Responses of Regulator of G Protein Signaling Proteins and Circadian Clock Components to Sustained Depolarization-Induced Dedifferentiation in MIN6 β-Cells.

Preeclampsia is a multifaceted pregnancy-associated hypertensive disorder that poses a major threat to maternal and fetal health. Though the etiology is not fully understood, syncytiotrophoblast stress is postulated to be a major driver of maternal symptomology. We previously demonstrated that regulator of G protein signaling-2 (RGS2) expression decreases in human preeclamptic placenta and has a transcriptional dependence on histone deacetylase 9 (HDAC9) in trophoblast cells. Furthermore, experimental reductions of Rgs2 expression in the mouse fetoplacental unit are sufficient to induce preeclampsia-like features, including placental stress, in C57BL/6J dams. Here, we examined the hypotheses that HDAC9 and RGS2 are both expressed within syncytiotrophoblasts, that HDAC9 and RGS2 expression are positively correlated within these cells, and that expression of each is reduced within syncytiotrophoblasts during preeclampsia. HDAC9 and RGS2 mRNA were localized and quantified in syncytiotrophoblast cells of human placental samples from pregnancies with and without preeclampsia, using laser-capture microdissection and in situ hybridization methods. Expression of Hdac9 and Rgs2 was similarly localized in the syncytiotrophoblast of the mouse placenta. Throughout, HDAC9/Hdac9 and RGS2/Rgs2 were detected and positively correlated in syncytiotrophoblasts, but expression of each was substantially reduced during preeclampsia. These results document reduced HDAC9 and RGS2 expression specifically in syncytiotrophoblast cells during preeclampsia and provide additional correlative support of HDAC9-mediated control of RGS2 expression within this population of trophoblasts. This work provides rationale to further explore cell-specific disruptions in HDAC9 and RGS2 control and function as a cause of syncytiotrophoblast stress and ultimately preeclampsia.NEW & NOTEWORTHY Syncytiotrophoblast stress contributes to the pathogenesis of preeclampsia, but many of the underlying causes remain undetermined. Previous work has implicated the loss of placental HDAC9-mediated Rgs2 transcription in the disorder. Extending these findings, we report that HDAC9 and RGS2 were abundant and localized primarily to syncytiotrophoblast cells of the control placenta. Expression of both targets was attenuated in these cells during preeclampsia and thus may be an underappreciated source of syncytiotrophoblast stress, warranting further investigation.

Klotho is a recently discovered antiaging gene. Klotho is expressed in mouse pancreatic islets and in insulinoma β-cells (MIN6 β-cells). The purpose of this study was to investigate whether Klotho plays a role in the regulation of insulin secretion in MIN6 β-cells by overexpression and silencing of Klotho. It is interesting that overexpression of Klotho increased glucose-induced insulin secretion in MIN6 β-cells. Overexpression of mouse Klotho protein also significantly increased plasma membrane levels of transient receptor potential V2 (TRPV2), calcium entry, and the glucose-induced increase in intracellular calcium. On the other hand, knockdown of Klotho by siRNA significantly decreased plasma membrane levels of TRPV2 and attenuated glucose-induced calcium entry and insulin secretion. Tranilast, a selective inhibitor of TRPV2, abolished the promoting effects of overexpression of Klotho on glucose-induced calcium entry and insulin secretion in MIN6 cells. Thus, TRPV2 lies in the downstream of Klotho in the regulation of glucose-induced insulin secretion. This study demonstrated, for the first time, that Klotho may enhance glucose-induced insulin secretion by up-regulating plasma membrane levels of TRPV2 and thus glucose-induced calcium responses. These findings reveal a previously unidentified role of Klotho in the regulation of glucose-induced insulin secretion in MIN6 β-cells.

Alterations in cardiac G protein-mediated signaling, most prominently G(q/11) signaling, are centrally involved in hypertrophy and heart failure development. Several RGS proteins that can act as negative regulators of G protein signaling are expressed in the heart, but their functional roles are still poorly understood. RGS expression changes have been described in hypertrophic and failing hearts. In this study, we report a marked decrease in RGS2 (but not other major cardiac RGS proteins (RGS3-RGS5)) that occurs prior to hypertrophy development in different models with enhanced G(q/11) signaling (transgenic expression of activated Galpha(q)(*) and pressure overload due to aortic constriction). To assess functional consequences of selective down-regulation of endogenous RGS2, we identified targeting sequences for effective RGS2 RNA interference and used lipid-based transfection to achieve uptake of fluorescently labeled RGS2 small interfering RNA in >90% of neonatal and adult ventricular myocytes. Endogenous RGS2 expression was dose-dependently suppressed (up to 90%) with no major change in RGS3-RGS5. RGS2 knockdown increased phenylephrine- and endothelin-1-induced phospholipase Cbeta stimulation in both cell types and exacerbated the hypertrophic effect (increase in cell size and radiolabeled protein) in neonatal myocytes, with no major change in G(q/11)-mediated ERK1/2, p38, or JNK activation. Taken together, this study demonstrates that endogenous RGS2 exerts functionally important inhibitory restraint on G(q/11)-mediated phospholipase Cbeta activation and hypertrophy in ventricular myocytes. Our findings point toward a potential pathophysiological role of loss of fine tuning due to selective RGS2 down-regulation in G(q/11)-mediated remodeling. Furthermore, this study shows the feasibility of effective RNA interference in cardiomyocytes using lipid-based small interfering RNA transfection.

Of all partners involved in G-protein coupled receptor (GPCR) signalling, the regulator of G-protein signalling (RGS) proteins are the only ones showing fast gene expression changes after various stimuli. These expression changes can offer feedback regulation to GPCR signalling as RGS accelerate the return of G-proteins to their inactive form and exert regulatory functions on intracellular effectors. However, it is not yet known which RGS regulate which receptor transduction pathways in the brain. To start to answer this question, we studied the influence of specific agonists and antagonists of the dopamine D1 and D2 receptors on the gene expression of the five most abundant RGS in the striatum: RGS2, RGS4, RGS8, RGS9 and RGS10. Only changes in RGS2 and RGS4 mRNA levels were observed. The D1 agonist SKF82958 and D2 antagonist haloperidol caused an up-regulation of RGS2 (+ 38.0% and + 41.6%, respectively). The D1 antagonist SCH23390 and D2 agonist quinpirole caused a down-regulation of RGS2 (- 25.0% and - 35.0%) and an up-regulation of RGS4 (+ 57.2% and + 52.5%). D1 and D2 receptors exert opposite effects on RGS2 expression, as they do on cAMP levels, suggesting a cAMP-mediated transcription of RGS2. This was confirmed by the unique induction of RGS2 (+ 111.1%) observed in the periventricular zone of the striatum after intracerebroventricular injection of forskolin. RGS4 was up-regulated only when RGS2 was down-regulated. This suggests that both RGS exert distinct functions. Considering the coupling of D1 and D2 receptors to the intracellular effector adenylate cyclase 5 (AC5) through their respective Galpha subunits in the striatum, our data allow us to suggest that RGS2 regulates the D1/Galphaolf/AC5 pathway and RGS4 the D2/Galphao/AC5 pathway.

This study determined the effect of chronic isoproterenol (ISO) treatment on cardiac structure and function in male mice without or with dual loss of regulator of G protein signaling (RGS) 2 and 5. RGS2 and 5 act as GTPase-activating proteins (GAPs) that preferentially terminate signaling via G q/11 - and G i/o class G proteins, by accelerating GTP hydrolysis. Deletion of either RGS2 or 5 increases susceptibility to cardiovascular disease. However, the effects of dual absence of the two RGS proteins on normal physiology and disease are unknown. Using mice concurrently lacking RGS2 and 5 ( Rgs2/5 dbKO) and wild type (WT) cohort, we determined how the dual absence of both RGS proteins affects cardiac response to chronic β-adrenergic receptor stimulation. WT and Rgs2/5 dbKO mice were infused with saline or 30 μg/g/day of ISO for 3 or 14 days. At baseline, Rgs2/5 dbKO mice showed cardiac hypertrophy (WT: 9.06 ± 0.34 vs. dbKO: 10.16 ± 0.32 mg/mm tibia length; p <0.05) and left ventricular chamber dilation by echocardiography, without tissue fibrosis. ISO infusion for 3 days caused and augmented cardiac hypertrophy in WT (SAL: 9.65 ± 0.38 vs. ISO: 11.68 ± 0.45 mg/mm tibia length; p <0.05) and Rgs2/5 dbKO (SAL: 9.97 ± 0.43 vs. ISO: 12.57 ± 0.69 mg/mm tibia length; p <0.01) mice, respectively, as well as interstitial fibrosis and increased expression of hypertrophic and heart failure gene markers, including Nppa , Serca , Mybpc3 and Tnni3 . Sub-chronic ISO infusion also caused a greater decrease in percent fractional shortening by day 3, in Rgs2/5 dbKO relative to WT mice (WT: -4.22 ± 4.15 vs. dbKO: -7.69 ± 3.86 %; p <0.05). In WT mice, cardiac hypertrophy and left ventricular dilation were lower after 14-day compared to 3-day ISO infusion, but similar to saline-treated control levels (ΔSAL to 3d-ISO: 21.04 ± 0.82 vs. ΔSAL to 14d-ISO: 15.05 ± 0.66 %; p <0.05). This was accompanied by a robust increase in Rgs5 but not Rgs2 mRNA expression in WT mice. In contrast, Rgs2/5 dbKO mice continued to show abnormal cardiac structure and function after 14-day ISO infusion. Together, these data suggest that increased expression of RGS5 compensates for the lack of change in RGS2 expression and/or function and protects against transition from ISO-induced cardiac hypertrophy to heart failure.

The “regulators of g-protein signalling” (RGS) comprise a large family of proteins that limit by virtue of their GTPase accelerating protein domain the signal transduction of G-protein coupled receptors. RGS proteins have been implicated in various neuropsychiatric diseases such as schizophrenia, drug abuse, depression and anxiety and aggressive behaviour. Since conditions associated with a large increase of adenosine in the brain such as seizures or ischemia were reported to modify the expression of some RGS proteins we hypothesized that adenosine might regulate RGS expression in neural cells. We measured the expression of RGS-2,-3, and -4 in both transformed glia cells (human U373 MG astrocytoma cells) and in primary rat astrocyte cultures stimulated with adenosine agonists. Expression of RGS-2 mRNA as well as RGS2 protein was increased up to 30-fold by adenosine agonists in astrocytes. The order of potency of agonists and the blockade by the adenosine A2B-antagonist MRS1706 indicated that this effect was largely mediated by adenosine A2B receptors. However, a smaller effect was observed due to activation of adenosine A2A receptors. In astrocytoma cells adenosine agonists elicited an increase in RGS-2 expression solely mediated by A2B receptors. Expression of RGS-3 was inhibited by adenosine agonists in both astrocytoma cells and astrocytes. However while this effect was mediated by A2B receptors in astrocytoma cells it was mediated by A2A receptors in astrocytes as assessed by the order of potency of agonists and selective blockade by the specific antagonists MRS1706 and ZM241385 respectively. RGS-4 expression was inhibited in astrocytoma cells but enhanced in astrocytes by adenosine agonists.

The glucose-dependent insulinotropic peptide receptor (GIP-R) is a member of the G protein-coupled receptors. Recent studies have indicated that elevated serum GIP concentrations in type II diabetic patients might induce desensitization of the GIP-R, and this mechanism could contribute to impaired insulin secretion. The cellular and molecular mechanisms governing GIP desensitization are unknown. Here, we report the results of studies on a new family of proteins known as regulators of G protein signaling (RGS) that have been shown to mediate the desensitization process of other receptors. GIP-R and RGS1, -2, -3, and -4 complementary DNAs were cotransfected into human embryonic kidney cells (L293). GIP-stimulated cAMP generation in control cells and in those coexpressing RGS1, -3, and -4 displayed a dose-dependent increase 10 min after GIP treatment. In contrast, RGS2 expression inhibited the GIP-induced cAMP response by 50%, a response similar to that of cells desensitized by preincubation with 10(-7) M GIP. In betaTC3 cells, preincubation of GIP attenuated GIP-induced insulin release by 45% at 15 min and by 55% at 30 min. Expression of RGS2 in the betaTC3 cells significantly decreased GIP-stimulated insulin secretion, whereas glucose-induced insulin release was not affected. RGS2 messenger RNA was identified by Northern blot analysis to be expressed endogenously in betaTC3 and L293 cells, and its level was significantly induced by GIP treatment in betaTC3 cells. Moreover, RGS2 bound Gs alpha protein in an in vitro system, suggesting that RGS2 attenuated the Gs-adenylate cyclase signaling pathway. These results suggest a potential role for RGS2 in modulating GIP-mediated insulin secretion in pancreatic islet cells.

Regulator of G protein signaling (RGS) proteins attenuate signaling of G protein‐coupled receptors associated with breast cancer metastasis. Using quantitative real‐time PCR, we found that RGS4 mRNA was selectively and significantly up‐regulated in highly metastatic breast cancer MDA‐MB‐231 and −436 cells. However, despite this high level of RGS4 transcription, RGS4 protein was barely detectable in these cells and additional studies determined that this was due to proteasome‐dependent degradation of RGS4 protein. Proteasome inhibitor‐induced blockade of endogenous RGS4 protein degradation attenuated the migration and invasion of theses cells in transwell assays. Similarly, transient expression of RGS4, but not other RGS proteins, in MDA‐MB‐231 cells also caused marked inhibition of these indices of tumor metastasis. Stable expression of RGS4, but not its function deficient N128A mutant, in MDA‐MB‐231 cells effectively attenuated cancer cell invasiveness both in vitro and in a mouse xenograft model. Other in vitro studies showed that RGS4 selectively inhibits Gi‐dependent activation of small G protein Rac, a key activator of breast cancer metastasis. Finally, immunohistochemistry studies of human breast carcinoma specimens indicated that RGS4 protein levels are down‐regulated in the cancer cells and that this down‐regulation is a relatively late event associated with the invasive phenotype.

Acting as GTPase activating proteins promoting the silencing of activated G-proteins, regulators of G protein signaling (RGSs) are generally considered negative modulators of cell signaling. In the CNS, the expression of RGS4 is altered in diverse pathologies and its upregulation was reported in astrocytes exposed to an inflammatory environment. In a model of cultured cortical astrocytes, we herein investigate the influence of RGS4 on intracellular calcium signaling mediated by type 5 metabotropic glutamate receptor (mGluR5), which is known to support the bidirectional communication between neurons and glial cells. RGS4 activity was manipulated by exposure to the inhibitor CCG 63802 or by infecting the cells with lentiviruses designed to achieve the silencing or overexpression of RGS4. The pharmacological inhibition or silencing of RGS4 resulted in a decrease in the percentage of cells responding to the mGluR5 agonist DHPG and in the proportion of cells showing typical calcium oscillations. Conversely, RGS4-lentivirus infection increased the percentage of cells showing calcium oscillations. While the physiological implication of cytosolic calcium oscillations in astrocytes is still under investigation, the fine-tuning of calcium signaling likely determines the coding of diverse biological events. Indirect signaling modulators such as RGS4 inhibitors, used in combination with receptor ligands, could pave the way for new therapeutic approaches for diverse neurological disorders with improved efficacy and selectivity.

G protein-coupled receptors (GPCRs) and their ligands are critical regulators of neural progenitor differentiation, and GPCR signaling pathways are regulated by regulator of G protein signaling (RGS) proteins. RGS protein expression is dynamically regulated, and we have recently described the epigenetic regulation of RGS transcript expression. Given the potential of RGS proteins to regulate GPCR signaling and the established role of epigenetic regulation in progenitor differentiation, we explored the impact of epigenetic regulation of RGS transcripts during in vitro differentiation of human neural progenitors. Here, we demonstrate robust upregulation of the RGS transcripts RGS4, RGS5, RGS6, RGS7, and RGS11 during neuronal differentiation, while DNA methyltransferase (DNMT) and histone deacetylase enzyme expression is suppressed during differentiation. Transcripts encoding R7 subfamily RGS proteins and the R7-binding partners R7BP and R9AP showed the greatest upregulation. Further, we showed that direct pharmacological inhibition of DNMT activity enhances expression of RGS2, RGS4, RGS5, RGS6, RGS7, RGS8, RGS9L, RGS10, and RGS14 as well as R7BP and R9AP transcripts in progenitors, consistent with regulation by DNMTs. Our results reveal marked upregulation of RGS expression during neuronal differentiation and suggest that decreased expression of DNMT enzymes during differentiation contributes to upregulation. © 2014 S. Karger AG, Basel

Simple SummaryRecent advances in molecular medicine have indicated G-protein coupled receptors (GPCRs) as possible therapeutic targets in ovarian cancer. The cellular effects of GPCRs are determined by regulator of G protein signaling (RGS) proteins. Especially RGS2 has currently moved into focus of cancer therapy. Therefore, we retrospectively analyzed RGS2 and its association with the prognosis of high-grade serous ovarian cancer (HGSOC). Here, we provide in situ and in silico analyses regarding the expression patterns and prognostic value of RGS2. In silico we found that RGS2 is barely detectable in tumor cells on the mRNA level in bulk and single-cell data. Applying immunohistochemistry in 519 HGSOC patients, we detected moderate to strong protein expression of RGS2 in situ in approximately half of the cohort, suggesting regulation by post translational modification. Furthermore, low protein expression of RGS2 was associated with an inferior overall- and progression-free survival. These results warrant further research of its role and related new therapeutic implications in HGSOC.RGS2 regulates G-protein signaling by accelerating hydrolysis of GTP and has been identified as a potentially druggable target in carcinomas. Since the prognosis of patients with high-grade serous ovarian carcinoma (HGSOC) remains utterly poor, new therapeutic options are urgently needed. Previous in vitro studies have linked RGS2 suppression to chemoresistance in HGSOC, but in situ data are still missing. In this study, we characterized the expression of RGS2 and its relation to prognosis in HGSOC on the protein level by immunohistochemistry in 519 patients treated at Charité, on the mRNA level in 299 cases from TCGA and on the single-cell level in 19 cases from publicly available datasets. We found that RGS2 is barely detectable on the mRNA level in both bulk tissue (median 8.2. normalized mRNA reads) and single-cell data (median 0 normalized counts), but variably present on the protein level (median 34.5% positive tumor cells, moderate/strong expression in approximately 50% of samples). Interestingly, low expression of RGS2 had a negative impact on overall survival (p = 0.037) and progression-free survival (p = 0.058) on the protein level in lower FIGO stages and in the absence of residual tumor burden. A similar trend was detected on the mRNA level. Our results indicated a significant prognostic impact of RGS2 protein suppression in HGSOC. Due to diverging expression patterns of RGS2 on mRNA and protein levels, posttranslational modification of RGS2 is likely. Our findings warrant further research to unravel the functional role of RGS2 in HGSOC, especially in the light of new drug discovery.

Cellular stress increases RGS2 mRNA and decreases RGS4 mRNA levels in SH-SY5Y cells

Regulators of G-protein signalling: multifunctional proteins with impact on signalling in the cardiovascular system

BackgroundA critical therapeutic challenge in epithelial ovarian carcinoma is the development of chemoresistance among tumor cells following exposure to first line chemotherapeutics. The molecular and genetic changes that drive the development of chemoresistance are unknown, and this lack of mechanistic insight is a major obstacle in preventing and predicting the occurrence of refractory disease. We have recently shown that Regulators of G-protein Signaling (RGS) proteins negatively regulate signaling by lysophosphatidic acid (LPA), a growth factor elevated in malignant ascites fluid that triggers oncogenic growth and survival signaling in ovarian cancer cells. The goal of this study was to determine the role of RGS protein expression in ovarian cancer chemoresistance.ResultsIn this study, we find that RGS2, RGS5, RGS10 and RGS17 transcripts are expressed at significantly lower levels in cells resistant to chemotherapy compared with parental, chemo-sensitive cells in gene expression datasets of multiple models of chemoresistance. Further, exposure of SKOV-3 cells to cytotoxic chemotherapy causes acute, persistent downregulation of RGS10 and RGS17 transcript expression. Direct inhibition of RGS10 or RGS17 expression using siRNA knock-down significantly reduces chemotherapy-induced cell toxicity. The effects of cisplatin, vincristine, and docetaxel are inhibited following RGS10 and RGS17 knock-down in cell viability assays and phosphatidyl serine externalization assays in SKOV-3 cells and MDR-HeyA8 cells. We further show that AKT activation is higher following RGS10 knock-down and RGS 10 and RGS17 overexpression blocked LPA mediated activation of AKT, suggesting that RGS proteins may blunt AKT survival pathways.ConclusionsTaken together, our data suggest that chemotherapy exposure triggers loss of RGS10 and RGS17 expression in ovarian cancer cells, and that loss of expression contributes to the development of chemoresistance, possibly through amplification of endogenous AKT signals. Our results establish RGS10 and RGS17 as novel regulators of cell survival and chemoresistance in ovarian cancer cells and suggest that their reduced expression may be diagnostic of chemoresistance.

Second messengers regulate RGS2 expression which is targeted to the nucleus