Luciferase Expression Vector Research Articles

Repression of NFAT1 Protein Expression by MicroRNA-184 in Umbilical Cord Blood-Derived CD4+ T-Cells as a Mechanism Underlying Lower Incidence of aGVHD

Clinical benefits of umbilical cord blood (UCB) grafts include lower incidence of acute graft-versus-host disease (aGVHD) following allogeneic transplantation. The dramatically lower expression of the transcription factor Nuclear Factor of Activated T-cells 1 (NFAT1) in UCB-derived CD4+ T-cells compared to adult blood (AB) results in reduced production of inflammatory cytokines following stimulation, in part underlying this advantage. Here we present a novel mechanism by which NFAT1 protein expression is restrained in these cells. Importantly, NFAT1 mRNA (NFATc2) is not significantly reduced in UCB CD4+ T-cells. Gradient separation and cellular fractionation reveal that NFAT1 is indeed translationally repressed in UCB through early stimulation timepoints, which we hypothesized might be due to the activity of microRNA. A search of known microRNA species reveals that miR- 184 is strongly predicted to bind to the NFATc2 3′ untranslated region (UTR). We have confirmed this interaction through luciferase expression and Western Blot analysis. First, luciferase expression vectors containing the short miR-184 binding sequence or the cloned 3′ UTR were constructed and transfected into UCB and AB CD4+ T-cells. In UCB, insertion of the miR-184 binding sequence alone reduced luciferase expression to 38% of control. The cloned 3′ UTR vector exhibited 60% expression compared to control. Both these negative effects were fully reversible by cotransfection with an excess of blocking antisense to miR- 184. Likewise, the miR-184 site did not impact luciferase expression in AB CD4+ T-cells, but expression was reduced by 61% in AB upon cotransfection with miR-184 mimic. miR-184 mimic produced a 23% decrease in luciferase activity from vector containing the NFATc2 3′ UTR. Both these effects were attenuated by cotransfection with an excess of blocking antisense to miR-184. Blocking antisense increased endogenous NFAT1 protein expression in UCB CD4+ T-cells by 86% as measured by quantified Western Blot, without producing a significant change in NFATc2 mRNA quantity. Conversely, miR-184 mimic produced a 31% decrease in NFAT1 protein expression by AB CD4+ T-cells. We confirmed by quantitative RT-PCR (qRT-PCR) that miR-184 exhibited an average of 58.4-times greater expression in UCB than in AB CD4+ T-cells (p=0.005). We have additionally demonstrated via gain-of-function and loss-of-function analyses that the effects on NFAT1 expression by miR-184 are sufficient to affect transcription of the NFAT-dependent pro-inflammatory cytokine IL-2 through 16 hours of in vitro stimulation. This study comprises the first identification of miR-184 activity in primary human lymphocytes and reveals its function in repressing NFAT1 protein expression uniquely in UCB CD4+ T-cells. These findings hold important implications for our further understanding of GVHD, autoimmunity, and neonatal lymphocyte development, and suggest miR-184 as a useful biomarker or target of ex vivo graft analysis.

Cooperation Between IL-7 and TNF-Alpha Results in Suppression of Runx2/Cbfa1 Expression and Inhibition of Osteoblast Differentiation in Myeloma.

Multiple myeloma (MM) is a plasma cell malignancy in which almost all patients develop osteolytic lesions characterized by enhanced osteoclast (OCL) activity and reduced bone formation. Although much is known about the mechanisms responsible for the increased OCLs activity, the mechanisms responsible for osteoblast (OBL) inhibition are still not clearly understood. However, it is now clear that OBL suppression represents an important feature of the disease. Factors reported to inhibit OBL differentiation in MM include Dickkopf-1 (DKK-1), secreted Frizzle-Related Protein-2 (sFRP2), which are inhibitors of the Wingless (Wnt) signaling pathway that is critical for the OBL differentiation, IL-3, IL-7 and tumor necrosis factor-alpha (TNF-alpha). To further define the mechanisms responsible for OBL suppression in MM, we co-cultured MC4 cells, a MC-3T3-derived mouse marrow OBL precursor cell line, with 5TGM1 cells, an aggressive murine MM cell line known to reproduce MM bone disease in vivo, and measured the expression levels of OBL differentiation markers by Real-time PCR. Type I collagen, bone sialoprotein and osteocalcin mRNA expression were downregulated by 10-fold after the co-culture for 48 hours, and mineralization was inhibited with longer co-culture. Further, expression of mRNAs for two major transcription factors that regulate OBL differentiation, Runx2 and osterix were suppressed 10-fold, while activating transcription factor-4 (ATF4) was unaffected. Time-course experiments demonstrated that Runx2 mRNA downregulation was detectable within 2 hours of co-culture. To identify the promoter region responsible for the suppression of Runx2, MC4 cells were then transfected with Runx2-promoter luciferase expression vectors carrying different deletions in the Runx2 promoter and co-cultured with 5TGM1 cells. 5TGM1 cells strongly downregulated Runx2 promoter activity and a 900 bp region was shown to be the region required for Runx2 suppression. Within this promoter region, several potential transcription factor sites were identified including GFI-1 that can suppress Runx2 expression. However, GFI-1 mRNA expression was only increased after 48 hours of co-culture, suggesting another mechanism was involved. To determine if OBL suppression was mediated by cell-cell interaction or by soluble factors, we then co-cultured MC4 and 5TGM1 cells in a transwell system and found that 5TGM1 cells were still capable of suppressing Runx2 expression, indicating a soluble inhibitor was responsible for suppression of OBL differentiation. Importantly, DKK-1, sFRP2 or IL-3 expression was not detectable in 5TGM1 cells, but the cells did produce IL-7 and TNF-alpha. Using blocking antibodies for IL-7 and TNF-alpha in the co-culture system, Runx2 expression increased to 90% of normal levels with either anti-TNF or anti-IL-7 and to 100% when both antibodies were added. These results suggest that IL-7 and TNF-alpha cooperate to suppress Runx2 expression. Moreover, when MC4 cells were treated with low dose (0.1ng/ml) of TNF-alpha or IL-7, no effect on Runx2 expression was detected. However, when low concentrations of IL-7, and TNF-alpha were both added, Runx2 was downregulated almost 80% within 2 hours. These results demonstrate that TNF-alpha and IL-7 can cooperate to rapidly suppress OBL differentiation in MM.

High glucose induces transactivation of the human paraoxonase 1 gene in hepatocytes

Human serum paraoxonase 1 (PON1) is associated with high-density lipoprotein and inhibits oxidative modification of low-density lipoprotein in vitro. Therefore, PON1 is expected to protect against atherosclerosis in vivo. We and other investigators have shown that PON1 enzymatic activity is decreased in diabetic patients; however, an alteration in hepatic PON1 synthesis under hyperglycemic conditions remains unclear. We previously demonstrated that Sp1 is a positive regulator of PON1 transcription and that an interaction between Sp1 and protein kinase C (PKC) is a crucial mechanism for the effect of Sp1 on PON1 transcription in cultured HepG2 cells. Because several PKC isoforms are activated under hyperglycemic conditions, we examined the effect of d-glucose, which can activate the diacylglycerol-PKC pathway, on the transcription and expression of PON1. For a reporter gene assay, Huh7 human hepatocyte cell line incorporated with PON1 (−1232/−6)–luciferase expression vector was established using a cationic lipid method. d-Glucose dose dependently enhanced PON1 promoter activity. d-Glucose also enhanced both messenger RNA and protein expression of PON1. Increased PON1 expression was also detected in primary human hepatocytes treated with high d-glucose concentrations. Bisindolylmaleimide, a PKC inhibitor, significantly inhibited d-glucose–induced transactivation of PON1; and mithramycin, an inhibitor of Sp1, completely abrogated the transactivation. Our data suggest that high glucose concentrations transactivate the PON1 gene through Sp1 activation by PKC in cultured hepatocytes. Up-regulated hepatic PON1 expression under high glucose conditions may be a compensatory mechanism in diabetes in which antioxidant capacity, including PON1 enzymatic activity, is attenuated.

STAT6 Inhibits TGF-β1-mediated Foxp3 Induction through Direct Binding to the Foxp3 Promoter, Which Is Reverted by Retinoic Acid Receptor

It has been shown that transforming growth factor beta1 (TGF-beta1) is critical in the generation of CD4(+)CD25(+)Foxp3(+)-inducible regulatory T cells (iTregs) from naïve CD4(+)T cells. However, in contrast to natural Tregs, TGF-beta1-induced iTregs rapidly lose both Foxp3 expression and suppression activity. We found that TGF-beta1-induced Foxp3 levels were maintained by the addition of the anti-interleukin 4 (IL-4) antibody or by STAT6 gene deletion. Thus, IL-4 is an important suppressor of Foxp3 induction, and T helper 2 development is a major cause for the disappearance of iTreg during long culture. Using promoter analysis in EL4 cells and primary T cells, we identified a silencer region containing a STAT6 binding site. STAT6 binding to this site reduced TGF-beta1-mediated Foxp3 promoter activation and chromatin modification. Retinoic acid has also been shown to suppress loss of Foxp3 induced by TGF-beta1. Retinoic acid in the presence of TGF-beta1 reduced STAT6 binding to the Foxp3 promoter and enhanced histone acetylation, thereby reverting the effect of IL-4. We propose that antagonistic agents for neutralizing IL-4 could be a novel strategy to facilitate inducible Treg cell generation and the promotion of tolerance in Th2-dominated diseases such as allergy.

Effects of microbubbles on ultrasound-mediated gene transfer in human prostate cancer PC3 cells: Comparison among Levovist, YM454, and MRX-815H

We compared the enhancement effects of three different echo contrast agents (ECAs); Levovist, YM454, and MRX-815H as artificial microbubbles on ultrasound mediated gene transfection (USMGT) with 1 MHz ultrasound at 0.2 MPa using a luciferase expression vector in PC3 cells and elucidated the mechanisms of differences of USMGT facilitation by these ECAs. At a concentration of each ECA that induced iso-survival, ECAs with lipid shell (YM454 and MRX-815H) facilitated USMGT higher than those without shell (Levovist), and the order of the ECAs facilitating free radical formation by sonication was; YM454 > MRX-815H > Levovist. These results suggested that the lipid shell type ECAs facilitated gene transfer higher than that by the non-shell type ECA.

Androgen Receptor Is Causally Involved in the Homeostasis of the Human Prostate Endothelial Cell

Androgen deprivation causes a reduction of blood flow in the prostate gland that precedes temporally apoptosis of the epithelium. The acute response of prostate endothelial cells to androgen deprivation suggested they represent a primary target for androgen. However, rat prostate endothelial cells were reported not to express androgen receptor (AR), and the role of the androgen axis in human prostate endothelial cell (HPEC) homeostasis was poorly characterized. In this study AR expression was detected in HPEC in vivo in clinical specimens of benign prostate and prostate cancer, and AR function as a transcription factor was demonstrated in HPEC in primary xenografts of human benign prostate tissue transplanted into severe combined immunodeficient mice by iv administration of adenoviral mouse mammary tumor virus-driven luciferase expression vector. AR expression and functionality were maintained in vitro in primary cultures of HPEC that coexpressed CD31, CD34, von Willebrand factor, intercellular adhesion molecule, vascular endothelial growth factor receptor 1, and vascular endothelial growth factor receptor 2 but did not express prostate-specific antigen. AR expression in primary cultures of HPEC isolated from surgical specimens of benign prostate was validated using RT-PCR, cDNA sequencing, immunocytochemistry, and Western blot analyses. Scatchard analyses demonstrated a single ligand-binding site for R1881 in primary cultures of HPEC, with dissociation constant of 0.25 nm, and AR-mediated transcriptional activity was demonstrated using adenoviral mouse mammary tumor virus-driven luciferase reporters. Dihydrotestosterone increased proliferation in primary cultures of HPEC in a dose-dependent manner without modulating endothelial tube formation in Matrigel (BD Biosciences, Bedford, MA). Therefore, HPECs express functional AR, and androgen plays a direct role in modulating HPEC biology.

S-Phase-specific Activation of PKCα Induces Senescence in Non-small Cell Lung Cancer Cells

Protein kinase C (PKC) has been widely implicated in positive and negative control of cell proliferation. We have recently shown that treatment of non-small cell lung cancer (NSCLC) cells with phorbol 12-myristate 13-acetate (PMA) during G1 phase inhibits the progression into S phase, an effect mediated by PKC delta-induced up-regulation of the cell cycle inhibitor p21 Cip1. However, PMA treatment in asynchronously growing NSCLC cells leads to accumulation of cells in G2/M. Studies in post-G1 phases revealed that PMA induced an irreversible G2/M cell cycle arrest in NSCLC cells and conferred morphological and biochemical features of senescence, including elevated SA-beta-Gal activity and reduced telomerase activity. Remarkably, this effect was phase-specific, as it occurred only when PKC was activated in S, but not in G1, phase. Mechanistic analysis revealed a crucial role for the classical PKC alpha isozyme as mediator of the G2/M arrest and senescence, as well as for inducing p21(Cip1) an obligatory event for conferring the senescence phenotype. In addition to the unappreciated role of PKC isozymes, and specifically PKC alpha, in senescence, our data introduce the paradigm that discrete PKCs trigger distinctive responses when activated in different phases of the cell cycle via a common mechanism that involves p21 Cip1 up-regulation.

A high-throughput reporter gene assay to prove the ability of natural compounds to modulate glutathione peroxidase, superoxide dismutase and catalase gene promoters in V79 cells

The aim of the study was to establish a 96-well microtiter plate-based reporter gene assay to test the influence of natural compounds on the promoter activities of rat catalase, human glutathione peroxidase and human superoxide dismutase expressed in V79 cells. Luciferase expression vectors with the promoter regions of the genes coding for the three above-mentioned enzymes were constructed and transfected into V79 cells. Thereafter the ability of sodium ascorbate, L-carnitine, catechin, epigallocatechin gallate, genistein, paraquat, quercetin, 12-O-tetradecanoylphorbol-13-acetate and Trolox to enhance the promoter activities was evaluated. Genistein, paraquat and quercetin led to a statistically significant increase in the glutathione peroxidase and superoxide dismutase gene promoter activities. None of the compounds tested enhanced the catalase gene promoter activity. The reporter gene assay described in this report is easy to perform, fast and allows one to test a high number of compounds and different concentrations of a single compound at the same time.

FIBER MODIFICATION AUGMENTS ANTITUMOR EFFECT OF CONDITIONALLY REPLICATIVE ADENOVIRUS UPON SYSTEMIC ADMINISTRATION IN ORTHOTOPIC PANCREATIC CANCER MODEL

Background: Pancreatic cancer, which is most often unresectable upon diagnosis, demands the development of novel therapeutic modalities for systemic usage. While conditionally replicative adenovirus (CRAd) is promising candidate, poor expression of adenoviral primary receptor impedes getting requisite tumor transduction in this tumor context. We have reported that adenoviral fiber modifications (RGD modification and Ad5/3 modification) enhance the CRAd efficiency upon intratumoral injection. In this study, we analyzed the fiber modified CRAds upon systemic vector administration in orthotopic pancreatic cancer xenograft model. Method: An Hs766T subcutaneous xenograft was established in a nude mouse first. Then, the small pieces (1mm3) made from this parental tumor were transplanted into pancreatic bed of other nude mice to establish the orthotopic tumor. Luciferase expression vectors with fiber modifications were used for comparing tumor transduction efficiency. The cyclooxygenase (COX)-2 promoter-based CRAd and its fiber modified versions were used for analyzing CRAd anti-tumor effect. The viruses were injected from tail vein 3 days after tumor transplantation. Mice are sacrificed 2 days and 28 days after vector administration for evaluating tumor transduction and the anti-tumor effect. Results: As for in vivo tumor transduction, Ad5/3 modified vector showed significantly (8 times) higher transduction compared to the background. This tumor transduction enhancement was also observed in pancreatic cancer subcutaneous xenografts upon systemic vector administration. When CRAds were injected systemically to assess the therapeutic effect in the mice with orthotopic tumors, the volume of the tumor treated with 5/3 CRAd COX-2 was significantly lower than that with non-replicative control virus, and comparable to non-selectively replicating wild type Ad. In the harvested tumors, the viral copy number of the 5/3 COX-2 CRAd was significantly high, even in comparison with non-selective wild type Ad. Conclusion: Ad5/3 modification enhanced the therapeutic effect of systemically administered CRAd. This suggests the benefits of adenoviral tropism modification as a way to achieve therapeutic effect in clinically relevant settings.

Vascular Endothelial Growth Factor Trap Blocks Tumor Growth, Metastasis Formation, and Vascular Leakage in an Orthotopic Murine Renal Cell Cancer Model

Angiogenesis inhibitors have shown clinical benefit in patients with advanced renal cell cancer, but further therapeutic improvement is needed. Vascular endothelial growth factor (VEGF) Trap is a newly developed VEGF-blocking agent with stronger affinity and broader activity than the anti-VEGF antibody bevacizumab. In this study, we tested the activity of VEGF Trap in an orthotopic murine model of renal cancer with spontaneous lung metastases. Murine syngeneic renal cell carcinoma cells (RENCA) transfected with a luciferase-expressing vector were injected into the renal capsule of BALB/c mice. I.p. treatment with VEGF Trap or control protein (10 or 25 mg/kg twice weekly) was started shortly after tumor injection to prevent tumor development (prevention model) or after established tumors were formed to inhibit tumor growth and metastasis formation (intervention model). In the prevention model, VEGF Trap inhibited tumor growth by 87 +/- 14% compared with control (P=0.007) and significantly prolonged survival. In the intervention model, VEGF Trap inhibited tumor growth by 74 +/- 9% (P<0.001) and the formation of lung metastases was inhibited by 98% (P<0.004). Microvascular density was reduced by 66% due to VEGF Trap treatment (P<0.001). In addition, VEGF Trap prevented fibrinogen leakage into the tumor microenvironment representative for reduced vascular leaking as shown by immunohistochemical staining. VEGF Trap is a potent inhibitor of RENCA tumor growth and metastasis formation and blocks the biological function of VEGF in vivo. These results support further clinical development of VEGF Trap for renal cell cancer and other cancer types.

Activation of the c-fos promoter is modulated by PKA in PC12 cells

(A) Schematic representation of the human c-fos-promoter-driven firefly luciferase (GL3) expression vector. The c-fos gene construct contains 360 base pairs of c-fos upstream regulatory sequence, two exons containing the coding region and first intron. The start of transcription is indicated by an arrow. GL3 gene is expressed as a fusion protein with N-terminal (63 a.a.) of the c-fos gene product.

Lack of Noggin Expression by Cancer Cells Is a Determinant of the Osteoblast Response in Bone Metastases

Prostate and mammary cancer bone metastases can be osteoblastic or osteolytic, but the mechanisms determining these features are unclear. Bone morphogenetic and Wnt proteins are osteoinductive molecules. Their activity is modulated by antagonists such as noggin and dickkopf-1. Differential expression analysis of bone morphogenetic and Wnt protein antagonists in human prostate and mammary cancer cell lines showed that osteolytic cell lines constitutively express in vitro noggin and dickkopf-1 and at least one of the osteolytic cytokines parathyroid hormone-related protein, colony-stimulating factor-1, and interleukin-8. In contrast, osteoinductive cell lines express neither noggin nor dickkopf-1 nor osteolytic cytokines in vitro. The noggin differential expression profile observed in vitro was confirmed in vivo in prostate cancer cell lines xenografted into bone and in clinical samples of bone metastasis. Forced noggin expression in an osteoinductive prostate cancer cell line abolished the osteoblast response induced in vivo by its intraosseous xenografts. Basal bone resorption and tumor growth kinetics were marginally affected. Lack of noggin and possibly dickkopf-1 expression by cancer cells may be a relevant mechanism contributing to the osteoblast response in bone metastases. Concomitant lack of osteolytic cytokines may be permissive of this effect. Noggin is a candidate drug for the adjuvant therapy of bone metastasis.

Association between the 120‐bp duplication of the dopamine D4 receptor gene and attention deficit hyperactivity disorder: Genetic and molecular analyses

Abnormalities of the dopamine neurotransmission have been hypothesized to play an important role in the pathophysiology of attention deficit hyperactivity disorder (ADHD). Promoter variants of the dopamine D4 receptor gene (DRD4) have attracted particular interest due to their possible role in regulation of gene transcription. Here we describe the haplotype analysis of the 120 base pair duplication (120-bp dup) and three SNPs (-616C/G, -615A/G, -521C/T) in the 5' region of the DRD4 gene among children with ADHD. We observed a trend (chi(2) = 14.905, df = 9, P = 0.093) in the four-locus haplotype distribution between ADHD probands (N = 173) and controls (N = 284). The homozygote genotype of the 1-repeat form of the 120-bp dup (1-1) had a significantly higher frequency among ADHD children than in controls (8.1% vs. 3.2%, chi(2) = 5.526, df = 1, P = 0.019, Odds Ratio = 2.71). In addition, a novel, 4-repeat allele was identified among ADHD patients. This particular allele has been cloned to the luciferase expression vector and its transcriptional activity has been compared to the 1- and 2-repeat allele. The number of repeats of the 120-bp dup was found to have an effect on transcriptional activity in both neuroblastoma and retinoblastoma cell lines in a dose-dependent manner (1-repeat > 2-repeat > 4-repeat). These results suggest that the 1-repeat form of the 120-bp dup might be a risk factor of ADHD, especially in the homozygous form and/or in the context of certain haplotypes.

Molecular mechanisms involved in interleukin‐8 production by normal human oesophageal epithelial cells

SummaryBackgroundIncrease in interleukin‐8 in the oesophageal mucosa has been associated with the pathogenesis of reflux oesophagitis.AimTo assess the effect of bile acids on the interleukin‐8 expression in normal human oesophageal epithelial cells and to determine its molecular mechanisms.MethodsHuman oesophageal epithelial cells were stimulated with unconjugated bile acids, conjugated bile acids and inflammatory cytokines. Protein and mRNA of interleukin‐8 were measured by enzyme‐linked immunosorbent assay and quantitative real‐time polymerase chain reaction, respectively. In addition, we examined protein kinases and transcription factors involved in interleukin‐8 synthetic pathways using protein kinase inhibitors and luciferase expression vectors, respectively.ResultsUnconjugated bile acids induced interleukin‐8 production from human oesophageal epithelial cells stronger than conjugated bile acids. However, conjugated bile acids in acidic media resulted in remarkable increase of interleukin‐8 production compared with those in neutral‐pH media. Mutation of the binding site of NF‐kB, AP‐1 and NF‐IL6 abrogated the induction of luciferase activities by 100%, 70% and 30%, respectively. Inhibitor of protein kinase A, protein kinase C or p38 mitogen‐activated protein kinase attenuated the production of interleukin‐8 by cholic acid.ConclusionsThese results indicate that bile acids induce interleukin‐8 expression from oesophageal epithelial cells mainly via the activation of NF‐kB as well as AP‐1.

Molecular mechanisms involved in interleukin‐8 production by normal human oesophageal epithelial cells

SummaryBackgroundIncrease in interleukin‐8 in the oesophageal mucosa has been associated with the pathogenesis of reflux oesophagitis.AimTo assess the effect of bile acids on the interleukin‐8 expression in normal human oesophageal epithelial cells and to determine its molecular mechanisms.MethodsHuman oesophageal epithelial cells were stimulated with unconjugated bile acids, conjugated bile acids and inflammatory cytokines. Protein and mRNA of interleukin‐8 were measured by enzyme‐linked immunosorbent assay and quantitative real‐time polymerase chain reaction, respectively. In addition, we examined protein kinases and transcription factors involved in interleukin‐8 synthetic pathways using protein kinase inhibitors and luciferase expression vectors, respectively.ResultsUnconjugated bile acids induced interleukin‐8 production from human oesophageal epithelial cells stronger than conjugated bile acids. However, conjugated bile acids in acidic media resulted in remarkable increase of interleukin‐8 production compared with those in neutral‐pH media. Mutation of the binding site of NF‐kB, AP‐1 and NF‐IL6 abrogated the induction of luciferase activities by 100%, 70% and 30%, respectively. Inhibitor of protein kinase A, protein kinase C or p38 mitogen‐activated protein kinase attenuated the production of interleukin‐8 by cholic acid.ConclusionsThese results indicate that bile acids induce interleukin‐8 expression from oesophageal epithelial cells mainly via the activation of NF‐kB as well as AP‐1.

CREB-AP1 Protein Complexes Regulate Transcription of the Collagen XXIV Gene (Col24a1) in Osteoblasts

Collagen XXIV is a newly discovered and poorly characterized member of the fibril-forming family of collagen molecules, which displays unique structural features of invertebrate fibrillar collagens and is expressed predominantly in bone tissue. Here we report the characterization of the proximal promoter of the mouse gene (Col24a1) and its regulation in osteoblastic cells. Using well characterized murine models of osteoblast differentiation, we found that the Col24a1 gene is activated sometime before onset of the late differentiation marker osteocalcin. Additional analyses revealed that Col24a1 produces equal amounts of two alternatively spliced products with different 5'-untranslated sequences that originate from distinct transcriptional start sites. Cell transfection experiments in combination with DNA binding assays demonstrated that Col24a1 promoter activity in ROS17/2.8 osteosarcoma cells is under the control of an upstream cis-acting element, which is shared by both transcripts and is recognized by specific combinations of c-Jun, CREB1, ATF1, and ATF2 dimers. Consistent with these results, overexpression of c-Jun, ATF1, ATF2, or CREB1 in transiently transfected osteoblastic cells stimulated transcription from reporter gene constructs driven by the Col24a1 promoter to different degrees. Moreover, chromatin immunoprecipitation experiments showed that these nuclear factors bind the same upstream sequence of the endogenous Col24a1 gene. Collectively these data provide new information about transcriptional control of collagen fibrillogenesis, in addition to implicating for the first time CREB-AP1 protein complexes in the regulation of collagen gene expression in osteoblasts.

The miscarriage-associated HLA-G –725G allele influences transcription rates in JEG-3 cells

HLA-G is a non-classical HLA with important immunomodulatory roles in pregnancy. A polymorphism in the promoter region, -725G, was previously associated with sporadic miscarriage in women who were unselected with respect to reproductive history. In this study, the transcription levels of different HLA-G promoter haplotypes were examined to determine whether the miscarriage-associated -725G allele influences transcription. Five naturally occurring promoter haplotypes and three variant haplotypes created by site-directed mutagenesis were sub-cloned into luciferase expression vectors and transfected into JEG-3 cells. Expression levels of these eight haplotypes were examined in cultured cells before and after treatment with interferon-beta (IFN-beta), cytosine-5-DNA methyltransferase (M. SssI) and 5-aza-2'-deoxycytidine. Differences in expression levels between haplotypes were determined by analysis of variance (ANOVA). Promoter haplotypes with the miscarriage-associated -725G allele were expressed at significantly higher levels in all culture conditions compared with otherwise identical haplotypes that had a -725C or -725T allele. Variation in the HLA-G promoter region influences transcription rates. Contrary to expectations, increased expression of HLA-G may be disadvantageous in some pregnancies.

Sustained Activation of Phosphatidylinositol 3-Kinase/Akt/Nuclear Factor κB Signaling Mediates G Protein-coupled δ-Opioid Receptor Gene Expression

Expression of the delta-opioid receptor gene (dor) is tightly controlled during neuronal differentiation and developmental stages. Such distinct temporal and spatial expression of dor during development suggests a role for the delta-opioid receptor in early developmental events. However, little is known about intracellular signaling pathways that control dor expression. A well established cell line model for the study of gene expression during neuronal differentiation is the rat adrenal pheochromocytoma PC12 cell line. Here we found that the constitutively activated TrkA/phosphatidylinositol 3-kinase/Akt (protein kinase B)/NF-kappaB survival cascade mediates dor expression during nerve growth factor (NGF)-induced differentiation of PC12h cells. Biochemical experiments showed that constitutive phosphorylation of Akt and IkappaBalpha correlates with NGF-induced dor expression. Overexpression of the transcriptional activator NF-kappaB/p65 increased dor promoter activity. Overexpression of the NF-kappaB signaling super inhibitor mutant IkappaBalpha (S32A/S36A) abolished the effect of p65 and blocked NGF-induced activation of NF-kappaB signaling, resulting in a significant reduction in dor promoter activity. Treatment with SN50, an NF-kappaB-specific nuclear translocation peptide inhibitor, inhibited the translocation of NF-kappaB, resulting in a reduction of dor mRNA. The gel shift assay supported the fact that there exists an NF-kappaB-binding site on the dor promoter. RNA interference experiments using NF-kappaB/p65 small interfering RNA confirmed that NF-kappaB signaling is required for dor expression. Our findings not only provide a new mechanistic explanation for NGF-induced dor expression but also shed some light on the molecular mechanism of the temporal and spatial expression of dor and the roles of the delta-opioid receptor during neuronal differentiation.

Firefly luciferase terminally degraded by mild heat exposure: Implications for reporter assays

Luciferase reporter constructs are an accurate method of assessing gene promoter activity and vectors constitutively expressing luciferase are useful in quantifying transfection efficiency. Common methodologies for examining the induction of the heat shock (stress) response require exposure of cells transfected with luciferase-expressing vectors to a mild heat stress. Here we re-examine the under-recognised phenomenon that luciferase is exquisitely sensitive to small temperature changes. In cells subjected to mild heat exposure following transfection with both luciferase and β-galactosidase reporter vectors, a marked reduction in luciferase activity was observed compared with β-galactosidase activity. On exposing recombinant firefly luciferase to small increases in temperature in vitro, a time and temperature dependent decrease in luciferase activity was demonstrated. Loss of luciferase activity following mild heat exposure will result in misinterpretation of reporter activity. This vastly underappreciated effect is worthy of further emphasis and luciferase reporter vectors should be used with caution in protocols that involve exposure to temperatures outside the physiological range.

284 IDENTIFICATION AND CHARACTERIZATION OF THE 5′-FLANKING REGION OF THREE MOUSE MATERNAL GENES (HISTONE H1OO, NUCLEOPLASMIN 2, AND ZYGOTE ARREST 1): TRANSCRIPTIONAL ACTIVITY IN MOUSE OOCYTES

Maternal transcripts that accumulate during oocyte growth are involved in the meiotic maturation, the initiation of the first mitosis, and the later pre-implantation development. Although the conserved E-box sequences in promoter region of some maternal genes (for example, Zp3 and Gdf-9), which are important in regulating gene transcripts as binding sites of transcriptional factors, may play a role in the oocyte-specific expression in ovary, the molecular mechanism that regulates the expression of the maternal genes is still not known. In this study, we have focused on the transcriptional activity of promoter regions to clarify the molecular mechanism of transcriptional regulation of these maternal genes [Histone H1oo (H1oo), Nucleoplasmin 2 (Npm2), and Zygote arrest 1 (Zar1)]. First, we observed the expression of firefly luciferase expression vectors under promoter regions of 3 maternal genes in oocytes isolated from 10- to 12-day-old mice, which is mainly NSN-type, transcriptionally active form (Bouniol-Baly et al. 1999 Biol. Reprod. 60, 580–587). Transcriptional activities of H1oo (-3975), Npm2 (-2610), and Zar1 (-5187) promoters were detected in oocytes, the relative luciferase activities being an average of 70, 130, and 12, respectively. On the other hand, these promoter activities were not detected in embryos at the 2-cell stage. Furthermore, deletion analysis of promoter elements (E-boxes) of H1oo and Npm2 was done by microinjecting deletion constructs into oocytes. In the H1oo promoter, deletion of sequences between -3975 and -72 bp from the transcription start site resulted in one-third of the level obtained in the full H1oo (-3975) promoter. In addition, deletion of sequence -68 bp resulted in no detection of luciferase activity. These findings indicate that the putative distal promoter sequences exist at the 52-flanking region (-3975 to -759) of the H1oo gene and that the region (-314 to -68) including the E-box region (-72) may be required for high-level transcriptional activity of the H1oo promoter. In the Npm2 promoter, deletion of sequences between -2610 and -180 bp from the transcription start site resulted in one-third of the level of wild-type activity of the Npm2 (-2610) promoter. In addition, deletion of sequence -101 bp resulted in no detection of luciferase activity. These findings also indicate that 3 putative distal promoter sequences exist at the 52-flanking region (-2610 to -210) of the Npm2 gene and that the region (-210 to -101) that includes the E-box region (-180) is crucial for high-level transcriptional activity of the Npm2 promoter. In conclusion, the E-box may be a key regulatory region for the expression of two of the maternal genes (H1oo and Npm2) examined. Currently, we are attempting to identify the transcriptional factor binding sites by DNase I footprint analysis and gel mobility shift assay.