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Endocrinology Vol. 138, No. 9 3711-3718
Copyright © 1997 by The Endocrine Society

ARTICLES

1,25-Dihydroxyvitamin D3 Regulates Estrogen Metabolism in Cultured Keratinocytes1

Susan V. Hughes, Elaine Robinson, Rosemary Bland, Helen M. Lewis, Paul M. Stewart and Martin Hewison

Department of Medicine, Queen Elizabeth Hospital, University of Birmingham, Birmingham, United Kingdom B15 2TH; and the Department of Dermatology, Selly Oak Hospital (H.M.L.), Birmingham, United Kingdom B29 6JD

Address all correspondence and requests for reprints to: Dr. M. Hewison, Department of Medicine, Queen Elizabeth Hospital, University of Birmingham, Birmingham, United Kingdom B15 2TH. E-mail: M.Hewison{at}bham.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Local estrogen metabolism may play an important role in modulating cell development in peripheral tissues such as breast, adipose, and bone. C19 androgens are converted to C18 estrogens by the enzyme aromatase, overexpression of which is associated with breast cancer. Interconversion of active estradiol (E2) to inactive estrone is controlled by various isoforms of the enzyme 17ß-hydroxysteroid dehydrogenase (17ßHSD). We have studied the expression of these two enzymes in human keratinocytes and report rapid changes in 17ßHSD activity in response to treatment with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Keratinocytes cultured in serum-free medium showed aromatase activity of 2.5 fmol/h·mg cell protein, which was unaffected by any culture treatment. A much higher level of 17ßHSD activity was observed in the keratinocytes, predominantly conversion of E2 to estrone (~120 pmol/h·mg cell protein). This inactivation of E2 increased in a dose-dependent fashion after treatment of the cells with antiproliferative doses of 1,25-(OH)2D3 (0.1–200 nM). The effect of 1,25-(OH)2D3 on 17ßHSD activity was enhanced by simultaneous treatment with dexamethasone, which also increased the antiproliferative action of 1,25-(OH)2D3. Reverse transcription-PCR and Northern analysis showed that keratinocytes expressed messenger RNA for three 17ßHSD isoenzymes (types I, II, and IV). Treatment with 1,25-(OH)2D3 (10 nM for 20 h) resulted in the up-regulation of messenger RNA levels for type 2 17ßHSD. Further RNA studies combined with E2 binding experiments demonstrated the presence of estrogen receptors in the cultured keratinocytes. These data indicate that keratinocytes are potential targets for systemically or locally produced estrogens, which may, in turn, play a key role in the development of normal skin. In particular, we propose that 17ßHSD isoenzymes are key target genes for 1,25-(OH)2D3 in keratinocytes and may be an important feature of the antipsoriatic effects of vitamin D and its analogs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ACTIVE form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], is a seco-steroid that influences a wide variety of tissues and functions (1). Classically, 1,25-(OH)2D3 modulates calcium homeostasis by stimulating calcium transport across the gut and calcium mobilization from bone (2). However, in recent years it has become clear that 1,25-(OH)2D3 can influence tissue and functions not immediately involved in calciotropic activity (1). In part, this has stemmed from studies that have described the ubiquitous nature of the receptor for 1,25-(OH)2D3 [vitamin D receptor (VDR)] (3). Other groups have reported synthesis, via the enzyme 1{alpha}-hydroxylase, of 1,25-(OH)2D3 at extrarenal sites (4). Key cell types that express both VDR and 1{alpha}-hydroxylase activity are keratinocytes and monocytes, highlighting a possible paracrine or autocrine role for 1,25-(OH)2D3 in modulating the functions of skin and the immune system (5, 6). The most important action of 1,25-(OH)2D3 in these tissues is its ability to act as an antiproliferative/differentiative agent. This has raised the possibility of the clinical application of 1,25-(OH)2D3 as both an inhibitor of keratinocyte proliferation and an immunosuppresive agent (7).

Several difficulties are associated with the therapeutic use of 1,25-(OH)2D3, the most obvious being its potent hypercalcemic side-effects. This has in part been addressed by the development of vitamin D analogs that have low calciotropic activity while retaining the antiproliferative effects of 1,25-(OH)2D3 (8). The use of these analogs as therapy for immune proliferative disorders such as leukemia has met with limited success. Indeed, recent studies of animal models suggest that a more effective use for 1,25-(OH)2D3 may be as a T cell-modulating immunosuppresive agent (7). However, 1,25-(OH)2D3-like analogs such as calcipotriol (MC903) and tacalcitol are now widely and successfully used in the topical treatment of psoriasis (9). Most studies have shown a marked decrease in epidermal proliferation in response to vitamin D treatment, albeit without any significant effect on the inflammatory infiltrate associated with psoriasis (10).

Although there is much interest in the clinical use of vitamin D analogs, it is clear that our understanding of the mechanisms by which 1,25-(OH)2D3 achieves its antiproliferative effects is far from complete. In this study we have investigated a possible role for novel steroid hormone interactions in mediating nonclassical effects of 1,25-(OH)2D3. Specifically, we have characterized local estrogen production by keratinocytes and have sought to determine whether this is a target for the antiproliferative agent, 1,25-(OH)2D3. Data indicate that both aromatase and 17ß-hydroxysteroid dehydrogenase (17ßHSD) enzymes are expressed by keratinocytes, with the predominant activity being 17ßHSD-catalyzed conversion of estradiol (E2) to estrone (E1). Further studies have shown that the antiproliferative effect of 1,25-(OH)2D3 is associated with rapid stimulation of 17ßHSD messenger RNA (mRNA) expression and activity. As well as highlighting the potential importance of vitamin D-estrogen interactions in regulating cell proliferation, these studies suggest that both systemically and locally produced estrogens may play a key role in directing keratinocyte development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Purified human foreskin keratinocytes (Promocell, Heidelburg, Germany) were cultured in phenol red-free, serum-free, defined medium based on a modified MCDB 153 preparation with 28 mM HEPES buffer and containing the following additives: calcium chloride (0.15 mM), epidermal growth factor (recombinant human; 0.1 ng/ml), insulin (bovine; 5.0 µg/ml), hydrocortisone (0.5 µg/ml), gentamicin (50 µg/ml), and amphotericin B (2.5 µg/ml). Each batch of keratinocytes was cultured for no more than five passages. Cells were seeded at a density of 10,000 cell/cm2, and medium was changed every 2 days. Experimental cultures were grown to approximately 70% confluency in 24-well plates before the addition of the various treatments, including 1,25-(OH)2D3 (0.1–200 nM; a gift from Dr. M. Uskukovic, Hoffman LaRoche, Nutley, NJ), 9-cis-retinoic acid (10 nM), dexamethasone (DEXA; 100 nM), E2 (100 nM), and E1 (100 nM; all from Sigma Chemical Co., St. Louis, MO).

Estrogen metabolic assays
Aromatase activity was assessed by a water release assay using [1ß-3H]androstenedione ([3H]1ßA; SA, 30 Ci/mmol; New England Nuclear, Boston, MA) as substrate. Assays were carried out in 24-well plates using 0.5-ml aliquots of medium supplemented with 40 nM [3H]1ßA. Monolayers were incubated with substrate for 5 h, after which the culture medium was removed, and steroids were extracted using chloroform. The aqueous layer was retained and contained 3H2O produced in a molar equivalent to E1 after aromatase metabolism of [3H]1ßA. Residual substrate contamination of the aqueous layer was further limited by addition of dextran-activated charcoal and centrifugation at 1000 x g for 15 min. Aliquots of the resulting supernatant were then removed and added to 5 ml scintillant before scintillation counting. Data were reported as femtomoles per h/mg cellular protein.

17ßHSD activity was assessed using [3H]E1 or [3H]E2 as substrate (SA for both, ~80 Ci/mmol; Amersham, Aylesbury, UK). Assays were carried out in 24-well plates using 0.5-ml aliquots of medium supplemented with 100 nM [3H]E1 or [3H]E2. Cells were incubated with substrate for 5 h, after which the culture medium was removed, and steroids were extracted using chloroform. Vacuum-dried chloroform extracts were then plated on silica TLC plates (Fluka), and estrogen metabolites were separated using 80% chloroform-20% ethyl acetate as a running solvent. Quantification of the conversion of [3H]E1 to [3H]E2 and of [3H]E2 to [3H]E1 was carried out by scanning radioactive analysis using a Bioscan System 200 imaging scanner (Lab Logic, Sheffield, UK). Data were reported as picomoles per h/mg cellular protein.

Analysis of keratinocyte proliferation
Data for aromatase and 17ßHSD assays were normalized by analyzing changes in the protein content of keratinocyte monolayers. After removal of culture medium for enzyme assay, cell monolayers were lysed in 1 ml water, and 0.2-ml aliquots were used for standard protein assays (Bio-Rad, Richmond, CA). Enzyme activity values are shown per mg cellular protein. Quantification of changes in keratinocyte proliferation was carried out by measuring the incorporation of [3H]thymidine (80 Ci/mmol; Amersham). Cell cultures were supplemented with 1 µCi [3H]thymidine for 5 h and then pulsed with 1 mmol unlabeled thymidine to eliminate nonspecific incorporation of the nucleotide. Isolated keratinocyte nuclei were dissolved in sodium hydroxide and added to scintillation fluid, and radioactivity was counted by standard methods.

Analysis of mRNA expression
Total RNA was isolated from keratinocyte cultures using RNeasy preparative minicolumns as described by the manufacturer (Qiagen, Chatsworth, CA). An optional incubation step to reduce DNA contamination was included using QiaShredder minicolumns (Qiagen).

Reverse transcription-PCR (RT-PCR) analysis was carried out using 1 µg total RNA, which was reverse transcribed using Promega’s AMV reverse transcriptase kit, at 42 C (1 h), according to the manufacturer’s protocol. At the end of the reaction, samples were heated to 95 C for 5 min. An aliquot of this RT reaction was then used as template for subsequent PCR reactions. PCR analysis of complementary DNAs (cDNAs) for 17ßHSD isoenzymes (types I–IV) was carried out as described previously (11). The following primers were used: 17ßHSD I: 5'-primer, AGG CTT ATG CGA GAG TCT GG; 3'-primer, CAT GGC GGT GAC GTA GTT GG; 17ßHSD II: 5'-primer, CTG AGG AAT TGC GAA GAA CC; 3'-primer, GAA GTC CTT GCT GGC TAA CG; 17ßHSD III: 5'-primer, ACA ATG TCG GAA TGC; 3'-primer, AGG TTG AAG TGC TGG TCT GC; and 17ßHSD IV: 5'-primer CTA TTG GCC AGA AAC TCC CT; 3'-primer, GGA CCT TGG TTT GAA AAT GA. PCR primers for estrogen receptor (ER) were as follows: 5'-primer, TGA CTA TGC TTC AGG CTA C; and 3'-primer, CTT TCA TCA TTC CCA CTT C. PCR reactions (20 µl) were set up using 2-µl aliquots of cDNA, primers at a final concentration of 0.5 µM, and 1 U Taq polymerase (Promega). PCR buffer contained deoxy-NTPs (0.2 mM), MgCl2 (1.5 mM), KCl (50 mM), Tris-HCl (10 mM; pH 8.3), and gelatin (0.01%, wt/vol). Samples were amplified using an initial denaturation cycle of 95 C for 3 min, followed by 30 cycles of 95 C (1 min), 55 C (1 min; ER and 17ßHSD IV) or 60 C (1 min; 17ßHSD I–III), and 72 C (2 min). A final elongation step of 72 C for 7 min was also included.

Northern blotting analysis was carried out using aliquots (15 µg) of RNA from control and 1,25-(OH)2D3-treated keratinocytes. RNA was separated by denaturing gel electrophoresis and blotted onto Hybond N-plus nylon filters (Amersham). After fixation by UV irradiation, filters were prehybridized for 3 h using a modified phosphate buffer containing 0.78 M sodium phosphate (pH 7.2; 0.2 M NaH2PO4·H2O and 0.58 M Na2HPO4), 5 mM EDTA, 7% SDS, and sonicated salmon sperm DNA (100 ng/ml). Radiolabeled probes for 17ßHSD I, II, and IV were derived from the amplified cDNA fragments produced in the RT-PCR studies. DNA bands were isolated from low melting point agarose gels and used as templates (25–50 ng) in random priming labeling preparations (Multiprime, Amersham, UK). After hybridization for 16 h at 65 C, filters were washed three times in 1 x standard sodium citrate (1 x SSC) with 0.1% SDS at room temperature, followed by a 0.3 x SSC-0.1% SDS wash at 55 C (30 min) and a 0.1 x SSC-0.1% SDS wash at 65 C. Washed filters were then exposed to DuPont Cronex film (Wilmington, DE) for various time periods before development of autoradiographs.

Analysis of nuclear binding of E2 and E1
The number of intracellular receptors for E2 was assessed by whole cell nuclear association assays using [3H]E2 and [3H]E1 as ligands (SA, 80–100 Ci/mmol; Amersham). Assays were carried out as follows. Monolayers of keratinocytes were trypsinized and washed twice with serum-free medium to remove traces of trypsin. Cell pellets were resuspended in serum-free medium to give 0.5 x 107 cells/ml. Aliquots of this cell suspension (0.2 ml) were then added to glass tubes containing increasing doses of [3H]E2 or [3H]E1 (0.2–10 nM) in the presence or absence of a 200-fold excess of unlabeled E2 or E1 (to determine nonspecific binding). Cells were then incubated at 37 C for 1 h to allow association of ligand-ER complexes with keratinocyte nuclei. Cells were washed three times with 0.5-ml aliquots of cold PBS including 5-min centrifugation steps at 500 x g. The final cell pellets were incubated with 0.5 ml lysis buffer (containing 0.25 M sucrose, 20 mM HCl, 1.1 mM magnesium chloride, and 0.5% Triton X-100) and centrifuged for 5 min at 1000 x g to isolate crude nuclear pellets. This step was repeated again, and the resulting nuclei were resuspended in 0.1 ml PBS and 0.5 ml ethanol and then transferred to 5 ml scintillation fluid before scintillation counting.

Statistics
Assays for hormone metabolism and keratinocyte proliferation were carried out in quadruplicate and reported as the mean ± SD. Analysis of differences between values for various treatments was carried out using unpaired Student’s t tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Preliminary investigations indicated that aromatase and 17ßHSD activities were both detectable in monolayers of human keratinocytes cultured in defined medium containing 0.15 mM calcium. Enzyme assays were optimized for 5-h incubations using 20–200 nM [3H]A (aromatase) and 20–500 nM [3H]E1 and [3H]E2 (17ßHSD; data not shown). Parallel studies also indicated that substrate conversion was similar when using either medium or cell monolayers (data not shown). However, additional assays were carried out using only cell medium, as this contained higher levels of both substrate and product, thereby providing more reproducible data. Both aromatase and 17ßHSD showed a linear increase in activity over respective concentration ranges, and subsequent assays were carried out using 40 nM [3H]A and 100 nM [3H]E1 or -E2. At these substrate concentrations, aromatase and 17ßHSD activities remained linear for incubation periods up to 9 h. Subsequent assays were routinely carried out using incubation periods of 5 h. Results in Fig. 1Go show typical TLC analyses of 17ßHSD activity in control cultures and cells treated with 10 nM 1,25-(OH)2D3 for 20 h. Control keratinocytes were able to metabolize both [3H]E1 and [3H]E2. The predominant activity was conversion of E2 to E1 (20% conversion of substrate). Metabolism of E1 to E2 was approximately 4% of substrate (27 ± 8 pmol/h·mg protein), which was unaffected by treatment with 1,25-(OH)2D3 (25 ± 10 pmol/h·mg protein). In contrast, conversion of E2 to E1 increased by approximately 2-fold after treatment with 10 nM 1,25-(OH)2D3 for 20 h.



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  		Figure 1. Effect of 1,25-(OH)2D3 on the interconversion of E2 and E1 in cultured keratinocytes. Cells were cultured in the absence or presence of 1,25-(OH)2D3 (10 nM) for 20 h and then incubated with either 100 nM [3H]E2 or 100 nM [3H]E1 for an additional 5 h. Supernatants were chloroform extracted and separated by TLC. The data shown are typical scanning analyses of TLC plates, showing conversion of E2 to E1 and of E1 to E2.

 
Further studies of E2 inactivation by 17ßHSD showed that conversion to E1 was stimulated in a dose-dependent fashion after treatment with increasing concentrations of 1,25-(OH)2D3 (Fig. 2BGo). This effect paralleled the dose-dependent inhibition of keratinocyte proliferation induced by 1,25-(OH)2D3 (Fig. 2AGo). The putative relationship between keratinocyte proliferation and E2 inactivation was assessed in more detail by the data shown in Fig. 3Go. Analysis of 17ßHSD activity (Fig. 3AGo) indicated that conversion of E2 to E1 was unaffected by the addition of 9-cis-retinoic acid (10 nM), although the retinoid produced a significant decrease in cell proliferation. Cotreatment with 1,25-(OH)2D3 and 9-cis-retinoic acid did not enhance the effects of 1,25-(OH)2D3 on cell proliferation or 17ßHSD activity. In contrast, DEXA (100 nM) induced a significant rise in 17ßHSD activity, which was additive when 1,25-(OH)2D3 and DEXA were used as cotreatments. DEXA on its own had no effect on keratinocyte proliferation, but significantly enhanced the antiproliferative effects of 1,25-(OH)2D3 when the two hormones were added together (Fig. 3BGo).



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  		Figure 2. Dose-dependent changes in keratinocyte proliferation and estrogen metabolism. Parallel cultures of keratinocytes were treated with 1,25-(OH)2D3 (0.1–200 nM) for 20 h. Cells were then assessed for proliferation (A) and E2 to E1 conversion (B). 17ßHSD activity was normalized per h/mg cellular protein for each assay replicate. Values are the mean ± SD of quadruplicate samples from a single assay. Experiments were carried out at least twice.

 


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  		Figure 3. Effects of modulators of keratinocyte proliferation on E2 inactivation. Log-phase cultures of keratinocytes were treated with the following agents in the absence or presence of 10 nM 1,25-(OH)2D3: vehicle only (control cells), 10 nM 9-cis-retinoic acid (9-CIS), 100 nM DEXA, 100 nM E1, and 100 nM E2. Each treatment was then assessed for changes in proliferation (A) and E2 to E1 inactivation (B). E1 and E2 treatments were assessed for changes in proliferation only (B). Values are the mean ± SD of quadruplicate samples from a single assay. Each experiment was carried out at least twice. *, Significantly different from untreated control (P < 0.01); **, significantly different from 1,25-(OH)2D3-treated cells (P < 0.01).

 
Analysis of aromatase-catalyzed conversion of androstenedione to E1 indicated a much lower level of activity compared with 17ßHSD (2.1 ± 0.4 fmol/h·mg protein). Furthermore, this activity was unaffected by 20-h exposure to 1,25-(OH)2D3 or any of the other culture treatments. RT-PCR analysis indicated that keratinocytes expressed several of the variable aromatase promoter mRNA species, including the 1.4-III transcript, which has been shown to be enhanced in breast cancer cell lines after treatment with glucocorticoids (data not shown).

The results presented in Fig. 4Go indicated that keratinocytes expressed mRNA for several 17ßHSD isoforms. RT-PCR analysis identified keratinocyte transcripts for 17ßHSD types I, II, and IV, which were also present in appropriate positive controls (Fig. 4AGo). All three transcripts were detectable in control and 1,25-(OH)2D3-treated keratinocytes. Type III 17ßHSD mRNA was undetectable in keratinocytes, but was faintly expressed in rat testis and more strongly in SW620 colonic cancer cells (routinely used by our laboratory as a control for this isoenzyme). Changes in the levels of mRNA expression for the 17ßHSD isoforms were assessed by Northern analysis using radiolabeled PCR fragments. The results in Fig. 4BGo indicated that treatment with 1,25-(OH)2D3 for 20 h stimulated expression of type I and II 17ßHSD mRNA. Relative densitometry analysis (corrected for 18S ribosomal RNA expression) indicated that 1,25-(OH)2D3 increased type I expression by a mean of 45% and increased type II expression by a mean of 105% (data not shown).



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  		Figure 4. RT-PCR and Northern analysis of 17ßHSD isoenzymes in keratinocytes. A, RNA was reverse transcribed using oligo(deoxythymidine) primers, and cDNAs were amplified using primers specific for 17ßHSD isoforms 1–4. Sample organization for each set of PCR reactions was as follows: 1) DNA markers; 2 and 4) control keratinocytes (-); 3 and 5) keratinocytes plus 10 nM 1,25-(OH)2D3 (+); 6) placenta; 7) testis; and 8) water blank. Analysis of 17ßHSD type III also included RNA from SW620 colonic cancer cells as a positive control. B, Northern analysis of RNA from control and 1,25-(OH)2D3-treated keratinocytes was carried out using radiolabeled probes for 17ßHSD types I, II, and IV generated from the RT-PCR fragments shown in A. Sample loading was normalized by probing for 18S ribosomal RNA.

 
Further proliferation studies suggested that estrogens themselves may influence keratinocyte development. Cells treated with 100 nM E1 or 100 nM E2 showed a small, but significant, decrease in proliferation (Fig. 3BGo). In particular, the effect of E1 (22% inhibition of proliferation) was similar to that observed with 10 nM 1,25-(OH)2D3. E1 and 1,25-(OH)2D3 did not have an additive effect when used as cotreatments. To investigate further the impact of estrogens on keratinocytes, we assessed ER mRNA expression and E1 and E2 binding in untreated cells (Fig. 5Go). RT-PCR analysis (Fig. 5AGo) revealed the presence of an ER transcript similar in size to that found in human placenta. Whole cell nuclear association assays using radiolabeled E2 (the preferred ligand for ER) indicated that there was specific estrogen binding in keratinocytes. The mean Kd value derived from Scatchard plots (2.1 nM) was similar to that previously described for ER, as were the numbers of receptors per cell (mean, 40,200). Parallel studies indicated that keratinocytes were also able to bind radiolabeled E1, although this was at a lower affinity as reflected by the higher mean Kd value (4.1 nM). The maximal binding capacity for E1 was slightly higher than that for E2 (mean, 45,900 ER/cell).



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  		Figure 5. ER expression in keratinocytes. A, RNA from untreated keratinocytes (-) and cells treated with 10 nM 1,25-(OH)2D3 (+) was reverse transcribed using oligo(deoxythymidine) primers. The resulting cDNA was amplified using ER-specific primers. The positive control was placental RNA (plac). B, Binding of [3H]E2 or [3H]E1 was determined by whole cell nuclear association assay using ligand concentrations of 0.1–20 nM in the presence or absence of a 200-fold excess of unlabeled E2 or E1. Data were assessed by Scatchard analysis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As the largest organ in the human body, the skin may make a significant contribution to both local and systemic hormone production. An established endocrine role for skin is in the photochemical conversion of 7-dehydrocholesterol to the parental vitamin D3 molecule. However, it now clear that the skin has much wider steroidogenic activity. In particular, skin cells have been reported to express a range of enzymes involved in the synthesis of androgens (12). Previous studies have shown that keratinocytes and fibroblasts are able to convert inactive androgens such as dehydroepiandrosterone to active androgens such as dihydrotestosterone via the enzymes 3ßHSD and 5{alpha}-reductase (13, 14). More recent reports indicate that components of the pilosebaceous unit, including keratinocytes from the inner and outer root sheaths, express mRNA for several steroidogenic enzymes, with 17ßHSD type II present in particularly high levels (15). These data suggest a possible role for local androgen metabolism in fetal sebaceous gland development as well as the pathogenesis of acne and androgen-dependent hair loss.

The presence of aromatase and 17ßHSD in skin components represents additional potential steps in the synthesis of active sex hormones. 17ßHSD modulates the activity of both estrogens and androgens by catalyzing the metabolism of 17-ketosteroids and their 17ß-hydroxysteroid counterparts. This includes the interconversion of androstenedione and testosterone and that of E1 and E2. In both of these cases reduction to the 17ß-hydroxysteroid (e.g. testosterone and E2) produces the more active configuration (16). Four different human 17ßHSD isoforms have been cloned to date, each of which may play a specific role in modulating estrogen and androgen levels (17, 18, 19, 20). Previous studies have shown that antiproliferative and differentiative agents may have modulatory effects on 17ßHSD enzymes. Glucocorticoids have been shown to increase 17ßHSD IV in THP-1 leukemic monocytes (21), whereas retinoic acid appears to stimulate 17ßHSD I in breast cancer cells (22, 23). In this study we have characterized estrogen metabolism in keratinocytes, a key target tissue for the antiproliferative agent 1,25-(OH)2D3. Our data indicate that regulation of 17ßHSD (primarily type II) is associated with the antiproliferative effects of 1,25-(OH)2D3, highlighting a possible role for estrogens as modulators of keratinocyte proliferation.

The observed aromatase and 17ß-dehydrogenase activities in keratinocytes show for the first time that keratinocytes have a capacity for estrogen metabolism similar to that of more well characterized peripheral tissue such as breast. Furthermore, the predominant oxidative activity of 17ßHSD is in agreement with other estrogen target tissues, in which regulation of E2 availability may influence the mitogenic impact of the hormone. The relationship among peripheral 17ßHSD activity, ER expression, and E2 concentrations has been studied extensively in breast tissue (24, 25, 26). Oxidative pathways tend to be dominant in ER-negative, nonresponsive cells, whereas reductive activity is higher in ER-positive, estrogen-responsive cells (27). The situation in keratinocytes is clearly different in view of the significant numbers of ER detected in these cells. This may be a feature of primary keratinocyte cultures per se. However, our data were derived using serum- and phenol red-free medium, which allowed us to quantify 17ßHSD activity in cultured intact cells rather than tissue homogenates. This approach appears to provide a more accurate representation of estrogen/androgen metabolism in vivo (27). With this is mind, we can perhaps speculate that the role of 17ßHSD activity in keratinocytes is a protective one, modulating the potentially mitogenic effects of E2.

Overall, our findings support the idea of skin being an estrogen-responsive tissue, in which effects of the hormone are tightly regulated by local 17ßHSD activity. Furthermore, the rapid stimulation of 17ßHSD activity after treatment with antiproliferative doses of 1,25-(OH)2D3 raises the possibility of a new nonclassical function for this pleiotropic hormone. Significant changes in 17ßHSD activity were observed with concentrations as low as 1 nM, suggesting physiological importance. This is emphasized by previous reports that have suggested that endogenously produced 1,25-(OH)2D3 may play a key role in regulating keratinocyte development in vivo (28). Cotreatment studies indicate that a mechanism linking 1,25-(OH)2D3, keratinocyte proliferation, and estrogen metabolism is likely to be very complex. For example, the actions of 1,25-(OH)2D3 were unaffected by simultaneous treatment with 9-cis-retinoic acid (the ligand for VDR’s heterodimer partner, retinoid X receptor). In contrast, combined treatment with DEXA enhanced the estrogenic and antiproliferative effects of 1,25-(OH)2D3. A precise mechanism for this action remains to be determined, but it is likely that this will involve other steroidogenic processes known to be influenced by glucocorticoids. It was particularly important to note that although treatment with DEXA alone up-regulated 17ßHSD activity, there was no parallel change in cell proliferation. This would suggest that the link between E2 inactivation and keratinocyte proliferation is not direct, although clearly this requires further investigation.

The Northern analyses shown in this study indicate that keratinocytes express inducible 1.3-kilobase (kb) mRNAs for both 17ßHSD I and II; increased expression of these transcripts provides the first direct evidence for regulation of estrogen metabolism by 1,25-(OH)2D3. These findings are in agreement with previous studies that have shown that the 1.3-kb mRNA for 17ßHSD I is up-regulated by retinoic acid, whereas the 2.3-kb transcript remains uninduced (22, 23). The likely gene target for the estrogenic effects of 1,25-(OH)2D3 is 17ßHSD II, which showed the most significant increase in expression after vitamin D treatment. A small change in 17ßHSD I was also observed in keratinocytes after treatment with 1,25-(OH)2D3, but there was no parallel up-regulation of reductive activity. Disparity between changes in mRNA expression and actual activity may, of course, simply reflect the relative abundance of one cofactor vs. another. In other words, although type I activity is NADPH dependent, type II activity is NAD dependent. Keratinocytes cultured as described in this system may thus be predisposed toward estrogen inactivation via more efficient NAD utilization.

The data presented here have extended the original concept of skin as a key source of steroid hormones, and in particular, we have highlighted the sensitive control of estrogen metabolism in keratinocytes. Recent studies of the effects of exogenously added estrogen and progesterone on keratinocyte proliferation suggested a biphasic effect, in which low doses of hormone (<0.1 nM) were stimulatory and higher doses (up to 100 nM) were inhibitory (29). The doses of E2 and E1 used in our study were relatively high (100 nM) and were growth inhibitory. Interestingly, the effect of E2 on keratinocyte proliferation was only marginal, whereas E1 was as potent as 10 nM 1,25-(OH)2D3. This is perhaps surprising in view of the established role of E1 as an inactive estrogen. Scatchard analyses of ER binding characteristics suggest that in keratinocytes, the receptor has a 2-fold greater affinity for E2 compared with E1, although maximal binding values were similar. This difference in Kd values agrees with the findings of recent studies that examined ligand binding specificity for ER {alpha} and ER ß (30). Our data, therefore, suggest that the product of estrogen metabolism in keratinocytes, E1, can itself have cell modulatory effects. These may be mediated via either classical or nonclassical estrogenic pathways.

These findings are clearly important when considering the development of normal skin function in vivo. The proliferation (and differentiation) of keratinocytes is likely to be heavily influenced by estrogenic activity. Thus, naturally occurring variations in estrogen and androgen levels may have a substantial impact on keratinocyte function in both the normal and the psoriatic epidermis. Previous studies have reported peaks of psoriasis at puberty and the menopause or improvement in psoriasis during pregnancy, followed by deterioration postpartum (31, 32). Regulation of local 17ßHSD and aromatase activity may also play a role in the homeostasis of hair follicles and sebaceous glands; disorders such as acne and androgen-dependent hair loss are known to involve abnormal control of androgen function. Furthermore, an established feature of the inherited disorder vitamin D-resistant rickets is partial or total alopecia (33). In summary, the data presented here confirm that there is considerable potential for local estrogen metabolism in skin. We have also shown that the expression and activity of estrogenic enzymes are regulated by 1,25-(OH)2D3, a known antipsoriasis agent. Future studies will seek to clarify the contributions of both aromatase and 17ßHSD activities to the development of normal skin as well as diseases such as psoriasis.


   Acknowledgments
 
We thank Drs. E. R. Simpson and W. E. Rainey for their help in initiating the RT-PCR studies.


   Footnotes
 
1 This work was supported by a project grant from the Psoriasis Association. Back

Received February 24, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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