Using the human CYP26A1 gene promoter as a suitable tool for the determination of RAR-mediated retinoid activity

Reza Zolfaghari, Floyd J. Mattie, Cheng Hsin Wei, David R. Chisholm, Andrew Whiting, A. Catharine Ross

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

We have used a shortened construct form of the CYP26A1 gene promoter, in a promoter-less vector with either luciferase (known as E4) or a red fluorescent protein, RFP (known as E4.2) as the reporter gene and examined their responses to retinoids in transfected HepG2 and HEK293T cells. The promoter responded linearly to a wide concentration range of at-RA in cells cotransfected with retinoic acid receptors (RAR). The promoter also responded quantitatively to retinol and various other retinoids. An isolated clonal line of HEK293T cells that was permanently transfected with the promoter driving the expression of RFP responded to both at-RA and retinol, and the responses could be measured by fluorescence microscopy and flow cytometry. The promoter was also used to assess the retinoid activity of 3 novel synthetic retinoid analogues. Among them, EC23 was shown to be more potent than at-RA at lower concentrations and also more stable than at-RA. The promoter was also used to estimate the retinoid activities of intact rat serum samples as well as extracts of rat liver and lung, using retinol and at-RA as the reference standards. The retinoid activities could be measured in control rat serum samples and were increased in the serum of at-RA-treated rats. The total retinol and at-RA levels in the rat liver and lung samples determined by this promoter-based assay were compared with total retinol levels determined by the UPLC as the conventional methods. This system should offer a biologically-based alternative to mass-based retinoid analysis.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
DOIs
StateAccepted/In press - Jan 1 2020

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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    Zolfaghari, R., Mattie, F. J., Wei, C. H., Chisholm, D. R., Whiting, A., & Ross, A. C. (Accepted/In press). Using the human CYP26A1 gene promoter as a suitable tool for the determination of RAR-mediated retinoid activity. In Methods in Enzymology (Methods in Enzymology). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2020.03.013