepatic ischemia-reperfusion injury.

Hepatic ischemia-reperfusion (IR) injury is a common clinical issue lacking effective therapy and validated pharmacological targets. Here, using integrative 'omics' analysis, we identified an arachidonate 12-lipoxygenase (ALOX12)-12-hydroxyeicosatetraenoic acid (12-HETE)-G-protein-coupled receptor 31 (GPR31) signaling axis as a key determinant of the hepatic IR process. We found that ALOX12 was markedly upregulated in hepatocytes during ischemia to promote 12-HETE accumulation and that 12-HETE then directly binds to GPR31, triggering an inflammatory response that exacerbates liver damage. Notably, blocking 12-HETE production inhibits IR-induced liver dysfunction, inflammation and cell death in mice and pigs. Furthermore, we established a nonhuman primate hepatic IR model that closely recapitulates clinical liver dysfunction following liver resection. Most strikingly, blocking 12-HETE accumulation effectively attenuated all pathologies of hepatic IR in this model. Collectively, this study has revealed previously uncharacterized metabolic reprogramming involving an ALOX12-12-HETE-GPR31 axis that functionally determines hepatic IR procession. We have also provided proof of concept that blocking 12-HETE production is a promising strategy for preventing and treating IR-induced liver damage.

Targeting RAS – will GPR31 deliver us a new path forward?

ABSTRACTEffective anti-rat sarcoma viral oncogene (RAS) therapies have remained the holy grail of cancer treatment. Mutant Kirsten rat sarcoma viral oncogene homolog (KRAS) sustains tumorigenesis when linked to the plasma membrane (PM). The G protein-coupled receptor 31 (GPR31) is now identified to mediate KRAS membrane association and is crucial for proliferation, survival and macropinocytosis of KRAS-dependent cancer cells, suggesting that GPR31 is a druggable target for anti-RAS therapy.

12-HETER1/GPR31, a high-affinity 12(S)-hydroxyeicosatetraenoic acid receptor, is significantly up-regulated in prostate cancer and plays a critical role in prostate cancer progression.

Previously we identified and deorphaned G-protein-coupled receptor 31 (GPR31) as the high-affinity 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] receptor (12-HETER1). Here we have determined its distribution in prostate cancer tissue and its role in prostate tumorigenesis using in vitro and in vivo assays. Data-mining studies strongly suggest that 12-HETER1 expression positively correlates with the aggressiveness and progression of prostate tumors. This was corroborated with real-time PCR analysis of human prostate tumor tissue arrays that revealed the expression of 12-HETER1 positively correlates with the clinical stages of prostate cancers and Gleason scores. Immunohistochemistry analysis also proved that the expression of 12-HETER1 is positively correlated with the grades of prostate cancer. Knockdown of 12-HETER1 in prostate cancer cells markedly reduced colony formation and inhibited tumor growth in animals. To discover the regulatory factors, 5 candidate 12-HETER1 promoter cis elements were assayed as luciferase reporter fusions in Chinese hamster ovary (CHO) cells, where the putative cis element required for gene regulation was mapped 2 kb upstream of the 12-HETER1 transcriptional start site. The data implicate 12-HETER1 in a critical new role in the regulation of prostate cancer progression and offer a novel alternative target for therapeutic intervention.-Honn, K. V., Guo, Y., Cai, Y., Lee, M.-J., Dyson, G., Zhang, W., Tucker, S. C. 12-HETER1/GPR31, a high-affinity 12(S)-hydroxyeicosatetraenoic acid receptor, is significantly up-regulated in prostate cancer and plays a critical role in prostate cancer progression.

Expression and clinical significance of G protein-coupled receptor 31 in colorectal cancer tissue

To investigate the expression and clinical significance of G protein-coupled receptor 31 (GPR31) in colorectal cancer tissue. Cancer tissues and adjacent normal tissues of 321 cases with colorectal cancer confirmed by pathology and undergoing resection in the First Affiliated Hospital of Sun Yat-sen University from January 1996 to December 2008 were collected. The expression of GPR31 was examined by immunohistochemical staining. According to the expression level of GPR31 (A value=0.051), all the patients were divided into low GPR31 expression group and high GPR31 expression group. Clinicopathology and prognosis between the two groups were compared. Risk factors affecting prognosis were investigated. GPR31 expression was significantly higher in colorectal cancer tissues compared to adjacent normal tissues (mean A, 0.063±0.014 vs. 0.045±0.020, P<0.001). A total of 197 cancer tissue samples were defined as low expression and 124 as high expression. Significant difference was observed in the number of patient in pM classification between the two groups (P=0.007). High expression group had obviously higher distant metastasis rate than low expression group [12.1% (15/124) vs. 4.1% (8/197), P=0.007]. The 5-year survival rate and tumor-free survival rate were 84.3% and 82.2% in the low expression group, and both 59.7% in high expression group (all P<0.05). Multivariate analysis revealed elderly, abnormal CEA, lymphatic metastasis, distant metastasis and up-regulated GPR31 expression were independent risk factors of overall survival and disease-free survival in colorectal cancer patients (all P<0.05). GPR31 expression is significantly up-regulated in colorectal cancer tissues. High GPR31expression indicates poor prognosis of colorectal cancer, and may be used as a predictive marker.