The transcription factor Ap-1 regulates monkey 20α-hydroxysteroid dehydrogenase promoter activity in CHO cells

Abstract

BackgroundMonkey 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.ResultsA 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.ConclusionsOur 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.

Pro‐inflammatory signals induce 20α‐HSD expression in myometrial cells: A key mechanism for local progesterone withdrawal

“…41 The AP-1 family of TFs were previously implicated in regulation of 20α-HSD gene promoter activity in the monkey. 42 In this study, we determined that the 5′ flanking region of AKR1C1 (−860 to +43) is responsive…”

“…41 The AP-1 family of TFs were previously implicated in regulation of 20α-HSD gene promoter activity in the monkey. 42 In this study, we determined that the 5′ flanking region of AKR1C1 (−860 to +43) is responsive to LPS and TPA. Moreover, blocking NF-кB or AP-1 activity, using specific inhibitors, attenuated AKR1C1 promoter activity suggesting that both TFs regulate human AKR1C1 transcription.…”

Identification and Functional Analysis of Cynoglossus semilaevis Z-Linked E3 Ubiquitin Ligase rnf34

“…In accordance, apoptosis signals were stronger in pseudomales than in males. Moreover, the rnf34 promoter clone has an obvious activity, and it is expected that there are multiple transcription factor binding sites related to apoptosis, such as JUN and SP1 [32,33]. Whether W spermatocyte/spermatozoa undergo apoptosis needs further study.…”

Data on the localization of EGFP and 20α-hydroxysteroid dehydrogenase (20α-HSD) in the placenta and testes of transgenic mice

“…The PCR conditions were: 1 cycle of pre-denaturation at 95 °C for 5 min; 30 cycles of denaturation at 94 °C for 40 s, annealing at 64 °C for 40 s, extension at 72 °C for 50 s; 1 final cycle of extension at 72 °C for 10 min; and cooling to 4 °C. Primers for the glyceraldehyde 3-phosphate gene ( GAPDH ) were used for normalization of EGFP expression, and the primer sequences were forward primer (5′-ACC ACA GTC CAT GCC ATC AC-3′); and reverse primer (5′-TCC ACC ACC CTG TTG CTG TA-3′) [2] . The PCR product (10 uL) was analyzed using gel electrophoresis, and the expected size was 400 bp.…”