Differential effectiveness of selected non‐psychotropic phytocannabinoids on human sebocyte functions implicates their introduction in dry/seborrhoeic skin and acne treatment

Acne is a common skin disease characterized by elevated sebum production and inflammation of the sebaceous glands. We have previously shown that a non‐psychotropic phytocannabinoid ((–)‐cannabidiol [CBD]) exerted complex anti‐acne effects by normalizing ‘pro‐acne agents’‐induced excessive sebaceous lipid production, reducing proliferation and alleviating inflammation in human SZ95 sebocytes. Therefore, in this study we aimed to explore the putative anti‐acne effects of further non‐psychotropic phytocannabinoids ((–)‐cannabichromene [CBC], (–)‐cannabidivarin [CBDV], (–)‐cannabigerol [CBG], (–)‐cannabigerovarin [CBGV] and (–)‐Δ⁹‐tetrahydrocannabivarin [THCV]). Viability and proliferation of human SZ95 sebocytes were investigated by MTT and CyQUANT assays; cell death and lipid synthesis were monitored by DilC₁(5)‐SYTOX Green labelling and Nile Red staining, respectively. Inflammatory responses were investigated by monitoring expressions of selected cytokines upon lipopolysaccharide treatment (RT‐qPCR, ELISA). Up to 10 μm , the phytocannabinoids only negligibly altered the viability of the sebocytes, whereas high doses (≥50 μm ) induced apoptosis. Interestingly, basal sebaceous lipid synthesis was differentially modulated by the substances: CBC and THCV suppressed it, and CBDV had only minor effects, whereas CBG and CBGV increased it. Importantly, CBC, CBDV and THCV significantly reduced arachidonic acid (AA)‐induced ‘acne‐like’ lipogenesis. Moreover, THCV suppressed proliferation, and all phytocannabinoids exerted remarkable anti‐inflammatory actions. Our data suggest that CBG and CBGV may have potential in the treatment of dry‐skin syndrome, whereas CBC, CBDV and especially THCV show promise to become highly efficient, novel anti‐acne agents. Moreover, based on their remarkable anti‐inflammatory actions, phytocannabinoids could be efficient, yet safe novel tools in the management of cutaneous inflammations.     

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.     

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.     

Enzymes involved in the conversion of arachidonic acid to eicosanoids in the skin of atopic dogs

Please cite this paper as: Enzymes involved in the conversion of arachidonic acid to eicosanoids in the skin of atopic dogs. Experimental Dermatology 2010; 19 : e317–e319. Abstract: Canine atopic dermatitis (AD), a chronic inflammatory skin disease, shares characteristics with its human counterpart. To get insight into the role of enzymes involved in production of prostaglandin E₂ (PGE2) and leukotriene B₄ (LTB₄), potent inflammatory mediators originating from membrane‐derived arachidonic acid (AA), expression of genes encoding these enzymes and receptors was quantified by qPCR in non‐lesional and lesional skin from atopic dogs and in healthy skin. Significantly higher mRNA expression of the key enzymes 5‐lipoxygenase (5‐LO), 5‐LO activating protein (FLAP), leukotriene A₄ hydrolase (LTA₄H) and prostaglandin E synthase 1 (mPGES‐1) and their receptors (PGE receptors 2 and 3) were observed. Being responsible for elevated levels of metabolites of the 3‐series prostaglandins and the 5‐series leukotrienes these enzymes may be interesting targets for therapy that should result in amelioration of clinical signs in canine atopic dermatitis.     

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.     

Isotretinoin increases skin-surface levels of neutrophil gelatinase-associated lipocalin in patients treated for severe acne

Background A clear‐cut need exists for safe and effective alternatives to the use of isotretinoin in severe acne. Lack of data regarding the specifics of isotretinoin’s mechanism of action has hampered progress in this area. Recently, the protein neutrophil gelatinase‐associated lipocalin (NGAL) has been identified as a mediator of the apoptotic effect of isotretinoin on sebocytes. Objectives To establish further the clinical relevance of NGAL and to elucidate the factors that induce NGAL expression in sebocytes. Methods Methods were developed to isolate and quantify skin‐surface levels of NGAL from normal subjects and patients with acne undergoing treatment with isotretinoin. Results Patients with acne were found to have higher skin levels of NGAL compared with normal subjects. Studies in SEB‐1 sebocytes indicate that NGAL expression is increased in response to Propionibacterium acnes and interleukin (IL)‐1β. In patients, isotretinoin increases NGAL levels by 2·4‐fold on the skin surface and this increase precedes decreases in sebum and P. acnes counts. Conclusions These data support the hypothesis that NGAL is an important mediator of the early effects of isotretinoin on the sebaceous glands and provide insights into the mechanisms that regulate NGAL expression in the skin.     

Endocrinology and immunology of acne: Two sides of the same coin

Current experimental research on acne pathophysiology has revealed a more complicated background than the classically reported four-factor aetiology. Cells of the pilosebaceous unit, which represent the template for the development of acne lesions, seem to be parallelly affected by endocrinological/metabolic factors as well as inflammatory/immunological ones that cooperate in sebocyte differentiation and lipogenesis. Indeed, the unique programme of sebocyte terminal differentiation and death, the so called holocrine secretion, is influenced by inflammatory and metabolic (lipid) signalling with common denominator the selective regulation of peroxisome proliferator-activated receptors. Autophagy provides substrates for energy generation and biosynthesis of new cell structure proteins contributing to the normally increased sebaceous gland metabolic functions, which are also regulated by extracellular calcium signalling, essential lipids and hormones. The ultimate differentiation product of human sebocytes, sebum, co-regulates the inflammatory sebocyte status. Sebum composition is controlled among others by Propionibacterium acnes and other bacteria, sexual hormones, neuropeptides, endogenous opioids and environmental agents, which may function as endocrine disruptors. Diet may also be an important source of substrates for the synthesis of pro-inflammatory and anti-inflammatory sebaceous lipids. Sebum changes might induce inflammation and initiate underlying immune mechanisms leading to acne lesions. Current new therapeutic efforts on acne concentrate on anti-inflammatory/immunologically active concepts, which are able to regulate sebaceous lipogenesis. At last, current molecular studies based on published molecular data sets confirmed the major role of inflammation in acne development.     

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.     

Zileuton, an oral 5-lipoxygenase inhibitor, directly reduces sebum production

BACKGROUND: Zileuton, a 5-lipoxygenase inhibitor, reduces the number of inflammatory lesions in moderate acne and inhibits the synthesis of sebaceous lipids. OBJECTIVE: To detect whether zileuton directly reduces sebum synthesis. METHODS: A 40-year-old female with mild disseminated sebaceous gland hyperplasia and seborrhea was treated with zileuton 4 x 600 mg/day over 2 weeks, was followed-up for 6 weeks after discontinuation of zileuton and was re-treated with low-dose isotretinoin 10 mg/2nd day over 5 weeks. Casual skin surface lipids and sebum synthesis were determined. RESULTS: Under treatment with zileuton increased casual skin surface lipids were normalized and synthesis of facial sebum was decreased. Six weeks after discontinuation of treatment casual skin surface lipids were increased again and synthesis of sebum returned to baseline. Subsequent low-dose isotretinoin treatment led to similar changes of casual skin surface lipids and sebum synthesis with zileuton already after 2 weeks. CONCLUSION: Zileuton directly inhibits sebum synthesis in a transient manner with a potency similar to low-dose isotretinoin at least in our patient.     

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.     

Arachidonic acid metabolism in HEL/30 murine epidermal Cell Line

Summary The established mouse epidermis-derived cell line HEL/30 was incubated in the presence of ³H arachidonic acid (AA) for 1 h. After medium removal, cells were reincubated with fresh medium in the presence or absence of the calcium ionophore A23187 and tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The AA metabolites formed were extracted from cell-free medium and analyzed using TLC and HPLC. The distribution of the recovered radioactivity showed PGE₂, 15-hydroxy-eicosatetraenoic acid (15-HETE), and leukotriene B₄ (LTB₄), as major products of AA metabolism. The presence of calcium ionophore A23187 increased the release of radioactivity, without affecting the profile of metabolites present in the medium. TPA elicited a preferential increase of cycloxygenase metabolism, this effect being reversed by indomethacin. 5,8,11,14-eicosatetraynoic acid (ETYA) almost completely inhibited LT and HETE formation in A23187 and TPA-treated cells. The results show that HEL/30 cells are able to metabolize AA via both cyclo-and lipoxygenase pathways and that these activities can be modified by chemical means. This cell line might be a suitable tool for studying the involvement of arachidonic acid cascade in cell response to exogenous stimuli.     

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.     

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.     

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.