Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UNIPROT:P42345 (mTOR)
26,049 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The membrane type 1 matrix metalloproteinase (MT1-MMP) has been identified as a major activator of MMP-2 - a process involving the formation of a trimolecular complex with TIMP-2. We previously identified the IGF-I receptor as a positive regulator of MMP-2 synthesis. Here, we investigated the role of IGF-IR in the regulation of MT1-MMP. Highly invasive Lewis lung carcinoma subline H-59 cells express MT1-MMP and utilize it to activate their major extracellular matrix degrading proteinase-MMP-2. These cells were transiently transfected with a plasmid vector expressing a luciferase reporter gene downstream of the mouse MT1-MMP promoter. IGF-I treatment increased luciferase activity in the transfected cells by up to 10-fold and augmented endogenous MT1-MMP mRNA and protein synthesis by up to 2-3-fold, relative to controls. MT1-MMP induction and invasion were blocked by the PI 3-kinase inhibitors LY294002 and wortmannin and by rapamycin, but not by the MEK inhibitor PD98059. Overexpression of a dominant negative Akt mutant or of the tumor suppressor phosphatase and tensin homologue, PTEN, in these cells also caused a significant reduction in MT1-MMP expression and invasion. The results demonstrate that IGF-IR controls tumor cell invasion by coordinately regulating MMP-2 expression and its MT1-MMP-mediated activation and identify PI 3-kinase/Akt/mTOR signaling as critical to this regulation.
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PMID:Type 1 insulin-like growth factor regulates MT1-MMP synthesis and tumor invasion via PI 3-kinase/Akt signaling. 1259 84

Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta (TGF-beta) have been shown to be oncogenic and tumor suppressive, respectively, on prostate epithelial cells. Here we show that IGF-I inhibits the ability of TGF-beta to regulate expression of several genes in the non-tumorigenic rat prostatic epithelial line, NRP-152. In these cells, IGF-I also inhibits TGF-beta-induced transcriptional responses, as shown by several promoter reporter constructs, suggesting that IGF-I intercepts an early step in TGF-beta signaling. We show that IGF-I does not down-regulate TGF-beta receptor levels, as determined by both receptor cross-linking and Western blot analyses. However, Western blot analysis reveals that IGF-I selectively inhibits the TGF-beta-triggered activation Smad3 but not Smad2, while not altering expression of total Smads 2, 3, or 4. The phosphatidylinositol 3-kinase (PI3K) inhibitor, LY29004 reverses the ability of IGF-I to inhibit TGF-beta-induced transcriptional responses and the activation of Smad3, suggesting that the suppression of TGF-beta signaling by IGF-I is mediated through activation of PI3K. Moreover, we show that enforced expression of dominant-negative PI3K (DN-p85alpha) or phosphatidylinositol 3-phosphate-phosphatase, PTEN, also reverse the suppressive effect of IGF-I on TGF-beta-induced 3TP-luciferase reporter activity, whereas constitutively active PI3K (p110alphaCAAX) completely blocks TGF-beta-induced 3TP-luciferase reporter activity. Further transfection experiments including expression of constitutively active and dominant-negative Akt and rapamycin treatment suggest that suppression of TGF-beta signaling/Smad3 activation by IGF-I occurs downstream of Akt and through mammalian target of rapamycin activation. In summary, our data suggest that IGF-I inhibits TGF-beta transcriptional responses through selective suppression of Smad3 activation via a PI3K/Akt-dependent pathway.
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PMID:Insulin-like growth factor-I inhibits transcriptional responses of transforming growth factor-beta by phosphatidylinositol 3-kinase/Akt-dependent suppression of the activation of Smad3 but not Smad2. 1287 89

The increased levels of c-Myc protein observed previously in an ovarian carcinoma cell line stably transfected to express HER2 has suggested a role for the HER2 pathway in c-Myc expression. Analysis of HER2-transfected cells stimulated with heregulin beta1 (HRG) revealed increased c-Myc protein levels but not a corresponding increase in c-Myc mRNA expression or any change in c-Myc protein half-life. Transfection of HER2-overexpressing cells with a construct containing the 5' untranslated region (5'UTR) of c-Myc mRNA originated from the P2 promoter and placed upstream of the Renilla luciferase gene, enhanced reporter expression upon stimulation with HRG. The HRG-mediated increase in reporter activity correlated with the HRG-mediated induction observed for c-Myc protein, identifying the P2-derived leader (P2L) of c-Myc mRNA as the cis-element involved in c-Myc translational induction. Both the increase in c-Myc protein levels and P2L-enhanced translational activity were inhibited by the PI3K inhibitor wortmannin. Together, these results demonstrate that HRG stimulation of HER2 overexpressing cells leads to enhanced c-Myc protein synthesis through activation of the PI3K/Akt/mTOR pathway and that the P2L of c-Myc mRNA is the element responsible for induction of c-Myc translation.
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PMID:HER2 signaling enhances 5'UTR-mediated translation of c-Myc mRNA. 1513 60

Signaling pathways regulating proliferation, differentiation, and apoptosis are commonly mediated through protein-protein interactions as well as reversible phosphorylation of proteins. To facilitate the study of regulated protein-protein interactions in cells and living animals, we optimized firefly luciferase protein fragment complementation by screening incremental truncation libraries of N- and C-terminal fragments of luciferase. Fused to the rapamycin-binding domain (FRB) of the kinase mammalian target of rapamycin and FK506-binding protein 12 (FKBP), respectively, the optimized FRB-N-terminal luciferase fragment (NLuc)/C-terminal luciferase fragment (CLuc)-FKBP luciferase complementation imaging (LCI) pair reconstituted luciferase activity in cells upon single-site binding of rapamycin in an FK506-competitive manner. LCI was used in three independent applications. In mice bearing implants of cells expressing the FRB-NLuc/CLuc-FKBP LCI pair, dose- and time-dependent luciferase activity allowed target-specific pharmacodynamic analysis of rapamycin-induced protein-protein interactions in vivo. In cells expressing a Cdc25C-NLuc/CLuc-14-3-3epsilon LCI pair, drug-mediated disruption of cell cycle regulated protein-protein interactions was demonstrated with the protein kinase inhibitor UCN-01 in a phosphoserine-dependent manner. When applied to IFN-gamma-dependent activation of Janus kinase/signal transducer and activator of transcription 1 (STAT1), LCI revealed, in the absence of ligand-induced phosphorylation, STAT1 proteins existing in live cells as preformed dimers. Thus, optimized LCI provides a platform for near real-time detection and characterization of regulated and small molecule-induced protein-protein interactions in intact cells and living animals and should enable a wide range of novel applications in drug discovery, chemical genetics, and proteomics research.
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PMID:Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. 1528 40

IL-8 plays an integral role in promoting the malignant phenotype in breast cancer, and its production is directly influenced by inflammatory cytokines in the tumor microenvironment. Here, we show that activation of IL-1beta receptors on malignant HS578t and MDA-MB-231 breast cancer cells strongly induces IL-8 expression and that RNA stabilization is persistently activated at least 12-24 hr after stimulation. SB 203580 and rapamycin reversed the RNA stabilization effect of IL-1beta in a dose-dependent manner, suggesting involvement of the p38/MAP kinase and mTOR pathways. A luciferase reporter assay indicated that the stabilization effect was dependent on cis elements in the 3'-untranslated region (UTR) of the IL-8 transcript. By UV cross-linking, we identified multiple cellular factors that interact with the IL-8 3'UTR, ranging 34-76 kDa. Immunoprecipitation analysis indicated that HuR, KSRP and TIAR bound to one or more loci in the 3'UTR. While the cross-linking patterns were similar, quantitative immunoprecipitation of native IL-8 RNA from IL-1beta-stimulated cytoplasmic extract revealed a 20-fold greater association of transcript with the stabilizing factor HuR vs. the destabilizing factor KSRP. In conclusion, IL-1beta is a potent cytokine stimulus for IL-8 RNA stabilization in breast cancer cells, possibly by enhanced binding of cytoplasmic HuR to the 3'UTR.
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PMID:IL-1beta induces stabilization of IL-8 mRNA in malignant breast cancer cells via the 3' untranslated region: Involvement of divergent RNA-binding factors HuR, KSRP and TIAR. 1551 71

The Akt kinase is a serine/threonine protein kinase that has been implicated in mediating a variety of biological responses. Studies show that high Akt activity in breast carcinoma is associated with a poor pathophenotype, as well as hormone and chemotherapy resistance. Additionally, high Akt activity is associated with other features of poor prognosis. Thus, a chemotherapeutic agent directed specifically toward tumors with high Akt activity could prove extremely potent in treating those breast tumors with the most aggressive phenotypes. Several studies have demonstrated that rapamycin, which inhibits mammalian target of rapamycin (mTOR), a downstream target of Akt, sensitizes certain resistant cancer cells to chemotherapeutic agents. This study evaluated the efficacy of mTOR inhibition in the treatment of tamoxifen-resistant breast carcinoma characterized by high Akt activity. We found that MCF-7 breast cancer cell lines expressing a constitutively active Akt are able to proliferate under reduced estrogen conditions and are resistant to the growth inhibitory effects of tamoxifen, both in vitro as well as in vivo in xenograft models. Cotreatment with the mTOR inhibitor rapamycin in vitro, or the ester of rapamycin, CCI-779 (Wyeth) in vivo, inhibited mTOR activity and restored sensitivity to tamoxifen, suggesting that Akt-induced tamoxifen resistance is mediated in part by signaling through the mTOR pathway. Although the mechanism underlying the synergism remains to be understood, the results were associated with rapamycin's ability to block transcriptional activity mediated by estrogen receptor alpha, as assessed by reporter gene assays with estrogen-responsive element luciferase. These data corroborate prior findings indicating that Akt activation induces resistance to tamoxifen in breast cancer cells. Importantly, these data indicate a novel mechanism for tamoxifen resistance and suggest that blockage of the phosphatidylinositol 3'-kinase/Akt signaling pathway by mTOR inhibition effectively restores the susceptibility of these cells to tamoxifen. These data may have implication for future clinical studies of mTOR inhibition in breast carcinoma.
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PMID:Inhibition of mTOR activity restores tamoxifen response in breast cancer cells with aberrant Akt Activity. 1558 41

Tauroursodeoxycholic acid (TUDCA) is a cytoprotective bile acid frequently prescribed to patients with cholestatic diseases. Several mechanisms of action have been investigated, but the possibility that cyclic adenosine monophosphate responsive element binding protein (CREB), a transcription factor promoting cell survival, mediates TUDCA's protective effects has not been considered. We examined whether TUDCA activates CREB and whether this activation can protect biliary epithelial cells. Cholangiocytes were stressed by exposure to CCI-779, which inhibits signaling though the kinase mTOR (mammalian target of rapamycin), resulting in cell cycle arrest and apoptosis. Incubation of normal rat cholangiocytes (NRC) cells, with TUDCA resulted in phosphorylation of CREB (Western blotting analysis) and activation of CREB transcription activity (luciferase reporter assay). Inhibition of calcium signals and inhibition of protein kinase C prevented the TUDCA-induced activation of CREB. CCI-779 decreased the viability of rat cholangiocytes in a dose-dependent manner (MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay). TUDCA protected against CCI-779 cytotoxicity. A dominant negative form of CREB was stably transduced in NRC cells (NRC-M1). TUDCA protection was decreased in NRC-M1. While CCI-779 induced apoptosis in NRC cells as determined by caspase 3 activity, TUDCA attenuated CCI-779-induced apoptosis, an effect absent in NRC-M1. Finally, CCI-779 blocked proliferation of both NRC and NRC-M1 (thymidine incorporation) and this was unaffected by TUDCA. In conclusion, TUDCA activates CREB in cholangiocytes, reducing the apoptotic effect of CCI-779. These findings suggest a novel cytoprotective mechanism for this bile acid.
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PMID:Activation of CREB by tauroursodeoxycholic acid protects cholangiocytes from apoptosis induced by mTOR inhibition. 1586 31

Peroxisome proliferator-activated receptor (PPAR)-gamma and retinoic acid X receptor (RXR) heterodimer regulates cell growth and differentiation. Zinc finger transcription factor-9 (Zf9), whose phosphorylation promotes target genes, is a transcription factor essential for transactivation of the transforming growth factor (TGF)-beta1 gene. This study investigated whether activation of PPARgamma-RXR heterodimer inhibits TGFbeta1 gene transcription and Zf9 phosphorylation and, if so, what signaling pathway regulates it. Either 15-deoxy-delta(12,14)-prostaglandin J2 (PGJ2) or 9-cis-retinoic acid (RA) treatment decreased the TGFbeta1 mRNA level in L929 fibroblasts. PGJ2 + RA, compared with individual treatment alone, synergistically inhibited the TGFbeta1 gene expression, which was abrogated by PPARgamma antagonists. Likewise, PGJ2 + RA decreased luciferase expression from the TGFbeta1 gene promoter. Promoter deletion analysis of the TGFbeta1 gene revealed that pGL3-323 making up to -323-base pair region, but lacking PPAR-responsive elements, responded to PGJ2 + RA. PGJ2 + RA treatment inhibited the activity of p70 ribosomal S6 kinase-1 (S6K1), abolishing Zf9 phosphorylation at serine as did rapamycin [a mammalian target of rapamycin (mTOR) inhibitor]. Zf9 dephosphorylation by PGJ2 + RA was reversed by transfection of cells with the plasmid encoding constitutively active S6K1 (CA-S6K1). Transfection with dominant negative S6K1 inhibited the TGFbeta1 gene. TGFbeta1 gene repression by PGJ2 + RA was consistently antagonized by CA-S6K1. Ectopic expression of PPARgamma1 and RXRalpha repressed pGL3-323 transactivation with S6K1 inhibition, which was abrogated by CA-S6K1 transfection. PGJ2 + RA induced phosphatase and tensin homolog deleted on chromosome 10 (PTEN), whose overexpression repressed the TGFbeta1 gene through S6K1 inhibition, decreasing extracellular signal-regulated kinase 1/2-90-kDa ribosomal S6 kinase 1 and Akt-mTOR phosphorylations. Data indicate that activation of PPARgamma-RXR heterodimer represses the TGFbeta1 gene and induces Zf9 dephosphorylation via PTEN-mediated S6K1 inhibition, providing insight into pharmacological manipulation of the TGFbeta1 gene regulation.
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PMID:Peroxisome proliferator-activated receptor-gamma and retinoic acid X receptor alpha represses the TGFbeta1 gene via PTEN-mediated p70 ribosomal S6 kinase-1 inhibition: role for Zf9 dephosphorylation. 1661 54

Cigarette smoke is a powerful inducer of inflammatory responses resulting in disruption of major cellular pathways with transcriptional and genomic alterations driving the cells towards carcinogenesis. Cell culture and animal model studies indicate that (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol present in green tea, possesses potent anti-inflammatory and antiproliferative activity capable of selectively inhibiting cell growth and inducing apoptosis in cancer cells without adversely affecting normal cells. Here, we demonstrate that EGCG pretreatment (20-80 microM) of normal human bronchial epithelial cells (NHBE) resulted in significant inhibition of cigarette smoke condensate (CSC)-induced cell proliferation. Nuclear factor-kappaB (NF-kappaB) controls the transcription of genes involved in immune and inflammatory responses. In most cells, NF-kappaB prevents apoptosis by mediating cell survival signals. Pretreatment of NHBE cells with EGCG suppressed CSC-induced phosphorylation of IkappaBalpha, and activation and nuclear translocation of NF-kappaB/p65. NHBE cells transfected with a luciferase reporter plasmid containing an NF-kappaB-inducible promoter sequence showed an increased reporter activity after CSC exposure that was specifically inhibited by EGCG pretreatment. Immunoblot analysis showed that pretreatment of NHBE cells with EGCG resulted in a significant downregulation of NF-kappaB-regulated proteins cyclin D1, MMP-9, IL-8 and iNOS. EGCG pretreatment further inhibited CSC-induced phosphorylation of ERK1/2, JNK and p38 MAPKs and resulted in a decreased expression of PI3K, AKT and mTOR signaling molecules. Taken together, our data indicate that EGCG can suppress NF-kappaB activation as well as other pro-survival pathways such as PI3K/AKT/mTOR and MAPKs in NHBE cells, which may contribute to its ability to suppress inflammation, proliferation and angiogenesis induced by cigarette smoke.
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PMID:Green tea polyphenol EGCG suppresses cigarette smoke condensate-induced NF-kappaB activation in normal human bronchial epithelial cells. 1686 72

The post-transcriptional mechanisms by which feeding and insulin increase leptin production are poorly understood. Starvation of 6-7-week-old rats for 14 h decreased leptin mRNA level by only 22% but decreased plasma levels, adipose tissue leptin content, and release by over 75%. The decreased leptin with starvation was explained by >85% decrease in relative rates of leptin biosynthesis measured by metabolic labeling and immunoprecipitation. In vitro insulin treatment of adipose tissue from fed or starved rats for 2 h increased relative rates of leptin biosynthesis by 2-3-fold, and the effect was blocked by inhibition of phosphatidylinositol 3-kinase or mammalian target of rapamycin. Consistent with the hypothesis that feeding/insulin increases leptin translation, more leptin mRNA was associated with polysomes in adipose tissue of fed than starved rats, and in vitro incubation of adipose tissue of starved rats with insulin shifted leptin mRNA into polysomes. To assess the mechanisms regulating leptin translation, chimeric human leptin untranslated region (UTR) reporter constructs were transiently transfected into differentiated 3T3-L1 adipocytes. The 5'-UTR of leptin mRNA increased luciferase reporter activity 2-3-fold, whereas the full-length 3'-UTR (nucleotides 1-2804) was inhibitory (-65%). Sequences between nucleotides 462 and 1130 of the leptin 3'-UTR conferred most of the inhibitory effect. Insulin stimulated the expression of constructs that included both the full-length 5'-UTR and the inhibitory 3'-UTR, and the effect was blocked by inhibition of phosphatidylinositol 3-kinase or mammalian target of rapamycin. Our data suggest that insulin derepresses leptin translation by a mechanism that requires both the 5'-UTR and the 3'-UTR and may contribute to the increase in leptin production with feeding.
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PMID:Feeding and insulin increase leptin translation. Importance of the leptin mRNA untranslated regions. 1708 42


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