Human facial sebaceous gland cells were transfected with a PBR-322-based plasmid containing the coding region for the Simian virus-40 large T antigen. The resulting proliferating cell cultures have been passaged over 50 times to date, have been cloned, and show no signs of senescence after 4&DF;1 2 y in vitro, whereas normal human sebocytes can only be grown for three to six passages. The immortalized transfected cells, termed SZ95, expressed the Simian virus-40 large T antigen and presented an hyper-diploid-aneuploid karyotype with a modal chromosome number of 64.5. The SZ95 cell line exhibited epithelial, polymorphous characteristics with different cell sizes of up to 3.25-fold during proliferation and 6-fold at confluence, showing numerous cytoplasmic lipid droplets. The cells showed large cytoplasm profiles with abundant organelles, including vacuoles and myelin figures which indicated lipid synthesis. Lack of or only few desmosomal areas were observed. SZ95 cells expressed molecules typically associated with human sebocytes, such as keratins 7, 13, and 19, and several proteins of the polymorphous epithelial mucin family. Functional studies revealed synthesis of the sebaceous lipids squalene and wax esters as well as of triglycerides and free fatty acids, even after 25-40 passages; active lipid secretion; population doubling times of 52.4 +/- 1.6 h; reduced growth but maintenance of lipid synthesis under serum-free conditions; and retrieval of cell proliferation after addition of 5alpha-dihydrotestosterone. Retinoids significantly inhibited proliferation of certain SZ95 cell clones in the expected magnitude 13-cis-retinoic acid > all-trans-retinoic acid > > acitretin. Thus SZ95 is an immortalized human sebaceous gland cell line that shows the morphologic, phenotypic and functional characteristics of normal human sebocytes.

Acne is a common skin disorder of the pilosebaceous unit. In addition to genetic, hormonal and environmental factors, abnormal colonization by Propionibacterium acnes has been implicated in the occurrence of acne via the induction of inflammatory mediators. To gain more insight into the role that sebocytes play in the innate immune response of the skin, particularly in acne, we compared the antimicrobial peptide and proinflammatory cytokine/chemokine expression at mRNA and protein levels, as well as the viability and differentiation of SZ95 sebocytes in response to co-culture with representative isolates of P. acnes type IA and type IB as well as Escherichia coli-derived lipopolysaccharide (LPS). We found that, in vitro, P. acnes type IA and IB isolates and LPS induced human beta-defensin-2 and proinflammatory cytokine/chemokine expression, and influenced sebocyte viability and differentiation. Our results provide evidence that sebocytes are capable of producing proinflammatory cytokines/chemokines and antimicrobial peptides, which may have a role in acne pathogenesis. Furthermore, since P. acnes types IA and IB differentially affect both the differentiation and viability of sebocytes, our data demonstrate that different strains of P. acnes vary in their capacity to stimulate an inflammatory response within the pilosebaceous follicle.

Sebaceous glands may be involved in a pathway conceptually similar to that of the hypothalamic-pituitary-adrenal (HPA) axis. Such a pathway has been described and may occur in human skin and lately in the sebaceous glands because they express neuropeptide receptors. Corticotropin-releasing hormone (CRH) is the most proximal element of the HPA axis, and it acts as central coordinator for neuroendocrine and behavioral responses to stress. To further examine the probability of an HPA equivalent pathway, we investigated the expression of CRH, CRH-binding protein (CRH-BP), and CRH receptors (CRH-R) in SZ95 sebocytes in vitro and their regulation by CRH and several other hormones. CRH, CRH-BP, CRH-R1, and CRH-R2 were detectable in SZ95 sebocytes at the mRNA and protein levels: CRH-R1 was the predominant type (CRH-R1͞CRH-R2 ‫؍‬ 2). CRH was biologically active on human sebocytes: it induced biphasic increase in synthesis of sebaceous lipids with a maximum stimulation at 10 ؊7 M and up-regulated mRNA levels of 3␤-hydroxysteroid dehydrogenase͞⌬ 5-4 isomerase, although it did not affect cell viability, cell proliferation, or IL-1␤-induced IL-8 release. CRH, dehydroepiandrosterone, and 17␤-estradiol did not modulate CRH-R expression, whereas testosterone at 10 ؊7 M down-regulated CRH-R1 and CRH-R2 mRNA expression at 6 to 24 h, and growth hormone (GH) switched CRH-R1 mRNA expression to CRH-R2 at 24 h. Based on these findings, CRH may be an autocrine hormone for human sebocytes that exerts homeostatic lipogenic activity, whereas testosterone and growth hormone induce CRH negative feedback. The findings implicate CRH in the clinical development of acne, seborrhea, androgenetic alopecia, skin aging, xerosis, and other skin disorders associated with alterations in lipid formation of sebaceous origin.

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Seborrhoea and acne are exclusively human diseases and sebaceous gland differentiation is species specific. Therefore, fundamental research on human sebaceous cell function and control requires human in vitro models. The human sebocyte culture model, introduced in 1989, has been used in several studies to elucidate sebaceous gland activity and its regulation at the cellular level. Cultured human sebocytes have been shown to preserve important sebocytic characteristics, although they undergo an incomplete terminal differentiation in vitro. In vitro synthesis of free fatty acids without bacterial involvement and marked interleukin 1α expression at the mRNA and protein levels with no further induction by lipopolysaccharides lead to the assumption that human sebocytes may initiate acne lesions by an intrinsic mechanism. Androgens affected sebocyte activity in vitro in a manner dependent on the localization of the sebaceous glands. In vitro stimulation of sebocyte proliferation by androgens could be completely abolished by spironolactone. Cultured sebocytes strongly expressed type 1 5α-reductase and metabolized testosterone to androstenedione, 5α-androstanedione, 5α-dihydrotestosterone, androsterone and 5α-androstanediol, whereas the levels of 5α-reductase activity were probably not feedback regulated. 4,7β-Dimethyl-4-aza-5αcholestan-3-one, a type 1 5α-reductase inhibitor, induced an early, marked down-regulation of 5α-reductase activity in human sebocytes in vitro, while hydrofinasteride, a type 2 inhibitor, required 10³-fold higher concentrations to induce similar effects. Stimulation of sebocyte proliferation by insulin, thyroid-stimulating hormone and hydrocortisone indicates that the hormonal control of the sebaceous gland could be a complex mechanism. Retinoids inhibited sebocyte proliferation in a dose-dependent manner and down-regulated lipid synthesis and sebocyte differentiation in vitro. Isotretinoin was the most potent compound. On the other hand, vitamin A was found essential for sebocyte activity and differentiation in vitro and could be partially substituted by synthetic retinoids. The inhibitory effect of isotretinoin on sebocyte proliferation was barely affected by the presence of vitamin A. The low persistent isotretinoin levels or, more likely, the considerably elevated tretinoin concentrations detected in human sebocytes after treatment with isotretinoin in vitro may be responsible for the inhibitory effect of this compound on sebocyte activity.

Increased cell volume, accumulation of lipid droplets in the cytoplasm, and nuclear degeneration are phenomena indicating terminal differentiation of human sebocytes followed by holocrine secretion and cell death. The molecular pathways of natural and induced sebocyte elimination are still unknown, however. In this study, SZ95 sebocytes were found to exhibit DNA fragmentation after a 6 h culture followed by increased lactate dehydrogenase release after 24 h, indicating cell damage. With the help of morphologic studies and using Oil Red detection of cellular lipids, cell enlargement, accumulation of lipid droplets in the cytoplasm, and nuclear fragmentation could be observed under treatment with arachidonic acid. Staurosporine, a potent inhibitor of phospholipid Ca2+-dependent protein kinase, increased externalized phosphatidylserine levels on SZ95 sebocytes, detected by annexin V/propidium iodide flow cytometry, as early as after 1 h, whereas dose-dependent reduction of bcl-2 mRNA and protein expression, enhanced DNA fragmentation, and increased caspase 3 levels, detected by caspase 3 inhibitor/propidium iodide flow cytometry, were found after 6 h of treatment. SZ95 sebocyte death was detected as early as after 6 h of SZ95 sebocyte treatment with high staurosporine concentrations (10(-6)-10(-5) M). 5Alpha-dihydrotestosterone (10(-8)-10(-5) M) did not affect externalized phosphatidylserine levels and DNA fragmentation in SZ95 sebocytes but slightly decreased lactate dehydrogenase cell release. Neither acitretin nor 13-cis retinoic acid (10(-8)-10(-5) M) affected externalized phosphatidylserine levels, DNA fragmentation, and lactate dehydrogenase cell release, despite the increased caspase 3 levels under treatment with 13-cis retinoic acid. The combined staurosporine and 13-cis retinoic acid treatment enhanced DNA fragmentation in SZ95 sebocytes to the same magnitude as in cells only treated with staurosporine. In conclusion, SZ95 sebocytes in vitro undergo apoptosis, which can be enhanced by the terminal differentiation inductor arachidonic acid or by staurosporine and leads to cell death. 5Alpha-dihydrotestosterone inhibits SZ95 sebocyte death without involving apoptotic pathways, and retinoids did not affect the programmed death of human sebocytes. The latter result fits well with the currently reported inability of normal skin cells to undergo apoptosis after treatment with retinoids, in contrast to their malignant counterparts.

Lipid synthesis and accumulation represent a major step in sebocyte differentiation and it may be of importance for sebocytes to express two families of transcription factors, CCAAT/enhancer binding proteins (c/EBPs) and peroxisome proliferator-activated receptors (PPARs), which were found to play a crucial role in the differentiation of adipocytes. Using the immortalized human sebaceous gland cell line SZ95 we examined the expression of the molecules before and after treatment with testosterone, 5alpha-dihydrotestosterone, dexamethasone, 17beta-estradiol and genistein, at 6, 12, 24, and 48 h, respectively. Reverse transcription-PCR analysis showed expression of peroxisome proliferator-activated receptors -alpha, -delta, -gamma1, -gamma2 and CCAAT/enhancer binding proteins-alpha, -beta, -gamma-delta in native SZ95 sebocytes. In western blot studies, high levels of CCAAT/enhancer binding proteins-alpha and -beta, and peroxisome proliferator-activated receptors-gamma were expressed at 6, 24, and 12 h, respectively. Immunostaining of the cultured sebocytes showed the CCAAT/enhancer binding proteins-alpha and -beta mainly localized within nuclei, whereas peroxisome proliferator-activated receptors-gamma in the cytoplasm. Strong staining of sebocytes was immunohistochemically revealed in the basal layer of sebaceous glands in human scalp and sebaceous nevus. Genistein down-regulated the expression of CCAAT/enhancer binding proteins-alpha and -beta, and peroxisome proliferator-activated receptors-gamma on the protein level. Treatment with linoleic acid for 48 h induced further differentiation of sebocytes leading to abundant lipid synthesis.

Many lines of evidence indicate that the activity of sebaceous glands can be modulated by neuropeptides. Direct evidence in man, however, is still missing. We show that SZ95 sebocytes, an immortalized human sebaceous gland cell line, express receptors for alpha-melanocyte-stimulating hormone. Reverse transcription polymerase chain reaction with primers against the five melanocortin receptors and immunofluorescence studies using an antibody directed against a peptide corresponding to the amino acids 2-18 of the human melanocortin-1 receptor disclosed specific transcripts and immunoreactivity for melanocortin-1 receptor in these cells. Melanocortin-1 receptor expression was confirmed in sebocytes of normal human skin by immunohistochemistry. In contrast, no immunostaining for the melanocortin-5 receptor could be detected in sebocytes in situ, in accordance with the lack of specific transcripts for this melanocortin receptor in SZ95 sebocytes. As cytokines play an important role in the recruitment of inflammatory cells in acne and related disorders and alpha-melanocyte-stimulating hormone exerts immunomodulatory effects in many other cell types, we investigated the effect of alpha-melanocyte-stimulating hormone on interleukin-8 secretion by SZ95 sebocytes. Treatment with interleukin-1beta resulted in a marked increase in interleukin-8 release that was partially blocked by coincubation with alpha-melanocyte-stimulating hormone in a dose-dependent manner. Taken together, we show here that the melanocortin-1 receptor is expressed in vitro and in situ in human sebocytes. By modulating interleukin-8 secretion, alpha-melanocyte-stimulating hormone may act as a modulator of inflammatory responses in the pilosebaceous unit.