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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 18, 2004 as doi:10.1096/fj.03-1088fje. |
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* Department of Dermatology and
Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts, USA
1 Correspondence: Boston University School of Medicine, Department of Dermatology, J-508 609 Albany Street Boston, MA 02118-2394, USA. E-mail: bgilchre{at}bu.edu
SPECIFIC AIMS
The role of estrogens in homeostasis of nonreproductive tissues, including skin, is under intensive investigations. Estrogen affects target tissue function by binding to intracellular nuclear receptors characterized as estrogen receptor-
(ER-) and estrogen receptor-ß (ER-ß) that regulate gene transcription. An increasing body of evidence suggests that estrogens also elicit rapid nontranscriptional effects through membrane associated ERs, identified as ER-. We investigated effects of estradiol on proliferation of neonatal keratinocytes, expression of estrogen receptor isoforms in these cells, and signaling mechanisms by which estradiol mediates its activity.
PRINCIPAL FINDINGS
1. Equilibrium binding of estradiol to keratinocyte cellular extract
Estradiol bound to keratinocytes’ total cellular extracts with high affinity and limited capacity. Scatchard analysis of binding data demonstrated a Kd of 5.2 nM and maximum binding sites of 14.2 fmol/mg protein.
2. Estradiol induces estrogen receptor expression in keratinocytes
Subconfluent cultures were stimulated with increasing doses of estradiol for 48 h and expression of ERs was determined by Western blot analysis. ER- level, barely detectable at baseline, increased with physiologic doses of estradiol (Fig. 1
A, B). ER-ß was constitutively expressed, and its induction appeared less dependent on estradiol dose (Fig. 1C, D
).
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3. Estradiol stimulates keratinocyte proliferation
To determine estradiol effect on keratinocytes, 17ß-estradiol (10–10M) was added to medium and 48 h later 3H-thymidine was added for an additional 24 h. 3H-thymidine uptake was significantly higher (P<0.03) in cultures stimulated with estradiol and the effect was abrogated when pure anti-estrogen ICI 182,780, a compound that competes with estradiol for binding to estrogen receptors, was added to culture at the time of estradiol supplementation, demonstrating that estrogen effect on keratinocyte proliferation requires the presence of free estrogen receptors.
4. Estradiol induces phosphorylation and activation of ERK1 and ERK2
To determine the effect of estradiol on intracellular signaling pathways, keratinocytes were maintained in basal medium lacking growth factors and hormones for 24 h. Estradiol (0.1 nM) was then added to medium and ERK1 and ERK2 protein levels and phosphorylation status were determined (Fig. 2
A, B). Within 15 min the phosphorylated level of ERK1 and ERK2 was induced with maximal increases above baseline levels 30 and 60 min, respectively, after stimulation. As expected, there were no modulations in ERK1 and ERK2 protein levels as a result of estradiol stimulation (Fig. 2A, B
).
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5. Estradiol induces expression of c-fos and c-jun
To determine the effect of estradiol on c-fos and c-jun, keratinocytes were stimulated as above and processed for Northern blot analysis. Estradiol (0.1 nM) up-regulated c-fos and c-jun mRNA levels above their respective baseline levels within 30 min of stimulation.
6. Estradiol up-regulates cyclin D1 protein level
To determine the effect of estradiol on cyclin D1 level, keratinocytes were maintained as above and total cellular proteins were harvested up to 24 h after stimulation. Estradiol (0.1 nM) up-regulated cyclin D1 as early as 4 h after stimulation, with maximal induction observed 8 h after stimulation.
CONCLUSIONS AND SIGNIFICANCE
The present study demonstrates that normal newborn human keratinocytes express functional ERs that bind estradiol with a Kd of 5.2 nM. This high-affinity Kd falls within the range reported for estradiol binding to its receptors in various target tissues. Using homogenates derived from facial skin of postmenopausal women, Hasselquist et al. reported presence of high-affinity estradiol binding sites. However, the latter used whole skin homogenates containing keratinocytes, fibroblasts and other minor cellular components. Our study is first to document high-affinity estradiol binding in a pure population of epidermal keratinocytes. Binding of estradiol to ER in keratinocytes is of limited capacity with an estimated Bmax of 14.2 fmol/mg of protein. This value is 
5-fold less than that reported in reproductive tissues such as the uterus. It is well documented that in nonreproductive tissues such as bone and the cardiovascular system the total number of estradiol binding sites is often lower than that of reproductive tissues.
We found that normal neonatal foreskin-derived human keratinocytes express both ER- and ER-ß, although baseline level of ER- was very low. Using normal human neonatal foreskin keratinocytes, Kanda et al. reported presence of ER-ß but not ER-. The latter study examined 20% of the amount of proteins we used, a likely explanation of it not being detected. In another study, using immunohistochemical techniques and scalp skin of both males and females, Thornton demonstrated strong nuclear immunostaining for ER-ß in the epidermis, but no staining for ER-. The level of ER- may be below the level of detection for this technique, as even in the more sensitive Western blot analysis ER- was hardly detectable at baseline. Alternatively, estrogen receptor type in the epidermis may vary with donor age or body site.
We found that estradiol stimulates synthesis of its receptors. Particularly, there was an effect on ER-, a receptor that was hardly detectable at baseline. Bayard et al. reported modulations in ER levels in human endometrium during the menstrual cycle and in MCF-7 breast cancer cells, and similar to keratinocytes, physiologic estradiol concentrations (10–11–10–10 M) elevate ER- levels. Our findings suggest that estrogens may maintain or even increase keratinocyte responsiveness to the hormone and that estrogen depletion, such as that occurring in postmenopausal women, may adversely affect keratinocyte proliferation.
Regarding estrogen effect on keratinocyte proliferation, there are two previous conflicting reports. Using cultured epidermal keratinocytes from adult skin, Urano et al. have demonstrated that physiologic concentrations of estradiol (0.01–100 nM) increased keratinocyte proliferation. In contrast, Tammi, using skin from postmenopausal women maintained in organ culture reported that estradiol (1 and 100 nM) had no effect on keratinocyte proliferation. The differing effect of estradiol on cultured keratinocytes compared with keratinocytes in organ culture may be explained by the differing culture conditions. Organ culture is an in vitro model closely mimicking in vivo intact epidermis in which few keratinocytes are rapidly proliferating, whereas keratinocytes in culture are more similar to the more proliferative keratinocytes of the wound bed. Thus, our results suggest that during wound healing, estrogen could increase keratinocyte proliferation and facilitate the re-epithelialization process. It was reported that estradiol plays a crucial role in cutaneous wound healing and that repair is significantly delayed in its absence. Ashcroft et al. reported that topical estrogens accelerate wound healing in aged patients, and two additional studies have demonstrated that hormone replacement therapy prevented development of both pressure ulcers and venous ulcers in postmenopausal women.
We have also shown that within 15 min estradiol induces ERK1and ERK2 activation as determined by phosphorylation of proteins. It was reported in uterus and mammary gland that estradiol stimulates production and secretion of growth factors including epidermal growth factor, transforming growth factor-, and insulin-like growth factor 1, leading to ERK phosphorylation and autocrine growth stimulation. We do not believe that in keratinocytes growth factor synthesis and/or secretion led to ERK1 and ERK2 phosphorylation, as these enzymes became phosphorylated within minutes of estradiol supplementation, whereas an indirect estradiol effect would require at least a few hours, as previously reported. Our findings suggest that estradiol directly activates the MAPK pathway in keratinocytes, consistent with findings demonstrating that estradiol effects in nonreproductive tissues are mediated through membrane associated receptors. Our findings are also in agreement with a recent publication showing presence of ER in keratinocyte membrane.
We have also observed up-regulation of early immediate genes c-fos and c-jun and induction of cyclin D1 protein. Although ERK1- and ERK2-mediated signaling is reported to up-regulate c-fos and c-jun transcription, promoters of these genes also contain an ERE. In our study, c-fos and c-jun were induced within 30 min of estradiol stimulation, a time frame consistent for induction either by phosphorylated ERK1 or by estradiol-ER complexes binding to c-fos and c-jun ERE or by both. Similarly, although cyclin D1 promoter does not display an ERE, it contains an AP-1 binding site. Without further experiments we cannot conclusively determine whether c-fos, c-jun, and cyclin D1 up-regulation in keratinocytes is the result of genomic signaling, nongenomic signaling, or both.
We have shown that keratinocytes are estrogen-responsive cells that display high estradiol binding affinity and express both estrogen receptors. Our study suggests that estrogen modulates epidermal function in premenopausal women and may improve skin quality and function in postmenopausal women.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1088fje; doi: 10.1096/fj.03-1088fje
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