Luciferase Reporter Vector Research Articles

Abstract 5133: Copy number variation in 13q32.1 associated with esophageal squamous cell carcinoma

Abstract Background: Esophageal squamous cell carcinoma (ESCC) is the major lethal upper gastrointestinal tract cancer in China. The highly regional and family aggregative incidence of ESCC in China suggests that genetic susceptibility contributed to pathogenesis. Copy number variation (CNV) is an important form of gene structure variations and has been associated with multiple complex diseases, such as malignant tumors. This study aimed to identify the common CNVs correlated with ESCC susceptibility and demonstrate the functional significances. Method: A genome-wide associated study was conducted on 128 discordant sibling pairs of ESCC, which were native residents in Yangquan area, Shan Xi Province of China. The genomic DNA of peripheral blood leukocytes were extracted and genotyping was detected using the Illumina HumanHap 610Q BeadChips. After stringent quality control filtering, the CNVs-penotype association analysis were performed using the paired Hidden Markov Model calling algorithm in the Partek Genomic Suite with settings of length above 1kb and minimum 10 consecutive probes. Another 223 Northern Chinese Han people (111 ESCC patients and 112 health controls) were chosen to validate the CNVs using quantitative real-time PCR assays. Then the paraffin-embedded tissue samples of 16 ESCC patients with known genotyping data were collected for immunohistochemistry. Last, the highest frequency of variation region was cloned into luciferase reporter vectors to investigate its biological significance in ESCC cells. Results: A total of 57,774 individual CNVs covering all autosomes were identified. Of them, 13q32.1 was identified as a critical enrichment region, and it mainly was involved in one candidate gene. Conditional logistic regression test showed that this region was significantly associated with ESCC with the odd ratio being 65.289 (95%CI: 11.177-381.366; P<0.001). Quantitative real-time PCR assays replicated CNV in this gene. The frequencies of the three genotypes in cases were 3.6% (1 copy), 74.8% (2 copies), and 21.7% (3 copies), which differed significantly from those in controls (91.1% [2 copies], and 8.9% [3 copies]). Logistic regression analysis showed that the CNV in this gene increased the risk of ESCC (odd ratio: 3.891; 95% CI: 1.53-9.91 P=0.004). Additionally, 9 out of 11 patients containing 3 copies had positive expression of the gene (2 positive and 7 strong positive), whereas only 1 out of 5 patients containing 2 copies had positive expression. Chi-Square analysis showed that there was significant difference between two groups of patients (P=0.018). The subsequently luciferase asaay further comfirmed the enhancer activity of this CNV region. Conclusion: Common CNVs at 13q32.1 was associated with ESCC susceptibility probably due to its potential enhancer activity on regulating the gene expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5133. doi:1538-7445.AM2012-5133

Chronic effect of parathyroid hormone on Na+/H+ exchanger isoform 3 gene proximal promoter

Previous studies have shown that parathyroid hormone (PTH) chronically inhibits NHE3 in opossum kidney cells (OKP) by reducing total NHE3 protein and mRNA levels by modulation of the transcription. The purpose of this study was to elucidate the inhibitory mechanisms of PTH on the NHE3 gene promoter. Different segments of the proximal promoter (−157/+31; −85/+31; −65/+31 and −44/+31) were inserted in the pGL3‐ basic luciferase reporter vector, and point mutations were introduced in some regulatory elements, which were also evaluated by gel shift assays (GMSA). OKP cells were transiently transfect with the vector constructs and kept in serum‐free media for 24 h, then treated with 10−7M PTH for 24 h. PTH decreased the promoter activity of the fragments −65 and −44 (~21% and 31% respectively). Mutations on Sp1/Egr‐1 and AP2 elements completely abolished the PTH inhibitory effect. Inhibition of JAK/STAT pathway abolished the suppressor effect of PTH on fragment −65. In GMSA, PTH reduced the protein‐DNA affinity of the segment −44/+31.In conclusion, these data suggest that the cis‐element(s) required for PTH responsiveness must be localized in the proximal promoter and the JAK/STAT pathway is involved in this inhibitory response.Financial Support: FAPESP and CAPES

MiR-181a sensitizes a multidrug-resistant leukemia cell line K562/A02 to daunorubicin by targeting BCL-2

The aim of this study was to investigate whether miR-181a could modulate the sensitivity of the leukemia drug-resistant cell line K562/A02 to the chemotherapeutic agent daunorubicin (DNR), and explore the mechanism of miR-181a on the DNR sensitivity of K562/A02 cells. MicroRNA microarray and stem-loop reverse transcription-polymerase chain reaction were used to detect the expression of miR-181a. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay was performed to quantify the effect of miR-181a on K562 cells growth and viability. Apoptotic cells were quantitatively detected using Annexin V/FITC and PI apoptosis detection kit. BCL-2 protein expression was measured by western blot. Luciferase reporter vector with the putative BCL-2 3' untranslated region was constructed to explore whether BCL-2 was a direct target gene of miR-181a. BCL-2 siRNA was transfected into the cell to explore the relationship between BCL-2 and DNR resistance. The miR-181a expression level was lower in the K562/A02 cells than in the K562 cells (P< 0.05). K562 cells that were transfected with miR-181a inhibitor had a significantly higher survival than K562 cells, and K562/A02 cells that were transfected with the miR-181a mimic had a significantly lower survival than K562/A02 cells (P< 0.05). miR-181a could enhance DNR-induced apoptosis in K562/A02 cells. BCL-2 siRNA transfected K562/A02 cells had decreased survival compared with the K562/A02 control group. In conclusion, miR-181a could play a role in the development of DNR resistance in K562/A02 cells and the over-expression of miR-181a could sensitize K562/A02 cells to DNR by targeting BCL-2.

Matrix metalloprotease 16 expression is downregulated by microRNA‐146a in spontaneously differentiating Caco‐2 cells

Cellular differentiation in the gut is vital in maintaining the cellular and functional specialization of the epithelial layer. MicroRNAs (miRNAs) have recently emerged as one of the key players in orchestrating the differentiation process in the gut. Using the spontaneously differentiating Caco-2 cell line, we observed an increased expression of miR-146a but not miR-146b in the course of differentiation. Bioinformatic analyses revealed that the membrane type matrix metalloprotease 16 (MMP16, MT3-MMP) was a predicted target of miR-146a and a decrease in the mRNA and protein expression of MMP16 was observed in the course of differentiation. Transfection of a luciferase reporter vector containing the 3'UTR of MMP16 showed decreased luciferase activity due to miR-146a expression. With forced expression of miR-146a in undifferentiated Caco-2 cells, a decrease in the mRNA and protein levels of MMP16 and a lower gelatinase activity in a gelatin zymogram were observed. Additionally, forced expression of miR-146a in HT-29 colon cancer cells also resulted in decreased expression of MMP16, along with a decrease in the invasion through Matrigel. Taken together, we have shown here that MMP16 is regulated by miR-146a in spontaneously differentiated Caco-2 cells. As MMP16 activates the zymogen of MMP2, which is known to degrade extracellular matrix proteins, the regulation of MMP16 by miR-146a may account, at least in part, for lower motility of well-differentiated cells.

Cold-Shock-Domain Protein A (CSDA) Contributes Posttranscriptionally to Gonadotropin-Releasing Hormone-Regulated Expression of Egr1 and Indirectly to Lhb1

Gonadotropin-releasing hormone (GnRH), a hypothalamic neurohormone, regulates transcription of Lhb in gonadotrophs indirectly through transient induction and accumulation of EGR1, a zinc finger transcription factor. AlphaT3 and LbetaT2 cell lines model gonadotrophs at two distinct stages of development, prenatal and postnatal expression of Lhb. Although GnRH induces EGR1 in both cell lines, the levels of the DNA-binding protein are lower and disappear more quickly in alphaT3 than in LbetaT2 cells. Herein we show that overexpression of Egr1 in alphaT3 cells rescues activity of a transfected LHB promoter-reporter, suggesting that its transcription is dependent on EGR1 crossing a critical concentration threshold. We also show that Csda, a gene that encodes an RNA-binding protein and is a member of the cold-shock-domain (CSD) family, is expressed at higher levels in LbetaT2 compared to alphaT3 cells. Transient expression studies indicate that at least one Csd element, residing in the 3' untranslated region of Egr1 mRNA, increases activity of a chimeric pGL3 luciferase reporter vector in LbetaT2 cells. Additional experiments indicate that CSDA physically interacts with Egr1 mRNA. Furthermore, siRNA-mediated reduction of endogenous Csda mRNA attenuates GnRH regulation of a transiently transfected LHB reporter vector. Taken together, these studies suggest that CSDA contributes posttranscriptionally to GnRH-regulated expression of Egr1, thereby enabling the transcription factor to cross a critical concentration threshold necessary for maximal accumulation of Lhb mRNA in response to the neurohormone.

Monofunctional Platinum–DNA Adducts Are Strong Inhibitors of Transcription and Substrates for Nucleotide Excision Repair in Live Mammalian Cells

To overcome drug resistance and reduce the side effects of cisplatin, a widely used antineoplastic agent, major efforts have been made to develop next generation platinum-based anticancer drugs. Because cisplatin-DNA adducts block RNA polymerase II unless removed by transcription-coupled excision repair, compounds that react similarly but elude repair are desirable. The monofunctional platinum agent pyriplatin displays antitumor activity in mice, a cytotoxicity profile in cell cultures distinct from that of cisplatin, and a unique in vitro transcription inhibition mechanism. In this study, we incorporated pyriplatin globally or site specifically into luciferase reporter vectors to examine its transcription inhibition profiles in live mammalian cells. Monofunctional pyriplatin reacted with plasmid DNA as efficiently as bifunctional cisplatin and inhibited transcription as strongly as cisplatin in various mammalian cells. Using repair-defective nucleotide excision repair (NER)-, mismatch repair-, and single-strand break repair-deficient cells, we show that NER is mainly responsible for removal of pyriplatin-DNA adducts. These findings reveal that the mechanism by which pyriplatin generates its antitumor activity is very similar to that of cisplatin, despite the chemically different nature of their DNA adducts, further supporting a role for monofunctional platinum anticancer agents in human cancer therapy. This information also provides support for the validity of the proposed mechanism of action of cisplatin and provides a rational basis for the design of more potent platinum anticancer drug candidates using a monofunctional DNA-damaging strategy.

Expression and clinical significance of miR-23a and metastasis suppressor 1 in colon carcinoma

To investigate the expression of miR-23a and metastasis suppressor 1 (MTSS1) and their clinical significance in colon carcinoma. A total of 92 cases of colon carcinomas were collected with both the tumor and paired normal tissue samples for the study. The miR-23a targeting MTSS1 was evaluated by luciferase reporter vector. Cell invasion potential was evaluated by trans-well invasion assay. In-situ hybridization and immunohistochemistry were used to detect miR-23a and MTSS1 expression. MiR-23a downregulated the expression of MTSS protein and enhanced the invasiveness of colon carcinoma. The expression rates of miR-23a and MTSS1 were 87.0% (80/92) and 17.4% (16/92) in colon carcinoma cases, respectively (P < 0.01). The up-regulation of miR-23a expression was associated with an advanced clinical stage (P = 0.029) and depth of invasion (P = 0.000). The expression of miR-23a was higher in the tumors with lymph node metastasis than those without (P = 0.041). Down-regulation of MTSS1 expression was associated with an advanced clinical stage (P = 0.027) and depth of invasion (P = 0.017). The expression of MTSS1 was lower in the tumors with lymph node metastasis than those without (P = 0.009). The expression of miR-23a had significantly negative correlation with that of MTSS1 (r = -0.594, P = 0.013). MiR-23a expression promotes colon carcinoma cell growth, invasion and metastasis through inhibition of MTSS gene. Both the low expression of MTSS1 and high expression of miR-23a may serve as important biological markers for the malignant phenotypes of colon cancer, such as invasion and metastasis.

Transcriptional Regulation of RKIP Expression by Androgen in Prostate Cells

Background/Aims: Raf kinase inhibitory protein (RKIP) is a scaffolding molecule in the PEBP family that sequesters certain signaling molecules away from their pathways, thereby abrogating intracellular growth signals. RKIP has been assigned multiple functions and is associated with an increasing number of diseases through its involvement with signal transduction pathways. We previously demonstrated that RKIP is highly expressed in human normal prostate epithelial cells and plays a pivotal role during prostate cancer (PCa) progression. Whether RKIP is subject to endocrine regulation has not been reported. Methods: The effect of dihydrotestosterone (DHT) on RKIP expression in normal prostate epithelial cells was determined by real-time RT-PCR and Western blot. Report assay was performed to determine whether the regulation of RKIP by androgens is at the transcriptional level. The binding of androgen receptor (AR) to the RKIP promoter was determined by EMSA and Chromatin Immunoprecipitation (ChIP) assays. To determine whether RKIP was regulated by androgen in vivo, we examined RKIP expression level in response to castration in 6-8 week old C57BL/6 male mice. Results: Here we report that DHT positively regulates the transcription of RKIP in the normal prostate epithelial cells. The anti-androgen bicalutamide blocked androgen-mediated regulation of RKIP, which indicates that this regulation is mediated through AR. Transfection of the cells with a RKIP promoter-driven luciferase reporter vector showed that DHT increased RKIP promoter activity in parallel with changes in expression. EMSA demonstrates that AR binds to a putative ARE in the RKIP promoter, which was further validated by ChIP assay. Importantly, these data are further supported by our in vivo experiment where castrated mice had less RKIP expression in their prostate glands than sham-operated mice. Conclusions: Collectively, the results establish RKIP as a novel androgen target gene. Androgens induce RKIP expression through AR-mediated transcriptional modulation of the RKIP promoter in the prostate. This is the first demonstration of endocrine regulation of the metastasis suppressor gene RKIP.

The pendrin anion exchanger gene is transcriptionally regulated by uroguanylin: a novel enterorenal link

The pendrin/SLC26A4 Cl(-)/HCO(3)(-) exchanger, encoded by the PDS gene, is expressed in cortical collecting duct (CCD) non-A intercalated cells. Pendrin is essential for CCD bicarbonate secretion and is also involved in NaCl balance and blood pressure regulation. The intestinal peptide uroguanylin (UGN) is produced in response to oral salt load and can function as an "intestinal natriuretic hormone." We aimed to investigate whether UGN modulates pendrin activity and to explore the molecular mechanisms responsible for this modulation. Injection of UGN into mice resulted in decreased pendrin mRNA and protein expression in the kidney. UGN decreased endogenous pendrin mRNA levels in HEK293 cells. A 4.2-kb human PDS (hPDS) promoter sequence and consecutive 5' deletion products were cloned into luciferase reporter vectors and transiently transfected into HEK293 cells. Exposure of transfected cells to UGN decreased hPDS promoter activity. This UGN-induced effect on the hPDS promoter occurred within a 52-bp region encompassing a single heat shock element (HSE). The effect of UGN on the promoter was abolished when the HSE located between nt -1119 and -1115 was absent or was mutated. Furthermore, treatment of HEK293 cells with heat shock factor 1 (HSF1) small interfering RNA (siRNA) reversed the UGN-induced decrease in endogenous PDS mRNA level. In conclusion, pendrin-mediated Cl(-)/HCO(3)(-) exchange in the renal tubule may be regulated transcriptionally by the peptide hormone UGN. UGN exerts its inhibitory activity on the hPDS promoter likely via HSF1 action at a defined HSE site. These data define a novel signaling pathway involved in the enterorenal axis controlling electrolyte and water homeostasis.

Ets-1 Activates Overexpression of JunB and CD30 in Hodgkin's Lymphoma and Anaplastic Large-Cell Lymphoma

Overexpression of CD30 and JunB is a hallmark of tumor cells in Hodgkin's lymphoma (HL) and anaplastic large-cell lymphoma (ALCL). We reported that CD30-extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) signaling induces JunB, which maintains constitutive activation of the CD30 promoter. Herein, we localize a cis-acting enhancer in the JunB promoter that is regulated by Ets-1. We show that E26 transformation-specific-1 (Ets-1) (-146 to -137) enhances JunB promoter activation in a manner that is dependent on CD30 or the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-ERK1/2 MAPK pathway. Ets-1 knockdown reduces the expression of both JunB and CD30, and CD30 knockdown significantly reduces JunB expression in HL and ALCL cell lines. NPM-ALK knockdown also reduces JunB expression in ALCL cell lines expressing NPM-ALK. Collectively, these results indicate that CD30 and NPM-ALK cooperate to activate the ERK1/2 MAPK-Ets-1 pathway. Ets-1, constitutively activated by ERK1/2-MAPK, plays a central role in the overexpression of JunB and CD30, which are both involved in the pathogenesis of HL and ALCL.

TOSO-Deficient B Cells Show An Impaired NF-κB and Migration Signaling In Vivo

Abstract 1779 Background:Our group firstly demonstrated that TOSO (FAIM3) is over-expressed in CLL compared to healthy B cell subsets as well as other B cell lymphomas. Furthermore, we detected a significant correlation of high TOSO expression to high lymphocyte count, unmutated IgVH status and Binet C, which are all markers for poor prognosis. TOSO has been described as pro-survival gene also in other settings. However, its mode of action is discussed controversially.Therefore, we aimed to elucidate the role of TOSO in B-cell specific gene expression by creating a knockdown mouse model. CD40 ligation and B cell receptor (BCR) activation influences TOSO expression and the fact that transcriptional regulation of TOSO is still unknown, we were eager to determine transcriptional factors that are directly responsible for the alterable TOSO levels. Methods:Faim3-floxed C57BL/6 FLP deleter mice were crossbred with CD19 specific Cre recombinase expressing mice. B-cells from the TOSOCD19−/− (KO) mice were isolated and gene expression was analyzed via mRNA based Illumina microchip array. Convincing results were verified by flow cytometry and blood count was carried out in addition.To determine the promoter region of TOSO, three overlapping DNA fragments (containing either NF-κB, Bcl-6 or both binding sites) upstream of the transcription start site of the first TOSO exon were cloned into a luciferase reporter vector lacking a promoter. Those constructs were transfected into HeLa cells. After 24 hours luciferase assays were performed. The involvement of NF-κB in the regulation of TOSO transcription was measured by TNFα stimulation of transfected cells prior to luminescence measurement. Targeted mutagenesis of the NF-κB binding site was performed to confirm the data. In addition, Bcl-6 expression vector was co-transfected for evaluation of repressing influence on TOSO expression. Results:In order to cover the functional part of TOSO, we generated a B-cell specific TOSOCD19−/− mouse model. Downstream effects of TOSO were validated via microarray-based gene expression analysis. Results displayed a clear clustering of deregulated genes compared to control mice. Nearly 400 genes showed expression alterations; genes involved in the NF-κB pathway and migration processes were downregulated in TOSOCD19−/−. These results were confirmed by flow cytometry analysis. The TOSO KO displayed also relevant effects on the hematopoietic system. Lymphocyte (p=0,0048), neutrophil (p=0,0007) and red blood cell counts (p=0,0051) were significantly decreased in the TOSOCD19−/− mice. Most important, the B-cell count was significantly reduced in TOSO-deficient settings (n=9; p=0,032).Since TOSO level seems to be so important for such fundamental pathways, investigation of gene expression regulation is mandatory. In situ analysis of the TOSO promoter region revealed NF-κB and Bcl-6 as promising results. Luciferase reporter assays including targeted mutagenesis confirmed the positive regulation of NF-κB and the repressing influence of Bcl-6 on TOSO expression significantly. Conclusions:We reveal for the first time a TOSO-dependent expression profile. We identified TOSO-dependent deregulated genes, which were involved in NF-κB signaling and migration, suggesting that TOSO represents an important factor in these pathways. Additionally, TOSO KO caused a decrease of peripheral B-cells in vivo. Furthermore, we identified NF-κB and Bcl-6 to regulate the TOSO expression in an opposite manner. Disclosures:No relevant conflicts of interest to declare.

Nuclear receptor NR5A2 and bone: gene expression and association with bone mineral density

There is growing evidence for a link between energy and bone metabolism. The nuclear receptor subfamily 5 member A2 (NR5A2) is involved in lipid metabolism and modulates the expression of estrogen-related genes in some tissues. The objective of this study was to explore the influence of NR5A2 on bone cells and to determine whether its allelic variations are associated with bone mineral density (BMD). Analyses of gene expression by quantitative PCR and inhibition of NR5A2 expression by siRNAs were used to explore the effects of NR5A2 in osteoblasts. Femoral neck BMD and 30 single nucleotide polymorphisms (SNPs) were first analyzed in 935 postmenopausal women and the association of NR5A2 genetic variants with BMD was explored in other 1284 women in replication cohorts. NR5A2 was highly expressed in bone. The inhibition of NR5A2 confirmed that it modulates the expression of osteocalcin, osteoprotegerin, and podoplanin in osteoblasts. Two SNPs were associated with BMD in the Spanish discovery cohort (rs6663479, P=0.0014, and rs2816948, P=0.0012). A similar trend was observed in another Spanish cohort, with statistically significant differences across genotypes in the combined analysis (P=0.03). However, the association in a cohort from the United States was rather weak. Electrophoretic mobility assays and studies with luciferase reporter vectors confirmed the existence of differences in the binding of nuclear proteins and the transcriptional activity of rs2816948 alleles. NR5A2 modulates gene expression in osteoblasts and some allelic variants are associated with bone mass in Spanish postmenopausal women.

Micro RNA -214,-150,-146a and-125b target Huntingtin gene

We observed earlier that expressions of several micro RNAs (miRNAs) are altered in a cell model of Huntington’s disease (HD). As genes involved in different neurodegenerative diseases are targeted by miRNAs, we searched databases to find out whether Huntingtin gene (HTT), mutation to which causes HD, is a target of any miRNA. Among many miRNAs that are predicted to target HTT, we showed using various experimental approaches that miR-214, miR-150, miR-146a and miR-125b could target both human HTT and mouse Htt. Luciferase reporter vectors containing 3’-UTRs of mouse and human HTT showed reduction in relative luciferase activities on exogenous expression of cloned miRNAs. Loss of function studies with miRNA inhibitors led to the revival of luciferase activities of cloned 3’-UTR of HTT. Exogenous expression of these miRNAs reduced the expression of endogenous Htt in mouse cells. Expression of miRNAs with mutations at seed sequences neither reduced the reporter luciferase activities nor the endogenous expression of Htt. Taking together, we showed that miR-214, miR-150, miR-146a and miR-125b targeted HTT. Besides, the exogenous expression of wild type miRNAs reduced HTT aggregates formed by the recombinant exon1 of HTT gene that codes for 83Q tagged with 3’-UTR of Htt, as observed by filter retardation assay and confocal microscopy. In summary, regulation of HTT by miRNAs might provide a new mechanism for the modulation of HD.

Testicular Nuclear Receptor 4 (TR4) Regulates UV Light-induced Responses via Cockayne Syndrome B Protein-mediated Transcription-coupled DNA Repair

UV irradiation is one of the major external insults to cells and can cause skin aging and cancer. In response to UV light-induced DNA damage, the nucleotide excision repair (NER) pathways are activated to remove DNA lesions. We report here that testicular nuclear receptor 4 (TR4), a member of the nuclear receptor family, modulates DNA repair specifically through the transcription-coupled (TC) NER pathway but not the global genomic NER pathway. The level of Cockayne syndrome B protein (CSB), a member of the TC-NER pathway, is 10-fold reduced in TR4-deficient mouse tissues, and TR4 directly regulates CSB at the transcriptional level. Moreover, restored CSB expression rescues UV hypersensitivity of TR4-deficient cells. Together, these results indicate that TR4 modulates UV sensitivity by promoting the TC-NER DNA repair pathway through transcriptional regulation of CSB. These results may lead to the development of new treatments for UV light-sensitive syndromes, skin cancer, and aging.

Cis-regulatory functions of overlapping HIF-1alpha/E-box/AP-1-like sequences of CD164

BackgroundCD164 (also known as MGC-24v or endolyn) is a sialomucin which has been suggested to participate in regulating the proliferation, cell adhesion and differentiation of hematopoietic stem and progenitor cells. CD164 is also involved in the development of cancer. The functions of cis-regulatory elements of CD164 remain relatively unknown.MethodsIn this study, we investigated the function of cis-regulatory elements within the promoter of CD164. We fused the 5'-flanking region of CD164 to a luciferase reporter vector. The minimal promoter region was confirmed by luciferase reporter assay. Using in silico analysis, we found the presence of one HIF-1alpha (HIF-1A) motif (5_-RCGTG-3_) overlapping E-box (CACGTG) and two AP-1-like binding sites (CGCTGTCCC, GTCTGTTG), one of which is also overlapped with HIF-1alpha sequence. Dual-luciferase assay was performed to examine the transcriptional activity of AP-1 and HIF-1alpha of CD164 promoter. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to measure CD164 expression. Chromatin Immunoprecipitation was used to confirm the binding of HIF-1alpha and CD164.ResultsCo-transfection of c-jun, HIF-1alpha and minimal promoter region construct demonstrated that c-jun and HIF-1alpha bound the CD164 promoter and promoted CD164 expression. Hypoxia treatment also led to the up-regulation of CD164 expression. The mutation of overlapping sequences resulted in the reduced expression of CD164 induced by HIF-1alpha. Chromatin Immunoprecipitation demonstrated that the HIF-1alpha bound the minimal promoter region.ConclusionsDetermination of the optimal promoter region and transcription factors governing CD164 expression is useful in understanding CD164 functions. These results suggest that cis-regulatory elements of CD164 overlapping HIF-1alpha/E-box/AP-1-like sequences may play important regulatory roles.

Post-transcriptional regulation of Transforming Growth Factor Beta-1 by microRNA-744.

Transforming Growth Factor Beta-1 (TGF-β1) is a pleiotropic cytokine that is of central importance in wound healing, inflammation, and in key pathological processes including cancer and progressive tissue fibrosis. TGF-β1 is post-transcriptionally regulated, but the underlying mechanisms remain incompletely defined. Previously, we have extensively delineated post-transcriptional regulation of TGF-β1 synthesis in the kidney, with evidence for relief of translational repression in proximal tubular cells in the context of diabetic nephropathy. In this study, we have investigated the role of the TGF-β1 3′Untranslated Region (3′UTR). Two different 3′UTR lengths have been reported for TGF-β1, of 543 and 137 nucleotides. Absolute quantification showed that, while both UTR lengths were detectable in various human cell types and in a broad range of tissues, the short form predominated in the kidney and elsewhere. Expression of both forms was up-regulated following auto-induction by TGF-β1, but the short:long UTR ratio remained constant. Incorporation of the short UTR into a luciferase reporter vector significantly reduced reporter protein synthesis without major effect on RNA amount, suggesting post-transcriptional inhibition. In silico approaches identified multiple binding sites for miR-744 located in the proximal TGF-β1 3′UTR. A screen in RNA from human tissues showed widespread miR-744 expression. miR-744 transfection inhibited endogenous TGF-β1 synthesis, while direct targeting of TGF-β1 was shown in separate experiments, in which miR-744 decreased TGF-β1 3′UTR reporter activity. This work identifies miR-744-directed post-transcriptional regulation of TGF-β1 which, given the pleiotropic nature of cellular responses to TGF-β1, is potentially widely significant.

Aryl hydrocarbon receptor-dependent induction of the IgA receptor FcαRI by the environmental contaminant benzo( a)pyrene in human macrophages

Polycyclic aromatic hydrocarbons (PAHs), such as benzo( a)pyrene (B aP), are widely distributed toxic environmental contaminants well known to regulate gene expression through activation of the aryl hydrocarbon receptor (AhR). In the present study, we demonstrated that the IgA receptor FcαRI/CD89 constitutes a molecular target for PAHs. Indeed, in vitro exposure to B aP markedly increased mRNA and protein expression of FcαRI in primary human macrophages; intratracheal instillation of B aP to rats also enhanced mRNA expression of FcαRI in alveolar macrophages. B aP concomitantly increased activity of the previously uncharacterized −1734 to −42 fragment of the Fc aRI promoter that we subcloned in a luciferase reporter vector. Three-methylcholanthrene, a PAH known to activate AhR like B aP, induced FcαRI expression, in contrast to benzo( e)pyrene, a PAH known to poorly interact with AhR. Moreover, FcαRI induction in B aP-exposed human macrophages was fully prevented by down-regulating AhR expression through small interference RNA transfection. In addition, B aP increased nuclear protein binding to a consensus AhR-related xenobiotic-responsive element found in the FcαRI gene promoter, as revealed by electrophoretic mobility shift assay. Overall, these data highlight an AhR-dependent up-regulation of FcαRI in response to B aP, which may contribute to the deleterious effects of environmental PAHs toward the immune/inflammatory response and which also likely emphasizes the role played by AhR in the regulation of genes involved in immunity and inflammation.

Hepatitis C Virus Is Primed by CD81 Protein for Low pH-dependent Fusion

Hepatitis C virus (HCV) entry into permissive cells is a complex process that involves interactions with at least four co-factors followed by endocytosis and low pH-dependent fusion with endosomes. The precise sequence of receptor engagement and their roles in promoting HCV E1E2 glycoprotein-mediated fusion are poorly characterized. Because cell-free HCV tolerates an acidic environment, we hypothesized that binding to one or more receptors on the cell surface renders E1E2 competent to undergo low pH-induced conformational changes and promote fusion with endosomes. To test this hypothesis, we examined the effects of low pH and of the second extracellular loop (ECL2) of CD81, one of the four entry factors, on HCV infectivity. Pretreatment with an acidic buffer or with ECL2 enhanced infection through changing the E1E2 conformation, as evidenced by the altered reactivity of these proteins with conformation-specific antibodies and stable association with liposomes. However, neither of the two treatments alone permitted direct fusion with the cell plasma membrane. Sequential HCV preincubation with ECL2 and acidic buffer in the absence of target cells resulted in a marked loss of infectivity, implying that the receptor-bound HCV is primed for low pH-dependent conformational changes. Indeed, soluble receptor-pretreated HCV fused with the cell plasma membrane at low pH under conditions blocking an endocytic entry pathway. These findings suggest that CD81 primes HCV for low pH-dependent fusion early in the entry process. The simple triggering paradigm and intermediate conformations of E1E2 identified in this study could help guide future vaccine and therapeutic efforts to block HCV infection.

Identifying the regulatory element for human angiotensin-converting enzyme 2 (ACE2) expression in human cardiofibroblasts

Angiotensin-converting enzyme 2 (ACE2) has been proposed as a potential target for cardioprotection in regulating cardiovascular functions, owing to its key role in the formation of the vasoprotective peptides angiotensin-(1–7) from angiotensin II (Ang II). The regulatory mechanism of ace2 expression, however, remains to be explored. In this study, we investigated the regulatory element within the upstream of ace2. The human ace2 promoter region, from position −2069 to +20, was cloned and a series of upstream deletion mutants were constructed and cloned into a luciferase reporter vector. The reporter luciferase activity was analyzed by transient transfection of the constructs into human cardiofibroblasts (HCFs) and an activating domain was identified in the −516/−481 region. Deletion or reversal of this domain within ace2 resulted in a significant decrease in promoter activity. The nuclear proteins isolated from the HCFs formed a DNA–protein complex with double stranded oligonucleotides of the −516/−481 domain, as detected by electrophoretic mobility shift assay. Site-directed mutagenesis of this region identified a putative protein binding domain and a potential binding site, ATTTGGA, homologous to that of an Ikaros binding domain. This regulatory element was responsible for Ang II stimulation via the Ang II–Ang II type-1 receptor (AT1R) signaling pathway, but was not responsible for pro-inflammatory cytokines TGF-β1 and TNF-α. Our results suggest that the nucleotide sequences −516/−481 of human ace2 may be a binding domain for an as yet unidentified regulatory factor(s) that regulates ace2 expression and is associated with Ang II stimulation.

In Vitro and Ex Vivo Analysis of CHRNA3 and CHRNA5 Haplotype Expression

Genome-wide association studies implicate variations in CHRNA5 and CHRNA3 as being associated with nicotine addiction (NA). Multiple common haplotypes (“risk”, “mixed” and “protective”) exist in Europeans; however, high linkage disequilibrium between variations in CHRNA5 and CHRNA3 makes assigning causative allele(s) for NA difficult through genotyping experiments alone. We investigated whether CHRNA5 or CHRNA3 promoter haplotypes, associated previously with NA, might influence allelic expression levels. For in vitro analyses, promoter haplotypes were sub-cloned into a luciferase reporter vector. When assessed in BE(2)-C cells, luciferase expression was equivalent among CHRNA3 haplotypes, but the combination of deletion at rs3841324 and variation at rs503464 decreased CHRNA5 promoter-derived luciferase activity, possibly due to loss of an SP-1 and other site(s). Variation within the CHRNA5 5’UTR at rs55853698 and rs55781567 also altered luciferase expression in BE(2)-C cells. Allelic expression imbalance (AEI) from the “risk” or “protective” haplotypes was assessed in post-mortem brain tissue from individuals heterozygous at coding polymorphisms in CHRNA3 (rs1051730) or CHRNA5 (rs16969968). In most cases, equivalent allelic expression was observed; however, one individual showed CHRNA5 AEI that favored the “protective” allele and that was concordant with heterozygosity at polymorphisms ∼13.5 kb upstream of the CHRNA5 transcription start site. Putative enhancer activity from these distal promoter elements was assessed using heterologous promoter constructs. We observed no differences in promoter activity from the two distal promoter haplotypes examined, but found that the distal promoter region strongly repressed transcription. We conclude that CHRNA5 promoter variants may affect relative risk for NA in some heterozygous individuals.