Metabolism and regulation of gene expression by 4‐oxoretinol versus all‐trans retinoic acid in normal human mammary epithelial cells

Abstract

We previously demonstrated that 4-oxoretinol (4-oxo-ROL) activated retinoic acid receptors (RARs) in F9 stem cells. We showed that 4-oxo-ROL inhibited the proliferation of normal human mammary epithelial cells (HMECs). To understand the mechanisms by which 4-oxo-ROL regulates HMEC growth we examined gene expression profiles following 4-oxo-ROL or all-trans retinoic acid (tRA). We also compared growth inhibition by tRA, 4-oxo-ROL, or 4-oxo-RA. All three retinoids inhibited HMEC proliferation. Gene expression analyses indicated that 4-oxo-ROL and tRA modulated gene expression in closely related pathways. The expression of many genes, e.g. ATP-binding cassette G1 (ABCG1); adrenergic receptorβ2 (ADRB2); ras-related C3 botulinum toxin substrate (RAC2); and short-chain dehydrogenase/reductase 1 gene (SDR1) was changed after 4-oxo-ROL or tRA. Metabolism of these retinoids was analyzed by high-performance liquid chromatography(HPLC). In 1 μM tRA treated HMECs all of the tRA was found intracellularly, and tRA was the predominant intracellular retinoid. In 1 μM 4-oxo-ROL treated HMECs most 4-oxo-ROL was esterified to 4-oxoretinyl esters, no tRA was detected, and 4-oxo-ROL and 4-oxo-RA were observed intracellularly. In 1 μM 4-oxoretinoic acid (4-oxo-RA) treated HMECs little intracellular 4-oxo-RA was detected; most 4-oxo-RA was in the medium. Our results indicate that: (a) 4-oxo-ROL regulates gene expression and inhibits proliferation of HMECs; (b) 4-oxo-ROL and tRA regulate some of the same genes; (c) more tRA is found in cells, as compared to 4-oxoretinoic acid, when each drug is added at the same concentration in the medium; and (d) the mechanism by which 4-oxo-ROL exerts its biological activity does not involve intracellular tRA production.

Vitamin A Deficiency and the Lung

“…Moreover, it has become increasingly evident that, in addition to RA, others vitamin A derivatives, among them oxoretinoids, are also biological active vitamin A compounds. Interestingly, for example, all- trans -4-oxoretinol, first considered to be an inactive metabolite of RA, has been identified as a physiologically retinoid signalling molecule that exerts an important biological activity and regulates some of the same genes as RA, independently of its intracellular transformation into RA [ 67 , 68 ]. Furthermore, nuclear RARs are targets for S -4-oxo-9- cis -13,14-dihydro-RA that activates the transcription of RARE-containing genes in several cell types both in vitro and in vivo.…”

Retinoids regulate stem cell differentiation

“…Retinol itself is also enzymatically converted to polar metabolites with biological activity (Achkar et al, 1996; Blumberg et al, 1996; Lane et al, 2008; Liu et al, 2009) and retinol can also induce the differentiation of cultured ES cells, though it is not as potent as RA (Lane et al, 2008). In fact, the major bioactive retinoid stored in the Xenopus egg and early embryo is all‐trans 4‐oxoretinaldehyde, which is capable of binding to and transactivating RARs.…”

The retinol signaling pathway in mouse pluripotent P19 cells

“…The bulk of retinol taken up by P19 cells, therefore, is probably metabolized. In addition to retinal and atRA, retinol has been reported to be metabolized to a number of transcriptionally active and inactive compounds in various cell types [Buck et al, 1991;Derguini et al, 1995;Achkar et al, 1996;Ross et al, 2000;Liu et al, 2009;Moise et al, 2009] and, although the profile of retinol metabolites in P19 cells has not been determined, both active and inactive metabolites would be expected to be produced in this cell as well. The generation of high levels of inactive retinol metabolites might be a contributing factor for the low potency of retinol, relative to atRA, in the present study.…”

“…atRA, the major active metabolite of vitamin A, is further oxidized by Cyp26 cytochrome p450 enzymes to polar metabolites for elimination or other biological functions. Additional natural metabolites of retinol and atRA have been identified, some of which are biologically active in some assays [Ross et al, 2000;Liu et al, 2009;Moise et al, 2009]. To maintain the cellular atRA concentration within defined limits, a balance appears to exist between its synthesis and metabolism.…”