Regulator Of G-protein Signaling 2 Expression Research Articles

RGS2 attenuates alveolar macrophage damage by inhibiting the Gq/11-Ca2+ pathway during cowshed PM2.5 exposure, and aberrant RGS2 expression is associated with TLR2/4 activation

Staff and animals in livestock buildings are constantly exposed to fine particulate matter (PM2.5), which affects their respiratory health. However, its exact pathogenic mechanism remains unclear. Regulator of G-protein signaling 2 (RGS2) has been reported to play a regulatory role in pneumonia. The aim of this study was to explore the therapeutic potential of RGS2 in cowshed PM2.5-induced respiratory damage. PM2.5 was collected from a cattle farm, and the alveolar macrophages (NR8383) of the model animal rat were stimulated with different treatment conditions of cowshed PM2.5. The RGS2 overexpression vector was constructed and transfected it into cells. Compared with the control group, cowshed PM2.5 significantly induced a decrease in cell viability and increased the levels of apoptosis and proinflammatory factor expression. Overexpression of RGS2 ameliorated the above-mentioned cellular changes induced by cowshed PM2.5. In addition, PM2.5 has significantly induced intracellular Ca2+ dysregulation. Affinity inhibition of Gq/11 by RGS2 attenuated the cytosolic calcium signaling pathway mediated by PLCβ/IP3R. To further investigate the causes and mechanisms of action of differential RGS2 expression, the possible effects of oxidative stress and TLR2/4 activation were investigated. The results have shown that RGS2 expression was not only regulated by oxidative stress-induced nitric oxide during cowshed PM2.5 cells stimulation but the activation of TLR2/4 had also an important inhibitory effect on its protein expression. The present study demonstrates the intracellular Ca2+ regulatory role of RGS2 during cellular injury, which could be a potential target for the prevention and treatment of PM2.5-induced respiratory injury.

RGS2 promotes estradiol biosynthesis by trophoblasts during human pregnancy

Production of estradiol (E2) by the placenta during human pregnancy ensures successful maintenance of placental development and fetal growth by stimulating trophoblast proliferation and the differentiation of cytotrophoblasts into syncytiotrophoblasts. Decreased levels of E2 are closely associated with obstetrical diseases such as preeclampsia (PE) in the clinic. However, the mechanisms underlying the inhibition of placental E2 biosynthesis remain poorly understood. Here, we report that regulator of G-protein signaling 2 (RGS2) affects E2 levels by regulating aromatase, a rate-limiting enzyme for E2 biosynthesis, by using human trophoblast-derived JEG-3 cells and human placental villus tissues. RGS2 enhanced the protein degradation of the transcription factor heart and neural crest derivatives expressed 1 (HAND1) by suppressing ubiquitin-specific protease 14 (USP14)-mediated deubiquitination of HAND1, resulting in the restoration of HAND1-induced trans-inactivation of the aromatase gene and subsequent increases in E2 levels. However, aromatase bound to RGS2 and repressed RGS2 GTPase activating protein (GAP) activity. Moreover, we observed a positive correlation between RGS2 and aromatase expression in clinical normal and preeclamptic placental tissues. Our results uncover a hitherto uncharacterized role of the RGS2-aromatase axis in the regulation of E2 production by human placental trophoblasts, which may pinpoint the molecular pathogenesis and highlight potential biomarkers for related obstetrical diseases.

Gq Signaling in the Placental Syncytiotrophoblast Layer During Preeclampsia

Preeclampsia is a pregnancy‐associated cardiovascular disorder characterized by hypertension and proteinuria after 20 weeks of gestation. Generalized cellular ‘stress’ (oxidative, mitochondrial, etc.) within the syncytiotrophoblast (STB) layer of placenta is theorized to drive its pathogenesis. Hormones implicated in preeclampsia such as angiotensin, endothelin, and vasopressin signal via receptors that typically couple to the Gq second‐messenger cascade, and Regulator of G protein Signaling‐2 (RGS2) buffers this signaling. We have published that RGS2 expression is decreased in human preeclamptic placenta, and reducing RGS2 expression in placenta is sufficient to cause development of key features of this disorder in mice. Unpublished in situ hybridization data indicate that in both humans and mice, RGS2 is present in the STB layer – and that RGS2 expression in the STB layer is reduced during preeclampsia in humans. Collectively, these findings lead us to generally hypothesize that excess Gq signaling specifically within the STB layer, whether due to excessive hormonal activation or loss of RGS2‐mediated buffering, causes STB‐Stress and thereby contributes to the pathogenesis of preeclampsia. Therefore, the objective of this study was to test whether artificial, exogenous activation of Gq signaling only within STB cells is sufficient to induce placental and maternal phenotypes of preeclampsia in mice.CAG‐FLEX‐hM3Dq+/+ (Gαq DREADD, Jax 026943) or C57BL/6J dams were bred with Gcm1‐Cre+/‐ sires (Jax 026625) or non‐transgenic littermates to elicit STB‐restricted hM3Dqexpression in ≍50% or 0% of placentas. The synthetic DREADD ligand clozapine N‐oxide (CNO) or saline vehicle was administered (2 mg/kg/d, ip) mid‐gestation (GD 12.5‐14.5). Placental and maternal phenotypes were compared between Cre– and Cre+ placentas within a dam, CNO‐ vs saline‐injected double‐transgenic dams, and double‐ vs single/non‐transgenic dams treated with CNO. Activation of Gq in the STB layer caused maternal proteinuria (n=4‐9, 49±6 mg/day vs 28±4 mg/day; p=0.01). Labyrinth vascularization, assessed by percentage of CD31+ area, was reduced with Gq activation (n=5, Cre+ 21±1%, Cre– 27±2%; p=0.01), while other regions remained unaffected (Decidua: Cre+ 14±4%, Cre– 14±3%; Junctional Zone: Cre+ 11±4%, Cre– 12±3%). Possible reductions in placental mass (n=5 Cre+ 0.12±0.01g, Cre– 0.15±0.01g; p=0.06), and placental vascular endothelial growth factor protein levels (n=6‐7 117±67 ng/g vs 151±12 ng/g; p=0.06) were noted, but no obvious trend in fetal growth restriction was observed (n=5 Cre+ 0.21±0.06g, Cre– 0.29±0.04g). These data support the pathological significance of excess Gq signaling specifically within the STB layer. Ongoing studies are aimed at further characterizing maternal phenotypes (blood pressure, circulating factors) in these models, and the synergistic effects of simultaneous STB‐specific deletion of RGS2 upon sensitivity to Gq stimulation in this cell type.

Abstract P227: Gq Signaling In The Placental Syncytiotrophoblast Layer During Preeclampsia

Hormones implicated in preeclampsia (PE) such as angiotensin, endothelin, and vasopressin signal via receptors coupled to the Gq cascade, and Regulator of G protein Signaling-2 (RGS2) buffers this signaling. We have published that RGS2 expression is decreased in human PE placenta, and reducing RGS2 in placenta causes development of key features of PE in mice. New in situ hybridization data indicate that in both humans and mice, RGS2 is abundant among many cell types in the placenta, including the syncytiotrophoblast (STB) layer. In addition, RGS2 expression in the human STB layer is reduced during PE. As this layer is strongly implicated in PE, these data lead us to hypothesize a critical Gq-buffering role for RGS2 in STB cells to prevent PE. To explore the effect of excess Gq signaling within the STB layer, we utilized a Cre-Lox approach to cause expression of the Gq-coupled hM3Dq DREADD throughout the fetoplacental unit (dam: hM3Dq+, sire: Actb-Cre+) or only within the STB layer (dam: hM3Dq+, sire: Gcm1-Cre+), and then activated the hM3Dq receptor via clozapine N-oxide (CNO, 0.5 to 2 mg/kg) injection in mid-gestation (GD12.5-14.5) before tissue collection at GD14.5. Gαq activation throughout the fetoplacental unit (Actb-Cre model) severely restricted fetoplacental growth compared to saline-injected controls (n=2 vs 3; placenta: 0.027±0.006 vs 0.115±0.021 g; p<0.05, and fetus: 0.048±0.007 vs 0.268±0.010 g; p<0.05). Similarly, placentas expressing hM3Dq only in STB cells (Gcm1-Cre model) had reduced placental (n=3 0.116±0.022 vs 0.201±0.036 g; p=0.05) and possibly fetal (n=3 0.1112±0.036 vs 0.247±0.028 g; p=0.06) masses after CNO. Vascularization (assessed by CD31 immunostain) was disproportionately reduced in the labyrinth layer of the Actb-Cre model after CNO (n=2 vs 3; 20.189±3.382 vs 35.762±1.976 % area; p<0.05), despite no relative change in layer (ie, decidua/junctional zone/labyrinth) thicknesses. Preliminary results indicate similar findings in the Gcm1-Cre model (n=1 17 vs 25 % area). These data highlight the pathological consequence of excess Gq signaling in the STB layer. Ongoing studies are aimed at characterizing maternal phenotypes in these models and the consequence of STB-specific deletion of RGS2 upon sensitivity to Gq stimulators.

Effect of the regulator of G-protein signaling 2 on the proliferation and invasion of oral squamous cell carcinoma cells and its molecular mechanism.

This study aims to investigate the effect of the regulator of G-protein signaling 2 (RGS2) on the proliferation and invasion of oral squamous cell carcinoma (OSCC) cells and its potential molecular mechanism. Metho⁃ds The expression status and clinical significance of RGS2 in head and neck squamous cell carcinomas and matched adjacent normal tissues were evaluated using TCGA database. Three OSCC cell lines (i.e., SCC-9, Cal27, and Fadu) were overexpressed with RGS2, and the effect of RGS2 on cell proliferation and invasion was determined using the Transwell, clone formation, and cell counting kit (CCK)-8 assays. Moreover, the yeast two-hybrid scree-ning and co-immunoprecipitation (Co-IP) assays were conducted to detect the correlation of RGS2, four and a half LIM domains protein 1 (FHL1), and damage DNA-binding protein 1 (DDB1). The expression level of RGS2 in OSCC was significantly lower than that in matched adjacent normal tissues (P=0.023). The high RGS2 expression level was negatively correlated with lymphovascular invasion (P<0.001). After transfection with lentiv-RGS2, the expression of RGS2 was increased, and the invasion and proliferation abilities of OSCC cell lines were evidently inhibited. FHL1 could competitively bind with RGS2, which decreased the integration of DDB1 and RGS2, inhibited the ubiquitination process of RGS2, and maintained the stability of the RGS2 protein. RGS2 plays an important role in the inhibition of OSCC proliferation and invasion. The structure stability of RGS2 is competitively regulated by FHL1 and DDB1.

The Increased Expression of Regulator of G-Protein Signaling 2 (RGS2) Inhibits Insulin-Induced Akt Phosphorylation and Is Associated with Uncontrolled Glycemia in Patients with Type 2 Diabetes.

Experimental evidence in mice models has demonstrated that a high regulator of G-protein signaling 2 (RSG2) protein levels precede an insulin resistance state. In the same context, a diet rich in saturated fatty acids induces an increase in RGS2 protein expression, which has been associated with decreased basal metabolism in mice; however, the above has not yet been analyzed in humans. For this reason, in the present study, we examined the association between RGS2 expression and insulin resistance state. The incubation with palmitic acid (PA), which inhibits insulin-mediated Akt Ser473 phosphorylation, resulted in the increased RGS2 expression in human umbilical vein endothelial-CS (HUVEC-CS) cells. The RGS2 overexpression without PA was enough to inhibit insulin-mediated Akt Ser473 phosphorylation in HUVEC-CS cells. Remarkably, the platelet RGS2 expression levels were higher in type 2 diabetes mellitus (T2DM) patients than in healthy donors. Moreover, an unbiased principal component analysis (PCA) revealed that RGS2 expression level positively correlated with glycated hemoglobin (HbA1c) and negatively with age and high-density lipoprotein cholesterol (HDL) in T2DM patients. Furthermore, PCA showed that healthy subjects segregated from T2DM patients by having lower levels of HbA1c and RGS2. These results demonstrate that RGS2 overexpression leads to decreased insulin signaling in a human endothelial cell line and is associated with poorly controlled diabetes.

RGS2 is prognostic for development of castration resistance and cancer-specific survival in castration-resistant prostate cancer.

Regulator of G-protein signaling 2 (RGS2) is a multifaceted protein with a prognostic value in hormone-naïve prostate cancer (PC). It has previously been associated with the development of castration resistance. However, RGS2 expression in clinical specimens of castration-resistant prostate cancer (CRPC) and its clinical relevance has not been explored. In the present study, RGS2 was assessed in CRPC and in relation to the development of castration resistance. In the present study, RGS2 expression was evaluated with immunohistochemistry in patient materials of hormone-naïve and castration-resistant primary tumors, also in matched specimens before and after 3 months of androgen deprivation therapy (ADT). Cox regression and Kaplan-Meier curves were used to evaluate the clinical significance of RGS2 expression. RGS2 expression in association to castration-resistant growth was assessed experimentally in an orthotopic xenograft mouse model of CRPC. In vitro, hormone depletion of LNCaP and enzalutamide treatment of LNCaP, 22Rv1, and VCaP was performed to evaluate the association between RGS2 and the androgen receptor (AR). Stable RGS2 knockdown was used to evaluate the impact of RGS2 in association to PC cell growth under hormone-reduced conditions. Gene and protein expression were evaluated with quantitative polymerase chain reaction and Western blot analysis, respectively. RGS2 expression is increased in CRPC and enriched under ADT. Furthermore, a high RGS2 level is prognostic for poor cancer-specific survival for CRPC patients and significantly reduced failure-free survival (FFS) after an initiated ADT. Additionally, the prognostic value of RGS2 outperforms prostate-specific antigen (PSA) in terms of FFS. The present study furthermore suggests that RGS2 expression is reflective of AR activity. Moreover, low RGS2-expressing cells display hampered growth under hormone-reduced conditions, in line with the poor prognosis associated with high RGS2 expression. High levels of RGS2 are associated with aggressive forms of castration-resistant PC. The results demonstrate that a high level of RGS2 is associated with poor prognosis in association with castration-resistant PC growth. RGS2 alone, or in association with PSA, has the potential to identify patients that require additional treatment at an early stage during ADT.

OR20-05 Regulation of Mouse Uterine Contractility by Regulator of G-Protein Signalling 2

INTRODUCTION: Uterine contractions in labour are produced by activation of receptors such as FP (PGF2α) and OXTR (oxytocin). Such receptors signal via Gαq proteins that can be selectively turned off by the regulator of G-protein signaling 2 (RGS2). RGS2 expression can be upregulated by Gαs-coupled receptor signaling. PGE2 elicits various uterine effects, some via activation of Gαs-coupled receptors. In primary human myometrial cells, PGE2 enhances RGS2 expression that attenuates subsequent oxytocin-stimulated calcium responses associated with contraction. RGS2 expression is higher in preterm non-labour versus labour human myometrium, suggesting that RGS2 promotes quiescence in the pregnant uterus. To directly study whether RGS2 dampens uterine contraction, we analyzed the ex vivo contractility of pregnant mouse uteri. We hypothesize that loss of RGS2 expression enhances oxytocin and PGF2α-stimulated uterine contractility. METHODS: Wildtype (WT) and RGS2 knockout (KO) female mice (8-14 weeks) were time-mated and euthanized on days 15-20 of pregnancy or during active labour (n=5-11 per group). Uteri were snap frozen for RNA/protein expression analysis by qPCR and western blot. Uterine sections from d18-19 were dissected into longitudinal strips for ex vivo contractility measurement. Tissues were submerged in Krebs-Henseleit buffer and tied to force transducers to measure isometric force development. Tissues were treated with or without 1μM PGE2 for 2 hours. In parallel, pretreated and non-pretreated tissues were exposed to increasing concentrations of oxytocin or PGF2α (0.1-10uM). All tissues were treated with 100mM KCl to determine the maximum contraction at the end of the experiment. Data were analyzed by ANOVA (Bonferroni post-hoc), or Student’s t-test, as appropriate. RESULTS: RGS2 expression in WT uteri sharply decreases at day 19 (p<0.05), remaining low during labour. OXTR and FP receptor expression increase at day 19 (p<0.01 and p<0.05, respectively) and are sustained through labour. Compared to WT, uteri from KO mice produced larger oxytocin-stimulated contractile amplitudes (p<0.05), and more frequent PGF2α-stimulated contractions (p<0.05). PGE2 pretreatment enhanced RGS2 expression in WT uteri, which reduced the peak amplitudes and integrals elicited by subsequent oxytocin treatment, compared to non-pretreated uteri (p<0.05). CONCLUSION: RGS2 may play a pro-quiescent role in pregnancy, since RGS2 loss enhances uterine contractility. Concomitant upregulation of pro-contractile receptors and RGS2 downregulation may facilitate labour activation. This mechanisms may be involved in the etiology of spontaneous preterm labour; loss of RGS2 expression observed in human preterm labour may allow for early contractile activation. Understanding RGS2’s role in quiescence is important to developing more effective strategies for managing preterm labour.

Reduced mRNA Expression of RGS2 (Regulator of G Protein Signaling-2) in the Placenta Is Associated With Human Preeclampsia and Sufficient to Cause Features of the Disorder in Mice.

Cascade-specific termination of G protein signaling is catalyzed by the RGS (regulator of G protein signaling) family members, including RGS2. Angiotensin, vasopressin, and endothelin are implicated in preeclampsia, and RGS2 is known to inhibit G protein cascades activated by these hormones. Mutations in RGS2 are associated with human hypertension and increased risk of developing preeclampsia and its sequelae. RGS family members are known to influence maternal vascular function, but the role of RGS2 within the placenta has not been explored. Here, we hypothesized that reduced expression of RGS2 within the placenta represents a risk factor for the development of preeclampsia. Although cAMP/CREB signaling was enriched in placentas from human pregnancies affected by preeclampsia compared with clinically matched controls and RGS2 is known to be a CREB-responsive gene, RGS2 mRNA was reduced in placentas from pregnancies affected by preeclampsia. Experimentally reducing Rgs2 expression within the feto-placental unit was sufficient to induce preeclampsia-like phenotypes in pregnant wild-type C57BL/6J mice. Stimulation of RGS2 transcription within immortalized human HTR8/SVneo trophoblasts by cAMP/CREB signaling was discovered to be dependent on the activity of histone deacetylase activity, and more specifically, HDAC9 (histone deacetylase-9), and HDAC9 expression was reduced in placentas from human pregnancies affected by preeclampsia. We conclude that reduced expression of RGS2 within the placenta may mechanistically contribute to preeclampsia. More generally, this work identifies RGS2 as an HDAC9-dependent CREB-responsive gene, which may contribute to reduced RGS2 expression in placenta during preeclampsia.

OR08-6 RGS2 Expression Attenuates Contractile Signaling in Myometrial Smooth Muscle In Vitro

Labour activation is regulated by a shift in the balance of pro-quiescent mechanisms that maintain pregnancy, and inflammatory and endocrine signals that stimulate uterine contractility. The prostaglandins (PG) and oxytocin act at Gq protein coupled receptors thought to drive labour contractions. However, emerging evidence suggests a twofold effect of PGE2 on the pregnant uterus, driving pro-labour or pro-quiescent signaling depending on the receptor subtype activated. It is possible that PGE2, abundant within the pregnant uterus, mediates pro-quiescent effects by activating Gs coupled receptors to upregulate regulator of G-protein signaling 2 (RGS2), a GTPase activating protein that can dampen smooth muscle contractility by turning off Gq signals. Approaching labour, inflammatory signals may downregulate RGS2 expression to derepress contractility. Understanding the regulatory role of RGS2 on uterine contractility may provide insights for improved clinical management of spontaneous preterm labour, to prevent premature birth. We hypothesise that RGS2, regulated by mediators that control quiescence and labour, reduces contractile functions of Gq coupled signals in the pregnant uterus. To test this hypothesis, we used primary myometrial smooth muscle (MSM) cells isolated from uterine biopsies collected at term (>37 weeks) non-labour caesarean deliveries. Treatment with 1μM PGE2 increased RGS2 mRNA and protein expression, peaking at 1 and 2 hours respectively. Pretreatment with 1ng/ml IL-1β or TNFα partially repressed the PGE2 effect, suggesting RGS2 may be downregulated in the inflammatory milieu that accompanies labour. To study the effect of RGS2 on MSM function, cells were loaded with Fluo-4 NW to detect intracellular calcium changes as a measure of contractility following challenge with 100nM oxytocin. Enhanced RGS2 expression, following PGE2 treatment or adenoviral overexpression, reduced oxytocin-mediated calcium flux, suggesting RGS2 attenuates this contractile signal. In addition to oxytocin, labour contractions likely integrate activation of many other GPCRs, signaling of which may also be regulated by RGS2. A PCR-based array analysis revealed expression of >300 GPCRs in MSM cells and tissue. The highly expressed histamine receptor 1 was targeted to begin exploring additional signals attenuated by RGS2 in MSM. Histamine (1μM) challenged cells generated a calcium response that was attenuated by PGE2-induced RGS2, and RGS2 adenoviral overexpression. Together our results show RGS2 is upregulated by pro-quiescent signals abundant in pregnancy, and downregulated by inflammatory mediators implicated in labour. Enhanced RGS2 expression reduces contractile responses to oxytocin and histamine, which we propose is consistent with a pro-quiescent role in pregnancy. Funding: CIHR, NSERC Project Grants; AIHS MD-PhD Studentship.

Uterine RGS2 expression is regulated by exogenous estrogen and progesterone in ovariectomized mice, and downregulation of RGS2 expression in artificial decidualized ESCs inhibits trophoblast spreading in vitro.

Embryo implantation is a complicated event that relies on two critical factors: the competent blastocyst and the receptive uterus. Successful implantation results from tight coordination of these two factors. The maternal hormone environment of the uterus and molecular cross-talk between the embryo and uterine tissue play pivotal roles in implantation. Here we showed that regulator of G-protein signaling 2 (RGS2), a member of ubiquitous family of proteins that regulate G-protein activation, plays an important role in embryo implantation by interfering in the cross-talk between the embryo and uterine tissue. RGS2 expression increased during the implantation process, and was higher in the implant site than at the nonimplantation site. Meanwhile, ovariectomized (OVX) mice exhibited higher expression of RGS2 in the uterus. Exogenous 17β-estradiol and progesterone in OVX mice downregulated the expression of RGS2. Treatment with exogenous 17β-estradiol alone caused uterine RGS2 messenger RNA levels of OVX mice to return to those of normal female mice; when these mice were treated with progesterone or 17β-estradiol plus progesterone, RGS2 levels rose. Downregulation of Rgs2 by small interfering RNA in an in vitro coculture system of decidualized endometrial stromal cells and blastocysts inhibited blastocyst outgrowth by restricting trophoblast spreading, suggesting a mechanism by which RGS2 regulates embryo implantation.

Abstract 015: Reduced Placental Expression of Regulator of G Protein Signaling-2 (RGS2) in Preeclampsia: Association, Consequence, and Cause

To date, the early-gestational mechanisms driving the pathogenesis of preeclampsia (PreE) remain largely unclear. However, altered G protein signaling has been implicated in PreE, including alterations in GPCR agonists such as vasopressin, endothelin, and angiotensin. Regulator of G protein Signaling 2 (RGS2) is an endogenous terminator of Gαq signaling, and mutations in the Rgs2 gene are linked to hypertension and increased risk of developing PreE. Therefore, we hypothesized reduced placental RGS2 may increase the risk of developing PreE by disinhibiting Gαq signaling. In silico reanalysis of a publicly-available microarray dataset (GSE75010) revealed a significant reduction in Rgs2 mRNA in placentas from PreE pregnancies compared to controls (Con n=35, PreE n=49, p&lt;0.05). We confirmed this reduction in Rgs2 mRNA by qPCR using human placental tissue samples (PreE 19% of Con, n=11 vs 9, p&lt;0.05). To examine if reduced feto-placental RGS2 was sufficient to induce PreE phenotypes, wildtype C57BL/6J female mice were mated with Rgs2 -deficient ( Rgs2 -KO) sires or wildtype littermate sires. Dams mated with Rgs2 -KO sires developed diastolic hypertension (Con 92 ± 2 vs Rgs2 -KO 98.2 ± 2 [24 hr avg mmHg]; p&lt;0.05 vs pregnant control and pre-pregnancy Rgs2 -KO) and increased proteinuria compared to dams mated with littermate sires (18.2 ± 2.2, n=7 vs 28.4 ± 2.8, n=10 mg/day, p&lt;0.05). Preliminary histological analysis of placentas from dams mated with an Rgs2 -KO sire suggest decreased spiral artery number and diameter (SA Number: Con 7.7 ± 0.2 vs Rgs2 -KO 5.6 ± 0.4, SA Diameter: Con 128 ± 3 vs Rgs2 -KO 86 ± 12). Previous studies have outlined a CRE element in the Rgs2 promoter that is critical for transcriptional regulation of Rgs2 . Thus, we hypothesized loss of cAMP/CREB regulation may lead to reduced RGS2 in human PreE. Indeed, reduced phosphorylated CREB (p-CREB) binding was observed in PreE placentas (Con 0.146 ± 0.024 vs PreE 0.070 ± 0.021 p&lt;0.05). However, loss of p-CREB was not due to decreases in cAMP levels (Con 1.69 ± 0.35 vs PreE 2.18 ± 0.18 nM). These data support a role for reduced placental RGS2 in the pathogenesis of PreE, and demonstrate that changes in p-CREB regulation may explain the loss of RGS2 expression in placental trophoblasts.

Regulation of the regulator of G protein signaling 2 expression and cellular localization by PKA and PKC pathways in mouse granulosa cells

G protein-coupled receptor (GPCR) activation-mediated PKA and PKC pathways have been recognized to be important in ovarian physiology. Expression of regulator of G-protein signaling 2 (RGS2) has been reported in ovarian granulosa cells. The detailed mechanisms in PKA- and PKC-regulated RGS2 expression and cellular translocation in granulosa cells remain mostly unclear. PKA activator 8-bromo-cAMP and PKC activator phorbol-12, 13-didecanoate appeared to rapidly elevate both protein and mRNA levels and promoter activation of RGS2 gene. Two consensus Sp1 elements within the shortest 78 bp fragment of RGS2 promoter sequence were essential for the full responsiveness to PKA and PKC. PKC activation appeared to increase the RGS2 translocation from nucleus to cytosol. PKA- and PKC-mediated RGS2 transcription in a Sp-1-dependent manner and a PKC-mediated RGS2 intracellular translocation were noted in granulosa cells.

Reduced Placental Expression of Regulator of G‐Protein Signaling‐2 (RGS2) and Preeclampsia

Although late‐gestational mechanisms of preeclampsia (PreE) have been described, the genetic risk factors and mechanisms driving early‐gestational pathogenesis remain largely unknown. Various activators of Gαq‐mediated signaling pathways have been identified as possible early mediators of PreE, including vasopressin, endothelin, and angiotensin. Regulator of G‐protein Signaling 2 (RGS2) acts as an endogenous terminator of Gαq signaling and previous studies have identified a single nucleotide polymorphism (rs4606) in the RGS2 gene that is associated with increased risk for PreE, and which may reduce RGS2 mRNA. We therefore hypothesize that reduced placental expression of RGS2 increases the risk of PreE due to the subsequent disinhibition of Gαq‐mediated signaling. In silico reanalysis of a publicly‐available dataset (GSE75010) describing the transcriptomes of human placentas uncovered a significant reduction in RGS2 mRNA in placentas from pre‐term PreE pregnancies compared to control pre‐term pregnancies (Con n=35, PreE n=49, p&lt;0.05). This reduction in RGS2 mRNA was confirmed by qPCR using human placental tissue samples from the University of Iowa Maternal‐Fetal Tissue Bank (PreE 19% of Con, n=11 vs 9, p&lt;0.05), though no significant correlation was observed between the rs4606 SNP and PreE in this cohort. In silico reanalysis of a separate dataset (GSE93839) revealed RGS2 mRNA is among the highest‐expressed RGS family members in normal human placenta, and that it may be selectively reduced in both syncytiotrophoblast (73% of Con) and invasive cytotrophoblast (68% of Con) layers of the placenta during PreE. To examine the sufficiency of feto‐placental‐specific reduction in RGS2 on the development of PreE phenotypes, wildtype C57BL/6J female mice were mated with RGS2‐deficient (RGS2‐KO) sires, which results in a wildtype dam carrying all heterozygous feto‐placental units, or wildtype littermates of the RGS2‐KO males. Dams mated with RGS2‐KO sires developed diastolic hypertension (Con 92 ± 2 vs RGS2‐KO 98.2 ± 2 [24 hr avg mmHg]; p&lt;0.05 vs pre‐pregnancy RGS2‐KO, and pregnant control) and increased proteinuria compared to dams mated with littermate males (18.2 ± 2.2, n=7 vs 28.4 ± 2.8, n=10 mg/day, p&lt;0.05), although no intrauterine growth restriction, changes in placental PlGF (Con n=6, 9.8 ± 1.1 vs KO n=12, 11.8 ± 0.8 μg/g) or FLT1 protein (103 ± 30.2 vs 114.6 ± 42.7 ng/g), or markers of hypoxia were observed. Previous studies have indicated that placentas from PreE patients exhibit reduced cAMP content compared to normal placentas, and in vascular smooth muscle, transcription of RGS2 is promoted by CREB occupancy at a cAMP Response Element (CRE) within its promoter. Thus we hypothesized a potential role for cAMP/CREB control of RGS2 in trophoblasts. Forskolin treatment increased CREB binding to the RGS2 promoter (by chromatin immunoprecipitation) and RGS2 mRNA expression (by qPCR) in cultured human HTR8/SVneo trophoblasts. Preliminary data support reduced CREB occupancy at the RGS2 promoter in PreE placenta. These data support a role for reduced placental RGS2 expression in the pathogenesis of PreE, and demonstrate that reduced placental cAMP levels may contribute to the loss of RGS2 expression in placental trophoblasts regardless of rs4606 SNP genotype.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 144: Reduced Placental Regulator of G-Protein Signaling-2 (RGS2) and Preeclampsia

The early mechanisms and genetic risk factors driving the pathogenesis of preeclampsia (PE), a cardiovascular disorder of pregnancy, remain largely unclear. Various hormone activators of Gα q second-messenger signaling pathways have been implicated in PE. Regulator of G-protein Signaling 2 (RGS2) acts as an endogenous terminator of Gα q signaling and previous studies identified a SNP (rs4606), which results in reduced RGS2 mRNA, as a risk factor for development of PE and its sequelae. We hypothesized reduced placental expression of RGS2 may precipitate the development of PE due to disinhibited Gα q signaling. In silico reanalysis of publically available dataset GSE75010 revealed RGS2 mRNA is reduced in placentas from pre-term PE pregnancies compared to normal pre-term pregnancies (con: 8.73 ± 0.07 n=35, PE: 8.37 ± 0.055 n=49, p&lt;0.05). Using human placental tissue samples from the University of Iowa Maternal-Fetal Tissue Bank, we confirmed RGS2 mRNA is reduced in PE placentas (19% of control, p&lt;0.05), despite a lack of correlation between the rs4606 SNP and PE. Additionally, in further reanalysis of other datasets, RGS2 mRNA is among the highest-expressed RGS member in normal human placenta, and appears to be selectively reduced in syncytio- and invasive cytotrophoblasts during PE (GSE93839, -26.3%, -23.3% of control). We next examined RGS2 expression in mouse placenta by FISH and found RGS2 mRNA colocalizes with markers of syncytiotrophoblast II (GCMA) and spongiotrophoblast (Tpbpα) layers. To test the effect of RGS2 loss during pregnancy, wildtype C57BL/6J female mice were mated with RGS2-deficient sires and developed diastolic hypertension, placental hypoxia by HIF1α ELISA (con 0.144±0.004, RGS2-KO 0.155±0.004 AU, p&lt;0.05, n=5 each), and reduced placental PlGF mRNA (fold; con=1.0 n=7, RGS2-KO=0.23 n=12, p&lt;0.05), compared to females mated with RGS2 littermate sires. These data support the concept that loss of RGS2 may contribute to the pathogenesis of PE rather than simply correlating with the disorder. Taken together, we have shown placental RGS2 is suppressed in PE, RGS2 is present in cytoplasm of specific layers of trophoblasts, and loss of feto-placental RGS2 is sufficient to cause placental hypoxia and maternal diastolic hypertension.

Reduced Placental Regulator of G‐Protein Signaling‐2 (RGS2) and Preeclampsia

Though mid‐ and late‐gestational mechanisms of the late‐pregnancy cardiovascular disorder preeclampsia (PreE) are relatively well characterized, genetic risk factors and mechanisms driving the early gestational pathogenesis of PreE remain largely unclear. Various hormone activators of Gαq second‐messenger signaling pathways (eg ‐ vasopressin, endothelin, and angiotensin) have been implicated in preeclampsia. Regulator of G‐protein Signaling 2 (RGS2) acts as an endogenous terminator of Gαq signaling, and previous association studies have identified an increased risk for PreE and its sequelae in women carrying a single nucleotide polymorphism that results in reduced RGS2. We hypothesized that RGS2 is expressed in placental trophoblasts, and that reduced expression of RGS2 in placental tissue may represent a risk factor for the development of PreE due to disinhibited Gαq signaling. Placental tissue samples from PreE and clinically‐matched control pregnancies were obtained from the University of Iowa Maternal‐Fetal Tissue Bank and examined for mRNA of the B/R4 family of RGS proteins, including RGS2. Of the members examined (RGS2, ‐3, ‐4.2, ‐4.3, ‐4.4, ‐4.5, and ‐ 5) in control placentas (n=9), only RGS2 (Ct 28.8±0.7 vs 18S Ct 12.1±0.4) and RGS4.3 (Ct 23.0±0.4 vs 18S Ct 13.3±0.3) transcripts were detected above background levels. In PreE placenta (n=11), RGS2 mRNA may be suppressed (1.0±0.4 vs 0.2±0.3‐fold, p=0.1) while RGS4.3 remains unchanged (1.0±0.4 vs 1.1±0.4 fold, p=0.8). Immunohistochemical (IHC) detection similarly confirms cytoplasmic localization of RGS2 protein in trophoblasts of placentas from control and PreE human pregnancies. RGS2 positive trophoblasts are found in the junctional zone and labyrinth of placentas from wildtype C57BL/6J mice. We next examined the cardiovascular consequences of reducing feto‐placental RGS2 within a wildtype mother (wildtype C57BL/6J dam bred with either an RGS2‐knockout (KO) or littermate control sire). Reduced RGS2 in the feto‐placental unit caused increased HIF1α binding to chromatin (control 0.144±0.004 vs RGS2‐KO 0.155±0.004 AU, p=0.05, n=5 each), supporting placental hypoxia. Further, dams exhibited isolated diastolic hypertension by telemetry (24 hr mean: pre‐pregnancy control n=3, 92±2 vs RGS2‐KO n=5, 92±2; late‐pregnancy control 92±2 vs RGS2‐KO 98.2±2 mmHg; p&lt;0.05 vs pre‐pregnancy RGS2‐KO, and pregnant control), consistent with microvascular dysfunction. We conclude that RGS2 is present in the cytoplasm of trophoblasts, that placental RGS2 is suppressed in PreE, and that loss of feto‐placental RGS2 is sufficient to cause placental hypoxia and maternal diastolic hypertension. Collectively these studies provide evidence supporting the novel concepts that disinhibited Gαq signaling in the placenta may contribute to PreE, and that a father's genetics may contribute to the risk for PreE.

Epigenetic regulation of RGS2 (Regulator of G-protein signaling 2) in chemoresistant ovarian cancer cells

Regulator of G-protein signaling 2 (RGS2) is a GTPase-activating protein functioning as an inhibitor of G-protein coupled receptors (GPCRs). RGS2 dysregulation was implicated in solid tumour development and RGS2 downregulation has been reported in prostate and ovarian cancer progression. However, the molecular mechanism by which RGS2 expression is suppressed in ovarian cancer remains unknown. The expression and epigenetic regulation of RGS2 in chemosensitive and chemoresistant ovarian cancer cells were determined by qRT-PCR and chromatin immunoprecipitation assays, respectively. In the present study, the molecular mechanisms contributing to the loss of RGS2 expression were determined in ovarian cancer. The data indicated that suppression of RGS2 gene in chemoresistant ovarian cancer cells, in part, due to accumulation of histone deacetylases (HDACs) and DNA methyltransferase I (DNMT1) at the promoter region of RGS2. Inhibition of HDACs or DNMTs significantly increases RGS2 expression. These results suggest that epigenetic changes in histone modifications and DNA methylation may contribute to the loss of RGS2 expression in chemoresistant ovarian cancer cells. The results further suggest that class I HDACs and DNMT1 contribute to the suppression of RGS2 during acquired chemoresistance and support growing evidence that inhibition of HDACs/DNMTs represents novel therapeutic approaches to overcome ovarian cancer chemoresistance.

RGS2 expression predicts amyloid-β sensitivity, MCI and Alzheimer's disease: genome-wide transcriptomic profiling and bioinformatics data mining.

Alzheimer's disease (AD) is the most frequent cause of dementia. Misfolded protein pathological hallmarks of AD are brain deposits of amyloid-β (Aβ) plaques and phosphorylated tau neurofibrillary tangles. However, doubts about the role of Aβ in AD pathology have been raised as Aβ is a common component of extracellular brain deposits found, also by in vivo imaging, in non-demented aged individuals. It has been suggested that some individuals are more prone to Aβ neurotoxicity and hence more likely to develop AD when aging brains start accumulating Aβ plaques. Here, we applied genome-wide transcriptomic profiling of lymphoblastoid cells lines (LCLs) from healthy individuals and AD patients for identifying genes that predict sensitivity to Aβ. Real-time PCR validation identified 3.78-fold lower expression of RGS2 (regulator of G-protein signaling 2; P=0.0085) in LCLs from healthy individuals exhibiting high vs low Aβ sensitivity. Furthermore, RGS2 showed 3.3-fold lower expression (P=0.0008) in AD LCLs compared with controls. Notably, RGS2 expression in AD LCLs correlated with the patients' cognitive function. Lower RGS2 expression levels were also discovered in published expression data sets from postmortem AD brain tissues as well as in mild cognitive impairment and AD blood samples compared with controls. In conclusion, Aβ sensitivity phenotyping followed by transcriptomic profiling and published patient data mining identified reduced peripheral and brain expression levels of RGS2, a key regulator of G-protein-coupled receptor signaling and neuronal plasticity. RGS2 is suggested as a novel AD biomarker (alongside other genes) toward early AD detection and future disease modifying therapeutics.

Abstract P177: Reduced Expression of Regulator of G-Protein Signaling-2 (RGS2) in the Placenta During Preeclampsia

Preeclampsia (PE) is a serious cardiovascular condition of late pregnancy. Genetic risk factors and the early-gestational etiology remain largely unclear, though evidence supports excessive activation of Gαq signaling within the placenta in response to various hormones including vasopressin, endothelin, and angiotensin. Regulator of G-protein Signaling 2 (RGS2) acts as an endogenous terminator of Gαq signaling, and previous association studies have identified an increased risk for PE and its sequelae in women carrying a single nucleotide polymorphism that is expected to reduce levels of RGS2. We hypothesized that RGS2 is expressed in placental trophoblasts, and that reduced expression of RGS2 in placental tissue may represent a risk factor for the development of PE. Whole placenta samples and clinical data from preeclamptic and clinically-matched control pregnancies were obtained from the University of Iowa Maternal-Fetal Tissue Bank (IRB#200910784) and examined for mRNA levels of the B/R4 family of RGS proteins, including RGS2. Of the members examined (RGS2, -3, -4.2, -4.3, -4.4, -4.5, and -5) in control placentas (n=9), only RGS2 (Ct 28.8±0.7 vs 18S Ct 12.1±0.4) and RGS4.3 (Ct 23.0±0.4 vs 18S Ct 13.3±0.3) transcripts were expressed above background levels. RGS2 protein expression was then confirmed in human placental tissues by Western blot. RGS2 mRNA expression was 3-fold higher in fetal (amniotic, p&lt;0.05) layers than maternal (decidual) layers. In preeclamptic placenta (n=11), RGS2 may be suppressed (1.0±0.4 vs 0.2±0.3-fold, p=0.1) while RGS4.3 remains unchanged (1.0±0.4 vs 1.1±0.4 fold, p=0.8). Initial immunohistochemical detection confirms cytoplasmic localization of RGS2 in trophoblasts of wildtype mouse placenta, despite exclusive nuclear localization in other tissues. We conclude that human placenta expresses RGS2, and that this expression may be suppressed during preeclampsia. Loss of RGS2 expression may result in disinhibited trophoblast Gαq signaling, and ultimately placental insufficiency.

MicroRNA hsa-miR-4717-5p regulates RGS2 and may be a risk factor for anxiety-related traits.

Regulator of G-protein Signaling 2 (RGS2) is a key regulator of G-protein-coupled signaling pathways involved in fear and anxiety. Data from rodent models and genetic analysis of anxiety-related traits and disorders in humans suggest down-regulation of RGS2 expression to be a risk factor for anxiety. Here we investigated, whether genetic variation in microRNAs mediating posttranscriptional down-regulation of RGS2 may be a risk factor for anxiety as well. 75 microRNAs predicted to regulate RGS2 were identified by four bioinformatic algorithms and validated experimentally by luciferase reporter gene assays. Specificity was confirmed for six microRNAs (hsa-miR-1271-5p, hsa-miR-22-3p, hsa-miR-3591-3p, hsa-miR-377-3p, hsa-miR-4717-5p, hsa-miR-96-5p) by disrupting their seed sequence at the 3' untranslated region of RGS2. Hsa-miR-4717-5p showed the most robust effect on RGS2 and regulated two other candidate genes of anxiety disorders (CNR1 and IKBKE) as well. Two SNPs (rs150925, rs161427) within and 1,000 bp upstream of the hostgene of hsa-miR-4717-5p (MIR4717) show a minor allele frequency greater than 0.05. Both were in high linkage disequilibrium (r(2) = 1, D' = 1) and both major (G) alleles showed a trend for association with panic disorder with comorbid agoraphobia in one of two patient/control samples (combined n(patients) = 497). Dimensional anxiety traits, as described by Anxiety Sensitivity Index (ASI) and Agoraphobic Cognitions Questionnaire (ACQ) were significantly higher among carriers of both major (G) alleles in a combined patient/control sample (n(combined) = 831). Taken together, data indicate that MIR4717 regulates human RGS2 and contributes to the genetic risk towards anxiety-related traits.