Zileuton prevents the activation of the leukotriene pathway and reduces sebaceous lipogenesis

Please cite this paper as: Zileuton prevents the activation of the leukotriene pathway and reduces sebaceous lipogenesis. Experimental Dermatology 2010; 19: 148–150. Abstract: Arachidonic acid (AA) activates the 5‐lipoxygenase, induces leukotriene‐B₄ (LTB₄) synthesis, enhances interleukin‐6 (IL‐6) release and increases intracellular neutral lipids in human sebocytes. Moreover, the enzymes of LTB₄ biosynthesis are activated in acne‐involved sebaceous glands. Zileuton a 5‐lipoxygenase inhibitor, reduces the number of inflammatory acne lesions and lipogenesis in patients with acne. In this study, we investigated the activity of zileuton on LTB₄ generation, lipid content and IL‐6 and ‐8 release from human SZ95 sebocytes in vitro. Pretreatment with zileuton partially prevented the AA‐induced LTB₄ and IL‐6 release and increased neutral lipid content. IL‐6 release and neutral lipid content were also reduced under long‐term zileuton treatment. In conclusion, zileuton prevents the activation of the leukotriene pathway and enhancement of lipogenesis by AA in human sebocytes in vitro.     

Sebocytes differentially express and secrete adipokines

In addition to producing sebum, sebocytes link lipid metabolism with inflammation at a cellular level and hence, greatly resemble adipocytes. However, so far no analysis was performed to identify and characterize the adipocyte‐associated inflammatory proteins, the members of the adipokine family in sebocytes. Therefore, we determined the expression profile of adipokines [adiponectin, interleukin (IL) 6, resistin, leptin, serpin E1, visfatin, apelin, chemerin, retinol‐binding protein 4 (RBP4) and monocyte chemoattractant protein 1 (MCP1)] in sebaceous glands of healthy and various disease‐affected (acne, rosacea, melanoma and psoriasis) skin samples. Sebaceous glands in all examined samples expressed adiponectin, IL6, resistin, leptin, serpin E1 and visfatin, but not apelin, chemerin, RBP4 and MCP1. Confirming the presence of the detected adipokines in the human SZ95 sebaceous gland cell line we further characterized their expression and secretion patterns under different stimuli mimicking bacterial invasion [by using Toll‐like receptor (TLR)2 and 4 activators], or by 13‐cis retinoic acid (13CRA; also known as isotretinoin), a key anti‐acne agent. With the exception of resistin, the expression of all of the detected adipokines (adiponectin, IL6, leptin, serpin E1 and visfatin) could be further regulated at the level of gene expression, showing a close correlation with the secreted protein levels. Besides providing further evidence on similarities between adipocytes and sebocytes, our results strongly suggest that sebocytes are not simply targets of inflammation but may exhibit initiatory and modulatory roles in the inflammatory processes of the skin through the expression and secretion of adipokines.     

Testosterone synthesized in cultured human SZ95 sebocytes derives mainly from dehydroepiandrosterone

Please cite this paper as: Testosterone synthesized in cultured human SZ95 sebocytes derives mainly from dehydroepiandrosterone. Experimental Dermatology 2010; 19: 470–472. Abstract: Human sebaceous gland possesses all the steroidogenic enzymes required for androgen synthesis. It remains unclear whether the testosterone produced in situ mainly derives from circulating dehydroepiandrosterone (DHEA) or from de novo synthesis utilizing serum cholesterol. Using testosterone radioimmunoassay, we found that testosterone was barely detectable in the supernatant of cultured human SZ95 sebocytes when cholesterol was added alone, indicating a low basal expression of steroidogenic acute regulatory protein (StAR) in SZ95 cells. Human chorionic gonadotropin and fibroblast growth factor‐9 were as potent as forskolin in activating StAR to enhance testosterone production, while interleukin‐1β, dexamethasone, insulin and insulin‐like growth factor‐1 showed no stimulatory effect. A two‐fold increase of testosterone production was observed in supplementation of DHEA as compared to pregnenolone, progesterone or 17α‐hydroxyprogesterone. Based on our findings, testosterone synthesized in cultured sebocytes derived mainly from DHEA and inhibition of 3β‐hydroxysteroid dehydrogenase and 17β‐hydroxysteroid dehydrogenase may be a new target of androgen suppression for acne treatment.     

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.     

Perilipin 3 modulates specific lipogenic pathways in SZ95 sebocytes

Lipid droplets (LD) are dynamic organelles that manage cellular lipid synthesis, storage and retrieval. Although LD‐associated proteins, including the perilipin family (PLIN1–PLIN5), are essential for these functions, they have been poorly characterized in sebocytes. Here, we employed siRNAs to downregulate PLIN3 in SZ95 sebaceous gland cells and evaluated the consequences in lipid accumulation by nile red staining and mass spectrometry. Nile red staining revealed that siRNA‐mediated downregulation of PLIN3 significantly impaired linoleic acid‐induced lipid accumulation in SZ95 sebocytes. Mass spectrometry revealed that PLIN3 was implicated in the metabolism of linoleic acid, a lipid source used in the build‐up of triglycerides, among other acyl lipids. Furthermore, the expression of key enzymes of sebaceous lipogenesis was altered in PLIN3‐deficient sebocytes, consistent with the changes observed in the neutral lipid abundance, suggesting that PLIN3 functions are intertwined with the lipogenic pathways implicated in sebaceous lipogenesis, such as desaturation and triglyceride synthesis.     

Enhancement of lipid content and inflammatory cytokine secretion in SZ95 sebocytes by palmitic acid suggests a potential link between free fatty acids and acne aggravation

A relationship between acne and free fatty acids (FFAs) has been suggested recently. However, the effects of FFAs on sebaceous glands are still largely unknown. At the same time, the role of FFAs during chronic inflammation is well established. Considering that FFAs are also a major component of sebum, it is likely that changes in FFA affect both the synthesis of sebum and the inflammatory response in sebaceous glands. In this study, we examined a hypothesis that FFAs increase the production of sebum and induce inflammation in the sebaceous glands. We found that treatment of SZ95 sebocytes with exogenously applied palmitic acid (PA), a major saturated FFA, induced a significant increase in intracellular lipid levels. Moreover, PA treatment also increased the expression and secretion of the proinflammatory cytokines in SZ95 sebocytes. We also found that Toll‐like receptors were required for the inflammatory response triggered by PA. The results of our study strengthen the notion about the link between acne and FFAs and suggest the mechanism underlying this relationship. Our results serve as a foundation for future work that will explore the association between FFA and acne and pave way to the development of novel treatment options for acne.     

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.     

Ex vivo human skin and SZ95 sebocytes exhibit a homoeostatic interaction in a novel coculture contact model

The sebaceous gland displays key functions of the human skin, such as hormone synthesis in situ, antimicrobial activity and participation to inflammatory responses. Consequently, there is an emerging need of advanced in vitro models to study complex interactions between the sebaceous gland and the other skin compartments. Despite the evolution of both full‐skin organ culture and reconstructed three‐dimensional skin models, no satisfactory solutions have been provided for the integration of sebaceous glands and/or sebaceous gland cells in those models, probably due to their problematic maintenance both in vitro and ex vivo. We have developed a coculture model of explant skin in direct contact with immortalized SZ95 sebocytes, which resulted in overall improved structural integrity of the epidermis, higher percentage of proliferating basal epidermal cells and reduced apoptosis of differentiating keratinocytes after 6 days, as detected by Ki67 and TUNEL staining, respectively. Furthermore SZ95 sebocytes exhibited morphological and biochemical signs of normal differentiation and lipid accumulation, while interleukin‐6 expression in the supernatant of the cocultures was decreased in comparison with the control. The data provide evidence of a beneficial interaction between sebocytes and skin explants and provide the rationale for their integration in future three‐dimensional skin models.     

LXRalpha 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.     

In situ hybridization analysis of CRABP II expression in sebaceous follicles from 13-cis retinoic acid-treated acne patients

The aim of this study was to investigate the effects of 13-cis retinoic acid treatment on cellular retinoic acid binding protein II (CRABP II) mRNA expression in sebaceous follicles from acne patients, using in situ hybridization. Biopsies were taken from uninvolved skin areas in close juxtaposition to inflamed comedos before therapy, and at 2–4 or 14-16 weeks of treatment. Paraffin sections were used for in situ hybridization study with riboprobes transcribed from human CRABP II cDNA. After oral treatment with l 3-cis retinoic acid, sebaceous glands were reduced in size and atrophic, and the ratio of sebum-free to fully differentiated (sebum-producing) sebocytes was dramatically increased. The CRABP II expression in the sebaceous gland, and to some extent in infundtbular structures, was strongly increased compared with the level of expression in the epidermis. The maximum signal was always found in layers of suprabasal sebocytes lacking lipid droplets. but never in the basal layers. These findings indicate a selective activity of 13-cis retinoic acid on CRABP II mRNA expression in the sebaceous glands of acne patients.     

SZ95 sebocytes induce epidermal melanocyte dendricity and proliferation in vitro

The regulatory effects of sebocytes on melanocytes (HMel) are unknown. In this study, SZ95 sebocytes co-cultured with HMel, whether in direct cell contact or with SZ95 sebocytes in inserts, resulted in epidermal HMel flattening with increase in surface area and multiple small dendrites formation. Only in high Ca(2+) level and direct cell contact co-culture, the HMel dendrites were remarkably long and preferentially targeted and attached to SZ95 sebocytes. Likewise, only high Ca(2+) SZ95 sebocyte conditioned medium stimulated HMel proliferation in a time-dependent manner at days 9 (142.9%, P < 0.01) and 12 (179.2%, P < 0.0001) of incubation when compared with day 0. In contrast, melanin contents significantly decreased on incubation with high Ca(2+) SZ95 sebocytes in comparison with low Ca(2+) SZ95 sebocytes at days 6 (P < 0.01) and 9 (P < 0.05) of incubation. These results denote that sebocytes also modulate HMel functions and may contribute to skin colour in sebaceous glands-rich body regions.     

Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95)

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.     

TRAIL contributes to the apoptotic effect of 13-cis retinoic acid in human sebaceous gland cells

Background The full mechanism of action of isotretinoin [13‐cis retinoic acid (13‐cis RA)] in treating acne is unknown. 13‐cis RA induces key genes in sebocytes that are involved in apoptosis, including Tumor necrosis factor Related Apoptosis Inducing Ligand (TRAIL). Objectives In this study, we investigated the role of 13‐cis RA‐induced TRAIL within SEB‐1 sebocytes. Methods Using 13‐cis RA and recombinant human TRAIL (rhTRAIL) protein, we assessed induction of TRAIL and apoptosis in SEB‐1 sebocytes, normal keratinocytes and patient skin biopsies. Results Treatment with rhTRAIL protein increased TUNEL‐positive staining in SEB‐1 sebocytes. TRAIL siRNA significantly decreased the percentage of TUNEL‐positive SEB‐1 sebocytes in response to 13‐cis RA treatment. Furthermore, TRAIL expression increased in the skin of patients with acne after 1 week of isotretinoin therapy compared with baseline. TRAIL expression localized within sebaceous glands. Unlike sebocytes, TRAIL protein expression was not increased in normal human epidermal keratinocytes in response to 13‐cis RA, nor did rhTRAIL induce apoptosis in keratinocytes, suggesting that TRAIL is key in the sebocyte‐specific apoptotic effects of 13‐cis RA. Conclusions Taken together, our data suggest that TRAIL, like the neutrophil gelatinase‐associated lipocalin, is involved in mediating 13‐cis RA apoptosis of sebocytes.     

Topically applied L-carnitine effectively reduces sebum secretion in human skin

Background Oily skin condition is caused by an excessive sebaceous gland activity, resulting in an overproduction of sebum, giving the skin an undesired shiny, oily appearance. Aims To identify an active substance that reduces sebum production in human sebaceous glands by regulating fat metabolism in a natural way. Patients/Methods The effects of l ‐carnitine on β‐oxidation and intracellular lipid content were investigated in vitro using the human sebaceous cell line SZ95. Penetration experiments utilizing pig skin as a model system were performed with a cosmetic formulation containing radioactively labeled l ‐carnitine. To determine the in vivo effects, a vehicle‐controlled, randomized study was carried out using a cosmetic formulation containing 2%l ‐carnitine for 3 weeks. Sebum production was investigated utilizing the lipid‐absorbent Sebutape^®. Results SZ95 cells treated with 0.5% or 1%l ‐carnitine demonstrated a significant concentration‐dependent increase in β‐oxidation compared to control cells. Following the treatment with l ‐carnitine, intracellular lipid concentrations decreased significantly in a dose‐dependent manner compared with untreated control cells. In skin penetration experiments, topically applied l ‐carnitine reached the dermis. In addition, topical in vivo application of a formulation containing 2%l ‐carnitine for 3 weeks significantly decreased the sebum secretion rate compared to the treatment with vehicle. Conclusions Our results show that the treatment of human sebocytes with l ‐carnitine significantly augments β‐oxidation and significantly decreases intracellular lipid content in human sebocytes. Topically applied l ‐carnitine is bioavailable and leads to a significant sebum reduction in vivo. In conclusion, l ‐carnitine represents a valuable compound, produced naturally within the body, for the topical treatment of oily skin in humans.     

Transient receptor potential vanilloid-1 signaling as a regulator of human sebocyte biology

Transient receptor potential vanilloid-1 (TRPV1), originally described as a central integrator of nociception, is expressed on human epidermal and hair follicle keratinocytes and is involved in regulation of cell growth and death. In human pilosebaceous units, we had shown that TRPV1 stimulation inhibits hair shaft elongation and matrix keratinocyte proliferation, and induces premature hair follicle regression and keratinocyte apoptosis. In the current study, we have explored the role of TRPV1-mediated signaling in sebaceous gland (SG) biology, using a human sebocyte cell culture model (SZ95 sebocytes). Demonstrating that human skin SG in situ and SZ95 sebocytes in vitro express TRPV1, we show that the prototypic TRPV1 agonist, capsaicin, selectively inhibits basal and arachidonic acid-induced lipid synthesis in a dose-, time-, and extracellular calcium-dependent and a TRPV1-specific manner. Low-dose capsaicin stimulates cellular proliferation via TRPV1, whereas higher concentrations inhibit sebocyte growth and induce cell death independent of TRPV1. Moreover, capsaicin suppresses the expression of genes involved in lipid homeostasis and of selected proinflammatory cytokines. Collectively, these findings support the concept that TRPV1 signaling is a significant, previously unreported player in human sebocyte biology and identify TRPV1 as a promising target in the clinical management of inflammatory SG disorders (for example, acne vulgaris).     

Polyphenol‐rich apple extract inhibits dexamethasone‐induced sebaceous lipids production by regulating SREBP1 expression

Sebum production and excretion is a primary function of the sebaceous glands, but abnormally increased sebum production is a major cause of acne vulgaris. To identify a new candidate that regulates sebum production, we investigated the possible inhibitory effects of apple polyphenols (APP) purified from unripe apples on primary cultured human sebocytes and in patients with acne vulgaris. Dexamethasone (Dex) increased lipid synthesis and expression of the sterol response element‐binding protein 1 (SREBP 1) and its target enzymes, acetyl‐CoA carboxylase (ACC) and fatty acid synthase (FAS), in the sebocytes. However, APP inhibited Dex‐induced lipid production and expression of SREBP‐1, ACC and FAS. APP also inhibited the increase in the expression and activation of glucocorticoid receptor in the sebocytes. Taken together, these results suggest that APP may be useful to regulate sebum production and may alleviate sebum‐involved skin disease, such as acne vulgaris.