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PubMed2005

Inhibition of replication and infection of severe acute respiratory syndrome-associated coronavirus with plasmid-mediated interference RNA.

Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease caused by a novel coronavirus (SARS-CoV), which spread to over 30 countries in early 2003. Until recently, no specific vaccines and effective drugs have been available to protect patients from infection by this virus. To exploit a new strategy to fight this disease, we investigated the effect of interference RNA (RNAi) on the virus infection and replication with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), plaque-forming, Western-blot and real-time PCR assays. Results showed that the plasmid-derived siRNAs targeting the non-structural protein 1 (NSP1) sequence of the SARS-CoV genome could specifically inhibit the expression of the NSP1 sequence and effectively suppress the replication and propagation of SARS-CoV in cultured Vero E6 cell lines. The expression of the Spike and Nucleoprotein genes of SARS-CoV at mRNA and protein levels in small interfering (si)RNA-expressing cells was significantly less than that in controls when analysed with PCR and Western-blot assays, 3 days post SARS-CoV infection. Our study provides strong evidence that the NSP1 sequence in the SARS-CoV genome is a valid target for RNAi and the effect of the siRNAs probably mainly resulted from effects on global reduction of subgenome synthesis and subsequent protein expression of SARS-CoV.

Animals↗
PubMed2006

Arabidopsis separase AESP is essential for embryo development and the release of cohesin during meiosis.

To investigate how and when sister chromatid cohesion is released from chromosomes in plants, we isolated the Arabidopsis thaliana homolog of separase (AESP) and investigated its role in somatic and meiotic cells. AESP is similar to separase proteins identified in other organisms but contains several additional structural motifs. The characterization of two Arabidopsis T-DNA insertion alleles for AESP demonstrated that it is an essential gene. Seeds homozygous for T-DNA insertions in AESP exhibited embryo arrest at the globular stage. The endosperm also exhibited a weak titan-like phenotype. Transgenic plants expressing AESP RNA interference (RNAi) from the meiosis-specific DMC1 promoter exhibited alterations in chromosome segregation during meiosis I and II that resulted in polyads containing from one to eight microspores. Consistent with its predicted role in the release of sister chromatid cohesion, immunolocalization studies showed that the removal of SYN1 from chromosome arms and the centromeres is inhibited in the RNAi mutants. However, the release of SYN1 during diplotene occurred normally, indicating that this process is independent of AESP. Therefore, our results demonstrate that AESP plays an essential role in embryo development and provide direct evidence that AESP is required for the removal of cohesin from meiotic chromosomes.

Amino Acid Motifs↗
PubMed2006

Telomerase inhibition by stable RNA interference impairs tumor growth and angiogenesis in glioblastoma xenografts.

Telomerase is highly expressed in advanced stages of most cancers where it allows the clonal expansion of transformed cells by counteracting telomere erosion. Telomerase may also contribute to tumor progression through still undefined cell growth-promoting functions. Here, we inhibited telomerase activity in 2 human glioblastoma (GBM) cell lines, TB10 and U87MG, by targeting the catalytic subunit, hTERT, via stable RNA interference (RNAi). Although the reduction in telomerase activity had no effect on GBM cell growth in vitro, the development of tumors in subcutaneously and intracranially grafted nude mice was significantly inhibited by antitelomerase RNAi. The in vivo effect was observed within a relatively small number of population doublings, suggesting that telomerase inhibition may hinder cancer cell growth in vivo prior to a substantial shortening of telomere length. Tumor xenografts that arose from telomerase-inhibited GBM cells also showed a less-malignant phenotype due both to the absence of massive necrosis and to reduced angiogenesis.

Animals↗
PubMed2006

Testing the efficacy of RNA interference in Haemonchus contortus.

RNA interference (RNAi) is widely used in Caenorhabditis elegans to identify gene function and has been adapted as a high throughput screening method to identify genes involved in essential processes. We have been examining whether RNAi could also be used on the strongylid parasitic nematode Haemonchus contortus to study gene function. Eleven genes were targeted in L1 and exsheathed L3 H. contortus larvae with RNAi methodologies which have been shown to be effective in C. elegans and parasitic nematodes-feeding, soaking and electroporation. Reverse transcriptase-PCR and, where possible, protein assays were carried out to examine decreases in mRNA and protein levels. RNAi soaking in dsRNA to beta-tubulin and sec-23, a gene involved in vesicle transport, resulted in specific decreases in mRNA levels in exsheathed L3 larvae. No signs of specific decreases in expression levels were observed for the other nine genes tested. Following electroporation of dsRNA in L1 stage larvae, significant decreases were observed for two out of four genes tested. These findings suggest that the RNAi pathway is functional in H. contortus and that, under certain conditions, it is possible to suppress gene expression by RNAi. However, it only works on a limited number of genes and in some cases the effect is small and difficult to reproduce. This indicates that the RNAi approaches established for C. elegans and other nematodes have limited efficacy in H. contortus. This may reflect differences between nematode species in dsRNA uptake and transport into cells and between cells.

Animals↗
PubMed2006

Aminopeptidase N (APN/CD13) is selectively expressed in vascular endothelial cells and plays multiple roles in angiogenesis.

Proteolytic enzyme-mediated degradation of the extracellular matrix (ECM) is crucial for the formation of both tumor metastasis and angiogenesis. Recently, several reports have suggested that aminopeptidases are involved in this process, but precisely how is largely unknown. We found here that aminopeptidase N (APN/CD13) was selectively expressed in vascular endothelial cells including human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC), and was not detectable in a majority of normal cells and tumor cell lines we examined. RNA interference (RNAi) of APN resulted in the inhibition of capillary tube formation of HUVEC on Matrigel. APN siRNA suppressed the migration of HUVEC through a fibronectin-coated Transwell membrane, and reduced the cellular adhesion to Matrigel and various adhesion molecules including type IV collagen, type I collagen and fibronectin. These findings suggest that APN is a multifunctional protein with important roles in vascular endothelial morphogenesis during angiogenesis.

CD13 Antigens↗
PubMedDate not supplied

RNA-interference silencing of the adenosine transporter-1 gene in Trypanosoma evansi confers resistance to diminazene aceturate.

Drug resistance of trypanosomes is now a problem, but its underlying mechanisms are not fully understood. Cellular uptake of the major trypanocidal drugs is thought to occur through an adenosine transporter. The adenosine transporter-1 gene, TbAT1, encoding a P2-like nucleoside transporter has previously been cloned from Trypanosoma brucei brucei, and when expressed in yeast, it showed very similar substrate specificity to the P2-nucleoside transporter, but could not transport diamidines (pentamidine and diminazene). We have cloned and sequenced a similar gene (TevAT1) from Trypanosoma evansi and found it to have 99.7% identity to the TbAT1 gene. To elucidate the role of the TevAT1 gene on diamidine trypanocidal effect, we genetically engineered T. evansi for conditional knock-out of the TevAT1 gene by RNA interference (RNAi). Induction of the RNAi resulted in 10-fold depletion of TevAT1 mRNA, with concomitantly significant resistance to diminazene aceturate (berenil). The induced parasites propagated normally and attained peak cell density at an in vitro concentration of berenil, 5.5-fold higher than the IC(100) of the wild-type. TevAT1 knock-out had no effect on the trypanocidal activity of suramin and antrycide, but conferred some resistance to samorin. Our findings validate the significance of the TevAT1 adenosine transporter-1 gene in mediating the trypanocidal effect of diamidines in T. evansi. Further, we show for the first time that RNAi gene silencing in T. evansi can be induced using plasmids designed for T. brucei. We also demonstrate the usefulness of real-time PCR in rapidly quantifying mRNA levels in trypanosomes.

Animals↗
PubMed2006

ATP synthase is responsible for maintaining mitochondrial membrane potential in bloodstream form Trypanosoma brucei.

The mitochondrion of Trypanosoma brucei bloodstream form maintains a membrane potential, although it lacks cytochromes and several Krebs cycle enzymes. At this stage, the ATP synthase is present at reduced, although significant, levels. To test whether the ATP synthase at this stage is important for maintaining the mitochondrial membrane potential, we used RNA interference (RNAi) to knock down the levels of the ATP synthase by targeting the F1-ATPase alpha and beta subunits. RNAi-induced cells grew significantly slower than uninduced cells but were not morphologically altered. RNAi of the beta subunit decreased the mRNA and protein levels for the beta subunit, as well as the mRNA and protein levels of the alpha subunit. Similarly, RNAi of alpha subunit decreased the alpha subunit transcript and protein levels, as well as the beta-subunit transcript and protein levels. In contrast, alpha and beta RNAi knockdown resulted in a 60% increase in the F0 complex subunit 9 protein levels without a significant change in the steady-state transcript levels of this subunit. The F0-32-kDa subunit protein expression, however, remained stable throughout induction of RNAi for alpha or beta subunits. Oligomycin-sensitive ATP hydrolytic and synthetic activities were decreased by 43 and 44%, respectively. Significantly, the mitochondrial membrane potential of alpha and beta RNAi cells was decreased compared to wild-type cells, as detected by MitoTracker Red CMXRos fluorescence microscopy and flow cytometry. These results support the role of the ATP synthase in the maintenance of the mitochondrial membrane potential in bloodstream form T. brucei.

Animals↗
PubMed2005

Neurite extension in central neurons: a novel role for the receptor tyrosine kinases Ror1 and Ror2.

Neurite elongation and branching are key cellular events during brain development as they underlie the formation of a properly wired neuronal network. Here we report that the receptor tyrosine kinases Ror1 and Ror2 modulate the growth of neurites as well as their branching pattern in hippocampal neurons. Upon Ror1 or Ror2 suppression using antisense oligonucleotides or RNA interference (RNAi), neurons extended shorter and less branched minor processes when compared to those in control cells. In addition, Ror-depleted cells elongated longer, albeit less branched, axons than seen in control cells. Conversely, Ror overexpression both in non-neuronal cells and in hippocampal neurons resulted in the enhanced extension of short and highly branched processes. These phenotypes were accompanied by changes in the microtubule-associated proteins MAP1B and MAP2. Taken together, these results support a novel role for Ror receptors as modulators of neurite extension in central neurons.

Animals↗
PubMed2005

The superoxide-producing NAD(P)H oxidase Nox4 in the nucleus of human vascular endothelial cells.

The superoxide-producing NAD(P)H oxidase Nox4 was initially identified as an enzyme that is highly expressed in the kidney and is possibly involved in oxygen sensing and cellular senescence. Although the oxidase is also abundant in vascular endothelial cells, its role remains to be elucidated. Here we show that Nox4 preferentially localizes to the nucleus of human umbilical vein endothelial cells (HUVECs), by immunocytochemistry and immunoelectron microscopy using three kinds of affinity-purified antibodies raised against distinct immunogens from human Nox4. Silencing of Nox4 by RNA interference (RNAi) abrogates nuclear signals given with the antibodies, confirming the nuclear localization of Nox4. The nuclear fraction of HUVECs exhibits an NAD(P)H-dependent superoxide-producing activity in a manner dependent on Nox4, which activity can be enhanced upon cell stimulation with phorbol 12-myristate 13-acetate. This stimulant also facilitates gene expression as estimated in the present transfection assay of HUVECs using a reporter regulated by the Maf-recognition element MARE, a DNA sequence that constitutes a part of oxidative stress response. Both basal and stimulated transcriptional activities are impaired by RNAi-mediated Nox4 silencing. Thus Nox4 appears to produce superoxide in the nucleus of HUVECs, thereby regulating gene expression via a mechanism for oxidative stress response.

Base Sequence↗
PubMed2006

The RACK1 homologue from Trypanosoma brucei is required for the onset and progression of cytokinesis.

The receptor for activated C kinase 1 (RACK1) is a conserved scaffold protein that helps regulate a range of cell activities including cell growth, shape, and protein translation. We report that a homologue of RACK1 is required for cytokinesis in pathogenic Trypanosoma brucei. The protein, referred to as TRACK, is comprised of WD repeat elements and can complement cpc2 null mutants of Schizosaccharomyces pombe. TRACK is expressed throughout the trypanosome life cycle and is distributed predominantly in a perinuclear region and the cytoplasm but not along the endoplasmic reticulum, mitochondrion, or cleavage furrow of dividing cells. When tetracycline-inducible RNA interference (RNAi) is used to deplete the cellular content of TRACK, the cells remain metabolically active, but growth is inhibited. In bloodstream forms, growth arrest is due to a delay in the onset of cytokinesis. By contrast, procyclic forms are able to initiate cytokinesis in the absence of TRACK but arrest midway through cell cleavage. The RNAi cells undergo multiple rounds of partial cytokinesis and accumulate nuclei and cytoplasmic extensions with attached flagella. The TRACK RNAi construct is also inducible within infected mice. Under these conditions parasites are eliminated from peripheral blood within 3 days post-infection. Taken as a whole, these data indicate that trypanosomes utilize a RACK1 homologue to regulate the final stages of mitosis. Moreover, disrupting the interaction between TRACK and its partners might be targeted in the design of novel therapies.

Animals↗
PubMed2003

A functional genomic analysis of cell morphology using RNA interference.

BACKGROUND: The diversity of metazoan cell shapes is influenced by the dynamic cytoskeletal network. With the advent of RNA-interference (RNAi) technology, it is now possible to screen systematically for genes controlling specific cell-biological processes, including those required to generate distinct morphologies. RESULTS: We adapted existing RNAi technology in Drosophila cell culture for use in high-throughput screens to enable a comprehensive genetic dissection of cell morphogenesis. To identify genes responsible for the characteristic shape of two morphologically distinct cell lines, we performed RNAi screens in each line with a set of double-stranded RNAs (dsRNAs) targeting 994 predicted cell shape regulators. Using automated fluorescence microscopy to visualize actin filaments, microtubules and DNA, we detected morphological phenotypes for 160 genes, one-third of which have not been previously characterized in vivo. Genes with similar phenotypes corresponded to known components of pathways controlling cytoskeletal organization and cell shape, leading us to propose similar functions for previously uncharacterized genes. Furthermore, we were able to uncover genes acting within a specific pathway using a co-RNAi screen to identify dsRNA suppressors of a cell shape change induced by Pten dsRNA. CONCLUSIONS: Using RNAi, we identified genes that influence cytoskeletal organization and morphology in two distinct cell types. Some genes exhibited similar RNAi phenotypes in both cell types, while others appeared to have cell-type-specific functions, in part reflecting the different mechanisms used to generate a round or a flat cell morphology.

Animals↗
PubMed2004

Resistance of ovarian carcinoma cells to docetaxel is XIAP dependent and reversible by phenoxodiol.

Although several pathways have been proposed to explain chemoresistance, all lead to some specific defect in the mechanism of apoptosis. The objective of this study was to characterize the molecular mechanisms of drug resistance to docetaxel in epithelial ovarian cancer cells (EOC) and the use of phenoxodiol as a chemosensitizer. Four established and 12 primary cultures of ovarian carcinoma cell lines (EOC) were treated with docetaxel (5-500 ng/ml) for 24 and/or 48 h. In all the studied cell lines, the best response was seen using 500 ng/ml of docetaxel. Sensitive cell lines were identified as those with IC50 < 100 ng/ml for 48 h while resistant cell lines were identified as those with IC50 > 100 ng/ml. The morphological features of apoptosis and the activation of caspases were seen only in the sensitive cell lines determined by Hoechst staining and Caspase Glo assay. Although X-linked inhibitor of apoptosis protein (XIAP) was expressed in all EOC cells, it was only inactivated in chemosensitive cells. We confirmed the role of XIAP in docetaxel resistance by downregulation of XIAP expression using RNA interference (RNAi) as well as by pretreatment with phenoxodiol. Our results indicate that 1) docetaxel induces its cytotoxic effect through the activation of apoptosis; 2) caspase activation relies on the removal of XIAP; and 3) phenoxodiol restores sensitivity in docetaxel-resistant EOC cells. We demonstrate that phenoxodiol, by interfering with XIAP activity, functions as a chemosensitizer to docetaxel and could provide a more effective treatment for refractory ovarian cancer.

Antineoplastic Agents, Phytogenic↗
PubMed2006

Survivin gene RNA interference inhibits proliferation, induces apoptosis, and enhances radiosensitivity in HeLa cells.

OBJECTIVE: Survivin is a new member of the inhibitors of apoptosis (IAPs) family. It is upregulated in various malignancies including human cervical carcinomas. Reduction of this molecule has resulted in chemosensitization, but it is uncertain whether it can lead to radiosensitization. We observed the effect of survivin gene RNA interference (RNAi) on the proliferation, apoptosis, and radiosensitivity of the human cervical carcinoma cells HeLa. STUDY DESIGN: Human cervical carcinoma cells (HeLa) were transfected with the specific siRNA expression vector (pSilencer2.1-s2) designed to target survivin mRNA. A corresponding site-mutated vector was constructed as a negative control (pSilencer2.1-NC). The expression of survivin mRNA and its protein among the stable transfected cells and the untransfected ones was detected by semi-quantitative RT-PCR and Western blotting respectively. The cell growth was examined by methyl thiazolyl tetrazolium (MTT) assay. The cell cycle distribution and cell apoptosis were measured by flow cytometry. The changes in cell radiosensitivity were observed by clonogenic survival assay. RESULTS: Three stable transfected cell lines: HeLa-s2 (with pSilencer2.1-s2), HeLa-NC (with pSilencer2.1-NC), and HeLa-U6 neo (with empty vector pSilencer2.1-U6 neo) were established. The expression levels of survivin gene mRNA and protein in HeLa-s2 were significantly lower than in HeLa-NC, HeLa-U6 neo, and those untransfected HeLa cells. The expression inhibitory rates were 62.8% and 60.1%. The cell proliferation of HeLa-s2 was inhibited, and the highest inhibitory rate was 57.8+/-2.1%. The changes in cell cycle distribution in HeLa-s2 compared with the other three cell lines were obvious, many cells were blocked in the G(0)/G(1) phase 72.7+/-3.1% (P<0.05), reduced sharply in the G(2)/M phase (5.1+/-2.9)% (P<0.05), and also the apoptotic rate was 29.2+/-1.4%, obviously increasing (P<0.05). At the same dose of radiation, the cloning efficiency of HeLa-s2 declined notably (P<0.05); the cell survival curve showed a significant decrease in D(0) and D(q), which were 3.15 and 1.21, respectively (P<0.05), and the radiation enhancement ratios were 2.01 (a ratio of D(0)) and 1.77 (a ratio of D(q)). CONCLUSIONS: Survivin gene RNAi not only could inhibit the proliferation of human cervical carcinoma cells (HeLa), but also could significantly enhance the radiosensitivity of those cells through the reduction of its mRNA and protein. Therefore, an RNAi-targeted survivin gene strategy would be a potential approach to radiosensitization therapy in human cervical carcinomas.

Apoptosis↗
PubMed2006

Role of the kinetochore/cell cycle checkpoint protein ZW10 in interphase cytoplasmic dynein function.

Zeste white 10 (ZW10) is a mitotic checkpoint protein and the anchor for cytoplasmic dynein at mitotic kinetochores, though it is expressed throughout the cell cycle. We find that ZW10 localizes to pericentriolar membranous structures during interphase and cosediments with Golgi membranes. Dominant-negative ZW10, anti-ZW10 antibody, and ZW10 RNA interference (RNAi) caused Golgi dispersal. ZW10 RNAi also dispersed endosomes and lysosomes. Live imaging of Golgi, endosomal, and lysosomal markers after reduced ZW10 expression showed a specific decrease in the frequency of minus end-directed movements. Golgi membrane-associated dynein was markedly decreased, suggesting a role for ZW10 in dynein cargo binding during interphase. We also find ZW10 enriched at the leading edge of migrating fibroblasts, suggesting that ZW10 serves as a general regulator of dynein function throughout the cell cycle.

Animals↗
PubMed2002

Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication.

OBJECTIVES: Duplexes of 21 base pair RNA, known as short-interfering RNA (siRNA), have been shown to inhibit gene expression by a sequence-specific RNA degradation mechanism termed RNA interference (RNAi). The objective of our study was to evaluate the effect of chemokine receptor gene suppression by RNAi on the entry and replication of HIV-1. METHODS: A flow cytometry and microscopy evaluation of HIV co-receptor expression of cells transfected with siRNA. An evaluation of the effect of siRNA on HIV entry and replication by intracellular p24 antigen detection, and virus production by infected cells, respectively. RESULTS: siRNA that target CXCR4 and CCR5 could effectively impede cell surface protein expression and their consequent function as HIV co-receptors. The inhibitory effect of RNAi directed to CXCR4 was detected 48 h after transfection of CXCR4+ U87-CD4+ cells. The expression of CXCR4 and CCR5 was blocked in 63 and 48% of positive cells by the corresponding siRNA. However, siRNA directed to CXCR4 or CCR5 did not have an effect on CD4 cells or green fluorescence protein expression. siRNA directed to CXCR4 did not suppress CCR5 expression or vice versa. The suppression of HIV-1 co-receptor expression effectively blocked the acute infection of CXCR4+ or CCR5+ U87-CD4+ cells by X4 (NL4-3) or R5 (BaL) HIV-1 strains. Inhibition of virus replication occurred regardless of the multiplicity of infection employed. CONCLUSION: Our results demonstrate that RNAi may be used to block HIV entry and replication through the blockade of cellular gene expression. Gene silencing by siRNA may become a valid alternative for HIV intervention.

CCR5 Receptor Antagonists↗
PubMed2006

Permanent, lowered HLA class I expression using lentivirus vectors with shRNA constructs: Averting cytotoxicity by alloreactive T lymphocytes.

Transplantation of many tissues requires histocompatibility matching of human leukocyte antigens (HLA) to prevent graft rejection, to reduce the level of immunosuppression needed to maintain graft survival, and to minimize the risk of graft-versus-host disease, particularly in the case of bone marrow transplantation. However, recent advances in fields of gene delivery and genetic regulation technologies have opened the possibility of engineering grafts that display reduced levels of HLA expression. Suppression of HLA expression could help to overcome the limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors, necessitate the maintenance of large donor registries, and complicate the logistics of procuring and delivering matched tissues and organs to the recipient. Accordingly, we investigated whether knockdown of HLA by RNA interference (RNAi), a ubiquitous regulatory system that can efficiently and selectively inhibit the expression of specific gene products, would enable allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors, which provide a delivery system that can achieve integration into genomic DNA, thereby permanently modifying transduced graft cells. Our results show that lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA can achieve efficient and dose-dependent reduction in surface expression of HLA in human cells, associated with enhanced resistance to alloreactive T lymphocyte-mediated cytotoxicity, while avoiding MHC-non-restricted killing. We hypothesize that RNAi-induced silencing of HLA expression has the potential to create histocompatibility-enhanced, and, eventually, perhaps "universally" compatible cellular grafts.

Base Sequence↗
PubMed2002

Inhibition of viral gene expression and replication in mosquito cells by dsRNA-triggered RNA interference.

Mosquitoes transmit numerous viral pathogens to humans including dengue virus which affects approximately 50 million individuals per year. Inhibition of viral gene expression within an insect host could be used to block virus replication and subsequent transmission of the pathogen to humans. A naturally occurring gene silencing mechanism triggered by double-stranded RNA (dsRNA), RNA interference (RNAi), has recently been described in a number of species including Drosophila. To ascertain if dsRNA-triggered RNAi is present in mosquito cells, we used Aedes albopictus C6/36 cells, and to investigate the feasibility of blocking viral gene expression and replication, we used two mosquito-borne viruses, Semliki Forest virus (SFV) and the serotype 1 dengue virus (DEN1). We demonstrate that dsRNA can specifically inhibit transgene expression in C6/36 cells from both plasmid and SFV replicons and can significantly modify the kinetics of DEN1 RNA and virus replication. The inhibition mediated by dsRNA was sequence-specific and either equal or superior to that induced by antisense single-stranded RNA (ssRNA). This study demonstrates dsRNA-triggered inhibition of gene expression and virus replication in mosquito cells and suggests that this mechanism could be used to block pathogen replication within an insect host and, thus, block disease transmission.

Animals↗
PubMed2007

RGS12 is essential for RANKL-evoked signaling for terminal differentiation of osteoclasts in vitro.

UNLABELLED: How RANKL evokes [Ca(2+)](i) oscillations and leads to osteoclast differentiation is unclear. We identified a new signaling protein, RGS12, and found that RGS12 is essential for [Ca(2+)](i) oscillations and osteoclast differentiation induced by RANKL. RGS12 may play a critical role in the RANKL-evoked PLCgamma-calcium channels-[Ca(2+)](i) oscillation-NFAT2 pathway. INTRODUCTION: RANKL-induced [Ca(2+)](i) oscillations play a switch-on role in NFAT2 expression and osteoclast differentiation. However, RANKL evokes [Ca(2+)](i) oscillations and leads to osteoclast differentiation by an unknown mechanism. In this study, we identified a new RANKL-induced signaling protein, regulator of G signaling protein 12 (RGS12), and investigated its effect on osteoclast differentiation in vitro. MATERIALS AND METHODS: We used a genome-wide screening approach to identify genes that are specifically or prominently expressed in osteoclasts. To study the role of the RGS12 in osteoclast differentiation, we used vector and lentivirus-based RNAi gene silencing technology to silence the RGS12 gene in the monocyte progenitor cell lines and primary bone marrow-derived monocytes (BMMs). The interaction between RGS12 and N-type calcium channels was elucidated using co-immunoprecipitation and immunoblotting. RESULTS: We found that RGS12 was prominently expressed in osteoclast-like cells (OLCs) induced by RANKL. This result was further confirmed at both the mRNA and protein level in human osteoclasts and mouse OLCs. Silence of RGS12 expression using vector and lentivirus based RNA interference (RNAi) impaired phosphorylation of phospholipase C (PLC)gamma and blocked [Ca(2+)](i) oscillations, NFAT2 expression, and osteoclast differentiation in RANKL-induced RAW264.7 cells and BMMs. We further found that N-type calcium channels were expressed in OLCs after RANKL stimulation and that RGS12 directly interacted with the N-type calcium channels. CONCLUSIONS: These results reveal that RGS12 is essential for the terminal differentiation of osteoclasts induced by RANKL. It is possible that RGS12 regulates osteoclast differentiation through a PLC gamma-calcium channel-[Ca(2+)](i) oscillation-NFAT2 pathway.

Animals↗