Estrogen regulates the expression of retinoic acid synthesis enzymes and binding proteins in mouse skin
Introduction
Estrogen regulates the hair cycle. There are 4 main stages of the hair cycle: anagen (growth), catagen (regression), telogen (rest) [1], and exogen (release) [2]. Topical 17-beta-estradiol (E2) applied during telogen inhibited hair growth by arresting hair follicles in telogen in CD-1 and C57BL/6 mice [3,4]. In contrast, the synthetic estrogen antagonist ICI 182,780 (ICI) increased anagen. Topical E2 applied during late anagen induced early catagen, but ICI did not reverse this [4]. Estrogen receptor alpha (ESR1) regulated both of these effects [4,5]; while estrogen receptor beta (ESR2) apposed the action of ESR1 on the anagen to catagen transition [4]. Further studies suggested that E2 increased transforming growth factor beta 2 to induce premature catagen and bone morphogenetic protein 4 to maintain telogen [6].
E2 is synthesized within the pilosebaceous unit (PSU; hair follicle and sebaceous gland) [[7], [8]-9]. Aromatase (cytochrome P450, family 19, subfamily a, polypeptide 1; CYP19A1) is the rate-limiting enzyme that converts testosterone/androstenedione to E2/estrone respectively [10]. Inhibiting CYP19A1 with letrozole significantly reduced E2 and estrone [7]. CYP19A1 localized to the PSU, but only one stage of the hair cycle was examined [11]. Estrone and E2 were also synthesized in anagen hair follicles independent of CYP19A1 [9]. Thus, E2 synthesized within the PSU may impact the PSU and regulate the hair cycle.
Vitamin A and synthetic retinoids also altered hair, the hair cycle, and sebaceous gland function in humans and rodents [12], [13], [14], [15], [16], [17]. In addition, alterations in retinoids led to alopecia [13,[18], [19], [20], [21], [22], [23], [24], [25], [26]]. Pharmacological doses of synthetic retinoids etretinate and acitretin arrested hair follicles in telogen leading to telogen effluvium in humans [25]. Dietary vitamin A (retinyl esters) also regulated the hair cycle in a dose dependent manner in C57BL/6J and C3H/HeJ mice and impacted both central centrifugal cicatricial alopecia and alopecia areata [21,24,27]. In addition, depletion of 2 different vitamin A metabolism proteins lead to accelerated anagen, suggesting that these proteins are important in maintaining telogen [22,28].
Retinoic acid (RA) is an active metabolite of vitamin A and is argued to be synthesized at or near the site of action. Precise levels of RA are regulated by a set of key enzymes and binding proteins. Synthesis of RA from retinol occurs by the action of 2 enzyme families [29]. Retinol dehydrogenases convert retinol to retinal, while retinal dehydrogenases (aldehyde dehydrogenase family 1, subfamily A1, A2, and A3; ALDH1A1-3) convert retinal to RA. Dehydrogenase/reductase SDR family member 9 (DHRS9) is a retinol dehydrogenase regulated by estrogen in the uterus [30]. Cellular RA binding protein type II (CRABP2) also associated with the ability of tissues to synthesize RA [31], [32], [33], [34]. CRABP2 transports RA to the nucleus, and delivers it to RA receptors alpha, and possibly beta, and gamma (RARA, RARB, RARG) [35,36]. RARs are part of the nuclear hormone superfamily of ligand activated transcription factors [37,38]. RA is degraded by Cytochrome P450, family 26, subfamily a, polypeptide 1 (CYP26A1) and subfamily b, polypeptide 1 (CYP26B1) [39]. DHRS9, ALDH1A1-3, CRABP2 and RARA, B, and G localized to the PSU in a hair cycle dependent manner [40].
Estrogen regulates RA synthesis proteins in a tissue specific manner. Estrogen increased ALDH1A2 and CRABP2 in the rat uterus, human endometrium, hippocampus, and rat choroid plexus [31,[41], [42], [43], [44], [45], [46]-47]. Estrogen also increased DHRS9 and RA synthesis in the rat uterus; as well as DHRS9 and ALDH1A1 in the rat choroid plexus [31,41,42,47]. In contrast, estrogen reduced ALDH1A1 in the rat uterus and prolactin cells of the rat anterior pituitary gland [31,41-43,48,49]. E2 also increased Cyp26b1 in the hippocampus [45,46]. ESR1 was likely responsible for most of this regulation. ESR1 specific agonists reduced Aldh1a1 in the pituitary [49]. In addition, ESR1 directly bound a classical estrogen response element (ERE) in the promoter of Cyp26b1 and the first intron of RARA [45,50]. Crabp2 promoter analysis found no classical EREs; but ESR1 bound a region of DNA containing an imperfect ERE, ERE half sites, and Sp1 sites [41]. The Aldh1a2 promoter also contains a classical ERE, but studies have not confirmed ER binding [51]. Thus, estrogen directly regulated several RA synthesis proteins via ESR1 in many tissues.
The purpose of this experiment is to determine if estrogen regulates the expression of RA synthesis proteins in the cycling hair follicle. The rationale for this experiment is that estrogen is produced in the PSU and regulates the hair cycle. RA synthesis components localized to the PSU and retinoids regulated the hair cycle. In addition, estrogen regulated RA synthesis components in other tissues. The hypothesis tested is that locally produced estrogen regulates RA synthesis in the cycling hair follicle in an autocrine or paracrine fashion by inducing expression of DHRS9, ALDH1A2, and CRABP2, but possibly not ALDH1A3. This study found that both exogenous and endogenous estrogen regulates several RA synthesis enzymes and binding proteins. However, more significant effects were seen with the E2 antagonist (ICI) than the CYP19A1 inhibiter letrozole. This report also identified locations where E2 and its inhibitors regulated RA synthesis enzymes and binding proteins.
Section snippets
Mice
The Jackson Laboratory Institutional Animal Care and Use Committee approved all mouse work (protocol number 05003) in compliance with the US National Research Council's Guide from the Care and Use of Laboratory Animals, the US Public Health Service's Policy on Humane Care, and Use of Laboratory Animals, and Guide for the Care and Use of Laboratory Animals. C57BL/6J wild-type (+/+) (hereafter referred to as B6) mice (JR# 664, The Jackson Laboratory, Bar Harbor, ME) were used. Mice were fed the
Estrogen impacted RA synthesis components in telogen, but with considerable variability
To test the hypothesis that locally produced estrogen regulates RA synthesis component expression a pilot study was performed. Male C57BL/6J (B6) mice (n = 3) with hair follicles in telogen were shaved and topically treated with 17-β-estradiol (E2), E2 receptor antagonist ICI 182-780 (ICI), epigallocatechin gallate (EGCG), EGCG plus E2, or vehicle (acetone). EGCG is a non-specific inhibitor of CYP19A1 [60,61]. The EGCG plus E2 group was included to see if E2 could rescue the loss of E2 produced
Discussion
The original hypothesis that locally produced estrogen regulates RA synthesis in the cycling hair follicle in an autocrine or paracrine fashion by inducing expression of DHRS9, ALDH1A2, and CRABP2, but possibly not ALDH1A3 was accepted in some locations, but rejected in other locations. Exogenous or endogenous estrogen regulated DHRS9, ALDH1A1, ALDH1A2, ALDH1A3, CRABP2, RARA, and RARB in a sex specific manner (Fig. 6). Female control mice expressed more DHRS9, ALDH1A2, ALDH1A3, and CRABP2:
Author contributions
HBE, DEO, LEK, and JPS conceptualized this study; HBE curated the data and administered the project; HBE, DEO, LEK, and JPS acquired funding, provided resources, and supervised the project; HBE, KAS, SO, DEO, LEK, and JPS developed methods; HBE, KAS, ANS, FJD, and JPS performed investigation; HBE, FJD, and WW performed statistical analysis; HBE and DEO validated antibodies, and SO validated QPCR. HBE wrote majority of the document. AHS wrote original draft of the estrogen receptor localization
Acknowledgment
We thank Latha Raju and the Skin Disease Research Center Molecular Biology and Mouse Pathology cores at Vanderbilt University Medical Center; and Wanyi Wang in the Texas Woman's University Center for Research Design and Analysis for their assistance. This work was supported in part by grants from the North American Hair Research Society and NIH (AR052009, AR041943, AR041943-14 5254, and AR052710). The authors declared no conflicts of interest.
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David E. Ong is deceased.