Sex Differences in Photoprotective Responses to 1,25-Dihydroxyvitamin D3 in Mice Are Modulated by the Estrogen Receptor-β

Abstract

Susceptibility to photoimmune suppression and photocarcinogenesis is greater in male than in female humans and mice and is exacerbated in female estrogen receptor-beta knockout (ER-β-/-) mice. We previously reported that the active vitamin D hormone, 1,25-dihydroxyvitamin D3 (1,25(OH)2D), applied topically protects against the ultraviolet radiation (UV) induction of cutaneous cyclobutane pyrimidine dimers (CPDs) and the suppression of contact hypersensitivity (CHS) in female mice. Here, we compare these responses in female versus male Skh:hr1 mice, in ER-β-/-/-- versus wild-type C57BL/6 mice, and in female ER-blockaded Skh:hr1 mice. The induction of CPDs was significantly greater in male than female Skh:hr1 mice and was more effectively reduced by 1,25(OH)2D in female Skh:hr1 and C57BL/6 mice than in male Skh:hr1 or ER-β-/- mice, respectively. This correlated with the reduced sunburn inflammation due to 1,25(OH)2D in female but not male Skh:hr1 mice. Furthermore, although 1,25(OH)2D alone dose-dependently suppressed basal CHS responses in male Skh:hr1 and ER-β-/- mice, UV-induced immunosuppression was universally observed. In female Skh:hr1 and C57BL/6 mice, the immunosuppression was decreased by 1,25(OH)2D dose-dependently, but not in male Skh:hr1, ER-β-/-, or ER-blockaded mice. These results reveal a sex bias in genetic, inflammatory, and immune photoprotection by 1,25(OH)2D favoring female mice that is dependent on the presence of ER-β.

Keywords: 1α,25-dihydroxyvitaminD3; DNA damage; ER-β knockout; cyclobutane pyrimidine dimers; edema; female vs. male mice; photoimmune suppression; photoprotection.

Figure 1

Protection against CPDs at 3 h post-SSUV by 1,25(OH)2D in female and male Skh:hr1 mice. Mice were exposed to 1 × 2.5 MED of SSUV followed immediately with topical vehicle or 1,25(OH)2D. (a) Skin sections, SSUV + vehicle or 1,25(OH)2D (23 pmol/cm²), stained immunohistochemically for CPD-positive nuclei. (b) Quantitation of the average percentage epidermal CPD-positive nuclear staining (n = 3 mice, 9 sections per group) following SSUV + vehicle or 1,25(OH)2D (4.6, 23 or 46 pmol/cm²). CPD staining in unirradiated skin was <0.1% positive nuclei. Results are representative of at least 2 experiments. *** significantly different from vehicle controls, p < 0.001. ###, @@@ significantly different among each concentration tested in the group, p < 0.001. <<< significantly different between females and males treated with vehicle, p < 0.001. <<< significantly different between females and males treated with the same concentration of 1,25(OH)2D, p < 0.001.

Figure 1

Protection against CPDs at 3 h post-SSUV by 1,25(OH)2D in female and male Skh:hr1 mice. Mice were exposed to 1 × 2.5 MED of SSUV followed immediately with topical vehicle or 1,25(OH)2D. (a) Skin sections, SSUV + vehicle or 1,25(OH)2D (23 pmol/cm²), stained immunohistochemically for CPD-positive nuclei. (b) Quantitation of the average percentage epidermal CPD-positive nuclear staining (n = 3 mice, 9 sections per group) following SSUV + vehicle or 1,25(OH)2D (4.6, 23 or 46 pmol/cm²). CPD staining in unirradiated skin was <0.1% positive nuclei. Results are representative of at least 2 experiments. *** significantly different from vehicle controls, p < 0.001. ###, @@@ significantly different among each concentration tested in the group, p < 0.001. <<< significantly different between females and males treated with vehicle, p < 0.001. <<< significantly different between females and males treated with the same concentration of 1,25(OH)2D, p < 0.001.

Figure 2

Protection against SSUV-induced inflammatory edema by 1,25(OH)2D in female and male Skh:hr1 mice. Mice (n = 5 per group) were exposed to 1 MED of SSUV for 3 consecutive days followed daily with topical vehicle, or 1,25(OH)2D (23 pmol/cm²). Average daily dorsal skinfold measurements ± SEM indicate edema compared to non-irradiated skinfold thickness. The data are representative of two independent experiments. *** matching symbols indicate statistically significant differences, p < 0.001.

Figure 3

The effect of 1,25(OH)2D on the CHS reaction to oxazolone in female and male Skh:hr1 mice. (a) Effect of 1,25(OH)2D alone. Mice (n = 5 per group) were treated topically on the dorsal skin with vehicle or 1,25(OH)2D (23 or 46 pmol/cm²) for 3 consecutive days. They were sensitized a week later, challenged after a further week, and the CHS was assessed by ear swelling at 18 h. Data are pooled from four independent experiments. (SEM are too small to be visible on the graph). # significantly different from vehicle females p < 0.05. *** from vehicle males p < 0.001; * p < 0.05. ^^^ from 1,25(OH)2D (46 ρmol/cm²) females p < 0.001. (b) Effect of 1,25(OH)2D (23 pmol/cm²) post-SSUV. Mice were exposed to 1 MED of SSUV followed immediately with topical vehicle or 1,25(OH)2D (23 pmol/cm²) for 3 consecutive days, then sensitized one week later followed by challenge after a further week. Data are representative of two independent experiments (n = 5 mice per group). *** significantly different from non-UV controls p < 0.001. # from UV vehicle females p < 0.05. ### from UV vehicle males p < 0.001.

Figure 3

The effect of 1,25(OH)2D on the CHS reaction to oxazolone in female and male Skh:hr1 mice. (a) Effect of 1,25(OH)2D alone. Mice (n = 5 per group) were treated topically on the dorsal skin with vehicle or 1,25(OH)2D (23 or 46 pmol/cm²) for 3 consecutive days. They were sensitized a week later, challenged after a further week, and the CHS was assessed by ear swelling at 18 h. Data are pooled from four independent experiments. (SEM are too small to be visible on the graph). # significantly different from vehicle females p < 0.05. *** from vehicle males p < 0.001; * p < 0.05. ^^^ from 1,25(OH)2D (46 ρmol/cm²) females p < 0.001. (b) Effect of 1,25(OH)2D (23 pmol/cm²) post-SSUV. Mice were exposed to 1 MED of SSUV followed immediately with topical vehicle or 1,25(OH)2D (23 pmol/cm²) for 3 consecutive days, then sensitized one week later followed by challenge after a further week. Data are representative of two independent experiments (n = 5 mice per group). *** significantly different from non-UV controls p < 0.001. # from UV vehicle females p < 0.05. ### from UV vehicle males p < 0.001.

Figure 4

Effect of ICI 182,780 on protection by1,25(OH)2D against the SSUV-induced suppression of CHS in female Skh:hr1 mice. Mice (n = 5 per group) were pretreated topically with ICI 182,780 or vehicle for 2 weeks, then exposed to 1 MED of SSUV, followed immediately by topical vehicle or 1,25(OH)2D (23 pmol/cm²) on 3 consecutive days. ICI 182,780 or vehicle treatment was continued until sensitization with oxazolone one week later. Mice were challenged after a further week, and CHS assessed by ear swelling at 18. h. Data are representative of two independent experiments. *** significantly different from relevant non-UV control p < 0.001; ## p < 0.01.

Figure 5

The effect of 1,25(OH)2D on the CHS reaction to oxazolone in female C57BL/6 wild-type and ER-β−/− mice. Mice (n = 5 per group) were exposed to 1 × 2.5 MED of SSUV (or not) and treated topically immediately with vehicle or 1,25(OH)2D (0.46, 4.6, 23, or 46 pmol/cm². They were sensitized one week later, then challenged after a further week. Ear swelling was measured 18 h post-challenge. (a) Mean ear swelling in non-irradiated and SSUV-irradiated C57BL/6 wild-type mice. *** significantly different from relevant non-UV control p < 0.001. ### from UV vehicle p < 0.001; # p < 0.05. (b) Mean ear swelling in non-irradiated and SSUV-irradiated ER-β−/− mice. Data are representative of two independent experiments. *** significantly different from relative non-UV group p < 0.001; ### from UV vehicle p < 0.001; ## p < 0.01.