Luciferase Activity Reporter Assay Research Articles

MiR-204 suppresses cochlear spiral ganglion neuron survival in vitro by targeting TMPRSS3

Sensorineural hearing loss (SNHL) is the most common cause of hearing impairment. One of the essential steps to prevent progressive hearing loss is to protect spiral ganglion neurons (SGNs) from ongoing degeneration. MicroRNAs and TMPRSS3 (transmembrane protease, serine 3) have been reported to be involved in development of SGNs and genesis of SNHL. The aim of this study was to investigate the role of miR-204 and TMPRSS3 in SGNs. Effect of miR-204 on cell viability of SGNs was first examined using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Expression of TMPRSS3 in SGNs with or without addition of miR-204 was assessed by real-time PCR and western blot further. A luciferase reporter activity assay was conducted to confirm target association between miR-204 and 3′-UTR of TMPRSS3. Finally, role of TMPRSS3 on cell viability of SGNs was evaluated by transfection of TMPRSS3 siRNA. Cell viability of SGNs was suppressed by miR-204 in a concentration-dependent manner. Overexpression of miR-204 reduced expression of TMPRSS3 in SGNs at both mRNA and protein levels. Binding to the 3′-UTR of TMPRSS3 by miR-204 was identified by luciferase assay. Knockdown of TMPRSS3 by siRNA significantly inhibits cell viability of SGNs. miR-204 could be a potential therapeutic target in sensorineural hearing loss.

MicroRNA-7a/b Protects against Cardiac Myocyte Injury in Ischemia/Reperfusion by Targeting Poly(ADP-Ribose) Polymerase

ObjectivesMicroRNA-7 (miR-7) is highly connected to cancerous cell proliferation and metastasis. It is also involved in myocardial ischemia-reperfusion (I/R) injury and is upregulated in cardiomyocyte under simulated I/R (SI/R). We aimed to investigate the role of miR-7 during myocardial I/R injury in vitro and in vivo and a possible gene target.Methods and ResultsReal-time PCR revealed that miR-7a/b expression was upregulated in H9c2 cells after SI/R. Flow cytometry showed SI/R-induced cell apoptosis was decreased with miR-7a/b mimic transfection but increased with miR-7a/b inhibitor in H9c2 cells. In a rat cardiac I/R injury model, infarct size determination and TUNEL assay revealed that miR-7a/b mimic decreased but miR-7a/b inhibitor increased cardiac infarct size and cardiomyocyte apoptosis as compared with controls. We previously identified an important gene connected with cell apoptosis -- poly(ADP-ribose) polymerase (PARP) -- as a candidate target for miR-7a/b and verified the target by luciferase reporter activity assay and western blot analysis.ConclusionsmiR-7a/b is sensitive to I/R injury and protects myocardial cells against I/R-induced apoptosis by negatively regulating PARP expression in vivo and in vitro. miR-7a/b may provide a new therapeutic approach for treatment of myocardial I/R injury. Poly(ADP-ribose) polymerase.

MiR‐138 inhibits proliferation by targeting 3‐phosphoinositide‐dependent protein kinase‐1 in non‐small cell lung cancer cells

Underlying mechanisms of non-small cell lung cancer (NSCLC) development remain poorly understood. miR-138 and 3-phosphoinositide-dependent protein kinase-1 (PDK1) have been reported to be involved in the genesis of NSCLC. The aim of this study was to investigate the role and mechanisms of miR-138 and PDK1 in human NSCLC cells. The effect of miR-138 on proliferation of A549 lung cancer cells was first examined using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay. The expression of PDK1 in A549 lung cancer cells was assessed by real-time polymerase chain reaction further. A luciferase reporter activity assay was conducted to confirm target association between miR-138 and 3' untranslated region (3'-UTR) of PDK1. Finally, the role of PDK1 on proliferation of A549 cells was evaluated by transefection of PDK1 small interfering RNA (siRNA). Proliferation of A549 lung cancer cells was suppressed by miR-138 in a concentration-dependent manner. Furthermore, miR-138 can bind to the 3'-UTR of PDK1 and downregulate expression of PDK1 at both mRNA and protein levels. Knockdown of PDK1 by siRNA significantly inhibits the proliferation of A549 lung cancer cells. These findings suggest that miR-138 as a potential tumor suppressor could inhibit cell proliferation by targeting PDK1 in NSCLC cells, which could be employed as a potential therapeutic target for miRNA-based NSCLC therapy.

WLIP derived from Lasiosphaera fenzlii Reich exhibits anti-tumor activity and induces cell cycle arrest through PPAR-γ associated pathways

White-line-inducing principle (WLIP), a lipodepsipeptide isolated from Lasiosphaera fenzlii Reich., which is one of the puffballs, for the first time, has been reported to exhibit anti-microbial activity. However, there are no reports regarding the anti-tumor effects of WLIP. In this study, we reported the anti-cancer effects of WLIP on K562 cells, and its potential effect on the PPAR-γ activation pathway. The obtained results showed that WLIP exerted strong anti-proliferative effect on K562 cells. Moreover, WLIP was found to increase apoptosis and induce G0/Gl arrest. Modeling results from the Surflex-Dock program suggested that PPAR-γ might be the potential anti-tumor target of WLIP, which was confirmed by the experiments that WLIP increased the activity in luciferase reporter assay and the expression of PPAR-γ in Western blot. Besides, WLIP was able to down-regulate the expression of Bcl-xL, Cyclin-D1 in K562 cells. In summary, our novel observations suggested that WLIP might have a potential implication in cancer prevention and treatment, and also showed for the first time that the anti-tumor effect of WLIP might be mediated through modulation of the PPAR-γ activation pathway.

MicroRNA-124 (miR-124) Regulates Ku70 Expression and is Correlated with Neuronal Death Induced by Ischemia/Reperfusion

MicroRNAs are small, non-coding RNA molecules that regulate gene expression, and miR-124 is the most abundant miRNA in the brain. Studies have shown that miR-124 is clearly reduced in the ischemic brain after stroke; however, the role of miR-124 after stroke is less well studied. Using TargetScan, MicroCosm Targets version 5, and microRNA.org databases, we identified miR-124 as a possible regulator of the DNA repair protein Ku70. We validated that Ku70 is a target for miR-124 with a luciferase reporter activity assay. Moreover, adult rats subjected to focal cerebral ischemia exhibited a substantial reduction of miR-124 expression, which was inversely upregulated by Ku70 expression. In vivo treatment with miR-124 antagomir effectively enhanced Ku70 mRNA and protein levels in the ischemic region. Furthermore, knockdown of cerebral miR-124 reduced cell death and infarct size and improved neurological outcomes. Our data demonstrate that miR-124 is an endogenous regulator of Ku70 that improves ischemia/reperfusion (I/R)-induced brain injury and dysfunction.

730 Cyclic GMP Kinase II (CKII) Inhibits NHE3 by Affecting Trafficking via Phosphorylating NHE3 At Three Sites: Identification of a Multifunctional Phosphorylation Site

was measured by luciferase reporter activity assays after transiently transfecting Caco-2 cells with DRA promoter construct (-1183/+144). Treatment of Caco-2 cells with 10 μM concentration of ATRA significantly induced DRA mRNA levels within 8 h of incubation, which increased to ~3 fold by 24 h. A concomitant increase (140 ± 5 % of control P ,0.001) in DRA protein expression was also observed. In addition, ATRA treatment significantly increased DRA promoter activity (258.8 ± 3.4 % of control, P,0.001) indicating that ATRA effects are transcriptionally mediated. ATRA is known to mediate its effects via specific retinoic acid receptors (RAR's) α, β and γ. A remarkable increase (~11 fold) in mRNA levels of RAR-β receptor subtype were found in response to ATRA treatment with no change in levels of RAR'-α and γ. Treatment of cells with RAR-β agonist (CH-55, 1μM) mimicked ATRA effects on DRA promoter. RAR-β antagonist (LE-135, 1μM) blocked the stimulatory effects of ATRA on DRA promoter activity. Since, DRA expression is known to be modulated by hepatic nuclear factors (HNF's), the potential mediation of ATRA effects via HNFs was examined. A significant increase in HNF-1β mRNA levels (but not HNF-1α or HNF4) in response to ATRA treatment was observed. The ability of ATRA to induce DRA expression was blocked by silencing HNF-1β using siRNA. In summary, ATRA increased DRA expression via RAR-β through HNF-1β activation. We speculate that ATRA may act as a potential therapeutic tool under inflammatory conditions where DRA expression is altered (Supported by NIDDK and Dept. of Veteran Affairs).

Endothelial CSN5 impairs NF-κB activation and monocyte adhesion to endothelial cells and is highly expressed in human atherosclerotic lesions

The COP9 signalosome (CSN), a multifunctional protein complex involved in the regulation of cullin-RING-E3 ubiquitin ligases (CRLs), has emerged as a regulator of NF-κB signalling. As NF-κB drives the expression of pro-inflammatory and pro-atherosclerotic genes, we probed the yet unknown role of the CSN, in particular CSN5, on NF-κB-mediated atherogenic responses in endothelial cells. Co-immunoprecipitation in human umbilical vein endothelial cells (HUVECs) revealed the presence of a super-complex between IKK and CSN, which dissociates upon TNF-α stimulation. Furthermore, CSN5 silencing enhanced TNF-α-induced IκB-α degradation and NF-κB activity in luciferase reporter assays. This was paralleled by an increased NF-κB-driven upregulation of atherogenic chemokines and adhesion molecules, as measured by qPCR and flow cytometry, and translated into an enhanced arrest of THP-1 monocytes on TNF-α-stimulated, CSN5-depleted HUVECs. Reverse effects on NF-κB activity and THP-1 arrest were seen upon CSN5 overexpression. Finally, double-immunostaining confirmed the expression of CSN subunits in the endothelium of human atherosclerotic lesions, and revealed an increased expression of CSN5 which correlated with atheroprogression. In conclusion, endothelial CSN5 attenuates NF-κB-dependent pro-inflammatory gene expression and monocyte arrest on stimulated endothelial cells in vitro, suggesting that CSN5 might serve as a negative regulator of atherogenesis.

Effect of Genetic Polymorphism of ALOX15 on Aspirin-Exacerbated Respiratory Disease

Background: Aspirin-exacerbated respiratory disease (AERD) is a clinical syndrome associated with chronic inflammation in the airways coincident with chronic rhinitis, sinusitis, recurrent polyposis and asthma. Eosinophils are the key inflammatory cells in the development of AERD. AERD has been attributed to abnormalities of the arachidonic acid metabolism, but the pathogenesis of AERD is not fully understood. Our aim was to investigate the genetic contribution of the arachidonate 15-lipoxygenase gene (ALOX15) to the development of AERD. Methods: We enrolled 171 patients with AERD, 229 patients with aspirin-tolerant asthma, and 195 normal healthy controls in a Korean population. Three polymorphisms (–427G/A, –272C/A, –217G/C) in the promoter region of ALOX15 were genotyped. The functional variability of the promoter polymorphisms were analyzed by luciferase reporter activity assay. Result: No significant difference in the genotype frequency of the ALOX15 genetic polymorphism was found. Peripheral total eosinophil count was significantly higher in the patients carrying the GG genotype of the –427G/A polymorphism (p = 0.016). Similarly, the patients carrying haplotype 1 (ht1) (GCG) of –427G/A, –272C/A and –217G/C showed a significantly higher total eosinophil count compared to the other haplotypes (p = 0.008) in the AERD group. The promoter activity of the ht1 (GCG) construct was significantly higher compared to that of the ht3 (AGG) construct in A549 and U937 cells (both p < 0.001). Conclusion: These results suggest that the promoter polymorphisms of the ALOX15 gene affect ALOX15 activity leading to increased eosinophil infiltration in AERD patients.

383 Ablation of TGFbeta Receptor II Inhibits Liver Injury, Inflammation, Fibrosis and Carcinogenesis in Hepatocyte Specific TAK1 Deleted Mice

Background and Aims: Smad7 has been reported to inhibit inflammation. The role of Smad7 in the pathogenesis of hepatocellular carcinoma (HCC) is unknown. We investigated the functional significance of Smad7 in HCC both In Vivo and In Vitro. Methods: Smad7 knockout (KO) and wildtype (WT) littermates were single injected with diethylnitrosamine (DEN), a liver carcinogen, at age 15 days to induce HCC. The biological function of Smad7 was determined In Vitro in human HCC cell line (Hep3B) and primary cultures established from resected HCCs from KO and WT mice. Cell growth was evaluated by cell viability and colony formation, cell apoptosis by Annexin V/7-AAD double staining, cell cycle distribution by flow cytometry and cell invasion ability by Matrigel invasion assay. Signaling pathways and downstream effectors of Smad7 in HCC was evaluated by luciferase reporter activity assay and pathway-PCR-array. Results: Smad7 KO mice were more susceptible to DENinduced HCC than WT mice (78% vs 22%, P=0.018). HCCs from KO mice displayed a greater proliferative activity (P<0.01), and a reduced apoptotic index compared with the WT littermates (P<0.05). Smad7 was down-regulated in human HCC tissues compared to their adjacent non-tumor tissues (n=11, P<0.05). These In Vivo results indicated that Smad 7 may function as a potential tumor suppressor, which was further functionally tested In Vitro. Ectopic expression of Smad7 in Hep3B cells suppressed cell growth as evidenced by retarded cell growth curve (P<0.0001) and colony formation assay (P<0.01). Conversely, Smad7 increased cell apoptosis (P<0.05), consistent with enhanced cleaved caspase-9, caspase-3 and PARP, indicative of Smad7-induced apoptosis through the intrinsic caspase dependent pathway. Cell cycle analysis in Heb3B transfected with Smad7 revealed an increased proportion of cells arrested at G0/G1 phase (P<0.001), concomitant with a reduction in the proportion of cells in S-phase (P<0.001) as compared with control. Smad7 also repressed cell invasive ability (P<0.05). Moreover, primary culture cells form HCCs of KO mice showed a increased cell growth curve (P<0.01) and colony formation (P<0.01) compared with the cells generated from HCCs of WT mice. Moreover, pathway luciferase reporter assay indicated that Smad7 inhibited NF-κB and TGF-β signalings. Co-immunoprecipitation analysis showed that Smad7 directly interacts with TAB2, an upstream activator of NF-κB. The downstream targets of Smad7 was further revealed by TGF-β and NF-κB pathway PCR array as shown in Figure 1, which contribute to the tumor suppressive effect. Conclusion: Loss of Smad7 is sufficient to enhance susceptibility to HCC. Smad7 suppresses HCC growth through reducing cell proliferation and invasion, inducing G0/G1 phase arrest and apoptosis by inhibiting NF-κB and TGF-β signalings. Thus, Smad7 acts as a tumor-suppressor in the liver.

SRY-box-containing Gene 2 Regulation of Nuclear Receptor Tailless (Tlx) Transcription in Adult Neural Stem Cells

Adult neurogenesis is maintained by self-renewable neural stem cells (NSCs). Their activity is regulated by multiple signaling pathways and key transcription factors. However, it has been unclear whether these factors interplay with each other at the molecular level. Here we show that SRY-box-containing gene 2 (Sox2) and nuclear receptor tailless (TLX) form a molecular network in adult NSCs. We observed that both Sox2 and TLX proteins bind to the upstream region of Tlx gene. Sox2 positively regulates Tlx expression, whereas the binding of TLX to its own promoter suppresses its transcriptional activity in luciferase reporter assays. Such TLX-mediated suppression can be antagonized by overexpressing wild-type Sox2 but not a mutant lacking the transcriptional activation domain. Furthermore, through regions involved in DNA-binding activity, Sox2 and TLX physically interact to form a complex on DNAs that contain a consensus binding site for TLX. Finally, depletion of Sox2 revealed the potential negative feedback loop of TLX expression that is antagonized by Sox2 in adult NSCs. These data suggest that Sox2 plays an important role in Tlx transcription in cultured adult NSCs.

Abstract B67: Phenotype-specific role for protein kinase CK2 in luminal and basal-like breast cancer cells

Abstract Protein Kinase CK2 is a hetero-tetrameric serine/threonine kinase which has been shown to have increased expression in most types of cancer. Previous studies have identified roles for CK2 in transcriptional regulation, proliferation and survival, as well as in epithelial mesenchymal transition. Our previous studies have shown that CK2 is responsible for the phosphorylation of the estrogen receptor (ERα) at two previously unidentified sites. Here we sought to delineate the specific functions of CK2 in ERα (+) luminal and ERα (−) basal-like breast cancer. Breast tissue microarrays studies show that CK2 is expressed in both ERα (+) and ERα (−) breast tumors. In ER dependent cell lines, inhibition of CK2 causes a substantial inhibition of transcriptional activity in luciferase reporter assays. Furthermore, CK2 was found to interact with the promoters of estrogen responsive genes in an ER-dependent fashion. Despite the selective effects on ER dependent gene expression, inhibition of CK2 attenuated proliferation/ cell viability both ERα (+) and ERα (−) cell lines, suggesting that CK2 may have distinct roles in the function of specific breast cancer cell phenotypes. Indeed, Inhibition of CK2 caused cell-type specific changes in the expression of vimentin and E-cadherin, as determined by flow cytometry, as well as cell morphology. Taken together, these studies suggest that CK2 utilizes multiple molecular mechanisms to promote tumorigenesis. Inhibition of CK2 may be a viable therapeutic targeting strategy for treatment of patients with ERα (+) and ERα (−) breast tumor phenotypes. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B67.

Fatty acid desaturase 2 promoter mutation is not responsible for Δ6-desaturase deficiency

Dietary essential polyunsaturated fatty acids (PUFAs) require fatty acid desaturases (FADS) for conversion to long-chain PUFAs (LCPUFAs), which are critical for many aspects of human health. A Δ6-desaturase deficiency in a single patient was attributed to an insertion mutation in the FADS2 promoter. Later population studies have shown this thymidine nucleotide (T) insertion to be a common polymorphism (rs3834458). We examined correlations between rs3834458 variants and fatty acid evidence of FADS2 activity in a cohort of rheumatoid arthritis patients selected for low or nil consumption of n-3 LCPUFA as fish or fish oil. The presence of the T allele was associated with higher FADS2 activity, as indicated by higher conversion of plasma n-3 PUFA to LCPUFA. However, the T-insertion/deletion polymorphism did not affect FADS2 promoter activity in luciferase reporter assays in HepG2 or NIH/3T3 cells. Our results indicate that the polymorphism rs3834458 does not appear to directly affect FADS2 promoter activity and is not responsible for a previously reported Δ6-desaturase deficiency.

L1CAM expression in endometrial carcinomas is regulated by usage of two different promoter regions

BackgroundThe L1 cell adhesion molecule (L1CAM) was originally identified as a neural adhesion molecule involved in axon guidance. In many human epithelial carcinomas L1CAM is overexpressed and thereby augments cell motility, invasion and metastasis formation. L1CAM positive carcinomas are associated with bad prognosis. Recent data point out that L1CAM is regulated in a fashion similar to epithelial-mesenchymal transition (EMT). Previous studies have implied the transcription factors Slug and/or β-catenin in L1CAM transcriptional regulation. However, the regulation of human L1CAM expression at the transcriptional level is not well understood.ResultsTo better understand the molecular basis of L1CAM transcriptional regulation, we carried out a detailed characterization of the human L1CAM promoter. We identified two transcription start sites, the first in front of a non-translated exon 0 (promoter 1) and the other next to the first protein-coding exon 1 (promoter 2). Both sites could be verified in endometrial carcinoma (EC) cell lines and appear to be used in a cell-type specific manner. The two identified promoter regions showed activity in luciferase reporter assays. Chromatin-IP analyses confirmed the in silico predicted E-boxes, binding sites for transcription factors Snail and Slug, as well as Lef-1 sites, which are related to β-catenin-mediated transcriptional regulation, in both promoters. Overexpression of β-catenin exclusively augmented activity of promoter 1 whereas Slug enhanced promoter 1 and 2 activity suggesting that both promoters can be active. Overexpression of β-catenin or Slug could upregulate L1CAM expression in a cell-type specific manner.ConclusionsOur results, for the first time, provide evidence that the L1CAM gene has two functionally active promoter sites that are used in a cell-type specific manner. Slug and β-catenin are involved L1CAM transcriptional regulation. Nevertheless, Slug rather than β-catenin levels are correlated with L1CAM expression in EC cell lines. Our findings suggest that the L1CAM transcriptional regulation is more complex than anticipated and this study provides the basis for a better understanding of L1CAM regulation in non-neuronal/tumor cells.

EGR-1 regulates Ho-1 expression induced by cigarette smoke

As an anti-oxidant molecule, heme oxygenase-1 (HO-1) has been implicated in the protection of lung injury by cigarette smoke (CS). The mechanisms regulating its expression have not been defined. In this report, the role of early growth response 1 (EGR-1) in the regulation of Ho-1 expression was investigated. In C57BL/6 mice with CS exposure, HO-1 was greatly increased in bronchial epithelial cells and alveolar inflammatory cells. In primary cultured mouse lung fibroblasts and RAW264.7 cells exposed to cigarette smoke water extract (CSE), an increase in HO-1 protein level was detected. In addition, CSE induced HO-1 expression was decreased in Egr-1 deficient mouse embryo fibroblasts ( Egr-1 −/− MEFs). Nuclear localization of EGR-1 was examined in mouse lung fibroblasts after exposure to CSE. Luciferase reporter activity assays showed that the enhancer region of the Ho-1 gene containing a proposed EGR-1 binding site was responsible for the induction of HO-1. A higher increase of alveolar mean linear intercept (Lm) was observed in lung tissues, and a larger increase in the number of total cells and monocytes/macrophages from bronchial alveolar lavage fluid was found in CS-exposed mice by loss of function of EGR-1 treatment. In summary, the present data demonstrate that EGR-1 plays a critical role in HO-1 production induced by CS.

Glucocorticoids regulate the human occludin gene through a single imperfect palindromic glucocorticoid response element

The 65 kDa protein occludin is an essential element of the blood-brain barrier. This integral membrane protein represents an important part of the tight junctions, which seal and protect the blood brain barrier against paracellular diffusion of solutes to the brain parenchyme and are therefore responsible for the high resistance and low permeability between cerebral capillary endothelial cells. However, the molecular basis for the regulation of occludin gene expression is only incompletely understood. In former projects we showed that treatment of a brain microvascular cell line, cEND, with glucocorticoids resulted in increased occludin expression in cell-cell-contacts Induction of occludin expression by glucocorticoids was shown to be dependent on the glucocorticoid receptor. This study aims to identify the underlying molecular mechanism of gene expression and to identify potential glucocorticoid receptor binding sites within the occludin promoter, the glucocorticoid response elements (GRE). We identified one candidate GRE within the distal part of the occludin promoter that differs from the consensus GRE by the presence of a 4-basepair instead of a 3-basepair spacer between two highly degenerate halfsites (5'-ACATGTGTTTACAAAT-3'). Chromatin immunoprecipitation assay and site-directed mutagenesis confirmed binding of the glucocorticoid receptor to this site. We synthesized plasmid containing four copies of this GRE in the vector pGL-3 basic. After stimulation of the cells with hydrocortisone we could observe up to 8-fold increased activity in luciferase reporter assay. The need for glucocorticoid receptor dimerization to induce gene expression was further confirmed by transfection studies using wild type and glucocorticoid receptor dimerization-deficient expression vectors, indicating that transactivation of occludin occur through the GRE.

A single anti-microRNA antisense oligodeoxyribonucleotide (AMO) targeting multiple microRNAs offers an improved approach for microRNA interference

Anti-miRNA antisense inhibitors (AMOs) have demonstrated their utility in miRNA research and potential in miRNA therapy. Here we report a modified AMO approach in which multiple antisense units are engineered into a single unit that is able to simultaneously silence multiple-target miRNAs, the multiple-target AMO or MTg-AMO. We validated the technique with two separate MTg-AMOs: anti-miR-21/anti-miR-155/anti-miR-17-5p and anti-miR-1/anti-miR-133. We first verified the ability of the MTg-AMOs to antagonize the repressive actions of their target miRNAs using luciferase reporter activity assays and to specifically knock down the levels of their target miRNAs using real-time RT-PCR methods. We then used the MTg-AMO approach to identify several tumor suppressors—TGFBI, APC and BCL2L11 as the target genes for oncogenic miR-21, miR-155 and miR-17-5p, respectively, and two cardiac ion channel genes HCN2 (encoding a subunit of cardiac pacemaker channel) and CACNA1C (encoding the α-subunit of cardiac L-type Ca2+ channel) for the muscle-specific miR-1 and miR-133. We further demonstrated that the MTg-AMO targeting miR-21, miR-155 and miR-17-5p produced a greater inhibitory effect on cancer cell growth, compared with the regular single-target AMOs. Moreover, while using the regular single-target AMOs excluded HCN2 as a target gene for either miR-1 or miR-133, the MTg-AMO approach is able to reveal HCN2 as the target for both miR-1 and miR-133. Our findings suggest the MTg-AMO as an improved approach for miRNA target finding and for studying function of miRNAs. This approach may find its broad application for exploring biological processes involving multiple miRNAs and multiple genes.

Potent Antidiabetic Effects of Rivoglitazone, a Novel Peroxisome Proliferator–Activated Receptor-γ Agonist, in Obese Diabetic Rodent Models

The pharmacological effects of rivoglitazone, a novel thiazolidinedione-derivative peroxisome proliferator–activated receptor (PPAR)-γ agonist, were characterized in vitro and in vivo. Rivoglitazone activated human PPARγ more potently compared with rosiglitazone and pioglitazone and had little effect on PPARα and PPARδ activity in luciferase reporter assays. In Zucker diabetic fatty (ZDF) rats, 14-day administration of rivoglitazone decreased the plasma glucose and triglyceride (TG) levels in a dose-dependent manner. The glucose-lowering effect of rivoglitazone was much more potent than those of pioglitazone (ED50: 0.19 vs. 34 mg/kg) and rosiglitazone (ED50: 0.20 vs. 28 mg/kg). In addition, rivoglitazone showed potent antidiabetic effects in diabetic db/db mice. In Zucker fatty rats, rivoglitazone at a dose of 0.1 mg/kg clearly ameliorated insulin resistance and lowered plasma TG levels by accelerating the clearance of plasma TG. Gene expression analysis in the liver and heart of ZDF rats treated with rivoglitazone for 14 days suggested that rivoglitazone may reduce hepatic glucose production and modulate the balance of the cardiac glucose/fatty acid metabolism in diabetic animals. In summary, we showed that rivoglitazone is a potent and selective PPARγ agonist and has a potent glucose-lowering effect via improvement of the insulin resistance in diabetic animal models.

Identification of a stress inducible activating region in human IRS-2 promoter depending on SP1, NF1 binding and Erk activation in HepG2 cells

Transcriptional regulation of Insulinreceptorsubstrates (IRS) is not well understood on a molecular level. IRS-2 knockout mice develop severe Type 2 diabetes after 9 weeks. Consequently, the regulation of the amount of IRS-2 and even its ratio to IRS-1 and IRS-4 is physiologically important. Based on luciferase promoter reporter assays we aimed to identifiy transcription factor binding sites in the human IRS-2 promoter in HepG2 cells. We found a region 688 bp upstream of translation start codon, which is responsible for the basal transcription of IRS-2 in liver. After truncating this region activity decreased about two-third. We postulated potential transcription factor binding motives in this region and confirmed binding of SP1 and NF1 using electro mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. After mutating both SP1 and NF1 binding sites, basal activity was diminished. Preincubation with PD 98,059 which inactivates Erk1 signaling inhibited IRS-2 promoter activity in luciferase reporter assays in HepG2 cells. This revealed an involvement of MAP kinase signaling cascade in IRS-2 transcription regulation that might finally gears to activate SP1. Activating this cascade with oxidative stress showed a twofold increase in IRS-2 promoter activity based on luciferase reporter assay and RT-PCR in HepG2 cells. Activity could be raised even more after additional inhibition of p38 MAPK with SB202190. This reveals an inhibitory effect of p38MAPK on Erk1 activity, and consequently on IRS-2 transcription. Recently a similar mechanism has been described for IL-2 promoter activation. After SP1 and NF1 binding site mutation on IRS-2 promoter these effects were diminished. In conclusion we discovered a new region (-688 to -611 bp) in the distal human IRS-2 promoter that is essential for its full basal and stress induced activation and is dependent on SP1 and NF1 binding.

The transcription factor CREMτ and cAMP regulate promoter activity of the Na,K‐ATPase α4 isoform

The Na,K-ATPase is an essential enzyme of the plasma membrane that plays a key role in numerous cell processes that depend on the transcellular gradients of Na(+) and K(+). Among the various isoforms of the catalytic subunit of the Na,K-ATPase, alpha4 exhibits the most limited pattern of expression, being restricted to male germ cells. Activity of alpha4 is essential for sperm function, and alpha4 is upregulated during spermatogenesis. The present study addressed the transcriptional control of the human Na,K-ATPase alpha4 gene, ATP1A4. We describe that a 5' untranslated region of the ATP1A4 gene (designated -339/+480 based on the ATP1A4 transcription initiation site) has promoter activity in luciferase reporter assays. Computer analysis of this promoter region revealed consensus sites (CRE) for the cyclic AMP (cAMP) response element modulator (CREM). Accordingly, dibutyryl cAMP (db-cAMP) and ectopic expression of CREMtau, a testis specific splice variant of CREM were able to activate the ATP1A4 promoter driven expression of luciferase in HEK 293 T, JEG-3 and GC-1 cells. Further characterization of the effect of db-cAMP and CREMtau on deleted constructs of the ATP1A4 promoter (-339/+80, and +25/+480), and on the -339/+480 region carrying mutations in the CRE sites showed that db-cAMP and CREMtau effect required the CRE motif located 263 bp upstream the transcription initiation site. EMSA experiments confirmed the CRE sequence as a bonafide CREMtau binding site. These results constitute the first demonstration of the transcriptional control of ATP1A4 gene expression by cAMP and by CREMtau, a transcription factor essential for male germ cell gene expression.

Chemical Inducers of Rodent GlutathioneS-Transferases Down-Regulate HumanGSTA1Transcription through a Mechanism Involving Variant Hepatic Nuclear Factor 1-C

The regulation of human GSTA1 by chemical inducers of rodent glutathione S-transferases (GSTs) and the regulatory role of hepatic nuclear factor (HNF) 1 was investigated in Caco-2 cells. Treatment of preconfluent and confluent cells with 12-O-tetra-decanoyl phorbol-13-acetate (TPA), 3-methylcholanthrene (3-MC), 2-tert-butyl-4-hydroxy-anisol (BHA), and phenobarbital (PB) reduced GSTA1 mRNA levels in preconfluent and confluent cells. Constitutive levels of GSTA1 and HNF1alpha mRNA were elevated 6.25- and 50-fold, respectively, in postconfluent cells compared with preconfluent cells. Overexpression of HNF1alpha in cells transfected with a GSTA1 promoter-luciferase construct (pGSTA1-1591-luc) resulted in dose-related increases in reporter activity not observed when an HNF1 response element (HRE) in the proximal promoter was mutated (pGSTA1-DeltaHNF1-luc). TPA, 3-MC, BHA, and PB reduced HNF1alpha mRNA levels in preconfluent and confluent cells and caused marked reductions in luciferase activity in pGSTA1-1591-luc transfectants. Transcriptional repression was abrogated with pGSTA1-DeltaHNF1-luc and with truncated constructs that eliminated a functional HRE. Moreover, cotransfection of pHNF1alpha with pGSTA1-1591-luc partially prevented the reduction in luciferase activity by rodent GST inducers. Immunoblot analysis of DNA binding studies indicate that variant (v)HNF1-C binding to HRE is increased in preconfluent cells treated with 3-MC, BHA, and PB. In addition, overexpression of vHNF1-C repressed GSTA1 transcriptional activity in luciferase reporter assays. Finally, treatment with 3-MC, BHA, and PB increased vHNF1-C mRNA levels in preconfluent cells. These data demonstrate that repression of human GSTA1 transcription by chemical inducers of rodent GSTs occurs, in part, through a mechanism involving the repressive action of vHNF1-C.