The CCAAT-box binding transcription factor, nuclear factor-Y (NF-Y) regulates transcription of human aldo-keto reductase 1C1 (AKR1C1) gene

Abstract

Dihydrodiol dehydrogenases are a family of aldo-keto reductases (AKR1Cs) involved in the metabolism of steroid hormones and xenobiotics. Whilst, several phase II drugs as well as endogenous & exogenous steroids/steroid metabolites have been identified as inducers of gene transcription, the cellular transcription factors controlling the expression of AKR1C1 are incompletely elucidated. Herein, we have cloned and characterized the proximal promoter region of the human AKR1C1 gene that controls its transcription. The 5’ flanking proximal promoter region of the AKR1C1 gene consists of a TATA box and an inverted CCAAT binding site. Deletion analysis of the 5’-flanking, ~3.0 kb region of the human AKR1C1 gene identified the region between -128 to -88 as the minimal proximal promoter essential for basal transcription of AKR1C1 in human ovarian (2008 & 2008/C13*), lung (H23 & A549) and liver carcinoma (HepG2) cells. Antioxidant response elements (ARE) have been shown to modulate the transcription ofv genes coding for phase II drug metabolizing enzymes. Cloning of the ARE upstream of the AKR1C1 proximal promoter resulted in increased transcription in human lung adenocarcinoma and liver hepatoblastoma cells but not in human ovarian carcinoma cells. Further, ARE increased the induction of the AKR1C1 gene in response to treatment with phase II drug inducers. However, ARE did not induce the transcription of AKR1C1 gene promoter in the presence of cisplatin in any of the cell lines. A computational analysis utilizing the Alibaba 2.0 on the proximal AKR1C1 gene promoter region was performed to identify potential transcription factor binding sites. Based on this analysis, a set of potential, putative transcription factor binding sites for Oct1, Sp1, Cp-1/NF-Y, CEBP, p40X, USF, NF1 and AP-2 were identified in the region -180 to -88 of the AKR1C1 gene promoter. Site-directed mutagenesis studies indicated that the transcription factor binding sites for NF-Y/CEBP were involved in controlling the basal transcription of AKR1C1 in all the cancer cells studied. Electrophoretic mobility shift (EMSAs) and gel supershift assays demonstrated that the transcription factor NF-Y preferentially binds to the inverted CCAAT box at -109ATTGG-105 of the AKR1C1 gene. Chromatin immunoprecipitation (ChIP) analysis confirmed the in vivo association between NF-Y and human AKR1C1 gene promoter in human ovarian, lung and liver carcinoma cells. Further, ectopic expression of NF-Y’s increased the AKR1C1 gene transcription, whereas expression of a dominant-negative NF-YA or knockdown of NF-YA by siRNA transfection, decreased the AKR1C1 gene transcription. A 2-fold increase in AKR1C1 transcription was observed specifically in cisplatin-treated 2008 cells that was CCAAT box-dependent. These results indicate that NF-Y regulates basal transcription of AKR1C1 in human ovarian, lung and liver carcinoma cells and cisplatin-induced transcription in human ovarian carcinoma cells.