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Results for “RNA, Small Interfering”

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PubMed2005

Regulation of alpha5beta1 integrin conformation and function by urokinase receptor binding.

Urokinase-type plasminogen activator receptors (uPARs), up-regulated during tumor progression, associate with beta1 integrins, localizing urokinase to sites of cell attachment. Binding of uPAR to the beta-propeller of alpha3beta1 empowers vitronectin adhesion by this integrin. How uPAR modifies other beta1 integrins remains unknown. Using recombinant proteins, we found uPAR directly binds alpha5beta1 and rather than blocking, renders fibronectin (Fn) binding by alpha5beta1 Arg-Gly-Asp (RGD) resistant. This resulted from RGD-independent binding of alpha5beta1-uPAR to Fn type III repeats 12-15 in addition to type III repeats 9-11 bound by alpha5beta1. Suppression of endogenous uPAR by small interfering RNA in tumor cells promoted weaker, RGD-sensitive Fn adhesion and altered overall alpha5beta1 conformation. A beta1 peptide (res 224NLDSPEGGF232) that models near the known alpha-chain uPAR-binding region, or a beta1-chain Ser227Ala point mutation, abrogated effects of uPAR on alpha5beta1. Direct binding and regulation of alpha5beta1 by uPAR implies a modified "bent" integrin conformation can function in an alternative activation state with this and possibly other cis-acting membrane ligands.

Amino Acid Sequence↗

PubMed2004

Nedd4-2 functionally interacts with ClC-5: involvement in constitutive albumin endocytosis in proximal tubule cells.

Constitutive albumin uptake by the proximal tubule is achieved by a receptor-mediated process in which the Cl(-) channel, ClC-5, plays an obligate role. Here we investigated the functional interaction between ClC-5 and ubiquitin ligases Nedd4 and Nedd4-2 and their role in albumin uptake in opossum kidney proximal tubule (OK) cells. In vivo immunoprecipitation using an anti-HECT antibody demonstrated that ClC-5 bound to ubiquitin ligases, whereas glutathione S-transferase pull-downs confirmed that the C terminus of ClC-5 bound both Nedd4 and Nedd4-2. Nedd4-2 alone was able to alter ClC-5 currents in Xenopus oocytes by decreasing cell surface expression of ClC-5. In OK cells, a physiological concentration of albumin (10 mug/ml) rapidly increased cell surface expression of ClC-5, which was also accompanied by the ubiquitination of ClC-5. Albumin uptake was reduced by inhibiting either the lysosome or proteasome. Total levels of Nedd4-2 and proteasome activity also increased rapidly in response to albumin. Overexpression of ligase defective Nedd4-2 or knockdown of endogenous Nedd4-2 with small interfering RNA resulted in significant decreases in albumin uptake. In contrast, pathophysiological concentrations of albumin (100 and 1000 mug/ml) reduced the levels of ClC-5 and Nedd4-2 and the activity of the proteasome to the levels seen in the absence of albumin. These data demonstrate that normal constitutive uptake of albumin by the proximal tubule requires Nedd4-2, which may act via ubiquitination to shunt ClC-5 into the endocytic pathway.

Albumins↗

PubMed2006

Gene expression response to cisplatin treatment in drug-sensitive and drug-resistant ovarian cancer cells.

The molecular pathways activated in response to acute cisplatin exposure, as well as the mechanisms involved in the long-term development of cisplatin-resistant cancer cells remain unclear. Using whole genome oligonucleotide microarrays, we have examined the kinetics of gene expression changes in a cisplatin-sensitive cell line, A2780, and its cisplatin-resistant derivative, ACRP. Both sensitive and resistant cell lines exhibited a very similar response of p53-inducible genes as early as 16 h after treatment. This p53 response was further increased at the 24-h time point. These experiments identify p53 as the main pathway producing a large-scale transcriptional response after cisplatin treatment in these cells containing wild-type p53. Consistent with a role for the p53 response in cisplatin sensitivity, knockdown of the p53 protein with small interfering RNA led to a twofold decrease in cell survival in the resistant cells. In addition, our analysis also allowed the identification of several genes that are differentially expressed between sensitive and resistant cells. These genes include GJA1 (encoding connexin 43 (Cx43)) and TWIST1, which are highly upregulated in cisplatin-resistant cells. The importance of Cx43 in drug resistance was demonstrated through functional analyses, although paradoxically, inhibition of Cx43 function in high expressing cells led to an increase in drug resistance. The pathways important in cisplatin response, as well as the genes found differentially expressed between cisplatin-resistant and -sensitive cells, may represent targets for therapy aimed at reversing drug resistance.

Cisplatin↗

PubMed2003

Stimulation of signal transducer and activator of transcription-1 (STAT1)-dependent gene transcription by lipopolysaccharide and interferon-gamma is regulated by mammalian target of rapamycin.

Mammalian target of rapamycin (mTOR) and phosphatidylinositol 3-kinase (PI3K) regulate cell growth, protein synthesis, and apoptosis in response to nutrients and mitogens. As an important source of nitric oxide during inflammation, human inducible nitric oxide synthase also plays a role in the regulation of cytokine-driven cell proliferation and apoptosis. The role of mTOR and PI3K in the activation of human inducible nitric oxide synthase transcription by cytokines and lipopolysaccharide (LPS) was investigated in lung epithelial adenocarcinoma (A549) cells. LY294002, a dual mTOR and PI3K inhibitor, blocked human inducible nitric oxide synthase (hiNOS) promoter activation and mRNA induction by cytokines and LPS in a PI3K-independent fashion. On gene expression analysis, LY294002 selectively blocked the induction of a subset of 14 LPS/interferon-gamma (IFN-gamma)-induced genes, previously characterized as signal transducer and activator of transcription-1 (STAT1)-dependent. LY294002, but not wortmannin, inhibited LPS/IFN-gamma-dependent STAT1 phosphorylation at Ser-727 and STAT1 activity. Consistent with dual inhibition of mTOR and PI3K by LY294002, dominant-negative mTOR, anti-mTOR small interfering RNA, or rapamycin each inhibited phosphorylation of STAT1 only in the presence of wortmannin. LPS/IFN-gamma led to the formation of a macromolecular complex containing mTOR, STAT1, as well as protein kinase C delta, a known STAT1alpha kinase. Thus, LPS and IFN-gamma activate the PI3K and mTOR pathways, which converge to regulate STAT1-dependent transcription of pro-apoptotic and pro-inflammatory genes in a rapamycin-insensitive manner.

Androstadienes↗

PubMed2006

Microenvironmental regulation of membrane type 1 matrix metalloproteinase activity in ovarian carcinoma cells via collagen-induced EGR1 expression.

Late stage ovarian cancer is characterized by disseminated intraperitoneal metastasis as secondary lesions anchor in the type I and III collagen-rich submesothelial matrix. Ovarian carcinoma cells preferentially adhere to interstitial collagen, and collagen-induced integrin clustering up-regulates the expression of the transmembrane collagenase membrane type 1 matrix metalloproteinase (MT1-MMP). Collagenolytic activity is important in intraperitoneal metastasis, potentiating invasion through the mesothelial cell layer and colonization of the submesothelial collagen-rich matrix. The objective of this study was to elucidate a potential mechanistic link between collagen adhesion and MT1-MMP expression. Our results indicate that culturing cells on three-dimensional collagen gels, but not thin layer collagen or synthetic three-dimensional hydrogels, results in rapid induction of the transcription factor EGR1. Integrin signaling through a SRC kinase-dependent pathway is necessary for EGR1 induction. Silencing of EGR1 expression using small interfering RNA abrogated collagen-induced MT1-MMP expression and inhibited cellular invasion of three-dimensional collagen gels. These data support a model for intraperitoneal metastasis wherein collagen adhesion and clustering of collagen binding integrins activates integrin-mediated signaling via SRC kinases to induce expression of EGR1, resulting in transcriptional activation of the MT1-MMP promoter and subsequent MT1-MMP-catalyzed collagen invasion. This model highlights the role of unique interactions between ovarian carcinoma cells and interstitial collagens in the ovarian tumor microenvironment in inducing gene expression changes that potentiate intraperitoneal metastatic progression.

Cell Adhesion↗

PubMed2005

Overexpression of glucose-6-phosphate dehydrogenase is associated with lipid dysregulation and insulin resistance in obesity.

Glucose-6-phosphate dehydrogenase (G6PD) produces cellular NADPH, which is required for the biosynthesis of fatty acids and cholesterol. Although G6PD is required for lipogenesis, it is poorly understood whether G6PD in adipocytes is involved in energy homeostasis, such as lipid and glucose metabolism. We report here that G6PD plays a role in adipogenesis and that its increase is tightly associated with the dysregulation of lipid metabolism and insulin resistance in obesity. We observed that the enzymatic activity and expression levels of G6PD were significantly elevated in white adipose tissues of obese models, including db/db, ob/ob, and diet-induced obesity mice. In 3T3-L1 cells, G6PD overexpression stimulated the expression of most adipocyte marker genes and elevated the levels of cellular free fatty acids, triglyceride, and FFA release. Consistently, G6PD knockdown via small interfering RNA attenuated adipocyte differentiation with less lipid droplet accumulation. Surprisingly, the expression of certain adipocytokines such as tumor necrosis factor alpha and resistin was increased, whereas that of adiponectin was decreased in G6PD overexpressed adipocytes. In accordance with these results, overexpression of G6PD impaired insulin signaling and suppressed insulin-dependent glucose uptake in adipocytes. Taken together, these data strongly suggest that aberrant increase of G6PD in obese and/or diabetic subjects would alter lipid metabolism and adipocytokine expression, thereby resulting in failure of lipid homeostasis and insulin resistance in adipocytes.

3T3-L1 Cells↗

PubMed2006

The role of mitogen-activated protein kinase phosphatase-1 in oxidative damage-induced cell death.

Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) is a member of the MAPK phosphatase family that functions as a negative regulator of MAPK signaling. MKP-1 is induced by oxidative stress, but the role of its induction in cell death is not fully understood. Here, we show that hydrogen peroxide (H(2)O(2)) induces MKP-1 and activates MAPKs. Induction of MKP-1 by H(2)O(2) correlated with inactivation of p38 and c-Jun-NH(2)-kinase (JNK). Overexpression of MKP-1 increased cell resistance to H(2)O(2)-induced death. Furthermore, we show by small interfering RNA silencing that down-regulation of MKP-1 increases phosphorylated p38 and JNK and subsequent cell death induced by H(2)O(2). More importantly, primary embryonic fibroblasts from mice lacking MKP-1 had a higher level of phosphorylated p38 and JNK and were more sensitive to H(2)O(2)-induced cell death compared with corresponding cells with MKP-1, indicating that p38 and JNK pathways may play important roles in H(2)O(2)-mediated cell death. Thus, these results suggest that activation of MKP-1 is a survival mechanism against oxidative damage.

Breast Neoplasms↗

PubMed2006

ARF6 and EFA6A regulate the development and maintenance of dendritic spines.

The cellular and molecular mechanisms underlying the development and maintenance of dendritic spines are not fully understood. ADP-ribosylation factor 6 (ARF6) is a small GTPase known to regulate actin remodeling and membrane traffic. Here, we report involvement of ARF6 and exchange factor for ARF6 (EFA6A) in the regulation of spine development and maintenance. An active form of ARF6 promotes the formation of dendritic spines at the expense of filopodia. EFA6A promotes spine formation in an ARF6 activation-dependent manner. Knockdown of ARF6 and EFA6A by small interfering RNA decreases spine formation. Live imaging indicates that ARF6 knockdown decreases the conversion of filopodia to spines and the stability of early spines. The spine-promoting effect of ARF6 is partially blocked by Rac1. ARF6 and EFA6A protect mature spines from inactivity-induced destabilization. These results suggest that ARF6 and EFA6A may regulate the conversion of filopodia to spines and the stability of both early and mature spines.

ADP-Ribosylation Factor 6↗

PubMed2005

Activation of AMP-activated protein kinase enhances angiotensin ii-induced proliferation in cardiac fibroblasts.

AMP-activated kinase (AMPK) is a highly conserved heterotrimeric kinase that functions as a metabolic regulator of cellular enzymes involved in carbohydrate and fat metabolism, which regulate ATP conservation and synthesis. Here, we investigated whether AMPK signaling has a role in the regulation of angiotensin II (Ang II)-induced proliferation in rat cardiac fibroblasts. Aminoimidazole-4-carboxamide-1-beta-ribofuranoside (AICAR) activated AMPK in rat cardiac fibroblasts and increased Ang II-induced extracellular signal-regulated kinase 1/2 phosphorylation and activity. AICAR also increased Ang II-induced c-fos mRNA expression in the cells. [3H]-thymidine and [3H]-proline incorporation by cardiac fibroblasts treated with Ang II was enhanced when the cells were pretreated with AICAR. Inhibition of AMPK by small interfering RNA for AMPKalpha1 suppressed Ang II-induced extracellular signal-regulated kinase activity, c-fos mRNA expression, and cell proliferation. Treatment of rats with AICAR (1 mg/g body weight per day) for 1 week significantly enhanced Ang II-induced hypertrophy of the myocardium. Our findings indicate that AMPK works as a stimulator of the Ang II-induced proliferative pathway in cardiac fibroblasts. Inhibition of AMPK signaling might serve as a new therapeutic target of remodeling of the hypertrophic myocardium.

AMP-Activated Protein Kinases↗

PubMed2004

[Inhibitory effect of siRNA on bcr-abl gene expression in K562 cell line].

To explore a new way to treat CML, inhibitory effect of small interfering RNA (SiRNA) on bcr-abl fusion gene expression of K562 cell line was studied. SiRNA for bcr-abl gene was designed and transfected into K562 cells, bcr-abl gene expression was tested by RT-PCR. The results showed that bcr-abl gene expression was inhibited by using siRNA in dose-dependent manner and reduced to 19.9% and 26.6% of the control at 24 and 48 hours after transfection with 0.2 micro g siRNA respectively. K562 cells proliferation was suppressed finally, but bcr-abl gene expression restored at 72 hours. In conclusion, anti-bcr-abl siRNA can effectively inhibit bcr-abl gene expression of K562 cell line.

Dose-Response Relationship, Drug↗

PubMed2006

NFV, an HIV-1 protease inhibitor, induces growth arrest, reduced Akt signalling, apoptosis and docetaxel sensitisation in NSCLC cell lines.

HIV-1 protease inhibitor (PI), nelfinavir (NFV) induced growth arrest and apoptosis of NCI-H460 and -H520, A549, EBC-1 and ABC-1 non-small-cell lung cancer (NSCLC) cells in association with upregulation of p21waf1, p27kip1 and p53, and downregulation of Bcl-2 and matrix metalloproteinase (MMP)-2 proteins. We found that NFV blocked Akt signalling in these cells as measured by Akt kinase assay with glycogen synthase kinase-3alpha/beta (GSK-3alpha/beta) as a substrate. To explore the role of Akt signalling in NFV-mediated growth inhibition of NSCLC cells, we blocked this signal pathway by transfection of Akt small interfering RNA (siRNA) in these cells; transient transfection of Akt siRNA in NCI-H460 cells decreased the level of Bcl-2 protein and slowed their proliferation compared to the nonspecific siRNA-transfected cells. Conversely, forced-expression of Akt partially reversed NFV-mediated growth inhibition of these cells, suggesting that Akt may be a molecular target of NFV in NSCLC cells. Also, we found that inhibition of Akt signalling by NFV enhanced the ability of docetaxel to inhibit the growth of NCI-H460 and -H520 cells, as measured by MTT assay. Importantly, NFV slowed the proliferation and induced apoptosis of NCI-H460 cells present as tumour xenografts in nude mice without adverse systemic effects. Taken together, this family of compounds might be useful for the treatment of individuals with NSCLC.

Adenocarcinoma↗

PubMed2004

Methylation in the core-promoter region of the chondromodulin-I gene determines the cell-specific expression by regulating the binding of transcriptional activator Sp3.

Transcriptional regulation of cell- and stage-specific genes is a crucial process in the development of mesenchymal tissues. Here we have investigated the regulatory mechanism of the expression of the chondromodulin-I (ChM-I) gene, one of the chondrocyte-specific genes, in osteogenic cells using osteosarcoma (OS) cells as a model. Methylation-specific sequence analyses revealed that the extent of methylation in the core-promoter region of the ChM-I gene was correlated inversely with the expression of the ChM-I gene in OS primary tumors and cell lines. 5-Aza-deoxycytidine treatment induced the expression of the ChM-I gene in ChM-I-negative OS cell lines, and the induction of expression was associated tightly with the demethylation of cytosine at -52 (C(-52)) in the middle of an Sp1/3 binding site to which the Sp3, but not Sp1, bound. The replacement of C(-52) with methyl-cytosine or thymine abrogated Sp3 binding and also the transcription activity of the genomic fragment including C(-52). The inhibition of Sp3 expression by small interfering RNA reduced the expression of the ChM-I gene in ChM-I-positive normal chondrocytes, indicating Sp3 as a physiological transcriptional activator of the ChM-I gene. These results suggest that the methylation status of the core-promoter region is one of the mechanisms to determine the cell-specific expression of the ChM-I gene through the regulation of the binding of Sp3.

5-Methylcytosine↗

PubMed2006

Select cancer testes antigens of the MAGE-A, -B, and -C families are expressed in mast cell lines and promote cell viability in vitro and in vivo.

MAGE antigens are proteins that are normally expressed only in gametes but are often aberrantly expressed in melanomas, hematopoietic malignancies, and other "cancers". The functions of most MAGE proteins are unknown. Data have accumulated suggesting expression of MAGE proteins by malignant cells may contribute to advanced disease or resistance to chemotherapy, but direct evidence supporting this hypothesis is lacking. We show here that small interfering RNA (siRNA) suppression of MAGE-A, -B, and -C gene expression slows proliferation and induces caspase independent apoptosis in human and murine mast cell lines. Furthermore, treatment with MAGE specific siRNA suppresses growth of malignant cells in an in vivo murine model of mastocytosis. These observations demonstrate that MAGE protein expression can contribute to the development of tumors by permitting proliferation and prolonging the survival of malignant cells. We suggest a shift of the current clinical paradigm from one that envisions MAGE proteins solely as targets for immunologic attack to one in which MAGE genes and proteins are also targets for functional manipulation.

Animals↗

PubMed2006

Inhibition of the mitotic kinesin Eg5 up-regulates Hsp70 through the phosphatidylinositol 3-kinase/Akt pathway in multiple myeloma cells.

The microtubule-dependent motor protein Eg5 plays a critical role in spindle assembly and maintenance in mitosis. Herein we show that the suppression of Eg5 by a specific inhibitor arrested mitosis, induced apoptosis, and up-regulated Hsp70 in human multiple myeloma cells. Mechanistically, Hsp70 induction occurred at the transcriptional level via a cis-regulatory DNA element in Hsp70 promoter and was mediated by the phosphatidylinositol 3-kinase/Akt pathway. Eg5 inhibitor-mediated Hsp70 up-regulation is cytoprotective because blocking Hsp70 induction directly by antisense or small interfering RNA or indirectly by inhibiting the phosphatidylinositol 3-kinase/Akt pathway significantly increased Eg5 inhibitor-induced apoptosis. Furthermore, a farnesyltransferase inhibitor interacted synergistically with the Eg5 inhibitor in inducing apoptosis through disrupting the Akt/Hsp70 signaling axis. These findings provide the first evidence for Eg5 inhibitor activity in hematologic malignancy and identify Hsp70 up-regulation as a critical mechanism responsible for modulating myeloma cell sensitivity to Eg5 inhibitors. In addition, these findings suggest that a combination of Eg5 inhibitors with agents abrogating Hsp70 induction would be useful for myeloma therapy in the clinic.

Apoptosis↗

PubMed2006

Differential regulation of the cell-surface targeting and function of beta- and alpha1-adrenergic receptors by Rab1 GTPase in cardiac myocytes.

The molecular mechanism underlying the export from the endoplasmic reticulum (ER) to the cell surface and its role in the regulation of signaling of adrenergic receptors (ARs) remain largely unknown. In this report, we determined the role of Rab1, a Ras-like GTPase that coordinates protein transport specifically from the ER to the Golgi, in the cell surface targeting and function of endogenous beta- and alpha1-ARs in neonatal rat ventricular myocytes. Adenovirus-driven expression of Rab1 into myocytes selectively increased the cell-surface number of alpha1-AR, but not beta-AR, whereas the dominant-negative mutant Rab1N124I significantly reduced the cell-surface expression of beta-AR and alpha1-AR. Brefeldin A inhibited beta-AR and alpha1-AR export and antagonized the Rab1 effect on alpha1-AR expression. Manipulation of Rab1 function similarly influenced the transport of alpha1A- and alpha1B-ARs as well as beta1- and beta2-ARs. Fluorescent microscopy analysis demonstrated that expression of Rab1N124I and Rab1 small interfering RNA induced a marked accumulation of GFP-tagged beta2-AR and alpha1B-AR in the ER. Consistent with the effects on receptor cell-surface targeting, Rab1 selectively enhanced ERK1/2 activation and hypertrophic growth in response to the alpha1-AR agonist phenylephrine but not to the beta-AR agonist isoproterenol. Rab1N124I inhibited both agonist-mediated ERK1/2 activation and hypertrophic growth in neonatal myocytes. These results demonstrate that the cell-surface targeting and signaling of beta- and alpha1-ARs require Rab1 and are differentially modulated by augmentation of Rab1 function. Our data provide strong evidence implicating the ER-to-Golgi traffic as a site for selective manipulation of distinct AR function in cardiac myocytes.

Animals↗

PubMed2006

Inhibition of TRIP1/S8/hSug1, a component of the human 19S proteasome, enhances mitotic apoptosis induced by spindle poisons.

Mitotic spindle poisons (e.g., Taxol and vinblastine), used as chemotherapy drugs, inhibit mitotic spindle function, activate the mitotic spindle checkpoint, arrest cells in mitosis, and then cause cell death by mechanisms that are poorly understood. By expression cloning, we identified a truncated version of human TRIP1 (also known as S8, hSug1), an AAA (ATPases associated with diverse cellular activities) family ATPase subunit of the 19S proteasome regulatory complex, as an enhancer of spindle poison-mediated apoptosis. Stable expression of the truncated TRIP1/S8/hSug1 in HeLa cells [OP-TRIP1(88-406)] resulted in a decrease of measurable cellular proteasome activity, indicating that OP-TRIP1(88-406) had a dominant-negative effect on proteasome function. OP-TRIP1(88-406) revealed an increased apoptotic response after treatment with spindle poisons or with proteasome inhibitors. The increased apoptosis coincided with a significant decrease in expression of BubR1, a kinase required for activation and maintenance of the mitotic spindle checkpoint in response to treatment with spindle poisons. Small interfering RNA (siRNA)-mediated knockdown of TRIP1/S8/hSug1 resulted in a reduction of general proteasome activity and an increase in mitotic index. The siRNA treatment also caused increased cell death after spindle poison treatment. These results indicate that inhibition of TRIP1/S8/hSug1 function by expression of a truncated version of the protein or by siRNA-mediated suppression enhances cell death in response to spindle poison treatment. Current proteasome inhibitor drugs in trial as anticancer agents target elements of the 20S catalytic subcomplex. Our results suggest that targeting the ATPase subunits in 19S regulatory complex in the proteasome may enhance the antitumor effects of spindle poisons.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2006

Gonadotropin-releasing hormone promotes ovarian cancer cell invasiveness through c-Jun NH2-terminal kinase-mediated activation of matrix metalloproteinase (MMP)-2 and MMP-9.

Gonadotropin-releasing hormone (GnRH) receptor is present in 80% of ovarian cancer, and numerous studies have provided evidence for a role of GnRH in cell proliferation. In this study, the effect of GnRH on the invasion potential of ovarian cancer cells was investigated. In vitro migration and cell invasion assays with the ovarian cancer cell lines Caov-3 and OVCAR-3 revealed the biphasic nature of GnRH; low concentrations of GnRH agonist (GnRHa) increased the cell motility and invasiveness of these cells, but at increased concentrations, the stimulatory effect was insignificant. Reverse transcription-PCR, Western blot, and gelatin zymography showed that the expression of metastasis-related proteinases, matrix metalloproteinase (MMP)-2 and MMP-9, was up-regulated and activated by GnRHa. Moreover, we observed that GnRHa was able to transactivate the MMP-2 and MMP-9 promoters. The invasive/migratory phenotype activated by GnRHa can be blocked by specific inhibitors or neutralizing antibodies to MMP-2 and MMP-9. Knockdown of the GnRH receptor using small interfering RNA significantly inhibited the GnRH-induced MMP activation, invasion, and migration. In addition, we showed that the c-Jun NH(2)-terminal kinase, but not extracellular signal-regulated kinase 1/2 or p38 mitogen-activated protein kinase, signaling pathway was critical for GnRH-mediated up-regulation of MMP, cell invasion, and motility. These results indicate for the first time an expanded role for GnRH in other aspects of ovarian tumor progression, such as metastasis, via activation of MMP and the subsequent increase in cell migration and invasion.

Cell Line, Tumor↗

PubMed2006

Characterization and functional consequences of underexpression of clusterin in rheumatoid arthritis.

We previously compared by microarray analysis gene expression in rheumatoid arthritis (RA) and osteoarthritis (OA) tissues. Among the set of genes identified as a molecular signature of RA, clusterin (clu) was one of the most differentially expressed. In the present study we sought to assess the expression and the role of CLU (mRNA and protein) in the affected joints and in cultured fibroblast-like synoviocytes (FLS) and to determine its functional role. Quantitative RT-PCR, Northern blot, in situ hybridization, immunohistochemistry, and Western blot were used to specify and quantify the expression of CLU in ex vivo synovial tissue. In synovial tissue, the protein was predominantly expressed by synoviocytes and it was detected in synovial fluids. Both full-length and spliced isoform CLU mRNA levels of expression were lower in RA tissues compared with OA and healthy synovium. In synovium and in cultured FLS, the overexpression of CLU concerned all protein isoforms in OA whereas in RA, the intracellular forms of the protein were barely detectable. Transgenic overexpression of CLU in RA FLS promoted apoptosis within 24 h. We observed that CLU knockdown with small interfering RNA promoted IL-6 and IL-8 production. CLU interacted with phosphorylated IkappaBalpha. Differential expression of CLU by OA and RA FLS appeared to be an intrinsic property of the cells. Expression of intracellular isoforms of CLU is differentially regulated between OA and RA. We propose that in RA joints, high levels of extracellular CLU and low expression of intracellular CLU may enhance NF-kappaB activation and survival of the synoviocytes.

Arthritis, Rheumatoid↗