Open Access
Zhang, Yao
Graduate Program:
Master of Science
Document Type:
Master Thesis
Date of Defense:
November 13, 2008
Committee Members:
  • A C Ross, Thesis Advisor
  • A Catharine Ross, Thesis Advisor
  • RARE
  • CYP26A1
  • all-trans retinoic acid
  • promoter
All-trans retinoic acid (ATRA) is considered as the major active metabolite of vitamin A. ATRA regulates many physiological processes, such as embryonic development, cellular proliferation and differentiation, testicular function, and immune function. Clinically, retinoids have been demonstrated to be useful for cancer treatment and chemoprevention. Oral ATRA have been effective in the treatment of the patients with acute promyelocytic leukemia (APL). ATRA concentration in tissues is tightly controlled in a spatial and temporal manner, accomplished by a balance of synthesizing and metabolizing enzymes. Several retinaldehyde dehydrogenases have been characterized for the irreversible conversion of retinaldehyde to ATRA. Metabolism of ATRA is mediated by cytochrome P450 activities, and is known to be induced by RA itself in many tissues. CYP26A1 (P450RA1) is a cytochrome P450 enzyme, and specifically catalyzes the conversion of ATRA to polar metabolites including 4-OH-RA, 4-oxo-RA, and 18-OH-RA. Moreover, CYP26A1 is remarkable for its strong inducibility by ATRA in vivo. In the liver of intact ATRA-treated rats, CYP26A1 mRNA increased nearly 2000-fold within 10 hours and then declined rapidly. Loudig et al. have previously reported on one retinoic acid response element (RARE, referred as RARE1), proximal to the transcription start site, which cooperated with guanine-guanine-rich element (GGRE) in the induction of CYP26A1 promoter activation in F9 and P19 (murine embryonal or teratocarcinoma cells). To better understand the mechanism of the strong regulation of CYP26A1 gene expression in the liver, we have measured endogenous levels of CYP26A1 mRNA in ATRA-treated human liver cells (HepG2 cells), and further characterized the CYP26A1 promoter region extending 2.2 kb upstream from the translational start site. Whereas HepG2 cells expressed very little endogenous CYP26A1 mRNA prior to retinoid treatment, they responded rapidly and dose-dependently to 1-1000 nM RA, and therefore closely resembled intact liver. Examination of the putative promoter of CYP26A1 extending 2.2 kb upstream from the translation state site revealed three additional potential regulatory elements: two 5-bp-spaced direct repeat (DR-5) RAREs (RARE2, RARE3) and a nearby half-site (RARE4). Sequence analysis shows that all three RAREs and the half site are highly conserved among mouse, rat and human. Based on the human CYP26A1 nucleotide sequence, we prepared a series of luciferase reporter constructs that contained either nucleotide deletions or mutations within RARE1, 2, 3, and the half site, RARE4, of the full-length construct. Relative luciferase activity for the full-length construct was dose-dependently regulated by RA in HepG2 cells, and correlated with the level of endogenously expressed CYP26A1 mRNA. Comparative studies revealed that RA increased CYP26A1 promoter activity significantly more in HepG2 cells as compared to HEK293T cells, and that all three of the RAREs and the half site synergistically regulated RA-mediated CYP26A1 expression in HepG2 cells. Cotransfection of each of the individual retinoic acid receptors (RARa, b, g) significantly increased relative luciferase activity. Interestingly, in electrophoretic mobility shift assays, similar patterns of nuclear factor complex binding to RARE2 and RARE3 and to the RARE4 half site sequence in conjunction with proximal DNA were observed. RAR was likely to play a major role in CYP26A1 expression in HepG2 cells. Together, these experiments demonstrate that additional RA-responsive elements upstream of the CYP26A1 coding sequence are both active and necessary for the strong inducibility of CYP26A1 expression in HepG2 liver cells.