Search Results (37)

Osteoclastogenesis—the activation and differentiation of osteoclasts—is one of the pivotal processes of bone remodeling and is regulated by RANKL/RANK signaling, the decoy function of osteoprotegerin (OPG), and a cascade of pro- and anti-inflammatory cytokines. The disruption of this balance leads to pathological bone loss in diseases such as osteoporosis and rheumatoid arthritis. FFAR4 (Free Fatty Acid Receptor 4), a G protein-coupled receptor for long-chain omega-3 fatty acids, has been confirmed as a key mediator of metabolic and anti-inflammatory effects. This review focuses on how FFAR4 acts as the selective receptor for the omega-3 fatty acid docosahexaenoic acid (DHA). It activates two divergent signaling pathways. The Gαq-dependent cascade facilitates intracellular calcium mobilization and ERK1/2 activation. Meanwhile, β-arrestin-2 recruitment inhibits NF-κB. These collective actions reshape the cytokine environment. In macrophages, DHA–FFAR4 signaling lowers the levels of TNF-α, interleukin-6 (IL-6), and IL-1β while increasing IL-10 secretion. Consequently, the activation of NFATc1 and NF-κB p65 is profoundly suppressed under TNF-α or RANKL stimulation. Additionally, DHA modulates the RANKL/OPG axis in osteoblastic cells by suppressing RANKL expression, thereby reducing osteoclast differentiation in an inflammatory mouse model. Full article

Cardiac remodeling, a critical process that can lead to heart failure, is primarily characterized by cardiac hypertrophy. Studies have shown that transgenic mice with Gαq receptor blockade exhibit reduced hypertrophy under induced pressure overload. GQ262, a novel Gαq/11 inhibitor, has demonstrated good biocompatibility and specific inhibitory effects on Gαq/11 compared to other inhibitors. However, its role in cardiac remodeling remains unclear. This study aims to explore the anti-cardiac remodeling effects and mechanisms of GQ262 both in vitro and in vivo, providing data and theoretical support for its potential use in treating cardiac remodeling diseases. Cardiac hypertrophy was induced in mice via transverse aortic constriction (TAC) for 4 weeks and in H9C2 cells through phenylephrine (PE) induction, confirmed with WGA and H&E staining. We found that GQ262 improved cardiac function, inhibited the protein and mRNA expression of hypertrophy markers, and reduced the levels of apoptosis and fibrosis. Furthermore, GQ262 inhibited the Akt/mTOR signaling pathway activation induced by TAC or PE, with its therapeutic effects disappearing upon the addition of the Akt inhibitor ARQ092. These findings reveal that GQ262 inhibits cardiomyocyte hypertrophy and apoptosis through the Akt/mTOR signaling pathway, thereby reducing fibrosis levels and mitigating cardiac remodeling. Full article

Serotonin or 5-hydroxytryptamine (5-HT) is a monoamine that plays a critical role in insulin secretion, energy metabolism, and mitochondrial biogenesis. However, the action of serotonin in insulin production and secretion by pancreatic β cells has not yet been elucidated. Here, we investigated how exogenous nanomolar serotonin concentrations regulate insulin synthesis and secretion in rat insulinoma INS-1E cells. Nanomolar serotonin concentrations (10 and 50 nM) significantly increased insulin protein expression above the constant levels in untreated control cells and decreased insulin protein levels in the media. The reductions in insulin protein levels in the media may be associated with ubiquitin-mediated protein degradation. The levels of membrane vesicle trafficking-related proteins including Rab5, Rab3A, syntaxin6, clathrin, and EEA1 proteins were significantly decreased by serotonin treatment compared to the untreated control cells, whereas the expressions of Rab27A, GOPC, and p-caveolin-1 proteins were significantly reduced by serotonin treatment. In this condition, serotonin receptors, Gαq-coupled 5-HT2b receptor (Htr2b), and ligand-gated ion channel receptor Htr3a were significantly decreased by serotonin treatment. To confirm the serotonylation of Rab3A and Rab27A during insulin secretion, we investigated the protein levels of Rab3A and Rab27A, in which transglutaminase 2 (TGase2) serotonylated Rab3A but not Rab27A. The increases in ERK phosphorylation levels were consistent with increases in the expression of p-Akt. Also, the expression level of the Bcl-2 protein was significantly increased by 50 and 100 nM serotonin treatment compared to the untreated control cells, whereas the levels of Cu/Zn-SOD and Mn-SOD proteins decreased. These results indicate that nanomolar serotonin treatment regulates the insulin protein level but decreases this level in media through membrane vesicle trafficking-related proteins (Rab5, Rab3A, syntaxin6, clathrin, and EEA1), the Akt/ERK pathway, and Htr2b/Htr3a in INS-1E cells. Full article

Transient receptor potential melastatin-8 (TRPM8) is a cation channel that is activated by cold and “cooling agents” such as menthol and icilin, which induce a cold sensation. The stimulation of TRPM8 activates an intracellular signaling cascade that ultimately leads to a change in the gene expression pattern of the cells. Here, we investigate the TRPM8-induced signaling pathway that links TRPM8 channel activation to gene transcription. Using a pharmacological approach, we show that the inhibition of phosphatidylinositol 4-phosphate 5 kinase α (PIP5K), an enzyme essential for the biosynthesis of phosphatidylinositol 4,5-bisphosphate, attenuates TRPM8-induced gene transcription. Analyzing the link between TRPM8 and Gq proteins, we show that the pharmacological inhibition of the βγ subunits impairs TRPM8 signaling. In addition, genetic studies show that TRPM8 requires an activated Gα subunit for signaling. In the nucleus, the TRPM8-induced signaling cascade triggers the activation of the transcription factor AP-1, a complex consisting of a dimer of basic region leucine zipper (bZIP) transcription factors. Here, we identify the bZIP protein c-Jun as an essential component of AP-1 within the TRPM8-induced signaling cascade. In summary, with PIP5K, Gq subunits, and c-Jun, we identified key molecules in TRPM8-induced signaling from the plasma membrane to the nucleus. Full article

Blue light is the higher-energy region of the visible spectrum. Excessive exposure to blue light is known to induce oxidative stress and is harmful to the eyes. The stems of Dendrobium nobile Lindl. (Orchidaceae), named Jinchaishihu, have long been used in traditional Chinese medicine (TCM) for nourishing yin, clearing heat, and brightening the eyes. The polysaccharide is one of the major components in D. nobile. However, the effect on ocular cells remains unclear. This study aimed to investigate whether the polysaccharide from D. nobile can protect the eyes from blue light-induced injury. A crude (DN-P) and a partially purified polysaccharide (DN-PP) from D. nobile were evaluated for their protective effects on blue light-induced damage in ARPE-19 and 661W cells. The in vivo study investigated the electroretinographic response and the expression of phototransduction-related genes in the retinas of a Drosophila model. The results showed that DN-P and DN-PP could improve blue light-induced damage in ARPE-19 and 661W cells, including cell viability, antioxidant activity, reactive oxygen species (ROS)/superoxide production, and reverse opsin 3 protein expression in a concentration-dependent manner. The in vivo study indicated that DN-P could alleviate eye damage and reverse the expression of phototransduction-related genes, including ninaE, norpA, Gαq, Gβ76C, Gγ30A, TRP, and TRPL, in a dose-dependent manner in blue light-exposed Drosophila. In conclusion, this is the first report demonstrating that D. nobile polysaccharide pretreatment can protect retinal cells and retinal photoreceptors from blue light-induced damage. These results provide supporting evidence for the beneficial potential of D. nobile in preventing blue light-induced eye damage and improving eyesight. Full article

The glycoprotein hormones LH, FSH, TSH and chorionic gonadotropin consist of a common α-subunit and a hormone-specific β-subunit. The α-subunit is expressed in the pituitary and the placental cells, and its expression is regulated by extracellular signal molecules. Much is known about the regulation of the α-subunit gene in the pituitary, but few studies have addressed the regulation of this gene in trophoblasts. The aim of this study was to characterize the molecular mechanism of stimulus-induced α-subunit gene transcription in JEG-3 cells, a cellular model for human trophoblasts, using chromatin-embedded reporter genes under the control of the α-subunit promoter. The results show that increasing the concentration of the second messengers cAMP or Ca²⁺, or expressing the catalytic subunit of cAMP-dependent protein kinase in the nucleus activated the α-subunit promoter. Similarly, the stimulation of p38 protein kinase activated the α-subunit promoter, linking α-subunit expression to stress response. The stimulation of a Gαq-coupled designer receptor activated the α-subunit promoter, involving the transcription factor CREB, linking α-subunit expression to hormonal stimulation and an increase in intracellular Ca²⁺. Deletion mutagenesis underscores the importance of a tandem cAMP response element within the glycoprotein hormone α-subunit promoter, which acts as a point of convergence for a multiple signaling pathway. Full article

Melanocortin receptor accessory protein 2 (MRAP2) is a membrane protein that binds multiple G protein-coupled receptors (GPCRs) involved in the control of energy homeostasis, including prokineticin receptors. These GPCRs are expressed both centrally and peripherally, and their endogenous ligands are prokineticin 1 (PK1) and prokineticin 2 (PK2). PKRs couple all G-protein subtypes, such as Gαq/11, Gαs, and Gαi, and recruit β-arrestins upon PK2 stimulation, although the interaction between PKR2 and β-arrestins does not trigger receptor internalisation. MRAP2 inhibits the anorexigenic effect of PK2 by binding PKR1 and PKR2. The aim of this work was to elucidate the role of MRAP2 in modulating PKR2-induced β-arrestin-2 recruitment and β-arrestin-mediated signalling. This study could allow the identification of new specific targets for potential new drugs useful for the treatment of the various pathologies correlated with prokineticin, in particular, obesity. Full article

In this study, we reported that novel single-chain fusion proteins linking thromboxane A₂ (TXA₂) receptor (TP) to a selected G-protein α-subunit q (SC-TP-Gαq) or to α-subunit s (SC-TP-Gαs) could be stably expressed in megakaryocytes (MKs). We tested the MK-released platelet-linked particles (PLPs) to be used as a vehicle to deliver the overexpressed SC-TP-Gαq or the SC-TP-Gαs to regulate human platelet function. To understand how the single-chain TP-Gα fusion proteins could regulate opposite platelet activities by an identical ligand TXA₂, we tested their dual functions—binding to ligands and directly linking to different signaling pathways within a single polypeptide chain—using a 3D structural model. The immature MKs were cultured and transfected with cDNAs constructed from structural models of the individual SC-TP-Gαq and SC-TP-Gαs, respectively. After transient expression was identified, the immature MKs stably expressing SC-TP-Gαq or SC-TP-Gαs (stable cell lines) were selected. The stable cell lines were induced into mature MKs which released PLPs. Western blot analysis confirmed that the released PLPs were carrying the recombinant SC-TP-Gαq or SC-TP-Gαs. Flow cytometry analysis showed that the PLPs carrying SC-TP-Gαq were able to perform the activity by promoting platelet aggregation. In contrast, PLPs carrying SC-TP-Gαs reversed Gq to Gs signaling to inhibit platelet aggregation. This is the first time demonstrating that SC-TP-Gαq and SC-TP-Gαs were successfully overexpressed in MK cells and released as PLPs with proper folding and programmed biological activities. This bio-engineering led to the formation of two sets of biologically active PLP forms mediating calcium and cAMP signaling, respectively. As a result, these PLPs are able to bind to identical endogenous TXA₂ with opposite activities, inhibiting and promoting platelet aggregation as reprogrammed for therapeutic process. Results also demonstrated that the nucleus-free PLPs could be used to deliver recombinant membrane-bound GPCRs to regulate cellular activity in general. Full article

Despite G protein-coupled receptors (GPCRs) being important theapeutic targets, the signaling properties of many GPCRs remain poorly characterized. GPCR activation primarily initiates heterotrimeric G protein signaling. To detect ligand-induced G protein activation, Bioluminescence Resonance Energy Transfer (BRET)-based biosensors were previously developed. Here, we designed a novel set of Nanoluciferase (NLuc) BRET-based biosensors (REGA-SIGN) that covers all Gα protein families (i.e., Gα_i/o, Gα_Ss/L, Gα_12/13 and Gα_q/15). REGA-SIGN uses NLuc as a bioluminescent donor and LSS-mKATE2, a red-shifted fluorophore, as an acceptor. Due to the enhanced spectral separation between donor and acceptor emission and the availability of a stable substrate for NLuc, this donor–acceptor pair enables sensitive kinetic assessment of G protein activity. After optimization, the NLuc integration sites into the Gα subunit largely corresponded with previously reported integration sites, except for Gα_Ss/L for which we describe an alternative NLuc insertion site. G protein rescue experiments validated the biological activity of these Gα donor proteins. Direct comparison between EGFP and LSS-mKATE2 as acceptor fluorophores revealed improved sensitivity for nearly all G protein subtypes when using the latter one. Hence, REGA-SIGN can be used as a panel of kinetic G protein biosensors with high sensitivity. Full article

The selectivity of drugs for G protein-coupled receptor (GPCR) signaling pathways is crucial for their therapeutic efficacy. Different agonists can cause receptors to recruit effector proteins at varying levels, thus inducing different signaling responses, called signaling bias. Although several GPCR-biased drugs are currently being developed, only a limited number of biased ligands have been identified regarding their signaling bias for the M1 muscarinic acetylcholine receptor (M1mAChR), and the mechanism is not yet well understood. In this study, we utilized bioluminescence resonance energy transfer (BRET) assays to compare the efficacy of six agonists in inducing Gαq and β-arrestin2 binding to M1mAChR. Our findings reveal notable variations in agonist efficacy in the recruitment of Gαq and β-arrestin2. Pilocarpine preferentially promoted the recruitment of β-arrestin2 (∆∆RAi = −0.5), while McN-A-343 (∆∆RAi = 1.5), Xanomeline (∆∆RAi = 0.6), and Iperoxo (∆∆RAi = 0.3) exhibited a preference for the recruitment of Gαq. We also used commercial methods to verify the agonists and obtained consistent results. Molecular docking revealed that certain residues (e.g., Y404, located in TM7 of M1mAChR) could play crucial roles in Gαq signaling bias by interacting with McN-A-343, Xanomeline, and Iperoxo, whereas other residues (e.g., W378 and Y381, located in TM6) contributed to β-arrestin recruitment by interacting with Pilocarpine. The preference of activated M1mAChR for different effectors may be due to significant conformational changes induced by biased agonists. By characterizing bias towards Gαq and β-arrestin2 recruitment, our study provides insights into M1mAChR signaling bias. Full article

Uveal Melanoma (UM) is a rare and malignant intraocular tumor with dismal prognosis. Even if radiation or surgery permit an efficient control of the primary tumor, up to 50% of patients subsequently develop metastases, mainly in the liver. The treatment of UM metastases is challenging and the patient survival is very poor. The most recurrent event in UM is the activation of Gαq signaling induced by mutations in GNAQ/11. These mutations activate downstream effectors including protein kinase C (PKC) and mitogen-activated protein kinases (MAPK). Clinical trials with inhibitors of these targets have not demonstrated a survival benefit for patients with UM metastasis. Recently, it has been shown that GNAQ promotes YAP activation through the focal adhesion kinase (FAK). Pharmacological inhibition of MEK and FAK showed remarkable synergistic growth-inhibitory effects in UM both in vitro and in vivo. In this study, we have evaluated the synergy of the FAK inhibitor with a series of inhibitors targeting recognized UM deregulated pathways in a panel of cell lines. The combined inhibition of FAK and MEK or PKC had highly synergistic effects by reducing cell viability and inducing apoptosis. Furthermore, we demonstrated that these combinations exert a remarkable in vivo activity in UM patient-derived xenografts. Our study confirms the previously described synergy of the dual inhibition of FAK and MEK and identifies a novel combination of drugs (FAK and PKC inhibitors) as a promising strategy for therapeutic intervention in metastatic UM. Full article

Angiotensin II (Ang II) upregulates transforming growth factor-beta1 (TGF-β1) and endothelin-1 (ET-1) in various types of cells, and all of them act as profibrotic mediators. However, the signal transduction of angiotensin II receptor (ATR) for upregulation of TGF-β1 and ET-1, and their effectors that play an essential role in myofibroblast differentiation, are not fully understood. Therefore, we investigated the ATR networking with TGF-β1 and ET-1 and identified the signal transduction of these mediators by measuring the mRNA expression of alpha-smooth muscle actin (α-SMA) and collagen I using qRT-PCR. Myofibroblast phenotypes were monitored by α-SMA and stress fiber formation with fluorescence microscopy. Our findings suggested that Ang II induced collagen I and α-SMA synthesis and stress fiber formation through the AT₁R/G_αq axis in adult human cardiac fibroblasts (HCFs). Following AT₁R stimulation, G_αq protein, not G_βγ subunit, was required for upregulation of TGF-β1 and ET-1. Moreover, dual inhibition of TGF-β and ET-1 signaling completely inhibited Ang II-induced myofibroblast differentiation. The AT₁R/G_αq cascade transduced signals to TGF-β1, which in turn upregulated ET-1 via the Smad- and ERK1/2-dependent pathways. ET-1 consecutively bound to and activated endothelin receptor type A (ET_AR), leading to increases in collagen I and α-SMA synthesis and stress fiber formation. Remarkably, dual blockade of TGF-β receptor and ETR exhibited the restorative effects to reverse the myofibroblast phenotype induced by Ang II. Collectively, TGF-β1 and ET-1 are major effectors of AT₁R/G_αq cascade, and therefore, negative regulation of TGF-β and ET-1 signaling represents a targeted therapeutic strategy for the prevention and restoration of cardiac fibrosis. Full article

Endothelin-1 (ET-1) has been implicated in the pathogenesis of cardiac fibrosis. Stimulation of endothelin receptors (ETR) with ET-1 leads to fibroblast activation and myofibroblast differentiation, which is mainly characterized by an overexpression of α-smooth muscle actin (α-SMA) and collagens. Although ET-1 is a potent profibrotic mediator, the signal transductions and subtype specificity of ETR contributing to cell proliferation, as well as α-SMA and collagen I synthesis in human cardiac fibroblasts are not well clarified. This study aimed to evaluate the subtype specificity and signal transduction of ETR on fibroblast activation and myofibroblast differentiation. Treatment with ET-1 induced fibroblast proliferation, and synthesis of myofibroblast markers, α-SMA, and collagen I through the ET_AR subtype. Inhibition of G_αq protein, not G_αi or G_βγ, inhibited these effects of ET-1, indicating the essential role of G_αq protein-mediated ET_AR signaling. In addition, ERK1/2 was required for ET_AR/G_αq axis-induced proliferative capacity and overexpression of these myofibroblast markers. Antagonism of ETR with ETR antagonists (ERAs), ambrisentan and bosentan, inhibited ET-1-induced cell proliferation and synthesis of α-SMA and collagen I. Furthermore, ambrisentan and bosentan promoted the reversal of myofibroblasts after day 3 of treatment, with loss of proliferative ability and a reduction in α-SMA synthesis, confirming the restorative effects of ERAs. This novel work reports on the ET_AR/G_αq/ERK signaling pathway for ET-1 actions and blockade of ETR signaling with ERAs, representing a promising therapeutic strategy for prevention and restoration of ET-1-induced cardiac fibrosis. Full article

Background: Atrial fibrillation (AF) is promoted by various stimuli like angiotensin II, endothelin-1, epinephrine/norepinephrine, vagal activation, or mechanical stress, all of which activate receptors coupled to G-proteins of the Gα_q/Gα₁₁-family (G_q). Besides pro-fibrotic and pro-inflammatory effects, G_q-mediated signaling induces inositol trisphosphate receptor (IP₃R)-mediated intracellular Ca²⁺ mobilization related to delayed after-depolarisations and AF. However, direct evidence of arrhythmogenic G_q-mediated signaling is absent. Methods and results: To define the role of G_q in AF, transgenic mice with tamoxifen-inducible, cardiomyocyte-specific Gα_q/Gα₁₁-deficiency (G_q-KO) were created and exposed to intracardiac electrophysiological studies. Baseline electrophysiological properties, including heart rate, sinus node recovery time, and atrial as well as AV nodal effective refractory periods, were comparable in G_q-KO and control mice. However, inducibility and mean duration of AF episodes were significantly reduced in G_q-KO mice—both before and after vagal stimulation. To explore underlying mechanisms, left atrial cardiomyocytes were isolated from G_q-KO and control mice and electrically stimulated to study Ca²⁺-mobilization during excitation–contraction coupling using confocal microscopy. Spontaneous arrhythmogenic Ca²⁺ waves and sarcoplasmic reticulum content-corrected Ca²⁺ sparks were less frequent in G_q-KO mice. Interestingly, nuclear but not cytosolic Ca²⁺ transient amplitudes were significantly decreased in G_q-KO mice. Conclusion: G_q-signaling promotes arrhythmogenic atrial Ca²⁺-release and AF in mice. Targeting this pathway, ideally using G_q-selective, biased receptor ligands, may be a promising approach for the treatment and prevention of AF. Importantly, the atrial-specific expression of the G_q-effector IP₃R confers atrial selectivity mitigating the risk of life-threatening ventricular pro-arrhythmic effects. Full article

The renin-angiotensin system (RAS) is one of the main regulatory systems of cardiovascular homeostasis. It is mainly composed of angiotensin-converting enzyme (ACE) and angiotensin II receptors AT1 and AT2. ACE and AT1 are targets of choice for the treatment of hypertension, whereas the AT2 receptor is still not exploited due to the lack of knowledge of its physiological properties. Peptide toxins from venoms display multiple biological functions associated with varied chemical and structural properties. If Brazilian viper toxins have been described to inhibit ACE, no animal toxin is known to act on AT1/AT2 receptors. We screened a library of toxins on angiotensin II receptors with a radioligand competition binding assay. Functional characterization of the selected toxin was conducted by measuring second messenger production, G-protein activation and β-arrestin 2 recruitment using bioluminescence resonance energy transfer (BRET) based biosensors. We identified one original toxin, A-CTX-cMila, which is a 7-residues cyclic peptide from Conus miliaris with no homology sequence with known angiotensin peptides nor identified toxins, displaying a 100-fold selectivity for AT1 over AT2. This toxin shows a competitive antagonism mode of action on AT1, blocking Gαq, Gαi3, GαoA, β-arrestin 2 pathways and ERK_1/2 activation. These results describe the first animal toxin active on angiotensin II receptors. Full article