Abstract
Androgens are well known to influence sebum synthesis and secretion. Various factors related to androgen biosynthesis are expressed in human sebaceous glands. In this study, immunohistochemical analysis of human skin specimens from 43 subjects indicated that various androgen-producing and -metabolizing enzymes were functionally localized to sebocytes accumulating lipid droplets and that the exclusive expression of 17β-hydroxysteroid dehydrogenase type 2 (17β-HSD2 (HSD17B2)) in sebaceous glands was negatively correlated with that of peroxisome proliferator-activated receptor gamma (PPARγ (PPARG)), which also significantly changed in an age-dependent manner. We also demonstrated that the changes of 17β-HSD2 expression in human immortalized sebocytes (SZ95) influenced the expressions of sebogenesis-related factors. In addition, the overexpression of 17β-HSD2 in SZ95 significantly increased the androstenedione production and markedly decreased the amounts of testosterone and dihydrotestosterone when DHEA was added externally. On the other hand, the phosphorylation of mammalian target of rapamycin, which is well known to induce sebum secretion and the onset and/or aggravation of acne, was increased by the addition of testosterone in the presence of IGF1 in hamster sebocytes. These results all indicated that local androgen biosynthesis and metabolism in human sebaceous glands could play a pivotal role in sebum synthesis and secretion.
Introduction
Sebum is known to be mainly composed of squalene, wax esters, fatty acids, cholesterol, cholesterol esters, and triacylglycerol and is synthesized in sebaceous glands, followed by secretion into the skin surface (Montagna 1974, Nikkari 1974). Sebum has also been reported to exhibit an anti-bacterial activity (Wille & Kydonieus 2003, Georgel et al. 2005). In addition, decreased hydration of stratum corneum and enhanced cutaneous inflammation were found in sebaceous gland-deficient (Asebia) mice (Fluhr et al. 2003, Georgel et al. 2005), indicating the pivotal role of sebum in innate skin immunity and skin homeostasis. On the other hand, excessive sebum secretion from activated sebaceous glands causes various skin disorders including acne (Zouboulis et al. 1998). In particular, patients with acne exhibit sustainable rates of sebum secretion compared with healthy subjects (Harris et al. 1983). Therefore, abnormal sebum secretion from sebaceous glands is currently considered to be one of the pivotal causes of the onset and/or the aggravation of acne. More than 60% of adolescents who are 16–20 years old suffer from acne in all ethnic groups but the correlation between ethnicity and acne has not been elucidated (Ghodsi et al. 2009, Perkins et al. 2011, Bhate & Williams 2013). This age period seems to correspond with the development of secondary sex characteristics and the initiation of sex steroid synthesis in adrenal glands and gonads, which influence sebum secretion (Pochi & Strauss 1974). Both testosterone and estradiol (E2) have been reported to increase and decrease sebum secretion respectively (Pochi & Strauss 1974). Consistently, androgen receptor (AR) blockers have been reported to decrease sebum secretion (Makrantonaki & Zouboulis 2007, George et al. 2008), resulting in the clinical improvement of acne. These findings all indicated the importance of sex steroids in the pathogenesis and development of acne.
Sex steroids are primarily synthesized and secreted from the adrenal cortex, ovaries, and testes and are subsequently transported to peripheral target tissues via circulation (Labrie 1991). The abundance of precursor steroids in the circulation and the presence of their synthetic enzymes have been reported for a variety of peripheral tissues (Belanger et al. 2002, Yanase et al. 2003), which indicates that sex steroids are locally biosynthesized in an ‘intracrine’ manner. This peripheral production of sex steroids has been considered to play important roles in the homeostasis of peripheral tissues (Sasano et al. 2008). In human skin, various androgen synthetic and metabolic enzymes have been reported to be abundantly expressed in human sebaceous glands (Fritsch et al. 2001, Thiboutot et al. 2003, Zouboulis 2004), suggesting the possible involvement of local androgen production in the function of human sebaceous glands.
In this study, we examined the hypothesis that local androgen synthesis may be involved in sebogenesis and we characterized the correlations between various androgen-producing/metabolizing factors and sebogenesis markers in human facial skin specimens. We then evaluated the influence of androgenesis factors on sebum secretion using human immortalized sebocytes and hamster sebocytes in order to further elucidate the mechanisms underlying the intracrine-based androgen regulation of sebum secretion in human sebaceous glands.
Materials and methods
Human skin specimens
Facial skin specimens from 43 subjects who had undergone surgery to remove nevi during the period from 1999 to 2008 were retrieved from surgical pathology files at the Department of Pathology, Tohoku University Hospital, Sendai, Japan. The subjects were all Japanese and there were no histopathological signs of inflammation in any of these specimens. The relevant information on the specimens studied is summarized in Table 1. All specimens were fixed in 10% formalin and embedded in paraffin. Two further skin specimens that were used for laser capture microdissection (LCM) were obtained from two male patients (61- and 68-year-old) after surgery, who showed no clinical or histopathological signs of skin abnormalities. All protocols in this study were approved by the Ethics Committee at the Tohoku University Graduate School of Medicine, Sendai, Japan (approval number 2008-124).
Summary of age and sex of the subjects studied
| No. of cases (n=43) | |
|---|---|
| Sex | |
| Male | 19 |
| Female | 24 |
| Age (years) | |
| 10–20 | 10 |
| 20–30 | 11 |
| 30–40 | 6 |
| 40–50 | 5 |
| 50–60 | 7 |
| 60–70 | 1 |
| 70–80 | 3 |
Immunohistochemistry
Streptavidin–biotin amplification was performed according to the manufacturer's instructions (Histofine Kit, Nichiei, Tokyo, Japan). StAR was detected by immunostaining following a 20 min microwave antigen retrieval in citric acid buffer (2 mmol/l citric acid and 9 mmol/l trisodium citrate dihydrate, pH 6.0), whereas the immunohistochemistry for AR (Dako, Copenhagen, Denmark) and peroxisome proliferator-activated receptor gamma (PPARγ (PPARG)) (Perseus Proteomics, Tokyo, Japan) was performed by heating the slides in an autoclave at 121 °C for 5 min. Antigen retrieval was not performed for the immunohistochemistry of 17β-hydroxysteroid dehydrogenase type 2 (17β-HSD2 (HSD17B2)) (Proteintech; Chicago, IL, USA), 3β-HSD (a gift from Dr Ian Mason at the University of Edinburgh, Edinburgh), 5α-reductase type 1 (5α-red1) (a gift from Dr D W Russell at the University of Texas Southwestern Medical Center, Dallas), and epithelial membrane antigen (EMA) (Dako). Each primary antibody was used at the following dilution based upon the results of preliminary studies determining the optimum conditions for immunostaining: AR, 1/50; 17β-HSD2, 1/200; 3β-HSD, 1/2500; StAR, 1/500; 5α-red1, 1/2000; PPARγ, 1/700; and EMA, 1/600. Antigen–antibody complexes were visualized using a 3,3′-diaminobenzidine (DAB) solution composed of 1 mM DAB, 50 mM Tris–HCl buffer, pH 7.6, and 0.006% H2O2 and were counterstained with hematoxylin. As a negative control for immunohistochemistry, normal rabbit or mouse IgG was used.
Evaluation of immunoreactivity
All evaluations were carried out in healthy skin regions. The immunoreactivity of StAR, 3β-HSD, 5α-red1, and EMA was evaluated according to the modified H-score method as described previously (Zhenhuan et al. 2002). Briefly, the cytoplasmic staining intensity in sebaceous glands was tentatively scored as follows: 0, negative; 1, weak; 2, intermediate; or 3, strong. Five areas in each tissue section were randomly selected by two of the authors (T I and Y M). The H-score was calculated according to the following formula: H-score=(the percentage of area occupied by cells scored as 1)×1+(the percentage of area occupied by cells scored as 2)×2+(the percentage of area occupied by cells scored as 3)×3. The immunoreactivity of AR, PPARγ, and 17β-HSD2 was also assessed by evaluating the percentage of positive cells using 300 sebaceous gland cells, i.e. the labeling index (LI), as described previously (Suzuki et al. 2007). The evaluation was also performed independently by two of the authors (T I and Y M) and inter- and intra-observer differences were confirmed to be less than 5%.
LCM/real-time PCR
Skin specimens obtained from two Japanese males, 61 and 68 years old, were embedded in optimal cutting temperature compound (Sakura Finetek, Tokyo, Japan) and were kept at −80 °C until use. The frozen sections stained with toluidine blue were carefully separated into the following components: i) epidermis, ii) sebaceous glands, iii) sweat glands, and iv) the remaining dermis except for hair follicles using Laser Scissors CRI-337 (Cell Robotics, Inc., Albuquerque, NM, USA) under light microscopy. Total RNAs of the tissue sections described as (i) to (iv) above were extracted according to the protocol of the RNA Microisolation Kit (Qiagen). cDNAs were synthesized using a QuantiTect RT Kit (Qiagen) according to the manufacturer's instructions. Real-time PCR was also performed in order to quantify the 17β-HSD2 expression level. The expression of large ribosomal protein P0 (RPLP0) was used as an internal control.
Cell culture
The immortalized human sebocyte cell line SZ95 (Zouboulis et al. 1999) was maintained in Sebomed medium (Biochrom, Berlin, Germany) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS, Life Technologies Japan), 50 U/ml penicillin, 50 μg/ml streptomycin (Life Technologies Japan), and 5 ng/ml human recombinant epidermal growth factor (EGF) (Funakoshi, Tokyo, Japan) at 37 °C in an atmosphere of 5% (v/v) CO2 (Wróbel et al. 2003).
Primary hamster sebocytes (Kurabo, Osaka, Japan) were cultured with Humedia BG (Kurabo) at 37 °C in an atmosphere of 5% (v/v) CO2.
Establishment of cell lines stably over-expressing 17β-HSD2
The open reading frame of 17β-HSD2 was amplified from SZ95 cDNA using the PrimeSTAR GXL DNA Polymerase (Takara Bio, Shiga, Japan) with the following primers: 5′-GTAGAATTCATGAGCACTTTCTTCTCGGACA-3′ and 5′-GTACTCGAGCTAGGTGGCCTTTTTCTTGTAG-3′. After the amplified fragments were inserted into the multiple cloning site of pcDNA3.1 (−) (Life Technologies) using an In-Fusion HD Cloning Kit (Takara Bio), the plasmid (pcDNA3.1 (17β-HSD2)) was extracted and purified using an EndoFree Plasmid Purification Kit (Qiagen). The consequent pcDNA3.1 (mock) and pcDNA3.1 (17β-HSD2) cut by PvuI were transfected in SZ95 sebocytes using Lipofectamine 2000 (Life Technologies Japan) and cells expressing the neomycin resistance gene were selected by cultivation in medium containing 200 μg/ml G418- for 7 days.
Total RNA extraction, cDNA synthesis, and real-time PCR
SZ95 sebocytes (SZ95 (cont)) transfected with pcDNA3.1 (mock) and SZ95 sebocytes (SZ95 (17β)), which were modified with pcDNA3.1 (17β-HSD2), were seeded in 24-well plates at a density of 0.1×104 cells/well and were cultured for 7 days in a medium (10% FBS, 50 U/ml penicillin, 50 μg/ml streptomycin, and 5 ng/ml EGF) without any medium change. After total RNAs were isolated from SZ95 (cont) and SZ95 (17β) cells using an RNeasy Mini Kit (Qiagen), cDNAs were synthesized using a QuantiTect RT Kit (Qiagen) according to the manufacturer's instructions. The expressions of PPARγ, 17β-HSD2, stearoyl-CoA desaturase (SCD), glycerol-3-phosphate acyltransferase, mitochondrial (GPAM), EMA, sterol regulatory element-binding 1 (SREBP1), perilipin1 (PLIN1), and carnitine palmitoyltransferase 1 (CPT1 (CPT1A)) were quantified by real-time PCR using TaqMan gene expression assays and an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems) according to the manufacturer's instructions. The expression of RPLP0 was used as an internal control.
Western blotting analysis
After the seeding of hamster sebocytes into six-well plates, these cells were cultured using Humedia BB (Kurabo) in the absence or presence of 1000 ng/ml insulin-like growth factor 1 (IGF1) (Sigma–Aldrich) with 0, 1, 10, and 100 nM testosterone for 24 h. Following their lyses in RIPA buffer (Sigma–Aldrich Japan), 10 μg of each sample were separated on 10% SDS gels (Bio-Rad Laboratories). Samples were then transferred onto PVDF membranes (Bio-Rad Laboratories) and were incubated with antibodies specific for human phospho-mammalian target of rapamycin (mTOR) (Ser2448), mTOR (Cell Signaling Technology, Inc.), or human β-actin (Sigma). Subsequent visualization of antibodies was performed using ECL Plus (GE Healthcare; Amersham, Buckinghamshire, UK) according to the manufacturer's instructions.
siRNA transfection
SZ95 cells seeded in a 24-well plate were transfected using Stealth Select RNAi siRNA with or without any specificity for 17β-HSD2 (Life Technologies Japan) and Lipofectamine 2000 reagent (Life Technologies Japan) according to the manufacturer's instructions. After SZ95 cells were cultured in a medium supplemented with 10% FBS for 3 days, the aforementioned procedures for total RNA extraction, cDNA synthesis, and real-time PCR were also performed in these experiments.
Cell culture and liquid chromatography/electrospray tandem mass spectrometry
SZ95 cells were seeded at a density of 8.0×105 cells/dish onto dishes that were 100 mm in diameter and then were incubated for 24 h, followed by transfection of pcDNA3.1 (mock) or pcDNA3.1 (17β-HSD2) in the presence of Lipofectamine 2000 (Life Technologies Japan). SZ95 cells were cultured in the absence or presence of 1000 nM DHEA in the media containing 5% charcoal serum for 3 days, after 12 h of transfection. The media and cells were then harvested and subsequently homogenized using a homogenizer for the quantification of androstenedione, testosterone, and dihydrotestosterone concentrations. The concentration of androgens was measured by liquid chromatography/electrospray tandem mass spectrometry (LC–MS/MS) at ASKA Pharma Medical Co. Ltd (Kawasaki, Kanagawa, Japan) as described previously (Inoue et al. 2012). Testosterone, androstenedione, and dihydrotestosterone concentrations were all measured by LC (Nexera, Shimazdu, Kyoto, Japan) coupled with an API 4000 triple-stage quadrupole mass spectrometer (ABsciex, Foster City, CA, USA) operated with electrospray ionization in the positive-ion mode; the chromatographic separation was performed on a Kinetex C18 column (2.1×150 mm, 1.7 μm, Phenomenex, Torrance, CA, USA). The injected volume of each sample was 20 μl. The mobile phase consisting of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) was used for gradient elution of A:B=65:35–0:100 (0–4.9 min) and isocratic elution of A:B=0:100 (4.9–5.4 min) and 65:35 (5.4–6.0 min). The flow rates were 0.5 ml/min (0–4.9 min), 0.9 ml/min (4.9–5.6 min), and 0.75 ml/min (5.6–6.0 min). The ion spray voltage was 5.5 kV, and the turbo gas temperature was 550 °C under ionization conditions. The testosterone derivatives were quantified by evaluating the transition of m/z 394.3 (internal standard (IS) 397.3)–253.2 (IS 147.2), the androstenedione derivatives were quantified by monitoring the transition of m/z 287.4 (IS 294.2)–97.2 (IS 100.0), and the dihydrotestosterone derivatives were quantified by monitoring the transition of m/z 396.3 (IS 399.4)–203.3 (IS 203.3), using a multiple reaction monitoring system.
Statistical analysis
Student's t-test was used to compare the expression levels of sebogenesis and lipid synthesis genes between SZ95 (cont) and SZ95 (17β) cells. The Kruskal–Wallis test was also used to compare the sex steroid concentrations. The Mann–Whitney U test was used to compare the sex differences in LIs of AR and 17β-HSD2 expression and H-scores for StAR, 3β-HSD, and 5α-red1 expressions. The relationships between immunoreactivity for androgen-related proteins and that for sebogenic proteins were analyzed using the Pearson product-moment correlation coefficient or Spearman's coefficient of rank correlation. A P value of <0.05 indicates statistical significance in this study.
Results
Immunohistochemistry of androgen- and sebogenesis-related factors in human sebaceous glands
AR and PPARγ were detected in the nuclei of basal and early differentiated sebocytes, while 3β-HSD and 5α-red1 were all detected in the cytoplasm of early, advanced, and fully differentiated cells (Fig. 1). On the other hand, StAR was detected in the cytoplasm harboring mitochondria of early, advanced, and fully differentiated cells, which is consistent with the results of a previous study (Stocco 2001). In addition, EMA was detected only in the cytoplasm of advanced and fully differentiated cells, whereas 17β-HSD2 was detected in the cytoplasm of several differentiated cells.
Localization of androgen-metabolizing enzymes, androgen receptor (AR), and sebogenesis-related proteins in human sebaceous glands. Immunohistochemistry for androgen-metabolizing enzymes (StAR, 3β-HSD, 17β-HSD2, and 5α-red1), AR, and sebogenesis-related factors (PPARγ and EMA) was performed on human facial skin specimens from 43 subjects, which had been fixed in 10% formalin and embedded in paraffin. Antigen–antibody complexes were visualized with 3,3′-diaminobenzidine solution and were counterstained with hematoxylin. (A) Localization of StAR, 3β-HSD, 17β-HSD2, 5α-red1, and AR expression. (B) Localization of PPARγ and EMA expression. Bars=100 μm.
Citation: Journal of Endocrinology 222, 3; 10.1530/JOE-14-0323
Sex- and age-related changes of immunoreactivity
Regarding the sex differences in AR and 17β-HSD2 immunoreactivity, males had significantly higher levels than females, whereas there were no significant differences in 3β-HSD and StAR immunoreactivity levels between males and females (Table 2). In addition, the levels of 5α-red1 expression also tended to be higher in males than in females (Table 2). However, there were no age-related differences in any of the androgen-related factors (data not shown), although the expression of PPARγ was significantly upregulated in subjects in their twenties compared with those in their teens (Fig. 2).
Sex-dependent changes in the LIs of AR and 17β-HSD2, and in the H-score for 3β-HSD, STAR, and 5α-red1
| Male (mean±s.d.) | Female (mean±s.d.) | P | |
|---|---|---|---|
| Labeling index (%) | |||
| AR | 77.5±11.5 | 64.6±9.67 | <0.01 |
| 17β-HSD2 | 18.0±7.53 | 4.00±6.48 | <0.001 |
| H-score | |||
| 3β-HSD | 91.9±66.6 | 128.5±70.0 | NS |
| StAR | 127.7±46.0 | 130.8±54.9 | NS |
| 5α-red1 | 177.8±42.8 | 133.3±81.6 | 0.06 |
Statistical analysis of the H-score was performed using the Mann–Whitney U test, and that of the LI was performed using Student's t-test. P values <0.05 are considered significant in this study. NS, not statistically significant (P≧0.05).
Age-related differences in PPARγ LI. The data for each age group are represented as box-and-whisker plots. The median value is indicated by the horizontal line in the box plot and the white box denotes the 75th (upper margin) and 25th percentiles of the values (lower margin). The upper and lower bars indicate the maximum and minimum values excluding outliers respectively. The statistical analysis was performed by the Kruskal–Wallis and/or Steel–Dwass tests. *P<0.05 (the teens vs twenties groups).
Citation: Journal of Endocrinology 222, 3; 10.1530/JOE-14-0323
Correlations between androgen- and sebogenesis-related factors
We evaluated the correlations between the immunohistochemical levels of androgen-related factors (StAR, AR, 3β-HSD, 17β-HSD2, and 5α-red1) and sebogenesis-related factors (PPARγ and EMA) to examine the influence of androgen synthesis on sebum production in human sebaceous glands. The LI of AR was positively correlated with that of PPARγ in males in all cases, and with the H-score for EMA in all cases examined (Table 3). A significant negative correlation was also detected between the LIs of 17β-HSD2 and PPARγ in males (Table 3). In addition, the H-score for 5α-red1 expression was positively correlated with that of EMA when the data from males, females, and all subjects were analyzed (Table 3).
Correlations among immunoreactivities of androgenesis- and sebogenesis-related proteins
| PPARγ | EMA | |||
|---|---|---|---|---|
| R | P | R | P | |
| AR | ||||
| Male | 0.58 | <0.01 | 0.35 | NS |
| Female | 0.15 | NS | 0.14 | NS |
| All | 0.31 | <0.05 | 0.37 | <0.05 |
| 17β-HSD2 | ||||
| Male | −0.69 | <0.01 | −0.11 | NS |
| Female | −0.34 | NS | −0.17 | NS |
| All | −0.3 | NS | 0.19 | NS |
| 3β-HSD | ||||
| Male | −0.01 | NS | −0.17 | NS |
| Female | −0.01 | NS | −0.31 | NS |
| All | −0.03 | NS | −0.33 | NS |
| STAR | ||||
| Male | −0.32 | NS | −0.17 | NS |
| Female | 0.27 | NS | 0.13 | NS |
| All | 0.03 | NS | −0.02 | NS |
| 5α-red1 | ||||
| Male | 0.03 | NS | 0.56 | <0.05 |
| Female | 0.35 | NS | 0.45 | <0.05 |
| All | 0.21 | NS | 0.52 | <0.001 |
The Pearson product-moment correlation coefficient was used in order to analyze the correlation coefficients (R) and P values between LIs. Spearman's coefficient of rank correlation was applied to evaluate R and P between H-scores or between H-score and LI.
Localization of 17β-HSD2 in human skin
The influence of 17β-HSD2 levels, which correlated with PPARγ immunoreactivity, on sebogenesis has not been reported previously. Therefore, we focused upon the functions of 17β-HSD2 in sebogenesis. The localization and expression levels of 17β-HSD2 mRNA in human skin were evaluated using LCM/real-time PCR. The mRNA expression of 17β-HSD2 was assessed in sebaceous glands compared with epidermis, dermis, and sweat glands and its substantial expression was exclusively discovered in sebaceous glands (Fig. 3a). Consistently, 17β-HSD2 immunoreactivity was also detected in mature cells of sebaceous glands, but not in any cells in the epidermis, hair, dermis, or sweat glands (Fig. 3b).
Localization and mRNA expression levels of 17β-HSD2 in human skin. (a) Skin specimens obtained from two Japanese males, 61- and 68-year-old (M61 and M68), were snap-frozen and sectioned (10 μm) using a cryostat. Using a microscope, the frozen sections were carefully dissected into epidermis, sebaceous glands, sweat glands, and dermis by LCM and the expression of 17β-HSD2 mRNA was quantified by real-time PCR using RPLP0 expression as an internal control. (b) 17β-HSD2 immunohistoreactivity in sebaceous glands, epidermis, hair, dermis, and sweat glands using formalin-fixed paraffin-embedded tissues. Bars=100 μm.
Citation: Journal of Endocrinology 222, 3; 10.1530/JOE-14-0323
Effects of the alteration in 17β-HSD2 expression on the expression of sebogenesis- and lipid synthesis-related factors in SZ95 sebocytes
17β-HSD2 was stably overexpressed in SZ95 sebocytes in order to study the effect of 17β-HSD2 expression on sebogenesis as described above. The mRNA expression level of 17β-HSD2 was demonstrated to be stably 16 times higher in SZ95 (17β) cells than that in SZ95 (cont) cells (data not shown). The mRNA expression level of PPARγ, one of the sebogenesis-related factors, in SZ95 (17β) cells was significantly decreased compared with SZ95 (cont) cells (Fig. 4a). In addition, the expression levels of GPAM, SCD, and SREBP1, all reported to be indispensable factors in lipid synthesis (Ntambi et al. 2002, Rosignoli et al. 2003, Harrison et al. 2007), and PLIN1, protecting oil droplets from degradation by lipase (Tansey et al. 2001), as well as CPT1A, transporting fatty acids into mitochondria (Louet et al. 2001) were evaluated. The expressions of GPAM and SREBP1 in SZ95 (17β) cells were also significantly suppressed, whereas those of SCD, PLIN1, and CPT1A were not significantly changed (Fig. 4a). Conversely, when the mRNA expression level of 17β-HSD2 was decreased by ∼50% by the use of siRNA for 17β-HSD2, the mRNA expression levels of EMA, GPAM, and SREBP1 in SZ95 were significantly increased compared with those in SZ95 that had been transfected with control siRNA (Fig. 4b). However, there were no significant changes in mRNA expression of PPARγ, SCD, PLIN1, and CPT1A (Fig. 4b).
mRNA expression levels of sebogenesis-related and lipid-synthesis-related factors. (a) SZ95 (cont), which were transfected with a mock plasmid, and SZ95 (17β), which were instead treated with a 17β-HSD2 plasmid to stably overexpress 17β-HSD2, were cultured for 7 days in the medium supplemented with 10% FBS, followed by the examination of mRNA transcript expression of the interested factors using real-time PCR. (b) After SZ95 cells were transfected with control siRNA or 17β-HSD2 siRNA, they were cultured in the medium containing 10% FBS for 3 days and their mRNA expressions were quantified. The P value was analyzed using Student's t-test. NS, not significant.
Citation: Journal of Endocrinology 222, 3; 10.1530/JOE-14-0323
Local androgen synthesis in sebocytes
The levels of androstenedione, testosterone, and dihydrotestosterone (DHT) produced in the presence or absence of DHEA were measured using LC–MS/MS in order to evaluate the ability to locally synthesize androgens in sebaceous cells. In SZ95 cells transfected with a mock plasmid, androstenedione, testosterone, and DHT concentrations were hardly detectable in the absence of DHEA (Mock), but all androgens were detected in the presence of DHEA (Mock+DHEA) (Fig. 5). When 17β-HSD2 was overexpressed under such a condition, the amounts of testosterone and DHT produced in SZ95 (17β-HSD2+DHEA) cells were significantly decreased compared with those in SZ95 cells transfected with a mock plasmid (Mock+DHEA). Consistently, the androstenedione concentration was significantly increased in 17β-HSD2+DHEA cells when compared with that in Mock+DHEA cells (Fig. 5).
The effect of DHEA and 17β-HSD2 on the production of androstenedione, testosterone, and DHT in SZ95. After SZ95 cells were transfected with pcDNA3.1 (mock) or pcDNA3.1 (17β-HSD2), plasmids were cultured for 24 h and were cultured in the absence or presence of 1000 nM DHEA for 3 days. The concentrations of androstenedione, testosterone, and DHT in 1 g of the mixture of cells and the media were measured using LC–MS/MS. The P value was analyzed using the Kruskal–Wallis test.
Citation: Journal of Endocrinology 222, 3; 10.1530/JOE-14-0323
Effect of testosterone upon the phosphorylation of mTOR
After the inoculation of hamster sebocytes, these cells were cultured with testosterone in the presence or absence of IGF1 for 24 h. Western blotting analysis demonstrated that testosterone markedly increased the expression of phosphorylation of mTOR (p-mTOR) in a dose-dependent manner under stimulation with IGF1, whereas there were no significant changes in its expression in the absence of IGF1 (Fig. 6).
The effect of testosterone on the phosphorylation of mTOR. Hamster sebocytes were cultured using Humedia-BB in the absence or presence of 1000 ng/ml IGF1 with 0, 1, 10, and 100 nM testosterone for 24 h.
Citation: Journal of Endocrinology 222, 3; 10.1530/JOE-14-0323
Discussion
Sebaceous glands have been reported to be able to synthesize steroid hormones from cholesterol, suggesting a paracrine and/or autocrine (intracrine) involvement of these sex steroids in sebum production and/or secretion (Labrie et al. 2000a, Thiboutot et al. 2003). SZ95 sebocytes have been reported to express mRNAs encoding various androgen-metabolizing enzymes, including 5α-red1, 3β-HSD, 17β-HSD2, and 17β-HSD3 (Fritsch et al. 2001, Thiboutot et al. 2003, Harrison et al. 2007), and to be able to convert DHEA into testosterone and testosterone into DHT (Fritsch et al. 2001, Makrantonaki & Zouboulis 2007, Chen et al. 2010). In addition, the local activation of 5α-red1 has also been recently reported to regulate sebum secretion via DHT synthesis (Li et al. 2010). The expression of 17β-HSD2 mRNA among 17β-HSD isozymes was reported to be markedly detected in sebaceous glands (Thiboutot et al. 1998) and 3β-HSD is considered to play an important role in the synthesis of sex steroid hormones in skin tissue (Labrie et al. 2000a). Furthermore, we have previously demonstrated that the status of 3β-HSD1 mRNA expression was correlated with the androstenedione concentration in human skin (Inoue et al. 2012). All of these findings indicate that local or in situ androgen synthesis in sebaceous glands may play an important role in skin homeostasis, especially in the process of sebogenesis. In this study, we first examined the correlation between the expression of androgen-producing/metabolizing enzymes and receptors, and the expression of sebogenesis-related factors in human facial skin specimens obtained from 43 subjects by surgery. Our results demonstrated that 5α-red1, 3β-HSD, 17β-HSD2, and StAR were all abundantly expressed in sebaceous glands, exclusively or especially in sebocytes accumulating lipid droplets (differentiating sebocytes). In addition, results of a previous study by Chen et al. (2010), demonstrating the ability of SZ95 to synthesize testosterone from DHEA, were consistent with our findings that SZ95 could synthesize not only testosterone but also DHT and androstenedione from the external DHEA. These results indicate that androgens are actively synthesized and/or metabolized in differentiated sebocytes via various enzymes in human sebaceous glands.
Levels of sebum secretion have been reported to be higher in males than in females after puberty mainly because of the increased blood concentration of androgens in males (Pochi & Strauss 1974). In addition, activation of PPARγ, one of the key induction factors of adipocyte differentiation, has been reported to increase sebum secretion in human skin (Trivedi et al. 2006). Our results also indicate that PPARγ immunoreactivity was significantly increased at puberty when sebum secretion is dramatically upregulated, a finding also consistent with the results of the studies mentioned above. In addition to PPARγ, EMA has been used to study the grade of sebocyte differentiation, because EMA has been reported to be abundantly expressed in differentiated sebocytes (Zouboulis et al. 1999, Eisinger et al. 2011). Therefore, we also evaluated the correlations between levels of androgen-producing/metabolizing enzymes and AR with levels of PPARγ and EMA in human sebaceous glands in order to further elucidate the roles of in situ androgen production in the regulation of sebogenesis in human skin. The levels of AR and 5α-red1 immunoreactivity in sebaceous glands were significantly higher in males than in females and were also positively correlated with PPARγ and EMA immunoreactivity. These results all indicate that AR and 5α-red1 are closely associated with each other, especially in male sebaceous glands, which could promote the supply of DHT via 5α-red1 to activate AR, subsequently resulting in the acceleration of sebum synthesis and secretion.
In this study, we also demonstrated for the first time, to our knowledge, that 17β-HSD2 was more abundantly expressed in males than in females and is negatively correlated with PPARγ in sebaceous glands. 17β-HSD2 has been proposed to be a key regulator of various functions of steroid hormones, especially in peripheral tissues (Labrie et al. 2000b), because of its multiple functions in catalyzing the NADP+-dependent oxidation of the most potent estrogen (17β-E2) into the weak estrogen (estrone) and in converting testosterone into androstenedione (Wu et al. 1993). In addition, transgenic mice overexpressing 17β-HSD2 have been reported to display markedly disturbed skeletal development (Shen et al. 2008) and it has been suggested that human kidney cells transiently overexpressing 17β-HSD2 actively convert testosterone into androstenedione (Suzuki et al. 2000). In this study, the experiments using SZ95 stably overexpressing 17β-HSD2 or PPARγ did demonstrate that the increase in 17β-HSD2 expression downregulated the expression of PPARγ, whereas the enhancement of PPARγ expression did not affect the expression of 17β-HSD2 (data not shown). Based on this result, we subsequently examined the effects of androgen on the expression of PPARγ in order to further explore the causes of the change in PPARγ expression under the augmentation of 17β-HSD2. It is entirely true that the externally administered testosterone, which was mostly metabolized by the action of 17β-HSD2, did not affect the level of PPARγ expression in this study (data not shown). In previous studies, 17β-HSD2 has been reported to metabolize not only androgens but also estrogens (Puranen et al. 1999) and to be involved in retinoid action in addition to sex steroid metabolism (Zhongyi et al. 2007, Shen et al. 2009), and both E2 and retinoic acid have been reported to upregulate the expression of PPARγ (Zhu et al. 1998, Sato et al. 2013). These findings indicated that 17β-HSD2 could influence the expression of PPARγ by exploiting the functions of estrogens and/or retinoids rather than androgens. In relation to this, we then employed SZ95 sebocytes to further evaluate the effects of 17β-HSD2 levels on sebum production in human skin. When 17β-HSD2 was overexpressed or decreased in SZ95 sebocytes, pivotal factors of sebum and lipid synthesis were significantly changed. Additionally, the expressions of GPAM and SREBP1 were also demonstrated to be enhanced by the decrease in 17β-HSD2 expression in SZ95 sebocytes consistent with results of an experiment on hamster's sebaceous glands showing their increases in the presence of androgens (Rosignoli et al. 2003). Parallel to our results, lipid droplet secretion into enterocytes was also reported to be augmented when 17β-HSD2 expression was decreased (Beilstein et al. 2013). In addition, we attempted to investigate 17β-HSD2 function in local androgen synthesis of SZ95, suggesting that 17β-HSD2 contributed to regulation of the testosterone and DHT concentration. These findings all indicated that 17β-HSD2 played important roles in the local metabolism of androgen, consequently resulting in the regulation of lipid synthesis via the alteration of expression of sebogenesis factors, such as SREBP1 and GPAM, in human sebaceous glands.
In addition to the local androgen regulation in human sebaceous glands, the actions of the central hormone driving puberty and sexual differentiation should also be considered, as IGF1, whose concentration is dramatically increased during puberty in conjunction with androgen, has been reported to play an important role in the onset and aggravation of acne (Melnik & Schmitz 2009). Western diets, associated with a higher glycemic load and dairy protein consumption, have been proposed to induce and/or aggravate acne accompanied by the activation of IGF1 signaling, followed by the promotion of mTOR signaling (Melnik 2012, Melnik & Zouboulis 2013). In addition, mTORC1 stimulation has been reported to cause PPARγ stimulation in adipose tissue (Blanchard et al. 2012) and promote lipid synthesis by activating SREBP1 (Bakan & Laplante 2012), while testosterone has been demonstrated to increase the phosphorylation of mTOR in cardiomyocytes (Altamirano et al. 2009). In addition, co-stimulation of androgen signaling with IGF1 signaling has been previously demonstrated to maximize sebum synthesis in sebocytes (Rosignoli et al. 2003). Thus, we examined the influence of androgen on p-mTOR in order to reveal the relationship between androgen and IGF1 signaling in SZ95. However, testosterone did not affect the p-mTOR in SZ95 (data not shown). Considering that androgen has been reported to induce sebum secretion and accumulation in human sebaceous glands in vivo and in hamster's sebocytes, but not in SZ95 (Rosignoli et al. 2003, Makrantonaki & Zouboulis 2007), androgen was considered not to be able to activate mTOR signaling in SZ95 because of the lack of androgen-responsive functions. Our experiments evaluating the role of androgen in p-mTOR activation using hamster's sebocytes, which have androgen-responsive functions, indicted that testosterone stimulated p-mTOR expression in the presence of IGF1. These findings above all indicated that the synergistic effects of local androgen production with circulating IGF1 play pivotal roles in sebum secretion and initiation and/or deterioration of acne.
In conclusion, we demonstrated that local androgen production and metabolism in human sebaceous glands are closely correlated with sebum synthesis and/or secretion. These results also provide new insights into the mechanisms underlying the involvement of intracrine-based androgenesis in the sebogenesis of peripheral tissues. In particular, the inverse correlations between 17β-HSD2 expression and sebogenesis markers could contribute to the development of an efficient strategy for treating acne-related disorders through the manipulation of local androgen production and metabolism.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Acknowledgements
The authors appreciate the skillful technical assistance of Mr Katsuhiko Ono (Department of Pathology, Tohoku University School of Medicine).
References
Altamirano F, Oyarce C, Silva P, Toyos M, Wilson C, Lavandero S, Uhlén P & Estrada M 2009 Testosterone induces cardiomyocyte hypertrophy through mammalian target of rapamycin complex 1 pathway. Journal of Endocrinology 202 299–307. (doi:10.1677/JOE-09-0044)
Bakan I & Laplante M 2012 Connecting mTORC1 signaling to SREBP-1 activation. Current Opinion in Lipidology 23 226–234. (doi:10.1097/MOL.0b013e328352dd03)
Beilstein F, Bouchoux J, Rousset M & Demignot S 2013 Proteomic analysis of lipid droplets from Caco-2/TC7 enterocytes identifies novel modulators of lipid secretion. PLoS ONE 8 e53017. (doi:10.1371/journal.pone.0053017)
Belanger C, Luu-The V, Dupont P & Tchernof A 2002 Adipose tissue intracrinology: potential importance of local androgen/estrogen metabolism in the regulation of adiposity. Hormone and Metabolic Research 34 737–745. (doi:10.1055/s-2002-38265)
Bhate K & Williams HC 2013 Epidemiology of acne vulgaris. British Journal of Dermatology 168 474–485. (doi:10.1111/bjd.12149)
Blanchard PG, Festuccia WT, Houde VP, St-Pierre P, Brûlé S, Turcotte V, Côté M, Bellmann K, Marette A & Deshaies Y 2012 Major involvement of mTOR in the PPARγ-induced stimulation of adipose tissue lipid uptake and fat accretion. Journal of Lipid Research 53 1117–1125. (doi:10.1194/jlr.M021485)
Chen W, Tsai SJ, Sheu HM, Tsai JC & Zouboulis CC 2010 Testosterone synthesized in cultured human SZ95 sebocytes derives mainly from dehydroepiandrosterone. Experimental Dermatology 19 470–472. (doi:10.1111/j.1600-0625.2009.00996.x)
Eisinger M, Li WH, Anthonavage M, Pappas A, Zhang L, Rossetti D, Huang Q & Seiberg M 2011 A melanocortin receptor 1 and 5 antagonist inhibits sebaceous gland differentiation and the production of sebum-specific lipids. Journal of Dermatological Science 63 23–32. (doi:10.1016/j.jdermsci.2011.04.001)
Fluhr JW, Mao-Qiang M, Brown BE, Wertz PW, Crumrine D, Sundberg JP, Feingold KR & Elias PM 2003 Glycerol regulates stratum corneum hydration in sebaceous gland deficient (Asebia) mice. Journal of Investigative Dermatology 120 728–737. (doi:10.1046/j.1523-1747.2003.12134.x)
Fritsch M, Orfanos CE & Zouboulis CC 2001 Sebocytes are the key regulators of androgen homeostasis in human skin. Journal of Investigative Dermatology 116 793–800. (doi:10.1046/j.1523-1747.2001.01312.x)
George R, Clarke S & Thiboutot D 2008 Hormonal therapy for acne. Seminars in Cutaneous Medicine and Surgery 27 188–196. (doi:10.1016/j.sder.2008.06.002)
Georgel P, Crozat K, Lauth X, Makrantonaki E, Seltmann H, Sovath S, Hoebe K, Du X, Rutschmann S & Beutler B et al. 2005 A TLR2-responsive lipid effector pathway protects mammals against Gram-positive bacterial skin infections. Infection and Immunity 73 4512–4521. (doi:10.1128/IAI.73.8.4512-4521.2005)
Ghodsi SZ, Orawa H & Zouboulis CC 2009 Prevalence, severity and severity risk factors of acne in high school pupils: a community-based study. Journal of Investigative Dermatology 129 2136–2141. (doi:10.1038/jid.2009.47)
Harris HH, Downing DT, Stewart ME & Strauss JS 1983 Sustainable rates of sebum secretion in acne patients and matched normal control subjects. Journal of the American Academy of Dermatology 8 200–203. (doi:10.1016/S0190-9622(83)70023-X)
Harrison WJ, Bull JJ, Seltmann H, Zouboulis CC & Philpott MP 2007 Expression of lipogenic factors galectin-12, resistin, SREBP-1 and SCD in human sebaceous glands and cultured sebocytes. Journal of Investigative Dermatology 127 1309–1317. (doi:10.1038/sj.jid.5700743)
Inoue T, Miki Y, Abe K, Hatori M, Hosaka M, Kariya Y, Kakuo S, Fujimura T, Hachiya A & Honma S et al. 2012 Sex steroid synthesis in human skin in situ: the roles of aromatase and steroidogenic acute regulatory protein in the homeostasis of human skin. Molecular and Cellular Endocrinology 362 19–22. (doi:10.1016/j.mce.2012.05.005)
Labrie F 1991 Intracrinology. Molecular and Cellular Endocrinology 78 C113–C118. (doi:10.1016/0303-7207(91)90116-A)
Labrie F, Luu-The V, Labrie C, Pelletier G & El-Alfy M 2000a Intracrinology and the skin. Hormone Research 54 218–229. (doi:10.1159/000053264)
Labrie F, Luu-The V, Lin SX, Simard J, Labrie C, El-Alfy M, Pelletier G & Belanger A 2000b Intracrinology: role of the family of 17β-hydroxysteroid dehydrogenases in human physiology and disease. Journal of Molecular Endocrinology 25 1–16. (doi:10.1677/jme.0.0250001)
Li L, Tang L, Baranov E, Yang M, Amoh Y, Katsuoka K & Hoffman RM 2010 Selective induction of apoptosis in the hamster flank sebaceous gland organ by a topical liposome 5-α-reductase inhibitor: a treatment strategy for acne. Journal of Dermatology 37 156–162. (doi:10.1111/j.1346-8138.2009.00778.x)
Louet JF, Le May C, Pégorier JP, Decaux JF & Girard J 2001 Regulation of liver carnitine palmitoyltransferase I gene expression by hormones and fatty acids. Biochemical Society Transactions 29 310–316. (doi:10.1042/BST0290310)
Makrantonaki E & Zouboulis CC 2007 Testosterone metabolism to 5α-dihydrotestosterone and synthesis of sebaceous lipids is regulated by the peroxisome proliferator-activated receptor ligand linoleic acid in human sebocytes. British Journal of Dermatology 156 428–432. (doi:10.1111/j.1365-2133.2006.07671.x)
Melnik BC 2012 Dietary intervention in acne: attenuation of increased mTORC1 signaling promoted by Western diet. Dermato-Endocrinology 4 20–32. (doi:10.4161/derm.19828)
Melnik BC & Schmitz G 2009 Role of insulin, insulin-like growth factor-1, hyperglycaemic food and milk consumption in the pathogenesis of acne vulgaris. Experimental Dermatology 18 833–841. (doi:10.1111/j.1600-0625.2009.00924.x)
Melnik BC & Zouboulis CC 2013 Potential role of FoxO1 and mTORC1 in the pathogenesis of Western diet-induced acne. Experimental Dermatology 22 311–315. (doi:10.1111/exd.12142)
Montagna W 1974 An introduction to sebaceous glands. Journal of Investigative Dermatology 62 120–123. (doi:10.1111/1523-1747.ep12676775)
Nikkari T 1974 Comparative chemistry of sebum. Journal of Investigative Dermatology 62 257–267. (doi:10.1111/1523-1747.ep12676800)
Ntambi JM, Miyazaki M, Stoehr JP, Lan H, Kendziorski CM, Yandell BS, Song Y, Cohen P, Friedman JM & Attie AD 2002 Loss of stearoyl-CoA desaturase-1 function protects mice against adiposity. PNAS 99 11482–11486. (doi:10.1073/pnas.132384699)
Perkins AC, Cheng CE, Hillebrand GG, Miyamoto K & Kimball AB 2011 Comparison of the epidemiology of acne vulgaris among Caucasian, Asian, Continental Indian and African American women. Journal of the European Academy of Dermatology and Venereology 25 1054–1060. (doi:10.1111/j.1468-3083.2010.03919.x)
Pochi PE & Strauss JS 1974 Endocrinologic control of the development and activity of the human sebaceous gland. Journal of Investigative Dermatology 62 191–201. (doi:10.1111/1523-1747.ep12676783)
Puranen TJ, Kurkela RM, Lakkakorpi JT, Poutanen MH, Itäranta PV, Melis JP, Ghosh D, Vihko RK & Vihko PT 1999 Characterization of molecular and catalytic properties of intact and truncated human 17β-hydroxysteroid dehydrogenase type 2 enzymes: intracellular localization of the wild-type enzyme in the endoplasmic reticulum. Endocrinology 140 3334–3441. (doi:10.1210/endo.140.7.6861)
Rosignoli C, Nicolas JC, Jomard A & Michel S 2003 Involvement of the SREBP pathway in the mode of action of androgens in sebaceous glands in vivo. Experimental Dermatology 12 480–489. (doi:10.1034/j.1600-0625.2003.00014.x)
Sasano H, Suzuki T, Miki Y & Moriya T 2008 Intracrinology of estrogen and androgens in breast carcinoma. Journal of Steroid Biochemistry and Molecular Biology 108 181–185. (doi:10.1016/j.jsbmb.2007.09.012)
Sato H, Sugai H, Kurosaki H, Ishikawa M, Funaki A, Kimura Y & Ueno K 2013 The effect of sex hormones on peroxisome proliferator-activated receptor γ expression and activity in mature adipocytes. Biological & Pharmaceutical Bulletin 36 564–573. (doi:10.1248/bpb.b12-00868)
Shen Z, Peng Z, Sun Y, Vaananen HK & Poutanen M 2008 Overexpression of human hydroxysteroid (17β) dehydrogenase 2 induces disturbance in skeletal development in young male mice. Journal of Bone and Mineral Research 23 1217–1226. (doi:10.1359/jbmr.080322)
Shen Z, Saloniemi T, Rönnblad A, Järvensivu P, Pakarinen P & Poutanen M 2009 Sex steroid-dependent and -independent action of hydroxysteroid (17β) dehydrogenase 2: evidence from transgenic female mice. Endocrinology 150 4941–4949. (doi:10.1210/en.2009-0670)
Stocco DM 2001 StAR protein and the regulation of steroid hormone biosynthesis. Annual Review of Physiology 63 193–213. (doi:10.1146/annurev.physiol.63.1.193)
Suzuki T, Sasano H, Andersson S & Mason JI 2000 3β-Hydroxysteroid dehydrogenase/Δ5→4-isomerase activity associated with the human 17β-hydroxysteroid dehydrogenase type 2 isoform. Journal of Clinical Endocrinology and Metabolism 85 3669–3672. (doi:10.1210/jcem.85.10.6918)
Suzuki T, Miki Y, Moriya T, Akahira J, Ishida T, Hirakawa H, Yamaguchi Y, Hayashi S & Sasano H 2007 5α-Reductase type 1 and aromatase in breast carcinoma as regulators of in situ androgen production. International Journal of Cancer 120 285–291. (doi:10.1002/ijc.22317)
Tansey JT, Sztalryd C, Gruia-Gray J, Roush DL, Zee JV, Gavrilova O, Reitman ML, Deng CX, Li C & Kimmel AR et al. 2001 Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity. PNAS 22 6494–6499. (doi:10.1073/pnas.101042998)
Thiboutot D, Martin P, Volikos L & Gilliland K 1998 Oxidative activity of the type 2 isozyme of 17β-hydroxysteroid dehydrogenase (17β-HSD) predominates in human sebaceous glands. Journal of Investigative Dermatology 111 390–395. (doi:10.1046/j.1523-1747.1998.00322.x)
Thiboutot D, Jabara S, McAllister JM, Sivarajah A, Gilliland K, Cong Z & Clawson G 2003 Human skin is a steroidogenic tissue: steroidogenic enzymes and cofactors are expressed in epidermis, normal sebocytes, and an immortalized sebocyte cell line (SEB-1). Journal of Investigative Dermatology 120 905–914. (doi:10.1046/j.1523-1747.2003.12244.x)
Trivedi NR, Cong Z, Nelson AM, Albert AJ, Rosamilia LL, Sivarajah S, Gilliland KL, Liu W, Mauger DT & Gabbay RA et al. 2006 Peroxisome proliferator-activated receptors increase human sebum production. Journal of Investigative Dermatology 126 2002–2009. (doi:10.1038/sj.jid.5700336)
Wille JJ & Kydonieus A 2003 Palmitoleic acid isomer (C16:1Δ6) in human skin sebum is effective against Gram-positive bacteria. Skin Pharmacology and Physiology 16 176–187. (doi:10.1159/000069757)
Wróbel A, Seltmann H, Fimmel S, Müller-Decker K, Tsukada M, Bogdanoff B, Mandt N, Blume-Peytavi U, Orfanos CE & Zouboulis CC 2003 Differentiation and apoptosis in human immortalized sebocytes. Journal of Investigative Dermatology 120 175–181. (doi:10.1046/j.1523-1747.2003.12029.x)
Wu L, Einstein M, Geissler WM, Chan HK, Elliston KO & Andersson S 1993 Expression cloning and characterization of human 17β-hydroxysteroid dehydrogenase type 2, a microsomal enzyme possessing 20α-hydroxysteroid dehydrogenase activity. Journal of Biological Chemistry 268 12964–12969.
Yanase T, Suzuki S, Goto K & Nomura M 2003 Aromatase in bone: roles of vitamin D3 and androgens. Journal of Steroid Biochemistry and Molecular Biology 86 393–397. (doi:10.1016/S0960-0760(03)00349-2)
Zhenhuan Z, Hiroko Y, Tatsuya T, Yasuo H, Yoko O, Hiroshi S, Shunzo K, Nobuhiro H & Hirotaka I 2002 Semi-quantitative immunohistochemical analysis of aromatase expression in ductal carcinoma in situ of the breast. Breast Cancer Research and Treatment 74 47–53. (doi:10.1023/A:1016022314608)
Zhongyi S, Rantakari P, Lamminen T, Toppari J & Poutanen M 2007 Transgenic male mice expressing human hydroxysteroid dehydrogenase 2 indicate a role for the enzyme independent of its action on sex steroids. Endocrinology 148 3827–3836. (doi:10.1210/en.2007-0365)
Zhu L, Gong B, Bisgaier CL, Aviram M & Newton RS 1998 Induction of PPARγ1 expression in human THP-1 monocytic leukemia cells by 9-cis-retinoic acid is associated with cellular growth suppression. Biochemical and Biophysical Research Communications 251 842–848. (doi:10.1006/bbrc.1998.9567)
Zouboulis CC 2004 The human skin as a hormone target and an endocrine gland. Hormones 3 9–26. (doi:10.14310/horm.2002.11109)
Zouboulis CC, Xia L, Akamatsu H, Seltmann H, Fritsch M, Hornemann S, Ruhl R, Chen W, Nau H & Orfanos CE 1998 The human sebocyte culture model provides new insights into development and management of seborrhoea and acne. Dermatology 196 21–31. (doi:10.1159/000017861)
Zouboulis CC, Seltmann H, Neitzel H & Orfanos CE 1999 Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95). Journal of Investigative Dermatology 113 1011–1020. (doi:10.1046/j.1523-1747.1999.00771.x)
