Critical Role of Regulator G-Protein Signaling 10 (RGS10) in Modulating Macrophage M1/M2 Activation

Regulator of G protein signaling 10 (RGS10), a GTPase accelerating protein (GAP) for G alpha subunits, is a negative regulator of NF-κB in microglia. Here, we investigated the role of RGS10 in macrophages, a closely related myeloid-derived cell type. Features of classical versus alternative activation were assessed in Rgs10-/- peritoneal and bone marrow-derived macrophages upon LPS or IL-4 treatments, respectively. Our results showed that Rgs10-/- macrophages produced higher levels of pro-inflammatory cytokines including TNF, IL-1β and IL-12p70 in response to LPS treatment and exerted higher cytotoxicity on dopaminergic MN9D neuroblastoma cells. We also found that Rgs10-/- macrophages displayed a blunted M2 phenotype upon IL-4 priming. Specifically, Rgs10-/- macrophages displayed lower YM1 and Fizz1 mRNA levels as measured by QPCR compared to wild type macrophages upon IL-4 treatment and this response was not attributable to differences in IL-4 receptor expression. Importantly, phagocytic activities of Rgs10-/- macrophages were blunted in response to IL-4 priming and/or LPS treatments. However, there was no difference in chemotaxis between Rgs10-/- and WT macrophages. Our data indicate that Rgs10-/- macrophages displayed dysregulated M1 responses along with blunted M2 alternative activation responses, suggesting that RGS10 plays an important role in determining macrophage activation responses.     

Suppressor of Cytokine Signaling (SOCS) Genes Are Silenced by DNA Hypermethylation and Histone Deacetylation and Regulate Response to Radiotherapy in Cervical Cancer Cells

Suppressor of cytokine signaling (SOCS) family is an important negative regulator of cytokine signaling and deregulation of SOCS has been involved in many types of cancer. All cervical cancer cell lines tested showed lower expression of SOCS1, SOCS3, and SOCS5 than normal tissue or cell lines. The immunohistochemistry result for SOCS proteins in human cervical tissue also confirmed that normal tissue expressed higher level of SOCS proteins than neighboring tumor. Similar to the regulation of SOCS in other types of cancer, DNA methylation contributed to SOCS1 downregulation in CaSki, ME-180, and HeLa cells. However, the expression of SOCS3 or SOCS5 was not recovered by the inhibition of DNA methylation. Histone deacetylation may be another regulatory mechanism involved in SOCS1 and SOCS3 expression, however, SOCS5 expression was neither affected by DNA methylation nor histone deacetylation. Ectopic expression of SOCS1 or SOCS3 conferred radioresistance to HeLa cells, which implied SOCS signaling regulates the response to radiation in cervical cancer. In this study, we have shown that SOCS expression repressed by, in part, epigenetically and altered SOCS1 and SOCS3 expression could contribute to the radiosensitive phenotype in cervical cancer.     

Regulator of G-Protein Signaling 19 (RGS19) and Its Partner Gα-Inhibiting Activity Polypeptide 3 (GNAI3) Are Required for zVAD-Induced Autophagy and Cell Death in L929 Cells

Autophagy has diverse biological functions and is involved in many biological processes. The L929 cell death induced by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone (zVAD) was shown to be an autophagy-mediated death for which RIP1 and RIP3 were both required. It was also reported that zVAD can induce a small amount of TNF production, which was shown to be required for zVAD-induced L929 cell death, arguing for the contribution of autophagy in the zVAD-induced L929 cell death. In an effort to study RIP3 mediated cell death, we identified regulator of G-protein signaling 19 (RGS19) as a RIP3 interacting protein. We showed that RGS19 and its partner Gα-inhibiting activity polypeptide 3 (GNAI3) are involved in zVAD-, but not TNF-, induced cell death. The role of RGS19 and GNAI3 in zVAD-induced cell death is that they are involved in zVAD-induced autophagy. By the use of small hairpin RNAs and chemical inhibitors, we further demonstrated that zVAD-induced autophagy requires not only RIP1, RIP3, PI3KC3 and Beclin-1, but also RGS19 and GNAI3, and this autophagy is required for zVAD-induced TNF production. Collectively, our data suggest that zVAD-induced L929 cell death is a synergistic result of autophagy, caspase inhibition and autocrine effect of TNF.     

Regulator of G Protein Signaling 2 (RGS2) and RGS4 Form Distinct G Protein-Dependent Complexes with Protease Activated-Receptor 1 (PAR1) in Live Cells

Protease-activated receptor 1 (PAR1) is a G-protein coupled receptor (GPCR) that is activated by natural proteases to regulate many physiological actions. We previously reported that PAR1 couples to G_i, G_q and G₁₂ to activate linked signaling pathways. Regulators of G protein signaling (RGS) proteins serve as GTPase activating proteins to inhibit GPCR/G protein signaling. Some RGS proteins interact directly with certain GPCRs to modulate their signals, though cellular mechanisms dictating selective RGS/GPCR coupling are poorly understood. Here, using bioluminescence resonance energy transfer (BRET), we tested whether RGS2 and RGS4 bind to PAR1 in live COS-7 cells to regulate PAR1/Gα-mediated signaling. We report that PAR1 selectively interacts with either RGS2 or RGS4 in a G protein-dependent manner. Very little BRET activity is observed between PAR1-Venus (PAR1-Ven) and either RGS2-Luciferase (RGS2-Luc) or RGS4-Luc in the absence of Gα. However, in the presence of specific Gα subunits, BRET activity was markedly enhanced between PAR1-RGS2 by Gα_q/11, and PAR1-RGS4 by Gα_o, but not by other Gα subunits. Gα_q/11-YFP/RGS2-Luc BRET activity is promoted by PAR1 and is markedly enhanced by agonist (TFLLR) stimulation. However, PAR1-Ven/RGS-Luc BRET activity was blocked by a PAR1 mutant (R205A) that eliminates PAR1-G_q/11 coupling. The purified intracellular third loop of PAR1 binds directly to purified His-RGS2 or His-RGS4. In cells, RGS2 and RGS4 inhibited PAR1/Gα-mediated calcium and MAPK/ERK signaling, respectively, but not RhoA signaling. Our findings indicate that RGS2 and RGS4 interact directly with PAR1 in Gα-dependent manner to modulate PAR1/Gα-mediated signaling, and highlight a cellular mechanism for selective GPCR/G protein/RGS coupling.     

RGS6, but Not RGS4, Is the Dominant Regulator of G Protein Signaling (RGS) Modulator of the Parasympathetic Regulation of Mouse Heart Rate

Parasympathetic activity decreases heart rate (HR) by inhibiting pacemaker cells in the sinoatrial node (SAN). Dysregulation of parasympathetic influence has been linked to sinus node dysfunction and arrhythmia. RGS (regulator of G protein signaling) proteins are negative modulators of the parasympathetic regulation of HR and the prototypical M₂ muscarinic receptor (M₂R)-dependent signaling pathway in the SAN that involves the muscarinic-gated atrial K⁺ channel I_KACh. Both RGS4 and RGS6-Gβ5 have been implicated in these processes. Here, we used Rgs4^−/−, Rgs6^−/−, and Rgs4^−/−:Rgs6^−/− mice to compare the relative influence of RGS4 and RGS6 on parasympathetic regulation of HR and M₂R-I_KACh-dependent signaling in the SAN. In retrogradely perfused hearts, ablation of RGS6, but not RGS4, correlated with decreased resting HR, increased heart rate variability, and enhanced sensitivity to the negative chronotropic effects of the muscarinic agonist carbachol. Similarly, loss of RGS6, but not RGS4, correlated with enhanced sensitivity of the M₂R-I_KACh signaling pathway in SAN cells to carbachol and a significant slowing of M₂R-I_KACh deactivation rate. Surprisingly, concurrent genetic ablation of RGS4 partially rescued some deficits observed in Rgs6^−/− mice. These findings, together with those from an acute pharmacologic approach in SAN cells from Rgs6^−/− and Gβ5^−/− mice, suggest that the partial rescue of phenotypes in Rgs4^−/−:Rgs6^−/− mice is attributable to another R7 RGS protein whose influence on M₂R-I_KACh signaling is masked by RGS4. Thus, RGS6-Gβ5, but not RGS4, is the primary RGS modulator of parasympathetic HR regulation and SAN M₂R-I_KACh signaling in mice.     

Regulator of G-Protein Signalling-14 (RGS14) Regulates the Activation of αMβ2 Integrin during Phagocytosis

Integrin-mediated phagocytosis, an important physiological activity undertaken by professional phagocytes, requires bidirectional signalling to/from αMβ2 integrin and involves Rap1 and Rho GTPases. The action of Rap1 and the cytoskeletal protein talin in activating αMβ2 integrins, in a RIAM-independent manner, has been previously shown to be critical during phagocytosis in mammalian phagocytes. However, the events downstream of Rap1 are not clearly understood. Our data demonstrate that one potential Rap1 effector, Regulator of G-Protein Signalling-14 (RGS14), is involved in activating αMβ2. Exogenous expression of RGS14 in COS-7 cells expressing αMβ2 results in increased binding of C3bi-opsonised sheep red blood cells. Consistent with this, knock-down of RGS14 in J774.A1 macrophages results in decreased association with C3bi-opsonised sheep red blood cells. Regulation of αMβ2 function occurs through the R333 residue of the RGS14 Ras/Rap binding domain (RBD) and the F754 residue of β2, residues previously shown to be involved in binding of H-Ras and talin1 head binding prior to αMβ2 activation, respectively. Surprisingly, overexpression of talin2 or RAPL had no effect on αMβ2 regulation. Our results establish for the first time a role for RGS14 in the mechanism of Rap1/talin1 activation of αMβ2 during phagocytosis.     

Regulator of G-Protein Signaling 3 Isoform 1 (PDZ-RGS3) Enhances Canonical Wnt Signaling and Promotes Epithelial Mesenchymal Transition

The Wnt β-catenin pathway controls numerous cellular processes including cell differentiation and cell-fate decisions. Wnt ligands engage Frizzled receptors and the low-density-lipoprotein-related protein 5/6 (LRP5/6) receptor complex leading to the recruitment of Dishevelled (Dvl) and Axin1 to the plasma membrane. Axin1 has a regulator of G-protein signaling (RGS) domain that binds adenomatous polyposis coli and Gα subunits, thereby providing a mechanism by which Gα subunits can affect β-catenin levels. Here we show that Wnt signaling enhances the expression of another RGS domain-containing protein, PDZ-RGS3. Reducing PDZ-RGS3 levels impaired Wnt3a-induced activation of the canonical pathway. PDZ-RGS3 bound GSK3β and decreased its catalytic activity toward β-catenin. PDZ-RGS3 overexpression enhanced Snail1 and led to morphological and biochemical changes reminiscent of epithelial mesenchymal transition (EMT). These results indicate that PDZ-RGS3 can enhance signals generated by the Wnt canonical pathway and that plays a pivotal role in EMT.     

Nanog 基因在卵巢癌和卵巢肿瘤干细胞中的表达及意义

目的:探讨胚胎干细胞关键因子Nanog基因mRNA及其蛋白在卵巢癌和卵巢癌肿瘤干细胞中的表达及意义。方法:选取10例正常卵巢上皮组织、10例卵巢良性肿瘤及60例卵巢癌组织,采用逆转录酶-聚合酶链反应(RT-PCR)方法和免疫组织化学PV-6000两步法检测Nanog mRNA和蛋白表达水平;采用无血清悬浮培养法从SKOV-3卵巢癌细胞株中分离培养肿瘤干细胞,流式细胞术鉴定肿瘤干细胞CD117表达,采用RT-PCR和Western Blot方法检测SKOV-3卵巢癌细胞及肿瘤干细胞中NanogmRNA及其蛋白的表达水平。结果:Nanog mRNA在卵巢癌组织中的表达水平均高于正常卵巢组织和卵巢良性肿瘤组织(P<0.05);Nanog mRNA在不同分化程度及临床分期的卵巢癌组织中表达水平不同,低分化组高于高分化组(P<0.05);III-IV期高于I-II期(P<0.05);免疫组化结果同RT-PCR。从SKOV-3卵巢癌细胞株中成功分离出肿瘤干细胞,SKOV-3卵巢癌细胞和肿瘤干细胞Nanog mRNA相对含量分别为0.6044±0.0368,0.8736±0.0537,差异具有统计学意义(P<0.05),两种细胞Nanog蛋白相对含量分别为0.6364±0.0169 1.2788±0.0314,差别具有统计学意义(P<0.05)。结论:Nanog基因在卵巢癌组织和SKOV-3细胞系中均高表达,其在组织中的表达强度与临床分期及病理分级关系密切,且在肿瘤干细胞中表达高于一般卵巢癌细胞,其与卵巢癌的发生发展关系密切,可能是卵巢癌干细胞的表面标志物,有望成为新的标志物。     

Adenosine Receptors Differentially Regulate the Expression of Regulators of G-Protein Signalling (RGS) 2, 3 and 4 in Astrocyte-Like Cells

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.     

FHIT基因在宫颈癌细胞中的表达及其甲基化调控

目的：分析FHIT基因在宫颈癌细胞中表达情况以及甲基化的调控情况。方法：对RJC-1、SiHa、CS1213以及C4-1细胞进行培养,提取这些细胞的DNA并经过亚硫酸氢盐修饰,进行PCR反应和产物的检测。分析FHIT基因在宫颈癌细胞中表达情况以及甲基化的调控情况。结果：RJC-1、CS1213细胞仅有甲基化引物扩增出了目的条带,为完全甲基化状态。其他细胞则是甲基特异性引物与非甲基特异性引物共同扩增出73bp的目的条带,其状态为甲基化杂合性。通过5-aza-CdR处理细胞后,通过实时定量PCR检测FHIT mRNA的表达,显示处理后各种细胞中的FHIT mRNA的表达升高。结论：FHIT基因的甲基化是其表达下调的重要机制之一,是临床研究宫颈癌细胞的重要方向之一。     

Histone Deacetylase 10 Regulates DNA Mismatch Repair and May Involve the Deacetylation of MutS Homolog 2

MutS homolog 2 (MSH2) is an essential DNA mismatch repair (MMR) protein. It interacts with MSH6 or MSH3 to form the MutSα or MutSβ complex, respectively, which recognize base-base mispairs and insertions/deletions and initiate the repair process. Mutation or dysregulation of MSH2 causes genomic instability that can lead to cancer. MSH2 is acetylated at its C terminus, and histone deacetylase (HDAC6) deacetylates MSH2. However, whether other regions of MSH2 can be acetylated and whether other histone deacetylases (HDACs) and histone acetyltransferases (HATs) are involved in MSH2 deacetylation/acetylation is unknown. Here, we report that MSH2 can be acetylated at Lys-73 near the N terminus. Lys-73 is highly conserved across many species. Although several Class I and II HDACs interact with MSH2, HDAC10 is the major enzyme that deacetylates MSH2 at Lys-73. Histone acetyltransferase HBO1 might acetylate this residue. HDAC10 overexpression in HeLa cells stimulates cellular DNA MMR activity, whereas HDAC10 knockdown decreases DNA MMR activity. Thus, our study identifies an HDAC10-mediated regulatory mechanism controlling the DNA mismatch repair function of MSH2.     

Effects of Regulator of G Protein Signaling 19 (RGS19) on Heart Development and Function

Wnt/Wg genes play a critical role in the development of various organisms. For example, the Wnt/β-catenin signal promotes heart formation and cardiomyocyte differentiation in mice. Previous studies have shown that RGS19 (regulator of G protein signaling 19), which has Gα subunits with GTPase activity, inhibits the Wnt/β-catenin signal through inactivation of Gα_o. In the present study, the effects of RGS19 on mouse cardiac development were observed. In P19 teratocarcinoma cells with RGS19 overexpression, RGS19 inhibited cardiomyocyte differentiation by blocking the Wnt signal. Additionally, several genes targeted by Wnt were down-regulated. For the in vivo study, we generated RGS19-overexpressing transgenic (RGS19 TG) mice. In these transgenic mice, septal defects and thin-walled ventricles were observed during the embryonic phase of development, and the expression of cardiogenesis-related genes, BMP4 and Mef2C, was reduced significantly. RGS19 TG mice showed increased expression levels of brain natriuretic peptide and β-MHC, which are markers of heart failure, increase of cell proliferation, and electrocardiogram analysis shows abnormal ventricle repolarization. These data provide in vitro and in vivo evidence that RGS19 influenced cardiac development and had negative effects on heart function.     

NDRG2基因启动子甲基化与肺腺癌细胞中mRNA表达相关性的实验研究

目的:探讨肺腺癌细胞中NDRG2基因启动子甲基化状态及其与基因表达的关系。方法:甲基化焦磷酸测序技术检测启动子区域甲基化状态,荧光定量PCR技术检测不同药物浓度下培养细胞中NDRG2基因mRNA的表达水平,分析启动子区域甲基化与基因表达之间的关系。结果:在体外培养细胞中检测到NDRG2基因启动子区域呈现不同程度的甲基化,甲基化频率分别为肺癌A549细胞71.8%、GLC-82细胞86.1%、人脐静脉内皮ECV-304细胞36.8%、胃上皮GES-1细胞42.9%。NDRG2基因mRNA表达与其启动子甲基化程度成反比,甲基转移酶抑制剂5-杂氮-2-脱氧胞苷(5-Aza-CdR)作用于细胞后,A549和GLC-82细胞中NDRG2基因的mRNA转录明显上调,至72 h差异显著(P0.05)。结论:肺腺癌细胞中NDRG2基因启动子CpG岛存在高甲基化,甲基化程度与该基因的表达具有负相关性,5-Aza-CdR能在一定程度上提高NDRG2的转录水平。     

Targeted Methylation of the Epithelial Cell Adhesion Molecule (EpCAM) Promoter to Silence Its Expression in Ovarian Cancer Cells

The Epithelial Cell Adhesion Molecule (EpCAM) is overexpressed in many cancers including ovarian cancer and EpCAM overexpression correlates with decreased survival of patients. It was the aim of this study to achieve a targeted methylation of the EpCAM promoter and silence EpCAM gene expression using an engineered zinc finger protein that specifically binds the EpCAM promoter fused to the catalytic domain of the Dnmt3a DNA methyltransferase. We show that transient transfection of this construct increased the methylation of the EpCAM promoter in SKOV3 cells from 4–8% in untreated cells to 30%. Up to 48% methylation was observed in stable cell lines which express the chimeric methyltransferase. Control experiments confirmed that the methylation was dependent on the fusion of the Zinc finger and the methyltransferase domains and specific for the target region. The stable cell lines with methylated EpCAM promoter showed a 60–80% reduction of EpCAM expression as determined at mRNA and protein level and exhibited a significantly reduced cell proliferation. Our data indicate that targeted methylation of the EpCAM promoter could be an approach in the therapy of EpCAM overexpressing cancers.     

DNA甲基化及组蛋白修饰在精神分裂症中的改变

精神分裂症（schizophrellia）是一种严重的精神疾病,对双胞胎、家庭及认养的研究揭示,遗传与环境因素之间复杂的相互作用是精神分裂症的病因。表观遗传学（epigenetics）研究与基因序列变化无关的基因表达调控机制．为人们深入认识精神分裂症的病因提供了新的思路。表观遗传调控机制主要包括：DNA甲基化、组蛋白修饰和非编码RNA三种方式。该文介绍前两种在精神分裂症发病机制中的作用。