Regulator Of G-protein Signaling 10 Research Articles

RGS10 mitigates high glucose-induced microglial inflammation via the reactive oxidative stress pathway and enhances synuclein clearance in microglia.

Microglia play a critical role in maintaining brain homeostasis but become dysregulated in neurodegenerative diseases. Regulator of G-protein Signaling 10 (RGS10), one of the most abundant homeostasis proteins in microglia, decreases with aging and functions as a negative regulator of microglia activation. RGS10-deficient mice exhibit impaired glucose tolerance, and high-fat diet induces insulin resistance in these mice. In this study, we investigated whether RGS10 modulates microglia activation in response to hyperglycemic conditions, complementing our previous findings of its role in inflammatory stimuli. In RGS10 knockdown (KD) BV2 cells, TNF production increased significantly in response to high glucose, particularly under proinflammatory conditions. Additionally, glucose uptake and GLUT1 mRNA levels were significantly elevated in RGS10 KD BV2 cells. These cells produced higher ROS and displayed reduced sensitivity to the antioxidant N-Acetyl Cysteine (NAC) when exposed to high glucose. Notably, both BV2 cells and primary microglia that lack RGS10 exhibited impaired uptake of alpha-synuclein aggregates. These findings suggest that RGS10 acts as a negative regulator of microglia activation not only in response to inflammation but also under hyperglycemic conditions.

RGS10 suppression by DNA methylation is associated with low survival rates in colorectal carcinoma

Colorectal cancer is known as the third most common cancer in both women and men. Genetic and epigenetic changes are major players contributing to colorectal carcinogenesis. Regulator of G-protein signaling 10 (RGS10) is a member of the RGS proteins, which negatively regulate several signaling pathways including cell survival and proliferation. We and others have previously shown that RGS10 expression is modulated by epigenetic modifications in ovarian cancer and suppression of RGS10 partially contributes to chemoresistance. Here, we further analyzed the roles and regulation of RGS10 in colon adenocarcinoma (COAD), using broad bioinformatics tools. We analyzed the expression profiles, promoter methylation state, prognostic value and effect of a hypomethylating agent on RGS10 expression. Results showed that RGS10 expression is higher in normal colon tissues than in tumor tissues. In addition, there is a negative correlation between DNA methylation and RGS10 transcript expression. We also observed that gene expression and promoter methylation of RGS10 in colorectal carcinoma patients were differently expressed depending on the tumor stage and microsatellite stability. DNA methylation was significantly increased in 18 probes of RGS10, which belongs to the high-risk group in COAD. In addition, pharmacological inhibition of DNA methyltransferase with decitabine reduced the six CpGsite-specific RGS10 hypermethylation in COAD. We also experimentally confirmed that RGS10 promoter activity was inhibited by treatment with decitabine in the HT-29 colorectal cell line. We further showed that decitabine treatment increases the RGS10 transcript expression in three different colorectal carcinoma cell lines. These results suggest that RGS10 expression is suppressed in the development of colorectal cancer and inhibition of DNA methylation may contribute to increasing overall survival rates of COAD patients.

RGS10 Reduces Lethal Influenza Infection and Associated Lung Inflammation in Mice.

Seasonal influenza epidemics represent a significant global health threat. The exacerbated immune response triggered by respiratory influenza virus infection causes severe pulmonary damage and contributes to substantial morbidity and mortality. Regulator of G-protein signaling 10 (RGS10) belongs to the RGS protein family that act as GTPase activating proteins for heterotrimeric G proteins to terminate signaling pathways downstream of G protein-coupled receptors. While RGS10 is highly expressed in immune cells, in particular monocytes and macrophages, where it has strong anti-inflammatory effects, its physiological role in the respiratory immune system has not been explored yet. Here, we show that Rgs10 negatively modulates lung immune and inflammatory responses associated with severe influenza H1N1 virus respiratory infection in a mouse model. In response to influenza A virus challenge, mice lacking RGS10 experience enhanced weight loss and lung viral titers, higher mortality and significantly faster disease onset. Deficiency of Rgs10 upregulates the levels of several proinflammatory cytokines and chemokines and increases myeloid leukocyte accumulation in the infected lung, markedly neutrophils, monocytes, and inflammatory monocytes, which is associated with more pronounced lung damage. Consistent with this, influenza-infected Rgs10-deficent lungs contain more neutrophil extracellular traps and exhibit higher neutrophil elastase activities than wild-type lungs. Overall, these findings propose a novel, in vivo role for RGS10 in the respiratory immune system controlling myeloid leukocyte infiltration, viral clearance and associated clinical symptoms following lethal influenza challenge. RGS10 also holds promise as a new, potential therapeutic target for respiratory infections.

MiR‑199b‑5p mediates adriamycin‑induced podocyte apoptosis by inhibiting the expression of RGS10

Podocyte apoptosis is a key risk factor for the progression of kidney diseases. MicroRNA (miR)-199b-5p has been shown to be involved in cell apoptosis. However, the molecular mechanisms of miR-199b-5p in podocyte apoptosis remain uncertain. Thus, the present study aimed to investigate whether miR-199b-5p participates in the regulation of podocyte apoptosis and to elucidate the involved mechanisms of this process. A podocyte apoptosis model was constructed using adriamycin (ADR) in vitro. miR-199b-5p mimic and inhibitor were transfected in podocytes to change the expression level of miR-199b-5p. RNA expression was examined by reverse transcription-quantitative PCR. Western blotting was used to measure protein expression. Apoptosis was monitored via flow cytometry and detection of apoptosis-associated proteins. The results from the present study demonstrated that miR-199b-5p was upregulated and that regulator of G-protein signaling 10 (RGS10) was downregulated in ADR-stimulated podocytes. Overexpression of miR-199b-5p could inhibit RGS10 expression and stimulate podocyte apoptosis, whereas miR-199b-5p knockdown restored the levels of RGS10 and ameliorated podocyte apoptosis in ADR-induced podocytes. Furthermore, the effects of miR-199b-5p overexpression could be significantly reversed by RGS10 overexpression. In addition, podocyte transfection of miR-199b-5p activated the AKT/mechanistic target of rapamycin (mTOR) signaling, which was blocked following RGS10 overexpression. Taken together, the present study demonstrated that miR-199b-5p upregulation could promote podocyte apoptosis by inhibiting the expression of RGS10 through the activation of AKT/mTOR signaling.

Experimental colitis promotes sustained, sex-dependent, T-cell-associated neuroinflammation and parkinsonian neuropathology

BackgroundThe etiology of sporadic Parkinson’s disease (PD) remains uncertain, but genetic, epidemiological, and physiological overlap between PD and inflammatory bowel disease suggests that gut inflammation could promote dysfunction of dopamine-producing neurons in the brain. Mechanisms behind this pathological gut-brain effect and their interactions with sex and with environmental factors are not well understood but may represent targets for therapeutic intervention.MethodsWe sought to identify active inflammatory mechanisms which could potentially contribute to neuroinflammation and neurological disease in colon biopsies and peripheral blood immune cells from PD patients. Then, in mouse models, we assessed whether dextran sodium sulfate-mediated colitis could exert lingering effects on dopaminergic pathways in the brain and whether colitis increased vulnerability to a subsequent exposure to the dopaminergic neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We assessed the involvement of inflammatory mechanisms identified in the PD patients in colitis-related neurological dysfunction in male and female mice, utilizing mice lacking the Regulator of G-Protein Signaling 10 (RGS10)—an inhibitor of nuclear factor kappa B (NFκB)—to model enhanced NFκB activity, and mice in which CD8+ T-cells were depleted.ResultsHigh levels of inflammatory markers including CD8B and NFκB p65 were found in colon biopsies from PD patients, and reduced levels of RGS10 were found in immune cells in the blood. Male mice that experienced colitis exhibited sustained reductions in tyrosine hydroxylase but not in dopamine as well as sustained CD8+ T-cell infiltration and elevated Ifng expression in the brain. CD8+ T-cell depletion prevented colitis-associated reductions in dopaminergic markers in males. In both sexes, colitis potentiated the effects of MPTP. RGS10 deficiency increased baseline intestinal inflammation, colitis severity, and neuropathology.ConclusionsThis study identifies peripheral inflammatory mechanisms in PD patients and explores their potential to impact central dopaminergic pathways in mice. Our findings implicate a sex-specific interaction between gastrointestinal inflammation and neurologic vulnerability that could contribute to PD pathogenesis, and they establish the importance of CD8+ T-cells in this process in male mice.Graphical abstract

PI3K/ NF-κB-dependent TNF-α and HDAC activities facilitate LPS-induced RGS10 suppression in pulmonary macrophages

Regulator of G-protein signaling 10 (RGS10) is a member of the superfamily of RGS proteins that canonically act as GTPase activating proteins (GAPs). RGS proteins accelerate GTP hydrolysis on the G-protein α subunits and result in termination of signaling pathways downstream of G protein-coupled receptors. Beyond its GAP function, RGS10 has emerged as an anti-inflammatory protein by inhibiting LPS-mediated NF-κB activation and expression of inflammatory cytokines, in particular TNF-α. Although RGS10 is abundantly expressed in resting macrophages, previous studies have shown that RGS10 expression is suppressed in macrophages following Toll-like receptor 4 (TLR4) activation by LPS. However, the molecular mechanism by which LPS induces Rgs10 silencing has not been clearly defined. The goal of the current study was to determine whether LPS silences Rgs10 expression through an NF-κB-mediated proinflammatory mechanism in pulmonary macrophages, a unique type of innate immune cells. We demonstrate that Rgs10 transcript and RGS10 protein levels are suppressed upon LPS treatment in the murine MH-S alveolar macrophage cell line. We show that pharmacological inhibition of PI3K/ NF-κB/p300 (NF-κB co-activator)/TNF-α signaling cascade and the activities of HDAC (1–3) enzymes block LPS-induced silencing of Rgs10 in MH-S cells as well as microglial BV2 cells and BMDMs. Further, loss of RGS10 generated by using CRISPR/Cas9 amplifies NF-κB phosphorylation and inflammatory gene expression following LPS treatment in MH-S cells. Together, our findings strongly provide critical insight into the molecular mechanism underlying RGS10 suppression by LPS in pulmonary macrophages.

Tetrandrine partially reverses multidrug resistance of human laryngeal cancer cells.

ObjectiveStudies have demonstrated that tetrandrine reverses multidrug resistance (MDR) in animal models or cell lines derived from multiple cancer types. We examined the potential MDR reversal activity of tetrandrine in a multidrug-resistant variant of a human laryngeal cancer Hep-2 cell line and explored potential mechanisms involved.MethodsWe developed the multidrug-resistant variant cell line (Hep-2/v) by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (VCR). After Hep-2 or Hep-2/v cells were treated with tetrandrine (2.52 µg/mL), MDR was measured by MTT assay, rhodamine 123 retention was measured by flow cytometry, and mRNA and protein expression of multidrug resistance 1 (MDR1), regulator of G-protein signaling 10 (RGS10), high-temperature requirement protein A1 (HTRA1), and nuclear protein 1 (NUPR1) were detected by real-time reverse transcription-PCR and western blotting, respectively.ResultsTetrandrine significantly lowered the half-maximal inhibitory concentration (IC50) of VCR in Hep-2/v cells, resulting in a 2.22-fold reversal of MDR. Treatment with tetrandrine increased rhodamine 123 retention, downregulated the mRNA and protein expression of MDR1 and RGS10, and upregulated expression of HTRA1 in Hep-2/v cells.ConclusionWe showed that tetrandrine exerts anti-MDR activity in Hep-2/v cells, possibly by inhibiting MDR1 overexpression-mediated drug efflux and by altering expression of HTRA1 and RGS10.

Abstract P2011: Chronic Systemic Administration of Pro-Inflammatory Cytokines Causes Cardiovascular Dysfunction and Downregulates Brain Expression of Regulator of G-Protein Signaling-10 in Rats

Systemic pro-inflammatory cytokines (PICs) promote central oxidative stress and neuroinflammation that contribute to sympathetic excitation and cardiovascular dysfunction in hypertension and heart failure. However, the key molecular regulators of neuroinflammatory responses in the brain remain unclear. Regulator of G-protein Signaling-10 (RGS10), a GTPase-accelerating protein that is expressed predominantly in the brain, has recently emerged as an important counter regulator of PICs-induced downstream signaling in the central nervous system. RGS10 negatively regulates NF-kB-mediated inflammatory responses in microglia and antagonizes neurotoxic effects of peripheral inflammation. The present study examined whether peripherally administered PICs alter RGS10 expression in cardiovascular/autonomic brain regions like subfornical organ (SFO) and hypothalamic paraventricular nucleus (PVN). Male SD rats received a 2-week subcutaneous infusion of vehicle, interleukin (IL)-1β (400 ng/day) and tumor necrosis factor (TNF)-α (600 ng/day). Rats treated with IL-1β (n=6) or TNF-α (n=6) had significantly (*P<0.05) reduced mRNA level (fold change) of RGS10 in SFO (0.60±0.15* or 0.69±0.16* vs 1.03±0.28; IL-1β or TNF-α, vs vehicle treatment) and PVN (0.72±0.15* or 0.71±0.19* vs 1.02±0.24), and lessoned mRNA level of NF-kB inhibitor IkB-α (SFO: 0.60±0.21* or 0.55±0.16* vs 1.07±0.46; PVN: 0.70±0.31* or 0.82±0.16* vs 1.00±0.07). Treatments with IL-1β or TNF-α increased mRNA level of NADPH oxidase 2 (SFO: 1.96±0.91* or 1.62±0.47* vs 1.03±0.28; PVN: 2.00±0.82* or 1.74±0.70* vs 1.00±0.15) and cyclooxygenase-2 (SFO: 1.78±0.76* or 1.98±1.04* vs 1.00±0.13; PVN: 1.68±0.34* or 1.62±0.57* vs 1.02±0.24). Rats treated with IL-1β or TNF-α had elevated mean blood pressure (98±11* or 100±9* vs 84±5 mmHg) and increased sympathetic activity as indicated by pressure response to ganglionic blockade (38±7 * or 36±6* vs 28±4 mmHg). Additionally, IL-1β or TNF-α-treated rats had increased cardiac fibrotic markers and impaired cardiac diastolic function. These results suggest that circulating PICs reduce RGS10 expression in the brain, which may contribute to sympathetic excitation and cardiovascular dysfunction in inflammatory conditions.

Depletion of regulator-of-G-protein signaling-10 in mice exaggerates high-fat diet-induced insulin resistance and inflammation, and this effect is mitigated by dietary green tea extract

The interaction between insulin resistance and inflammation plays a central role in the development of chronic diseases, although the mechanism is not fully understood. We previously demonstrated that regulator of G-protein signaling-10 (RGS10) protein is a negative modulator of the inflammatory response in macrophages and microglia. Because inflammation is a critical component in the development of high fat diet-induced insulin resistance, in this study we investigated whether RGS10 is involved in the diet-dependent regulation of glucose tolerance and insulin sensitivity. We hypothesized that the absence of RGS10 would exaggerate high-fat diet (HFD)-induced insulin resistance and inflammation response. Our results showed that RGS10 knockout (KO) mice fed a HFD gained significantly more weight and developed severe insulin resistance compared to wild-type (WT) mice fed HFD. Furthermore, compared to WT HFD-fed mice, KO mice fed the HFD displayed inflammatory phenotypes such as decreased adipose tissue expression of the anti-inflammatory M2 markers YM1 and Fizz1 and increased expression of the proinflammatory M1 cytokine interleukin 6 in adipose and CD11b, CD68 and interleukin 1β in liver tissues. The impact of RGS10 deficiency on the exaggeration of HFD-induced insulin resistance and inflammation was ameliorated by oral consumption of green tea extract. Our results demonstrate that RGS10 is an important part of a protective mechanism involved in in regulating metabolic homeostasis by reducing inflammatory responses, which could potentially lead to an innovative new approach targeting inflammation and insulin resistance.

“Regulator of G‐protein Signaling 10 (RGS10) inhibits NF‐κB signaling, COX‐2 and inflammatory cytokine production in ovarian cancer cells”

Epithelial Ovarian Cancer (EOC) is the fifth leading cause of cancer‐related deaths among females in the United States. NF‐κB signaling and production of inflammatory mediators have been strongly linked to ovarian cancer progression and chemoresistance. Regulator of G‐protein Signaling 10 (RGS10) regulates ovarian cancer survival and chemoresistance, and has been linked to NF‐κB signaling in immune cells. The goal of the current study was to determine the ability of RGS10 to inhibit NF‐κB signaling and subsequent inflammatory effects in ovarian cancer. RGS10 is a negative regulator of Gαi signaling and multiple Gi coupled GPCRs are implicated in ovarian cancer. Therefore, we predicted that RGS10 regulates NF‐κB signaling and inflammatory cytokines downstream of Gαi mediated‐pathway. We found that knock‐down of RGS10 enhanced NF‐κB phosphorylation and produced significantly higher levels of COX‐2 and TNF‐α mRNA compared to cells transfected with control siRNA in SKOV‐3 EOC cells. Surprisingly, we found that Pertussis toxin (ptx), which is a Gαi inhibitor, had no effects on upregulation of p‐NF‐κB, COX‐2 and TNF‐α expressions‐mediated by RGS10 knock‐down. These results provide insight in the mechanism that RGS10 utilizes to regulate ovarian cancer survival and chemoresistance.Support or Funding Information National Institutes of Health Rivkin Center for Ovarian Cancer. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulator of G‐protein Signaling 10 (RGS10) Expression is Transcriptionally Silenced in Activated Microglia by Histone Deacetylase Activity

Regulator of G‐protein Signaling 10 (RGS10) has emerged as a key regulator of pro‐inflammatory cytokine production in microglia, functioning as an important neuroprotective factor. While RGS10 is normally expressed in microglia at high levels, expression is silenced in vitro following activation of TLR4 receptor‐activation. Given the ability of RGS10 to regulate inflammatory signaling, dynamic regulation of RGS10 expression in microglia may be an important mechanism to tune inflammatory responses. The goals of the current study were to confirm that RGS10 is silenced in an in vivo inflammatory model of microglial activation and to determine the mechanism for activation‐dependent silencing of RGS10 expression in microglia. We demonstrate that RGS10 is endogenously expressed in spinal cord microglia, and expression is suppressed in the spinal cord in a nerve injury induced neuropathic pain mouse model. We show that the histone deacetylase (HDAC) enzyme inhibitor Trichostatin A blocks the ability of Lipopolysaccharide (LPS) to suppress RGS10 transcription in BV‐2 and primary microglia, demonstrating that HDAC enzymes are required for LPS silencing of RGS10. Further, we used chromatin immunoprecipitation to demonstrate that H3 histones at the RGS10 proximal promoter are deacetylated in BV‐2 microglia following LPS activation, and HDAC1 association at the RGS10 promoter is enhanced following LPS stimulation. Finally, we have shown that sphingosine 1‐phosphate, an endogenous microglial signaling mediator which inhibits HDAC activity, enhances basal RGS10 expression in BV‐2 microglia, suggesting that RGS10 expression is dynamically regulated in microglia in response to multiple signals.Support or Funding InformationThis work was supported by grants from the National Multiple Sclerosis Society (SBH) and the National Institutes of Neurological Disorders and Stroke [RO1NS064289] (HRW).

Regulator of G-Protein Signaling 10 Negatively Regulates Cardiac Remodeling by Blocking Mitogen-Activated Protein Kinase-Extracellular Signal-Regulated Protein Kinase 1/2 Signaling.

Regulator of G-protein signaling 10 (RGS10) is an important member of the RGS family and produces biological effects in multiple organs. We used a genetic approach to study the role of RGS10 in the regulation of pathological cardiac hypertrophy and found that RGS10 can negatively influence pressure overload-induced cardiac remodeling. RGS10 expression was markedly decreased in failing human hearts and hypertrophic murine hearts. The extent of aortic banding-induced cardiac hypertrophy, dysfunction, and fibrosis in RGS10-knockout mice was exacerbated, whereas the heart of transgenic mice with cardiac-specific RGS10 overexpression exhibited an alleviated response to pressure overload. Consistently, RGS10 also inhibited an angiotensin II-induced hypertrophic response in isolated cardiomyocytes. Mechanistically, cardiac remodeling improvement elicited by RGS10 was associated with the abrogation of mitogen-activated protein kinase kinase 1/2-extracellular signal-regulated protein kinase 1/2 signaling. Furthermore, the inhibition of mitogen-activated protein kinase kinase-extracellular signal-regulated protein kinase 1/2 transduction abolished RGS10 deletion-induced hypertrophic aggravation. These findings place RGS10 and its downstream signaling mitogen-activated protein kinase kinase-extracellular signal-regulated protein kinase 1/2 as crucial regulators of pathological cardiac hypertrophy after pressure overload and identify this pathway as a potential therapeutic target to attenuate the pressure overload-driven cardiac remodeling.

Age-related changes in regulator of G-protein signaling (RGS)-10 expression in peripheral and central immune cells may influence the risk for age-related degeneration

Inflammation in the aging brain increases risk for neurodegenerative disease. In humans, the regulator of G-protein signaling-10 (RGS10) locus has been associated with age-related maculopathy. Chronic peripheral administration of lipopolysaccharide in the RGS10-null mice induces nigral dopaminergic (DA) degeneration, suggesting that RGS10 modulates neuroimmune interactions and may influence susceptibility to neurodegeneration. Because age is the strongest risk factor for neurodegenerative disease, we assessed whether RGS10 expression changes with age and whether aged RGS10-null mice have altered immune cell profiles. Loss of RGS10 in aged mice does not alter the regulation of nigral DA neurons but does alter B-cell, monocyte, microglial, and CD4+ T-cell populations and inflammatory cytokine levels in the cerebrospinal fluid. These results suggest that loss of RGS10 is associated with an age-dependent dysregulation of peripheral and central immune cells rather than dysregulation of DA neuron function.

Physiology of RGS10 in Neurons and Immune Cells.

Regulator of G protein signaling-10 (RGS10), a GTPase-activating protein (GAP) for Gαi3, Gαq, and Gαz, belongs to the D/R12 subfamily based on the homology within the RGS domain and is one of the smallest RGS proteins, outside the RGS box. Although RGS10 lacks the flanking domains or motifs, RGS10 can be targeted to membrane by palmitoylation which markedly increases its net GAP activity. The cAMP-dependent protein kinase A phosphorylates RGS10 on serine 168 (Ser(168)) which promotes translocation of RGS10 from the cytosol to nucleus. In addition to its GAP function, RGS10 modulates adenylyl cyclase (AC) activity as well as the G protein-gated inwardly rectifying potassium channels. Although the roles of various RGS proteins have been well addressed in previous reviews, the function and mechanism of RGS10 in particular has not been reviewed in-depth. Moreover, recent arrays of studies implicate a role for RGS10 in immune and neuronal cells. RGS10 has been localized to various cell compartments including the cytoplasm and nucleus. In this chapter, we will review its role in neurons and immune cells.

Silencing of regulator of G‐protein signaling 10 (RGS10) increases phosphorylation of eukaryotic initiation factor 4E binding protein (4E‐BP1), and enhances growth and survival signaling pathways in ovarian cancer (843.11)

Ovarian cancer is a devastating disease that is the 5th leading cause of cancer related deaths in women in the US. It is difficult to treat in that more than 70% of patients that are initially responsive to first‐line chemotherapeutics become refractory in less than 2 years. The problem of drug‐resistance or chemoresistance has plagued cancer researchers and hindered the success of cancer therapy development. In our previous studies examining computational bioinformatics, we demonstrated that reduced RGS gene expression develops with the occurrence of drug resistance in ovarian cancer cell lines. Specifically, we have shown that silencing of RGS10 protein increases viability of ovarian cancer cells in the presence of cycle arrest compounds such as paclitaxel. In our present study, we have shown that silencing RGS10 in ovarian cancer cell lines increases phosphorylation of Eukaryotic initiation factor (eLF) 4E, the mRNA 5’ cap‐binding protein (phosphorylated‐4E‐BP1). Phosphorylation of 4E‐BP1 has been shown to be a hallmark of more aggressive cancer phenotypes in prostate cancer and ovarian cancer. HeyA8 Parental and SKOV3 ovarian cancer cell lines were transiently transfected with siRNA non‐targeted control and human RGS10. Approximately 36 hrs post‐transfection cells were serum‐starved and treated with INK128 a selective mTOR inhibitor overnight. Cells were treated the following day with lysophosphatidic acid (LPA) and collected for western blot analysis. The same treatment conditions were also analyzed by immunofluorescence assays and imaged using a Cellomics ArrayScan VTI reader and HCS software. We found that when RGS10 was silenced in these ovarian cancer cell lines we observed changes in cell growth and size. Based on the results from these assays, we believe that we have elucidated a novel role of RGS10 in the growth and survival signaling pathways involved in the development of chemoresistance in ovarian cancer. Grant Funding Source: NIH

Transcriptional suppression, DNA methylation, and histone deacetylation of the Regulator of G‐protein Signaling 10 (RGS10) gene in ovarian cancer cells

Regulators of G‐Protein Signaling (RGS) Proteins suppress GPCR‐mediated growth and survival signaling in multiple cancers. We have shown that RGS10 is downregulated in ovarian cancer chemoresistance, and RGS10 expression levels alter ovarian cancer cell growth and sensitivity to cytotoxic drugs. This suggests that the suppression of RGS10 may contribute to ovarian cancer progression and the development of chemoresistance by indirectly amplifying GPCR‐mediated growth and survival signaling. The current study investigates the epigenetic regulation of RGS10 expression in ovarian cancer. RGS10 expression was reduced in clinical ovarian cancer and CAOV‐3 ovarian cancer cells compared to control immortalized ovarian surface epithelial (IOSE) cells. Histone acetylation at RGS10 promoters was decreased in CAOV‐3 compared to IOSE cells, with a corresponding increase in HDAC1 association. Similar loss of histone acetylation and increased HDAC1 binding was observed in chemoresistant versus chemosensitive ovarian cancer cells. Further, enhanced DNA methylation was observed in RGS10 promoter of chemoresistant cells. Our results suggest that epigenetic modifications correlate with the loss of RGS10 expression in ovarian cancer cells, and may contribute to the amplification of GPCR‐mediated growth and survival signaling. This research is funded by NIH and the Marsha Rivkin Center.

Transcriptional suppression, DNA methylation, and histone deacetylation of the regulator of G-protein signaling 10 (RGS10) gene in ovarian cancer cells.

RGS10 regulates ovarian cancer cell growth and survival, and RGS10 expression is suppressed in cell models of ovarian cancer chemoresistance. However, the mechanisms governing RGS10 expression in ovarian cancer are poorly understood. Here we report RGS10 suppression in primary ovarian cancer and CAOV-3 ovarian cancer cells compared to immortalized ovarian surface epithelial (IOSE) cells, and in A2780-AD chemoresistant cells compared to parental A2780 cells. RGS10-1 and RGS10-2 transcripts are expressed in ovarian cancer cells, but only RGS10-1 is suppressed in A2780-AD and CAOV-3 cells, and the RGS10-1 promoter is uniquely enriched in CpG dinucleotides. Pharmacological inhibition of DNA methyl-transferases (DNMTs) increased RGS10 expression, suggesting potential regulation by DNA methylation. Bisulfite sequencing analysis identified a region of the RGS10-1 promoter with significantly enhanced DNA methylation in chemoresistant A2780-AD cells relative to parental A2780 cells. DNA methylation in CAOV-3 and IOSE cells was similar to A2780 cells. More marked differences were observed in histone acetylation of the RGS10-1 promoter. Acetylated histone H3 associated with the RGS10-1 promoter was significantly lower in A2780-AD cells compared to parental cells, with a corresponding increase in histone deacetylase (HDAC) enzyme association. Similarly, acetylated histone levels at the RGS10-1 promoter were markedly lower in CAOV-3 cells compared to IOSE cells, and HDAC1 binding was doubled in CAOV-3 cells. Finally, we show that pharmacological inhibition of DNMT or HDAC enzymes in chemoresistant A2780-AD cells increases RGS10 expression and enhances cisplatin toxicity. These data suggest that histone de-acetylation and DNA methylation correlate with RGS10 suppression and chemoresistance in ovarian cancer. Markers for loss of RGS10 expression may identify cancer cells with unique response to therapeutics.

RGS10 exerts a neuroprotective role through the PKA/c‐AMP response‐element (CREB) pathway in dopaminergic neuron‐like cells

Regulator of G-protein signaling-10 (RGS10) is a GTPase activating protein for Gαi/q/z subunits that is highly expressed in the immune system and in a broad range of brain regions including the hippocampus, striatum, dorsal raphe, and ventral midbrain. Previously, we reported that RGS10-null mice display increased vulnerability to chronic systemic inflammation-induced degeneration of nigral dopaminergic (DA) neurons. Given that RGS10 is expressed in DA neurons, we investigated the extent to which RGS10 regulates cell survival under conditions of inflammatory stress. Because of the inherent limitations associated with use of primary DA neurons for biochemical analyses, we employed a well-characterized ventral mesencephalon DA neuroblastoma cell line (MN9D) for our studies. We found that stable over-expression of RGS10 rendered them resistant to TNF-induced cytotoxicity; whereas MN9D cells expressing mutant RGS10-S168A (which is resistant to phosphorylation by protein kinase A at a serine residue that promotes its nuclear translocation) showed similar sensitivity to TNF as the parental MN9D cells. Using biochemical and pharmacologic approaches, we identified protein kinase A and the downstream phospho-cAMP response element-binding signaling pathway (and ruled out ERK 1/2, JNK, and NFkB) as key mediators of the neuroprotective effect of RGS10 against inflammatory stress.

Evaluation of in vitro growth factor treatments on fibrochondrogenesis by synovial membrane cells from osteoarthritic and nonosteoarthritic joints of dogs

To determine the in vitro effects of selected growth factors on fibrochondrogenesis by synovial membrane cells from nonosteoarthritic (normal) and osteoarthritic joints of dogs. 5 dogs with secondary osteoarthritis of shoulder or stifle joints and 6 dogs with normal joints. Synovial membrane cells were harvested from normal and osteoarthritic joints and cultured in monolayer with or without (control) basic fibroblast growth factor, transforming growth factor-β1, and insulin-like growth factor-1. In the cultured cells, fibrochondrogenesis was measured by use of a real-time reverse transcriptase PCR assay to determine relative expressions of collagen I, collagen II, and aggrecan genes and of 3 genes involved in embryonic chondrogenesis: Sry-type homeobox protein-9 (SOX-9), frizzled-motif associated with bone development (Frzb), and regulator of G-protein signaling-10 (RGS-10). Tissue collagen content was measured via a hydroxyproline assay, and sulfated glycosaminoglycan content was measured via a 1,9-dimethylmethylene blue assay. Cellularity was determined via a double-stranded DNA assay. Immunohistochemical analysis for collagens I and II was also performed. In vitro collagen synthesis was enhanced by growth factor stimulation. Although osteoarthritic-joint synoviocytes could undergo a fibrocartilage-like phenotypic shift, their production of collagenous extracellular matrix was less than that of normal-joint synoviocytes. Gene expressions of SOX-9 and RGS-10 were highest in the osteoarthritic-joint cells; Frzb expression was highest in growth factor treated cells. Autogenous synovium may be a viable cell source for meniscal tissue engineering. Gene expressions of SOX-9 and RGS-10 may be potential future targets for in vitro enhancement of chondrogenesis.

Neuroinflammation in Parkinson's disease.

During the last two decades, a wealth of animal and human studies has implicated inflammation-derived oxidative stress and cytokine-dependent neurotoxicity in the progressive degeneration of the dopaminergic nigrostriatal pathway, the hallmark of Parkinson's disease (PD). In this review, we discuss the various hypotheses regarding the role of microglia and other immune cells in PD pathogenesis and progression, the inflammatory mechanisms implicated in disease progression from pre-clinical and clinical studies, the recent evidence that systemic inflammation can trigger microglia activation in PD-relevant central nervous system regions, the synergism between gene products linked to parkinsonian phenotypes (alpha-synuclein, parkin, Nurr1, and regulator of G-protein signaling-10) and neuroinflammation in promoting neurodegeneration of the nigrostriatal pathway, and the latest update on meta-analysis of epidemiological studies on the risk-lowering effects of anti-inflammatory drug regimens.