Regulator Of G-protein Signaling 2 Gene Research Articles

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<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<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<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<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.

Luteinizing Hormone Receptor Gene and Regulator of G-protein Signaling 2 Gene Expression Level and Association with Oocyte Maturity in In vitro Fertilization/Intracytoplasmic Sperm Injection Cycle.

Aims:The aim is to study the relation and distribution in gene expression level of the luteinizing hormone receptor (LHR) gene and regulator of G-protein signaling 2 (RGS2) gene expression with oocyte maturation.Setting and Design:This cross-sectional study was undertaken in an instruction-based tertiary care infertility unit, department of obstetrics and gynecology.Materials and Methods:After controlled ovarian hyperstimulation, cumulus granulosa cells (CCs) from 59 oocytes among 18 women being treated by in vitro fertilization/intracytoplasmic sperm injection cycle technique from November 2015 to January 2016 were collected on the day of oocyte retrieval. Total RNA was extracted and converted to cDNA in individual oocytes. LHR and RGS2 gene levels were measured and analyzed using digital droplet polymerase chain reaction.Statistical Analysis:Gene expression level was analyzed using software STATA, version 14.0 (College Station, TX: StataCorp LP, USA).Results:CCs were obtained from 59 cumulus-oocyte complexes (COC), 46 COC from metaphase II (CCMII), 13 COC from metaphase I, and GV oocyte (CCMI + GV). The RGS2 gene expression level, when compared with the housekeeping gene in CCMII and CCMI + GV, was 0.15 (0.05–0.52) and 0.08 (0.02–0.27), respectively. The LHR gene expression when compared with the housekeeping gene in CCMII and CCMI + GV did not differ and was quite in the same value that was 0.02 (0.00–0.11) and 0.02 (0.00–0.06), respectively.Conclusion:This study showed that LHR gene expression did not differ in between oocyte groups. Even though the median of RGS2 gene expression was more in the mature oocyte group, the result was inconclusive due to scattering and overlapping of gene expression data between oocyte groups.

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.

Epigenetic Regulation of RGS2 in Cystic Fibrosis

Cystic fibrosis (CF) is a recessive autosomal disease that affects the respiratory, gastrointestinal and reproductive systems. At pulmonary level, the absence of CFTR function (cystic fibrosis transmembrane conductance regulator encoding an ion channel chlorine) leads to a decrease in mucociliary clearance, resulting in mucus accumulation. A new approach based on epigenetic regulation of gene expression has been investigated and we identified a list of hypermethylated genes in dF508 cell lines, the most frequently observed mutation in the CF population. Among the CF hypermethylated genes, RGS2 (Regulator of G protein signaling 2) gene demonstrated either transcript or protein downregulation. RGS2 is a key modulator of bronchial hyperresponsiveness, plays a critical role in fibrosis‐related diseases and inflammation.We hypothesized that variation in RGS2 protein expression could be the result of modulation of promoter methylation and has an impact on cytokine regulation. The first objective was to verify that methylation observed on RGS2 promoter in dF508 homozygous cell lines induces a modulation of protein expression. Then, to assess the consequences of this downregulation of RGS2, we performed an shRNA‐mediated RGS2 knockdown followed by DMNQ treatment in non‐CF cells and monitored the expression of inflammatory mediators.We confirmed that methylation of RGS2 promoter has an impact on RGS2 gene expression using azacythidine and oxidative stress treatments and that RGS2 is downregulated at the protein level in CF cells and in dF508 homozygous patients. We observed a relation between RGS2 downregulation by shRNA and the upregulation of 3 modulators of early inflammation: CHI3L1, S100A12 and TNFa.

β 2 -Adrenoceptor agonist-induced RGS2 expression is a genomic mechanism of bronchoprotection that is enhanced by glucocorticoids

In asthma and chronic obstructive pulmonary disease, activation of G(q)-protein-coupled receptors causes bronchoconstriction. In each case, the management of moderate-to-severe disease uses inhaled corticosteroid (glucocorticoid)/long-acting β(2)-adrenoceptor agonist (LABA) combination therapies, which are more efficacious than either monotherapy alone. In primary human airway smooth muscle cells, glucocorticoid/LABA combinations synergistically induce the expression of regulator of G-protein signaling 2 (RGS2), a GTPase-activating protein that attenuates G(q) signaling. Functionally, RGS2 reduced intracellular free calcium flux elicited by histamine, methacholine, leukotrienes, and other spasmogens. Furthermore, protection against spasmogen-increased intracellular free calcium, following treatment for 6 h with LABA plus corticosteroid, was dependent on RGS2. Finally, Rgs2-deficient mice revealed enhanced bronchoconstriction to spasmogens and an absence of LABA-induced bronchoprotection. These data identify RGS2 gene expression as a genomic mechanism of bronchoprotection that is induced by glucocorticoids plus LABAs in human airway smooth muscle and provide a rational explanation for the clinical efficacy of inhaled corticosteroid (glucocorticoid)/LABA combinations in obstructive airways diseases.

Impact of RGS2 deficiency on the therapeutic effect of telmisartan in angiotensin II-induced aortic aneurysm

Regulator of G-protein signaling 2 (RGS2) negatively regulates the signaling of G-protein-coupled receptors, such as the angiotensin II (AngII) type 1 receptor by accelerating the inactivation of Gαq. Rgs2-deficient mice show increased sensitivity and prolonged responsiveness to vasoconstrictors, and genetic variations in the RGS2 gene are associated with hypertension in humans. This study aimed to clarify whether Rgs2 deficiency contributes to the development of vascular remodeling and therapeutic efficacy of the angiotensin receptor blocker telmisartan on atherosclerotic vascular damage. We treated Rgs2(+/+), Rgs2(+/-) and Rgs2(-/-) mice with saline (control group), AngII (1000 ng per kg per min, AngII group) or low-dose telmisartan (0.3 mg per kg per day) with AngII infusion (AngII+Telmi group) for 4 weeks. For all genotypes, the AngII groups exhibited significantly higher blood pressure, a higher mortality rate and a higher incidence of aortic aneurysm than the respective control group. Interestingly, aneurysm incidence was decreased in the AngII+Telmi group compared with the AngII group in Rgs2(-/-) mice (6.7 vs. 42.9%, P<0.05), but not in Rgs2(+/+) mice (38.9 vs. 40.0%). Moreover, in Rgs2(-/-) mice, the AngII+Telmi group exhibited significant improvement in survival, reduction of enlarged aortic diameter, inhibition of superoxide production and suppression of NAD(P)H oxidase activity compared with the AngII group. Thus, Rgs2 deficiency potentiates the vascular protection effect of low-dose telmisartan. Our results suggest that angiotensin receptor blocker may be useful for protection from cardiovascular events in hypertensive subjects with risk alleles in the RGS2 gene.

Promoter Polymorphism of RGS2 Gene Is Associated with Change of Blood Pressure in Subjects with Antihypertensive Treatment: The Azelnidipine and Temocapril in Hypertensive Patients with Type 2 Diabetes Study

We performed a prospective study to examine the genetic effect on the response to a calcium (Ca) channel blocker, azelnidipine and an ACE inhibitor, temocapril treatment in patients with hypertension, as a part of the prior clinical trial, the Azelnidipine and Temocapril in Hypertensive Patients with Type 2 Diabetes Study (ATTEST). Methods and Results. All subjects who gave informed consent for genetic research were divided into two groups: the subjects treated with azelnidipine or temocapril, for 52 weeks. We selected 18 susceptible genes for hypertension and determined their genotypes using TaqMan PCR method. RNA samples were extracted from peripheral blood, and quantitative real time PCR for all genes was performed using TaqMan method. One of the polymorphisms of the RGS2 gene was extracted as being able to influence the effect of these treatments to reduce BP. At eight weeks, BP change showed a significant interaction between the A-638G polymorphism of Regulator of G protein signaling-2 (RGS2) gene and treatment with azelnidipine or temocapril. There was no gene whose expression was associated with BP phenotypes or the polymorphisms of each gene. Conclusions. A-638G polymorphism of the RGS-2 gene could be a predictive factor for therapeutic performance of Ca channel blockers.

Association analysis between functional polymorphism of the rs4606 SNP in the RGS2 gene and antipsychotic-induced Parkinsonism in Japanese patients with schizophrenia: Results from the Juntendo University Schizophrenia Projects (JUSP)

Antipsychotic-induced extrapyramidal symptoms (AIEPSs) are commonly recognized side effects of typical 1st generation antipsychotics, and considerable variability is seen in the susceptibility of individual patients to AIEPSs. Regulator of G-protein signaling 2 (RGS2) proteins regulate intracellular signaling and second messenger activation of molecules including dopamine, serotonin, and acetylcholine receptors, all of which appear to be involved in the pathophysiology of AIEPSs. Previous studies have shown an association between AIEPSs in schizophrenia and RGS2, especially the minor G allele of single nucleotide polymorphism (SNP) rs4606 (+2971C>G) in RGS2, and have suggested a possible protective effect by the G allele on AIEPSs. In this study, we investigated whether the rs4606 SNP in RGS2 alone also showed an effect on AIEPSs by utilizing the Drug-Induced Extrapyramidal Symptom Scale (DIEPSS) in 103 Japanese patients with schizophrenia. In the assumed G allele recessive model, sialorrhea and total Parkinsonism scores were significantly higher in subjects with the GG genotype than in subjects with other genotypes. Other clinical variables were not significantly different among the various genotype groups. Controlling for clinical variables as covariates, a one-way analysis of covariance found no association between rs4606 genotypes and DIEPSS scores. Taken together, these results, although preliminary, suggest that rs4606 does not affect AIEPSs in Japanese subjects.

Polymorphism C1114G of Gene Encoding the Cardiac Regulator of G-Protein Signaling 2 May Be Associated with Number of Episodes of Neurally Mediated Syncope

The cardiac regulator of G-protein signaling 2 (RGS2) negatively regulates G-protein-coupled receptor signaling. The C1114G polymorphism reduces RGS2 gene expression. This molecular disorder may be one of the important factors influencing progress of neurally mediated syncope. The aim of the study was to evaluate the association between C1114G RGS2 polymorphism and tilting results and number of syncope episodes in patients with no other diseases. Of 214 tilted patients (39% males, 39.7 +/- 17.1 years of age), genomic DNA was extracted from cellular blood components. C1114G RGS2 polymorphism was diagnosed by designed primers. Clinical variables and genetic traits were introduced into multivariate stepwise regression. Analysis was performed as follows: positive tilting n = 145 vs. negative n = 69, positive passive n = 49 vs. nitroglycerin (NTG)-positive n = 96, dominant vasodepressive n = 111 vs. cardioinhibition n = 34; and in number of syncope groups with cut-off >or=10 vs. <10. No relationship was found between the studied polymorphism and outcome of tilting (p >0.05). In multivariate regression model, homozygosity G/G 1114 RGS2 was the only variable associated with a reduced number of episodes of syncope (95% CI 2.3-10.9; p = 0.04). Our preliminary results suggest the association of G/G 1114 RGS2 genotype with the number of episodes of neurally mediated syncope. Detailed molecular mechanism of the influence of the studied polymorphism on syncopal number is probably associated with the reduced expression of RGS2 gene.

Are RGS2 Gene Polymorphisms Associated With High Blood Pressure in an Ethnicity- and Gender-Specific Manner?

Polymorphisms in the Regulator of G-protein Signaling 2 (RGS2) gene have been reported to be associated with hypertension (HT) in Japanese women and black Americans of either gender but not in white Americans or Japanese men. We have tested whether these proposed ethnicity- and gender-specific associations between RGS2 gene polymorphisms and HT can be confirmed in an independent population of male and female blacks, whites, and south Asians. A population-based sample of 1379 black, white Dutch, and south Asian subjects from the Amsterdam area was genotyped for eight polymorphisms in the RGS2 gene. All analyses were done separately per ethnic group. The phenotype high blood pressure was defined as a dichotomous variable comparing HT vs. normotension (NT) and as a linear variable using systolic blood pressure (SBP) in a multiple regression analysis with concomitant antihypertensive medication, age and body mass index as coexplanatory variables. Ethnic differences in the frequency of polymorphisms and haplotypes (HAPs) derived thereof were in line with previous studies. Our data do not confirm previously reported ethnicity- or gender-specific associations regardless which phenotype definition was used. While the D allele of 1891-1892TC insertion/deletion polymorphism showed association in several groups, they differed from previously reported ones. Haplotype-phenotype analysis was not more sensitive to detect genotype-phenotype associations than individual alleles. Previously reported ethnicity- and gender-specific associations of RGS2 genotype and hypertensive phenotype are not robust. Nevertheless, the 1891-1892TC insertion/deletion polymorphism warrants further investigation.

Variant in RGS2 moderates posttraumatic stress symptoms following potentially traumatic event exposure

Polymorphisms in the RGS2 (regulator of G-protein signaling 2) gene were found to be associated with anxious behavior in mice and anxiety in humans. We examined whether rs4606, a single nucleotide polymorphism (SNP) of RGS2, and social support moderated risk for PTSD in an epidemiologic sample. The study examines 607 adults from the 2004 Florida Hurricanes study who returned buccal DNA samples via mail. rs4606 was associated with increased symptoms of posthurricane PTSD symptoms under conditions of high hurricane exposure and low social support ( P < .05). Further, this polymorphism was associated with lifetime PTSD symptoms under conditions of lifetime exposure to a potentially traumatic event, and low social support ( P < .001). These gene by environment interactions remained significant after adjustment for sex, ancestry, and age. RGS2 rs4606 modifies risk of postdisaster and lifetime PTSD symptoms under conditions of high stressor exposure. This is the first demonstration of gene–environment interaction for this locus.

RGS2and generalized anxiety disorder in an epidemiologic sample of hurricane-exposed adults

Generalized anxiety disorder (GAD) is a common and sometimes disabling condition often associated with stressful life events that involve significant loss or danger. The disorder appears moderately heritable. Polymorphisms in the RGS2 (regulator of G-protein signaling 2) gene were recently associated with anxious behavior in mice and panic disorder and trait anxiety in humans. We examined whether rs4606, a single nucleotide polymorphism (SNP) in the 3' UTR of RGS2, was associated with GAD in an epidemiologic sample of adults exposed to the 2004 Florida Hurricanes. The sample for the current study is 607 adults from the 2004 Florida Hurricane Study who returned buccal DNA samples via mail. Participants were selected via random digit dial procedures and interviewed via telephone about hurricane exposure, social support, and GAD symptoms. The outcome measure was DSM-IV diagnosis of GAD derived from structured interviews. RGS2 SNP rs4606 was significantly associated with GAD in this sample. In logistic regression analyses, each C allele was associated with a 100% (P=.026) increased risk of GAD after controlling for age, sex, ancestry, hurricane exposure, and social support. These findings are consistent with a previously published study showing a higher prevalence of the C allele among panic disorder patients than controls. This study points toward a relevant polymorphism for GAD at the 3' end of the RGS2 gene; and suggests that studying a recently disaster-exposed sample is both feasible and may improve power to find gene-disorder associations.

Further evidence for association of the RGS2 gene with antipsychotic-induced parkinsonism: protective role of a functional polymorphism in the 3′-untranslated region

RGS2 (regulator of G-protein signaling 2) modulates dopamine receptor signal transduction. Functional variants in the gene may influence susceptibility to extrapyramidal symptoms (EPS) induced by antipsychotic drugs. To further investigate our previous report of association of the RGS2 gene with susceptibility to antipsychotic-induced EPS, we performed a replication study. EPS were rated in 184 US patients with schizophrenia (115 African Americans, 69 Caucasian) treated for at least a month with typical antipsychotic drugs (n=45), risperidone (n=46), olanzapine (n=50) or clozapine (n=43). Six single nucleotide polymorphisms (SNPs) within or flanking RGS2 were genotyped (rs1933695, rs2179652, rs2746073, rs4606, rs1819741 and rs1152746). Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression. Our results indicate association of SNP rs4606 with antipsychotic-induced parkinsonism (AIP), as measured by the Simpson Angus scale, in the overall sample and in the African-American subsample, the G (minor) allele having a protective effect. ORs for AIP among rs4606 G-allele carriers were 0.23 (95% CI 0.10-0.54, P=0.001) in the overall sample, and 0.20 (0.07-0.57, P=0.003) in the African-American subsample. In the previously studied Israeli sample the OR was 0.31 (0.11-0.84, P=0.02). We completely sequenced the RGS2 gene in nine patients with AIP and nine patients without, from the Israeli sample. No common coding polymorphisms or additional regulatory variants were revealed, suggesting that association of the rs4606 C/G polymorphism with AIP is biologically meaningful and not a consequence of linkage disequilibrium with another functional variant. Taken together, the findings of the current study support the association of RGS2 with AIP and focus on a possible protective effect of the minor G allele of SNP rs4606. This SNP is located in the 3'-regulatory region of the gene, and is known to influence RGS2 mRNA levels and protein expression.

Association of RGS2 Gene Polymorphisms with Suicide and Increased RGS2 Immunoreactivity in the Postmortem Brain of Suicide Victims

Regulators of G-protein signaling are a family of proteins that negatively regulate the intracellular signaling of G protein-coupled receptors, such as the serotonin receptor. Recent studies have suggested that one of these proteins, the regulator of G-protein signaling 2 (RGS2), plays an important part in anxiety and/or aggressive behavior. To explore the involvement of the RGS2 gene in the vulnerability to suicide, we screened Japanese suicide victims for sequence variations in the RGS2 gene and carried out an association study of RGS2 gene polymorphisms with suicide victims. In the eight identified polymorphisms that were identified by mutation screening, we genotyped four common single-nucleotide polymorphisms (SNPs) in the RGS2 gene, and found significant differences in the distribution of the SNP3 (C+2971G, rs4606) genotypes and alleles of the SNP2 (C-395G, rs2746072) and the SNP3 between completed suicides and the controls. The distribution of the haplotype was also significantly different between the two groups (global p<0.0001). Furthermore, RGS2 immunoreactivity significantly increased in the amygdala and the prefrontal cortex (Brodmann area 9 (BA9)) of the postmortem brain of the suicide subjects. These findings suggest that RGS2 is genetically involved in the biological susceptibility to suicide in the Japanese population.

Reduced expression of regulator of G-protein signaling 2 (RGS2) in hypertensive patients increases calcium mobilization and ERK1/2 phosphorylation induced by angiotensin II

RGS2 (regulators of G-protein signaling) is a negative regulator of Galphaq protein signaling, which mediates the action of several vasoconstrictors. RGS2-deficient mouse line exhibits a hypertensive phenotype and a prolonged response to vasoconstrictors. To compare RGS2 expression in peripheral blood mononuclear cells (PBMs) and cultured fibroblasts from normotensive subjects and hypertensive patients. PBMs were isolated from 100 controls and 150 essential hypertensives. Additionally, fibroblasts were isolated from skin biopsy of 11 normotensives and 12 hypertensives and cultured up to the third passage. Quantitative mRNA and protein RGS2 expression were performed by real-time quantitative reverse transcriptase-polymerase chain reaction and by immunoblotting, respectively. Free Ca measurement was performed in monolayers of 24-h serum-deprived cells, using FURA-2 AM. Phosphorylation of the extracellular signal-regulated kinases ERK1/2 was measured by immunoblotting. Polymorphism (C1114G) in the 3' untranslated region of the RGS2 gene was investigated by direct sequencing and real-time polymerase chain reaction (PCR). RGS2 mRNA expression was significantly lower in PBM and in fibroblasts from hypertensives, in comparison to normotensives. C1114G polymorphism was associated with RGS2 expression, with the lowest values in GG hypertensives. The 1114G allele frequency was increased in hypertensives compared with normotensives. Angiotensin II-stimulated intracellular Ca increase and ERK1/2 phosphorylation were higher in fibroblasts from hypertensive patients compared with control subjects, and in those with the G allele, independently of the blood pressure status. The angiotensin II-stimulated Ca mobilization and ERK1/2 phosphorylation were negatively correlated with RGS2 mRNA expression. Low expression of RGS2 contributes to increased G-protein-coupled signaling in hypertensive patients. The allele G is associated with low RGS2 expression and blood pressure increase in humans.

Analysis of regulator of G-protein signaling-2 (RGS-2) expression and function in osteoblastic cells.

Regulator of G-protein signaling-2 (RGS-2) belongs to a novel family of GTPase-activating proteins that rapidly turn-off G-protein coupled receptor signaling. RGS proteins contain a characteristic RGS domain by which they interact with the alpha-subunit of G-proteins and drive them into their inactive GDP-bound forms. Previously, we have reported that RGS-2 mRNA is rapidly and transiently increased by PTH in rat bone and in osteoblast cultures in vitro. In this study, we further explored the molecular basis for the regulation of RGS-2 by cloning and functionally characterizing the RGS-2 gene promoter. We cloned 2.3- and 2.8-kb fragments of the 5'-flanking regions of the rat and mouse RGS-2 genes, respectively, and generated a stable clone of UMR106 osteoblastic cells containing the rat RGS-2 promoter driving the beta-gal reporter gene (p2.3RGS-2-beta-gal). Treatment of the stable clone with PTH resulted in a maximal 2.2- to 3.6-fold increase in promoter activity at 8 h, reminiscent of the early response observed with endogenous RGS-2 mRNA regulation. Further, PTH (1-38), (1-31), PTHrP (1-34), and forskolin, which elevate cAMP levels, stimulated the promoter, while PTH (3-34) and (7-34), which do not readily stimulate cAMP accumulation, and PMA that directly activates protein kinase C, had no effect on promoter activity. Taken together, these results implicate the involvement of the Galpha(s)-adenylate cyclase-protein kinase A pathway in stimulating RGS-2 expression. Maintenance of a hyperphosphorylated state via the inhibition of type 2A protein phosphatases by okadaic acid, resulted in a strong dose-dependent increase in transcriptional activity of the RGS-2 promoter as well as that of the endogenous RGS-2 gene. Furthermore, overexpression of the osteoblast-specific transcription factor Runx2 also led to a stimulation of RGS-2 promoter activity. Functional analysis using RGS-2 overexpression suggests the potential negative regulatory effects of RGS-2 on PTH- and forskolin-induced cAMP production in osteoblastic cells. In summary, our data suggest that PTH treatment results in a direct transcriptional stimulation of RGS-2 that in turn may play a role in modulating the duration/intensity of PTH receptor signaling.