No / The proopiomelanocortin (POMC)-derived peptides, ACTH and alpha-MSH, are the principal mediators of human skin pigmentation via their action at the melanocortin-1 receptor (MC-1R). Recent data have demonstrated the existence of a functionally active beta-endorphin/mu-opiate receptor system in both epidermal and hair follicle melanocytes, whereby beta-endorphin can regulate melanogenesis, dendricity, and proliferation in these cells. However, a role for ACTH and alpha-MSH in the regulation of the human follicular pigmentary unit has not been determined. This study was designed to examine the involvement of ACTH and the alpha-MSH/MC-1R system in human follicular melanocyte biology. To address this question we employed RT-PCR and immunohisto/cytochemistry, and a functional role for these POMC peptides was assessed in follicular melanocyte cultures. Human scalp hair follicle melanocytes synthesized and processed POMC. ACTH and alpha-MSH in association with their processing enzymes and MC-1R are expressed in human follicular melanocytes at the message level in vitro and at the protein level both in situ and in vitro. The expression of the POMC/MC-1R receptor system was confined only to subpopulations of poorly and moderately differentiated melanocytes. In addition, functional studies revealed that ACTH and alpha-MSH are able to promote follicular melanocyte differentiation by up-regulating melanogenesis, dendricity, and proliferation in less differentiated melanocyte subpopulations. Thus, these findings suggest a role for these POMC peptides in regulating human hair follicle melanocyte differentiation.

No / Although ATP-sensitive potassium (K(ATP)) channel openers, e.g., minoxidil and diazoxide, can induce hair growth, their mechanisms require clarification. Improved drugs are needed clinically. but the absence of a good bioassay hampers research. K(ATP) channels from various tissues contain subtypes of the regulatory sulfonylurea receptor, SUR, and pore-forming, K(+) inward rectifier subunits, Kir6.X, giving differing sensitivities to regulators. Therefore, the in vitro effects of established potassium channel openers and inhibitors (tolbutamide and glibenclamide), plus a novel, selective Kir6.2/SUR1 opener, NNC 55-0118, were assessed on deer hair follicle growth in serum-free median without streptomycin. Minoxidil (0.1-100 microM, p<0.001), NNC 55-0118 (1 mM, p<0.01; 0.1, 10, 100 microM, p<0.001), and diazoxide (10 microM, p<0.01) increased growth. Tolbutamide (1 mM) inhibited growth (p<0.001) and abolished the effect of 10 microM minoxidil, diazoxide and NNC 55-0118; glibenclamide (10 microM) had no effect, but prevented stimulation by 10 microM minoxidil. Phenol red stimulated growth (p<0.001), but channel modulator responses remained unaltered. Thus, deer follicles offer a practical, ethically advantageous in vitro bioassay that reflects clinical responses in vivo. The results indicate direct actions of K(ATP) channel modulators within hair follicles via two types of channels, with SUR 1 and SUR 2, probably SUR2B, sulfonylurea receptors.

No / The hair follicle is a cyclic biological system that progresses through stages of growth, regression, and quiescence, which involves dynamic changes in a program of gene regulation. Micro-RNAs (miRNAs) are critically important for the control of gene expression and silencing. Here, we show that global miRNA expression in the skin markedly changes during distinct stages of the hair cycle in mice. Furthermore, we show that expression of miR-31 markedly increases during anagen and decreases during catagen and telogen. Administration of antisense miR-31 inhibitor into mouse skin during the early- and midanagen phases of the hair cycle results in accelerated anagen development, and altered differentiation of hair matrix keratinocytes and hair shaft formation. Microarray, qRT-PCR and Western blot analyses revealed that miR-31 negatively regulates expression of Fgf10, the components of Wnt and BMP signaling pathways Sclerostin and BAMBI, and Dlx3 transcription factor, as well as selected keratin genes, both in vitro and in vivo. Using luciferase reporter assay, we show that Krt16, Krt17, Dlx3, and Fgf10 serve as direct miR-31 targets. Thus, by targeting a number of growth regulatory molecules and cytoskeletal proteins, miR-31 is involved in establishing an optimal balance of gene expression in the hair follicle required for its proper growth and hair fiber formation.

No / P-cadherin serves as a major topobiological cue in mammalian epithelium. In human hair follicles (HFs), it is prominently expressed in the inner hair matrix that harbors the HF pigmentary unit. However, the role of P-cadherin in normal human pigmentation remains unknown. As patients with mutations in the gene that encodes P-cadherin show hypotrichosis and fair hair, we explored the hypothesis that P-cadherin may control HF pigmentation. When P-cadherin was silenced in melanogenically active organ-cultured human scalp HFs, this significantly reduced HF melanogenesis and tyrosinase activity as well as gene and/or protein expression of gp100, stem cell factor, c-Kit, and microphthalmia-associated transcription factor (MITF), both in situ and in isolated human HF melanocytes. Instead, epidermal pigmentation was unaffected by P-cadherin knockdown in organ-cultured human skin. In hair matrix keratinocytes, P-cadherin silencing reduced plasma membrane β-catenin, whereas glycogen synthase kinase 3 beta (GSK3β) and phospho-β-catenin expression were significantly upregulated. This suggests that P-cadherin-GSK3β/Wnt signaling is required for maintaining the expression of MITF to sustain intrafollicular melanogenesis. Thus, P-cadherin-mediated signaling is a melanocyte subtype-specific topobiological regulator of normal human pigmentation, possibly via GSK3β-mediated canonical Wnt signaling.

No / Although the regulation of pigmentation is well characterized, it remains unclear whether cell-autonomous controls regulate the cyclic on–off switching of pigmentation in the hair follicle (HF). As human HFs and epidermal melanocytes express clock genes and proteins, and given that core clock genes (PER1, BMAL1) modulate human HF cycling, we investigated whether peripheral clock activity influences human HF pigmentation. We found that silencing BMAL1 or PER1 in human HFs increased HF melanin content. Furthermore, tyrosinase expression and activity, as well as TYRP1 and TYRP2 mRNA levels, gp100 protein expression, melanocyte dendricity, and the number gp100+ HF melanocytes, were all significantly increased in BMAL1 and/or PER1-silenced HFs. BMAL1 or PER1 silencing also increased epidermal melanin content, gp100 protein expression, and tyrosinase activity in human skin. These effects reflect direct modulation of melanocytes, as BMAL1 and/or PER1 silencing in isolated melanocytes increased tyrosinase activity and TYRP1/2 expression. Mechanistically, BMAL1 knockdown reduces PER1 transcription, and PER1 silencing induces phosphorylation of the master regulator of melanogenesis, microphthalmia-associated transcription factor, thus stimulating human melanogenesis and melanocyte activity in situ and in vitro. Therefore, the molecular clock operates as a cell-autonomous modulator of human pigmentation and may be targeted for future therapeutic strategies.

No / The development of hair follicle organ culture techniques is a significant milestone in cutaneous biology research. The hair follicle, or more accurately the "pilo-sebaceous unit", encapsulates all the important physiologic processes found in the human body; controlled cell growth/death, interactions between cells of different histologic type, cell differentiation and migration, and hormone responsitivity to name a few. Thus, the value of the hair follicle as a model for biological scientific research goes way beyond its scope for cutaneous biology or dermatology alone. Indeed, the recent and dramatic upturn in interest in hair follicle biology has focused principally on the pursuit of two of biology's holy grails; post-embryonic morphogenesis and control of cyclical tissue activity. The hair follicle organ culture model, pioneered by Philpott and colleagues, ushered in an exceptionally accessible way to assess how cells of epithelial (e.g., keratinocytes), mesenchymal (e.g., fibroblasts), and neuroectodermal (e.g., melanocytes) origin interact in a three-dimensional manner. Moreover, this assay system allows us to assess how various natural and pharmacologic agents affect complex tissues for growth modulation. In this article, I focus on the culture of the human hair follicle mini-organ, discussing both the practical issues involved and some possible research applications of this assay.

Yes / Alopecia areata (AA) is an autoimmune hair loss disease with infiltration of proinflammatory cells into hair follicles. Current therapeutic regimens are unsatisfactory mainly because of the potential for side effects and/or limited efficacy. Here we report that cultured, transduced fibroblasts, which express the immunomodulatory molecule indoleamine 2,3-dioxygenase (IDO), can be applied to prevent hair loss in an experimental AA model. A single intraperitoneal (IP) injection of IDO-expressing primary dermal fibroblasts was given to C3H/HeJ mice at the time of AA induction. While 60–70% of mice that received either control fibroblasts or vehicle injections developed extensive AA, none of the IDO-expressing fibroblast-treated mice showed new hair loss up to 20 weeks post injection. IDO cell therapy significantly reduced infiltration of CD4+ and CD8+ T cells into hair follicles and resulted in decreased expression of TNF-α, IFN-γ and IL-17 in the skin. Skin draining lymph nodes of IDO fibroblast-treated mice were significantly smaller, with more CD4+ CD25+ FoxP3+ regulatory T cells and fewer Th17 cells than those of control fibroblast and vehicle-injected mice. These findings indicate that IP injected IDO-expressing dermal fibroblasts can control inflammation and thereby prevent AA hair loss. / Canadian Institutes of Health Researches (Funding Reference Number: 134214 and 136945).

One of the most challenging questions in the study of cancer is the origin and the nature of the cells that initiate cancer. Accumulated studies have provided many molecular origins of cancers but we still do not know what kind of cells in the tissues transform to cancer cells. Therefore, identifying the cellular origin of these cells is critical for the development of better prognosis, diagnosis and treatment of cancer. A stem cell origin of cancer has been postulated over 150 years. Recent cancer stem cell studies have opened a new window on aspects of the cellular origin of cancer. In this communication, we will address two possible cellular origins of cancer in epithelial tumor development using mouse skin cancer model: tissue specific stem cells, and cells from other organs. To demonstrate contribution of the tissue specific stem cells in tumor development, we monitored the contribution of keratin-15 positive hair follicle bulge stem cells to skin tumor development in the multistage skin carcinogenesis model with Krtl- 15CrePRl;R26R transgenic mice. We found that labeled progeny of the keratin-15 positive bulge stem cells migrate into papillomas and these cells contribute to almost all papilloma samples by 20 weeks of promotion. Additionally, in contrast to the transient contribution of bulge-derived cells in skin wound healing, consistent percentage of the bulge-derived cells stay in the papillomas over 20 weeks. Furthermore, papillomas have heterogeneous expression of the codon 61 signature Ha-ras mutation, with approximately 30 percent of bulge-derived regions expressing the mutation. To determine the contribution of exogenous sources in skin tumor development, we examined bone marrow-derived cells (BMDCs) in the skin tumors from the allogeneic gender-mismatched bone marrow transplantation recipient mice after chemical skin carcinogenesis. We observed that genetically marked (EGFP) BMDCs were detected in the epithelial part of skin wounds and also skin tumors, and we found greater degree of BMDC contribution in chronic ulcer-related skin lesions. Lastly, an in-vitro assay demonstrated plasticity of BMDCs by inducing keratin-14 expressing cells from mesenchymal stem cells. These results demonstrated that hair follicle bulge stem cells and also BMDCs are able to contribute to skin tumor development.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O objetivo deste estudo foi investigar os nÃveis de RNA mensageiro (RNAm) para ativina-A em folÃculos prÃ-antrais e antrais caprinos e os efeitos do hormÃnio folÃculo estimulante (FSH), ativina-A e fator de crescimento e diferenciaÃÃo - 9 (GDF-9) sobre o crescimento e a expressÃo de RNAm para ativina-A, GDF-9, proteÃna morfogenÃtica Ãssea (BMP) -2, -4, -6, -7, -15 e receptor de FSH (R-FSH) em folÃculos prÃ-antrais caprinos cultivados in vitro. Inicialmente, folÃculos primordiais, primÃrios e secundÃrios, bem como complexos cumulus-oÃcito (COCs) e cÃlulas da granulosa mural/teca de pequenos e grandes folÃculos antrais foram isoladas mecanicamente a partir de ovÃrios de cabra e a expressÃo de RNAm para ativina-A foi avaliada por PCR em tempo real. Para os estudos in vitro, folÃculos secundÃrios caprinos foram isolados e cultivados por 6 dias na presenÃa de FSH sozinho (50 ng/mL) ou em combinaÃÃo com ativina-A (100 ng/mL) ou GDF-9 (200 ng/mL). Isoladamente ou em combinaÃÃo com FSH, a influÃncia de ativina-A na expressÃo de ativina-A e R-FSH, e o efeito do GDF-9 sobre os nÃveis de RNAm para GDF-9, R-FSH e BMP -2, -4 , -6, -7 e -15 em folÃculos secundÃrios apÃs 6 dias de cultivo foram testados. Para isso, apÃs a extraÃÃo do RNA total e sÃntese do cDNA, os nÃveis de RNAm para ativina-A, GDF-9, R-FSH e BMP -2, -4, -6, -7 e -15 foram quantificados por PCR em tempo real. Os resultados mostraram que folÃculos secundÃrios apresentavam nÃveis menores de RNAm para ativina-A comparado aos folÃculos primÃrios (p<0,05). NÃo houve diferenÃa nos nÃveis de RNAm para ativina-A entre folÃculos primordial e primÃrio ou secundÃrio (p>0,05). Aliado a isso, nÃo houve diferenÃa entre COCs de pequenos e grandes folÃculos antrais (p>0,05). AlÃm disso, as cÃlulas da granulosa e da teca de grandes folÃculos antrais apresentaram maiores nÃveis de RNAm para ativina-A do que de pequenos folÃculos antrais (p<0,05). Quando comparamos a expressÃo do RNAm para ativina-A entre COCs de pequenos e grandes folÃculos antrais e suas cÃlulas da granulosa e da teca, nenhuma diferenÃa nos nÃveis de expressÃo foi observada (p>0,05). ApÃs o cultivo in vitro de folÃculos secundÃrios, a presenÃa de FSH sozinho ou associado com ativina-A ou GDF-9 aumentou a sobrevivÃncia, crescimento folicular e formaÃÃo de antro (p<0,05). O FSH tambÃm aumentou o RNAm para ativina-A, enquanto os folÃculos cultivados com ativina-A apresentaram nÃveis aumentados de RNAm para R-FSH (p<0,05). AlÃm disso, o GDF-9 diminuiu a expressÃo de RNAm para BMP -2 e -15 (p<0,05), mas nÃo teve efeito sobre a expressÃo de BMP -4, -6 e -7 (p>0,05). Em conclusÃo, durante a transiÃÃo de folÃculo primÃrio para secundÃrio hà uma diminuiÃÃo do RNAm para ativina-A, enquanto ocorre um aumento da expressÃo deste fator durante o crescimento de folÃculos antrais. FSH, ativina-A e GDF-9 estimulam o crescimento de folÃculos secundÃrios caprinos apÃs 6 dias de cultivo. ApÃs cultivo folicular, FSH controla a expressÃo de ativina-A e a expressÃo do R-FSH à regulada por ativina-A. Ainda, GDF-9 reduz a expressÃo do RNAm para BMP -2 e -15. / The aim of this study was to investigate the levels of messenger RNA (mRNA) for activin-A on goat preantral and antral follicles and the effects of follicle stimulating hormone (FSH), activin-A and growth and differentiation factor - 9 (GDF-9) on growth and mRNA expression for activin-A, GDF-9, bone morphogenetic protein (BMP) -2, -4, -6, -7, -15 and FSH receptor (FSH-R) in goat preantral follicles cultured in vitro. Initially, primordial, primary and secondary follicles, as well as cumulus-oocyte complexes (COCs) and mural granulosa/theca cells of small and large antral follicles were isolated mechanically from goat ovaries and the expression of mRNA for activin-A was evaluated by real-time PCR. For in vitro studies, goat secondary follicles were isolated and cultured for 6 days in the presence of FSH alone (50 ng/mL) or in combination with activin-A (100 ng/mL) or GDF-9 (200 ng/mL). Alone or in combination with FSH, the influence of activin-A on expression of activin-A and FSH-R, and the effect of GDF-9 on the levels of mRNA for GDF-9, FSH-R and BMP -2, -4, -6, -7 and -15 in secondary follicles after 6 days of culture were tested. For this, after extraction of total RNA and cDNA synthesis, the levels of mRNA for activin-A, GDF-9, FSH-R and BMP -2, -4, -6, -7 and -15 were quantified by real time PCR. The results showed that secondary follicles had lower levels of mRNA for activin-A, than compared to primary follicles (p<0.05). There was no difference in levels of mRNA for activin-A between primordial and primary or secondary (p>0.05). Allied to this, there was no difference between COCs of small and large antral follicles (p>0.05). Moreover, granulosa and theca cells of large antral follicles showed higher levels of mRNA for activin-A than in small antral follicles (p<0.05). When comparing mRNA expression for activin-A between COCs from small and large antral follicles and their granulosa and theca cells, no difference in expression levels was observed (p>0.05). After in vitro culture of secondary follicles, the presence of FSH either alone or associated with activin-A or GDF-9 increased survival, follicular growth and antrum formation (p<0.05). The FSH increased mRNA for activin-A, while the follicles cultured with activin-A showed increased levels of mRNA for FSH-R (p<0.05). In addition, GDF-9 decreased mRNA expression for BMP -2 and -15 (p<0.05), but had no effect on expression of BMP -4, -6, and -7 (p>0.05). In conclusion, during the transition from primary to secondary follicles there is a reduction of mRNA for activin-A, whereas there is an increased expression of this factor during the growth of antral follicles. FSH, activin-A and GDF-9 stimulate growth of goat secondary follicles after 6-day culture. After follicle culture FSH controls the expression of activin-A, and the expression of FSH-R is regulated by activin-A. Furthermore, GDF-9 reduces the mRNA expression for BMP-2 and -15.

The wave-like developmental pattern of follicles ≥1 mm in temporal relationship with follicle stimulating hormone (FSH) and the existence of 2- and 3-waves of follicular development during an interovulatory interval (IOI) have been clearly defined in cattle. However, information about the developmental pattern of antral follicles <1 mm and the repeatability of the wave pattern (2- or 3-wave IOI) is lacking. Using approaches such as immunization against GnRH (to suppress circulating concentrations of FSH) and histomorphometric study of ovarian tissues collected from cyclic heifers on different days after ovulation, the developmental pattern of antral follicles <1 mm and the role of FSH in their development were studied in heifers. Ultrasonographically acquired follicular data were used to determine the repeatability of 2- and 3-wave patterns and the effect of season on the wave patterns. The ovulatory follicle in 3-wave IOI is exposed to a shorter term high-progesterone environment than that of 2-wave IOI, and it has been argued that the less-aged ovulatory follicle of 3-wave IOI yields a more fertile oocyte than the 2-wave IOI. The developmental competence of oocytes in preovulatory follicles of 2- versus 3-wave IOI was compared using in vivo environments created to mimic short-term low- and high-progesterone environments similar to 2- and 3-wave IOI, respectively. The developmental competence of oocytes in persistent dominant-type follicles was also determined.<p>The vaccination against GnRH attenuated FSH surges but did not suppress the basal circulating concentrations of FSH. The attenuation of FSH surges suppressed the wave-like emergence of follicles ≥4 mm but not of the antral follicles <4 mm. The study revealed an inverse relationship between the mean and peak circulating concentrations of FSH and the number of follicles recruited into ≥1 mm size category. Histomorphometric study revealed that antral follicles <1 mm developed in a wave-like fashion in response to a rise in the circulating concentrations of FSH. After treatment with exogenous FSH, the growth rate of follicles in GnRH-immunized heifers was similar to controls. <p>The duration of IOI was predictive of the wave pattern (i.e., 2- or 3-wave IOI), and the pattern was repeatable within individuals throughout the year. The dominant follicle of Wave 1 in 2-wave IOI had a longer duration of dominance than in 3-wave IOI. Hence, the dominant follicle of Wave 1 may have a primary role in the regulation of 2- and 3-wave patterns. Greater attrition of follicles in 3-wave IOI, due to the emergence of an extra wave compared to 2-wave IOI, may contribute to earlier follicular depletion and onset of reproductive senescence in heifers with primarily a 3-wave pattern. The fertilization capacity of oocytes that were exposed to the short-term low-progesterone environment (i.e., similar to the early growing phase of the ovulatory follicle of 3-wave IOI) was increased, but the developmental competence post-fertilization was not different from oocytes that were exposed to a short-term high-progesterone environment (i.e., similar to the early growing phase of preovulatory follicle of 2-wave IOI). Multiple follicles developed under the prolonged-low progesterone environment, but failed to ovulate.