Effects of oestrogen agonists on human dermal fibroblasts in an in vitro wounding assay

Abstract

Oestrogen and dehydroepiandrosterone (DHEA) improve wound healing, but circulating levels decline significantly with age. Recently, the selective oestrogen receptor modulators (SERMs) tamoxifen and raloxifene have been shown to improve age-associated impaired wound healing. Therefore, we have evaluated the effects of 17b-oestradiol, ERa and ERb agonists, tamoxifen, raloxifene and DHEA on human dermal fibroblasts using an in vitro wound assay. An ERa agonist, 17b-oestradiol and DHEA all significantly accelerated cell migration; the DHEA effect was blocked with an aromatase inhibitor. Tamoxifen, raloxifene and DHEA all significantly increased DNA synthesis; the DHEA stimulatory effect was reversed by an aromatase inhibitor. This study demonstrates that 17b-oestradiol, an ERa agonist, tamoxifen, raloxifene and DHEA (following conversion to oestrogen) all have significant effects on human fibroblasts, the key mesenchymal cell involved in the wound healing process. Further understanding of the mechanisms involved may have important implications for the management of age-related impaired wound healing.Key words: DHEA -oestrogen -raloxifene -SERMs -tamoxifen Please cite this paper as: Effects of oestrogen agonists on human dermal fibroblasts in an in vitro wounding assay. Experimental Dermatology 2009; 18: 988-990.

The role of estrogen deficiency in skin ageing and wound healing

“…Despite use of SERMs in age-and menopauseassociated pathologies (reviewed in Pickar et al 2010) knowledge of SERM activity in the skin is severely lacking. In vitro both tamoxifen and raloxifene stimulate fibroblast proliferation, but fail to promote fibroblast migration (Stevenson et al 2009). Preliminary studies in post-menopausal skin indicate increased elasticity and collagen content following raloxifene treatment (Sumino et al 2009) and improved dermal vascularisation and increased epidermal thickness following genistein treatment (Moraes et al 2009).…”

Intracrine sex steroid synthesis and signaling in human epidermal keratinocytes and dermal fibroblasts

“…Briefly, cells were seeded into 6-well plates at a density of 1 3 10 5 per well and grown to confluence, then mechanically wounded as previously described (28,30,37). After washing with PBS, they were incubated with serum-free medium containing 0.5 mCi [1b 3 H]-androstenedione (NEN Perkin Elmer, Waltham, MA, USA) for 2 or 24 h at 37°C.…”

“…Previous dose-response assays using this technique have shown that a range of concentrations of 17b-estradiol (10 27 -10 29 M) and DHEA (10 25 -10 28 M) all stimulated cell migration to a similar level (37,39). The ability of the cells to metabolize these steroids was determined by including 100 nM Arimidex (to block aromatase activity) in the presence and absence of DHEA or testosterone, and 100 nM STX64 (to block STS activity) in the presence and absence of DHEA-S. Migration was quantitated as previously described (28,30). The addition of 10 mg/ml of mitomycin C to block proliferation did not alter the migration of keratinocytes (n = 3) or fibroblasts (n = 5; data not shown).…”

“…The effect of steroid hormones on fibroblast and keratinocyte migration was assessed using the scratch wound assay as previously described (28,30,37). Mechanically wounded cells were assayed in phenol red-free, serum-free medium containing 10 mg/ml mitomycin C (to block proliferation) and the following steroids: DHEA-S (10 mM), DHEA (10 mM), testosterone (50 nM), 17b-estradiol (1 nM), or vehicle control (0.0001% ethanol).…”

“…For RNA extraction, each sample was transported in RNA later or frozen in liquid nitrogen; for cell culture, biopsies were kept in DMEM (GIBCO, Invitrogen, Milan, Italy, stored at 4°C and processed within 24 h of surgery. Primary cultures of dermal fibroblasts and epidermal keratinocytes were established as described previously (28)(29)(30). Briefly, small pieces of skin were washed in PBS containing amphotericin B (750 ml/ml); excess fat and dermis were removed before incubating overnight in dispase (2.4 U/ml) at 4°C, then at 37°C for 1 h. The epidermis was separated from the dermis with forceps, washed, and incubated with 0.05% trypsin/EDTA at 37°for 5-6 min.…”