Inhibition of retinoic acid-inducible transcription by COUP-TFI in human salivary gland adenocarcinoma cell line HSG

1999 ◽  
Vol 77 (6) ◽  
pp. 515-526 ◽  
Author(s):  
Seiko Kyakumoto ◽  
Minoru Ota ◽  
Nobuko Sato
Keyword(s):  
Transcription Factor ◽  
Salivary Gland ◽  
Retinoic Acid ◽  
Reporter Gene ◽  
Luciferase Activity ◽  
Transcription Activation ◽  
Luciferase Reporter ◽  
Response Element ◽  
Human Salivary Gland ◽  
Retinoic Acid Response Element

Human salivary gland adenocarcinoma cells (HSG) express nuclear receptors, all-trans-retinoic acid (at-RA) receptors (RARs), and retinoid X/9-cis-retinoic acid (9-c-RA) receptors (RXRs). In order to investigate whether the endogenous RARs or RXRs of HSG cells can induce transcription activation, the thymidine kinase promoter (TK)-driven luciferase reporter gene containing the retinoic acid response element (RARE), of RARβ, βRARE2-TK-Luc, was transfected into HSG cells and ligand-dependent transcription activation was examined. Luciferase activity of cell lysate increased by the treatment with either at-RA or 9-c-RA. Co-transfection of RARα and (or) RXRα-expression plasmids with the reporter gene enhanced the luciferase activity, suggesting that endogenous RARs and RXRs work as ligand-dependent transfactors in HSG cells. Reverse transcriptase - polymerase chain reaction analysis revealed that HSG cells express chicken ovalbumin upstream promoter - transcription factor I (COUP-TFI). Co-transfection of COUP-TFI-expression plasmid suppressed the at-RA-induced transcription activation of the reporter gene. Similar results were shown using a chromatin-integrated reporter gene system, using a stably transfected β-RARE2-TK-β-galactosidase (β-Gal) reporter gene. The at-RA-dependent increase in the β-Gal expression was completely inhibited by COUP-TFI. The transfection of antisense oligonucleotide of COUP-TFI squelched the RA-dependent growth inhibition induced by RAR-RXR heterodimers. Conclusively, RARs and RXRs of HSG cells are functional and play roles as transactivators in at-RA-sensitive processes such as the proliferation or differentiation of cells. COUP-TFI very likely regulates these processes by repressing the functions of these transactivators.Key words: retinoic acid receptor, retinoid X receptor, COUP-transcription factor (COUP-TF), retinoic acid response element.

Expression of retinoid X receptors and COUP-TFI in a human salivary gland adenocarcinoma cell line

1997 ◽  
Vol 75 (6) ◽  
pp. 749-758 ◽  
Author(s):  
Seiko Kyakumoto ◽  
Takayuki Nemoto ◽  
Nobuko Sato ◽  
Minoru Ota
Keyword(s):  
Transcription Factor ◽  
Salivary Gland ◽  
Retinoic Acid ◽  
Cell Line ◽  
Nuclear Extract ◽  
Retinoid X Receptor ◽  
Adenocarcinoma Cell Line ◽  
Response Element ◽  
Adenocarcinoma Cell ◽  
Retinoic Acid Response Element

The growth of the adenocarcinoma cell line derived from human salivary gland (HSG) is regulated by all-trans-retinoic acid (t-RA), which binds to its specific receptor, retinoic acid receptors (RARs), located in the nucleus, and thereby transactivates target genes. In this study, we examined the binding characteristics of the nuclear extract of HSG cells to the retinoic acid response element (RARE) compared with those of in vitro translated RAR alpha and retinoid X receptor alpha (RXR alpha ), a heterodimeric partner of RAR alpha . Gel shift analysis using anti-RAR alpha and anti-RXR alpha antibodies revealed that the translated RAR alpha bound to RARE as a heterodimer with RXR alpha . In contrast, the binding of the nuclear extract of HSG cells to RARE showed a heterogenous pattern, suggesting the existence of several species of RXRs as well as RARs in the nuclei of HSG cells. We therefore tried to clone these putative RXRs by the polymerase chain reaction using degenerated oligonucleotide primers conserved across the RXR family. The DNA sequencing of the recombinant clones revealed the expression of RXR alpha and RXR beta . In addition, chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI), which is also an RXR family member, was cloned. To evaluate the transcriptional activity of RARs and RXRs endogenously expressed in HSG cells, we performed a transient transfection analysis. When HSG cells were transfected with a luciferase reporter plasmid containing two repeats of either the RARE of the RAR beta gene or that of cellular retinol-binding protein II gene, positioned upstream of a thymidine kinase promoter fused to the luciferase sequence, a 2-3-fold induction of luciferase activity was observed in both cases. These results suggest that RARs and RXRs endogenously expressed in HSG cells were transcriptionally active in vivo. Thus, our findings showed that RXR alpha , RXR beta , and COUP-TFI are expressed in HSG cells and suggest that these molecules function as heterodimeric partners of RARs and (or) competitive repressors for RAREs and are involved in cellular responses mediated by retinoids. Key words: retinoid X receptor, retinoic acid receptor, retinoic acid response element, COUP-transcription factor (COUP-TF).

scholarly journals Identification of a retinoic acid response element upstream of the murine Hox-4.2 gene.

1993 ◽  
Vol 13 (1) ◽  
pp. 257-265 ◽  
Author(s):  
H Pöpperl ◽  
M S Featherstone
Keyword(s):  
Retinoic Acid ◽  
Hox Genes ◽  
Regulatory Element ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Response Element ◽  
Retinoic Acid Response Element ◽  
Ec Cells

Hox genes play an important role in the process of vertebrate pattern formation, and their expression is intricately regulated both temporally and spatially. All-trans-retinoic acid (RA), a physiologically active metabolite of vitamin A, affects the expression of a large number of Hox genes in vitro and in vivo. However, the regulatory mechanisms underlying the RA response of these genes have not been extensively studied, and no response element for RA receptors (RARs) has been characterized in a Hox regulatory region. The expression of murine Hox-4.2 and its human homolog, HOX4B, is increased in embryonal carcinoma (EC) cell lines upon RA treatment (M. S. Featherstone, A. Baron, S. J. Gaunt, M.-G. Mattei, and D. Duboule, Proc. Natl. Acad. Sci. USA 85:4760-4764, 1988; A. Simeone, D. Acampora, V. Nigro, A. Faiella, M. D'Esposito, A. Stornaiuolo, F. Mavilio, and E. Boncinelli, Mech. Dev. 33:215-228, 1991). Using transient expression assays, we showed that luciferase reporter gene constructs carrying genomic sequences located upstream of Hox-4.2 responded to RA in murine P19 EC cells. A 402-bp NcoI fragment was necessary for the RA responsiveness of reporter constructs. This fragment contained a regulatory element, 5'-AGGTGA(N)5AGGTCA-3', that closely resembles the consensus sequence for an RA response element. The Hox-4.2 RA response element was critical for the RA induction and specifically bound RARs. In addition, the response to RA could be inhibited by expressing a dominant negative form of RAR alpha in transfected P19 EC cells. These results suggested that Hox-4.2 is a target for RAR-mediated regulation by RA.

Expression of the murine Hoxa4 gene requires both autoregulation and a conserved retinoic acid response element

Development ◽  
1998 ◽  
Vol 125 (11) ◽  
pp. 1991-1998 ◽  
Author(s):  
A.I. Packer ◽  
D.A. Crotty ◽  
V.A. Elwell ◽  
D.J. Wolgemuth
Keyword(s):  
Retinoic Acid ◽  
Reporter Gene ◽  
Neural Tube ◽  
Hox Genes ◽  
Regulatory Region ◽  
Ectopic Expression ◽  
Response Element ◽  
Retinoic Acid Response Element ◽  
Transgenic Embryos

Analysis of the regulatory regions of the Hox genes has revealed a complex array of positive and negative cis-acting elements that control the spatial and temporal pattern of expression of these genes during embryogenesis. In this study we show that normal expression of the murine Hoxa4 gene during development requires both autoregulatory and retinoic acid-dependent modes of regulation. When introduced into a Hoxa4 null background, expression of a lacZ reporter gene driven by the Hoxa4 regulatory region (Hoxa4/lacZ) is either abolished or significantly reduced in all tissues at E10. 5-E12.5. Thus, the observed autoregulation of the Drosophila Deformed gene is conserved in a mouse homolog in vivo, and is reflected in a widespread requirement for positive feedback to maintain Hoxa4 expression. We also identify three potential retinoic acid response elements in the Hoxa4 5′ flanking region, one of which is identical to a well-characterized element flanking the Hoxd4 gene. Administration of retinoic acid to Hoxa4/lacZ transgenic embryos resulted in stage-dependent ectopic expression of the reporter gene in the neural tube and hindbrain. When administered to Hoxa4 null embryos, however, persistent ectopic expression was not observed, suggesting that autoregulation is required for maintenance of the retinoic acid-induced expression. Finally, mutation of the consensus retinoic acid response element eliminated the response of the reporter gene to exogenous retinoic acid, and abolished all embryonic expression in untreated embryos, with the exception of the neural tube and prevertebrae. These data add to the evidence that Hox gene expression is regulated, in part, by endogenous retinoids and autoregulatory loops.

Identification of a retinoic acid response element upstream of the murine Hox-4.2 gene

1993 ◽  
Vol 13 (1) ◽  
pp. 257-265 ◽  
Author(s):  
H Pöpperl ◽  
M S Featherstone
Keyword(s):  
Retinoic Acid ◽  
Hox Genes ◽  
Regulatory Element ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Response Element ◽  
Retinoic Acid Response Element ◽  
Ec Cells

Hox genes play an important role in the process of vertebrate pattern formation, and their expression is intricately regulated both temporally and spatially. All-trans-retinoic acid (RA), a physiologically active metabolite of vitamin A, affects the expression of a large number of Hox genes in vitro and in vivo. However, the regulatory mechanisms underlying the RA response of these genes have not been extensively studied, and no response element for RA receptors (RARs) has been characterized in a Hox regulatory region. The expression of murine Hox-4.2 and its human homolog, HOX4B, is increased in embryonal carcinoma (EC) cell lines upon RA treatment (M. S. Featherstone, A. Baron, S. J. Gaunt, M.-G. Mattei, and D. Duboule, Proc. Natl. Acad. Sci. USA 85:4760-4764, 1988; A. Simeone, D. Acampora, V. Nigro, A. Faiella, M. D'Esposito, A. Stornaiuolo, F. Mavilio, and E. Boncinelli, Mech. Dev. 33:215-228, 1991). Using transient expression assays, we showed that luciferase reporter gene constructs carrying genomic sequences located upstream of Hox-4.2 responded to RA in murine P19 EC cells. A 402-bp NcoI fragment was necessary for the RA responsiveness of reporter constructs. This fragment contained a regulatory element, 5'-AGGTGA(N)5AGGTCA-3', that closely resembles the consensus sequence for an RA response element. The Hox-4.2 RA response element was critical for the RA induction and specifically bound RARs. In addition, the response to RA could be inhibited by expressing a dominant negative form of RAR alpha in transfected P19 EC cells. These results suggested that Hox-4.2 is a target for RAR-mediated regulation by RA.

scholarly journals In vivo analysis of the myosin heavy chain IIB promoter region

1998 ◽  
Vol 274 (3) ◽  
pp. C681-C687 ◽  
Author(s):  
Steven J. Swoap
Keyword(s):  
Reporter Gene ◽  
Luciferase Activity ◽  
Fiber Type ◽  
Firefly Luciferase ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Base Pairs ◽  
Specific Expression ◽  
Mhc Iib

The myosin heavy chain (MHC) IIB gene is preferentially expressed in fast-twitch muscles of the hindlimb, such as the tibialis anterior (TA). The molecular mechanism(s) for this preferential expression are unknown. The goals of the current study were 1) to determine whether the cloned region of the MHC IIB promoter contains the necessary cis-acting element(s) to drive fiber-type-specific expression of this gene in vivo, 2) to determine which region within the promoter is responsible for fiber-type-specific expression, and 3) to determine whether transcription off of the cloned region of the MHC IIB promoter accurately mimics endogenous gene expression in a muscle undergoing a fiber-type transition. To accomplish these goals, a 2.6-kilobase fragment of the promoter-enhancer region of the MHC IIB gene was cloned upstream of the firefly luciferase reporter gene and coinjected with pRL-cytomegalovirus (CMV) (CMV promoter driving the renilla luciferase reporter) into the TA and the slow soleus muscle. Firefly luciferase activity relative to renilla luciferase activity within the TA was 35-fold greater than within the soleus. Deletional analysis demonstrated that only the proximal 295 base pairs (pGL3IIB0.3) were required to maintain this muscle-fiber-type specificity. Reporter gene expression of pGL3IIB0.3 construct was significantly upregulated twofold in unweighted soleus muscles compared with normal soleus muscles. Thus the region within the proximal 295 base pairs of the MHC IIB gene contains at least one element that can drive fiber-type-specific expression of a reporter gene.

Cloning and Characterization of the Murine PKC α Promoter: Identification of a Retinoic Acid Response Element

1999 ◽  
Vol 263 (1) ◽  
pp. 28-34 ◽  
Author(s):  
Dinakar S. Desai ◽  
Syu-ichi Hirai ◽  
William E. Karnes ◽  
Richard M. Niles ◽  
Shi-geo Ohno
Keyword(s):  
Retinoic Acid ◽  
Response Element ◽  
Retinoic Acid Response Element
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