Transient Cotransfection Assays Research Articles

MicroRNA-Mediated Suppression of Glial Cell Line-Derived Neurotrophic Factor Expression Is Modulated by a Schizophrenia-Associated Non-Coding Polymorphism.

Plasma levels of glial cell line-derived neurotrophic factor (GDNF), a pivotal regulator of differentiation and survival of dopaminergic neurons, are reportedly decreased in schizophrenia. To explore the involvement of GDNF in the pathogenesis of the disease, a case-control association analysis was performed between five non-coding single nucleotide polymorphisms (SNP) across the GDNF gene and schizophrenia. Of them, the 'G' allele of the rs11111 SNP located in the 3' untranslated region (3'-UTR) of the gene was found to associate with schizophrenia. In silico analysis revealed that the rs11111 'G' allele might create binding sites for three microRNA (miRNA) species. To explore the significance of this polymorphism, transient co-transfection assays were performed in human embryonic kidney 293T (HEK293T) cells with a luciferase reporter construct harboring either the 'A' or 'G' allele of the 3'-UTR of GDNF in combination with the hsa-miR-1185-1-3p pre-miRNA. It was demonstrated that in the presence of the rs11111 'G' (but not the 'A') allele, hsa-miR-1185-2-3p repressed luciferase activity in a dose-dependent manner. Deletion of the miRNA binding site or its substitution with the complementary sequence abrogated the modulatory effect. Our results imply that the rs11111 'G' allele occurring more frequently in patients with schizophrenia might downregulate GDNF expression in a miRNA-dependent fashion.

Transcription factor CgPOU3F4-like regulates expression of pheomelanin synthesis related gene CgB-aat1 in the Pacific oyster (Crassostrea gigas)

Previous study has found that b (0, +) -type amino acid transporter 1 (CgB-aat1) plays an essential role on mantle pigmentation in the Pacific oyster Crassostrea gigas. However, the molecular regulation of CgB-aat1 gene expression remains unclear. Herein, three POU domain family members, CgPOU2F1, CgPOU3F4-like and CgPOU4F3-X1 were characterized and they all had POUs and HOX domains, respectively, which were important in transcriptional regulation. CgPOU3F4-like gene expression was the highest in mantle edge. Subsequently, the dual-luciferase reporter result showed that the core regulatory region of CgB-aat1 gene was from −632 to −350 bp of promoter. In transient co-transfection assays, the strongest activity was activated only by CgPOU3F4-like, suggesting CgPOU3F4-like was a valid transcriptional activator of CgB-aat1 gene promoter. And the structural integrity of CgPOU3F4-like was essential for its activation function. In addition, site directed mutagenesis assay was applied to detect three key binding sites between CgPOU3F4-like and core region of CgB-aat1 gene promoter, and this interaction was verified by ChIP test. Furthermore, CgPOU3F4-like knockdown by RNA interference led to obvious decreases in CgB-aat1 and cystathionine beta-synthase (CgCbs) expressions at both mRNA and protein levels. Collectively, these results indicate that CgPOU3F4-like positively regulate CgB-aat1 gene expression and it may be a critical upstream transcriptional regulation factor in pheomelanin synthesis in C. gigas.

Transcription factor Pf-Rel regulates expression of matrix protein genes Prismalin-14 and MSI60 in the pearl oyster Pinctada fucata.

Molluscan shell is a biomineral that consists of a highly organized calcium carbonate composite. Organisms mainly use matrix proteins to elaborately control the biomineralization process, but knowledge of their regulatory mechanisms is limited. The transcription factor Pf-Rel, which belongs to the Rel/nuclear factor-κB family, was shown to regulate transcription at the Nacrein promoter in the pearl oyster Pinctada fucata. Here, we further explored the transcriptional regulation mechanisms of Pf-Rel on the matrix proteins Prismalin-14 and MSI60. The relative expression levels of Prismalin-14 and MSI60 were high in the mantle edge and mantle pallial tissues of P. fucata. These three genes were significantly up-regulated after shell notching, suggesting that they might play important roles during shell formation. Importantly, Pf-Rel gene knockdown by RNA interference led to down-regulation of Prismalin-14 and MSI60 expression. In transient co-transfection assays, Pf-Rel significantly up-regulated the promoter activities of the Prismalin-14 and MSI60 genes in a dose-dependent manner. Furthermore, the promoter regions of Prismalin-14 (-1794 to -1599bp) and MSI60 (-2244 to -1141bp) were required for the activation by Pf-Rel. Altogether, these results suggest that the transcription factor Pf-Rel can up-regulate the expression of the matrix protein genes Prismalin-14 and MSI60 during shell formation in P. fucata, which improves our understanding of transcription regulation at the molecular level during molluscan shell development.

Transcriptional regulation of the matrix protein Shematrin-2 during shell formation in pearl oyster

The molluscan shell is a fascinating biomineral consisting of a highly organized calcium carbonate composite. Biomineralization is elaborately controlled and involves several macromolecules, especially matrix proteins, but little is known about the regulatory mechanisms. The matrix protein Shematrin-2, expression of which peaks in the mantle tissues and in the shell components of the pearl oyster Pinctada fucata, has been suggested to be a key participant in biomineralization. Here, we expressed and purified Shematrin-2 from P. fucata and explored its function and transcriptional regulation. An in vitro functional assay revealed that Shematrin-2 binds the calcite, aragonite, and chitin components of the shell, decreases the rate of calcium carbonate deposition, and changes the morphology of the deposited crystal in the calcite crystallization system. Furthermore, we cloned the Shematrin-2 gene promoter, and analysis of its sequence revealed putative binding sites for the transcription factors CCAAT enhancer-binding proteins (Pf-C/EBPs) and nuclear factor-Y (NF-Y). Using transient co-transfection and reporter gene assays, we found that cloned and recombinantly expressed Pf-C/EBP-A and Pf-C/EBP-B greatly and dose-dependently up-regulate the promoter activity of the Shematrin-2 gene. Importantly, Pf-C/EBP-A and Pf-C/EBP-B knockdowns decreased Shematrin-2 gene expression and induced changes in the inner-surface structures in prismatic layers that were similar to those of antibody-based Shematrin-2 inhibition. Altogether, our data reveal that the transcription factors Pf-C/EBP-A and Pf-C/EBP-B up-regulate the expression of the matrix protein Shematrin-2 during shell formation in P. fucata, improving our understanding of the transcriptional regulation of molluscan shell development at the molecular level.

The Basic Helix-Loop-Helix Transcription Factor GubHLH3 Positively Regulates Soyasaponin Biosynthetic Genes in Glycyrrhiza uralensis.

Glycyrrhiza uralensis (licorice) is a widely used medicinal plant belonging to the Fabaceae. Its main active component, glycyrrhizin, is an oleanane-type triterpenoid saponin widely used as a medicine and as a natural sweetener. Licorice also produces other triterpenoids, including soyasaponins. Recent studies have revealed various oxidosqualene cyclases and cytochrome P450 monooxygenases (P450s) required for the biosynthesis of triterpenoids in licorice. Of these enzymes, β-amyrin synthase (bAS) and β-amyrin C-24 hydroxylase (CYP93E3) are involved in the biosynthesis of soyasapogenol B (an aglycone of soyasaponins) from 2,3-oxidosqualene. Although these biosynthetic enzyme genes are known to be temporally and spatially expressed in licorice, the regulatory mechanisms underlying their expression remain unknown. Here, we identified a basic helix-loop-helix (bHLH) transcription factor, GubHLH3, that positively regulates the expression of soyasaponin biosynthetic genes. GubHLH3 preferentially activates transcription from promoters of CYP93E3 and CYP72A566, the second P450 gene newly identified and shown to be responsible for C-22β hydroxylation in soyasapogenol B biosynthesis, in transient co-transfection assays of promoter-reporter constructs and transcription factors. Overexpression of GubHLH3 in transgenic hairy roots of G. uralensis enhanced the expression levels of bAS, CYP93E3 and CYP72A566. Moreover, soyasapogenol B and sophoradiol (22β-hydroxy-β-amyrin), an intermediate between β-amyrin and soyasapogenol B, were increased in transgenic hairy root lines overexpressing GubHLH3. We found that soyasaponin biosynthetic genes and GubHLH3 were co-ordinately up-regulated by methyl jasmonate (MeJA). These results suggest that GubHLH3 regulates MeJA-responsive expression of soyasaponin biosynthetic genes in G. uralensis. The regulatory mechanisms of triterpenoid biosynthesis in legumes are compared and discussed.

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

The transcription factor Pf-POU3F4 regulates expression of the matrix protein genes Aspein and Prismalin-14 in pearl oyster (Pinctada fucata).

Matrix proteins play key roles in shell formation in the pearl oyster, but little is known about how these proteins are regulated. Here, two POU domain family members, Pf-POU2F1 and Pf-POU3F4, were cloned and characterized. Functional domain analysis revealed that both them have conserved POUS and POUH domains; these domains are important for transcription factor function. The tissue distributions of Pf-POU2F1 and Pf-POU3F4 mRNAs in pearl oyster revealed different expression patterns, and the expression of Pf-POU3F4 mRNA was relatively high in the mantle. The promoters of the matrix protein genes Aspein and Prismalin-14 were cloned using genome-walking PCR. Relatively high transcriptional activities of these promoters were detected in HEK-293T cells. In transient co-transfection assays, Pf-POU3F4 greatly up-regulated the promoter activities of the Aspein and Prismalin-14 genes in a dose-dependent manner. Structural integrity of Pf-POU3F4 was essential for its activation function. One region of the Aspein gene promoter, -181 to -77bp, and two binding sites in the Prismalin-14 gene promoter, -359 to -337 bp and -100 to -73bp, were required for activation of Pf-POU3F4. An electrophoresis mobility shift assay demonstrated that Pf-POU3F4 directly bound these sites. Pf-POU3F4 knockdown led to a decrease in Aspein and Prismalin-14 gene expression. Furthermore, expression levels for the Pf-POU3F4 gene were similar to those of the Aspein and Prismalin-14 genes during five development stages. Taken together, these results suggest that the transcription factor Pf-POU3F4 regulates expression of the matrix protein genes Aspein and Prismalin-14 in pearl oyster. The nucleotide sequence data of Pf-POU2F1 is available in the GenBank databases under the accession number KM588196. The nucleotide sequence data of Pf-POU3F4 is available in the GenBank databases under the accession number KM519606. The nucleotide sequence data of Aspein gene promoter is available in the GenBank databases under the accession number KM519607. The nucleotide sequence data of Prismalin-14 gene promoter is available in the GenBank databases under the accession number KM519601.

Analyzing Synthetic Promoters Using Arabidopsis Protoplasts.

This chapter describes a transient protoplast co-transfection method that can be used to quantitatively study in vivo the activity and function of promoters and promoter elements (reporters), and their induction or repression by transcription factors (effectors), stresses, hormones, or metabolites. A detailed protocol for carrying out transient co-transfection assays with Arabidopsis At7 protoplasts and calculating the promoter activity is provided.

High expression of kinesin light chain-2, a novel target of miR-125b, is associated with poor clinical outcome of elderly non-small-cell lung cancer patients.

Background:MiR-125b has critical role in non-small-cell lung cancer (NSCLC) cell migration, and its target genes have not been elucidated. Kinesin-1 light chain (KLC)-2 was predicted as one of miR-125b's targets by bioinformatics analysis. This study is to identify the function of KLC2 and its interaction with miR-125b in NSCLC.Methods:Kinesin-1 light chain-2 protein expression and its clinical relevance were analysed in 140 matched NSCLC and adjacent non-neoplastic lung tissues. Both KLC2 gain- and loss-of-function analyses were performed in NSCLC cell lines by transient transfection. The direct interaction between KLC2 and miR-125b was confirmed by a luciferase reporter assay and a transient co-transfection assay as well as an analysis of eight matched clinical samples.Results:KLC2 protein was upregulated in NSCLC cell lines and tissues, and was an independent predictor of poor prognosis for elderly NSCLC patients. Kinesin-1 light chain-2 remarkably enhanced the invasive and migratory ability of NSCLC cells. MiR-125b inhibited KLC2 3′-untranslated region luciferase activity and protein expression, and inversely correlated with KLC2 expression in clinical samples. Kinesin-1 light chain-2 almost completely reversed miR-125b-induced inhibition on migration and invasion.Conclusions:Kinesin-1 light chain-2 protein overexpression predicts poor survival in elderly NSCLC patients. Kinesin-1 light chain-2 acts as a proto-oncogene and a functional target of miR-125b in NSCLC cells.

Differential nuclear localization of complexes may underlie in vivo intrabody efficacy in Huntington's disease.

Intrabodies offer attractive options for manipulating the protein misfolding that triggers neurodegenerative diseases. In Huntington's disease, where the expanded polyglutamine tract in the extreme N-terminal region of huntingtin exon1 misfolds, two lead intrabodies have been selected against an adjacent peptide, using slightly different approaches. Both are effective at preventing aggregation of a reporter fragment in transient co-transfection assays. However, after intracranial delivery to mutant mouse brains, VL12.3, which is mainly localized to the nucleus, appears to accelerate the mutant phenotype, while C4 scFv, which is largely cytoplasmic, shows partial phenotypic correction. This comparison highlights parameters that could inform intrabody therapeutics for multiple proteostatic diseases.

Targeting of Casein Kinase II (CK2) Enhances Ikaros-Mediated Control of the Cell Cycle Progression in Pre-Clinical Model of Pre-B-ALL.

Abstract 2431The Ikaros (IKZF1) gene encodes a DNA-binding zinc finger protein that functions as a tumor suppressor in leukemia. Defects in the Ikaros gene that lead to its decreased activity are associated with the development of B-cell precursor acute lymphoblastic leukemia (pre-B-ALL). However, the mechanisms by which Ikaros exerts its tumor suppressor activity, as well as the mechanisms that control the tumor suppressor function of Ikaros are poorly understood. Here, we report the identification of two novel Ikaros target genes, as well as a signal transduction pathway that regulates the Ikaros-mediated transcriptional control of these genes in pre-B ALL. We also present evidence that targeting the signal transduction pathway which regulates Ikaros transcriptional control is a potent therapeutic tool for treating pre-B ALL.Analysis of the promoter sequences of the CDC7 and the CDK6 genes revealed multiple evolutionarily-conserved Ikaros consensus binding sites. Using qChIP (quantitative chromatin immunoprecipitation assay) we found that Ikaros binds in vivo to the promoters of the CDC7 and CDK6 genes in the human Nalm6 pre-B ALL cell line, and in primary human pre-B ALL cells. This led to the hypothesis that Ikaros regulates transcription of CDC7 and CDK6 - genes whose expression is essential for cell cycle progression and cellular proliferation. The effect of Ikaros on CDC7 and CDK6 expression was studied using a transient co-transfection assay. Luciferase reporter plasmids containing the CDC7 or CDK6 promoter regions were co-transfected with or without Ikaros into 293T cells. Co-transfection of Ikaros led to decreased luciferase activity, suggesting that Ikaros acts as a repressor of the CDC7 and CDK6 upstream regulatory elements. Increased expression of Ikaros via retroviral transduction in Nalm6 cells resulted in decreased transcription of CDC7 and CDK6. Decreased transcription of these genes was associated with increased binding of Ikaros to their promoter regions as measured by qChIP. These results suggest that Ikaros negatively regulates transcription of CDC7 and CDK6, and thus negatively regulates cell cycle progression in pre-B cell leukemia.We have shown previously that phosphorylation of Ikaros by casein kinase II (CK2) inhibits Ikaros' ability to bind DNA and to regulate transcription of its target genes. CK2 activity is elevated in human leukemia. We tested whether the inhibition of CK2 affects the transcription of CDC7 and CDK6 in pre-B ALL in vitro and in vivo. In vitro treatment of Nalm6 pre-B ALL with TBB, a specific CK2 inhibitor, decreased transcription of CDC7 and CDK6 and was associated with increased binding of Ikaros to the promoters of these genes. Using an in vivo preclinical model of pre-B ALL we tested whether targeting of CK2 would affect transcription of CDC7 and CDK6. In vivo treatment of a human-mouse xenograft model of pre-B ALL with a CK2 inhibitor resulted in decreased transcription of CDC7 and CDK6 and strongly increased Ikaros binding to the promoters of these genes. The in vivo targeting of CK2 provided a strong anti-leukemia effect that resulted in the prolonged survival of treated mice as compared to controls.In summary, our results suggest that Ikaros exerts its tumor suppressor activity in pre-B ALL by repressing transcription of cell cycle-promoting genes. Targeting CK2 in vivo enhances Ikaros-mediated repression of cell cycle progression resulting in an anti-leukemia effect. These data demonstrate the efficacy of CK2 targeting as a treatment for pre-B ALL in a preclinical model. These results provide a mechanistic rationale and support for the use of CK2 inhibitors as a targeted treatment of pre-B ALL in early-stage clinical trials. Supported by National Institutes of Health R01 HL095120 and a St. Baldrick's Foundation Career Development Award (to S.D.). Disclosures:No relevant conflicts of interest to declare.

Critical Role of Klf5 in Regulating Gene Expression during Post-Eyelid Opening Maturation of Mouse Corneas

BackgroundKlf5 plays an important role in maturation and maintenance of the mouse ocular surface. Here, we quantify WT and Klf5-conditional null (Klf5CN) corneal gene expression, identify Klf5-target genes and compare them with the previously identified Klf4-target genes to understand the molecular basis for non-redundant functions of Klf4 and Klf5 in the cornea.Methodology/Principal FindingsPostnatal day-11 (PN11) and PN56 WT and Klf5CN corneal transcriptomes were quantified by microarrays to compare gene expression in maturing WT corneas, identify Klf5-target genes, and compare corneal Klf4- and Klf5-target genes. Whole-mount corneal immunofluorescent staining was employed to examine CD45+ cell influx and neovascularization. Effect of Klf5 on expression of desmosomal components was studied by immunofluorescent staining and transient co-transfection assays. Expression of 714 and 753 genes was increased, and 299 and 210 genes decreased in PN11 and PN56 Klf5CN corneas, respectively, with 366 concordant increases and 72 concordant decreases. PN56 Klf5CN corneas shared 241 increases and 98 decreases with those previously described in Klf4CN corneas. Xenobiotic metabolism related pathways were enriched among genes decreased in Klf5CN corneas. Expression of angiogenesis and immune response-related genes was elevated, consistent with neovascularization and CD45+ cell influx in Klf5CN corneas. Expression of 1574 genes was increased and 1915 genes decreased in WT PN56 compared with PN11 corneas. Expression of ECM-associated genes decreased, while that of solute carrier family members increased in WT PN56 compared with PN11 corneas. Dsg1a, Dsg1b and Dsp were down-regulated in Klf5CN corneas and their corresponding promoter activities were stimulated by Klf5 in transient co-transfection assays.Conclusions/SignificanceDifferences between PN11 and PN56 corneal Klf5-target genes reveal dynamic changes in functions of Klf5 during corneal maturation. Klf5 contributes to corneal epithelial homeostasis by regulating the expression of desmosomal components. Klf4- and Klf5-target genes are largely distinct, consistent with their non-redundant roles in the mouse cornea.

STAT3 activation and aberrant ligand-dependent sonic hedgehog signaling in human pulmonary adenocarcinoma

The Sonic hedgehog (SHH) signaling and STAT3 pathways play important roles during carcinogenesis with possible interaction. To determine the association of the activation of SHH signaling pathway and STAT3 pathway in carcinogenesis of human non-small cell lung carcinomas (NSCLC), firstly we examined the expression of SHH signaling molecules including SHH, Gli1(glioma-associated oncogene homolog 1) and HHIP (Hh interacting protein), as well as p-STAT3 (phosphorylation at Tyr705) by immunohistochemistry in 87 cases of NSCLC, 12 atypical adenomatous hyperplasia (AAH) and 20 adjacent normal lung tissues. The expression of SHH, Gli1, HHIP and p-STAT3 was detected respectively in 87/87(100%), 74/87(85.1%), 75/87(86.2%) and 54/87(62.1%) of the NSCLC cases, but not in the adjacent normal lung parenchyma. Ligand-dependent SHH pathway activation and STAT3 signaling activation were observed in most cases of NSCLC, and the high SHH pathway activation rate and STAT3 activation rate were significantly higher in adenocarcinoma compared with squamous cell carcinomas and large cell carcinomas (P<0.05). Both SHH and STAT3 pathway activation level correlated with histological grade in adenocarcinoma, being higher in well-differentiated types (P<0.05). Furthermore, high SHH pathway activation and p-STAT3 expression were also detected in 10/12(83.3%) of AAH cases as well as in most cases of early-stage adenocarcinoma — adenocarcinoma in situ (AIS) and minimally invasive adenocarcinomas (MIA). Correlation analysis showed that p-STAT3 protein expression level was correlated positively with ligand-dependent activation level of SHH signaling in adenocarcinoma (rs=0.585, P=0.000) and AAH (rs=0.996, P=0.000). We speculated that activation of STAT3 could up-regulate SHH gene expression directly or indirectly, and thereby activated SHH signaling resulting in lung tumor cell ontogeny. To explore the interactional mechanism, we then performed serial transient co-transfection assay in human pulmonary adenocarcinoma cell line H441 cells, and the results confirmed STAT3 activation can up-regulate SHH gene expression indirectly. In conclusion, aberrant ligand-dependent SHH signaling activation occurs frequently in NSCLC. The signaling plays a more active role in adenocarcinoma, and is an early event in carcinogenesis of lung adenocarcinoma. The involvement of STAT3 pathway activation might function in inducing the process.

Identification and expression analysis of a novel transcript of the human PRMT2 gene resulted from alternative polyadenylation in breast cancer

The arginine N-methyltransferase 2 protein (PRMT2, also known as HRMT1L1) is thought to act as a coactivator of ERα. The present results show the occurrence of a novel transcript by alternative polyadenylation in the human PRMT2 gene. We demonstrated that the newly identified intron-retaining PRMT2L2 transcript is functionally intact, efficiently translated into protein in vivo. PRMT2 and PRMT2L2 mRNA expression profiles overlap with the distribution of ERα, with the strongest abundance in estrogen target tissues. Transient co-transfection assays demonstrated that PRMT2L2 enhance ERα-mediated transactivation activity of ERE-Luc in a ligand-dependent manner. Confocal microscopy scanning revealed a distinct intra-cellular localization of their fusion proteins, suggesting that the C-terminal region absent in PRMT2L2 is critical for the localization. Statistical analysis further showed that both PRMT2 and PRMT2L2 mRNAexpressions were up-regulated in breast cancer tissues, and significantly associated with ERα positivity status. Thus, post-transcriptional processing mechanism as alternative polyadenylation and splicing may play a crucial role in regulating human PRMT2 gene expression.

JUN-CCAAT/Enhancer-Binding Protein Complexes Inhibit Surfactant-Associated Protein B Promoter Activity

The murine surfactant-associated protein B (Sftpb) gene promoter, spanning nucleotides -653 to +42, is composed of functionally distinct proximal and distal regions. Although both regions contain consensus/putative activator protein 1 (AP-1) sites, the distal, but not the proximal, region mediates the inhibition by jun proto-oncogene (JUN) of Sftpb promoter activity. In transient cotransfection assays, JUN inhibited the luciferase reporter activity of plasmid constructs containing Sftpb promoter fragments that lacked the distal putative AP-1 site, indicating that another regulatory motif mediates JUN-dependent inhibition. Electrophoretic mobility shift assays and in silico analyses identified a DNA target sequence (Sftpb nucleotides -339 to -316) and transcription factors that regulate Sftpb promoter activity. The identified sequence contains a CCAAT/enhancer-binding protein (C/EBP) consensus recognition element. Mutation of the site reduced Sftpb promoter activity and sensitivity to inhibition by JUN. Purified recombinant JUN, which did not recognize the -339 to -316 target sequence when added alone, supershifted the mobility of in vitro translated C/EBP-α and C/EBP-β proteins complexed with the identified cis-regulatory element. These findings support the idea that heterodimerization between JUN and C/EBP-α and/or C/EBP-β targets JUN to the Sftpb promoter, thereby mediating its inhibitory regulatory role.

Evidence that α-lipoic acid inhibits NF-κB activation independent of its antioxidant function

α-Lipoic acid (LA) exerts beneficial effects in cardiovascular diseases though its antioxidant and/or anti-inflammatory functions. It is postulated that the anti-inflammatory function of LA results from its antioxidant function. In this study we tested whether inhibition of NF-κB by LA is dependent on its antioxidant function. Human umbilical vein endothelial cells (HUVECs) were treated with tumor necrosis factor-α (TNFα) in the presence of various antioxidants, including LA, tiron, apocynin, and tempol. The activation of the nuclear factor-κB (NF-κB) signaling pathway was then analyzed. LA, but not other tested antioxidants, inhibited TNFα-induced inhibitor-kappaB-α (IκBα) degradation and VCAM-1 and COX2 expression in HUVECs. Although LA activated the phosphatidylinositol-3-kinase (PI3-kinase)/Akt pathway in HUVECs, inhibition of Akt by LY294002 did not affect inhibition of TNFα-induced IκBα degradation by LA. In transient co-transfection assays of a constitutively active mutant of IκB kinase-2 (IKK2), IKK2(EE), and a NF-κB luciferase reporter construct, LA dose-dependently inhibited IKK2(EE)-induced NF-κB activation in addition to inhibiting IKK activity in in vitro assays. Consistent with the effect on luciferase expression, LA inhibited IKK2(EE)-induced cyclo-oxygenase-2 (COX2) expression, suggesting that IKK2 inhibition by LA may be a relevant mechanism that explains its anti-inflammatory effects. LA inhibits NF-κB activation through antioxidant-independent and probably IKK-dependent mechanisms.

Stringent testing identifies highly potent and escape‐proof anti‐HIV short hairpin RNAs

RNA interference (RNAi) is a cellular mechanism that can be induced by small interfering RNAs to mediate sequence-specific gene silencing by cleavage of the targeted mRNA. RNAi can be used as an antiviral approach to silence the human immunodeficiency virus type 1 (HIV-1) through stable expression of short hairpin RNAs (shRNAs). Previously, we used a co-transfection assay in which shRNA constructs were transfected with an HIV-1 molecular clone to identify 20 shRNA inhibitors that target highly conserved HIV-1 sequences. In the present study, we selected the most potent shRNAs to formulate a combinatorial shRNA therapy and determine the best and easiest method for antiviral shRNA selection. We performed transient inhibition assays with either a luciferase reporter or HIV-1 molecular clone and also infected shRNA-expressing T cell lines with HIV-1 and monitored virus replication. The latter assay allows detection of viral escape. In addition, we also tested shRNA-expressing T cells upon challenge with increasing dosages of HIV-1, and measured the dose required to result in massive virus-induced syncytia formation in this 2-week assay. Extended culturing selected three highly effective shRNAs that do not allow viral replication for more than 100 days. This difference in potency was not observed in the transient co-transfection assays. The use of increased dosages of HIV-1 selected the same highly potent shRNAs as the laborious and extended escape study. These highly potent shRNAs could be used for a clinical vector and the comparison of the developed assays might help other researchers in their search for antiviral shRNAs.

Interaction between PHOX2B and CREBBP mediates synergistic activation: Mechanistic implications of PHOX2B mutants

Congenital central hypoventilation syndrome (CCHS) is a disorder of the autonomic nervous system. The genetic defect is usually caused by heterozygous mutations of the PHOX2B gene such as a 20-alanine tract (+5 to+13 alanines) expansion (approximately 95%) and occasional frameshift or missense mutations. Cytoplasmic aggregates were shown in PHOX2B proteins with longer alanine tract (+9 and longer) expansion and impaired DNA binding was observed in PHOX2B proteins with frameshift, missense, or longer alanine tract (+9 and longer) expansion. Defective transactivation activity was shown in certain PHOX2B mutants. However, PHOX2B proteins with short alanine tract (+5 to+7) expansion in the majority of patients (approximately 75%) did not have cytoplasmic aggregates or DNA binding defects. CREB-binding protein (CREBBP/CBP) is a transcriptional coactivator that interacts with multiple transcription factors to cause synergistic activation. Here we show that CBP interacted with PHOX2B and served as its coactivator to mediate synergistic activation. Wild-type PHOX2B and CBP used specific domains to interact with each other. The domains of CBP that interacted with different PHOX2B mutants were different compared to those interacting with wild-type PHOX2B. Transient cotransfection assays using different PHOX2B mutants and CBP showed the impaired synergistic activation caused by different PHOX2B mutants. An interfering effect was observed in certain PHOX2B mutants. These results demonstrated that aberrant interaction of PHOX2B mutants with CBP and/or an interfering effect of certain PHOX2B mutants may be the critical mechanism to impair synergistic activation, thereby contributing to the phenotypes of CCHS.

KLF4 gene expression is inhibited by the notch signaling pathway that controls goblet cell differentiation in mouse gastrointestinal tract

In Kruppel-like factor (KLF)-4-deficient mice, colonic goblet cell numbers are significantly reduced. Goblet cell development is regulated by the Notch signaling pathway. The aim of this study was to examine whether Notch represses KLF4 expression to regulate goblet cell differentiation. We first detected that KLF4 gene expression was upregulated in a human progastrin-overexpressing mouse model where goblet cell hyperplasia has been observed. We then found that mice treated with a gamma-secretase inhibitor (compound E, 10 micromol/kg) for 24 h, which inhibits the Notch signaling pathway, had significantly increased KLF4 mRNA levels in small intestine and colon, accompanied by an increased number of KLF4-expressing cells at the bottom of crypts in small intestine and colon. In a colon cancer cell line (HCT116 cells), KLF4 promoter activity was inhibited by a constitutively active form of Notch1 (ICN1) by transient cotransfection assays. This inhibition was significantly compromised by a dominant-negative RBPjk, a repressive mediator of the Notch signaling pathway. An ICN1-responsive element was then mapped in the human KLF4 promoter between -151 and -122 nucleotides upstream of the transcriptional start site. It was also found that an intact ICN1-responsive element is required for ICN1 to inhibit KLF4 promoter activity by transient cotransfection assays. Our findings thus reveal a possible mechanism by which KLF4 is inhibited by Notch, which controls goblet cell differentiation in mouse gastrointestinal tract.

Cooperative Synergy between NFAT and MyoD Regulates Myogenin Expression and Myogenesis

Calcineurin/NFAT signaling is involved in multiple aspects of skeletal muscle development and disease. The myogenic basic helix-loop-helix transcription factors, MyoD, myogenin, Myf5, and MRF4 specify the myogenic lineage. Here we show that calcineurin/NFAT (nuclear factor of activated T cells) signaling is required for primary myogenesis by transcriptional cooperation with the basic helix-loop-helix transcription factor MyoD. Calcineurin/NFAT signaling is involved in myogenin expression in differentiating myoblasts, where the myogenic regulatory factor MyoD synergistically cooperates with NFATc2/c3 at the myogenin promoter. Using gel shift and chromatin immunoprecipitation assays, we identified two conserved NFAT binding sites in the myogenin promoter that were occupied by NFATc3 upon skeletal muscle differentiation. The transcriptional integration between NFATc3 and MyoD is crucial for primary myogenesis in vivo, as myogenin expression is weak in myod:nfatc3 double null embryos, whereas myogenin expression is unaffected in embryos with null mutations for either factor alone. Thus, the combined findings provide a novel transcriptional paradigm for the first steps of myogenesis, where a calcineurin/NFATc3 pathway regulates myogenin induction in cooperation with MyoD during myogenesis.