Cotransfection Studies Research Articles

Mechanism of nuclear factor of activated T-cells mediated FasL expression in corticosterone -treated mouse Leydig tumor cells

BackgroundFas and FasL is important mediators of apoptosis. We have previously reported that the stress levels of corticosterone (CORT, glucocorticoid in rat) increase expression of Fas/FasL and activate Fas/FasL signal pathway in rat Leydig cells, which consequently leads to apoptosis. Moreover, our another study showed that nuclear factor of activated T-cells (NFAT) may play a potential role in up-regulation of FasL during CORT-treated rat Leydig cell. It is not clear yet how NFAT is involved in CORT-induced up-regulation of FasL. The aim of the present study is to investigate the molecular mechanisms of NFAT-mediated FasL expression in CORT-treated Leydig cells.ResultsWestern blot analysis showed that NFAT2 expression is present in mouse Leydig tumor cell (mLTC-1). CORT-induced increase in FasL expression in mLTC-1 was ascertained by Western Blot analysis and CORT-induced increase in apoptotic frequency of mLTC-1 cells was detected by FACS with annexin-V labeling. Confocal imaging of NFAT2-GFP in mLTC-1 showed that high level of CORT stimulated NFAT translocation from the cytoplasm to the nucleus. RNA interference-mediated knockdown of NFAT2 significantly attenuated CORT-induced up-regulation of FasL expression in mLTC. These results corroborated our previous finding that NFAT2 is involved in CORT-induced FasL expression in rat Leydig cells and showed that mLTC-1 is a suitable model for investigating the mechanism of CORT-induced FasL expression. The analysis of reporter constructs revealed that the sequence between -201 and +71 of mouse FasL gene is essential for CORT-induced FasL expression. The mutation analysis demonstrated that CORT-induced FasL expression is mediated via an NFAT binding element located in the -201 to +71 region. Co-transfection studies with an NFAT2 expression vector and reporter construct containing -201 to +71 region of FasL gene showed that NFAT2 confer a strong inducible activity to the FasL promoter at its regulatory region. In addition, chromatin immunoprecipitation assay further confirmed the results of reporter gene studies by showing the specific binding of NFAT2 to the -201 to +71 region.ConclusionIn the present study, we demonstrated that NFAT2 directly stimulates transcription of FasL in high level CORT-treated mLTC-1. In conclusion, the present study provides further evidence for our finding that CORT-induced FasL expression in Leydig cells is mediated by NFAT.

Enhancement of Glycoprotein Hormone Alpha Subunit Promoter Reporter Gene Activity in Co-transfection Studies – A Cautionary Reminder

Reporter constructs have a wide range of application in determining eucaryotic gene regulation and expression. IN VITRO models frequently necessitate co-transfection and there have been reports of interference, predominantly inhibition, between promoters. Studies investigating the biological activity of a mutant thyrotropin receptor involved co-transfection with receptor constructs and a cAMP responsive luciferase reporter driven by the glycoprotein hormone alpha subunit promoter. We observed considerable enhancement of basal luciferase activity by co-transfecting the empty expression vector, which contained the SV40 late promoter. We have investigated the mechanism using different concentrations and several viral promoters in COS cells, following transient co-transfection. The increase was dose dependent but plateaued at 50 ng of vector DNA. This was not due to an adjuvant effect since luciferase activity was unchanged by adding increasing amounts of a promoterless plasmid. Enhancement was maintained when truncating the promoter to -346, but eliminated in the promoter truncated upstream of -244, indicating a binding site for a putative repressor of glycoprotein hormone alpha expression between -244 and -346. Enhancement was maintained with the addition of a second constitutive reporter (bos beta-gal) to correct for transfection efficiency, although this was the consequence of enhanced luciferase activity by the bos beta-gal, which in itself was inhibited by the SV40 promoter. Artefactual enhancement of reporter activity can occur and highlights the need for careful choice of controls when performing transient co-transfection experiments. In silico analysis of the promoters identified a possible shared forkhead transcription factor binding site.

Sex determination involves synergistic action of SRY and SF1 on a specific Sox9 enhancer

The mammalian Y chromosome acts as a dominant male determinant as a result of the action of a single gene, Sry, whose role in sex determination is to initiate testis rather than ovary development from early bipotential gonads. It does so by triggering the differentiation of Sertoli cells from supporting cell precursors, which would otherwise give follicle cells. The related autosomal gene Sox9 is also known from loss-of-function mutations in mice and humans to be essential for Sertoli cell differentiation; moreover, its abnormal expression in an XX gonad can lead to male development in the absence of Sry. These genetic data, together with the finding that Sox9 is upregulated in Sertoli cell precursors just after SRY expression begins, has led to the proposal that Sox9 could be directly regulated by SRY. However, the mechanism by which SRY action might affect Sox9 expression was not understood. Here we show that SRY binds to multiple elements within a Sox9 gonad-specific enhancer in mice, and that it does so along with steroidogenic factor 1 (SF1, encoded by the gene Nr5a1 (Sf1)), an orphan nuclear receptor. Mutation, co-transfection and sex-reversal studies all point to a feedforward, self-reinforcing pathway in which SF1 and SRY cooperatively upregulate Sox9 and then, together with SF1, SOX9 also binds to the enhancer to help maintain its own expression after that of SRY has ceased. Our results open up the field, permitting further characterization of the molecular mechanisms regulating sex determination and how they have evolved, as well as how they fail in cases of sex reversal.

A-Kinase Anchoring Protein Targeting of Protein Kinase A and Regulation of HERG Channels

Adrenergic stimulation of the heart initiates a signaling cascade in cardiac myocytes that increases the concentration of cAMP. Although cAMP elevation may occur over a large area of a target-organ cell, its effects are often more restricted due to local concentration of its main effector, protein kinase A (PKA), through A-kinase anchoring proteins (AKAPs). The HERG potassium channel, which produces the cardiac rapidly activating delayed rectifying K+ current (I Kr), is a target for cAMP/PKA regulation. PKA regulation of the current may play a role in the pathogenesis of hereditary and acquired abnormalities of the channel leading to cardiac arrhythmia. We examined the possible role for AKAP-mediated regulation of HERG channels. Here, we report that the PKA-RII-specific AKAP inhibitory peptide AKAP-IS perturbs the distribution of PKA-RII and diminishes the PKA-dependent phosphorylation of HERG protein. The functional consequence of AKAP-IS is a reversal of cAMP-dependent regulation of HERG channel activity. In further support of AKAP-mediated targeting of kinase to HERG, PKA activity was coprecipitated from HERG expressed in HEK cells. Velocity gradient centrifugation of solubilized porcine cardiac membrane proteins showed that several PKA-RI and PKA-RII binding proteins cosediment with ERG channels. A physical association of HERG with several specific AKAPs with known cardiac expression, however, was not demonstrable in heterologous cotransfection studies. These results suggest that one or more AKAP(s) targets PKA to HERG channels and may contribute to the acute regulation of I Kr by cAMP.Electronic supplementary materialThe online version of this article (doi:10.1007/s00232-008-9118-4) contains supplementary material, which is available to authorized users.

Analysis of EphA4 receptor tyrosine kinase substrate specificity using peptide‐based arrays

Eph receptor tyrosine kinases regulate many important biological processes. In the present study, we explored the substrate specificity of the EphA4 receptor tyrosine kinase using peptide arrays. We define a consensus substrate motif for EphA4 and go on to identify and test a number of potential EphA4 substrates and map their putative site(s) of phosphorylation. Cotransfection studies validate two of the predicted substrates: Nck2 and Dok1. Our findings identify several potential EphA4 substrates and demonstrate the general utility of using peptide arrays to rapidly identify and map protein kinase phosphorylation sites.

Ser484 and Ser494 in REL Are the Major Sites of IKK Phosphorylation In Vitro: Evidence That IKK Does Not Directly Enhance GAL4-REL Transactivation

Human c-Rel (REL) is a member of the NF-kappa B family of transcription factors, and one of its primary physiological roles is in the regulation of B-cell proliferation and survival. Although REL is primarily regulated by cytoplasmic-nuclear translocation through interaction with I kappa B inhibitors, REL also undergoes several posttranslational modifications that have been proposed to modulate its transcriptional activation activity. For example, phosphorylation of C-terminal sequences of REL has been proposed to increase its transactivation activity. In this report, we have used immune complex kinase assays to identify Ser484 and Ser494 as the primary sites of IKK alpha- and IKK beta-mediated in vitro phosphorylation in the C-terminal transactivation domain of REL. However, in cotransfection studies in A293 cells we have failed to detect IKK beta-mediated phosphorylation of these sites on REL in vivo, nor does IKK beta appear to interact with REL in these cells. Ser-to-Ala mutation of Ser484 and Ser494 does not affect IKK's ability to enhance GAL4-REL transactivation in reporter gene assays in A293 cells. We also show that the previously reported effects of overexpressed IKK and tumor necrosis factor treatment on GAL4-REL transactivation are due to IKK-mediated activation of the endogenous NF-kappa B pathway, which increases transcription from kappa B sites in the promoter of a commonly used GAL4 expression vector. Taken together, these results do not support a role for IKK-mediated phosphorylation as means for regulating the activity of REL in vivo.

Effective Cellular Uptake and Efflux of Thyroid Hormone by Human Monocarboxylate Transporter 10

Cellular entry of thyroid hormone is mediated by plasma membrane transporters, among others a T-type (aromatic) amino acid transporter. Monocarboxylate transporter 10 (MCT10) has been reported to transport aromatic amino acids but not iodothyronines. Within the MCT family, MCT10 is most homologous to MCT8, which is a very important iodothyronine transporter but does not transport amino acids. In view of this paradox, we decided to reinvestigate the possible transport of thyroid hormone by human (h) MCT10 in comparison with hMCT8. Transfection of COS1 cells with hMCT10 cDNA resulted in 1) the production of an approximately 55 kDa protein located to the plasma membrane as shown by immunoblotting and confocal microscopy, 2) a strong increase in the affinity labeling of intracellular type I deiodinase by N-bromoacetyl-[(125)I]T(3), 3) a marked stimulation of cellular T(4) and, particularly, T(3) uptake, 4) a significant inhibition of T(3) uptake by phenylalanine, tyrosine, and tryptophan of 12.5%, 22.2%, and 51.4%, respectively, and 5) a marked increase in the intracellular deiodination of T(4) and T(3) by different deiodinases. Cotransfection studies using the cytosolic thyroid hormone-binding protein micro-crystallin (CRYM) indicated that hMCT10 facilitates both cellular uptake and efflux of T(4) and T(3). In the absence of CRYM, hMCT10 and hMCT8 increased T(3) uptake after 5 min incubation up to 4.0- and 1.9-fold, and in the presence of CRYM up to 6.9- and 5.8-fold, respectively. hMCT10 was less active toward T(4) than hMCT8. These findings establish that hMCT10 is at least as active a thyroid hormone transporter as hMCT8, and that both transporters facilitate iodothyronine uptake as well as efflux.

Expression studies of missense mutations p.D141Y, p.C275S located in the propeptide of von Willebrand factor in patients with type 3 von Willebrand disease

Missense mutations are not considered a common cause of type 3 von Willebrand's disease (VWD), the most severe defect of von Willebrand factor (VWF) characterized by undetectable levels of this protein in plasma and platelets. Nevertheless, several missense mutations have been identified in these patients. In this study, we report the cases of two Italian patients with type 3 VWD, both compound heterozygotes for different missense mutations and null alleles, p.D141Y/c.2016_2019del and p.C275S/p.W222X. We performed in vitro expression studies of the candidate missense mutations, both located in the D1 domain of VWF propeptide, to confirm their link with the disease and to understand the mechanisms of type 3 VWD responsible in these patients. Mutant and wild-type (WT) expression vectors were used for transient transfection and co-transfection studies in COS-7 cells. Single construct transfections of both missense mutations showed a strongly reduced but detectable secretion of recombinant (r)VWFs (approximately 15% of WT), with essentially only dimers being visualized on multimeric analysis. As expected, expression of a single construct of either mutation with the WT, showed mildly reduced secretion (approximately 40% of WT) and a full set of multimers. These expression studies indicate that the two amino acids D141 and C275 are key residues in the tertiary structure of the VWF propeptide. Their replacement with a tyrosine and a serine, respectively, might compromise propeptide folding, affecting both its intracellular survival and its capacity to mediate multimerization. Co-expression of hybrid rVWFs confirmed the recessive inheritance pattern of these missense mutations.

Monocyte Chemoattractant Protein-1 Expression Is Enhanced by Granulocyte-Macrophage Colony-stimulating Factor via Jak2-Stat5 Signaling and Inhibited by Atorvastatin in Human Monocytic U937 Cells

The proinflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is expressed in inflammatory and atherosclerotic lesions. GM-CSF is known to enhance monocytic expression of monocyte chemoattractant protein-1 (MCP-1). However, the molecular mechanism(s) by which GM-CSF up-regulates the MCP-1 expression remains to be clarified. Thus, in this study, we examined our hypothesis that GM-CSF up-regulates the MCP-1 expression via Jak2-Stat5 signaling pathway. In human monocytic cell line U937, GM-CSF increased MCP-1 expression in protein and mRNA levels. Furthermore, analysis of the GM-CSF promoter element revealed that the STAT5 (signal transducer and activator of transcription-5) transcription factor binding site, located between -152 and -144 upstream of the transcription start site, as well as Janus kinase-2-mediated Stat5 activation were necessary for the GM-CSF-induced transcriptional up-regulation of the MCP-1 gene. This GM-CSF-induced MCP-1 expression, measured as both protein and mRNA levels, was down-regulated by atorvastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor. However, this decrease in MCP-1 expression was not at the transcriptional level of MCP-1 gene but rather at the level of the stability of MCP-1 mRNA. These results indicate that GM-CSF regulates MCP-1 expression via Janus kinase-2-Stat5 pathway and by a novel regulatory mechanism of statins to reduce inflammatory reactions by down-regulating the expression of monocytic MCP-1, which promotes atherogenesis.

Cloning, characterization and promoter analysis of the common carp IGF-II gene

Insulin-like growth factors (IGFs) belong to a family of growth factors with structural homology to proinsulin. Up till now, no specific details regarding the transcriptional regulation by autocrine, paracrine or endocrine effector molecules in vivo have been described for the IGF-II gene. This is in big contrast to IGF-I gene transcription which has been studied more extensively. To better understand how the IGF-II gene is controlled at the gene transcription level, we have isolated the common carp IGF-II gene together with the 5′-flanking region by genomic library screening. The mature IGF-II protein was encoded by exon 2 and exon 3. Transient transfection of the 5′-flanking region containing a TATA box-like sequence into cultured eukaryotic cells revealed that it is a strong promoter with definitive tissue specificity. Nucleotides between − 301 and − 62 in the promoter are essential to drive the basal IGF-II gene expression; whereas nucleotides between − 891 and − 416 in the promoter are responsible for the growth hormone activation. Using electrophoretic mobility shift assay and yeast one-hybrid screening, it was demonstrated that α1-antitrypsin could bind specifically to the nucleotide position − 301 to − 262 of the gene promoter. Co-transfection studies revealed that the over-expression of α1-antitrypsin increased the IGF-II promoter activity by 3.4-fold, further confirming that α1-antitrypsin acts as a trans-acting factor on the IGF-II promoter.

Transcriptional regulation of the murine 1‐cys peroxiredoxin gene by the B cell‐specific activator protein, Pax5

Pax5, a member of the paired box gene family of transcription factors, is a B cell-specific activator protein (BSAP) that plays important roles in controlling the expression of lineage- and differentiation-stage specific genes during B lymphopoiesis. We identified two putative Pax5 binding sites in a 668 bp of the murine 1-cys peroxiredoxin (1-cysPrx) promoter region. These sites were located at positions -278 to -262 and -50 to -34 from the translation start site. Gel mobility shift assays showed that recombinant Pax5 protein bound specifically to the nucleotide regions -56 to -24 (MP1 probe) and -284 to -253 (MP2 probe). Furthermore, endogenous Pax5 protein from B lymphoblast cells (IM-9) formed a DNA-protein complex with MP1 and MP2 probes, indicating that Pax5 binding occurs specifically at these sequences in vivo. Transient transfection studies showed that expression of exogenous Pax5 resulted in dose-dependent increases in 1-cysPrx promoter activity, implying that Pax5 functions as a positive transcription factor for 1-cysPrx expression. Further transient cotransfection studies showed that coexpression of Pax5 and histone acetyltransferases (HATs), such as p300, cAMP-response-element-binding protein (CBP) and p300/CBP associated factor (PCAF) enhanced the Pax5-mediated 1-cysPrx promoter activity. Immunoprecipitation studies indicated that Pax5 directly binds to HATs. Chromatin immunoprecipitation assays showed that the recruitment of Pax5 to the promoter induced histone H3 and H4 hyperacetylation of chromatin. Lipopolysaccharides (LPS) stimulation of murine splenocytes resulted in coordinated expression of Pax5 and 1-cysPrx proteins. These findings suggest that Pax5 may function as a transactivator of 1-cysPrx gene expression.

Pseudomonas exotoxin antisense RNA selectively kills hepatitis B virus infected cells

To present an approach for selectively killing retrovirus-infected cells that combines the toxicity of Pseudomonas exotoxin (PE) and the presence of reverse transcriptase (RT) in infected cells. PE antisense toxin RNA has palindromic stem loops at its 5' and 3' ends enabling self-primed generation of cDNA in the presence of RT. The RT activity expressed in retrovirus-infected cells converts "antisense-toxin-RNA" into a lethal toxin gene exclusively in these cells. Using cotransfection studies with PE-expressing RNAs and beta-gal expressing reporter plasmids, we show that, in HepG2 and HepG2.2.15 hepatoma cells as well as in duck hepatitis B virus (DHBV) infected cells, HBV or DHBV-polymerase reverse transcribe a lethal cDNA copy of an antisense toxin RNA, which is composed of sequences complementary to a PE gene and eukaryotic transcription and translation signals. This finding may have important implications as a novel therapeutic strategy aimed at the elimination of HBV infection.

A Heterozygous Mutation in the Third Transmembrane Domain Causes a Dominant-Negative Effect on Signalling Capability of the MC4R

Background: Heterozygous MC4R mutation is the most frequent cause of monogenic obesity. For most MC4R mutations a gene dosage effect seems to be the underlying mechanism. However, a dominant negative effect of a heterozygous MC4R mutation was recently identified, pointing to an additional mechanism of MC4R inactivation. Methods: The complete loss-of-function mutation (Ser136Phe), identified in a cohort of obese Austrian patients, was characterized for cell surface expression, signal transduction and ligand binding properties. Co-transfection studies tested for a dominant negative effect. Dimerization was investigated by a sandwich ELISA and by fluorescence resonance energy transfer (FRET) approach. Potential intramolecular interactions of Ser136 were studied by homologous receptor modelling based on the crystal structure of the β2-adrenergic receptor. Results: The Ser136Phe mutation showed a dominant negative effect. The sandwich ELISA and FRET approach demonstrated dimerization of mutant and wild type receptor. Receptor modelling revealed an essential function of Ser136 at transmembrane helix 3 (TMH3) for establishing H-bonds between TMH2, TMH3, and TMH7. The mutation Ser136Phe most likely disrupts this network and leads to an incompetent helix-helix arrangement in the mutated receptor. Conclusion: Identification of dominant negative MC4R mutations is important to fully understand receptor function and to determine receptor regions that are involved in MC4R dimer activation.

Hypoxia transcriptionally induces macrophage-inflammatory protein-3α/CCL-20 in primary human mononuclear phagocytes through nuclear factor (NF)-κB

Hypoxia, a condition of low oxygen tension, occurring in many pathological processes, modifies the mononuclear phagocyte transcriptional profile. Here, we demonstrate hypoxic up-regulation of the CCL20 chemokine in primary human monocytes (Mn) and macrophages. mRNA induction was paralleled by protein secretion and dependent on gene transcription activation. Functional studies of the CCL20 promoter using a series of 5'-deleted and mutated reporter constructs demonstrated the requirement for the NF-kappaB-binding site located at position -92/-82 for gene transactivation by hypoxia, as 1) transcription was abrogated by a 3-bp mutation of the NF-kappaB motif; 2) three copies of the wild-type NF-kappaB-binding site conferred hypoxia responsiveness to a minimal heterologous promoter; and 3) hypoxia increased specific NF-kappaB binding to this sequence. Furthermore, we provide evidence of the specific role of a single NF-kappaB family member, p50, in mediating CCL20 gene transcription in hypoxic Mn. p50 homodimers were the only detectable NF-kappaB complexes binding the cognate kappaB site on the CCL20 promoter upon hypoxia exposure, and NF-kappaBp50 knockdown by lentiviral-mediated short hairpin RNA interference resulted in complete binding inhibition. NF-kappaBp50 overexpression in transient cotransfection studies promoted CCL20 gene transactivation, which was abrogated by mutation of the -92/-82 kappaB site. Moreover, nuclear expression of the other NF-kappaB family members was inhibited in hypoxic Mn. In conclusion, this study characterizes a previously unrecognized role for hypoxia as a transcriptional inducer of CCL20 in human mononuclear phagocytes and highlights the importance of the NF-kappaB pathway in mediating this response, with potential implications for inflammatory disease and cancer pathogenesis.

Mef2c is an essential regulatory element required for unique expression of the cardiac‐specific CARK gene

The cardiac ankyrin repeat kinase (CARK) gene, also named TNNI3K for its interaction with cardiac troponin I, is both a unique expression and heart-enriched gene. To understand the mechanisms of CARK gene expression and regulation, we first cloned the full-length mRNA sequence and mapped the transcription start site of the mouse CARK gene and characterized its promoter regions. Two transcriptional isoforms of the CARK gene were identified in mouse heart tissue. Truncation analysis of the CARK promoter identified a minimal 151 bp region that has strong basal transcription activity. Mutational analysis revealed five conserved cis-acting elements in this 151-bp long minimal promoter. Mutational and loss-of-functional analysis and co-transfection studies indicated that MEF2 binding region is the most critical cis-acting element in the CARK promoter, and CARK transcription level can be down-regulated by MEF2C antisense. Binding to the MEF2 sites by Mef2c protein was confirmed by electrophoretic mobility shift assay and competition and supershift electrophoretic mobility shift assays.

Kit- and FcɛRI-induced differential phosphorylation of the transmembrane adaptor molecule NTAL/LAB/LAT2 allows flexibility in its scaffolding function in mast cells

The transmembrane adaptor protein (TRAP), NTAL, is phosphorylated in mast cells following FcɛRI aggregation whereby it cooperates with LAT to induce degranulation. The Kit ligand, stem cell factor (SCF), enhances antigen-induced degranulation and this also appears to be NTAL-dependent. However, Kit and FcɛRI appear to utilize different mechanisms to induce NTAL phosphorylation. Thus, we examined whether the responsible kinases selectively phosphorylated distinct tyrosines in NTAL and explored the implications for downstream signaling. Whereas FcɛRI required Lyn and Syk for NTAL phosphorylation, Kit appeared to directly phosphorylate NTAL. Furthermore, co-transfection studies with NTAL constructs revealed that Lyn, Syk, and Kit phosphorylate different tyrosines in NTAL. The tyrosines principally phosphorylated by Syk were recognized as Grb2-binding sites, whereas Lyn and Kit phosphorylated other tyrosines, both inside and outside of these motifs. Pull down studies revealed that PLCγ 1 associated with the two terminal Syk-phosphorylated Grb2-binding sites, which would help to explain the observed decrease in antigen-induced calcium signal and degranulation in NTAL-knock down-human mast cells. The observations reported herein support the conclusion that NTAL may be differentially utilized by specific receptors for relaying alternative signals and this suggests a flexibility in the function of TRAPs not previously appreciated.

Determining the Transrepression Activity of Xenoestrogen on Nuclear Factor-κB in Cos-1 Cells by Estrogen Receptor-α

Functional assays have been used to define the estrogenicity of xenoestrogens in cotransfection studies employing estrogen receptors in various cell lines. It is known that estrogen is able to affect transcription from other nuclear transcription factors, especially the nuclear factor-kappa B (NF-kappa B). The ability of selected xenoestrogens (methoxychlor [MXC], dieldrin, and o',p'-DDT) to transrepress the NF-kappa B-mediated transcription in Cos-1 cells was evaluated by cotransfection of human estrogen receptor-alpha (hERalpha). These xenoestrogens have been described as comparably potent xenoestrogens, whereas their relative binding activity (RBA) has been relegated to a lower order as compare to estrogen. The two NF-kappa B response element-containing SV40 promoter and -242/+54 cytomegalovirus (CMV)-expressing firefly luciferase (2 x NRE-PV-Luc and 2 x NRE-CMV-Luc, respectively) were transfected into Cos-1 cells with pRL-tk, expressing the renilla luciferase as internal control. The estrogen receptor was expressed from cytomegalovirus major immediate early promoter (CMV-MIEP) (CMV5-hERalpha). Treatment with 1 nM estrogen (E(2)) (26.2%), 5 nM E(2) (41.4%; p < .05), and xenoestrogens (methoxychlor [1 nM: 29.6%, p < .05; 10 nM: 22.6%), dieldrin [1 nM: 10.3%; 10 nM: 36.06%, p < .05], and o',p'-DDT [1 nM: 17.0%; 10 nM: 7.15%]) repressed transcription from 2 x NREX-PV-Luc. The antiestrogen, ICI 182,780, failed to antagonize the effects of xenoestrogens. The effects of xenoestrogens in transrepression of NF-kappa B by ERalpha were similar when 2 x NRE-CMV-Luc was employed as reporter. Statistically significant (p < .01) repression by 1 nM E(2) (69.2%), 5 nM E(2) (69.1%), 1 nM o',p'-DDT (51.4%), 1 nM dieldrin (47.3%), and 1 nM MXC (73.3%) were observed. The effect of these xenoestrogens without ERalpha cotransfection on 2 x NRE-PV-Luc- and 2 x NRE-CMV-Luc-mediated NF-kappa B transcription was not affected by the treatment alone. It is concluded that xenoestrogens, like estrogens, are capable of producing transrepression of NF-kappa B by hERalpha.

The histone deacetylase inhibitor trichostatin A mediates upregulation of 5-lipoxygenase promoter activity by recruitment of Sp1 to distinct GC-boxes

The histone deacetylase inhibitor trichostatin A (TsA) potently induces 5-lipoxygenase (5-LO) promoter activity in reporter gene assays as well as 5-LO mRNA expression. We identified two proximal Sp1/Sp3 binding sites in the 5-LO gene promoter mediating the TsA effect in both 5-LO-negative HeLa cells and in 5-LO expressing Mono Mac 6 (MM6) cells, the tandem GC-boxes, by contrast, were not important for the TsA effect. TsA neither altered the protein expression levels of Sp1/Sp3 nor of the histone deacetylases HDAC1/2, nor did it apparently change the protein complex formation by these factors. Also, treatment of cells with TsA did not change the binding affinity of Sp1/Sp3 in cell extracts, as tested by DAPA analysis using probes containing the proximal GC boxes. However, in the living cell TsA induced Sp1, Sp3 and RNA polymerase II recruitment to the 5-LO promoter without changing the acetylation status of histone protein H4. Cotransfection studies suggest that both Sp1 and Sp3 can mediate the TsA effect. This is the first report demonstrating that Sp3 is involved in the regulation of 5-LO promoter activity. In summary, we show that TsA increases 5-LO promoter activity by the enhanced recruitment of Sp1 and Sp3 to the 5-LO promoter.

Phloretin enhances adipocyte differentiation and adiponectin expression in 3T3-L1 cells

Adipocyte dysfunction is strongly associated with the development of cardiovascular risk factors and diabetes. It is accepted that the regulation of adipogenesis or adipokines expression, notably adiponectin, is able to prevent these disorders. In this report, we show that phloretin, a dietary flavonoid, enhances 3T3-L1 adipocyte differentiation as evidenced by increased triglyceride accumulation and GPDH activity. At a molecular level, mRNA expression levels of both PPARγ and C/EBPα, the master adipogenic transcription factors, are markedly increased by phloretin. Moreover, mRNA levels of PPARγ target genes such as LPL, aP2, CD36 and LXRα are up-regulated by phloretin. We also show that phloretin enhances the expression and secretion of adiponectin. Co-transfection studies suggest the induction of PPARγ transcriptional activity as a possible mechanism underlying the phloretin-mediated effects. Taken together, these results suggest that phloretin may be beneficial for reducing insulin resistance through its potency to regulate adipocyte differentiation and function.

Novel function of the poly(C)‐binding protein αCP3 as a transcriptional repressor of the mu opioid receptor gene

The alpha-complex proteins (alphaCP) are generally known as RNA-binding proteins that interact in a sequence-specific fashion with single-stranded poly(C). These proteins are mainly involved in various post-transcriptional regulations (e.g., mRNA stabilization or translational activation/silencing). Here we report a novel function of alphaCP3, a member of the alphaCP family. alphaCP3 bound to the double-stranded poly(C) element essential for the mu opioid receptor (MOR) promoter and repressed the promoter activity at the transcriptional level. We identified alphaCP3 using affinity column chromatography containing the double-stranded poly(C) element and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. AlphaCP3 binding to the poly(C) sequence of the MOR gene was sequence specific, as confirmed by the supershift assay. In cotransfection studies, alphaCP3 repressed the MOR promoter only when the poly(C) sequence was intact. Ectopic expression of alphaCP3 led to repression of the endogenous MOR transcripts in NS20Y cells. When alphaCP3 was disrupted using small interfering RNA (siRNA) in NS20Y cells, the transcription of the endogenous target MOR gene was increased significantly. Our data suggest that alphaCP3 can function as a repressor of MOR transcription dependent on the MOR poly(C) sequence. We demonstrate for the first time a role of alphaCP3 as a transcriptional repressor in MOR gene regulation.