RGS10 deficiency facilitates distant metastasis by inducing epithelial-mesenchymal transition in breast cancer.

Distant metastasis is the major cause of death in patients with breast cancer. Epithelial-mesenchymal transition (EMT) contributes to breast cancer metastasis. Regulator of G protein-signaling (RGS) proteins modulates metastasis in various cancers. This study identified a novel role for RGS10 in EMT and metastasis in breast cancer. RGS10 protein levels were significantly lower in breast cancer tissues compared to normal breast tissues, and deficiency in RGS10 protein predicted a worse prognosis in patients with breast cancer. RGS10 protein levels were lower in the highly aggressive cell line MDA-MB-231 than in the poorly aggressive, less invasive cell lines MCF7 and SKBR3. Silencing RGS10 in SKBR3 cells enhanced EMT and caused SKBR3 cell migration and invasion. The ability of RGS10 to suppress EMT and metastasis in breast cancer was dependent on lipocalin-2 and MIR539-5p. These findings identify RGS10 as a tumor suppressor, prognostic biomarker, and potential therapeutic target for breast cancer.

Age-Dependent RGS5 Loss in Pericytes Induces Cardiac Dysfunction and Fibrosis.

BACKGROUND: Pericytes are capillary-associated mural cells involved in the maintenance and stability of the vascular network. Although aging is one of the main risk factors for cardiovascular disease, the consequences of aging on cardiac pericytes are unknown. METHODS: In this study, we have combined single-nucleus RNA sequencing and histological analysis to determine the effects of aging on cardiac pericytes. Furthermore, we have conducted in vivo and in vitro analysis of RGS5 (regulator of G-protein signaling 5) loss of function and finally have performed pericytes-fibroblasts coculture studies to understand the effect of RGS5 deletion in pericytes on the neighboring fibroblasts. RESULTS: Aging reduced the pericyte area and capillary coverage in the murine heart. Single-nucleus RNA sequencing analysis further revealed that the expression of Rgs5 was reduced in cardiac pericytes from aged mice. In vivo and in vitro studies showed that the deletion of RGS5 impaired cardiac function, induced fibrosis, and morphological changes in pericytes characterized by a profibrotic gene expression signature and the expression of different ECM (extracellular matrix) components and growth factors, for example, TGFB2 and PDGFB. Indeed, culturing fibroblasts with the supernatant of RGS5-deficient pericytes induced their activation as evidenced by the increased expression of αSMA (alpha smooth muscle actin) in a TGFß (transforming growth factor beta)2-dependent mechanism. CONCLUSIONS: Our results have identified RGS5 as a crucial regulator of pericyte function during cardiac aging. The deletion of RGS5 causes cardiac dysfunction and induces myocardial fibrosis, one of the hallmarks of cardiac aging.

Differential methamphetamine-induced behavioral effects in male and female mice lacking regulator of G Protein signaling 4.

Methamphetamine-induced behavioral effects are mediated by several neurotransmitters that act via the G-protein coupled receptors (GPCRs). The functioning of GPCRs are negatively regulated by regulators of G-protein signaling (RGS) proteins. The goal of this study was to assess the role of two specific RGS proteins namely the RGS2 and the RGS4 proteins in methamphetamine-induced behaviors. The effects of methamphetamine (1 mg/kg; i.p.) on conditioned place preference (CPP) and locomotor activity were assessed in genetically modified male and female mice lacking either RGS2 or RGS4 and their wildtype littermates to achieve the above goal. Locomotor activity after methamphetamine administration was assessed in both methamphetamine-naïve and -experienced mice. Methamphetamine-induced CPP at the tested dose was blocked in male, but not female, mice lacking RGS4 compared to respective controls. Interestingly, methamphetamine-induced increase in locomotor activity at the tested dose was observed in methamphetamine-experienced, but not in the methamphetamine-naïve, male mice lacking RGS4. However, methamphetamine-induced increase in locomotor activity at the tested dose was blocked in both methamphetamine-naïve and -experienced female mice lacking RGS4. Interestingly, methamphetamine-induced rewarding effects and methamphetamine-induced increase in locomotor activity at the tested dose were observed in mice lacking RGS2, irrespective of sex and/or history of methamphetamine exposure. Together, the data suggest that RGS4 plays a role in methamphetamine-induced behaviors and could serve as a potential target for medications intended to treat the acute effects of methamphetamine.

RGS5 Determines Neutrophil Migration in the Acute Inflammatory Phase of Bleomycin-Induced Lung Injury.

The regulator of G protein signaling (RGS) represents a widespread system of controllers of cellular responses. The activities of the R4 subfamily of RGSs have been elucidated in allergic pulmonary diseases. However, the R4 signaling in other inflammatory lung diseases, with a strong cellular immune response, remained unexplored. Thus, our study aimed to discern the functional relevance of the R4 family member, RGS5, as a potential modulating element in this context. Gene profiling of the R4 subfamily showed increased RGS5 expression in human fibrosing lung disease samples. In line with this, RGS5 was markedly increased in murine lungs following bleomycin injury. RGS knock-out mice (RGS-/-) had preserved lung function while control mice showed significant combined ventilatory disorders three days after bleomycin application as compared to untreated control mice. Loss of RGS5 was associated with a significantly reduced neutrophil influx and tissue myeloperoxidase expression. In the LPS lung injury model, RGS5-/- mice also failed to recruit neutrophils into the lung, which was accompanied by reduced tissue myeloperoxidase levels after 24 h. Our in-vitro assays showed impaired migration of RGS5-/- neutrophils towards chemokines despite preserved Ca2+ signaling. ERK dephosphorylation might play a role in reduced neutrophil migration in our model. As a conclusion, loss of RGS5 preserves lung function and attenuates hyperinflammation in the acute phase of bleomycin-induced pulmonary fibrosis and LPS-induced lung injury. Targeting RGS5 might alleviate the severity of exacerbations in interstitial lung diseases.

Regulator of G protein signaling 12 enhances osteoclastogenesis by suppressing Nrf2-dependent antioxidant proteins to promote the generation of reactive oxygen species.

Regulators of G-protein Signaling are a conserved family of proteins required in various biological processes including cell differentiation. We previously demonstrated that Rgs12 is essential for osteoclast differentiation and its deletion in vivo protected mice against pathological bone loss. To characterize its mechanism in osteoclastogenesis, we selectively deleted Rgs12 in C57BL/6J mice targeting osteoclast precursors using LyzM-driven Cre mice or overexpressed Rgs12 in RAW264.7 cells. Rgs12 deletion in vivo led to an osteopetrotic phenotype evidenced by increased trabecular bone, decreased osteoclast number and activity but no change in osteoblast number and bone formation. Rgs12 overexpression increased osteoclast number and size, and bone resorption activity. Proteomics analysis of Rgs12-depleted osteoclasts identified an upregulation of antioxidant enzymes under the transcriptional regulation of Nrf2, the master regulator of oxidative stress. We confirmed an increase of Nrf2 activity and impaired reactive oxygen species production in Rgs12-deficient cells. Conversely, Rgs12 overexpression suppressed Nrf2 through a mechanism dependent on the 26S proteasome, and promoted RANKL-induced phosphorylation of ERK1/2 and NFκB, which was abrogated by antioxidant treatment. Our study therefore identified a novel role of Rgs12 in regulating Nrf2, thereby controlling cellular redox state and osteoclast differentiation.

MiR-21-3p modulates lipopolysaccharide-induced inflammation and apoptosis via targeting TGS4 in retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is a major reason of blindness in the elderly. MicroRNAs are implicated in various pathological processes, including inflammation and apoptosis. In this study, we aim to investigate the biological functions of miR-21-3p in inflammation and apoptosis caused by lipopolysaccharide (LPS) in human retinal pigment epithelial (ARPE-19) cells. The miR-21-3p inhibitor and mimic were transfected into ARPE-19 cells for 48 hours, followed by exposed to LPS (10 µg/mL) for 24 hours. The mRNA and protein expression of IL-6 and MCP-1 were measured using real-time PCR (RT-PCR) and enzyme-linked immunosorbent assays. Cell viability, apoptosis, caspase 3 activity, cleaved caspase-3 and cleaved-PARP protein levels were detected to evaluate the effects of miR-21-3p on apoptosis. Additionally, the target relationship between miR-21-3p and regulator of G-protein signalling 4 (RGS4) was verified by dual luciferase reporter assay. RT-PCR analysis demonstrated that LPS induced miR-21-3p expression. Inhibition of miR-21-3p reduced the mRNA and protein levels of IL-6 and MCP-1. Apoptosis, caspase-3 activity, and cleaved-caspase 3 and cleaved PARP protein levels were repressed by the miR-21-3p inhibitor. However, overexpression of miR-21-3p showed the opposite results. Furthermore, we identified that miR-21-3p directly targeted the 3' untranslated region of RGS4. MiR-21-3p negatively regulated the expression of RGS4 both in mRNA and protein levels. Silencing RGS4 reduced the anti-inflammatory and anti-apoptotic effects of miR-21-3p inhibitor. Our results revealed that miR-21-3p inhibition targeted RGS4 to attenuate inflammatory responses and apoptosis caused by LPS in ARPE-19 cells.

The signaling proteins GPR158 and RGS7 modulate excitability of L2/3 pyramidal neurons and control A-type potassium channel in the prelimbic cortex.

Stress profoundly affects physiological properties of neurons across brain circuits and thereby increases the risk for depression. However, the molecular and cellular mechanisms mediating these effects are poorly understood. In this study, we report that chronic physical restraint stress in mice decreases excitability specifically in layer 2/3 of pyramidal neurons within the prelimbic subarea of the prefrontal cortex (PFC) accompanied by the induction of depressive-like behavioral states. We found that a complex between G protein-coupled receptor (GPCR) 158 (GPR158) and regulator of G protein signaling 7 (RGS7), a regulatory GPCR signaling node recently discovered to be a key modulator of affective behaviors, plays a key role in controlling stress-induced changes in excitability in this neuronal population. Deletion of GPR158 or RGS7 enhanced excitability of layer 2/3 PFC neurons and prevented the impact of stress. Investigation of the underlying molecular mechanisms revealed that the A-type potassium channel Kv4.2 subunit is a molecular target of the GPR158-RGS7 complex. We further report that GPR158 physically associates with Kv4.2 channel and promotes its function by suppressing inhibitory modulation by cAMP-protein kinase A (PKA)-mediated phosphorylation. Taken together, our observations reveal a critical mechanism that adjusts neuronal excitability in L2/3 pyramidal neurons of the PFC and may thereby modulate the effects of stress on depression.

Regulator of G-protein signaling 5 regulates the shift from perivascular to parenchymal pericytes in the chronic phase after stroke.

Poststroke recovery requires multiple repair mechanisms, including vascular remodeling and blood-brain barrier (BBB) restoration. Brain pericytes are essential for BBB repair and angiogenesis after stroke, but they also give rise to scar-forming platelet-derived growth factor receptor ß (PDGFR-ß)-expressing cells. However, many of the molecular mechanisms underlying this pericyte response after stroke still remain unknown. Regulator of G-protein signaling 5 (RGS5) has been associated with pericyte detachment from the vascular wall, but whether it regulates pericyte function and vascular stabilization in the chronic phase of stroke is not known. Using RGS5-knockout (KO) mice, we study how loss of RGS5 affects the pericyte response and vascular remodeling in a stroke model at 7 d after ischemia. Loss of RGS5 leads to a shift toward an increase in the number of perivascular pericytes and reduction in the density of parenchymal PDGFR-ß-expressing cells associated with normalized PDGFR-ß activation after stroke. The redistribution of pericytes resulted in higher pericyte coverage, increased vascular density, preservation of vessel lengths, and a significant reduction in vascular leakage in RGS5-KO mice compared with controls. Our study demonstrates RGS5 in pericytes as an important target to enhance vascular remodeling.-Roth, M., Gaceb, A., Enström, A., Padel, T., Genové, G., Özen, I., Paul, G. Regulator of G-protein signaling 5 regulates the shift from perivascular to parenchymal pericytes in the chronic phase after stroke.

Regulator of G-Protein Signaling 16 Is a Negative Modulator of Platelet Function and Thrombosis.

Background Members of the regulator of G-protein signaling ( RGS ) family inhibit G-protein coupled receptor signaling by modulating G-protein activity. In platelets, there are 3 different RGS isoforms that are expressed at the protein level, including RGS 16. Recently, we have shown that CXCL 12 regulates platelet function via RGS 16. However, the role of RGS 16 in platelet function and thrombus formation is poorly defined. Methods and Results We used a genetic knockout mouse model approach to examine the role(s) of RGS 16 in platelet activation by using a host of in vitro and in vivo assays. We observed that agonist-induced platelet aggregation, secretion, and integrin activation were much more pronounced in platelets from the RGS 16 knockout ( Rgs16 -/-) mice relative to their wild type ( Rgs16 +/+) littermates. Furthermore, the Rgs16 -/- mice had a markedly shortened bleeding time and were more susceptible to vascular injury-associated thrombus formation than the controls. Conclusions These findings support a critical role for RGS 16 in regulating hemostatic and thrombotic functions of platelets in mice. Hence, RGS 16 represents a potential therapeutic target for modulating platelet function.

Homeostatic cAMP regulation by the RGS7 complex controls depression-related behaviors.

Affective disorders arise from abnormal responses of the brain to prolonged exposure to challenging environmental stimuli. Recent work identified the orphan receptor GPR158 as a molecular link between chronic stress and depression. Here we reveal a non-canonical mechanism by which GPR158 exerts its effects on stress-induced depression by the complex formation with Regulator of G protein Signaling 7 (RGS7). Chronic stress promotes membrane recruitment of RGS7 via GPR158 in the medial prefrontal cortex (mPFC). The resultant complex suppresses homeostatic regulation of cAMP by inhibitory GPCRs in the region. Accordingly, RGS7 loss in mice induces an antidepressant-like phenotype and resiliency to stress, whereas its restoration within the mPFC is sufficient to rescue this phenotype in a GPR158-dependent way. These findings mechanistically link the unusual orphan receptor-RGS complex to a major stress mediator, the cAMP system and suggest new avenues for pharmacological interventions in affective disorders.

Selective Role of RGS9-2 in Regulating Retrograde Synaptic Signaling of Indirect Pathway Medium Spiny Neurons in Dorsal Striatum.

In the striatum, medium spiny neurons (MSNs) are heavily involved in controlling movement and reward. MSNs form two distinct populations expressing either dopamine receptor 1 (D1-MSN) or dopamine receptor 2 (D2-MSN), which differ in their projection targets and neurochemical composition. The activity of both types of MSNs is shaped by multiple neuromodulatory inputs processed by GPCRs that fundamentally impact their synaptic properties biasing behavioral outcomes. How these GPCR signaling cascades are regulated and what downstream targets they recruit in D1-MSN and D2-MSN populations are incompletely understood. In this study, we examined the cellular and molecular mechanisms underlying the action of RGS9-2, a key GPCR regulator in MSNs implicated in both movement control and actions of addictive drugs. Imaging cultured striatal neurons, we found that ablation of RGS9-2 significantly reduced calcium influx through NMDARs. Electrophysiological recordings in slices confirmed inhibition of NMDAR function in MSNs, resulting in enhanced AMPAR/NMDAR ratio. Accordingly, male mice lacking RGS9-2 displayed behavioral hypersensitivity to NMDAR blockade by MK-801 or ketamine. Recordings from genetically identified populations of striatal neurons revealed that these changes were selective to D2-MSNs. Surprisingly, we found that these postsynaptic effects resulted in remodeling of presynaptic inputs to D2-MSNs increasing the frequency of mEPSC and inhibiting paired-pulse ratio. Pharmacological dissection revealed that these adaptations were mediated by the NMDAR-dependent inhibition of retrograde endocannabinoid signaling from D2-MSNs to CB1 receptor on presynaptic terminals. Together, these data demonstrate a novel mechanism for pathway selective regulation of synaptic plasticity in MSNs controlled by GPCR signaling.SIGNIFICANCE STATEMENT This study identifies a role for a major G-protein regulator in controlling synaptic properties of striatal neurons in a pathway selective fashion.

Development of Full Sweet, Umami, and Bitter Taste Responsiveness Requires Regulator of G protein Signaling-21 (RGS21).

The mammalian tastes of sweet, umami, and bitter are initiated by activation of G protein-coupled receptors (GPCRs) of the T1R and T2R families on taste receptor cells. GPCRs signal via nucleotide exchange and hydrolysis, the latter hastened by GTPase-accelerating proteins (GAPs) that include the Regulators of G protein Signaling (RGS) protein family. We previously reported that RGS21, uniquely expressed in Type II taste receptor cells, decreases the potency of bitter-stimulated T2R signaling in cultured cells, consistent with its in vitro GAP activity. However, the role of RGS21 in organismal responses to GPCR-mediated tastants was not established. Here, we characterized mice lacking the Rgs21 fifth exon. Eliminating Rgs21 expression had no effect on body mass accumulation (a measure of alimentation), fungiform papillae number and morphology, circumvallate papillae morphology, and taste bud number. Two-bottle preference tests, however, revealed that Rgs21-null mice have blunted aversion to quinine and denatonium, and blunted preference for monosodium glutamate, the sweeteners sucrose and SC45647, and (surprisingly) NaCl. Observed reductions in GPCR-mediated tastant responses upon Rgs21 loss are opposite to original expectations, given that loss of RGS21-a GPCR signaling negative regulator-should lead to increased responsiveness to tastant-mediated GPCR signaling (all else being equal). Yet, reduced organismal tastant responses are consistent with observations of reduced chorda tympani nerve recordings in Rgs21-null mice. Reduced tastant-mediated responses and behaviors exhibited by adult mice lacking Rgs21 expression since birth have thus revealed an underappreciated requirement for a GPCR GAP to establish the full character of tastant signaling.

Enhanced longevity and metabolism by brown adipose tissue with disruption of the regulator of G protein signaling 14.

Disruption of the regulator for G protein signaling 14 (RGS14) knockout (KO) in mice extends their lifespan and has multiple beneficial effects related to healthful aging, that is, protection from obesity, as reflected by reduced white adipose tissue, protection against cold exposure, and improved metabolism. The observed beneficial effects were mediated by improved mitochondrial function. But most importantly, the main mechanism responsible for the salutary properties of the RGS14 KO involved an increase in brown adipose tissue (BAT), which was confirmed by surgical BAT removal and transplantation to wild-type (WT) mice, a surgical simulation of a molecular knockout. This technique reversed the phenotype of the RGS14 KO and WT, resulting in loss of the improved metabolism and protection against cold exposure in RGS14 KO and conferring this protection to the WT BAT recipients. Another mechanism mediating the salutary features in the RGS14 KO was increased SIRT3. This mechanism was confirmed in the RGS14 X SIRT3 double KO, which no longer demonstrated improved metabolism and protection against cold exposure. Loss of function of the Caenorhabditis elegans RGS-14 homolog confirmed the evolutionary conservation of this mechanism. Thus, disruption of RGS14 is a model of healthful aging, as it not only enhances lifespan, but also protects against obesity and cold exposure and improves metabolism with a key mechanism of increased BAT, which, when removed, eliminates the features of healthful aging.

Vascular wall regulator of G-protein signalling-1 (RGS-1) is required for angiotensin II-mediated blood pressure control.

G-Protein coupled receptors (GPCRs) activate intracellular signalling pathways by coupling to heterotrimeric G-proteins that control many physiological processes including blood pressure homeostasis. The Regulator of G-Protein Signalling-1 (RGS1) controls the magnitude and duration of downstream GPCR signalling by acting as a GTPase-activating protein for specific Gα-proteins. RGS1 has contrasting roles in haematopoietic and non-haematopoietic cells. Rgs1-/-ApoE-/- mice are protected from Angiotensin II (Ang II)-induced aortic aneurysm rupture. Conversely, Ang II treatment increases systolic blood pressure to a greater extent in Rgs1-/-ApoE-/- mice than ApoE-/- mice, independent of its role in myeloid cells. However the precise role of RGS1 in hypertension and vascular-derived cells remains unknown. We determined the effects of Rgs1 deletion on vascular function in ApoE-/- mice. Rgs1 deletion led to enhanced vasoconstriction in aortas and mesenteric arteries from ApoE-/- mice in response to phenylephrine (PE) and U46619 respectively. Rgs1 was shown to have a role in the vasculature, with endothelium-dependent vasodilation being impaired, and endothelium-independent dilatation to SNP being enhanced in Rgs1-/-ApoE-/- mesenteric arteries. To address the downstream signalling pathways in vascular smooth muscle cells (VSMCs) in response to Ang II-stimulation, we assessed pErk1/2, pJNK and pp38 MAPK activation in VSMCs transiently transfected with Rgs1. pErk1/2 signalling but not pJNK and pp38 signalling was impaired in the presence of Rgs1. Furthermore, we demonstrated that the enhanced contractile response to PE in Rgs1-/-ApoE-/- aortas was reduced by a MAPK/Erk (MEK) inhibitor and an L-type voltage gated calcium channel antagonist, suggesting that Erk1/2 signalling and calcium influx are major effectors of Rgs1-mediated vascular contractile responses, respectively. These findings indicate RGS1 is a novel regulator of blood pressure homeostasis and highlight RGS1-controlled signalling pathways in the vasculature that may be new drug development targets for hypertension.

Ultrahigh-Resolution Optical Coherence Elastography Images Cellular-Scale Stiffness of Mouse Aorta.

Cellular-scale imaging of the mechanical properties of tissue has helped to reveal the origins of disease; however, cellular-scale resolution is not readily achievable in intact tissue volumes. Here, we demonstrate volumetric imaging of Young's modulus using ultrahigh-resolution optical coherence elastography, and apply it to characterizing the stiffness of mouse aortas. We achieve isotropic resolution of better than 15 µm over a 1-mm lateral field of view through the entire depth of an intact aortic wall. We employ a method of quasi-static compression elastography that measures volumetric axial strain and uses a compliant, transparent layer to measure surface axial stress. This combination is used to estimate Young's modulus throughout the volume. We demonstrate differentiation by stiffness of individual elastic lamellae and vascular smooth muscle. We observe stiffening of the aorta in regulator of G protein signaling 5-deficient mice, a model that is linked to vascular remodeling and fibrosis. We observe increased stiffness with proximity to the heart, as well as regions with micro-structural and micro-mechanical signatures characteristic of fibrous and lipid-rich tissue. High-resolution imaging of Young's modulus with optical coherence elastography may become an important tool in vascular biology and in other fields concerned with understanding the role of mechanics within the complex three-dimensional architecture of tissue.

Essentiality of Regulator of G Protein Signaling 6 and Oxidized Ca2+/Calmodulin-Dependent Protein Kinase II in Notch Signaling and Cardiovascular Development.

BACKGROUND: Congenital heart defects are the most common birth defects worldwide. Although defective Notch signaling is the major cause of mouse embryonic death from cardiovascular defects, how Notch signaling is regulated during embryonic vasculogenesis and heart development is poorly understood. METHODS AND RESULTS: Regulator of G protein signaling 6 (RGS6)-/-/Ca2+/calmodulin-dependent protein kinase II (CaMKII)VV double mutant mice were developed by crossing RGS6-/- mice with mice expressing an oxidation-resistant CaMKIIδ (CaMKIIVV), and the resulting embryonic defects/lethality were investigated using E7.5 to E15.5 embryos. While loss of either RGS6 or oxidized CaMKIIδ does not alter embryogenesis, their combined loss causes defective Notch signaling, severe cardiovascular defects, and embryonic lethality (≈E10.5-11.5). Embryos lacking RGS6 and expressing oxidation-resistant CaMKIIδ exhibit reduced myocardial wall thickness, abnormal trabeculation, and arterial specification defects. Double mutants show vascular remodeling defects, including reduced neurovascularization, delayed neural tube maturation, and small dorsal aortae. These striking cardiovascular defects were accompanied by placental and yolk sac defects in angiogenesis, hematopoiesis, and vascular remodeling similar to what is seen with defective Notch1 signaling. Double mutant hearts, embryos, and yolk sacs exhibit profound downregulation of Notch1, Jagged 1, and Notch downstream target genes Hey1, Hey2, and Hey1L as well as impaired Notch1 signaling in embryos/hearts. CONCLUSIONS: RGS6 and oxidized CaMKIIδ together function as novel critical upstream modulators of Notch signaling required for normal cardiovascular development and embryo survival. Their combined need indicates that they function in parallel pathways needed for Notch1 signaling in yolk sac, placenta and embryos. Thus, dysregulated embryonic RGS6 expression and oxidative activation of CaMKII may potentially contribute to congenital heart defects.

RGS9-2 Modulates Responses to Oxycodone in Pain-Free and Chronic Pain States.

Regulator of G-protein signaling 9-2 (RGS9-2) is a striatal-enriched signal-transduction modulator known to have a critical role in the development of addiction-related behaviors following exposure to psychostimulants or opioids. RGS9-2 controls the function of several G-protein-coupled receptors, including dopamine receptor and mu opioid receptor (MOR). We previously showed that RGS9-2 complexes negatively control morphine analgesia, and promote the development of morphine tolerance. In contrast, RGS9-2 positively modulates the actions of other opioid analgesics, such as fentanyl and methadone. Here we investigate the role of RGS9-2 in regulating responses to oxycodone, an MOR agonist prescribed for the treatment of severe pain conditions that has addictive properties. Using mice lacking the Rgs9 gene (RGS9KO), we demonstrate that RGS9-2 positively regulates the rewarding effects of oxycodone in pain-free states, and in a model of neuropathic pain. Furthermore, although RGS9-2 does not affect the analgesic efficacy of oxycodone or the expression of physical withdrawal, it opposes the development of oxycodone tolerance, in both acute pain and chronic neuropathic pain models. Taken together, these data provide new information on the signal-transduction mechanisms that modulate the rewarding and analgesic actions of oxycodone.

Upregulation of RGS2: a new mechanism for pirfenidone amelioration of pulmonary fibrosis.

BACKGROUND: Pirfenidone was recently approved for treatment of idiopathic pulmonary fibrosis. However, the therapeutic dose of pirfenidone is very high, causing side effects that limit its doses and therapeutic effectiveness. Understanding the molecular mechanisms of action of pirfenidone could improve its safety and efficacy. Because activated fibroblasts are critical effector cells associated with the progression of fibrosis, this study investigated the genes that change expression rapidly in response to pirfenidone treatment of pulmonary fibroblasts and explored their contributions to the anti-fibrotic effects of pirfenidone. METHODS: We used the GeneChip microarray to screen for genes that were rapidly up-regulated upon exposure of human lung fibroblast cells to pirfenidone, with confirmation for specific genes by real-time PCR and western blots. Biochemical and functional analyses were used to establish their anti-fibrotic effects in cellular and animal models of pulmonary fibrosis. RESULTS: We identified Regulator of G-protein Signaling 2 (RGS2) as an early pirfenidone-induced gene. Treatment with pirfenidone significantly increased RGS2 mRNA and protein expression in both a human fetal lung fibroblast cell line and primary pulmonary fibroblasts isolated from patients without or with idiopathic pulmonary fibrosis. Pirfenidone treatment or direct overexpression of recombinant RGS2 in human lung fibroblasts inhibited the profibrotic effects of thrombin, whereas loss of RGS2 exacerbated bleomycin-induced pulmonary fibrosis and mortality in mice. Pirfenidone treatment reduced bleomycin-induced pulmonary fibrosis in wild-type but not RGS2 knockout mice. CONCLUSIONS: Endogenous RGS2 exhibits anti-fibrotic functions. Upregulated RGS2 contributes significantly to the anti-fibrotic effects of pirfenidone.

Potential Role of Regulator of G-Protein Signaling 5 in the Protection of Vagal-Related Bradycardia and Atrial Tachyarrhythmia.

BACKGROUND: The regulator of G-protein signaling 5 (Rgs5), which functions as the regulator of G-protein-coupled receptor (GPCR) including muscarinic receptors, has a potential effect on atrial muscarinic receptor-activated IKA ch current. METHODS AND RESULTS: In the present study, hearts of Rgs5 knockout (KO) mice had decreased low-frequency/high-frequency ratio in spectral measures of heart rate variability. Loss of Rgs5 provoked dramatically exaggerated bradycardia and significantly (P<0.05) prolonged sinus nodal recovery time in response to carbachol (0.1 mg/kg, intraperitoneally). Compared to those from wild-type (WT) mice, Langendorff perfused hearts from Rgs5 KO mice had significantly (P<0.01) abbreviated atrial effective refractory periods and increased dominant frequency after administration of acetylcholine (ACh; 1 µmol/L). In addition, whole patch clamp analyses of single atrial myocytes revealed that the ACh-regulated potassium current (IKA ch) was significant increased in the time course of activation and deactivation (P<0.01) in Rgs5 KO, compared to those in WT, mice. To further determine the effect of Rgs5, transgenic mice with cardiac-specific overexpression of human Rgs5 were found to be resistant to ACh-related effects in bradycardia, atrial electrophysiology, and atrial tachyarrhythmia (AT). CONCLUSION: The results of this study indicate that, as a critical regulator of parasympathetic activation in the heart, Rgs5 prevents vagal-related bradycardia and AT through negatively regulating the IKA ch current.

RGS10 deficiency ameliorates the severity of disease in experimental autoimmune encephalomyelitis.

BACKGROUND: Regulator of G-protein signaling (RGS) family proteins, which are GTPase accelerating proteins (GAPs) that negatively regulate G-protein-coupled receptors (GPCRs), are known to be important modulators of immune cell activation and function. Various single-nucleotide polymorphisms in RGS proteins highly correlate with increased risk for multiple sclerosis (MS), an autoimmune, neurodegenerative disorder. An in-depth search of the gene expression omnibus profile database revealed higher levels of RGS10 and RGS1 transcripts in peripheral blood mononuclear cells (PBMCs) in MS patients, suggesting potential functional roles for RGS proteins in MS etiology and/or progression. METHODS: To define potential roles for RGS10 in regulating autoimmune responses, we evaluated RGS10-null and wild-type (WT) mice for susceptibility to experimental autoimmune encephalomyelitis (EAE), a widely studied model of MS. Leukocyte distribution and functional responses were assessed using biochemical, immunohistological, and flow cytometry approaches. RESULTS: RGS10-null mice displayed significantly milder clinical symptoms of EAE with reduced disease incidence and severity, as well as delayed onset. We observed fewer CD3+ T lymphocytes and CD11b+ myeloid cells in the central nervous system (CNS) tissues of RGS10-null mice with myelin oligodendrocyte protein (MOG)35-55-induced EAE. Lymph node cells and splenocytes of immunized RGS10-null mice demonstrated decreased proliferative and cytokine responses in response to in vitro MOG memory recall challenge. In adoptive recipients, transferred myelin-reactive RGS10-null Th1 cells (but not Th17 cells) induced EAE that was less severe than their WT counterparts. CONCLUSIONS: These data demonstrate a critical role for RGS10 in mediating autoimmune disease through regulation of T lymphocyte function. This is the first study ever conducted to elucidate the function of RGS10 in effector lymphocytes in the context of EAE. The identification of RGS10 as an important regulator of inflammation might open possibilities for the development of more specific therapies for MS.