2,3,7,8-tetrachlorodibenzo-p-dioxin alters the differentiation of SZ95 sebocytes in vitro

Introduction:  Chloracne is an acneiform skin disease, which is considered to be the most specific and sensitive clinical condition of dioxin intoxication. Sebogenesis is decreased and skin xerosis is one of the most prominent clinical characteristics compared with acne vulgaris. However, the activity of dioxin on the sebaceous glands is still unclear. We studied the effects of 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD), which is a representative for a group of dioxins, on sebaceous lipid synthesis and expression of markers related to sebocyte differentiation in vitro .
Materials and Methods:  After pretreatment with and without linoleic acid (LA) 10⁻⁴  M for 3 days, SZ95 sebocytes were treated again with TCDD with and without linoleic acid 10⁻⁴  M for 3 additional days. Neutral lipids in SZ95 sebocytes were measured by the nile red microassay. Immunohistology and western blotting were used to detect expression of keratin 7 (a marker of undifferentiated sebocytes), EMA (a marker of differentiated sebocytes) and keratin 10 (a marker of keratinocyte differentiation).
Results:  The neutral lipid content of SZ95 sebocytes was markedly inhibited under treatment with TCDD in concentrations of 10⁻⁸  M , 10⁻⁹  M and 10⁻¹⁰  M ( P  < 0.001 respectively), in the presence of LA. SZ95 sebocyte lipid content was not affected by TCDD alone. Moreover, expression of keratin 7 and EMA decreased and keratin 10 increased under TCDD treatment.
Conclusions:  Dioxin affects sebaceous gland cell lipogenesis and differentiation in vitro , probably by switching the sebocyte into a kerationocyte lineage. These findings indicate that altered sebaceous gland differentiation is likely to be the major reason of decreased sebogenesis in patients with chloracne.     

Die Talgdrüse

The development and function of the sebaceous gland in the fetal and neonatal periods appear to be regulated by maternal androgens and by endogenous steroid synthesis, as well as by other morphogens. The most apparent function of the glands is to excrete sebum. A strong increase in sebum excretion occurs a few hours after birth; this peaks during the first week and slowly subsides thereafter. A new rise takes place at about age 9 years with adrenarche and continues up to age 17 years, when the adult level is reached. The sebaceous gland is a target organ but also an important formation site of hormones, and especially of active androgens. Hormonal activity is based on an hormone (ligand)-receptor interaction, whereas sebocytes express a wide spectrum of hormone receptors. Androgens are well known for their effects on sebum excretion, whereas terminal sebocyte differentiation is assisted by peroxisome proliferator-activated receptor ligands. Estrogens, glucocorticoids, and prolactin also influence sebaceous gland function. In addition, stress-sensing cutaneous signals lead to the production and release of corticotrophin-releasing hormone from dermal nerves and sebocytes with subsequent dose-dependent regulation of sebaceous nonpolar lipids. Among other lipid fractions, sebaceous glands have been shown to synthesize considerable amounts of free fatty acids without exogenous influence. Atopic dermatitis, seborrheic dermatitis, psoriasis and acne vulgaris are some of the disease on which pathogenesis and severity sebaceous lipids may or are surely involved.     

Acne and sebaceous gland function

The embryologic development of the human sebaceous gland is closely related to the differentiation of the hair follicle and the epidermis. The number of sebaceous glands remains approximately the same throughout life, whereas their size tends to increase with age. The development and function of the sebaceous gland in the fetal and neonatal periods appear to be regulated by maternal androgens and by endogenous steroid synthesis, as well as by other morphogens. The most apparent function of the glands is to excrete sebum. A strong increase in sebum excretion occurs a few hours after birth; this peaks during the first week and slowly subsides thereafter. A new rise takes place at about age 9 years with adrenarche and continues up to age 17 years, when the adult level is reached. The sebaceous gland is an important formation site of active androgens. Androgens are well known for their effects on sebum excretion, whereas terminal sebocyte differentiation is assisted by peroxisome proliferator-activated receptor ligands. Estrogens, glucocorticoids, and prolactin also influence sebaceous gland function. In addition, stress-sensing cutaneous signals lead to the production and release of corticotrophin-releasing hormone from dermal nerves and sebocytes with subsequent dose-dependent regulation of sebaceous nonpolar lipids. Among other lipid fractions, sebaceous glands have been shown to synthesize considerable amounts of free fatty acids without exogenous influence. Sebaceous lipids are responsible for the three-dimensional skin surface lipid organization. Contributing to the integrity of the skin barrier. They also exhibit strong innate antimicrobial activity, transport antioxidants to the skin surface, and express proinflammatory and anti-inflammatory properties. Acne in childhood has been suggested to be strongly associated with the development of severe acne during adolescence. Increased sebum excretion is a major factor in the pathophysiology of acne vulgaris. Other sebaceous gland functions are also associated with the development of acne, including sebaceous proinflammatory lipids; different cytokines produced locally; periglandular peptides and neuropeptides, such as corticotrophin-releasing hormone, which is produced by sebocytes; and substance P, which is expressed in the nerve endings at the vicinity of healthy-looking glands of acne patients. Current data indicate that acne vulgaris may be a primary inflammatory disease. Future drugs developed to treat acne not only should reduce sebum production and Propionibacterium acnes populations, but also should be targeted to reduce proinflammatory lipids in sebum, down-regulate proinflammatory signals in the pilosebaceous unit, and inhibit leukotriene B(4)-induced accumulation of inflammatory cells. They should also influence peroxisome proliferator-activated receptor regulation. Isotretinoin is still the most active available drug for the treatment of severe acne.     

Effects of 13-cis-retinoic acid, all-trans-retinoic acid, and acitretin on the proliferation, lipid synthesis and keratin expression of cultured human sebocytes in vitro.

The aim of this study was to determine the effects of 13-cis-RA, all-trans-RA, and acitretin on the proliferation, lipid synthesis, and keratin expression of human sebocytes in vitro and to elucidate possible mechanisms of retinoid action on sebaceous glands at the cellular level. It was found that 13-cis-RA and all-trans-RA decreased sebocyte proliferation in a dose- and time-dependent manner, with a 13-cis-RA-IC50 of 10(-5) M (after 7 d) and 10(-6) M (after 14 d) and an all-trans-RA-IC50 of 10(-7) M (after 14 d; no IC50 after 7 d). Acitretin inhibited sebocyte proliferation only at 10(-5) M. Furthermore, 13-cis-RA was the most potent inhibitor of acetate incorporation into lipids, which indicated lipid synthesis (48.2% reduction), followed by all-trans-RA (-38.6%), and by acitretin (-27.5%). All retinoids tested markedly decreased the synthesis of triglycerides, wax/stearyl esters, and free fatty acids in cultured sebocytes, whereas squalene synthesis remained uninfluenced and cholesterol synthesis slightly increased. On the other hand, keratin 5 was down-regulated, keratin 17 was up-regulated, and the expression of keratin 13 was virtually unaffected by all retinoids tested. Keratins 6 and 16 were down-regulated by 13-cis-RA and by all-trans-RA, keratin 14 was down-regulated by 13-cis-RA only, and keratin 19 was up-regulated by all-trans-RA. These investigations indicate that 13-cis-RA and, to a lesser extent, all-trans-RA are potent inhibitors of both cell proliferation and lipid synthesis in human sebocytes in vitro, whereas acitretin only decreases lipogenesis in this model. In addition, retinoids may modify the differentiation of sebocytes in vitro by modulating keratin expression. Models of cultured human sebocytes are useful tools for further investigations on the sebaceous gland and its activity at the cellular level.     

Epidermal growth factor receptor and the sebaceous gland

Side effects of EGFR inhibition do not include primary sebaceous gland changes. Dahlhoff et al. evaluated EGFR actions on the sebaceous glands in mutant mice with constitutively activated EGFR expression. Enlarged, hyperproliferative sebaceous glands and increased sebum levels were detected, and c‐myc levels were increased. These data indicate that EGFR‐induced sebocyte mechanisms are probably species‐specific. The detected SCD deregulation is compatible with previous reports, whereas SCD is a marker of sebaceous differentiation, and its expression is essential for lipogenesis. As SCD expression is upregulated by TLR2 activation, there is a link between SCD overexpression and sebaceous differentiation/lipogenesis with inflammation.     

Sebaceous gland: Milestones of 30-year modelling research dedicated to the “brain of the skin”

In 2018, Schneider and Zouboulis analysed the available tools for studying sebaceous gland pathophysiology in vitro. Since then, the interest in this field remains unbroken, as demonstrated by recent reviews on sebaceous gland physiology, endocrinology and neurobiology, the role of sebaceous glands beyond acne, and several original works on different areas of sebaceous gland function, including sebaceous lipogenesis. Landmark developments in the first part of the 30-year modelling research dedicated to the sebaceous gland, which is considered by several scientists as the brain of the skin, were the short-term culture of human sebaceous glands, the culture of human sebaceous gland cells and the development of immortalized sebaceous gland cell lines exhibiting characteristics of normal sebocytes. On the other hand, current developments represent the establishment of sebaceous gland spheroids, the 3D-SeboSkin model of viable skin explants ex vivo, the combination of culture-expanded epidermal stem cells of mice and adult humans to form de novo hair follicles and sebaceous glands, when they are transplanted into excisional wounds in mice, and 3D-printed scaffolds coated with decellularized matrix of adipose-derived mesenchymal stromal cells and SZ95 sebocytes. These novel tools may become useful platforms for better understanding of cellular and molecular mechanisms governing sebocyte biology and sebaceous gland homeostasis, such as the changes in sebum synthesis and composition, the infundibular differentiation and the influence of the innate immunity and the cutaneous microbiome and for identifying potential therapeutic targets of skin diseases affecting the sebaceous glands.     

5α-还原酶对雄激素诱发痤疮的研究进展

雄激素可以促进皮脂腺生长和脂质合成。5α-还原酶通过将雄激素睾酮转变为活性更强的双氢睾酮,进一步促进皮脂腺分泌皮脂,在痤疮的发病中具有重要作用。本文就雄激素与痤疮的相关研究的进展进行综述,主要包括雄激素在皮肤代谢的途径及5α-还原酶在该生理过程中的作用等。     

Acne vulgaris

Peripherally active androgens control cellular functions by binding to androgen receptors. Follicular keratinocytes and sebocytes are target cells for androgens, which, directly or indirectly, stimulate keratinocyte proliferation and the volume of sebaceous glands as well as the sebum secretion rate. Acne often begins with the adrenarche, namely with the up- regulation of adrenal synthesis of dehydroepiandrosterone sulfate, a hormone that is upstream to testosterone. The majority of acne patients exhibit normal levels of circulating androgens, while sebaceous glands from acne regions exhibit a stronger sensitivity to androgens than the sebaceous glands from other parts of the body. Hormone-like active lipids, hormones in diet and neuropeptides may also play a role in the development of acne lesions. The target of antiandrogen treatment of the skin is mainly the sebaceous gland and the primary effect is sebostasis.     

Steroid hormone receptors and their relevance for sebum production in the sebaceous gland ear model of the Syrian hamster

We determined the capacity of steroid hormone receptors in the sebaceous glands of intact nontreated, castrated, with testosterone substituted castrated male, intact female, and intact with testosterone substituted female animals using the animal ear model of the Syrian hamster. The steroid hormone binding capacity was compared with the sebaceous gland areas and sebogenesis. Intact male animals showed large sebaceous follicles, a high sebogenesis rate, and high capacity for sexual hormone binding proteins. In castrated males, the sebaceous gland areas and sebogenesis were both diminished, and androgen and estrogen receptors were decreased. When the castrated males were substituted with testosterone propionate, the sebaceous glands showed large volumes, high sebum production, and androgen binding activity again. In female animals having small sebaceous follicles and a low rate of sebogenesis, testosterone propionate enlarged the sebaceous glands and increased sebogenesis and the capacity of androgen binding. One can conclude from these data that testosterone is not only the main hormone for sebum production but also induces the synthesis of its own receptor.     

Effects of topically applied antiandrogenic compounds on sebaceous glands of hamster ears and flank organs

Growth of sebaceous glands in the ears and flank organs of castrated male hamsters is dependent on androgen substitution. Taking this for granted, a study was done to compare the effects of topical antiandrogenic treatment in vivo on the morphology and size of sebaceous glands with the concomitant changes in in vitro metabolism of 3H-testosterone. The role of dihydrotestosterone in sebaceous gland stimulation was thereby investigated. Topical treatment was carried out with the androgen antagonist 17 alpha-propylmesterolone (PM), with 4-androsten-3-one-17 beta-carboxylic acid (17 beta-C), and 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (4-MA), both described as specific 4-steroid-5 alpha-reductase inhibitors, and with progesterone (PRO), which is an androgen receptor antagonist with 5 alpha-reductase inhibiting properties. Regrowth of sebaceous glands after castration and substitution with testosterone propionate or dihydrotestosterone could be inhibited by topical PM and PRO. This occurred irrespective of the influence on testosterone metabolism and irrespective of the mode of substitution. 4-MA, on the other hand, while exhibiting strong 5 alpha-reductase inhibition in vitro, was ineffective in reducing sebaceous gland sizes in vivo. The compound 17 beta-C was ineffective in every respect. In no case were systemic antiandrogenic effects on prostates and seminal vesicles observed. Our results support the view that the DHT formation rate has no regulatory function for growth of sebaceous glands in hamsters and that PM and PRO counteract the androgenic stimulus by their competitive antagonistic binding to the androgen receptor, but not by their influence on testosterone metabolism.     

丹参酮ⅡA磺酸钠抗金黄地鼠皮脂腺增生的研究

目的 探讨丹参酮ⅡA磺酸钠（STS）外用对动物模型皮脂腺增生的影响。方法 选用成年雄性金黄地鼠侧腹皮脂腺斑作为动物模型,采用自身对照法,分别予丹参酮ⅡA磺酸钠、生理氯化钠溶液外涂一侧皮脂腺斑,一天3次,随机分用药0、10、20、30 d 4个时间组。在各时间点用游标卡尺测定两侧皮脂腺斑的面积。HE染色法观察皮脂腺斑组织结构的变化,取组织切片用免疫组化方法检测皮脂腺细胞增殖细胞核抗原（PCNA）的表达,TUNEL法检测皮脂腺细胞的凋亡情况。结果 用药前,两侧皮脂腺斑大小比较差异无统计学意义（P > 0.05）,皮脂腺结构完整,排列紧密,皮脂腺细胞的增殖与凋亡比较差异均无统计学意义（P > 0.05）。随着用药时间的延长,与生理氯化钠溶液对照侧比较,STS侧金黄地鼠皮脂腺斑的面积缩小（P < 0.05）;皮脂腺数目减少,体积变小,排列疏松,用药至30 d时,皮脂腺呈明显萎缩状态;皮脂腺细胞PCNA表达明显下调（P < 0.01）,用药至10 d、20 d时更为明显（P < 0.01）;皮脂腺细胞凋亡亦增加（P < 0.01）,用药至20 d时凋亡显著（P < 0.01）,且皮脂腺中央部细胞的凋亡较周围更为明显。结论 丹参酮ⅡA磺酸钠能缩小金地鼠皮脂腺斑的面积,改变皮脂腺斑的显微结构,可抑制皮脂腺增生。     

Hirsutism and the variable response of the pilosebaceous unit to androgen

The pilosebaceous unit (PSU) response to androgen is variable. Certain population of PSU respond to androgen in a distinctive pattern that results in sexual hair development in some, sebaceous gland development in others. Furthermore, androgen excess is variably manifest in women as hirsutism, acne vulgaris, seborrhea, or pattern alopecia. Although sebaceous cells act as intracrine cells, activating pro-hormones to potent androgens that act within the sebocyte, hair follicle metabolism predominantly inactivates testosterone. Androgen action in the sexual hair follicle appears to be mediated by the dermal papilla and possibly, by inducing expression of a specific keratin, hHa7, in the hair medulla. The data do not clearly support a relationship between idiopathic hirsutism, the hirsutism that occurs in the absence of androgen excess, and variations in androgen mechanism of action. Androgens are prominent among the hormones that modulate the biological mechanism regulating the hair cycle. However, the basis for the variable pattern of PSU response to androgen is unclear, as is the basis for the variable development of hirsutism in response to androgen excess and the incomplete reversal of hirsutism by anti-androgen treatment. Improved treatment of hirsutism awaits improved understanding of the nature of the interaction between androgens and other determinants of hair follicle biology.     

Inactivation of autophagy leads to changes in sebaceous gland morphology and function

We have reported recently that inactivation of the essential autophagy‐related gene 7 (Atg7) in keratinocytes has little or no impact on morphology and function of the epidermal barrier in experimental animals. When these mice aged, mutant males, (Atg7 ΔKC), developed an oily coat. As the keratin 14 promoter driven cre/LoxP system inactivates floxed Atg7 in all keratin 14 (K14) expressing cells, including sebocytes, we investigated whether the oily hair phenotype was the consequence of changes in function of the skin sebaceous glands. Using an antibody to the GFP‐LC3 fusion protein, autophagosomes were detected at the border of sebocyte disintegration in control but not in mutant animals, suggesting that autophagy was (a) active in normal sebaceous glands and (b) was inactivated in the mutant mice. Detailed analysis established that dorsal sebaceous glands were about twice as large in all Atg7 ΔKC mice compared to those of controls (Atg7 F/F), and their rate of sebocyte proliferation was increased. In addition, male mutant mice yielded twice as much lipid per unit hair as age‐matched controls. Analysis of sebum lipids by thin layer chromatography revealed a 40% reduction in the proportion of free fatty acids (FFA) and cholesterol, and a 5‐fold increase in the proportion of fatty acid methyl esters (FAME). In addition, the most common diester wax species (58‐60 carbon atoms) were increased, while shorter species (54‐55 carbon atoms) were under‐represented in mutant sebum. Our data show that autophagy contributes to sebaceous gland function and to the control of sebum composition.     

Therapeutic aspects of seborrhea oleosa and pityriasis simplex capillitii]

The treatment of seborrhoea oleosa capillitii should aim at inhibiting depletion of the sebaceous glands, lipid synthesis in the sebaceous gland and microbial lipolysis of the triglycerides in the scalp and hair lipids. The use of isopropyl alcohol as a vehicle in a hair tonic reduces sebum depletion. Coal tar and estrogens reduce the lipid synthesis in the sebaceous gland. The lipolysis can be inhibited by various surfactants, isopropyl alcohol, colloidal sulphur, selenium disulphide, Omadine MDS and benzoyl peroxide. In the treatment of pityriasis simplex capillitii (dandruff) one aims at achieving inhibition of mitosis in the epidermis and, if possible, an additional "keratolytic" effect. Inhibition of mitosis could be demonstrated for coal tar, corticosteroids, selenium disulphide, cadmium sulphide, Omadine MDS, Zn Omadine and ichthyol sodium. Colloidal sulphur, salicylic acid, Omadine MDS and various surfactants act "keratolytically".     

Immunohistochemical staining for androgen receptors: a sensitive marker of sebaceous differentiation.

Androgen receptors (AR) are present in normal skin being localized to the basal and differentiating cells of the sebaceous gland, and as such, sebaceous glands are androgen sensitive tissue. Androgen receptor expression was examined in 43 sebaceous neoplasms including 8 sebaceous carcinomas, 22 sebaceous adenomas, 12 specimens showing sebaceous hyperplasia, and 1 sebaceous epithelioma, as well as in 14 squamous cell carcinomas, 2 clear cell acanthomas, and 35 basal cell carcinomas. Epithelial membrane antigen (EMA) expression was also examined in all of the sebaceous neoplasms. All specimens were fixed in formalin and embedded in paraffin. Diffuse positive nuclear androgen receptor antibody immunohistochemical staining was observed in all samples of sebaceous neoplasms, whereas approximately 60% of basal cell carcinomas showed only focal positivity for nuclear androgen receptor immunoreactivity. Clear cell acanthomas and squamous cell carcinomas were uniformly negative. Whereas all sebaceous neoplasms exhibited immunoreactivity for androgen receptors, the staining pattern was more marked in the nuclei of seboblasts and differentiating sebocytes in the adenomatous, hyperplastic, and epitheliomatous lesions than in the nuclei of the less differentiated sebaceous carcinoma cells. All the sebaceous neoplasms except for sebaceous carcinomas exhibited immunoreactivity for EMA. In the sebaceous carcinomas, EMA staining was absent in the most poorly differentiated specimen, but with increasing differentiation, the carcinomas became immunoreactive to EMA. We have shown that the nuclei of sebaceous neoplasms, including sebaceous gland carcinomas, show immunoreactivity for androgen receptors (AR), that immunohistochemical staining for the presence of AR may be a reliable marker of sebaceous differentiation, and that the AR may be a better marker of sebaceous differentiation than EMA, particularly in poorly differentiated sebaceous carcinomas.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters sebaceous gland cell differentiation in vitro

Chloracne is a characteristic marker of intoxication by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or related compounds. Decreased lipogenesis is a prominent clinical sign in this disease. However, the activity of dioxins on human sebaceous glands is still unclear. In this study, the effects of TCDD on sebaceous gland differentiation were studied both in human skin samples maintained ex vivo and in cultured SZ95 sebocytes. Aryl hydrocarbon receptor (AhR) protein expression, the receptor for dioxin, was detected in SZ95 sebocytes. Its expression was markedly inhibited by TCDD. Furthermore, we detected a reduced release of neutral lipids (10(-10) -10(-8) M; P<0.001) and decreased expression of epithelial membrane antigen and keratin 7, all of which are specific markers of sebaceous differentiation. Markedly, increased expression of the keratinocyte differentiation marker keratin 10 and of peroxisome proliferators-activated receptor-δ was assessed in SZ95 sebocytes treated with TCDD. To corroborate these in vitro data, an ex vivo sebaceous gland-rich skin culture model was investigated. Obvious shrinkage of sebaceous glands with sebaceous duct hyperplasia and increased expression of keratin 10 in the atrophic sebaceous glands were observed on the 5th day of TCDD treatment. In conclusion, TCDD affects the differentiation of sebaceous gland cells probably by switching human sebaceous into keratinocyte-like differentiation. In addition and together with the results of a parallel study (J Dermatol Sci 58, 2010, 211), we provide evidence that TCDD effects on human sebocytes are mediated through the AhR signalling pathway.