Immunolocalization of enzymes, binding proteins, and receptors sufficient for retinoic acid synthesis and signaling during the hair cycle

Helen B. Everts, PhD. Vanderbilt University Medical Center, Nashville, TN

Vitamin A is essential for the development and maintenance of multiple epithelial tissues, including skin and hair (Frazier and Hu, 1931, Peck and DiGiovanna, 1994, Wolbach and Howe, 1925). The skin takes up circulating retinol and can either store it in the form of retinyl esters or metabolize it to retinoic acid (RA, the active form, (Roos et al, 1998). Cellular retinol binding protein (Crbp, tentative gene symbol Rbp1) binds this retinol and interacts with either lecithin: retinol acyltransferase (Lrat) to esterify it for storage or retinol dehydrogenase (Roldh) to oxidize it to retinal (Napoli, 1999). Dehydrogenase/reductase (SDR family) member 9 (Dhrs9; aka eRoldh, hRODH-E2) is one enzyme that catalyzes the oxidation of Crbp-bound, as well as free, retinol to retinal in numerous epithelial tissues (Everts et al, 2005, Markova et al, 2003, Rexer and Ong, 2002). Retinal is oxidized to RA by retinal dehygrogenases 1-3 (gene names Aldh1a1, 2 and 3, (Napoli, 1999). Expression of the cellular retinoic acid binding protein type II (Crabp2) was also associated with the ability to make RA (Bucco et al, 1997, Napoli, 1999). Crabp2 also carries RA to the nucleus, where it shuttles RA to retinoic acid receptors (Rars) and increases transcriptional efficiency (Budhu and Noy, 2002, Dong et al, 1999, Sessler and Noy, 2005). Three Rars have been described (Rar alpha-a, Rar beta-b, and Rar gamma-g), which bind DNA and regulate transcription of specific genes in an RA dependent manner. Therefore, it can be assumed that expression of these binding proteins, enzymes, and receptors indicate sites of RA synthesis and action. This assumption is supported by similar localization patterns between defects seen in vitamin A deficient rat embryos and Aldh1a2 and Aldh1a3 null mice with immunohistochemical and in situ hybridization localization patterns of enzymes and binding proteins involved in RA synthesis (Bavik et al, 1997, Blentic et al, 2003, Dupe et al, 2003, Niederreither et al, 2002, Niederreither et al, 1999). To obtain predictions of where endogenous RA fits into the signaling network that regulates the hair cycle, as well as to better understand the physiological pathway of RA biosynthesis and signaling we characterized the localization of the complete system for RA biosynthesis and signaling including Crbp, Dhrs9, Aldh1a1, Aldh1a2, Aldh1a3, Crabp2, Rara, Rarb, and Rarg during the hair cycle by immunohistochemistry in wax stripped C57BL/6J mice (Everts, Sundberg, King, Ong; J Invest Dermatol, in press). 

            We found that all components necessary for RA biosynthesis and signaling were present throughout the hair follicle in a hair cycle dependent manner. During telogen, Rars predominated in the cycling portion of the hair follicle. During anagen RA synthesis enzymes and Crabp2 increased. The whole system necessary for RA synthesis and signaling was present in several sites, including the sebaceous gland, dermal papilla, and most layers of the differentiating hair follicle. There were several changes in the RA synthesis and signaling localization pattern during the anagen to catagen transition. These include: a switch in Crabp2 localization from the cells outside the bulge (stem cell niche) to cells within the bulge (stem cells); a decrease in Rara in the dermal papilla and connective tissue sheath, except those outside the bulge (stem cell niche); and an increase in Crbp and Crabp2 within the lower, regressing, follicle. By catagen VII, Dhrs9 and Rars decreased in most of the cycling portion of the hair follicle. Components of RA synthesis and signaling were not always all in the same cell layer. Since retinal and RA are diffusible between cells it suggests that epithelial-mesenchymal interactions are important in this tissue as was seen for other epithelial tissues. In addition, in some sites expression of the RA synthesis and signaling pathway flowed through different stages of differentiation, with the most differentiated cells expressing the final proteins in this pathway- nuclear Crabp2 and Rars. This was most evident in the sebaceous gland during early-mid anagen. This localization pattern suggests that RA regulates epithelial-mesenchymal interactions in the stem cell niche and bulb, anagen induction, differentiation of all layers of the pilosebaceous unit during anagen, and catagen induction. Future studies will examine these functions in depth. These data predict that blocking RA synthesis would most likely also block differentiation of the entire pilosebaceous unit resulting in metaplasia as was seen during severe vitamin A deficiency in the rat (Wolbach and Howe, 1925). More mild reductions in endogenous RA synthesis may reduce sebum production, cause hair medulla defects and/or prolong anagen. The localization of endogenous RA synthesis and signaling also revealed sites where exogenous RA could disrupt the balance of liganded vs. unliganded Rara and Crabp2 leading to the reported toxic effects including hyperproliferation of the epidermis, skin fragility, and hair loss. For example, during anagen Rara may act to repress transcription within the connective tissue sheath and dermal papilla. The decrease of Rara in the connective tissue sheath and dermal papilla during anagen VI/catagen I would remove this inhibition and trigger catagen. When exogenous RA is given during anagen, it would disrupt the balance of liganded to unliganded Rara and activate transcription of RA target genes, such as Transforming growth factor, beta 2, to induce premature catagen, as was reported (Foitzik et al, 2005). This highlights the need for a better understanding of endogenous RA function and the regulation of endogenous RA synthesis, which could lead to better treatments without unwanted side effects. We are also examining the expression of this system of endogenous RA synthesis and signaling in the hair follicles, sebaceous glands, and epidermis of human skin affected by cicatricial alopecia and mouse models of this disease to provide clues as to the etiology and pathogenesis of this disfiguring disease, provide markers to distinguish CCCA from other primary cicatricial alopecias, and predict novel therapeutic targets.


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