Human Sebocytes Research Articles

Phospholipase stimulates lipogenesis in SZ95 sebocytes

Introduction: With progressing ageing human sebocytes reduce lipid production. However, the influence of certain aging mechanisms on sebaceous lipid synthesis as well as ways to influence the latter is not fully identified. Certain lipids act as ligands of nuclear receptors such as PPAR. Phospholipase (PLA2) catalyzes the hydrolysis of the sn‐2 fatty acyl bond of phospholipids to yield free fatty acid and lysophospholipid. It has been hypothesized that PPAR may be activated by hydrolysis products of phospholipids and also by eicosanoids obtained through PLA2 activity.Materials and Methods: A method to quantify sebaceous lipid synthesis of SZ95 sebocytes in vitro was established and the cells were treated by snake venom Bothrops moojeni gel filtration fractions (Botmo GF). Botmo GF fractions were further purified by RP‐HPLC, and a fraction with PLA2 activity (Botmo GF11‐117) and a fraction without enzymatic activity (Botmo GF11‐101) were identified and additionally tested.Results: Botmo GF fractions increased lipogenesis in SZ95 sebocytes without inducing apparent toxic or apoptotic effects. Botmo GF11‐101 (1 μg/ml) enhanced neutral lipid synthesis by up to 170% and polar lipid synthesis by up to 120%. The enzymatically active PLA2 Botmo GF11‐117 (1 μg/ml) increased synthesis of neutral lipids by up to 200%, and polar lipids by up to 120% compared to untreated SZ95 sebocytes.Conclusion: PLA2 activation or suppression could be important for human sebaceous lipogenesis. PLA2 modifiers may be attractive for skin lipid research and pharmacological/cosmetic products.

Truncated peptide derivates of the C‐terminal domain of alpha‐MSH (11–13) – emerging agents for anti‐inflammatory future therapy

There is increasing evidence that selected peptide fragments of the neuropeptide alpha‐melanocyte‐stimulating hormone (alpha‐MSH) have preserved immunomodulatory effects in various in vitro and in vivo models. However, until recently, emphasis has been attributed mostly to peptides related to the central pharmacophore of alpha‐MSH (6–9) as well as to peptides homologous to the C‐terminal tripeptide sequence, MSH (11–13). Here we investigated in detail the in vitro effects of an alpha MSH (11–13) derivative, K(D)PT, in which the last amino acid valine of alpha‐MSH (11–13) was substituted by threonine and by the D‐enantiomer of proline in position 2. Using the immortalized human sebocyte cell line SZ95 as an in vitro model we demonstrate that K(D)PDT has potent antagonistic effects against interleukin (IL)‐induced activation of NF‐kB presumably by inhibiting IkBalpha protein degradation. In contrast IL‐1‐mediated activation of the stress kinase p38 was not affected. The significance of the NF‐kB‐modulatory effect of K(D)PT was highlighted by suppression of IL‐1‐mediated mRNA expression and protein secretion of IL‐6 and IL‐8, two proinflammatory cytokines crucially implicated in the pathogenesis of acne vulgaris. Interestingly, K(D)PT likewise suppressed P. acnes‐induced expression of IL‐6 and IL‐8. Our in vitro data are promising towards the therapeutic exploitation of small peptide derivatives of the C‐terminal domain of alpha‐MSH, e.g. K(D)PT, not only for the treatment of acne but also for many other inflammatory skin diseases.

Frontiers in sebaceous gland biology and pathology

The development of experimental models for the in vitro study of human sebaceous gland turned down the theory of a phylogenetic relict and led to the identification of several, unknown or disregarded functions of this organ. Such functions are the production of foetal vernix caseosa, the influence of three-dimensional organization of the skin surface lipids and the integrity of skin barrier and the influence on follicular differentiation. In addition, the sebaceous gland contributes to the transport of fat-soluble antioxidants from and to the skin surface, the natural photoprotection, the pro- and antiinflammatory skin properties and to the innate antimicrobial activity of the skin. It is mainly responsible for skin's independent endocrine function, the hormonally induced skin ageing process, the steroidogenic function of the skin as well as its thermoregulatory and repelling properties and for selective control of the hormonal and xenobiotical actions of the skin. Interestingly, sebocytes, at least in vitro, preserve characteristics of stem-like cells despite their programming for terminal differentiation. This review reports on various sebaceous gland functions, which are currently under investigation, including its role on the hypothalamus-pituitary-adrenal-like axis of the skin, the impact of acetylcholine on sebocyte biology, the activity of ectopeptidases as new targets to regulate sebocyte function, the effects of vitamin D on human sebocytes, the expression of retinoid metabolizing cytochrome P450 enzymes and the possible role of sebum as vehicle of fragrances. These multiple homeostatic functions award the sebaceous gland the role 'brain of the skin' and the most important cutaneous endocrine gland.

LXRα Enhances Lipid Synthesis in SZ95 Sebocytes

Differentiation of sebocytes is strongly associated with enhanced lipid synthesis and accumulation in the cells. Liver X receptors (LXRs) are members of the nuclear receptor superfamily, which play a critical role in cholesterol homeostasis and lipid metabolism. We examined whether LXRalpha regulated lipid synthesis in the immortalized human sebaceous gland cell line SZ95. When the SZ95 sebocytes were treated with the ligand of LXR such as TO901317 or 22(R)-hydroxycholesterol, lipid droplets were accumulated in the majority of cells when examined by Oil Red O staining. The expression of the known LXR targets, such as fatty acid synthase and sterol regulatory-binding protein-1, was induced by TO901317. TO901317 treatment increased expression of LXRalpha but not that of LXRbeta. Transfection of antisense LXRalpha significantly decreased TO901317-induced target gene expression and lipid droplet accumulation, suggesting a major role of LXRalpha in differentiation of sebocytes. Further, TO901317 decreased the expression of cyclooxygenase-2 and inducible nitric oxide synthase that was induced by lipopolysaccharide treatment. Together, these results indicate that important roles of LXRalpha in differentiation and inflammatory signaling in sebaceous glands. Thus, we suggest that LXR ligands could provide a new class of therapeutic agents for sebaceous gland-associated disorders such as seborrhea and acne.

Characterization of Bipotential Epidermal Progenitors Derived from Human Sebaceous Gland: Contrasting Roles of c-Myc and β-Catenin

The current belief is that the epidermal sebaceous gland (SG) is maintained by unipotent stem cells that are replenished by multipotent stem cells in the hair follicle (HF) bulge. However, sebocytes can be induced by c-Myc (Myc) activation in interfollicular epidermis (IFE), suggesting the existence of bipotential stem cells. We found that every SZ95 immortalized human sebocyte that underwent clonal growth in culture generated progeny that differentiated into both sebocytes and cells expressing involucrin and cornifin, markers of IFE and HF inner root sheath differentiation. The ability to generate involucrin positive cells was also observed in a new human sebocyte line, Seb-E6E7. SZ95 xenografts differentiated into SG and IFE but not HF. SZ95 cells that expressed involucrin had reduced Myc levels; however, this did not correlate with increased expression of the Myc repressor Blimp1, and Blimp1 expression did not distinguish cells undergoing SG, IFE, or HF differentiation in vivo. Overexpression of Myc stimulated sebocyte differentiation, whereas overexpression of beta-catenin stimulated involucrin and cornifin expression. In transgenic mice simultaneous activation of Myc and beta-catenin revealed mutual antagonism: Myc blocked ectopic HF formation and beta-catenin reduced SG differentiation. Overexpression of the Myc target gene Indian hedgehog did not promote sebocyte differentiation in culture and cyclopamine treatment, while reducing proliferation, did not block Myc induced sebocyte differentiation in vivo. Our studies provide evidence for a bipotential epidermal stem cell population in an in vitro model of human epidermal lineage selection and highlight the importance of Myc as a regulator of sebocyte differentiation.

Vaccination Targeting a Surface Sialidase of P. acnes: Implication for New Treatment of Acne Vulgaris

BackgroundAcne vulgaris afflicts more than fifty million people in the United State and the severity of this disorder is associated with the immune response to Propionibacterium acnes (P. acnes). Systemic therapies for acne target P. acnes using antibiotics, or target the follicle with retinoids such as isotretinoin. The latter systemic treatment is highly effective but also carries a risk of side effects including immune imbalance, hyperlipidemia, and teratogenicity. Despite substantial research into potential new therapies for this common disease, vaccines against acne vulgaris are not yet available.Methods and FindingsHere we create an acne vaccine targeting a cell wall-anchored sialidase of P. acnes. The importance of sialidase to disease pathogenesis is shown by treatment of a human sebocyte cell line with recombinant sialidase that increased susceptibility to P. acnes cytotoxicity and adhesion. Mice immunized with sialidase elicit a detectable antibody; the anti-sialidase serum effectively neutralized the cytotoxicity of P. acnes in vitro and P. acnes-induced interleukin-8 (IL-8) production in human sebocytes. Furthermore, the sialidase-immunized mice provided protective immunity against P. acnes in vivo as this treatment blocked an increase in ear thickness and release of pro-inflammatory macrophage inflammatory protein (MIP-2) cytokine.ConclusionsResults indicated that acne vaccines open novel therapeutic avenues for acne vulgaris and other P. acnes-associated diseases.

Thematic review series: Skin Lipids. Sebaceous gland lipids: friend or foe?

Sebaceous glands are intriguing glands that are found throughout the human body except on the palms of the hands and soles of the feet. The true function of these glands has yet to be determined, but there are several theories, including antioxidant effects, antibacterial effects, and transport of pheromones. Sebaceous glands produce lipids that are involved in the pathogenesis of one of the most prevalent diseases of adolescence, acne. Although the majority of lipids produced by the sebaceous gland are also produced in other areas of the body, there are two that are characteristic of the sebaceous gland, wax esters and squalene. This review seeks to present an update on the physiology of the sebaceous glands, with particular emphasis on the production of sebaceous lipids.

GRO‐α

Age-regulated genes may serve as markers of aging, enabling assessment of physiological aging independent of chronological age. One gene with transcripts that increase in abundance with age in human organs, inter alia in epithelial skin cells, is the chemokine growth-regulated protein alpha (GRO-alpha). When chemokines, such as GRO-alpha, become disregulated so that they are chronically expressed, tissue damage, angiogenesis, and tumorigenesis can follow. To consider the role of GRO-alpha as a potential marker for aging and cancer, we compared the transient knockdown of GRO-alpha by RNA interference in the human sebaceous gland cell line SZ95, which behaves like normal human sebocytes, and in the melanoma cell line A375, which originates from a primary human tumor. The reduced GRO-alpha RNA expression, of about 75% in SZ95 sebocytes and 58% in A375 melanoma cells, has functional consequences in normal aged cells and in cancer cells. Silencing of the proangiogenic chemokine GRO-alpha is proportionally correlated with interleukin-6 (IL-6), IL-8 and vascular endothelial growth factor secretion in both cell types. Thus, GRO-alpha may be a novel diagnostic marker for age-related pathology, including cancer.

The skin as a mirror of the aging process in the human organism – State of the art and results of the aging research in the German National Genome Research Network 2 (NGFN-2)

As our society is growing older, the consequences of aging have begun to gain particular attention. Improvement of quality of life at old age and prevention of age-associated diseases have become the main focus of the aging research. The process of aging in humans is complex and underlies multiple influences, with the probable involvement of heritable and various environmental factors. In particular, hormones are decisively involved in the generation of aging. Over time, important circulating hormones decline due to a reduced secretion of the pituitary, the adrenal glands and the gonads or due to an intercurrent disease. Among them, serum levels of growth factors and sexual steroids show significant aging-associated changes. Within the scope of the Explorative Project ‘Genetic aetiology of human longevity’ supported by the German National Genome Research Network 2 (NGFN-2) an in vitro model of human hormonal aging has been developed. Human SZ95 sebocytes were maintained under a hormone-substituted environment consisting of growth factors and sexual steroids in concentrations corresponding to those circulating in 20- and in 60-year-old women. Eight hundred and ninety-nine genes showed a differential expression in SZ95 sebocytes maintained under the 20- and 60-year-old hormone mixture, respectively. Among them genes were regulated which are involved in biological processes which are all hallmarks of aging. The most significantly altered signaling pathway identified was that of the transforming growth factor-β (TGF-β). A disturbed function of this cascade has been associated with tumorigenesis, i.e. in pancreatic, prostate, intestine, breast, and uterine cancer. Interestingly, genes expressed in signaling pathways operative in age-associated diseases such as Huntington’s disease (HD), dentatorubral-pallidoluysian atrophy (DRPLA), and amyotrophic lateral sclerosis (ALS) were also identified. These data demonstrate that skin and its appendages may represent an adequate model for aging research. Hormones interact in a complex fashion, and aging may be partly attributed to the changes in their circulating blood levels. Furthermore, a disturbed hormone status may partially act towards the manifestation of neurodegenerative diseases. Thus, these results could be a basis for an integrated and interdisciplinary approach to the analysis of the aging process.

Inhibitors of Dipeptidyl Peptidase IV and Aminopeptidase N Target Major Pathogenetic Steps in Acne Initiation

Acne is a chronic disease hallmarked by sebaceous hyperplasia, follicular hyperkeratosis, and inflammation. Parallel targeting of these factors is required to treat acne effectively. Inhibitors of dipeptidyl peptidase IV (DP IV) and aminopeptidase N (APN) show strong anti-inflammatory effects on immune cells and therapeutic efficacy in autoimmune disorders. Our investigation focused on the expression and functional relevance of these ectopeptidases in three cell types which exhibit an altered phenotype in early acne lesions. We showed for the first time expression of DP IV and APN on human sebocytes. In the SZ95 sebocyte cell line, the DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrrolidide and the APN inhibitors actinonin and bestatin suppressed proliferation, enhanced terminal differentiation, and slightly decreased total neutral lipid production. The anti-inflammatory and differentiation-restoring cytokine IL-1 receptor antagonist was significantly upregulated in SZ95 sebocytes and the HaCaT keratinocyte cell line in the presence of inhibitors. Furthermore, the inhibitors suppressed proliferation and IL-2 production of Propionibacterium acnes-stimulated T cells ex vivo and enhanced the expression of the immunosuppressive cytokine transforming growth factor-beta1. Our data provide first evidence for a functional role of DP IV and APN in the sebaceous gland apparatus and for their inhibitors, used alone or in combination, as completely new substances possibly affecting acne pathogenesis in a therapeutic manner.

Corticotropin-Releasing Hormone Skin Signaling is Receptor-Mediated and is Predominant in the Sebaceous Glands

There is increasing evidence that the sebaceous gland expresses receptors for several neuropeptides and is involved in responses to stress. Among them, corticotropin-releasing hormone (CRH) was currently found to be produced also in the skin. In this study, the distribution of CRH, CRH receptors 1 and 2 (CRH-R1 and CRH-R2), and CRH binding protein (CRH-BP) in cultured human (SZ95) sebocytes was further characterized. Moreover, the effects of CRH and CRH-like peptides on proliferation and inflammatory signaling of CRH receptor-expressing SZ95 sebocytes IN VITRO were investigated. Urocortin (Uct), urotensin and sauvagine are recently described members of the family of structurally related CRH-like peptides, whereas Uct shares a 45% homology with CRH. CRH and Uct inhibited SZ95 sebocyte proliferation with CRH also stimulating interleukin-6 (IL-6) and interleukin-8 (IL-8) release from SZ95 sebocytes. However, CRH had no effect on interleukin-1alpha and interleukin-1beta production in these cells. alpha-Helical-CRF, a CRH antagonistic peptide, annulled the CRH effect on SZ95 sebocyte proliferation and interleukin secretion, while the non-peptidic CRH-R1 selective antagonist antalarmin inhibited the increased production of neutral lipids caused by CRH. In conclusion, CRH, and to a lesser extent Uct, may be involved in signaling of stress pathophysiology in the skin. However, further investigations into the downstream effects of CRH and Uct are required to elucidate the mechanism by which these neuropeptides could establish a stress-related pathophysiological condition in the skin.

Inhibition of the Androgen Receptor by Antisense Oligonucleotides Regulates the Biological Activity of Androgens in SZ95 Sebocytes

One novel strategy for the blockade of the androgen receptor could be the selective inhibition of androgen receptor by antisense oligonucleotides or small interfering RNA molecules. Here we describe the down regulation of the androgen receptor in cultured human SZ95 sebocytes with antisense oligonucleotides modified with phosphorothioates and 2'- O-methylribosyl residues. The ability of antisense oligonucleotides to cross the cellular membrane was enhanced by establishing a transient transfection system based on cationic lipid vesicles. Both antisense oligonucleotide types administered caused assumedly translational arrest. Dose-dependent inhibition of androgen receptor protein expression was observed after SZ95 sebocyte transfection with modified phosphorothioate oligonucleotides and modified 2'- O-methylribonucleotides which were directed against the translational start of the androgen receptor mRNA. The strongest transient inhibition of androgen receptor expression was detected after 14 hours with 1.0 muM antisense 2'- O-methylribonucleotides (88+/-1.3%, p<0.001). With longer recovery times than 24 hours, androgen receptor protein expression returned to the native control levels. Inhibition of the expression of androgen receptor by antisense oligonucleotides, reduced the enhanced proliferation of SZ95 sebocytes challenged by testosterone and 5alpha-dihydrotestosterone. This administration opens new therapeutic possibilities in androgen-associated skin diseases, since we could also show androgen inhibition with these antisense oligonucleotides in a reconstituted human epidermis model (Horm Metab Res 2007; 39:157-165).

Testosterone metabolism to 5?-dihydrotestosterone and synthesis of sebaceous lipids is regulated by the peroxisome proliferator-activated receptor ligand linoleic acid in human sebocytes

Despite the clinical evidence that androgens stimulate sebaceous lipids, androgens in vitro have shown no similar effects. This contradiction led to the assumption that cofactors may be required for lipid regulation and peroxisome proliferator-activated receptor (PPAR) ligands were suggested to be adequate candidates. The influence of testosterone and linoleic acid, a PPAR ligand, as single agents and in combination with of LY191704, a 5alpha-reductase type I inhibitor, was examined on 5alpha-dihydrotestosterone (5alpha-DHT) synthesis and lipid content in human SZ95 sebocytes. Cell proliferation and viability were measured by the 4-methylumbelliferyl heptanoate fluorescence assay and by the Boehringer Lactate Dehydrogenase Assay kit, respectively. 5alpha-DHT enzyme-linked immunosorbent assay was used for the detection of 5alpha-DHT synthesis in cell supernatants after treatment, whereas lipid production was documented by means of the Nile red lipid microassay and fluorescence microscopy. Testosterone promoted 5alpha-DHT synthesis (P < 0.001), whereas linoleic acid increased sebaceous lipids (P < 0.001). The combination of testosterone and linoleic acid exhibited a synergistic effect on the synthesis of 5alpha-DHT (P < 0.01 vs. testosterone) and sebaceous lipids (P < 0.01 vs. linoleic acid). Furthermore, LY191704 reduced 5alpha-DHT and sebaceous lipid levels (P < 0.01 and P < 0.001 in comparison with testosterone/linoleic acid, respectively). Cell proliferation and viability remained unchanged under treatment with all compounds tested. These data suggest a catalytic effect of PPAR ligands on cellular testosterone activation by 5alpha-reduction and the importance of the latter for the regulation of sebaceous lipids.

13-cis Retinoic Acid Induces Apoptosis and Cell Cycle Arrest in Human SEB-1 Sebocytes

Isotretinoin (13-cis retinoic acid (13-cis RA)) is the most potent inhibitor of sebum production, a key component in the pathophysiology of acne, yet its mechanism of action remains largely unknown. The effects of 13-cis RA, 9-cis retinoic acid (9-cis RA), and all-trans retinoic acid (ATRA) on cell proliferation, apoptosis, and cell cycle proteins were examined in SEB-1 sebocytes and keratinocytes. 13-cis RA causes significant dose-dependent and time-dependent decreases in viable SEB-1 sebocytes. A portion of this decrease can be attributed to cell cycle arrest as evidenced by decreased DNA synthesis, increased p21 protein expression, and decreased cyclin D1. Although not previously demonstrated in sebocytes, we report that 13-cis RA induces apoptosis in SEB-1 sebocytes as shown by increased Annexin V-FITC staining, increased TUNEL staining, and increased cleaved caspase 3 protein. Furthermore, the ability of 13-cis RA to induce apoptosis cannot be recapitulated by 9-cis RA or ATRA, and it is not inhibited by the presence of a retinoid acid receptor (RAR) pan-antagonist AGN 193109. Taken together these data indicate that 13-cis RA causes cell cycle arrest and induces apoptosis in SEB-1 sebocytes by a RAR-independent mechanism, which contributes to its sebosuppressive effect and the resolution of acne.

Differential Regulation of Toll-Like Receptor and CD14 Pathways by Retinoids and Corticosteroids in Human Sebocytes

active retinoid by slightly upregulating mRNA levels of TLR2, TLR4 and CD14, while 13- cis -RA and all- transRA were practically inactive. Retinoids did not affect mRNA levels of IL-1 � , IL-1 � and IL-8 mRNA and their release. However, IL-6 mRNA levels and release were significantly reduced under treatment with 13- cis -RA and all- trans -RA being more potent than retinol, indicating a selective, TLR-independent, anti-inflammatory effect of retinoids on SZ95 sebocytes. Hydrocortisone slightly upregulated TLR2 mRNA levels but markedly reduced TLR4 and CD14 mRNA levels. Furthermore, hydrocortisol induced an overall reduction of cytokine/chemokine expression, a finding that undersigns its global antiinflammatory activity. We provide evidence that the pharmacological regulation of TLR and CD14 expression may provide a novel mechanism in the treatment of inflammatory acne lesions. The combination of 13-cis- retinoic acid (RA) and corticosteroids has been shown to be markedly effective in severe inflammatory acne, such as acne fulminans. We have currently shown human epithelial cells to induce and modulate inflammatory signals by Toll-like receptors (TLRs) and CD14 in acne. By this pathway the innate immunity is able to recognize microbial components and to induce cytokine/chemokine synthesis. We examined the effects of retinoids, hydrocortisone and the microbial components lipopolysaccharide and lipoteichonic acid on TLR2, TLR4 and CD14 expression as well as on cytokine/chemokine expression in human SZ95 sebocytes. Human SZ95 sebocytes were found to constitutively express TLR2, TLR4, CD14, interleukin (IL)-1 � , IL-1 � , IL-6 and IL-8 that was augmented by exposure to components of Gram-negative (lipopolysaccharide) and Gram-positive (lipoteichonic acid) bacteria. Retinol was the most

Isotretinoin Revisited: Pluripotent Effects on Human Sebaceous Gland Cells

Nelson et al. confirmed the previously described antiproliferative effect of isotretinoin on human sebocytes. They attributed a portion of this decrease to cell cycle arrest and detected sebocyte apoptosis, which was not recapitulated by alitretinoin or tretinoin. These events were specific to sebocytes, as isotretinoin failed to induce apoptosis in keratinocytes. Isotretinoin-induced apoptosis was shown to be an RAR-independent mechanism.

Peroxisome Proliferator-Activated Receptors Increase Human Sebum Production

Sebum production is key in the pathophysiology of acne, an extremely common condition, which when severe, may require treatment with isotretinoin, a known teratogen. Apart from isotretinoin and hormonal therapy, no agents are available to reduce sebum. Increasing our understanding of the regulation of sebum production is a milestone in identifying alternative therapeutic targets. Studies in sebocytes and human sebaceous glands indicate that agonists of peroxisome proliferator-activated receptors (PPARs) alter sebaceous lipid production. The goal of this study is to verify the expression and activity of PPARs in human skin and SEB-1 sebocytes and to assess the effects of PPAR ligands on sebum production in patients. To investigate the contribution of each receptor subtype to sebum production, lipogenesis assays were performed in SEB-1 sebocytes that were treated with PPAR ligands and isotretinoin. Isotretinoin significantly decreased lipogenesis, while the PPARalpha agonist-GW7647, PPARdelta agonist-GW0742, PPARalpha/delta agonist-GW2433, PPARgamma agonist rosiglitazone, and the pan-agonist-GW4148, increased lipogenesis. Patients treated with thiazolidinediones or fibrates had significant increases in sebum production (37 and 77%, respectively) when compared to age-, disease-, and sex-matched controls. These data indicate that PPARs play a role in regulating sebum production and that selective modulation of their activity may represent a novel therapeutic strategy for the treatment of acne.

Decline of hormones with increasing age regulates the biological activity of human sebocytes

Hormone deficiency occurring with age leads among other systemic disorders to deterioration of the skin quality. Systemic substitution with estradiol in females and GH in males reversed the observed alterations and retarded the skin aging process in single controlled studies. In order to evaluate the importance of sex steroids and growth factors on skin aging in vitro, human sebocytes were treated with GH, IGF‐I, 17β‐estradiol, DHEA, testosterone and progesterone in combination and as single agents in concentrations corresponding to those circulating in young (f20) and postmenopausal women (f60). Cell proliferation and lipid synthesis were measured by means of the 4‐methylumbelliferyl heptanoate fluorescence assay and nile‐red microassay/fluorescence microscopy, respectively. Cells incubated with all hormones mentioned above at f60 showed significantly lower content of sebaceous lipids (P &lt; 0.001) vs. cells at f20 relating to in vivo observations, which have documented a decline of epidermal lipids in aged skin. While progesterone and DHEA, as single agents, showed no effect on lipid synthesis, after treatment with IGF‐I for 48 h at f20 and f60 concentrations, a significant dose‐dependent increase of neutral (P &lt; 0.001) and polar lipids (P &lt; 0.001) was observed at both concentrations tested. Cell size was also increased. On the other hand, GH at f20 and f60 enhanced significantly the production of neutral (P &lt; 0.01 and P &lt; 0.05, accordingly) and polar lipids (P &lt; 0.05 and P &lt; 0.01, accordingly) in sebocytes but not in such an extent as IGF‐I. 17β‐estradiol induced only polar lipid synthesis (P &lt; 0.01) and cell size. No treatment affected the cell proliferation. In conclusion, sex steroids and growth factors and their decline with age play a key role in the regulation of the lipid synthesis in human sebocytes. Furthermore, among all hormones tested, the GH/IGF‐I axis seems to be the most potent regulator of skin lipids.

The vitamin D endocrine system of human sebocytes

Locally produced vitamin D metabolites are of high importance for growth regulation in various tissues. Using real‐time PCR, we have characterized expression of key components of the vitamin D system vitamin D receptor (VDR), vitamin D‐25‐hydroxylase (25OHase), 25‐hydroxyvitamin D−1 α‐hydroxylase (1αOHase), and 1,25‐dihydroxyvitamin D‐24‐hydroxylase (24OHase) in the SZ95 human sebaceous gland cell line. Incubation of SZ95 cells with 1,25‐dihydroxyvitamin D resulted in a dose‐dependent suppression of cell proliferation. RNA levels for VDR, 25OHase, 1αOHase, and 24OHase were characterized in SZ95 cells treated with various vitamin D analogs. In conclusion, our findings demonstrate that key components of the vitamin D system are strongly expressed in the SZ95 human sebaceous gland cell line and that local synthesis or metabolism of vitamin D metabolites may be of importance for growth regulation and various other cellular functions in sebaceous glands. Moreover, we conclude that sebaceous glands represent promising targets for therapy with vitamin D analogs or for pharmacological modulation of calcitriol synthesis/metabolism.

Age‐specific hormonal decline is accompanied by transcriptional changes in human sebocytes in vitro

The importance of hormones in endogenous aging has been displayed by recent studies performed on animal models and humans. To decipher the molecular mechanisms involved in aging we maintained human sebocytes at defined hormone-substituted conditions that corresponded to average serum levels of females from 20 (f20) to 60 (f60) years of age. The corresponding hormone receptor expression was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunocytochemistry. Cells at f60 produced significantly lower lipids than at f20. Increased mRNA and protein levels of c-Myc and increased protein levels of FN1, which have been associated with aging, were detected in SZ95 sebocytes at f60 compared to those detected at f20 after 5 days of treatment. Expression profiling employing a cDNA microarray composed of 15 529 cDNAs identified 899 genes with altered expression levels at f20 vs. f60. Confirmation of gene regulation was performed by real-time RT-PCR. The functional annotation of these genes according to the Gene Ontology identified pathways related to mitochondrial function, oxidative stress, ubiquitin-mediated proteolysis, cell cycle, immune responses, steroid biosynthesis and phospholipid degradation - all hallmarks of aging. Twenty-five genes in common with those identified in aging kidneys and several genes involved in neurodegenerative diseases were also detected. This is the first report describing the transcriptome of human sebocytes and its modification by a cocktail of hormones administered in age-specific levels and provides an in vitro model system, which approximates some of the hormone-dependent changes in gene transcription that occur during aging in humans.