HS Yoon et al. Effects of Estrogen on UV Responses
assembles in large clusters, most likely by extensive cross-linking. As a first effect of the depletion of non-desmosomal Dsg1, intercellular widening occurs. Then, when the desmosomes also become depleted of Dsg1, they shrink in size and number. When the amount of anti-Dsg1 IgG increases, the IgG will spread further upward into the higher layers, also leading to intercellular widening and desmosomal reduction there. Finally, when the IgG reaches the layers where Dsg3 is absent and cannot compensate for the loss of Dsg1, desmosomes will no longer be able to form stable structures and will melt away with subcorneal acantholysis as the final result. The effects of anti-Dsg3 antibodies on the epidermis differ from those of antiDsg1 antibodies. IgG directed against Dsg3 spreads through the epidermis and leads to clustering and depletion of Dsg3 throughout the Dsg3-expressing layers. This does not, however, lead to intercellular widening or a reduction in the size and number of the desmosomes. Apparently, loss of Dsg3 is less devastating than loss of Dsg1 to the desmosomes as they retain their normal shape. This fits with observations in patients with mdPV, who have blistering
of the mucous membranes but a perfectly healthy and strong skin. Although their skin is loaded with anti–cell surface IgG deposits, it does not blister, even when it is firmly rubbed to elicit the Nikolsky sign. Next, when antibodies directed against Dsg1 are also present in addition to antibodies against Dsg3 (as in mcPV), the depletion of Dsg1 will affect the desmosomes, which then start to shrink. As the desmosomes can no longer compensate for the loss of both Dsg1 and Dsg3, they will melt away in the lower layers, which eventually leads to suprabasal acantholysis. We therefore conclude that Dsg1, but not Dgs3, is necessary for preserving the normal size and number of desmosomes in the human epidermis and that loss of Dsg1 is conditional for developing cutaneous acantholysis in pemphigus. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS This study was supported by the J.P. Nater Fund and the J.K. de Cock Foundation.
Gerda van der Wier1, Hendri H. Pas1, Duco Kramer1, Gilles F.H. Diercks1 and Marcel F. Jonkman1
1
Center for Blistering Diseases, Department of Dermatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands E-mail: [email protected]
SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid
REFERENCES Green KJ, Simpson CL (2007) Desmosomes: new perspectives on a classic. J Invest Dermatol 127:2499–515 Holthofer B, Windoffer R, Troyanovsky S et al. (2007) Structure and function of desmosomes. Int Rev Cytol 264:65–163 Odland GF (1958) The fine structure of the interrelationship of cells in the human epidermis. J Biophys Biochem Cytol 4: 529–38 Oktarina DA, van der Wier G, Diercks GF et al. (2011) IgG-induced clustering of desmogleins 1 and 3 in skin of patients with pemphigus fits with the desmoglein nonassembly depletion hypothesis. Br J Dermatol 165:552–62 Scothern A, Garrod D (2008) Visualization of desmosomes in the electron microscope. Methods Cell Biol 88:347–66 Van der Wier G, Jonkman MF, Pas HH et al. (2012) Ultrastructure of acantholysis in pemphigus foliaceus re-examined from the current perspective. Br J Dermatol 167:1265–71 Waschke J (2008) The desmosome and pemphigus. Histochem Cell Biol 130:21–54
Endogenous Estrogen Exacerbates UV-Induced Inflammation and Photoaging in Mice Journal of Investigative Dermatology (2014) 134, 2290–2293; doi:10.1038/jid.2014.160; published online 24 April 2014
TO THE EDITOR Estrogen regulates diverse cellular functions and affects various tissues including the skin (Hall and Phillips, 2005). Studies have reported hormone replacement therapy to increase the content of collagen in sun-protected human skin (Brincat et al., 1987a, b). In addition, estrogen could stimulate dermal glycosaminoglycan and may have a benefi-
cial effect on dermal matrix in mice (Rock et al., 2012). However, topical estrogen therapy failed to induce procollagen expression in sun-exposed skin of the forearms and face, regardless of its collagen-stimulating effect in sun-protected buttock skin (Rittie et al., 2008). Furthermore, hormone therapy failed to lessen the mild-tomoderate changes in photoaged facial
Abbreviations: MMP, matrix metalloproteinase; OVX, ovariectomized Accepted article preview online 27 March 2014; published online 24 April 2014
2290 Journal of Investigative Dermatology (2014), Volume 134
skin in postmenopausal women (Phillips et al., 2008). Therefore, the effects of estrogen on skin exposed to UV irradiation are likely to be different from skin protected from UV irradiation. Although the influence of estrogen on photosensitive dermatoses, such as polymorphic light eruption, has been studied (Aubin, 2004), the role of estrogen in the normal physiological response of skin to UV irradiation such as sunburn and photoaging remains unclear. Thus, this study investigated the effects of endogenous estrogen on
HS Yoon et al. Effects of Estrogen on UV Responses
the response of skin to UV irradiation in vivo. Ovariectomized (OVX) immunocompetent hairless mice were used as a model of estrogen deficiency. Sevenweek-old female albino hairless mice (HOS:HR-1) were bilaterally OVX under
general anesthesia. For the sham operation, a similar procedure was performed except that ovaries and oviducts were not removed. Successful ovariectomy was verified by atrophy of the uterine horn at necropsy and suppression of GREB1 (gene regulated by estrogen in
OVX
Sham
Sham
breast cancer) mRNA expression in the skin (Supplementary Figure S1a, b online). Every UV irradiation was started 4 weeks after operation. We examined the effect of endogenous estrogen on wrinkle formation using a murine model of photoaging.
Sham+UV OVX+UV
*
Wrinkle grade
5
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†
4 †
3 2
NS
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OVX
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†
*
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40 35 30 25 20 15 10 5 0
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†
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*
*
30
†
† NS
†
25 Sham OVX
20
*
15 10 5 0
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UV–
UV+
UV+
* †
3 Relative ratio
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† pg per mg protein
pg per mg protein
*
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*
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IL-1β
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UV+
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Sham
350 300 250 200 150 100 50 0
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* 2 Sham OVX
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UV+
Figure 1. Response of skin to UV irradiation is reduced in ovariectomized (OVX) mice after single and repetitive UV exposures. (a) Photographs taken at 3 weeks post irradiation during the 8-week photoaging experiment. Every sham-operated mouse developed obvious erythema on their dorsum. However, six of eight OVX mice developed only subtle erythema and the remaining two showed less severe erythema than sham-operated mice. (b) Wrinkle formation at 8 weeks after repetitive UV exposures was less pronounced in OVX mice than in sham-operated mice (n ¼ 8 for each group). (c) CD45-positive leukocytes were counted in three different fields from each animal. Scale bar ¼ 100 mm. Data are presented as means±SEM (n ¼ 6–7 for each group). (d) Levels of IL-1b, IL-6, and tumor necrosis factor-a in skin lysates were measured by Multiplex Bead-Based Cytokine Assay Kit. (e) Representative western blots of the matrix metalloproteinase-13 level in skin lysates normalized to the b-actin level. Data are presented as means±SEM (n ¼ 6–7 for each group). *Po0.05 vs. time-matched sham-operated mice; w Po0.05 vs. nonirradiated counterpart; NS, not significant; Mann–Whitney U-test.
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HS Yoon et al. Effects of Estrogen on UV Responses
When the UV dose was increased to 300 mJ cm 2, sham-operated mice developed bright erythema on the dorsal skin. However, all OVX mice experienced less severe erythema than shamoperated mice (Figure 1a). After completion of the 8-week course of UV irradiation, the wrinkle score was significantly lower in the UV-irradiated OVX group (2.4±0.2) than in the UV-irradiated sham-operated group (3.9±0.2; Po0.05; Figure 1b). To investigate whether estrogen exacerbates UV-induced inflammation in the skin, a single dose of UVB (200 mJ cm 2) was administered to the dorsal area of mice under general anesthesia. Biopsies of the dorsal skin were obtained from mice at 48 hours after UV irradiation. The increase in skin fold thickness after UV irradiation, which is indicative of UV-induced skin edema, was less in OVX mice than
OVX
Number of CD45+ cells/field
OVX+E2
UV–
UV+
*
140
†
120 †
100 80
OVX
NS
60
OVX+E2
40 20 0 UV–
200 100 0
†
10
pg per mg protein
*
†
pg per mg protein
8 †
6 4
NS
2
20
UV+ UV–
Actin
†
*
10
OVX OVX+E2
5 0
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*
15
0 UV–
*
4 Relative ratio
pg per mg protein
†
TNF-α
*
NS 300
UV+
IL-6
IL-1β 400
contrast, in nonirradiated mice, the levels of IL-1b and TNF-a were significantly higher in OVX than in shamoperated mice (Figure 1d). Matrix metalloproteinase (MMP)-1 is a key regulator of collagen degradation in humans that is involved in photoaging (Fisher et al., 2002), and rodents lack the MMP-1 gene, which is functionally replaced by the MMP-13 gene (Schorpp et al., 1995). Although a single dose of UV irradiation significantly increased the expression of MMP-13 in sham-operated mice (2.6-fold increase vs. non-irradiated mice), OVX mice showed no increase in MMP-13 expression after UV exposure (Figure 1e). As estrogen is not the only ovarian hormone, we administered 17-b-estradiol-3-benzoate, an exogenous estrogen, to OVX mice to verify whether estrogen contributes to UV-induced inflammation in the skin. The compound was
in sham-operated mice (Supplementary Figure S1c online). Furthermore, there were fewer CD45-positive inflammatory cells (Figure 1c) and TUNEL-positive apoptotic cells (Supplementary Figure S1d online) in OVX mice than in sham-operated mice at 48 hours after UV irradiation. To determine the effect of estrogen on the expression of proinflammatory mediators, the protein levels of IL-1b, IL-6, and tumor necrosis factor (TNF)-a in skin lysates were quantified simultaneously using the Procarta Cytokine Assay Kit (Panomics, Fremont, CA). After acute UV irradiation, the levels of IL-1b, IL-6, and TNF-a were significantly lower in OVX mice than in sham-operated mice (131.60±20.20 vs. 256.10± 44.86 pg ml 1 for IL-1b; IL-6 12.28± 2.66 vs. 27.70±8.38 pg ml 1 for IL-6; and 11.07±1.74 vs. 22.90±3.82 pg ml 1 for TNF-a, respectively). By
UV+
†
3 NS OVX OVX+E2
2 1 0 UV–
UV+
Figure 2. Exogenous estrogen restores UV-induced responses compared with vehicle-treated ovariectomized (OVX) mice. (a) Exogenous estrogen (E2) increased UV-induced infiltration of CD45-positive inflammatory cells. Scale bar ¼ 100 mm. (b) Levels of IL-1b, IL-6, and tumor necrosis factor-a (TNF-a) in skin lysates were measured using Multiplex Bead-Based Cytokine Assay Kit. (c) Representative western blots of the matrix metalloproteinase-13 (MMP-13) level normalized to the b-actin level. Data are presented as means±SEM (n ¼ 6 for each group). *Po0.05 vs. time-matched vehicle-treated OVX mice; wPo0.05 vs. nonirradiated counterpart; NS, not significant; Mann–Whitney U-test.
2292 Journal of Investigative Dermatology (2014), Volume 134
HS Yoon et al. Effects of Estrogen on UV Responses
dissolved in 0.1 ml of olive oil and administered to mice at an approximate physiological dose (0.4 mg per mouse, Sigma-Aldrich, St Louis, MO; Jansson et al., 1990). Estrogen or its vehicle was administered subcutaneously immediately after surgery and once every 4 days. Exogenous estrogen restored OVXinduced changes, such as uterine atrophy, and reduced GREB1 mRNA expression in the skin (Supplementary Figure S2a, b online). Exogenous estrogen to OVX mice increased skin edema after UV irradiation compared with vehicletreated mice (Supplementary Figure S2c online). In addition, there were significantly more CD45-positive inflammatory cells (Figure 2a) and TUNELpositive apoptotic cells (Supplementary Figure S2d online) in estrogen-treated OVX mice than in vehicle-treated OVX mice after UV irradiation. We examined the expressions of several proinflammatory cytokines and MMP-13 and found that exogenous estrogen reversed the induction in IL-1b, IL-6, and MMP-13 levels in OVX mice after UV irradiation (Figure 2b and c). These results illustrate that exogenous estrogen can partially restore UV-induced responses and inflammation in the skin of OVX mice. Chronic administration of estrogen to OVX mice was previously reported to increase the expression of IL-1b, IL-6, and IL-12p40 by peritoneal macrophages in response to lipopolysaccharides ex vivo (Calippe et al., 2008). In addition, estrogen can stimulate MMP activity in the presence of specific inflammatory stimuli (Richette et al., 2004; Kapila et al., 2009). Estrogen has long been thought to be an antiaging modality by stimulating collagen synthesis (Draelos, 2005; Hall and Phillips, 2005). However, skin aging is more complicated than other organs, because skin is continuously exposed to UV irradiation. Regardless of the beneficial effects of estrogen on different organs (Giraud et al.,
2010), our results demonstrate that in vivo exposure to estrogen in the presence of UV irradiation increased the production of inflammatory mediators in the skin and negatively affected the physiological response of skin to UV irradiation. These findings could partly explain why estrogen treatment had no effect on photodamaged human skin and suggest that estrogen might not be an appropriate strategy in the prevention or treatment of photoaging. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (no. 20090092835).
Hyun-Sun Yoon1,2,3, Chang-yup Shin1,2, Yeon Kyung Kim1,2, Se-Rah Lee1,2 and Jin Ho Chung1,2,4 1
Department of Dermatology, Seoul National University College of Medicine, Seoul, Korea; 2 Institute of Human–Environment Interface Biology, Seoul National University, Seoul, Korea; 3Department of Dermatology, Seoul National University Boramae Hospital, Seoul, Korea and 4Institute on Aging, Seoul National University, Seoul, Korea E-mail: [email protected] SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid
REFERENCES Aubin F (2004) Why is polymorphous light eruption so common in young women? Arch Dermatol Res 296:240–1 Brincat M, Moniz CF, Kabalan S et al. (1987a) Decline in skin collagen content and metacarpal index after the menopause and its prevention with sex hormone replacement. Br J Obstet Gynaecol 94:126–9 Brincat M, Versi E, O’Dowd T et al. (1987b) Skin collagen changes in post-menopausal women receiving oestradiol gel. Maturitas 9:1–5 Calippe B, Douin-Echinard V, Laffargue M et al. (2008) Chronic estradiol administration
in vivo promotes the proinflammatory response of macrophages to TLR4 activation: involvement of the phosphatidylinositol 3-kinase pathway. J Immunol 180:7980–8 Draelos ZD (2005) Topical and oral estrogens revisited for antiaging purposes. Fertil Steril 84:291–2 Fisher GJ, Kang S, Varani J et al. (2002) Mechanisms of photoaging and chronological skin aging. Arch Dermatol 138:1462–70 Giraud SN, Caron CM, Pham-Dinh D et al. (2010) Estradiol inhibits ongoing autoimmune neuroinflammation and NFkappaB-dependent CCL2 expression in reactive astrocytes. Proc Natl Acad Sci USA 107:8416–21 Hall G, Phillips TJ (2005) Estrogen and skin: the effects of estrogen, menopause, and hormone replacement therapy on the skin. J Am Acad Dermatol 53:555–68 Jansson L, Mattsson A, Mattsson R et al. (1990) Estrogen induced suppression of collagen arthritis. V: Physiological level of estrogen in DBA/1 mice is therapeutic on established arthritis, suppresses anti-type II collagen T-cell dependent immunity and stimulates polyclonal B-cell activity. J Autoimmun 3: 257–70 Kapila S, Wang W, Uston K (2009) Matrix metalloproteinase induction by relaxin causes cartilage matrix degradation in target synovial joints. Ann N Y Acad Sci 1160:322–8 Phillips TJ, Symons J, Menon S et al. (2008) Does hormone therapy improve age-related skin changes in postmenopausal women? A randomized, double-blind, double-dummy, placebo-controlled multicenter study assessing the effects of norethindrone acetate and ethinyl estradiol in the improvement of mild to moderate age-related skin changes in postmenopausal women. J Am Acad Dermatol 59:397–404. e3 Richette P, Dumontier MF, Francois M et al. (2004) Dual effects of 17beta-oestradiol on interleukin 1beta-induced proteoglycan degradation in chondrocytes. Ann Rheum Dis 63: 191–9 Rittie L, Kang S, Voorhees JJ et al. (2008) Induction of collagen by estradiol: difference between sun-protected and photodamaged human skin in vivo. Arch Dermatol 144:1129–40 Rock K, Meusch M, Fuchs N et al. (2012) Estradiol protects dermal hyaluronan/versican matrix during photoaging by release of epidermal growth factor from keratinocytes. J Biol Chem 287:20056–69 Schorpp M, Mattei MG, Herr I et al. (1995) Structural organization and chromosomal localization of the mouse collagenase type I gene. Biochem J 308(Pt 1):211–7
www.jidonline.org 2293
assembles in large clusters, most likely by extensive cross-linking. As a first effect of the depletion of non-desmosomal Dsg1, intercellular widening occurs. Then, when the desmosomes also become depleted of Dsg1, they shrink in size and number. When the amount of anti-Dsg1 IgG increases, the IgG will spread further upward into the higher layers, also leading to intercellular widening and desmosomal reduction there. Finally, when the IgG reaches the layers where Dsg3 is absent and cannot compensate for the loss of Dsg1, desmosomes will no longer be able to form stable structures and will melt away with subcorneal acantholysis as the final result. The effects of anti-Dsg3 antibodies on the epidermis differ from those of antiDsg1 antibodies. IgG directed against Dsg3 spreads through the epidermis and leads to clustering and depletion of Dsg3 throughout the Dsg3-expressing layers. This does not, however, lead to intercellular widening or a reduction in the size and number of the desmosomes. Apparently, loss of Dsg3 is less devastating than loss of Dsg1 to the desmosomes as they retain their normal shape. This fits with observations in patients with mdPV, who have blistering
of the mucous membranes but a perfectly healthy and strong skin. Although their skin is loaded with anti–cell surface IgG deposits, it does not blister, even when it is firmly rubbed to elicit the Nikolsky sign. Next, when antibodies directed against Dsg1 are also present in addition to antibodies against Dsg3 (as in mcPV), the depletion of Dsg1 will affect the desmosomes, which then start to shrink. As the desmosomes can no longer compensate for the loss of both Dsg1 and Dsg3, they will melt away in the lower layers, which eventually leads to suprabasal acantholysis. We therefore conclude that Dsg1, but not Dgs3, is necessary for preserving the normal size and number of desmosomes in the human epidermis and that loss of Dsg1 is conditional for developing cutaneous acantholysis in pemphigus. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS This study was supported by the J.P. Nater Fund and the J.K. de Cock Foundation.
Gerda van der Wier1, Hendri H. Pas1, Duco Kramer1, Gilles F.H. Diercks1 and Marcel F. Jonkman1
1
Center for Blistering Diseases, Department of Dermatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands E-mail: [email protected]
SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid
REFERENCES Green KJ, Simpson CL (2007) Desmosomes: new perspectives on a classic. J Invest Dermatol 127:2499–515 Holthofer B, Windoffer R, Troyanovsky S et al. (2007) Structure and function of desmosomes. Int Rev Cytol 264:65–163 Odland GF (1958) The fine structure of the interrelationship of cells in the human epidermis. J Biophys Biochem Cytol 4: 529–38 Oktarina DA, van der Wier G, Diercks GF et al. (2011) IgG-induced clustering of desmogleins 1 and 3 in skin of patients with pemphigus fits with the desmoglein nonassembly depletion hypothesis. Br J Dermatol 165:552–62 Scothern A, Garrod D (2008) Visualization of desmosomes in the electron microscope. Methods Cell Biol 88:347–66 Van der Wier G, Jonkman MF, Pas HH et al. (2012) Ultrastructure of acantholysis in pemphigus foliaceus re-examined from the current perspective. Br J Dermatol 167:1265–71 Waschke J (2008) The desmosome and pemphigus. Histochem Cell Biol 130:21–54
Endogenous Estrogen Exacerbates UV-Induced Inflammation and Photoaging in Mice Journal of Investigative Dermatology (2014) 134, 2290–2293; doi:10.1038/jid.2014.160; published online 24 April 2014
TO THE EDITOR Estrogen regulates diverse cellular functions and affects various tissues including the skin (Hall and Phillips, 2005). Studies have reported hormone replacement therapy to increase the content of collagen in sun-protected human skin (Brincat et al., 1987a, b). In addition, estrogen could stimulate dermal glycosaminoglycan and may have a benefi-
cial effect on dermal matrix in mice (Rock et al., 2012). However, topical estrogen therapy failed to induce procollagen expression in sun-exposed skin of the forearms and face, regardless of its collagen-stimulating effect in sun-protected buttock skin (Rittie et al., 2008). Furthermore, hormone therapy failed to lessen the mild-tomoderate changes in photoaged facial
Abbreviations: MMP, matrix metalloproteinase; OVX, ovariectomized Accepted article preview online 27 March 2014; published online 24 April 2014
2290 Journal of Investigative Dermatology (2014), Volume 134
skin in postmenopausal women (Phillips et al., 2008). Therefore, the effects of estrogen on skin exposed to UV irradiation are likely to be different from skin protected from UV irradiation. Although the influence of estrogen on photosensitive dermatoses, such as polymorphic light eruption, has been studied (Aubin, 2004), the role of estrogen in the normal physiological response of skin to UV irradiation such as sunburn and photoaging remains unclear. Thus, this study investigated the effects of endogenous estrogen on
HS Yoon et al. Effects of Estrogen on UV Responses
the response of skin to UV irradiation in vivo. Ovariectomized (OVX) immunocompetent hairless mice were used as a model of estrogen deficiency. Sevenweek-old female albino hairless mice (HOS:HR-1) were bilaterally OVX under
general anesthesia. For the sham operation, a similar procedure was performed except that ovaries and oviducts were not removed. Successful ovariectomy was verified by atrophy of the uterine horn at necropsy and suppression of GREB1 (gene regulated by estrogen in
OVX
Sham
Sham
breast cancer) mRNA expression in the skin (Supplementary Figure S1a, b online). Every UV irradiation was started 4 weeks after operation. We examined the effect of endogenous estrogen on wrinkle formation using a murine model of photoaging.
Sham+UV OVX+UV
*
Wrinkle grade
5
OVX
†
4 †
3 2
NS
1 0 Sham
OVX
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UV–
UV+
†
*
200 150
40 35 30 25 20 15 10 5 0
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Sham OVX
†
100 NS 50 0 UV–
UV+ TNF-α
*
*
30
†
† NS
†
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20
*
15 10 5 0
UV–
UV+
UV–
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* †
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Actin
†
pg per mg protein
† pg per mg protein
pg per mg protein
*
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*
250
IL-6
IL-1β
UV–
UV+
OVX
Sham
350 300 250 200 150 100 50 0
OVX
Sham
UV–
* 2 Sham OVX
1 0 UV–
UV+
Figure 1. Response of skin to UV irradiation is reduced in ovariectomized (OVX) mice after single and repetitive UV exposures. (a) Photographs taken at 3 weeks post irradiation during the 8-week photoaging experiment. Every sham-operated mouse developed obvious erythema on their dorsum. However, six of eight OVX mice developed only subtle erythema and the remaining two showed less severe erythema than sham-operated mice. (b) Wrinkle formation at 8 weeks after repetitive UV exposures was less pronounced in OVX mice than in sham-operated mice (n ¼ 8 for each group). (c) CD45-positive leukocytes were counted in three different fields from each animal. Scale bar ¼ 100 mm. Data are presented as means±SEM (n ¼ 6–7 for each group). (d) Levels of IL-1b, IL-6, and tumor necrosis factor-a in skin lysates were measured by Multiplex Bead-Based Cytokine Assay Kit. (e) Representative western blots of the matrix metalloproteinase-13 level in skin lysates normalized to the b-actin level. Data are presented as means±SEM (n ¼ 6–7 for each group). *Po0.05 vs. time-matched sham-operated mice; w Po0.05 vs. nonirradiated counterpart; NS, not significant; Mann–Whitney U-test.
www.jidonline.org 2291
HS Yoon et al. Effects of Estrogen on UV Responses
When the UV dose was increased to 300 mJ cm 2, sham-operated mice developed bright erythema on the dorsal skin. However, all OVX mice experienced less severe erythema than shamoperated mice (Figure 1a). After completion of the 8-week course of UV irradiation, the wrinkle score was significantly lower in the UV-irradiated OVX group (2.4±0.2) than in the UV-irradiated sham-operated group (3.9±0.2; Po0.05; Figure 1b). To investigate whether estrogen exacerbates UV-induced inflammation in the skin, a single dose of UVB (200 mJ cm 2) was administered to the dorsal area of mice under general anesthesia. Biopsies of the dorsal skin were obtained from mice at 48 hours after UV irradiation. The increase in skin fold thickness after UV irradiation, which is indicative of UV-induced skin edema, was less in OVX mice than
OVX
Number of CD45+ cells/field
OVX+E2
UV–
UV+
*
140
†
120 †
100 80
OVX
NS
60
OVX+E2
40 20 0 UV–
200 100 0
†
10
pg per mg protein
*
†
pg per mg protein
8 †
6 4
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2
20
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Actin
†
*
10
OVX OVX+E2
5 0
UV–
UV–
UV+
UV+
OVX OVX+E2 OVX OVX+E2 MMP-13
*
15
0 UV–
*
4 Relative ratio
pg per mg protein
†
TNF-α
*
NS 300
UV+
IL-6
IL-1β 400
contrast, in nonirradiated mice, the levels of IL-1b and TNF-a were significantly higher in OVX than in shamoperated mice (Figure 1d). Matrix metalloproteinase (MMP)-1 is a key regulator of collagen degradation in humans that is involved in photoaging (Fisher et al., 2002), and rodents lack the MMP-1 gene, which is functionally replaced by the MMP-13 gene (Schorpp et al., 1995). Although a single dose of UV irradiation significantly increased the expression of MMP-13 in sham-operated mice (2.6-fold increase vs. non-irradiated mice), OVX mice showed no increase in MMP-13 expression after UV exposure (Figure 1e). As estrogen is not the only ovarian hormone, we administered 17-b-estradiol-3-benzoate, an exogenous estrogen, to OVX mice to verify whether estrogen contributes to UV-induced inflammation in the skin. The compound was
in sham-operated mice (Supplementary Figure S1c online). Furthermore, there were fewer CD45-positive inflammatory cells (Figure 1c) and TUNEL-positive apoptotic cells (Supplementary Figure S1d online) in OVX mice than in sham-operated mice at 48 hours after UV irradiation. To determine the effect of estrogen on the expression of proinflammatory mediators, the protein levels of IL-1b, IL-6, and tumor necrosis factor (TNF)-a in skin lysates were quantified simultaneously using the Procarta Cytokine Assay Kit (Panomics, Fremont, CA). After acute UV irradiation, the levels of IL-1b, IL-6, and TNF-a were significantly lower in OVX mice than in sham-operated mice (131.60±20.20 vs. 256.10± 44.86 pg ml 1 for IL-1b; IL-6 12.28± 2.66 vs. 27.70±8.38 pg ml 1 for IL-6; and 11.07±1.74 vs. 22.90±3.82 pg ml 1 for TNF-a, respectively). By
UV+
†
3 NS OVX OVX+E2
2 1 0 UV–
UV+
Figure 2. Exogenous estrogen restores UV-induced responses compared with vehicle-treated ovariectomized (OVX) mice. (a) Exogenous estrogen (E2) increased UV-induced infiltration of CD45-positive inflammatory cells. Scale bar ¼ 100 mm. (b) Levels of IL-1b, IL-6, and tumor necrosis factor-a (TNF-a) in skin lysates were measured using Multiplex Bead-Based Cytokine Assay Kit. (c) Representative western blots of the matrix metalloproteinase-13 (MMP-13) level normalized to the b-actin level. Data are presented as means±SEM (n ¼ 6 for each group). *Po0.05 vs. time-matched vehicle-treated OVX mice; wPo0.05 vs. nonirradiated counterpart; NS, not significant; Mann–Whitney U-test.
2292 Journal of Investigative Dermatology (2014), Volume 134
HS Yoon et al. Effects of Estrogen on UV Responses
dissolved in 0.1 ml of olive oil and administered to mice at an approximate physiological dose (0.4 mg per mouse, Sigma-Aldrich, St Louis, MO; Jansson et al., 1990). Estrogen or its vehicle was administered subcutaneously immediately after surgery and once every 4 days. Exogenous estrogen restored OVXinduced changes, such as uterine atrophy, and reduced GREB1 mRNA expression in the skin (Supplementary Figure S2a, b online). Exogenous estrogen to OVX mice increased skin edema after UV irradiation compared with vehicletreated mice (Supplementary Figure S2c online). In addition, there were significantly more CD45-positive inflammatory cells (Figure 2a) and TUNELpositive apoptotic cells (Supplementary Figure S2d online) in estrogen-treated OVX mice than in vehicle-treated OVX mice after UV irradiation. We examined the expressions of several proinflammatory cytokines and MMP-13 and found that exogenous estrogen reversed the induction in IL-1b, IL-6, and MMP-13 levels in OVX mice after UV irradiation (Figure 2b and c). These results illustrate that exogenous estrogen can partially restore UV-induced responses and inflammation in the skin of OVX mice. Chronic administration of estrogen to OVX mice was previously reported to increase the expression of IL-1b, IL-6, and IL-12p40 by peritoneal macrophages in response to lipopolysaccharides ex vivo (Calippe et al., 2008). In addition, estrogen can stimulate MMP activity in the presence of specific inflammatory stimuli (Richette et al., 2004; Kapila et al., 2009). Estrogen has long been thought to be an antiaging modality by stimulating collagen synthesis (Draelos, 2005; Hall and Phillips, 2005). However, skin aging is more complicated than other organs, because skin is continuously exposed to UV irradiation. Regardless of the beneficial effects of estrogen on different organs (Giraud et al.,
2010), our results demonstrate that in vivo exposure to estrogen in the presence of UV irradiation increased the production of inflammatory mediators in the skin and negatively affected the physiological response of skin to UV irradiation. These findings could partly explain why estrogen treatment had no effect on photodamaged human skin and suggest that estrogen might not be an appropriate strategy in the prevention or treatment of photoaging. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (no. 20090092835).
Hyun-Sun Yoon1,2,3, Chang-yup Shin1,2, Yeon Kyung Kim1,2, Se-Rah Lee1,2 and Jin Ho Chung1,2,4 1
Department of Dermatology, Seoul National University College of Medicine, Seoul, Korea; 2 Institute of Human–Environment Interface Biology, Seoul National University, Seoul, Korea; 3Department of Dermatology, Seoul National University Boramae Hospital, Seoul, Korea and 4Institute on Aging, Seoul National University, Seoul, Korea E-mail: [email protected] SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid
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