Wen-Hwa Li

Melanocortin-5 receptor: A marker of human sebocyte differentiation

Melanocortin receptors (MC1R-MC5R) and their ligands (melanocyte-stimulating hormone (MSH) and adrenocorticotrophin hormone (ACTH)) have been shown to influence physiological functions of cells and organs, including exocrine glands. Since relatively little is known about MC5R expression and function in the human sebaceous gland, we examined expression of MC5R by immunohistochemistry and RT-PCR in human sebaceous cells in vivo and in vitro. In human skin, MC5R was detected only in differentiating, lipid-laden sebaceous cells but not in basal, undifferentiated sebaceous cells. Similarly, in cultured human sebocytes MC5R was only detectable at the onset of differentiation and in fully differentiated cells displaying prominent lipid granules. The lipid profile of the cultured and differentiated human sebocytes was shown to be human sebum-specific using (14)C-acetate labeling and high performance thin layer chromatography. Our studies suggest that MC5R is a marker of human sebocyte differentiation.

Proopiomelanocortin Peptides and Sebogenesis

Abstract: Previous animal studies have demonstrated that α‐melanocyte‐stimulating hormone (α‐MSH) is a sebotropic hormone in rats and that targeted disruption of melanocortin 5 receptor (MC5‐R) can down‐regulate sebum output in mice. To study the role of proopiomelanocortin (POMC) peptides in the regulation of human sebaceous lipid production and sebocyte differentiation, we established a primary human sebocyte culture system. Sebocytes were derived from normal human facial skin. Differentiation of sebocytes, induced by POMC‐derived peptides such as MSH, adrenocorticotropic hormone (ACTH), or bovine pituitary extract (BPE), resulted in the appearance of prominent cytoplasmic lipid droplets. Partial induction of sebocyte differentiation also was observed in serum‐depleted cultures, but there was very limited spontaneous differentiation in serum‐containing medium. Analysis by high‐performance thin‐layer chromatography (HPTLC) of 14C‐acetate‐labeled lipids showed a dose‐dependent increase in synthesis of sebaceous‐specific lipid (i.e., squalene) induced by NDP α‐MSH. Molecular studies using RT‐PCR showed a low level of human MC5‐R expression under serum‐free condition but a substantial increase after treatment with NDP α‐MSH or BPE. In contrast, MC1‐R expression remained the same, independent of treatment. Our data indicate that expression of MC5‐R correlates with sebocyte differentiation and suggest a regulatory role for MC5‐R in human sebaceous lipid production.

The role of keratinocyte growth factor in melanogenesis: a possible mechanism for the initiation of solar lentigines

Please cite this paper as: The role of keratinocyte growth factor in melanogenesis: a possible mechanism for the initiation of solar lentigines. Experimental Dermatology 2010; 19: 865–872.

Melanocortin-5 receptor and sebogenesis

The melanocortins (α-MSH, β-MSH, γ-MSH, and ACTH) bind to the melanocortin receptors and signal through increases in cyclic adenosine monophosphate to induce biological effects. The melanocortin MC(5) and MC(1) receptors are expressed in human sebaceous glands, which produce sebum, a lipid mixture of squalene, wax esters, triglycerides, cholesterol esters, and free fatty acids that is secreted onto the skin. Excessive sebum production is one of the major factors in the pathogenesis of acne. The expression of melanocortin MC(5) receptor has been associated with sebocyte differentiation and sebum production. Sebaceous lipids are down-regulated in melanocortin MC(5) receptor-deficient mice, consistent with the observation that α-MSH acts as a sebotropic hormone in rodents. These findings, which suggest that melanocortins stimulate sebaceous lipid production through the MC(5) receptor, led to our search for MC(5) receptor antagonists as potential sebum-suppressive agents. As predicted, an antagonist was shown to inhibit sebocyte differentiation in vitro, and to reduce sebum production in human skin transplanted onto immunodeficient mice. The melanocortin MC(5) receptor antagonists may prove to be clinically useful for the treatment of sebaceous disorders with excessive sebum production, such as acne.

A melanocortin receptor 1 and 5 antagonist inhibits sebaceous gland differentiation and the production of sebum-specific lipids

BACKGROUND The melanocortin receptor-5 (MC5R) is present in human sebaceous glands, where it is expressed in differentiated sebocytes only. The targeted disruption of MC5R in mice resulted in reduced sebaceous lipid production and a severe defect in water repulsion. OBJECTIVE To investigate the physiological function of MC5R in human sebaceous glands. METHODS A novel MC1R and MC5R antagonist (JNJ-10229570) was used to treat primary human sebaceous cells or human skins grafted onto severe combined immunodeficient (SCID) mice. Transcription profiling, lipid analyses, and histological and immunohistochemical staining were used to analyze the effect of MC5R inhibition on sebaceous gland differentiation and sebum production. RESULTS JNJ-10229570 dose dependently inhibited the production of sebaceous lipids in cultured primary human sebocytes. Topical treatment with JNJ-10229570 of human skins transplanted onto SCID mice resulted in a marked decrease in sebum-specific lipid production, sebaceous glands size and the expression of the sebaceous differentiation marker epithelial-membrane antigen (EMA). Treatment with flutamide, a known inhibitor of sebum production, gave similar results, validating the human skin/SCID mouse experimental system for sebaceous secretion studies. CONCLUSION Our data suggest that antagonists of MC1R and MC5R could be effective sebum suppressive agents and might have a potential for the treatment of acne and other sebaceous gland pathologies.

p38 MAP Kinase Inhibition Reduces Propionibacterium acnes-Induced Inflammation in Vitro

IntroductionPropionibacterium acnes, a ubiquitous skin bacterium, stimulates keratinocytes to produce a number of proinflammatory cytokines and may contribute to inflammatory acne. The aim of the study was to investigate whether P. acnes-induced proinflammatory cytokine release is mediated by P. acnes-induced activation of p38 mitogen-activated protein kinase (p38 MAPK or p38) in human keratinocytes.MethodsImmunohistochemistry was used to evaluate p38 phosphorylation in human skin samples with or without acne. Primary human keratinocytes and epidermal skin equivalents were exposed to viable P. acnes. Phosphorylation of MAPKs without or with p38 inhibitors was examined by Western blot and cytokine secretion was detected by Enzyme-Linked Immunosorbent Assay (ELISA).ResultsIncreased levels of phospho-p38 were observed in human acne lesions, predominantly in follicular and perifollicular keratinocytes. Exposure of cultured human keratinocytes to viable P. acnes resulted in phosphorylation of multiple members of the MAPK family, including rapid and transient activation of p38 and extracellular signal-related kinase (ERK1/2) and relatively slow but sustained activation of c-Jun N-terminal kinases (JNK1/2). Viable P. acnes induced the secretion of interleukin-1α (IL-1α), tumor necrosis factor-α (TNF-α), and IL-8 from human keratinocytes. The phosphorylation of p38 (phospho-p38) and the secretion of cytokines induced by P. acnes in cultured keratinocytes were inhibited by SB203580, a p38α/β inhibitor. Furthermore, SCIO-469, a selective inhibitor of p38α, showed similar effects in cultured keratinocytes. Topical treatment of SCIO-469 inhibited the P. acnes-induced phospho-p38 and cytokine secretion in human epidermal equivalents.ConclusionThe data demonstrate that P. acnes induces p38-dependent inflammatory responses in keratinocytes, and suggest that p38 may play an important role in the pathogenesis of inflammatory acne.FundingJohnson & Johnson.

Sebaceous Gland Regeneration in Human Skin Xenografts

TO THE EDITOR Human sebaceous glands are microscopic, multi-lobular glandular structures of the pilosebaceous units. Their holocrine secretion product, sebum, contains a unique mixture of lipids, including triglycerides, wax esters, squalene, cholesterol esters, and free fatty acids (reviewed in Zouboulis et al., 2003). Sebaceous glands have been shown to contribute to many skin functions (reviewed in Zouboulis, 2004; Zouboulis et al., 2008), but also are involved in the pathology of acne and seborrhea. Sebum is speciesspecific and acne is an exclusively human disease. Using primary and immortalized human sebaceous cells and mouse models, many pathways were shown to regulate sebaceous cell activities, including signaling molecules, transcription factors and growth factors (reviewed in Smith and Thiboutot, 2008; Zouboulis et al., 2008). To complement and extend the current data and broaden studies of human sebogenesis, we examined the use of the human skin/severe combined immunodeficient mouse model, originally developed by Juhasz et al. (1993); see Supplementary Material online for methods. In the course of our experiments we observed that split thickness human skin grafts (400 mm), originally containing none or very few sebaceous glands (Figure 1a and b), were filled with numerous, fully developed glands, secreting human sebum, within 3 months post-transplantation (Figure 1c and i). To assure the validity of this finding, multiple samples were collected at different times post grafting, and were compared with the original split-thickness skins. Interestingly, grafts examined within the first 10 days postgrafting showed no sebaceous glands, or the degeneration of transplanted glands, shown as eosinophilic tissue, void of nuclear material, and as remnants of lipid aggregates within the degenerated tissue (data not shown). However, at week 3 posttransplantation, small islands of sebaceous cells were first documented close to the epidermis (Figure 1d), with a gradual increase in gland size at weeks 5–12 (Figure 1e and f). At 7–8 weeks the sebaceous glands were fully formed. The regenerated glands were shown to express epithelial membrane antigen (Figure 1g), a specific marker of human differentiated sebocytes (Latham et al., 1989). Sebaceous cells loaded with lipid granules were visualized by Oil Red O staining as early as 18 days post-transplantation, and in the mature glands (Figure 1h). The glands produced human sebumspecific lipids, specifically squalene, and wax esters, as shown by highperformance thin-layer chromatography (HPTLC, Figure 1i). An identical high-performance thin-layer chromatography profile was documented from glands isolated before transplantation, and from punch biopsies of the grafts (not shown). We used Ki67 to identify actively proliferating cells. Before transplantation, a subpopulation of Ki67þ (cycling) cells was observed in the epidermis, hair follicles, and sebaceous gland (Figure 2a) of the human skin samples. In contrast, between days 3 and 10 post-grafting the cells in the graft were quiescent, with no detectable Ki67 expression (Figure 2b) possibly caused by insufficient vascularization, which could contribute to the degeneration of the few transplanted glands. By 15 days, almost all cells within the lower epidermis and the outer root sheath showed Ki67 staining (Figure 2c and d) with most seen at 28 days, (Figure 2e), coinciding with sebaceous gland regeneration. At 60 days and thereafter, Ki67 staining was similar to that of normal human skin (Figure 2f). In an attempt to understand the origin of the developing sebaceous glands, we examined expression of human stem cell markers in the transplanted tissues. Keratin 15 (K15), originally identified as a human and mouse hair follicle stem cell marker (Lyle et al., 1999) was found to co-localize with the regenerating sebaceous glands (Figure 2h). K15 cells were shown to generate murine sebaceous glands in vivo (Morris et al., 2004) and human sebaceous cells in vitro (Roh et al., 2008). Before transplantation, K15 was expressed in the basal layer of epidermis, outer root sheath, the basal layer of the mantle, and the apical part of the sebaceous gland, leading into the sebaceous duct (Figure 2g). At 2–15 days post-transplantation, K15 expression was retained in the deep rete ridges of the epidermis and the upper portion of the hair follicle. By day 28, the time of active cell-cycling and formation of sebaceous glands, multilayered aggregates of K15-expressing cells were seen at the apical part of the sebaceous glands, possibly representing sebaceous or ductal precursors (Figure 2h). At the time sebaceous glands were fully developed (3–5 months post-transplantation), K15 expression was similar to that of the tissues before transplantation (not shown). Colocalization of K15-positive cells with the developing sebaceous glands is not sufficient to confirm that these K15positive cells are sebaceous progenitors; more detailed studies are needed to conclusively elucidate the role of multi potential K15-positive cells in human sebaceous gland formation and maintenance. Although organ regeneration is rare in mammals (e.g., liver in man), the full

Low-level red LED light inhibits hyperkeratinization and inflammation induced by unsaturated fatty acid in an in vitro model mimicking acne: RED LIGHT DELIVERS ACNE-RELEVANT BENEFITS IN VITRO

Acne vulgaris is a chronic inflammatory disease of the pilosebaceous units (PSU), associated with increased sebum production, abnormal follicular keratinization (hyperkeratinization), follicular overgrowth of Propionibacterium acnes (P. acnes), and increased inflammatory mediator release. Light therapy has attracted medical interests as a safe alternative treatment for acne. Both blue and red light therapies at high doses >10 J/cm2 have demonstrated marked effects on inflammatory acne lesions. However, few studies have investigated the effects of lower doses of light. The aim of this study is to investigate the biological effects of lower doses of red light at 0.2–1.2 J/cm2 for acne using an in vitro model previously developed to mimic the inflammation and hyperkeratinization observed clinically in acne.

Topical stabilized retinol treatment induces the expression of HAS genes and HA production in human skin in vitro and in vivo

Skin Aging manifests primarily with wrinkles, dyspigmentations, texture changes, and loss of elasticity. During the skin aging process, there is a loss of moisture and elasticity in skin resulting in loss of firmness finally leading to skin sagging. The key molecule involved in skin moisture is hyaluronic acid (HA), which has a significant water-binding capacity. HA levels in skin decline with age resulting in decrease in skin moisture, which may contribute to loss of firmness. Clinical trials have shown that topically applied ROL effectively reduces wrinkles and helps retain youthful appearance. In the current study, ROL was shown to induce HA production and stimulates the gene expression of all three forms of hyaluronic acid synthases (HAS) in normal human epidermal keratinocytes monolayer cultures. Moreover, in human skin equivalent tissues and in human skin explants, topical treatment of tissues with a stabilized-ROL formulation significantly induced the gene expression of HAS mRNA concomitant with an increased HA production. Finally, in a vehicle-controlled human clinical study, histochemical analysis confirmed increased HA accumulation in the epidermis in ROL-treated human skin as compared to vehicle. These results show that ROL increases skin expression of HA, a significant contributing factor responsible for wrinkle formation and skin moisture, which decrease during aging. Taken together with the activity to increase collagen, elastin, and cell proliferation, these studies establish that retinol provides multi-functional activity for photodamaged skin.