Research ArticleImmunolocalization of retinoic acid biosynthesis systems in selected sites in rat☆
Introduction
Careful studies by Wolbach and Howe [1] found that when adult rats are made vitamin A deficient there is a focal metaplasia to a stratified squamous cornifying epithelium at sites that normally contain secretory epithelia and atrophy of associated glands. When these vitamin A deficient rats were then fed a vitamin A sufficient diet [2], there was simultaneous change of the cornified epithelia (events described were similar to apoptosis) and differentiation of basal cells towards the secretory epithelia normally found at these sites. The first tissues to be affected by vitamin A deficiency were the respiratory tract and the salivary glands. The next group of tissues involved was the urogenital tract as the vitamin A deficiency worsened. The ocular–paraocular group (meibomian gland ducts, lacrimal, and harderian glands), pancreatic ducts, hair follicles, and sebaceous glands were later affected in severe vitamin A deficiency. Subsequent studies confirmed the role of vitamin A in injury repair, metaplasia, differentiation, and apoptosis [3], [4], [5], [6], [7]. Until recently, a system for synthesis of retinoic acid (RA; the active metabolite of vitamin A) within all of these epithelia was not known.
The synthesis of RA from circulating retinol occurs by the activity of two enzyme families [8]. Retinol dehydrogenases (Roldhs) convert retinol to retinal, while retinal dehydrogenases (Raldhs) convert retinal to RA. There are four known rat Roldhs of the short chain dehydrogenase reductase family (SDR): Roldh 1, 2, and 3, and eRoldh [9], [10], [11], [12], [13]. Two human Roldh family members, hRDH4 (aka hRODH-E) and hRDH-TBE (aka hRODH-E2), have been identified [14], [15], [16], [17], [18], [19], [20]. Rat eRoldh, identified in this laboratory, is 85% identical at the amino acid level to the human hRDH-TBE. During the course of manuscript preparation, the gene name for eRoldh was changed to dehydrogenase/reductase (SDR) family member 9 (Dhrs9), but we will use the name we originally gave this enzyme throughout the work presented here.
Currently, there are three known Raldhs (gene names Aldh1a1, 2, and 3) found in mice, rat, and human that convert all-trans-retinal to atRA. Aldh1a2tm1Ipc null mice die in utero due to defects in heart development [21], and Aldh1a3tm1.Pcn null mice die within 10 h of birth due to defects in nasal development [22], implying that they are essential for RA synthesis. But Aldh1a1tm1Gdu null mice have no apparent phenotype and therefore may not be essential for RA biosynthesis [23]. Instead, it has been hypothesized that this enzyme is involved in the catabolism of excess retinol [23], [24]. Two binding proteins, cellular retinol binding protein (Crbp) and cellular retinoic acid binding protein type II (Crabp2), are also closely associated with RA synthesis [8], [25]. Crbp acts to chaperone retinol and retinal to retinoid metabolizing enzymes, while Crabp2 carries RA to the nucleus and channels it to RARs via direct protein interactions [26], [27]. In contrast, Crabp1 is not directed to the nucleus upon RA binding, does not bind RARs, but is instead believed to be involved in RA catabolism.
Previous work in our laboratory focused on the synthesis of RA in the rat female reproductive system, particularly the uterus. The uterus is sensitive to vitamin A deficiency [1], [2]. The RA synthesis system in the rat uterus consists of Crbp, eRoldh, Aldh1a2, and Crabp2 [12], [25], [28], [29]. This RA synthesis system is regulated by estrogen, and during estrus, Crbp, eRoldh, and Crabp2 are localized to the uterine epithelium, while Aldh1a2 is present within the uterine stromal cells, particularly those cells directly adjacent to the epithelium [28], [30]. This suggests that retinal is made within the uterine epithelial cell but needs to be transferred to its underlying stroma for RA to be produced. The present study was performed to determine if this RA synthesis system is unique to estrogen-regulated female reproductive organs or is also expressed in other vitamin A sensitive tissues. This study is not meant to be definitive for all locations, but rather a survey of where the newly discovered eRoldh and other members of this RA synthesis system are present. Crabp1 and other components of RA catabolism were not included in this study of RA biosynthesis.
We found that this RA synthesis system was present in all the main epithelial tissues defined as vitamin A sensitive by Wolbach and Howe [1]. However, the greater expression of another SDR family member (possibly Roldh 1, 2, or 3) in the rat ureter suggests that other Roldhs may be important in this tissue. In addition, the epithelial vs. mesenchymal expression of Aldh1a2 and Aldh1a3 in other tissues is more complex than what is seen in the uterus.
Section snippets
Rats
Adult Sprague–Dawley rats were purchased from Harlan (Indianapolis, IN), fed a standard chow diet (Purina, St. Louis, MO), and sacrificed by CO2 asphyxiation in compliance with the Vanderbilt University Institutional Animal Care and Use Committee. Tissues were quick frozen in liquid nitrogen or fixed in zinc alcohol buffered paraformaldehyde, paraffin embedded, and sectioned at 5 μM.
Western blot analysis
Tissues were homogenized in HEPES buffer and fractionated by differential centrifugation. 75 μg cytosolic or
Results
Western blot analysis and RT-PCR were performed to examine the expression pattern of RA synthesis enzymes and binding proteins in vitamin A sensitive tissues. This was followed by immunohistochemistry to confirm expression and determine localization within the various tissues. These results were then compared to the expression of the basal cell markers Keratin 5 and 14 (K5, K14). Table 1 shows that eRoldh and the rest of this system for RA synthesis are expressed in all epithelial tissues
Discussion
Vitamin A deficiency studies revealed a set of epithelial cells whose basal cells appear to be regulated by vitamin A [1], [2]. The present study provides further evidence that RA synthesis occurs at the site of its action and clearly shows that eRoldh and the system for RA synthesis previously described in the rat uterus are not restricted to the female reproductive system but are present in all vitamin A sensitive epithelial tissues examined. This RA synthesis system consists of Crbp, eRoldh,
Acknowledgments
The authors would like to thank Dr. M.T. Stahlman for help analyzing the trachea and lung sections, Dr. M.A. Gannon for help analyzing the pancreas section, M.-C. Orgebin-Crist for help analyzing the epididymis, B. Kakkad for assistance in antibody production, and J. Miller for some of the K5 and K14 IHC. This work was supported by NIH Grants: DK 32642, DK 26657 (to the Clinical Nutrition Research Unit Protein and Immunology core), HD 07043 (Reproductive Biology Training Grant), DK20593 (to the
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The mouse genome nomenclature was used (http://www.informatics.jax.org). Genes and RNA message expression is italicized, while proteins are not. First letter capitalized followed by lower case indicates a mouse or rat gene, while all capital letters indicates the human gene or protein.