Induction of Collagen by Estradiol Difference Between Sun-Protected and Photodamaged Human Skin in Vivo

STUDY Induction of Collagen by Estradiol Difference Between Sun-Protected and Photodamaged Human Skin In Vivo

Laure Rittie´, PhD; Sewon Kang, MD; John J. Voorhees, MD; Gary J. Fisher, PhD

Objective: To evaluate the effectiveness of topical es- protected aged hip skin in postmenopausal women and, tradiol in stimulating collagen I and III production in natu- to a lesser extent, in age-matched men. Surprisingly, no rally aged and photoaged human skin of postmeno- significant changes in production were observed in women pausal women and age-matched men. or men after 2-week estradiol treatment of photoaged forearm or face skin, despite similar expression of estrogen Design: Vehicle-controlled treatment followed by bio- receptors (ER-␣, ER-␤, and GPR30) in aged and photo- chemical and immunohistochemical analyses of skin bi- aged skin. Estradiol treatment induced the estrogen- opsy specimens. responsive gene GREB1, indicating that penetration of topical estradiol and genomic response to estrogen were Setting: Academic referral center. similar in the 3 anatomic sites. Participants: Seventy healthy volunteers (40 postmeno- Conclusions: Two-week topical estradiol treatment pausal women with a mean age of 75 years, and 30 men with a mean age of 75 years) with photodamaged skin. stimulates collagen production in sun-protected hip skin, but not in photoaged forearm or face skin, in postmeno- Interventions: Topical application of estradiol, 0.01%, pausal women and aged-matched men. These findings 0.1%, 1%, or 2.5% or vehicle on aged or photoaged skin, suggest that menopause-associated estrogen decline is in- with biopsy specimens taken after last treatment. volved in reduced collagen production in sun-protected skin. Interestingly, alterations induced by long-term sun Main Outcome Measures: De novo synthesis of col- exposure hinder the ability of topical 2-week estradiol lagen by quantitative polymerase chain reaction, immu- to stimulate collagen production in aged skin. nohistochemistry, and enzyme-linked immunosorbent assay. Trial Registration: clinicaltrials.gov Identifier: NCT00113100 Results: Topical estradiol increased procollagen I and III messenger RNA and collagen I protein levels in sun- Arch Dermatol. 2008;144(9):1129-1140

KIN AGING IS ASSOCIATED WITH structural protein of the dermis, and it pro- reduced skin function, in- vides strength and resiliency to the skin. Col- creased skin fragility, and lagen I is primarily produced by dermal fi- compromised wound heal- broblasts. Collagen production is regulated ing.1 On areas of the body that by a variety of mediators including growth are often not covered by clothing (ie, face, factors, cytokines, hormones, and mechani- S 4 nape of the neck, hands, and forearms), cal tension. In naturally aged skin, the com- long-term UV exposure from the sun dam- bination of reduced fibroblast number, re- ages the skin, causing it to look prema- duced fibroblast metabolic activity, and loss turely old. Photoaging is the main pro- of mechanical tension results in a 70% de- cess responsible for the worsening crease in new collagen (procollagen) pro- appearance of aged skin. duction by fibroblasts.5 In photoaged skin, Clinical signs of natural aging include loss of mechanical tension appears to be pri- fine wrinkles, skin laxity, and sagging, while marily responsible for reduced procolla- photoaged skin appears dry, with coarse gen production.6-8 This loss of mechanical wrinkles and uneven pigmentation. Histo- tension is due to accumulation of frag- logically, epidermis is thin in aged skin and mented collagen, which is generated by re- rather irregular in photoaged skin.2 How- peated exposures to UV radiation (re- ever, both processes share major biochemi- viewed by Rittie´etal9). Overall, decreased Author Affiliations: cal features such as reduced collagen con- quantity and quality of collagen fibers in the Department of Dermatology, tent in the dermis, resulting from increased dermis are associated with aged appear- University of Michigan Medical collagen degradation and decreased pro- ance of human skin. Hence, antiaging thera- School, Ann Arbor. collagen synthesis.3 Collagen I is the major pies include drugs (such as tretinoin) or pro-

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©2008 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 cedures (such as carbon dioxide laser resurfacing) that topical estradiol treatment increased procollagen I and III stimulate collagen production in human skin9,10 and thereby expression in sun-protected hip skin in a dose-dependent improve skin strength, resiliency, and function. manner. Surprisingly, 2-week estradiol treatment had no Estrogens exert a profound influence on skin, as high- effect on procollagen synthesis in photoaged skin (fore- lighted by regressive cutaneous changes that occur after arm or face). We found that ERs were expressed at similar menopause in women. Diminution of circulating estro- levels in sun-protected and photoaged skin and that estra- gens that accompanies menopause is associated with in- diol penetration and genomic activity were similar in all creased skin dryness11 and slackness,12 and decreased skin skin sites, as assessed by quantification of estrogen target elasticity,13 dermal thickness of thigh skin,14 and skin col- gene expression. Taken together, our results demonstrate lagen content as measured in thigh,15 abdomen,16 and pu- that estradiol-mediated collagen stimulation is indirect and bis.17 Conversely, various studies have shown that es- that alterations induced by long-term sun exposure hinder trogen therapy (ET) users have better skin hydration18-21 the ability of topical estradiol to stimulate collagen pro- and elasticity,20-23 higher density of collagen fibers,24 fewer duction in aged human skin in vivo. fine wrinkles,19,21 and thicker skin20,25 than nonusers. How- ever, other studies have failed to show any effect of ET METHODS on the prevalence or severity of cutaneous signs of meno- 11 pause on the photoaged face. Reasons for this discrep- HUMAN SUBJECT DESCRIPTION ancy are unknown. Effects of estrogens on skin collagen content are also not All procedures involving human subjects were approved by the clear. While some studies demonstrated that ET significantly University of Michigan Institutional Review Board, and all sub- increased skin and dermal thickness in postmenopausal jects provided written informed consent. Healthy volunteers, women26-28 (as measured on thigh and abdomen), a study not taking ET, were enrolled in the study with no racial or eth- of 3875 women did not demonstrate any impact of ET on nic distinction. Participants’ age distribution, degree of pho- postmenopausal-associatedskinatrophy19 (unspecifiedsite). todamage, and years after menopause are given in Table 1. Similarly, some studies demonstrated greater skin collagen content after ET15,16,24,27-29 (as measured on thigh, lower ab- ESTRADIOL TREATMENT AND domen, upper inner arm, and unspecified site), while oth- TISSUE PROCUREMENT ers did not show any beneficial effects of ET on collagen syn- thesis30,31 (upper arm and unspecified site). Volunteers were treated with estradiol and vehicle (95% ethanol– A few studies have used topical estrogens on human skin. propylene glycol, 7:3, vol/vol), under occlusion, on sun- Topical estradiol (17␤-estradiol) induces procollagen in ab- protected hip or photodamaged forearm skin sites. Treatment (0.1%-2.5%, ie, 5.4-135 µg of estradiol per square centimeter) dominal skin of postmenopausal women (38% increase in 32 31 was administered 3 times, every other day. Twenty-four hours total collagen and hydroxyproline content in estradiol- after the last treatment, 4-mm full-thickness punch biopsy speci- treated skin after 3 months) and hip skin of elderly men mens were obtained from each site. For face treatment, estra- and women (58% increase in procollagen after the use of diol was freshly dissolved in propylene glycol and incorpo- estradiol cream, 0.01%, twice a week for 2 weeks).33 When rated into moisturizing cream (Neutrogena; Neutrogena Corp, applied to the photoaged face, estrogens increase skin thick- Los Angeles, California) to a final concentration of 0.2% (wt/ ness (7.8% increase vs placebo),34 increase epidermal thick- vol). Estradiol treatment (570 µL of cream on the whole sur- ness,35 and decrease fine wrinkles34 but have no effect on face of the face; approximately 5.7 µg of estradiol per square epidermal hydration.36 To our knowledge, the effects of topi- centimeter) was applied twice a day for 14 days (participants cal estrogens on procollagen production in photoaged skin washed their face with soap and water before applying the cream in the morning and at bedtime). Biopsy specimens (2 mm) were have not been reported. However, despite the lack of sci- taken in the crow’s-foot area of the face before and 24 hours entific data, estrogen-based treatments are largely be- after the last treatment. For photoaging studies, all partici- lieved to be beneficial for maintaining a youthful appear- pants were asked to avoid sun exposure at least 48 hours be- ance of photoaged skin.19,37-41 fore and during treatment of photoaged areas. Face studies (with- Estrogens mediate their effects by interacting with and out occlusion) were performed in Michigan between November activating specific estrogen receptors (ERs), which func- and February to limit sun exposure during treatment. tion as ligand-activated transcription factors.42 Acti- Immediately after biopsy, skin samples were embedded in vated ERs bind to specific regions in target gene promot- low-temperature embedding medium (Tissue-Tek OCT com- ers and modulate their expression.42 Recently, estrogens pound; Miles, Naperville, Illinois), frozen in liquid nitrogen, have been described to transmit some of their effects and stored at −80°C until processing. through membrane-associated G-coupled protein receptor GPR30,43 the presence of which in skin has not been RNA EXTRACTION AND REAL-TIME reported. Mechanisms by which estradiol regulates col- REVERSE TRANSCRIPTION–POLYMERASE lagen production in human skin remain largely un- CHAIN REACTION known. Procollagen I gene promoter lacks estrogen response elements,44 and a direct effect of estrogens on Total RNA was extracted from whole punch skin biopsy specimens by means of a commercial kit (RNeasy; Qiagen, Chat- procollagen I gene expression has yet to be described. sworth, California) and quantified with dye (RiboGreen; Invit- In the present study, we quantified procollagen after ve- rogen, Carlsbad, California). Real-time reverse transcription– hicle or estradiol treatment of healthy volunteers (post- polymerase chain reaction (RT-PCR) was performed on 100 ng menopausal women and age-matched men) on sun- of total RNA as described previously,45 with the use of custom protected or photoaged skin in vivo. We found that 1-week primers and probe for collagen I and 36B4,45 and for collagen III

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©2008 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 Table 1. Age Distribution, Degree of Photodamage, and Postmenopausal Years of All Study Participants

Women Men

Sun-Protected Photodamaged Sun-Protected Photodamaged Total Skin Skin Total Skin Skin No. of subjects 40 17 23 30 13 17 Age, y Mean 75.2 76 74.6 74.7 75.1 74.4 Range 65-94 66-94 65-81 57-90 57-86 61-90 Photodamage distribution, No. Moderate NA NA 1 NA NA 1 Moderate to severe NA NA 10 NA NA 4 Severe NA NA 12 NA NA 12 Years after menopause, No. of subjects 10-20 20 713NANANA 21-30 16 610NANANA Ͼ30 4 4 0 NA NA NA

Abbreviation: NA, not applicable.

(COL3A1 sense primer, 5Ј-TCT-TGG-TCA-GTC-CTA-TGC- LASER CAPTURE MICRODISSECTION GGA-TA-3Ј; antisense primer, 5Ј-TCC-TAG-GCA-AGA-GAC- GCT-AC-3Ј;probe,5Ј-CCA-GAA-CCA-TGC-CAA-ATA-TGT- Laser capture microdissection was performed as previously de- GTC-TGT-GAC-T-3Ј). All other primer-probe sets were validated scribed46 to separate interfollicular epidermis (without hair fol- gene expression assays (TaqMan; Applied Biosystems, Foster City, licle infundibulum), dermis, and appendages (whole hair follicles, California). Efficiency of real-time RT-PCR reactions was deter- sebaceous glands, and sweat glands in a defined length of section). mined by using complementary DNA standards for ER-␣ (ESR1, UniGene Hs.208124) and ER-␤ (ESR2, UniGene Hs.660607) (gen- BLOOD ESTRADIOL MEASUREMENTS erous gifts from Peter J. Kushner, PhD, University of California, San Francisco). Results were normalized to the level of house- Blood estradiol measurements were obtained by radioimmu- keeping gene 36B4 (UniGene Hs.546285) (internal control). Re- noassay by the Clinical Chemistry Laboratory at the Univer- sults are presented as normalized fold change in estradiol- sity of Michigan Hospital. treated vs vehicle or untreated skin sample, or fold vs 36B4 −[CTtarget−CT36B4] (=2 , where CT indicates cycle threshold and is the STATISTICAL ANALYSIS end point of the real-time PCR reaction). Data are expressed as mean and standard error of the mean. Com- PROTEIN EXTRACTION AND PROCOLLAGEN I parisons among groups were made with the paired ttest. All Pval- ENZYME-LINKED IMMUNOSORBENT ASSAY ues are 2-tailed and were considered significant when less than .05.

Serial frozen sections of 7, 200, and 7 µm were prepared from RESULTS OCT-embedded skin biopsy specimens. Dermal areas of 7-µm sections were measured with Image ProPlus software (Media Cy- bernetics, Bethesda, Maryland) and used to calculate the volume INCREASED PROCOLLAGEN PRODUCTION of the 200-µm sample. Soluble proteins were extracted from the IN NATURALLY AGED SKIN AFTER TOPICAL 200-µm sample in ice-cold extraction buffer (50mM Tris hydro- ESTRADIOL TREATMENT chloride, pH 7.4; 0.15M sodium chloride; 1% Triton X-100; pro- tease inhibitors [Complete Mini; Roche Diagnostics, Indianapo- To assess the effects of topical estradiol on procollagen I lis, Indiana]). After 5-minute centrifugation at 10 000g at 4°C, and III expression, postmenopausal women were treated supernatants were assayed for procollagen I by means of a com- on hip skin with vehicle or various doses of estradiol for mercial enzyme-linked immunosorbent assay kit (Panvera, Madi- 1 week, and procollagen messenger RNA (mRNA) and son, Wisconsin). This enzyme-linked immunosorbent assay, which protein levels were measured in skin biopsy specimens. detects the C-terminal domain of procollagen I, quantifies only As shown in Figure 1A, estradiol increased procollagen newly made collagen I. Procollagen I concentrations were normalized to the volume of tissue used for each sample. I and III mRNA levels in a dose-dependent manner: 0.1%, 1%, and 2.5% increased procollagen I mRNA levels by 2.87-, 3.54-, and 3.67-fold vs vehicle, respectively (n=6-18; IMMUNOHISTOCHEMISTRY PϽ.05 for all concentrations). Similarly, 0.1%, 1%, and 2.5% estradiol treatment increased procollagen III mRNA Frozen skin sections (7 µm) were fixed in 2% paraformalde- levels by 2.10-, 3.19-, and 3.25-fold vs vehicle, respec- hyde and immunohistochemistry was performed by means of Ͻ anti–procollagen I antibody (Millipore, Billerica, Massachu- tively (P .05 for all concentrations). A concentration of setts) and an immunohistochemistry detection system (Link- 0.01% estradiol did not significantly increase procolla- Label; BioGenex Laboratories Inc, San Ramon, California). Slides gen I or III mRNA levels (n=6; P=.14 and .45, respec- were counterstained with hematoxylin (BioCare, Concord, Cali- tively). Estradiol treatment was also accompanied by a dose- fornia) and mounted with medium (Supermount; BioGenex). dependent increase in procollagen I protein (Figure 1B):

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5 4 COL1A1 ∗† ∗ COL3A1 ∗ † NS ∗ ∗ 4 † 3 ∗ NS ∗ NS 3 ∗ 2

1 mRNA Levels, Fold Change 1 Procollagen I Protein, Fold Change

0 0 Vehicle 0.01% 0.1% 1% 2.5% Vehicle 0.01% 1% 2.5% (n = 6) (n = 12) (n = 18) (n = 6)

Estradiol Estradiol

Figure 1. Effect of topical treatment in postmenopausal women on sun-protected hip skin with vehicle or estradiol for 1 week. Mean (SEM) levels of procollagen I (COL1A1) and III (COL3A1) messenger RNA (mRNA) (A) and procollagen I protein (B) were quantified in skin biopsy specimens by real-time reverse transcription–polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. NS indicates nonsignificant vs vehicle. *PϽ.05 vs vehicle. †Nonsignificant vs estradiol, 1%.

0.1%, 1%, and 2.5% estradiol stimulated procollagen I pro- RT-PCR. As shown in Figure 4A, 1% estradiol treat- duction by 1.53-, 2.33-, and 2.75-fold vs vehicle, respec- ment did not significantly alter procollagen I or III mRNA tively (n=6; PϽ.05 for 1% and 2.5% estradiol). levels in photodamaged forearm skin of postmeno- Collagen-producing cells were localized by immuno- pausal women in vivo (n=18). In contrast, estradiol treat- histochemistry. As shown in Figure 2, estradiol, 1%, ment of sun-protected hip skin increased procollagen I treatment induced a marked increase in procollagen I pro- mRNA levels by 3.54 (0.56)–fold (mean [SEM]) vs base- tein expression throughout the reticular and papillary der- line (n=18; PϽ.05, Figure 1A). Similarly, 2.5% estra- mis in sun-protected skin of postmenopausal women in diol did not alter procollagen mRNA levels in photodam- vivo. aged skin of postmenopausal women (1.61 [0.44]– and Histologic analyses of human skin sections did not 1.37 [0.16]–fold vs vehicle for procollagen I and III, re- show any effect of topical estradiol treatment on epider- spectively; n=6). Because type I collagen regulation in- mal or dermal thickness, as measured by image analysis cludes multiple posttranslational steps,47 procollagen I on hematoxylin-eosin–stained skin sections (Figure 2 and protein levels were measured in estradiol- and vehicle- data not shown). treated forearm skin samples of postmenopausal women. Topical estradiol also increased procollagen expres- As shown in Figure 4B, estradiol treatment had no sig- sion in sun-protected hip skin of men, although to a lesser nificant effect on procollagen I protein levels (n=12). extent. As shown in Figure 3A, procollagen I mRNA Results obtained in photoaged forearm skin of men levels were increased by topical estradiol by 1.69-, 1.58-, were similar to those obtained in postmenopausal women. and 2.02-fold vs vehicle after 0.1%, 1%, and 2.5% estra- Topical treatment with 1% or 2.5% estradiol did not al- diol treatment, respectively (n=12; PϽ.05 for 0.1% and ter procollagen I or III mRNA expression (Figure 4C; 1% estradiol). Similarly, procollagen III mRNA levels were n=12) or procollagen I protein (Figure 4D; n=6) com- increased by topical estradiol by 1.87-, 1.64-, and 2.11- pared with vehicle. fold vs vehicle after 0.1%, 1%, and 2.5% estradiol treat- Lack of effect of estradiol on collagen levels in pho- ment, respectively (n=12; PϽ.05 for all concentra- todamaged forearm skin was confirmed by immunohis- tions). Topical estradiol also induced procollagen I protein tochemistry in postmenopausal women (Figure 5) and (Figure 3B) by 1.25-, 1.3-, and 2.3-fold vs vehicle after age-matched men (not shown). 0.1%, 1%, and 2.5% estradiol treatment, respectively (n=9; PϽ.05 for 1% and 2.5%). LACK OF EFFECT OF ESTRADIOL TREATMENT ON COLLAGEN I PRODUCTION LACK OF EFFECT OF TOPICAL ESTRADIOL IN PHOTOAGED FACE TREATMENT ON PROCOLLAGEN PRODUCTION IN PHOTOAGED FOREARM SKIN To determine whether the observed lack of effect of estrogen on collagen synthesis in photoaged forearms is re- To determine the effect of estradiol on procollagen ex- lated to anatomic site or photoaging, we evaluated the pression in photoaged skin, we next treated postmeno- effect of topical estradiol on procollagen synthesis in the pausal women on photodamaged forearms with vehicle photoaged face. To this end, 5 postmenopausal women or estradiol for 1 week and measured procollagen I and and 5 age-matched men with clinical photoaging were III mRNA levels in skin biopsy specimens by real-time treated on the face with 0.2% estradiol cream twice daily

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Upper dermis

C D

Lower dermis

Vehicle Estradiol, 1%

Figure 2. Skin specimens showing effect of topical treatment in postmenopausal women on sun-protected hip skin with vehicle or estradiol, 1%, for 1 week. Skin samples were stained for procollagen I by immunohistochemistry. Positive staining appears red, and hematoxylin counterstaining appears blue (original magnification ϫ10; insets ϫ40). White squares in A and B indicate areas shown in insets. Insets in C and D show the deepest part of the dermis. Specimens are representative of 6 experiments.

for 2 weeks. The treatment regimen matched the quan- GPR30 mRNA levels were readily detected in human skin tity of estradiol per surface area that was used on fore- of all tested subjects (Figure 7). We did not find any dif- arms and hips. Levels of procollagen were compared in ferences in the levels of expression of ER-␣ (Figure 7A), skin samples obtained before and after estradiol appli- ER-␤ (Figure 7B), or GPR30 (Figure 7C) between sun- cation. No change in procollagen mRNA (Figure 6A) protected hip and photoaged forearm or face skin samples, or protein (Figure 6B) expression was observed after in women or men, nor were these levels affected by estra- 2-week treatment of photoaged faces of men or post- diol treatment. Interestingly, men had significantly less ER-␣ menopausal women. In parallel, blood estradiol concen- in hip skin (−49%) and less ER-␤ in forearm skin (−49%) trations were measured before and after treatment. Topi- than did site-matched women (n=6; PϽ.05). Direct com- cal estradiol treatment significantly increased estradiol parison of Figure 7A, B, and C shows that ER-␣ mRNA lev- blood levels in all treated subjects (Figure 6C). The in- els were approximately 10 times more abundant than those crease in estradiol blood levels was greater in postmeno- of ER-␤ or GPR30 in both men and women. pausal women than in men (12- vs 2-fold, respectively; Despite readily detectable ER mRNA levels, we were not P=.006 and P=.001, respectively). These results demonstrate that the 0.2% estradiol penetrated the skin and able to reliably detect the presence of ER protein in skin was chemically stable throughout the treatment. samples by immunohistochemistry, using various different antibodies (data not shown). We therefore used laser ESTROGEN RECEPTOR LEVELS IN NATURALLY capture microdissection to localize ER mRNA expression AGED AND PHOTOAGED SKIN in normal human skin. Expression of each of the 3 genes was extremely low in the epidermis (Figure 7D). The ER-␣ To determine whether lack of response to estradiol treat- and GPR30 were primarily expressed in dermal cells, while ment in photoaged skin was due to a lower level of ERs, expression was near the limit of detection in appendages. we quantified ER-␣, ER-␤, and GPR30 (GPER; UniGene In contrast, ER-␤ expression was localized in appendages Hs.20961) mRNA levels in vehicle- and estradiol-treated and, to a lesser extent, in dermal cells. These data indicate skin samples from hip, forearm, and face of postmeno- that all ERs are expressed in the dermal compartment of pausal women and age-matched men. The ER-␣, ER-␤, and human skin in vivo.

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5 5 COL1A1 COL3A1 4 4

∗ 3 3 NS ∗ ∗ ∗ ∗ ∗ 2 2 ∗ NS

mRNA Levels, Fold Change 1 1 Procollagen I Protein, Fold Change

0 0 Vehicle 0.1% 1% 2.5% Vehicle 0.01% 1% 2.5%

Estradiol Estradiol

Figure 3. Effect of topical estradiol treatment in men on sun-protected hip skin with vehicle or estradiol for 1 week. Mean (SEM) levels of procollagen I (COL1A1) and III (COL3A1) messenger RNA (mRNA) (A; n=12) and procollagen I protein (B; n=9) were quantified in skin biopsy specimens by real-time reverse transcription– polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. NS indicates nonsignificant vs vehicle. *PϽ.05 vs vehicle.

A B

3 3 COL1A1 COL3A1

NS 2 NS 2 NS NS NS

1 1 mRNA Levels, Fold Change Procollagen I Protein, Fold Change

0 0 Vehicle 1% 2.5% Vehicle 1% Estradiol Estradiol

C D

3 3 COL1A1 COL3A1

2 NS 2 NS NS NS NS

1 1 mRNA Levels, Fold Change Procollagen I Protein, Fold Change

0 0 Vehicle 1% 2.5% Vehicle 1% Estradiol Estradiol

Figure 4. Effect of topical treatment in postmenopausal women (A and B) and men (C and D) on photodamaged forearms with vehicle or estradiol for 1 week. Mean (SEM) levels of procollagen I (COL1A1) and III (COL3A1) messenger RNA (mRNA) in women (A; n=18) and men (C; n=12) and procollagen I protein in women (B; n=12) and men (D; n=6) were quantified in skin biopsy specimens by real-time reverse transcription–polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. NS indicates nonsignificant vs vehicle.

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Upper dermis

C D

Lower dermis

Vehicle Estradiol, 1%

Figure 5. Skin specimens showing effect of topical treatment in postmenopausal women on photodamaged forearm skin with vehicle or estradiol, 1%, for 1 week. Skin samples were stained for procollagen I by immunohistochemistry. Positive staining appears red, and hematoxylin counterstaining appears blue (original magnification ϫ10; insets ϫ40). White squares in A and B indicate areas shown in insets. Insets in C and D show the deepest part of the dermis. Specimens are representative of 6 experiments.

A B C

3 3 140 COL1A1 ∗ COL3A1 120

100 2 2 NS NS 80 NS NS NS NS 60 ∗ 1 1 40 mRNA Levels, Fold Change Estradiol Blood Levels, pg/mL

Procollagen I Protein, Fold Change 20

0 0 0 Before After Before After Before After Before After Before After Before After

Women Men Women Men Women Men

Figure 6. Effect of topical treatment in postmenopausal women and men on photodamaged face skin with estradiol, 0.2%, twice daily for 2 weeks. Mean (SEM) levels of procollagen I (COL1A1) and III (COL3A1) messenger RNA (mRNA) (A) and procollagen I protein (B) were quantified in skin biopsy specimens before and after treatment by real-time reverse transcription–polymerase chain reaction and enzyme-linked immunosorbent assay, respectively (n=5). NS indicates nonsignificant vs vehicle. C, In parallel, estradiol blood levels were determined before and after treatment (to convert estradiol to picomoles per liter, multiply by 3.671). *PϽ.05 vs before treatment.

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©2008 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 A B NS 20 Vehicle 2.5 Vehicle Estradiol, 1% Estradiol, 1% x 1000 x 1000 2.0 15 36B4 NS 36B4 NS 1.5 ∗ 10 NS 1.0

5 ∗ mRNA Levels, Fold vs mRNA Levels, Fold vs 0.5 β α ER- ER- 0 0 Hip Forearm Face Hip Forearm Face Hip Forearm Face Hip Forearm Face

Women Men Women Men

C D NS 2.5 Vehicle 30 Estradiol, 1% Epidermis 25 Dermis x 1000 2.0 Appendages x 1000 36B4 NS 20 1.5 36B4

1.0 10

0.5 5 mRNA Levels, Fold vs GPR30 mRNA Levels, Fold vs 0 0 Hip Forearm Face Hip Forearm Face ER-α ER-β GPR30 Women Men

Figure 7. Effect of topical estradiol treatment on expression of estrogen receptors in human skin in vivo. Mean (SEM) messenger RNA (mRNA) levels for estrogen receptor ␣ (ER-␣) (A), ER-␤ (B), and GPR30 (C) were measured by real-time reverse transcription–polymerase chain reaction in control- and estradiol-treated hip, forearm, and face skin of postmenopausal women and men (n=5-6). NS indicates nonsignificant for estradiol vs vehicle, hip vs forearm, or hip vs face. *PϽ.05 vs the same skin site in women. D, Localization of ER-␣, ER-␤, and GPR30 mRNA expression determined by laser capture microdissection–coupled real-time reverse transcription–polymerase chain reaction in sun-protected human hip skin (n=4).

LACK OF EFFECT OF TOPICAL ESTRADIOL The HSD17B2, HSD17B8, CYP1B1, and SULT1E1 TREATMENT ON ESTROGEN-METABOLIZING were expressed at similar levels, approximately 5 times ENZYME GENE EXPRESSION less than HSD17B4. The SULT1A1 mRNA levels were 3000 times lower than those of HSD17B4, whereas Sex steroid concentrations in human tissues are tightly SULT2A1 mRNA was not detected in human skin in vivo regulated, and excess estrogens are biologically inacti- (not shown). vated by specific enzymes.48 To determine whether As shown in Table 2, mRNA levels of estradiol me- lack of response to estradiol treatment in photoaged tabolizing enzymes were similar in hip and forearm skin skin was due to increased estrogen catabolism, we and were not altered by topical estradiol treatment in post- measured the levels of estrogen inactivating enzymes menopausal women. Expression of HSD17B2 tended to in human skin treated with vehicle or estradiol. be higher in face skin than in hip skin, although this dif- Estrogen-inactivating enzymes can be subdivided into ference (approximately 10-fold) did not reach statisti- 3 groups: (1) 17-␤-hydroxysteroid dehydrogenases cal significance (P=.09 and .06 for vehicle- and estradiol- (HSD17B) 2, 4, and 8 catalyze oxidation of the biologi- treated sites, respectively; n=5-6). In addition, CYP1B1 cally active estradiol into weaker estrone49; (2) sulfo- mRNA levels were 2.7-fold higher in face skin than in transferase (SULT) 1A1, SULT2A1, and SULT1E1 hip skin (P=.04). The level of HSD17B8 was approxi- catalyze sulfation of estradiol, preventing it from bind- mately 4 times lower in face skin than in hip skin of post- ing to, and thereby activating, ERs50,51; and (3) cyto- menopausal women (PϽ.001). chrome P450 1B1 (CYP1B1) is the major CYP450 All of the estradiol metabolizing enzymes that were enzyme inactivating estradiol by 4-hydroxylation.52 expressed in women were also expressed in men, and their We first assessed gene expression of each of these levels were not altered by estradiol treatment of hip, fore- enzymes in hip, forearm, and face skin in vivo. The arm, or face skin. Interestingly, levels of CYP1B1 mRNA HSD17B4 was the most highly expressed (Table 2). were significantly higher in hip and forearm skin of men

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©2008 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 Table 2. Gene Expression Levels of Estradiol-Degrading Table 2. Gene Expression Levels of Estradiol-Degrading Enzymes in Human Skin In Vivoa Enzymes in Human Skin In Vivo (cont)a

Vehicle Estradiol, 1% Vehicle Estradiol, 1% HSD17B2 SULT1A1 Hip Hip Women 1.29 (0.38) 3.08 (1.31) Women 0.045 (0.033) 0.045 (0.021) Men 2.35 (0.75) 3.46 (1.39) Men 0.054 (0.031) 0.026 (0.012) Forearm Forearm Women 3.11 (0.88) 6.98 (2.03) Women 0.035 (0.011) 0.039 (0.010) Men 9.25 (4.03) 9.67 (3.69) Men 0.005 (0.001) 0.005 (0.001) Face Face Women 27.34 (10.31) 36.50 (11.54) Women 0.012 (0.002) 0.008 (0.001)c Men 66.66 (23.97) 70.00 (18.64) Men 0.016 (0.004) 0.017 (0.004) HSD17B4 SULT1E1 Hip Hip Women 10.02 (3.88) 11.98 (4.72) Women 0.73 (0.28) 0.75 (0.32) Men 9.42 (1.00) 8.53 (1.03) Men 2.99 (1.25) 3.32 (1.55) Forearm Forearm Women 7.50 (0.91) 7.88 (0.76) Women 0.88 (0.23) 1.04 (0.20) Men 8.00 (1.27) 8.34 (1.59) Men 2.69 (1.27) 3.64 (1.78) Face Face Women 13.61 (3.95) 10.70 (4.02) Women 0.30 (0.07) 0.28 (0.05) Men 17.88 (5.62) 18.78 (5.76) Men 0.34 (0.11)b 0.38 (0.06) HSD17B8 Hip Abbreviations: CYP1B1, cytochrome P450 1B1; HSD17B2, 4, and 8, Women 2.04 (0.20) 1.99 (0.23) 17-␤-hydroxysteroid dehydrogenase 2, 4, and 8; SULT1A1 and SULT1E1, Men 2.44 (0.12) 2.19 (0.23) sulfotransferase 1A1 and 1E1. a Forearm Postmenopausal women and age-matched men were treated on Women 2.04 (0.24) 1.93 (0.29) sun-protected hip or photoaged forearm or face skin with vehicle or estradiol. Levels of estrogen-degrading enzymes (CYP1B1; HSD17B2, 4, and Men 7.38 (4.8) 6.99 (4.57) 8; SULT1A1; and SULT1E1) were quantified in skin by real-time reverse Face ϫ 3 b transcription–polymerase chain reaction. Values are fold vs 36B4 10 Women 0.49 (0.14) 0.53 (0.17) −[CT −CT36B4] 3 (=2 target ϫ 10 ), given as mean (SEM) (n = 5-6). (CT indicates cycle Men 0.90 (0.21) 0.96 (0.21) threshold). CYP1B1 b P Ͻ .05 in forearm or face vs hip. Hip c P Ͻ .05 in estradiol- vs vehicle-treated skin. Women 1.29 (0.32) 1.86 (0.37)c d P Ͻ .05 in men vs women. Men 4.71 (1.16)d 6.90 (2.66) Forearm Women 1.68 (0.27) 2.58 (0.84) COMMENT Men 6.40 (2.03) 7.51 (2.20) Face In the present study, we evaluated the effects of estra- Women 3.47 (0.70)b 3.39 (0.65) diol treatment on collagen production in sun-protected Men 3.73 (0.67) 4.77 (0.60) aged and photoaged skin of postmenopausal women and age-matched men. We demonstrated that 1-week topi- (continued) cal treatment with estradiol stimulates procollagen I and III expression in sun-protected aged skin of postmeno- compared with women (3.7- and 3.8-fold, respectively; pausal women in vivo. Surprisingly, we found that es- P=.03 for hip). tradiol has no effect on procollagen expression in photoaged skin (forearm or face) of postmenopausal women, EFFECT OF TOPICAL ESTRADIOL TREATMENT ON despite similar levels of expression of estrogen recep- TRANSCRIPTION OF ESTROGEN-RESPONSIVE tors in sun-protected and sun-exposed skin sites, and simi- GENE GREB1 IN NATURALLY AGED lar levels of expression of estradiol-inactivating en- AND PHOTOAGED SKIN zymes in hip and forearm skin. In addition, we provide evidence that topical estradiol penetration and activity To assess topical estradiol biological activity, we mea- are similar in sun-protected and sun-exposed skin sites, sured induction of GREB1 (gene regulated by estrogen as assessed by quantification of induction of GREB1 gene in breast cancer 1; Unigene Hs.467733), an estrogen- expression, which is directly regulated by ERs.53 Taken responsive gene that is directly regulated by ERs.53 As together, these findings support the concept that topi- shown in Figure 8, topical estradiol treatment of post- cal estradiol regulates procollagen I and III production menopausal women induced GREB1 mRNA levels with in sun-protected skin in women. Our results also indi- a 3.6-, 4.7-, and 9.9-fold increase vs control in hip, fore- cate that alterations induced by long-term sun exposure arm, and face skin, respectively (PϽ.05). In men, GREB1 hinder the ability of topical estradiol to stimulate colla- mRNA levels were significantly increased after estradiol gen production in aged human skin in vivo. treatment in hip skin (2.25-fold; n=6; P=.005) but not The study of estradiol responses in male skin yielded in forearm (n=12) or face (n=5) skin (Figure 8). interesting differences compared with women. Topical

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©2008 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 findings of Meyer et al,54 who showed that 17␣- 14 ∗ estradiol, which does not activate estrogen receptors, † stimulated skin procollagen production in rodent and 12 human skin. However, the mediators of estrogen action 10 on skin collagen remain to be identified. More recently, it was reported that the combination 8 of ET and topical estradiol increased procollagen im- ∗† 55 6 munostaining in facial skin in women. The study did ∗ NS not investigate the effects of either ET or topical estra- mRNA Levels, Fold Change 4 ∗ diol alone. It is possible that ET triggers systemic re- NS sponses that enable topical estradiol to promote colla- GREB1 2 gen production in photoaged skin, or vice versa. This 0 possibility raises interesting questions regarding mecha- Vehicle Hip Forearm Face Vehicle Hip Forearm Face nisms of estrogen actions in human skin. Women Men The presence of estrogen binding sites in skin has been demonstrated by means of radioactive estradiol.56,57 These Figure 8. Effect of topical estradiol treatment on estrogen-responsive gene studies found that thigh skin has approximately 3-fold lower GREB1 messenger RNA (mRNA) expression in sun-protected and photoaged human skin in vivo. Mean (SEM) GREB1 transcript levels were measured by ER levels than does face or abdominal skin (1.5, 4.3, and real-time reverse transcription–polymerase chain reaction in hip, forearm, 4.9 fmol of receptor per milligram of protein, respec- and face skin of postmenopausal women and men after treatment with tively56,57). In the present study, we did not find any sig- vehicle or estradiol (n=5-12). NS indicates nonsignificant vs vehicle. *PϽ.05 vs vehicle. †Nonsignificant vs hip. nificant difference in the level of ESR1, ESR2, or GPR30 gene expression in face, forearm, or hip skin in postmenopausal women. In addition, we did not find any significant difference in ER expression between vehicle- and es- estradiol increased procollagen production in naturally tradiol-treated samples, consistent with previous results.57 aged hip skin of men to a lesser extent than in postmeno- Detection of ERs in human skin has yielded discor- pausal women. Induction of GREB1 gene expression was dant results. Immunostaining-based studies reported that also weaker in men than in women. In contrast, topical ER-␣ was not detected or was weakly detected, whereas estradiol did not alter procollagen or GREB1 expres- ER-␤ was strongly detected in epidermis, eccrine glands, sions in photoaged forearm or face skin of men. Differ- sebaceous glands, and hair follicles. On the basis of these ences in estradiol responsiveness between men and reports, ER-␤ is often described as the predominant form women were not due to differences in ER expression. In- of ER in adult human skin,58-60 although the relative abun- terestingly, CYP1B1, which inactivates 17␤-estradiol by dance of the 2 ER isoforms has not been formally quan- 4-hydroxylation, was significantly more highly ex- tified. We quantified ER-␣, ER-␤, and GPR30 mRNA in pressed in photoaged forearm skin of men than of women, human skin and demonstrated that ER-␣ mRNA levels suggesting that reduced responsiveness of men to estra- were approximately 10 times greater than those of ER-␤ diol may be related to catabolism. or GPR30. We were unable to detect either protein by Our data are in agreement with previously pub- immunohistochemistry (using a variety of different an- lished studies that demonstrated that topical estrogen tibodies) because of absent or nonspecific staining. Using treatment stimulates procollagen production in laser capture microdissection, we found that most of the abdominal skin of postmenopausal women32 and hip ER-␣ and GPR30 mRNA was expressed in dermal cells, skin of elderly men and women.33 We did not observe whereas ER-␤ was expressed in both appendages and der- any correlation between subject age and magnitude of mal cells. More studies are needed to precisely identify estradiol-mediated induction of collagen mRNA or ER-expressing cells within dermis and appendages. protein (n=18). In summary, our study shows that estradiol stimu- To our knowledge, the present study is the first to lates collagen I and III production in sun-protected skin describe a lack of effect of topical estradiol treatment on but not in photoaged skin of postmenopausal women and procollagen production in photoaged skin. It is possible aged-matched men within 2 weeks. Because photoaging that a treatment time longer than 2 weeks would have is superimposed on natural aging in sun-exposed areas demonstrated effects similar to those in sun-protected of the skin, our results suggest that alterations induced skin. Additional studies will be necessary to test this by long-term sun exposure hinder the ability of topical possibility. However, GREB1 levels rose significantly estradiol to stimulate collagen production in aged hu- and similarly in all treated sites in postmenopausal man skin in vivo. women, indicating that estradiol penetrated the skin and activated ERs similarly in sun-protected and sun- Accepted for Publication: April 8, 2008. exposed skin. These results demonstrate that photoag- Correspondence: Gary J. Fisher, PhD, Department of Der- ing is not associated with a loss of estrogen genomic matology, University of Michigan Medical School, Medi- response, at least in postmenopausal women. Con- cal Science I, Room 6447, 1301 E Catherine, Ann Arbor, versely, our results suggest that the effect of estradiol on MI 48109-0609 ([email protected]). procollagen is indirect, and that photoaged skin lacks Author Contributions: Drs Rittie´ and Fisher had full ac- an essential component to the estrogen-mediated colla- cess to all of the data in the study and take responsibil- gen response. This hypothesis is in agreement with ity for the integrity of the data and the accuracy of the

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©2008 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 data analysis. Study concept and design: Rittie´, Kang, 19. Dunn LB, Damesyn M, Moore AA, Reuben DB, Greendale GA. Does estrogen pre- Voorhees, and Fisher. Acquisition of data: Rittie´, Kang, vent skin aging? results from the First National Health and Nutrition Examina- tion Survey (NHANES I). Arch Dermatol. 1997;133(3):339-342. and Fisher. Analysis and interpretation of data: Rittie´, Kang, 20. Sator PG, Schmidt JB, Sator MO, Huber JC, Honigsmann H. The influence of Voorhees, and Fisher. Drafting of the manuscript: Rittie´. hormone replacement therapy on skin ageing: a pilot study. Maturitas. 2001; Critical revision of the manuscript for important intellec- 39(1):43-55. tual content: Rittie´, Kang, Voorhees, and Fisher. Statisti- 21. Schmidt JB, Binder M, Demschik G, Bieglmayer C, Reiner A. Treatment of skin cal analysis: Rittie´.Obtaining funding: Voorhees and Fisher. aging with topical estrogens. Int J Dermatol. 1996;35(9):669-674. 22. Sumino H, Ichikawa S, Abe M, Endo Y, Ishikawa O, Kurabayashi M. Effects of Study supervision: Voorhees and Fisher. aging, menopause, and hormone replacement therapy on forearm skin elastic- Financial Disclosure: Dr Voorhees was a consultant for ity in women. J Am Geriatr Soc. 2004;52(6):945-949. Pfizer Inc and received consulting payments. 23. Wolff EF, Narayan D, Taylor HS. Long-term effects of hormone therapy on skin Funding/Support: This study was supported in part by rigidity and wrinkles. Fertil Steril. 2005;84(2):285-288. 24. Sauerbronn AV, Fonseca AM, Bagnoli VR, Saldiva PH, Pinotti JA. The effects of a grant from Pfizer Inc (Dr Fisher). systemic hormonal replacement therapy on the skin of postmenopausal women. Additional Contributions: Suzan Rehbine, LPN, helped Int J Gynaecol Obstet. 2000;68(1):35-41. with volunteer recruitment and tissue collection; Laura 25. Callens A, Vaillant L, Lecomte P, Berson M, Gall Y, Lorette G. Does hormonal VanGoor, BFA, assisted with graphic material; and Ste- skin aging exist? a study of the influence of different hormone therapy regimens phanie Cooke, BS, Kenne Currie, BS, and Craig Ham- on the skin of postmenopausal women using non-invasive measurement techniques. Dermatology. 1996;193(4):289-294. merberg, PhD, provided technical help. 26. Maheux R, Naud F, Rioux M, et al. A randomized, double-blind, placebo- controlled study on the effect of conjugated estrogens on skin thickness. Am J Obstet Gynecol. 1994;170(2):642-649. REFERENCES 27. Brincat M, Moniz CJ, Studd JW, et al. Long-term effects of the menopause and sex hormones on skin thickness. Br J Obstet Gynaecol. 1985;92(3):256-259. 28. Brincat M, Versi E, Moniz CF, Magos A, de Trafford J, Studd JW. Skin collagen 1. Yaar M, Eller MS, Gilchrest BA. Fifty years of skin aging. J Investig Dermatol Symp changes in postmenopausal women receiving different regimens of estrogen Proc. 2002;7(1):51-58. therapy. Obstet Gynecol. 1987;70(1):123-127. 2. Lavker RM. Cutaneous aging: chronologic versus photoaging. In: Gilchrest BA, 29. Punnonen R. Effect of castration and peroral estrogen therapy on the skin. Acta ed. Photodamage. Cambridge, MA: Blackwell Science; 1975:123-135. Obstet Gynecol Scand Suppl. 1972;21:3-44. 3. Rittie´ L, Fisher GJ. UV-light-induced signal cascades and skin aging. Ageing Res 30. Haapasaari KM, Raudaskoski T, Kallioinen M, et al. Systemic therapy with es- Rev. 2002;1(4):705-720. trogen or estrogen with progestin has no effect on skin collagen in postmeno- 4. Eckes B, Zweers MC, Zhang ZG, et al. Mechanical tension and integrin alpha 2 pausal women. Maturitas. 1997;27(2):153-162. beta 1 regulate fibroblast functions. J Investig Dermatol Symp Proc. 2006; 31. Oikarinen A. Systemic estrogens have no conclusive beneficial effect on human 11(1):66-72. skin connective tissue. Acta Obstet Gynecol Scand. 2000;79(4):250-254. 5. Varani J, Dame MK, Rittie´ L, et al. Decreased collagen production in chronologi- cally aged skin: roles of age-dependent alteration in fibroblast function and de- 32. Varila E, Rantala I, Oikarinen A, et al. The effect of topical oestradiol on skin col- fective mechanical stimulation. Am J Pathol. 2006;168(6):1861-1868. lagen of postmenopausal women. Br J Obstet Gynaecol. 1995;102(12):985- 6. Varani J, Perone P, Fligiel SE, Fisher GJ, Voorhees JJ. Inhibition of type I pro- 989. ␤ collagen production in photodamage: correlation between presence of high mo- 33. Son ED, Lee JY, Lee S, et al. Topical application of 17 -estradiol increases ex- ␤ lecular weight collagen fragments and reduced procollagen synthesis. J Invest tracellular matrix protein synthesis by stimulating TGF- signaling in aged hu- Dermatol. 2002;119(1):122-129. man skin in vivo. J Invest Dermatol. 2005;124(6):1149-1161. 7. Varani J, Spearman D, Perone P, et al. Inhibition of type I procollagen synthesis 34. Creidi P, Faivre B, Agache P, Richard E, Haudiquet V, Sauvanet JP. Effect of a by damaged collagen in photoaged skin and by collagenase-degraded collagen conjugated oestrogen (Premarin) cream on ageing facial skin: a comparative study in vitro. Am J Pathol. 2001;158(3):931-942. with a placebo cream. Maturitas. 1994;19(3):211-223. 8. Wang F, Garza LA, Kang S, et al. In vivo stimulation of de novo collagen produc- 35. Fuchs KO, Solis O, Tapawan R, Paranjpe J. The effects of an estrogen and gly- tion caused by cross-linked hyaluronic acid dermal filler injections in photodam- colic acid cream on the facial skin of postmenopausal women: a randomized his- aged human skin. Arch Dermatol. 2007;143(2):155-163. tologic study. Cutis. 2003;71(6):481-488. 9. Rittie´ L, Fisher GJ, Voorhees JJ. Retinoid therapy for photoaging. In: Gilchrest 36. Jemec GB, Serup J. Short-term effects of topical 17 beta-oestradiol on human BA, Krutman J, eds. Skin Ageing. Berlin, Germany: Springer-Verlag; 2006:143- post-menopausal skin. Maturitas. 1989;11(3):229-234. 156. 37. Taylor HS. Judging a book by its cover: estrogen and skin aging. Fertil Steril. 10. Orringer JS, Kang S, Johnson TM, et al. Connective tissue remodeling induced 2005;84(2):295. doi:10.1016/j.fertnstert.2005.04.017. by carbon dioxide laser resurfacing of photodamaged human skin. Arch Dermatol. 38. Draelos ZD. Topical and oral estrogens revisited for antiaging purposes. Fertil 2004;140(11):1326-1332. Steril. 2005;84(2):291-292. 11. Bolognia JL, Braverman IM, Rousseau ME, Sarrel PM. Skin changes in menopause. 39. Naftolin F. Prevention during the menopause is critical for good health: skin stud- Maturitas. 1989;11(4):295-304. ies support protracted hormone therapy. Fertil Steril. 2005;84(2):293-294. 12. Pie´rard GE, Letawe C, Dowlati A, Pie´rard-Franchimont C. Effect of hormone re- 40. Raine-Fenning NJ, Brincat MP, Muscat-Baron Y. Skin aging and menopause: im- placement therapy for menopause on the mechanical properties of skin. JAm plications for treatment. Am J Clin Dermatol. 2003;4(6):371-378. Geriatr Soc. 1995;43(6):662-665. 41. Sator PG, Schmidt JB, Rabe T, Zouboulis CC. Skin aging and sex hormones in 13. Sumino H, Ichikawa S, Abe M, et al. Effects of aging and postmenopausal hy- women—clinical perspectives for intervention by hormone replacement therapy. poestrogenism on skin elasticity and bone mineral density in Japanese women. Exp Dermatol. 2004;13(suppl 4):36-40. Endocr J. 2004;51(2):159-164. 42. Kushner PJ, Webb P, Uht RM, Liu MM, Price RH. Estrogen receptor action through 14. Chotnopparatpattara P, Panyakhamlerd K, Taechakraichana N, Tantivatana J, Chai- target genes with classical and alternative response elements. Pure Appl Chem. kittisilpa S, Limpaphayom KK. An effect of hormone replacement therapy on skin 2003;75(11-12):1757-1769 http://media.iupac.org/publications/pac/2003/pdf thickness in early postmenopausal women. J Med Assoc Thai. 2001;84(9): /7511x1757.pdf. Accessed August 27, 2007. 1275-1280. 43. Rae JM, Johnson MD. What does an orphan G-protein-coupled receptor have to 15. Brincat M, Moniz CF, Studd JW, Darby AJ, Magos A, Cooper D. Sex hormones do with estrogen? Breast Cancer Res. 2005;7(6):243-244. and skin collagen content in postmenopausal women. Br Med J (Clin Res Ed). 44. Jimenez SA, Varga J, Olsen A, et al. Functional analysis of human ␣1(I) procol- 1983;287(6402):1337-1338. lagen gene promoter: differential activity in collagen-producing and -nonpro-

16. Castelo-Branco C, Duran M, Gonzalez-Merlo J. Skin collagen changes related to ducing cells and response to transforming growth factor ␤1. J Biol Chem. 1994; age and hormone replacement therapy. Maturitas. 1992;15(2):113-119. 269(17):12684-12691. 17. Affinito P, Palomba S, Sorrentino C, et al. Effects of postmenopausal hypoes- 45. Quan T, He T, Kang S, Voorhees JJ, Fisher GJ. Solar ultraviolet irradiation re- trogenism on skin collagen. Maturitas. 1999;33(3):239-247. duces collagen in photoaged human skin by blocking transforming growth fac- 18. Pie´rard-Franchimont C, Letawe C, Goffin V, Pie´rard GE. Skin water-holding ca- tor-␤ type II receptor/Smad signaling. Am J Pathol. 2004;165(3):741-751. pacity and transdermal estrogen therapy for menopause: a pilot study. Maturitas. 46. Rittie´ L, Varani J, Kang S, Voorhees JJ, Fisher GJ. Retinoid-induced epidermal 1995;22(2):151-154. hyperplasia is mediated by epidermal growth factor receptor activation via spe-

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©2008 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 cific induction of its ligands heparin-binding EGF and amphiregulin in human skin tion of estrogen effect on collagen from other clinical and biochemical effects in in vivo. J Invest Dermatol. 2006;126(4):732-739. man. Res Commun Chem Pathol Pharmacol. 1976;13(4):685-695. 47. Nimni ME. Collagen: structure, function, and metabolism in normal and fibrotic 55. Patriarca MT, Goldman KZ, Dos Santos JM, et al. Effects of topical estradiol on tissues. Semin Arthritis Rheum. 1983;13(1):1-86. the facial skin collagen of postmenopausal women under oral hormone therapy: 48. Raftogianis R, Creveling C, Weinshilboum R, Weisz J. Estrogen metabolism by a pilot study. Eur J Obstet Gynecol Reprod Biol. 2007;130(2):202-205. conjugation. J Natl Cancer Inst Monogr. 2000;(27):113-124. 56. Hasselquist MB, Goldberg N, Schroeter A, Spelsberg TC. Isolation and charac- 49. Peltoketo H, Vihko P, Vihko R. Regulation of estrogen action: role of 17 beta- terization of the estrogen receptor in human skin. J Clin Endocrinol Metab. 1980; hydroxysteroid dehydrogenases. Vitam Horm. 1999;55:353-398. 50(1):76-82. 50. Iwamori M. Estrogen sulfatase. Methods Enzymol. 2005;400:293-302. 57. Punnonen R, Lövgren T, Kouvonen I. Demonstration of estrogen receptors in 51. Gamage N, Barnett A, Hempel N, et al. Human sulfotransferases and their role in the skin. J Endocrinol Invest. 1980;3(3):217-221. chemical metabolism. Toxicol Sci. 2006;90(1):5-22. 52. Zhu BT, Lee AJ. NADPH-dependent metabolism of 17␤-estradiol and estrone to 58. Pelletier G, Ren L. Localization of sex steroid receptors in human skin. Histol polar and nonpolar metabolites by human tissues and cytochrome P450 isoforms. Histopathol. 2004;19(2):629-636. Steroids. 2005;70(4):225-244. 59. Zouboulis CC, Chen WC, Thornton MJ, Qin K, Rosenfield R. Sexual hormones in 53. Bourdeau V, Deschenes J, Metivier R, et al. Genome-wide identification of high- human skin. Horm Metab Res. 2007;39(2):85-95. affinity estrogen response elements in human and mouse. Mol Endocrinol. 2004; 60. Hall G, Phillips TJ. Estrogen and skin: the effects of estrogen, menopause, and 18(6):1411-1427. hormone replacement therapy on the skin. J Am Acad Dermatol. 2005;53(4): 54. Meyer WJ III, Henneman DH, Keiser HR, Bartter FC. 17 Alpha estradiol: separa- 555-568.

Notable Notes

The Nikolskiy Sign

In 1894, Russian dermatologist Pyotr Vasiliyevich Nikolskiy, MD (Figure), reported a mechanical phenomenon that would later be internationally recognized by the ep- onym crediting his name—the Nikolsky sign.1 (His name was originally spelled Ni- kolskiy, but it is more commonly referenced as Nikolsky.1) In 1896, his thesis on pemphigus foliaceus described the clinical finding, and his later work suggested that the underlying pathologic process was acantholysis occurring in affected areas as well as in areas with intact, normal-appearing skin. A review of the literature on the Nikolsky sign interestingly reveals a history of dis- agreement about its exact definition and method of elicitation. In 2003, Grando et al1 suggested that the existence of the various different interpretations of the Nikolsky sign may be attributed to the difficulty of clinically differentiating between the many types of bullous diseases before immunofluorescence was available and to the inac- curate translation of the Nikolsky sign, along with its subsequent modifications, which were written in Russian, German, and French.1 Therefore, Grando and colleagues re- turned to the original sources to interpret the Nikolsky sign and other mechanical Figure. Pyotr Vasiliyevich Nikolskiy, signs associated with the bullous diseases that are often confused with the Nikolsky MD (1858-1940), was a professor sign, including the blister-spread sign (Asboe-Hansen/Lutz sign), perifocal subepi- and head of the Department of dermal separation (Sheklakov sign), and epidermal peeling (pseudo-Nikolsky sign). Dermatology at Warsaw University According to Nikolskiy, the Nikolsky sign can be elicited by the following 3 meth- (1898-1915) in Poland and subsequently served as head of the ods: (1) detaching the stratum corneum far beyond the preexisting erosion and over Department of Dermatology and visibly normal skin by pulling the remnant of a ruptured blister wall, known as the Venereology at North-Caucasian marginal Nikolsky sign, a term that was suggested by Sheklakov2 in 19671; (2) dis- University in Rostov-on-Don, Russia. placing or dislodging the stratum corneum to induce an erosion along the periphery of a preexisting erosion by applying lateral pressure with a finger1; and (3) displacing or dislodging the stratum corneum to induce an erosion on areas of visibly normal skin by applying lateral pressure with a finger, known as the direct Nikolsky sign. As elucidated by Grando and coauthors, the Nikolsky sign is usually only positive for diseases that involve epidermal acantholysis, including the pemphigus group of disorders and the staphylococcal scalded skin syndrome. The Nikolsky sign is typically negative for diseases with dermoepidermal separation, such as pemphigoid (bullous, cicatrical, and gestationis) and epidermolysis bullosa acquisita, as well as for diseases in which exfoliation of dead epidermis occurs, such as erythema multiforme (Stevens-Johnson syndrome) and toxic epidermal necrolysis. In 2006, Uzun and Durdu3 conducted a study that tested the specificity and sensitivity of the Nikolsky sign in the diagnosis of pemphigus in 123 patients using the elicitation methods described by Grando et al.1 They discovered that the Nikolsky sign offers a moderately sensitive but highly specific tool for the diagnosis of pemphigus. The marginal Nikolsky sign is more sensitive (69%) for the diagnosis of pemphigus, but the direct Nikolsky sign is more specific (100%). Jennifer Channual, BS; Jashin J. Wu, MD 1. Grando SA, Grando AA, Glukhenky BT, Doguzov V, Nguyen VT, Holubar K. History and clinical significance of mechanical symptoms in blistering dermatoses: a reappraisal. J Am Acad Dermatol. 2003;48(1):86-92. 2. Sheklakov ND. On so-called mechanical symptoms in certain bullous dermatoses [in Russian]. Vestn Dermatol Venereol. 1967;41(3): 28-31. 3. Uzun S, Durdu M. The specificity and sensitivity of Nikolskiy sign in the diagnosis of pemphigus. J Am Acad Dermatol. 2006;54(3):411-415.

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