title: The transcription factor Ap-1 regulates monkey 20α-hydroxysteroid dehydrogenase promoter activity in CHO cells.
abstract: BACKGROUND
  Monkey 20α-hydroxysteroid dehydrogenase (20α-HSD) is a catabolic enzyme responsible for converting progesterone into biologically inactive 20α-hydroxyprogesterone, thereby playing a key role in the estrous cycle or pregnancy and allowing ovulation and parturition to occur in most mammalian animals. Monkey 20α-HSD was highly abundant in ovarian and placental tissues during the pre-ovulation and pre-parturition phase and was primarily localized in the syncytiotrophoblast of the placenta. In this study, we focused on the molecular characterization of the monkey 20α-HSD promoter region by conducting reporter assays in Chinese hamster ovary (CHO) K1 cells.
  
  
  RESULTS
  A reporter assay using constructs of various lengths of the 5'-flanking region (-890-Luc, -513-Luc, -306-Luc, -273-Luc, and -70-Luc) revealed that a region corresponding to the activator protein 1 (Ap-1) located between -281 and -274 bp was essential for the transcriptional activity. Absence of the Ap-1 site in -273-Luc dramatically decreased the transcription levels to the control levels. When the reporter constructs were co-transfected with Ap-1 (Jun) and specificity protein (Sp-1) genes, the transcription activities of the constructs increased with the exception of -273 and -70, while that of the double construct was reduced compared to that of Ap-1 alone. Furthermore, mutational analysis demonstrated that a putative Ap-1 site played an important role in the expression of the reporter gene. These findings were confirmed by EMSA examining the interactions of the protein Ap-1 in a nuclear extract from CHO-K1 cells and the expression levels of the Ap-1 transcription factor in pre-parturition placenta and CHO-K1 cells. Although mut-1 and mut-2 of Ap-1 bound with nuclear extracts from CHO-K1 cells, the transcriptional activity of mut-3 was almost completely suppressed.
  
  
  CONCLUSIONS
  Our results indicate that the Ap-1 site (-281 → -274) (5'-TGTCTCAT-3') plays a crucial role in the activation of the monkey 20α-HSD gene. Thus, we demonstrated that monkey 20α-HSD promoter activity is regulated by the transcription factor Ap-1 in CHO-K1 cells.
authors: Tsevelmaa
authors: T
authors: Nanjidsuren
authors: Animal Biotechnology, Graduate School of Bio and Information Technology, Institute of Genetic Engineering, Hankyong National University, Ansung 456-749, Republic of Korea. ksmin@hknu.ac.kr.
authors: Kwan-Sik
authors: KS
authors: Min
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D017930:Genes, Reporter; D006913:Hydroxysteroid Dehydrogenases / Q000235:genetics*; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism*
chemical_list: D020121:5' Untranslated Regions; D016329:Sp1 Transcription Factor; D018808:Transcription Factor AP-1; D006913:Hydroxysteroid Dehydrogenases