Resveratrol ameliorates lipid accumulation and inflammation in human SZ95 sebocytes via the AMPK signaling pathways in vitro.

BACKGROUND: Acne vulgaris is a prevalent skin disease lacking effective and well-tolerated treatment. An earlier study indicated that resveratrol (RVT) has therapeutic effects in acne patients through unknown mechanisms. OBJECTIVES: To evaluate the effects of RVT on linoleic acid (LA)-induced lipogenesis and peptidoglycan (PGN)-induced inflammation in cultured SZ95 sebocytes in vitro, and to investigate the underlying mechanisms. METHODS: RNA-sequencing was used to analyze the whole transcriptome. Nile red staining was used to detect intracellular neutral lipids, whereas lipidomics was used to investigate changes in the lipid profile in sebocytes. Interleukin (IL)-1ß and IL-6 mRNA and protein levels were assessed through quantitative real-time PCR and Enzyme-linked immunosorbent assay, respectively. Western blot was used to evaluate the expression of lipogenesis-related proteins, the inflammatory signaling pathway, and the AMP-activated protein kinase (AMPK) pathway. Further, specific small interfering RNA (siRNA) was used to knockdown sirtuin-1 (SIRT1) expression. RESULTS: RVT inhibited the lipogenesis-related pathway and nuclear factor-kappa B (NF-κB) signaling pathway in SZ95 sebocytes. It also downregulated LA-induced lipogenesis, the expression of lipid-related proteins, and the contents of unsaturated fatty acids. Besides, RVT promoted SIRT1 expression and deacetylation of the NF-κB p65 subunit, thereby lowering IL-1ß and IL-6 secretion under PGN induction. Furthermore, pretreatment with AMPK inhibitor Compound C abolished RVT-mediated sebosuppressive and anti-inflammation effects. Meanwhile, SIRT1 silencing abrogated the anti-inflammatory potential of RVT. CONCLUSION: In human SZ95 sebocytes, RVT exhibits sebosuppressive and anti-inflammatory effects partially through the AMPK pathway, which may justify the role of RVT treatment in acne vulgaris.

Histone Deacetylase 1 Reduces Lipogenesis by Suppressing SREBP1 Transcription in Human Sebocyte Cell Line SZ95.

Proper regulation of sebum production is important for maintaining skin homeostasis in humans. However, little is known about the role of epigenetic regulation in sebocyte lipogenesis. We investigated histone acetylation changes and their role in key lipogenic gene regulation during sebocyte lipogenesis using the human sebaceous gland cell line SZ95. Sebocyte lipogenesis is associated with a significant increase in histone acetylation. Treatment with anacardic acid (AA), a p300 histone acetyltransferase inhibitor, significantly decreased the lipid droplet number and the expression of key lipogenic genes, including sterol regulatory-binding protein 1 (SREBP1), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC). In contrast, treatment with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, increased the expression of these genes. Global HDAC enzyme activity was decreased, and HDAC1 and HDAC2 expression was downregulated during sebaceous lipogenesis. Interestingly, HDAC1 knockdown increased lipogenesis through SREBP1 induction, whereas HDAC1 overexpression decreased lipogenesis and significantly suppressed SREBP1 promoter activity. HDAC1 and SREBP1 levels were inversely correlated in human skin sebaceous glands as demonstrated in immunofluorescence images. In conclusion, HDAC1 plays a critical role in reducing SREBP1 transcription, leading to decreased sebaceous lipogenesis. Therefore, HDAC1 activation could be an effective therapeutic strategy for skin diseases related to excessive sebum production.

Formalin-killed Propionibacterium acnes activates the aryl hydrocarbon receptor and modifies differentiation of SZ95 sebocytes in vitro.

BACKGROUND: Acne vulgaris is a common pilosebaceous disease associated with Propionibacterium acnes (P. acnes). Resolution of comedones may occur in association with shrunken sebaceous glands (SGs) containing de-differentiated cells, however the role of P. acnes is unclear. OBJECTIVES: To investigate the effects of P. acnes on aryl hydrocarbon receptor (AhR) activation, lipogenesis and differentiation in cultured immortalized human SZ95 sebocytes. MATERIALS & METHODS: Cultured sebocytes were incubated with formalin-killed (f-) P. acnes (f-P. acnes) at different ratios of multiplicity of infection. The mRNA levels of the AhR downstream cytochrome P450 (CYP) genes were measured by quantitative RT-PCR, nuclear translocation of AhR by western blot and immunofluorescence, lipogenesis and keratinization by gene set enrichment analysis (GSEA), lipid related analysis by Oil red O staining and Nile red staining, and sebaceous differentiation-related gene expression by western blot. RESULTS: f-P. acnes upregulated CYPs mRNA levels and induced translocation of AhR protein from the cytoplasm into the nucleus. GSEA revealed downregulation of lipogenesis and upregulation of keratinization. f-P. acnes inhibited linoleic acid-induced neutral lipid synthesis and expression of sebocyte markers, keratin 7 and mucin1/EMA, but increased expression of keratinocyte markers, keratin 10 and involucrin, which were abolished by AhR gene silencing. Inhibition of lipogenesis-related genes, such as sterol response element-binding protein, was also observed. CONCLUSION: f-P. acnes inhibits lipogenesis and induces terminal differentiation of sebocytes, into keratinocyte-like cells, via activation of the AhR pathway in vitro, suggesting that follicular P. acnes is not only acnegenic but also promotes acne remission through feedback regulation of sebum production.

Macrophage-activating lipopeptide-2 and corticotropin-releasing hormone stimulate the inflammatory signalling in human sebocytes through activation of stearoyl-CoA desaturase and fatty acid desaturase 2.

BACKGROUND: The macrophage-activating lipopeptide-2 (MALP-2) activates cells carrying a functional Toll-like receptor (TLR)-2/6. Human sebocytes express functional TLR-2, TLR-4 and CD14. Upregulation of stearoyl-CoA desaturase (SCD) and fatty acid desaturase-2 (FADS2) expression induces pro-inflammatory sebaceous activity. On the other hand, corticotropin-releasing hormone (CRH) is likely to serve as an autocrine stress hormone in human sebocytes. In addition to its antiproliferative, lipogenetic and androgen-activating functions, CRH exhibits a pro-inflammatory action and its expression is upregulated in acne-involved sebaceous glands. OBJECTIVE: Determination of the pro-inflammatory function of MALP-2 and CRH and clarification of the option that MALP-2 and/or CRH activity on human sebocytes might be mediated through SCD and/or FADS2. METHODS: SZ95 sebocytes were treated with MALP-2, CRH and the SCD inhibitor/ligand FPCA. SCD, FADS2, TLR-2 mRNA and protein levels and IL-6 and IL-8 secretion were investigated. Intracellular CRH levels were assessed under treatment with CRH, MALP-2, linoleic acid and arachidonic acid. Phorbol 12-myristate 13-acetate and dexamethasone served as positive and negative controls, respectively. RESULTS: MALP-2 upregulated SCD, FADS2, TLR-2 mRNA and protein levels and IL-6 and IL-8 secretion from SZ95 sebocytes. Co-incubation of SZ95 sebocytes with MALP-2/FPCA did not affect the MALP-2-induced SCD mRNA upregulation but reduced FADS2 mRNA levels and inhibited IL-8 secretion. CRH induced an early, low-level SCD and FADS2 upregulation and TLR-2 and IL-8 secretion. High intracellular CRH concentrations could be detected early after CRH treatment and persisted up to 24 h. MALP-2 stimulated intracellular CRH levels. CONCLUSIONS: MALP-2 stimulates the inflammatory signalling in human sebocytes through SCD and FADS2 activation. Inhibition of FADS2 mRNA levels and IL-8 secretion through MALP-2/FCPA co-incubation and diminution of fatty acid unsaturation might lead to a reduction of pro-inflammatory sebaceous lipids. CRH upregulates inflammatory signalling via the SCD/FADS2 pathway, and MALP-2 selectively enhances CRH levels in human sebocytes.

Contribution of GATA6 to homeostasis of the human upper pilosebaceous unit and acne pathogenesis.

Although acne is the most common human inflammatory skin disease, its pathogenic mechanisms remain incompletely understood. Here we show that GATA6, which is expressed in the upper pilosebaceous unit of normal human skin, is down-regulated in acne. GATA6 controls keratinocyte proliferation and differentiation to prevent hyperkeratinisation of the infundibulum, which is the primary pathological event in acne. When overexpressed in immortalised human sebocytes, GATA6 triggers a junctional zone and sebaceous differentiation program whilst limiting lipid production and cell proliferation. It modulates the immunological repertoire of sebocytes, notably by upregulating PD-L1 and IL10. GATA6 expression contributes to the therapeutic effect of retinoic acid, the main treatment for acne. In a human sebaceous organoid model GATA6-mediated down-regulation of the infundibular differentiation program is mediated by induction of TGFß signalling. We conclude that GATA6 is involved in regulation of the upper pilosebaceous unit and may be an actionable target in the treatment of acne.

Sebaceous gland: Milestones of 30-year modelling research dedicated to the "brain of the skin".

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

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.

Autophagy regulates lipid production and contributes to the sebosuppressive effect of retinoic acid in human SZ95 sebocytes.

BACKGROUND: Autophagy is a catabolic process for eliminating damaged organelles or proteins to maintain cellular homeostasis. Recently, lipids have been demonstrated to be targets for autophagosomal degradation. Therefore, autophagy might be involved in sebaceous gland homeostasis, however, relevant data are lacking. OBJECTIVES: We investigated the role of autophagy in sebaceous lipogenesis and its regulatory mechanisms in human SZ95 sebocytes. We also examined the possible role of autophagy in 13-cis-retinoic acid (13-cis-RA)-mediated sebosuppression. METHODS: Autophagy markers expression was examined by immunohistochemistry in normal and acne lesional skin. SZ95 sebocytes were treated with autophagy inhibitors under starvation or treated with a combination of testosterone and linoleic acid (testosterone/LA), with or without autophagy inducer rapamycin or 13-cis-RA. Lipids were assessed by BODIPY and quantitative Nile Red staining. Autophagy-related gene 7 small interference RNA was used to confirm the role of autophagy on the sebosuppressive effect of rapamycin or 13-cis-RA. RESULTS: Autophagy markers were strongly expressed in the maturing sebaceous gland cells in healthy skin, whereas downregulated in the acne-involved sebaceous glands. Testosterone/LA or insulin-like growth factor-1 inhibited starvation-induced sebocyte autophagy. Pharmacological inhibition of autophagy led to increased sebaceous lipid accumulation. Contrary, rapamycin inhibited the testosterone/LA-induced lipogenesis and expression of fatty acid synthesis genes via activating the autophagy pathway. 13-cis-RA increased autophagy in SZ95 sebocytes, partly via FoxO1 activation, and inhibition of autophagy abolished the sebosuppressive effect of 13-cis-RA. CONCLUSIONS: Autophagy plays an important role in the modulation of lipogenesis in human sebocytes and is involved in the sebostatic effect of 13-cis-RA.

GPR119 Is a Potent Regulator of Human Sebocyte Biology.

We have shown previously that endocannabinoids promote sebaceous lipogenesis, and sebocytes are involved in the metabolism of the endocannabinoid-like substance oleoylethanolamide (OEA). OEA is an endogenous activator of GPR119, a recently deorphanized receptor, which currently is being investigated as a promising antidiabetic drug target. In this study, we investigated the effects of OEA as well as the expression and role of GPR119 in human sebocytes. We found that OEA promoted differentiation of human SZ95 sebocytes (elevated lipogenesis, enhanced granulation, and the induction of early apoptotic events), and it switched the cells to a proinflammatory phenotype (increased expression and release of several proinflammatory cytokines). Moreover, we could also demonstrate that GPR119 was expressed in human sebocytes, and its small interfering RNA-mediated gene silencing suppressed OEA-induced sebaceous lipogenesis, which was mediated via c-Jun N-terminal kinase, extracellular signal-regulated kinase 1/2, protein kinase B, and CRE-binding protein activation. Finally, our pilot data demonstrated that GPR119 was downregulated in the sebaceous glands of patients with acne, arguing that GPR119 signaling may indeed be disturbed in acne. Collectively, our findings introduce the OEA/GPR119 signaling as a positive regulator of sebocyte differentiation and highlight the possibility that dysregulation of this pathway may contribute to the development of seborrhea and acne.

Neuroendocrinology and neurobiology of sebaceous glands.

The nervous system communicates with peripheral tissues through nerve fibres and the systemic release of hypothalamic and pituitary neurohormones. Communication between the nervous system and the largest human organ, skin, has traditionally received little attention. In particular, the neuro-regulation of sebaceous glands (SGs), a major skin appendage, is rarely considered. Yet, it is clear that the SG is under stringent pituitary control, and forms a fascinating, clinically relevant peripheral target organ in which to study the neuroendocrine and neural regulation of epithelia. Sebum, the major secretory product of the SG, is composed of a complex mixture of lipids resulting from the holocrine secretion of specialised epithelial cells (sebocytes). It is indicative of a role of the neuroendocrine system in SG function that excess circulating levels of growth hormone, thyroxine or prolactin result in increased sebum production (seborrhoea). Conversely, growth hormone deficiency, hypothyroidism, and adrenal insufficiency result in reduced sebum production and dry skin. Furthermore, the androgen sensitivity of SGs appears to be under neuroendocrine control, as hypophysectomy (removal of the pituitary) renders SGs largely insensitive to stimulation by testosterone, which is crucial for maintaining SG homeostasis. However, several neurohormones, such as adrenocorticotropic hormone and α-melanocyte-stimulating hormone, can stimulate sebum production independently of either the testes or the adrenal glands, further underscoring the importance of neuroendocrine control in SG biology. Moreover, sebocytes synthesise several neurohormones and express their receptors, suggestive of the presence of neuro-autocrine mechanisms of sebocyte modulation. Aside from the neuroendocrine system, it is conceivable that secretion of neuropeptides and neurotransmitters from cutaneous nerve endings may also act on sebocytes or their progenitors, given that the skin is richly innervated. However, to date, the neural controls of SG development and function remain poorly investigated and incompletely understood. Botulinum toxin-mediated or facial paresis-associated reduction of human sebum secretion suggests that cutaneous nerve-derived substances modulate lipid and inflammatory cytokine synthesis by sebocytes, possibly implicating the nervous system in acne pathogenesis. Additionally, evidence suggests that cutaneous denervation in mice alters the expression of key regulators of SG homeostasis. In this review, we examine the current evidence regarding neuroendocrine and neurobiological regulation of human SG function in physiology and pathology. We further call attention to this line of research as an instructive model for probing and therapeutically manipulating the mechanistic links between the nervous system and mammalian skin.

Holocrine Secretion Occurs outside the Tight Junction Barrier in Multicellular Glands: Lessons from Claudin-1-Deficient Mice.

Holocrine secretion is a specific mode of secretion involving secretion of entire cytoplasmic materials with remnants of dead cells, as observed in multicellular exocrine glands of reptiles, birds, and mammals. Here, we found that sebaceous glands in mice, representative of multicellular exocrine glands of mammals, exhibit a form of polarized stratified epithelium equipped with tight junctions (TJs), and found that holocrine secretion occurred outside the TJ barriers. Sebaceous glands share characteristics of stratified epithelia with interfollicular epidermis, including basal-layer-restricted cell proliferation, TJ barrier formation at a specific single layer of cells with apico-basolateral plasma membrane polarity, and cell death outside the TJ barrier. Knockout of claudin-1, a transmembrane adhesive protein in TJs, in mice caused leakage of the TJ barrier in sebaceous glands and incomplete degradation of the plasma membrane and nuclei during holocrine secretion. Claudin-1 knockout resulted in the accumulation of incompletely degenerated sebocytes in sebaceous ducts, suggesting that the TJ barrier was necessary for differentiation of holocrine secretion. The redefinition of sebaceous glands as TJ-forming stratified epithelia provides an important framework to understand the molecular mechanism of holocrine secretion.

Niche-Specific Factors Dynamically Regulate Sebaceous Gland Stem Cells in the Skin.

Oil-secreting sebaceous glands (SGs) are critical for proper skin function; however, it remains unclear how different factors act together to modulate SG stem cells. Here, we provide functional evidence that each SG lobe is serviced by its own dedicated stem cell population. Upon ablating Notch signaling in different skin subcompartments, we find that this pathway exerts dual counteracting effects on SGs. Suppressing Notch in SG progenitors traps them in a hybrid state where stem and differentiation features become intermingled. In contrast, ablating Notch outside of the SG stem cell compartment indirectly drives SG expansion. Finally, we report that a K14:K5→K14:K79 keratin shift occurs during SG differentiation. Deleting K79 destabilizes K14 in sebocytes, and attenuates SGs and eyelid meibomian glands, leading to corneal ulceration. Altogether, our findings demonstrate that SGs integrate diverse signals from different niches and suggest that mutations incurred within one stem cell compartment can indirectly influence another.

Differential antigen expression between human eccrine sweat glands and hair follicles/pilosebaceous units.

Eccrine sweat glands and hair follicles are two primary skin appendages that serve different functions. Although the two appendages exhibit unique morphological patterns in adults, it is difficult to distinguish them morphologically in the early stages of development and regeneration. To research and compare the development, differentiation and regeneration between eccrine sweat glands and hair follicles/pilosebaceous units, specific antigen markers must be found first. Human skin samples were fixed, paraffin-embedded, and cut. The expression of K5, K7, K8, K14, K27, K31, K73, AE13, α-smooth muscle actin (α-SMA), epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA), Na+/K+-ATPase α and Na+-K+-2Cl cotransporter 1 (NKCC1) in eccrine sweat glands, hair follicles and sebaceous glands was detected by immunofluorescence staining. The results showed that eccrine sweat glands expressed K5, K7, K8, K14, K31, α-SMA, CEA, EMA, Na+/K+-ATPase α and NKCC1, but did not express K27, K73 or K31. Hair follicles expressed K5, K8, K14, K27, K31, K73, α-SMA and AE13, but did not express K7, CEA, Na+/K+-ATPase α or NKCC1. Sebaceous glands expressed K5, K14, K73, and EMA, but did not express K7, K8, K31, α-SMA, CEA, EMA, Na+/K+-ATPase α or NKCC1. We concluded that K7, CEA, Na+/K+-ATPase and NKCC1 can be used as specific markers for eccrine sweat glands, K27 and AE13 can be used as specific markers for hair follicles, and K73 can be used as a specific marker for pilosebaceous unit. These specific markers may contribute to differentiate between eccrine sweat glands and hair follicle/pilosebaceous units.

Alterations in IL-4, IL-10 and IFN-γ levels synergistically decrease lipid content and protein expression of FAS and mature SREBP-1 in human sebocytes.

When anti-acne alternatives from dietary and plant sources are ingested, systemic alterations of interleukin (IL)-4, IL-10, IL-12 and interferon (IFN)-γ, individually or simultaneously, are induced at a 0.1-10.0-fold (×) range of normal physiological concentrations (1×). However, little is known about the effects of these cytokines on excess sebum, a pathophysiological factor of acne development. In this study, human sebocytes were treated with 0.1-10.0× of IL-4, IL-10, IL-12 and IFN-γ for 3 or 5 days to elucidate the effects on lipid content. Treatment with individual cytokines decreased the lipid content at specific concentrations rather than in a concentration-dependent manner. Specifically, 5.0× of IL-4, 5.0× of IFN-γ (5.0IFN), and 0.5×, 5.0× and 10.0× of IL-10 for 3 days, and 0.5× of IL-4 (0.5IL4) for 5 days decreased lipid content to 87.6-93.0% of the control. Treatment with other concentrations of IL-4, IL-10 and IFN-γ, and 0.1-10.0× of IL-12 did not alter lipid content. Combined treatment with 0.5IL4, 5.0IFN and 0.5× of IL-10 for 3 or 5 days decreased the lipid content more than each individual treatment. However, this effect was more evident after 3 days, in parallel with decreased levels of triglycerides, cholesterol esters and free fatty acids, the major lipid compositions of sebocytes, and decreased protein expression of fatty acid synthase (FAS) and mature sterol response element-binding protein-1 (SREBP-1), the lipogenesis-related factors, without altered cell proliferation. We demonstrated that suppressed IL-4 and IL-10 with enhanced IFN-γ synergistically decreased lipid content and protein expression of FAS and mature SREBP-1 in human sebocytes.

Cortexolone 17α-propionate (Clascoterone) Is a Novel Androgen Receptor Antagonist that Inhibits Production of Lipids and Inflammatory Cytokines from Sebocytes In Vitro

Cortexolone 17α-propionate (clascoterone) is a novel topical androgen antagonist that is being analyzed for its ability to treat acne. The pathogenesis of acne is attributed to multiple factors, including altered sebum production, inflammatory processes, dysregulation of the hormone microenvironment, and the proliferation of the skin commensal bacteria, Propionibacterium acnes (P. acnes). Androgens induce the proliferation and differentiation of sebocytes, (cells that comprise the sebaceous gland), help regulate the synthesis of the lipids that are incorporated into sebum and stimulate the production of cytokines that are found in inflammatory acne lesions. Several studies have established that clascoterone is a potent antiandrogen that is well tolerated and has selective topical activity. Its potency as an acne therapeutic is currently being analyzed in a large phase 3 clinical trial. The study described herein elucidates for the first time the mechanism of action of clascoterone. Clascoterone was found to bind the androgen receptor (AR) with high affinity in vitro, inhibit AR-regulated transcription in a reporter cell line, and antagonize androgen-regulated lipid and inflammatory cytokine production in a dose-dependent manner in human primary sebocytes. In particular, when compared to another AR antagonist, spironolactone, clascoterone was significantly better at inhibiting inflammatory cytokine synthesis from sebocytes. Therefore, clascoterone may be an excellent candidate to be the first topical antiandrogen to treat acne. J Drugs Dermatol. 2019;18(5):412-418.

Regeneration of a bioengineered 3D integumentary organ system from iPS cells.

Organ systems play essential roles in the physiological functions required for homeostasis. A 3D integumentary organ system (3D-IOS) comprises the skin and skin appendages such as hair follicles and sebaceous glands. This protocol describes how to induce the differentiation of murine induced pluripotent stem (iPS) cells into a 3D-IOS. First, iPS cells are grown for 7 d under conditions that encourage the formation of embryoid bodies (EBs). The iPS cell-derived EBs are stimulated by Wnt10b one day before transplantation of multiple EBs in vivo (a method we describe as the clustering-dependent embryoid body (CDB) transplantation method). After a further 30 d, the transplanted EBs will have differentiated into a 3D-IOS containing mature hair follicles and sebaceous glands. These can be removed and transplanted into wounds in the skin of other mice. After transplantation of a 3D-IOS, the organ system shows full physiological function in vivo starting 14 d following transplant. Thus, this protocol enables a whole functional organ system to be generated from pluripotent stem cells.

Salicylic acid treats acne vulgaris by suppressing AMPK/SREBP1 pathway in sebocytes.

Acne vulgaris is a prevalent cutaneous disease characterized by a multifactorial pathogenic process including hyperseborrhea, inflammation, over-keratinization of follicular keratinocytes and Propionibacterium acnes (P acnes) overgrowth. Salicylic acid (SA), a beta-hydroxy acid, is frequently used in the treatment of acne. SA has been found to decrease skin lipids and to possess anti-inflammatory properties. However, few studies have elucidated the mechanisms and pathways involved in such treatment of acne. In this study, we initially investigated the anti-acne properties of SA in human SEB-1 sebocytes. Treatment with SA decreased sebocyte lipogenesis by downregulating the adenosine monophosphate-activated protein kinase (AMPK)/sterol response element-binding protein-1 (SREBP-1) pathway and reduced inflammation by suppressing the NF-κB pathway in these cells. Salicylic acid also decreased the cell viability of SEB-1 by increasing apoptosis via the death signal receptor pathway. Subsequently, histopathological analysis of a rabbit ear acne model after application of SA for three weeks confirmed that SA suppressed the levels of cytokines and major pathogenic proteins around acne lesions, which supports the mechanisms suggested by our in vitro experiments. These results initially clarified that therapeutic activities of SA in acne vulgaris treatment could be associated with the regulation of SREBP-1 pathway and NF-κB pathway in human SEB-1 sebocytes.

VEGFR-2 Is in a State of Activation in Hair Follicles, Sebaceous Glands, Eccrine Sweat Glands, and Epidermis from Human Scalp: An In Situ Immunohistochemistry Study of Phosphorylated VEGFR-2.

BACKGROUND Recent research reports that VEGFR-2 is expressed in the whole hair follicle, sebaceous glands, eccrine sweat glands, and epidermis. However, phosphorylated VEGFR-2 was not found, and it could not be ascertained whether the activated form of VEGFR-2 actually participates in the biological control of epidermal appendages. In this study we aimed to determine whether the VEGFR-2 pathway is directly involved in the daily regulation of epidermal appendages biology. MATERIAL AND METHODS In this study, we investigated the expression of phosphorylation of VEGFR-2 by immunohistochemical analysis in the epidermis and epidermal appendages in normal human scalp skin. RESULTS Immunohistochemical analysis revealed phosphorylation of VEGFR-2 in a whole hair follicle, mainly in the infundibulum basal layer, hair cortex, and medulla in the isthmus, and matrix in the hair bulb. Phosphorylated VEGFR-2 also was found in the sebaceous glands, eccrine sweat glands, and epidermis. CONCLUSIONS Therefore, we suggest that VEGFR-2 activation is involved in routine regulation of human epidermal appendages.

Androgens modulate keratinocyte differentiation indirectly through enhancing growth factor production from dermal fibroblasts.

BACKGROUND: The main pathogenesis of acne vulgaris is increase in sebum production and abnormal keratinization of the hair infundibulum. The androgens are involved in acne pathogenesis by modulating sebaceous glands to enhance sebum production. However, the molecular mechanisms of abnormal keratinization of the hair infundibulum are not fully elucidated. OBJECTIVE: We hypothesized that the androgens affect the dermal fibroblasts, another androgen receptor-positive cells in the skin, resulting in abnormal keratinization through keratinocyte-fibroblast interaction. METHODS: We investigated effects of androgens and estrogens on growth factors expressions by RT-PCR and western blot analysis in human fibroblast (hFB), human keratinocyte (hKC), and fibroblast-keratinocyte co-culture. In vivo, we examined the growth factor expression in acne lesions compared to normal hair follicles by laser-assisted confocal microscope. RESULTS: In vitro, androgens but not estrogens significantly increased amphiregulin (AREG), epiregulin (EREG), fibroblast growth factor (FGF) 10, and insulin-like growth factor binding protein (IGFBP) 5 mRNA and protein expressions in human fibroblasts but not in keratinocytes. In vivo, AREG, EREG, FGF10, and IGFBP5 were more abundant in acne lesion compared to normal facial skin. FGF10 suppressed cytokeratin 1 and cytokeratin 10 expression in hKC, which was along with the decreased ratio of cytokeratin 10 against cytokeratin 14 in acne lesions compared to normal facial skin. Also, DHT suppressed cytokeratin 1 and cytokeratin 10, in fibroblast-keratinocyte co-culture similarly to the effect of FGF10 to hKC. CONCLUSION: These observations suggested that androgens enhance growth factors production from dermal fibroblasts, and growth factors from fibroblasts alter keratinocyte differentiation in acne lesion.