Gs Protein-coupled Receptor Research Articles

Intracellular cAMP signaling-induced Ca2+ influx mediated by calcium homeostasis modulator 1 (CALHM1) in human odontoblasts.

In odontoblasts, intracellular Ca2+ signaling plays key roles in reactionary dentin formation and generation of dentinal pain. Odontoblasts also express several Gs protein-coupled receptors that promote production of cyclic AMP (cAMP). However, the crosstalk between intracellular cAMP and Ca2+ signaling, as well as the role of cAMP in the cellular functions of odontoblasts, remains unclear. In this study, we measured intracellular cAMP levels and intracellular free Ca2+ concentration ([Ca2+]i). We also investigated the effect of intracellular cAMP on mineralization by the odontoblasts. In the presence of extracellular Ca2+, the application of forskolin (adenylyl cyclase activator) or isoproterenol (Gs protein-coupled beta-2 adrenergic receptor agonist) increased intracellular cAMP levels and [Ca2+]i in odontoblasts. The [Ca2+]i increases could not be observed by removing extracellular Ca2+, indicating that cAMP is capable to activate Ca2+ entry. Forskolin-induced [Ca2+]i increase was inhibited by a protein kinase A inhibitor in odontoblasts. The [Ca2+]i increase was sensitive to Gd3+, 2APB, or Zn2+ but not verapamil, ML218, or La3+. In immunofluorescence analyses, odontoblasts were immunopositive for calcium homeostasis modulator 1 (CALHM1), which was found close to ionotropic ATP receptor subtype, P2X3 receptors. When CALHM1 was knocked down, forskolin-induced [Ca2+]i increase was suppressed. Alizarin red and von Kossa staining showed that forskolin decreased mineralization. These findings suggest that activation of adenylyl cyclase elicited increases in the intracellular cAMP level and Ca2+ influx via protein kinase A activation in odontoblasts. Subsequent cAMP-dependent Ca2+ influx was mediated by CALHM1 in odontoblasts. In addition, the intracellular cAMP signaling pathway in odontoblasts negatively mediated dentinogenesis.

Intracellular cAMP Signaling Pathway via Gs Protein-Coupled Receptor Activation in Rat Primary Cultured Trigeminal Ganglion Cells.

G protein-coupled receptors in trigeminal ganglion (TG) neurons are often associated with sensory mechanisms, including nociception. We have previously reported the expression of P2Y12 receptors, which are Gi protein-coupled receptors, in TG cells. Activating P2Y12 receptors decreased the intracellular free Ca2+ concentration ([Ca2+]i). This indicated that intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels can mediate Ca2+ signaling in TG cells. Here, we report more extensive-expression patterns of Gs protein-coupled receptors in primary cultured TG neurons isolated from 7-day-old newborn Wistar rats and further examine the roles of these receptors in cAMP signaling using the BacMam sensor in these neurons. To identify TG neurons, we also measured [Ca2+]i using fura-2 in TG cells and measured intracellular cAMP levels. TG neurons were positive for Gαs protein-coupled receptors, beta-2 adrenergic (β2), calcitonin gene-related peptide (CGRP), adenosine A2A (A2A), dopamine 1 (D1), prostaglandin I2 (IP), and 5-hydroxytriptamine 4 (5-HT4) receptor. Application of forskolin (FSK), an activator of adenylyl cyclase, transiently increased intracellular cAMP levels in TG neurons. The application of a phosphodiesterase inhibitor augmented the FSK-elicited intracellular cAMP level increase. These increases were significantly suppressed by the application of SQ22536, an adenylyl cyclase inhibitor, in TG neurons. Application of agonists for β2, CGRP, A2A, D1-like, IP, and 5-HT4 receptors increased intracellular cAMP levels. These increases were SQ22536-sensitive. These results suggested that TG neurons express β2, CGRP, A2A, D1, IP, and 5-HT4 receptors, and the activations of these Gαs protein-coupled receptors increase intracellular cAMP levels by activating adenylyl cyclase.

Crystal structure of adenosine A2A receptor in complex with clinical candidate Etrumadenant reveals unprecedented antagonist interaction

The Gs protein-coupled adenosine A2A receptor (A2AAR) represents an emerging drug target for cancer immunotherapy. The clinical candidate Etrumadenant was developed as an A2AAR antagonist with ancillary blockade of the A2BAR subtype. It constitutes a unique chemotype featuring a poly-substituted 2-amino-4-phenyl-6-triazolylpyrimidine core structure. Herein, we report two crystal structures of the A2AAR in complex with Etrumadenant, obtained with differently thermostabilized A2AAR constructs. This led to the discovery of an unprecedented interaction, a hydrogen bond of T883.36 with the cyano group of Etrumadenant. T883.36 is mutated in most A2AAR constructs used for crystallization, which has prevented the discovery of its interactions. In-vitro characterization of Etrumadenant indicated low selectivity versus the A1AR subtype, which can be rationalized by the structural data. These results will facilitate the future design of AR antagonists with desired selectivity. Moreover, they highlight the advantages of the employed A2AAR crystallization construct that is devoid of ligand binding site mutations.

Functional Expression of IP, 5-HT4, D1, A2A, and VIP Receptors in Human Odontoblast Cell Line.

Odontoblasts are involved in sensory generation as sensory receptor cells and in dentin formation. We previously reported that an increase in intracellular cAMP levels by cannabinoid 1 receptor activation induces Ca2+ influx via transient receptor potential vanilloid subfamily member 1 channels in odontoblasts, indicating that intracellular cAMP/Ca2+ signal coupling is involved in dentinal pain generation and reactionary dentin formation. Here, intracellular cAMP dynamics in cultured human odontoblasts were investigated to understand the detailed expression patterns of the intracellular cAMP signaling pathway activated by the Gs protein-coupled receptor and to clarify its role in cellular functions. The presence of plasma membrane Gαs as well as prostaglandin I2 (IP), 5-hydroxytryptamine 5-HT4 (5-HT4), dopamine D1 (D1), adenosine A2A (A2A), and vasoactive intestinal polypeptide (VIP) receptor immunoreactivity was observed in human odontoblasts. In the presence of extracellular Ca2+, the application of agonists for the IP (beraprost), 5-HT4 (BIMU8), D1 (SKF83959), A2A (PSB0777), and VIP (VIP) receptors increased intracellular cAMP levels. This increase in cAMP levels was inhibited by the application of the adenylyl cyclase (AC) inhibitor SQ22536 and each receptor antagonist, dose-dependently. These results suggested that odontoblasts express Gs protein-coupled IP, 5-HT4, D1, A2A, and VIP receptors. In addition, activation of these receptors increased intracellular cAMP levels by activating AC in odontoblasts.

Abstract 3437: Characterization of a potent and selective A2aR monoclonal antibody antagonist for the treatment of cancer

Abstract Introduction: Adenosine-mediated suppression of anti-tumor immunity has been shown to be one of the mechanisms for immunosuppression and resistance to immune checkpoint blockade (ICB) therapies such as anti-PD-1 or anti-PD-L1 monoclonal antibodies (mAbs). Therapeutic candidates (biologics and small molecule inhibitors(SMIs)) targeting ectonucleotidases CD39 and CD73, enzymes responsible for hydrolyzing ATP to adenosine, and SMIs targeting adenosine receptors are currently under active preclinical and early clinical development. Here we present the characterization of a novel, selective anti-human A2aR antagonist mAb that showed high potency in inhibiting adenosine mediated immunosuppressive functions to enhance anti-tumor immunity. Methods: A panel of mAbs binding to human adenosine receptor A2aR (hA2aR) were generated via hybridoma technology. The specificity and cross-reactivity were measured by FACS using human A1R, A2aR, A2bR, A3R or mouse A2aR over-expressing cells, as well as human and cynomolgus monkey PBMC. The cellular potency of these mAbs was assessed by measuring decreased cAMP levels in engineered HEK293 cells stably over-expressing hA2aR, a Gs protein coupled receptor (GPCR), upon stimulation with agonist NECA. The ability of lead candidate AT-004 to reverse adenosine-mediated immune suppression of human CD8 T-cells was determined using standard CD3/CD28 activation conditions. The in vivo efficacy of humanized AT-004 is being evaluated using ICB treatment resistant solid tumor mouse models. Results: AT-004 represents a panel of novel, selective mAbs. These mAbs specifically bind to human A2aR, but not to family members including A1R, A2bR and A3R. Cross-reactivity to cynomolgus monkey PBMCs was also observed. AT-004 inhibited NECA-mediated A2aR activation with a potency over 100-fold higher than SMIs e.g. AZD4635. AT-004 significantly reversed the ability of adenosine to suppress CD8 T cell activation as indicated by restoration of cell surface marker expression and increased levels of cytokines such as IFN-γ in the supernatants. Conclusions: AT-004 is a potent, selective and highly potent antagonist of A2aR receptor. Considering its potential safety advantage as an mAb comparing to most known A2aR SMI antagonists in that it does not cross the blood brain barrier, AT-004 maybe a promising novel therapeutic candidate for immunotherapy, vital for enhancing anti-tumor responses in solid tumors that show an incomplete response or resistance to anti-PD-1 or anti-PD-L1 mAb treatment due to elevated adenosine level in tumor microenvironment (TME). Citation Format: Changyun Hu, Jiandong Zhang, Shuying Liu, Xinyan Zhao. Characterization of a potent and selective A2aR monoclonal antibody antagonist for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3437.

Aquaporin 2 regulation: implications for water balance and polycystic kidney diseases.

Targeting the collecting duct water channel aquaporin 2 (AQP2) to the plasma membrane is essential for the maintenance of mammalian water homeostasis. The vasopressin V2 receptor (V2R), which is a GS protein-coupled receptor that increases intracellular cAMP levels, has a major role in this targeting process. Although a rise in cAMP levels and activation of protein kinase A are involved in facilitating the actions of V2R, studies in knockout mice and cell models have suggested that cAMP signalling pathways are not an absolute requirement for V2R-mediated AQP2 trafficking to the plasma membrane. In addition, although AQP2 phosphorylation is a known prerequisite for V2R-mediated plasma membrane targeting, none of the known AQP2 phosphorylation events appears to be rate-limiting in this process, which suggests the involvement of other factors; cytoskeletal remodelling has also been implicated. Notably, several regulatory processes and signalling pathways involved in AQP2 trafficking also have a role in the pathophysiology of autosomal dominant polycystic kidney disease, although the role of AQP2 in cyst progression is unknown. Here, we highlight advances in the field of AQP2 regulation that might be exploited for the treatment of water balance disorders and provide a rationale for targeting these pathways in autosomal dominant polycystic kidney disease.

GPR3 sets brown fat on fire

Activation of thermogenic adipocytes, a process canonically driven by norepinephrine through β-adrenergic receptors, presents an appealing therapeutic approach to combat obesity. Recent work by Sveidahl Johansen et al., 2021 published in Cell has identified a noncanonical mechanism of brown adipocyte activation, in which lipolysis transcriptionally drives the constitutive activation of the Gs protein-coupled receptor, GPR3, to induce thermogenesis.

Cyclic AMP‐dependent Activation of ERK Via GLP‐1 Receptor Signaling Requires the Neuroendocrine Cell‐Selective Guanine Nucleotide Exchanger NCS‐RapGEF2

G-protein coupled receptors activate various intracellular processes upon ligand engagement depending on coupling to adenylyl cyclase (Gs, Gi/o), phospholipase C (Gq), or RhoGEFs (G12/13). Cyclic AMP activation of the Rap-Braf-MEK-ERK pathway after signaling initiated by the neuropeptide PACAP, via the Gs-protein coupled receptor (GsPCR) PAC1, occurs uniquely through the neuritogenic cAMP sensor Rap guanine nucleotide exchange factor 2 (NCS-RapGEF2) in NS-1 neuroendocrine cells. We examined the expression of other Family B GsPCRs in this cell line and assessed cAMP elevation and neuritogenesis after treatment with their cognate peptide ligands. Exposure of NS-1 cells to the VIPR1/2 agonist VIP, or the GLP1R agonist exendin-4, did not induce neuritogenesis, or elevation of cAMP, presumably due to insufficient receptor protein expression. VIP and exendin-4 did induce neuritogenesis after transduction of human VIPR1, VIPR2 and GLP1R into NS-1 cells. Exendin-4/GLP1R-stimulated neuritogenesis was MEK-ERK- dependent (blocked by U0126), indicating its use of the cAMP->RapGEF2->ERK neuritogenic signaling pathway previously identified for PACAP/PAC1 signaling in NS-1 cells. NCS- RapGEF2 is expressed in the beta cell tumor-derived cell lines MIN6 and INS-1, and in human pancreatic islets. As in NS-1 cells, exendin-4 caused ERK phosphorylation in INS-1 cells. Reduction in RapGEF2 expression after its specific shRNA treatment reduced exendin-4-induced ERK phosphorylation. Transcriptome analysis of INS-1 cells after one hour exposure to exendin- 4 revealed an immediate early-gene response that was composed of both ERK-dependent and ERK-independent signaling targets. ERK-dependent targets, and presumably involving NCS-RapGEF2 signaling, included Nr4a2, Btg1, Skil, Plkl2, Per1 and Dyrk3, while ERK-independent targets and presumably involving PKA or Epac signaling, included Nr4a1, Nr4a3, Crem, Fosl2, Sik1 and Rgs2. We propose that cAMP signaling initiated by GLP-1 in pancreatic beta cells causes parallel activation of multiple cAMP effectors, including NCS- RapGEF2, Epac, and protein kinase A, to separately control various facets of GLP-1 action, including insulin secretion and transcriptional modulation. Identification of coupling of GsPCRs to ERK activation via NCS-RapGEF2 provides a novel signaling mode for GLP-1 in pancreatic beta cells and potentially brain and elsewhere. RapGEF2-dependent ERK activation is likely to be a feature of additional nervous and endocrine targets of family B receptor ligands including PACAP, VIP, CRH, GIP, secretin, glucagon, PTH, calcitonin, and GHRH. Whether or not ligands for these receptors, including GLP-1, might activate the NCS-RapGEF2, PKA, or Epac cAMP-dependent pathways in a biased fashion, with differential physiological effects, has yet to be investigated. This abstract is dedicated to the memory of Andrew C. Emery.

Mechanisms of severe acute intermittent hypoxia-induced phrenic long-term facilitation.

Moderate acute intermittent hypoxia (mAIH; 35-55 mmHg PaO2) elicits phrenic long-term facilitation (pLTF) by a mechanism that requires activation of Gq protein-coupled serotonin type 2 receptors, MEK/ERK MAP kinase, and NADPH oxidase activity and is constrained by cAMP-PKA signaling. In contrast, severe AIH (sAIH; 25-35 mmHg PaO2) elicits Gs protein-coupled adenosine type 2 A receptor-dependent pLTF. Another Gs protein-coupled receptor, serotonin 7 receptors, elicits phrenic motor facilitation (pMF) by a mechanism that requires exchange protein activated by cyclic AMP (EPAC) and phosphatidylinositol 3-kinase/Akt (PI3K/Akt) activation and is constrained by NADPH oxidase activity. Here, we tested the hypothesis that the same downstream signaling mechanisms giving rise to serotonin 7 (vs. serotonin 2) receptor-induced pMF underlie sAIH-induced pLTF. In anesthetized rats, sAIH-induced pLTF was compared after pretreatment with intrathecal (C4) injections of inhibitors for: 1) EPAC (ESI-05); 2) MEK/ERK (UO126); 3) PKA (KT-5720); 4) PI3K/Akt (PI828); and 5) NADPH oxidase (apocynin). In partial agreement with our hypothesis, sAIH-induced pLTF was abolished by ESI-05 and PI828 and marginally enhanced by apocynin but, surprisingly, was abolished by UO126 and attenuated by KT-5720. Mechanisms of sAIH-induced pLTF reflect elements of both Gq and Gs pathways to pMF, likely as a consequence of the complex, cross-talk interactions between them.NEW & NOTEWORTHY Distinct mechanisms give rise to pLTF induced by moderate and severe AIH. We demonstrate that, unlike moderate AIH, severe AIH-induced pLTF requires EPAC and PI3K/Akt and is marginally constrained by NADPH oxidase activity. Surprisingly, sAIH-induced pLTF requires MEK/ERK activity similar to moderate AIH-induced pLTF and is reduced by PKA inhibition. We suggest sAIH-induced pLTF arises from complex interactions between dominant mechanisms characteristic of moderate versus severe AIH-induced pLTF.

MiR-18a promotes glioblastoma development by down-regulating ALOXE3-mediated ferroptotic and anti-migration activities

The development of glioblastoma (GBM) is typically accompanied by marked changes in lipid metabolism. Oxylipins and their catalyzed enzymes lipoxygenases (LOXs) have been shown to participate in the development of cancers via multiple pathways, while the understanding of LOXs in GBM remains enigmatic. Thus, we aimed to explore the expression and functional roles of LOXs in the development of GBM. Here we showed that ALOXE3 was markedly down-regulated in human GBM. Knockdown of ALOXE3 in GBM cells fostered the orthotopic tumor growth and shortened lifespan in mice. ALOXE3 deficiency rendered GBM cells resistant to p53-SLC7A11 dependent ferroptosis, promoting GBM cell survival. Mechanistically, miR-18a directly targeted ALOXE3 and suppressed its expression and functions in GBM cells. Furthermore, ALOXE3 silencing promoted 12-hydroxyeicosatetraenoic acids (12-HETE) secretion from GBM cells, in turn, 12-HETE enhanced migration of GBM cells by activating Gs-protein-coupled receptor (GsPCR)-PI3K-Akt pathway in an autocrine manner. Altogether, miR-18a/ALOXE3 axis exerts tumor promoting functions by regulating ferroptosis and migration of GBM cells. Targeting miR-18a/ALOXE3 axis may provide novel therapeutic approaches for GBM treatment.

A Cellular Assay for the Identification and Characterization of Connexin Gap Junction Modulators.

Connexin gap junctions (Cx GJs) enable the passage of small molecules and ions between cells and are therefore important for cell-to-cell communication. Their dysfunction is associated with diseases, and small molecules acting as modulators of GJs may therefore be useful as therapeutic drugs. To identify GJ modulators, suitable assays are needed that allow compound screening. In the present study, we established a novel assay utilizing HeLa cells recombinantly expressing Cx43. Donor cells additionally expressing the Gs protein-coupled adenosine A2A receptor, and biosensor cells expressing a cAMP-sensitive GloSensor luciferase were established. Adenosine A2A receptor activation in the donor cells using a selective agonist results in intracellular cAMP production. The negatively charged cAMP migrates via the Cx43 gap junctions to the biosensor cells and can there be measured by the cAMP-dependent luminescence signal. Cx43 GJ modulators can be expected to impact the transfer of cAMP from the donor to the biosensor cells, since cAMP transit is only possible via GJs. The new assay was validated by testing the standard GJ inhibitor carbenoxolon, which showed a concentration-dependent inhibition of the signal and an IC50 value that was consistent with previously reported values. The assay was demonstrated to be suitable for high-throughput screening.

Retraction: AKAP12 mediates PKA-induced phosphorylation of ATR to enhance nucleotide excision repair.

Loss-of-function in melanocortin 1 receptor (MC1R), a GS protein-coupled receptor that regulates signal transduction through cAMP and protein kinase A (PKA) in melanocytes, is a major inherited melanoma risk factor. Herein, we report a novel cAMP-mediated response for sensing and responding to UV-induced DNA damage regulated by A-kinase-anchoring protein 12 (AKAP12). AKAP12 is identified as a necessary participant in PKA-mediated phosphorylation of ataxia telangiectasia mutated and Rad3-related (ATR) at S435, a post-translational event required for cAMP-enhanced nucleotide excision repair (NER). Moreover, UV exposure promotes ATR-directed phosphorylation of AKAP12 at S732, which promotes nuclear translocation of AKAP12-ATR-pS435. This complex subsequently recruits XPA to UV DNA damage and enhances 5' strand incision. Preventing AKAP12's interaction with PKA or with ATR abrogates ATR-pS435 accumulation, delays recruitment of XPA to UV-damaged DNA, impairs NER and increases UV-induced mutagenesis. Our results define a critical role for AKAP12 as an UV-inducible scaffold for PKA-mediated ATR phosphorylation, and identify a repair complex consisting of AKAP12-ATR-pS435-XPA at photodamage, which is essential for cAMP-enhanced NER.

PKC- and PKA-dependent phosphorylation modulates TREK-1 function in naïve and neuropathic rats.

PKC and PKA phosphorylation inhibit TREK-1 channels downstream of Gs protein-coupled receptor activation in vitro. However, the role of phosphorylation of TREK-1 in neuropathic pain is unknown. The purpose of this study was to investigate whether altered TREK-1 channel function by PKA and PKC modulators contributes to antiallodynia in neuropathic rats. Furthermore, we investigated if the in vitro described sites for PKC and PKA phosphorylation (S300 and S333, respectively) participate in the modulation of TREK-1 in naïve and neuropathic rats. L5/L6 spinal nerve ligation (SNL) induced tactile allodynia. Intrathecal injection of BL-1249 (TREK-1 activator) reversed nerve injury-induced tactile allodynia, whereas spadin (TREK-1 blocker) produced tactile allodynia in naïve rats and reversed the antiallodynic effect induced by BL-1249 in neuropathic rats. Intrathecal administration of rottlerin or Rp-cAMPs (PKC and PKA inhibitors, respectively) enhanced the antiallodynia observed with BL-1249 in neuropathic rats. In contrast, pretreatment with PdBu or forskolin (PKC and PKA activators, respectively) reduced the BL-1249-induced antiallodynia. Intrathecal injection of two high-activity TREK-1 recombinant channels, using a in vivo transfection method with lipofectamine, with mutations at PKC/PKA phosphosites (S300A and S333A) reversed tactile allodynia in neuropathic rats, with no effect in naïve rats. In contrast, transfection of two low-activity TREK-1 recombinant channels with phosphomimetic mutations at those sites (S300D and S333D) produced tactile allodynia in naïve rats and interfered with antiallodynic effects of rottlerin/BL-1249 or Rp-cAMPs/BL-1249. Data suggest that TREK-1 channel activity can be dynamically tuned in vivo by PKC/PKA to provoke allodynia and modulate its antiallodynic role in neuropathic pain.

Aldosterone And Osteopontin Promote Cardiac Fibrosis Via GRK2‐dependent β2‐Adrenergic Receptor Dysfunction

BackgroundCardiac β2‐adrenergic receptors (ARs) are known to inhibit collagen production and fibrosis in cardiac fibroblasts and myocytes. β2AR is a Gs protein‐coupled receptor (GPCR) and, upon its activation, stimulates generation of cyclic 3′, 5′‐adenosine monophosphate (cAMP). cAMP has two effectors: protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac1/2). Epac1 has been shown to inhibit cardiac fibroblast activation and fibrosis. Osteopontin (OPN) is a ubiquitous pro‐inflammatory and pro‐fibrotic cytokine, including in the heart. OPN expression is known to be induced by the cardiotoxic hormone aldosterone in various tissues and OPN has been reported to dysregulate β2AR signaling and function in bone cells. Thus, we hypothesized here that aldosterone upregulates OPN in the heart in order to perturb β2AR anti‐fibrotic function.MethodsWe used the rat cardiomyoblast cell line H9c2 and human cardiac fibroblasts (HCFs). We measured OPN levels and fibrosis markers via real time PCR and β2AR function via cAMP accumulation studies and co‐immunoprecipitation/western blotting for several downstream signaling mediators. We also deleted OPN via CRISPR/Cas9 to examine its role in cardiac β2AR function and fibrosis.ResultsAldosterone upregulates OPN via the mineralocorticoid receptor (MR) in H9c2 cardiomyocytes. This is prevented by β2AR activation with salbutamol (albuterol), which stimulates GPCR‐kinase (GRK)‐5 to phosphorylate the cardiac MR and inhibit its transcriptional activity. Importantly, CRISPR/Cas9‐mediated OPN gene deletion enhances β2AR‐dependent cAMP generation in H9c2 cardiomyocytes and upregulates Epac1 protein levels. The functional result of this is that β2AR’s inhibition of transforming growth factor (TGF)‐β‐dependent fibrosis in H9c2 cardiomyocytes is robustly augmented. In addition, pharmacological inhibition of GRK2, the major GRK that phosphorylates and desensitizes the cardiac β2AR, with Cmpd101 or OPN CRIPSR/Cas9‐mediated knockout completely abrogates isoproterenol (agonist)‐induced β2AR functional desensitization in HCFs (Isoproterenol’s ΔEC50: 94±16 μM in control HCFs, p<0.05, n=4; 6±7.2 μM in Cmpd101‐treated HCFs, Not significant at p=0.05, n=4; 5.3+6.1 μM in OPN knockout HCFs, Not significant at p=0.05, n=4). Mechanistically, OPN dysregulates the β2AR via enhancement of GRK2‐mediated functional desensitization, since OPN forms a multi‐protein complex with GRK2 and GRK2‐interacting protein (GIT)‐1 in HCFs, thereby facilitating GRK2 plasma membrane recruitment. This reduces β2AR’s anti‐fibrotic cAMP signaling in the heart.ConclusionsWe have uncovered a direct inhibitory effect of OPN on cardiac β2AR’s anti‐fibrotic signaling: facilitation of GRK2‐mediated receptor desensitization. Since aldosterone/MR upregulates OPN, a combination of an MR blocker with OPN or GRK2 blockade could be of value in cardiac fibrosis treatment.Support or Funding Information1) American Heart Association (A.L.)2) NSU President’s Faculty Research & Development Grant (A.L.)

Abstract 012: Regulation of Cardiomyocyte Fibrosis by a Negative Feedback Loop Between Aldosterone-Induced Osteopontin Up-Regulation and beta 2 -Adrenergic Receptor Signaling

Cardiac β 2 -adrenergic receptors (ARs) are known to inhibit collagen production and fibrosis in cardiac fibroblasts and myocytes. β 2 AR is a Gs protein-coupled receptor (GPCR) and, upon its activation, stimulates generation of cyclic 3`, 5`-adenosine monophosphate (cAMP). cAMP has two effectors: protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac). Epac1 has been shown to inhibit cardiac fibroblast activation and fibrosis. Osteopontin (OPN) is a ubiquitous pro-inflammatory and pro-fibrotic cytokine, including in the heart. We report here that the cardiotoxic hormone aldosterone upregulates OPN via the mineralocorticoid receptor (MR) in H9c2 cardiomyoblasts. This is prevented by β 2 AR-activated GPCR-kinase (GRK)-5. GRK5 directly phosphorylates and inhibits the MR in cardiomyocytes. Additionally, CRISPR-mediated OPN deletion enhances β 2 AR-dependent cAMP generation in H9c2 cardiomyocytes and upregulates Epac1. OPN deletion also enables the β 2 AR to completely abrogate transforming growth factor (TGF)-β-dependent fibrosis in H9c2 cardiomyocytes. Mechanistically, OPN interacts with G α s subunits in H9c2 cardiomyocytes to facilitate recruitment of GRK2, the major GRK phosphorylating and desensitizing the cardiac β 2 AR. This, in turn, augments the GRK2-dependent functional desensitization of the β 2 AR, thereby opposing this receptor`s anti-fibrotic cAMP/Epac1 signaling. In conclusion, we have uncovered a direct inhibitory effect of OPN in cardiac β 2 AR`s anti-fibrotic signaling via facilitation of GRK2-mediated receptor desensitization. Thus, OPN blockade could be of value in the treatment and/or prevention of cardiac fibrosis.

Deletion of Osteopontin Enhances β₂-Adrenergic Receptor-Dependent Anti-Fibrotic Signaling in Cardiomyocytes.

Cardiac β2-adrenergic receptors (ARs) are known to inhibit collagen production and fibrosis in cardiac fibroblasts and myocytes. The β2AR is a Gs protein-coupled receptor (GPCR) and, upon its activation, stimulates the generation of cyclic 3′,5′-adenosine monophosphate (cAMP). cAMP has two effectors: protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac). Epac1 has been shown to inhibit cardiac fibroblast activation and fibrosis. Osteopontin (OPN) is a ubiquitous pro-inflammatory cytokine, which also mediates fibrosis in several tissues, including the heart. OPN underlies several cardiovascular pathologies, including atherosclerosis and cardiac adverse remodeling. We found that the cardiotoxic hormone aldosterone transcriptionally upregulates OPN in H9c2 rat cardiac myoblasts—an effect prevented by endogenous β2AR activation. Additionally, CRISPR-mediated OPN deletion enhanced cAMP generation in response to both β1AR and β2AR activation in H9c2 cardiomyocytes, leading to the upregulation of Epac1 protein levels. These effects rendered β2AR stimulation capable of completely abrogating transforming growth factor (TGF)-β-dependent fibrosis in OPN-lacking H9c2 cardiomyocytes. Finally, OPN interacted constitutively with Gαs subunits in H9c2 cardiac cells. Thus, we uncovered a direct inhibitory role of OPN in cardiac β2AR anti-fibrotic signaling via cAMP/Epac1. OPN blockade could be of value in the treatment and/or prevention of cardiac fibrosis.

Iloprost, a prostacyclin analog, inhibits the invasion of ovarian cancer cells by downregulating matrix metallopeptidase-2 (MMP-2) through the IP-dependent pathway

Recent studies have shown that a bioactive lipid prostacyclin (PGI2) plays a role in various cancers, including lung cancer. However, the specific function of PGI2 in ovarian cancer progression has not been determined. This study investigated the effects of PGI2 on cell growth, migration, and invasion in ovarian cancer cells using iloprost, a stable PGI2 analog. Iloprost significantly inhibited migration and invasion, but not cell growth, in a dose-dependent manner in human ovarian cancer cells (A2780 and SKOV3). Interestingly, the cell surface Gs protein-coupled PGI2 receptor IP was enhanced in human ovarian cancer cells. The inhibitory effect of iloprost on migration and invasion was entirely reversed by an IP antagonist (CAY10449) and IP siRNA, whereas the knockdown of peroxisome proliferator-activated receptor δ (PPARδ), a nuclear receptor of PGI2, did not rescue the effect of iloprost. Additionally, iloprost markedly decreased the expression of matrix metallopeptidase-2 and -9 (MMP-2 and MMP-9), which may be induced in the process of ovarian cancer metastasis. IP siRNA inhibited iloprost-reduced MMP-2 expression but not MMP-9 expression. Moreover, inhibition of protein kinase A (PKA) and overexpression of Akt and p38 rescued the inhibition of invasion and the reduction of MMP-2 expression by iloprost. Furthermore, iloprost-induced activation of PKA was associated with PKA-mediated Akt and p38 inactivation in ovarian cancer cells. Taken together, these results demonstrate that iloprost inhibits ovarian cancer cell invasion by downregulating MMP-2 expression via the IP-mediated PKA pathway. This study is the first to reveal a novel role for iloprost and to clarify its underlying mechanism in human ovarian cancer cells.

Human melanocortin 1 receptor-mediated ubiquitination of nonvisual arrestins. Role of Mahogunin Ring Finger 1 E3 ligase

Signaling from the melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor (GPCR) crucial for melanocyte proliferation and differentiation, is regulated by cytosolic β-arrestins (ARRBs). MC1R signaling is also negatively modulated by the E3-ubiquitin ligase Mahogunin Ring Finger-1 (MGRN1), whose mutation causes hyperpigmentation, congenital heart defects and neurodegeneration in mice. We showed previously that although MC1R interacts stably with human ARRB1 or ARRB2, only ARRB2 mediates receptor desensitization and internalization. We analyzed MC1R-dependent ARRB ubiquitination, and the possible role of MGRN1. ARRB1 expressed in heterologous cells or human melanoma cells migrated in SDS-PAGE as a 55kDa protein whereas ARRB2 migrated as two major bands of apparent molecular weight near 45 and 55kDa, with an intermediate mobility band occasionally detected. These forms were related by post-translational modification rather than by proteolysis. Presence of MC1R favored expression of the 45kDa protein, the form that interacted preferentially with MC1R. MC1R also mediated poly- or multimonoubiquitination of ARRB2. Ubiquitination was agonist-independent, but required a native MC1R conformation and/or normal receptor trafficking to the plasma membrane, as it was not observed for loss-of-function MC1R variants. In a heterologous expression system, MC1R-dependent ARRB ubiquitination was enhanced by overexpression of MGRN1 and was impaired by siRNA-mediated MGRN1 knockdown thus pointing to MGRN1 as the responsible E3-ligase. Co-immunoprecipitation experiments demonstrated interaction of MGRN1 and ARRBs in the presence of MC1R, suggesting a scaffolding role for the GPCR that may determine the selectivity of E3-ubiquitin ligase engagement and the functional outcome of ARRB ubiquitination.

Cannabidiol, a novel inverse agonist for GPR12

GPR12 is a constitutively active, Gs protein-coupled receptor that currently has no confirmed endogenous ligands. GPR12 may be involved in physiological processes such as maintenance of oocyte meiotic arrest and brain development, as well as pathological conditions such as metastatic cancer. In this study, the potential effects of various classes of cannabinoids on GPR12 were tested using a cAMP accumulation assay. Our data demonstrate that cannabidiol (CBD), a major non-psychoactive phytocannabinoid, acted as an inverse agonist to inhibit cAMP accumulation stimulated by the constitutively active GPR12. Thus, GPR12 is a novel molecular target for CBD. The structure-activity relationship studies of CBD indicate that both the free hydroxyl and the pentyl side chain are crucial for the effects of CBD on GPR12. Furthermore, studies using cholera toxin, which blocks Gs protein and pertussis toxin, which blocks Gi protein, revealed that Gs, but not Gi is involved in the inverse agonism of CBD on GPR12. CBD is a promising novel therapeutic agent for cancer, and GPR12 has been shown to alter viscoelasticity of metastatic cancer cells. Since we have demonstrated that CBD is an inverse agonist for GPR12, this provides novel mechanism of action for CBD, and an initial chemical scaffold upon which highly potent and efficacious agents acting on GPR12 may be developed with the ultimate goal of blocking cancer metastasis.

The GS Protein-coupled A2a Adenosine Receptor Controls T Cell Help in the Germinal Center

T follicular helper (TFH) cells have been shown to be critically required for the germinal center (GC) reaction where B cells undergo class switch recombination and clonal selection to generate high affinity neutralizing antibodies. However, detailed knowledge of the physiological cues within the GC microenvironment that regulate T cell help is limited. The cAMP-elevating, Gs protein-coupled A2a adenosine receptor (A2aR) is an evolutionarily conserved receptor that limits and redirects cellular immunity. However, the role of A2aR in humoral immunity and B cell differentiation is unknown. We hypothesized that the hypoxic microenvironment within the GC facilitates an extracellular adenosine-rich milieu, which serves to limit TFH frequency and function, and also promotes immunosuppressive T follicular regulatory cells (TFR). In support of this hypothesis, we found that following immunization, mice lacking A2aR (A2aRKO) exhibited a significant expansion of T follicular cells, as well as increases in TFH to TFR ratio, GC T cell frequency, GC B cell frequency, and class switching of GC B cells to IgG1. Transfer of CD4 T cells from A2aRKO or wild type donors into T cell-deficient hosts revealed that these increases were largely T cell-intrinsic. Finally, injection of A2aR agonist, CGS21680, following immunization suppressed T follicular differentiation, GC B cell frequency, and class switching of GC B cells to IgG1. Taken together, these observations point to a previously unappreciated role of GS protein-coupled A2aR in regulating humoral immunity, which may be pharmacologically targeted during vaccination or pathological states in which GC-derived autoantibodies contribute to the pathology.