- Email: [email protected]
193 Unveiling the role of estradiol in the pathogenesis of female pattern hair loss
ABSTRACTS | Hair and Other Adnexal Structures 192
193
194
195
MicroRNAs and their regulatory interactions in the human hair follicle LM Hochfeld, T Anhalt, A Hofmann, N Fricker, MM No¨then and S Heilmann-Heimbach Institute of Human Genetics, Dept. Genomics, University of Bonn, Bonn, Germany Human hair follicle (HF) cycling involves numerous signaling cascades and cellular processes that have been reported to be controlled by micro(mi)RNAs. However, our current knowledge on the expression and function of these miRNAs in hair biology is largely based on murine data or cell culture experiments and little is known about their exact role in the human HF. We therefore sought to gain a deeper understanding of the role of 9 previously reported miRNAs in human hair biology by systematically investigating their expression and regulatory interactions in HF samples from 25 healthy male donors. After nucleic acid extraction, we performed a genome-wide miRNA and mRNA expression profiling and subsequently correlated miRNA expression levels with mRNA-expression levels from the same samples to identify potential target genes. Moreover, we tested for an enrichment of target genes in biological pathways and searched for protein-protein interactions (PPIs). Our analysis confirmed the expression of 7 of the 9 candidate miRNAs. For 3 of them (miR-24, miR-31, miR-106a), significant miRNA/mRNA correlations were detected. The identified target genes included genes with known functions in hair biology (e.g. ITGB1, SOX9), genes whose described function matched the described function of their corresponding miRNA (e.g. RXRA) and numerous genes that had not previously been implicated in hair biology. The pathwayand PPI-based analysis revealed an enrichment of target genes in relevant pathways such as integrin and GnRH signaling and confirmed the interaction of target gene products in PPI-networks. In summary, our data provide novel insights into the role of miRNAs and their regulatory interactions in the human HF and will eventually contribute to a deeper understanding of healthy hair growth and the pathobiology of hair loss disorders.
Unveiling the role of estradiol in the pathogenesis of female pattern hair loss M Takahashi1, Y Endo1, Y Obayashi1, T Serizawa1, M Murakoshi1, R Ueki2 and M Ohyama3 1 Life Science Reserch Laboratories, Lion Corporation, Odawara, Japan, 2 Department of Dermatology, Juntendo University School of Medicine, Koto-ku, Japan and 3 Department of Dermatology, Kyorin University School of Medicine, Mitaka, Japan Female pattern hair loss (FPHL) shares major features with male androgenetic alopecia (AGA). Despite that major progress has been made in the investigation and management of AGA, our understanding of FPHL has been limited. As the incidence of FPHL is relatively high in postmenopausal females, we attempted to dissect the mechanism responsible for increased hair shedding in FPHL in the context of estradiol (E2) decrease. Thirty 25 to 58-year-old healthy women were evaluated for serum E2 levels, the frequency and the anchoring strength of telogen hairs. Interestingly, the decrease in E2 levels correlated with the increase in the proportion of telogen hairs. The extraction force required to pluck telogen hairs was significantly weaker in those with low E2 levels, suggesting that the reduction in E2 not only altered hair cycles but also affected hair shaft anchoring with resultant increase in hair shedding in low E2 individuals. PCR array analysis of human outer root sheath cells cultured with or without E2 detected up-regulation of adherence junction genes including desmocollin 3 and E-cadherin and down-regulation of metallopeptidase 10 and 15, implying that E2 may enhance hair follicle epithelia cell adhesion to suppress hair follicle shedding in homeostasis. Forced hair pluck experiments using C57BL/6 mice demonstrated marked decrease in hair anchoring strength in later telogen phase accompanied with altered expression of the genes detected in the experiment using human outer root sheath cells. Importantly, ovariectomized mice without E2 showed weaker telogen hair anchoring when compared to control mice. These findings suggested a previously unrecognized role of E2 in the pathophysiology of FPHL and may provide strategies to better manage postmenopausal FPHL by supplementing E2.
An underlying mechanism of hair loss in acrodermatitis enteropathica Y Ogawa, T Kawamura and S Shimada Dermatology, University of Yamanashi, Yamanashi, Japan The triad of acrodermatitis enteropathica (AE) is dermatitis, diarrhea and hair loss. We previously elucidated an underlying mechanism of characteristic dermatitis in AE using skin samples of AE patients and dietary Zinc (Zn)-deficient mice. We herein assessed underlying mechanisms of hair loss in AE using dietary Zn-adequate (ZA) and -deficient (ZD) mice. Fiveweek-old female Balb/c mice were continued ZA diet or switched to ZD diet for ZA or ZD mice, respectively. Although ZD mice did not exhibit obvious hair loss, the hair coat of ZD mice was sparse when compared to that of ZA mice. Hair morphology of ZD mice was stuck in telogen stage even after one week and thereafter of initiation of ZD diet, implying disruption of hair cycle. Moreover, depilation of telogen hair of ZA mice induced anagen hair, but not of ZD mice, in which hair did not regrow after depilation. Strikingly, loss of hair cycle and depilation-induced anagen initiation in ZD mice was completely restored by supplementation of Zn to ZD mice, suggesting that functions of some molecules related to hair cycle and regeneration was reversibly impaired by Zn deficiency. Immunofluorescence study revealed that lack of positivity of tissue-nonspecific alkaline phosphatase (TNAP) in dermal papilla (DP) and of P-cadherin in hair germ cells in ZD mice. TNAP, a zinc dependent enzyme, has been reported to be critical for hair regeneration by DP cells. Thus, the lack of TNAP activity in DP cells due to Zn deficiency appeared to be the major cause of loss of hair cycle and depilation-induced anagen initiation in ZD mice. Additionally, qPCR with dermis from ZA and ZD mice revealed downregulation of mRNA expression of pdgfa, pdgfb, fgf7 and fgf10. These data suggest that Zn deficiency decreases the production of PDGF and FGF in dermal cells including intra-dermal adipocytes, followed by downregulation of TNAP activity in DP cells, thereby resulting in loss of hair cycle and depilation-induced anagen initiation.
Androgen action and 3D culture conditions influence dermal papilla cells inductive properties: the role of Wnt agonists/antagonists balance JM Ceruti, GJ Leiro´s, AG Kusinsky and ME Balan˜a´ Fundacion Cassara´, ICT-MIlstein CONICET, Buenos Aires, Argentina In androgenetic alopecia, circulating androgens act on dermal papilla cells (DPC) and alter paracrine factors that influence hair epithelial cells leading to hair follicle (HF) miniaturization. We showed that androgens deregulate DPC-secreted factors involved in normal HF stem cell (HFSC) differentiation inhibiting the canonical Wnt signalling pathway reported as important for hair morphogenesis and for the maintenance of DPC inductive properties. In this work, we studied the effect of dihydrotestosterone (DHT) and culture conditions on Wnt agonists/antagonists balance in DPC and evaluated the role of the Wnt antagonist DKK-1 in dermal papilla-induced HFSC differentiation. To assess the effect that culture conditions could have on androgen-driven signals, monolayer and spheroids DPC cultures were established. The presence of DHT in both types of DPC cultures downregulated the expression of the agonists Wnt 5a and Wnt 10b, and upregulated the expression of Dkk1. Notably, in basal conditions, a higher expression of Wnt agonists was observed in DPC-spheroids culture system. HFSC differentiation was evaluated in presence of media conditioned by DPC after androgen treatment and in presence of DKK-1. DKK-1 presence in DPC culture abrogated the inductive ability of its conditioned medium mimicking the androgen effect previously reported. Furthermore, DPC-conditioned medium supplemented with Dkk-1 lost this HFSCdifferentiation inductive ability failing in the Wnt-b catenin pathway activation, evidenced by failure of accumulation of b catenin in the cytoplasm of these cells. We conclude that threedimensional-tissue architecture of DPC-spheroids appears to contribute in maintaining Wnt pathway activation, and probably DPC inductive properties. DHT can alter the balance of Wnt agonist/antagonist in DPC, reducing its ability to induce HFSC differentiation to hair lineage. The paracrine action of DKK-1 would be enough to abrogate HFSC differentiation, suggesting that DHT-inducible DKK-1 is involved in DHT-driven balding.
196
197
The endocannabinoid tone regulates human sebocyte biology A Ola´h1, N Za´ka´ny1, A Markovics1, S Nicolussi2, J Gertsch2, F Piscitelli3, V Di Marzo3, A´ Po´r6, C Zouboulis4 and T Bı´ro´5 1 Department of Physiology, University of Debrecen, Debrecen, Hungary, 2 Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland, 3 Consiglio Nazionale delle Ricerche, Pozzuoli, Italy, 4 Dessau Medical Center, Dessau, Germany, 5 Department of Immunology, University of Debrecen, Debrecen, Hungary and 6 Gyula Kene´zy Hospital, Debrecen, Hungary We have previously shown that i) endocannabinoids (eCB; e.g. anandamide [AEA]) are key players in the maintenance of homeostatic sebaceous lipid production (SLP) in sebaceous glands (SG); and ii) eCB treatment of sebocytes dramatically increases their lipid synthesis. Thus, here, we aimed to i) investigate the expression of the major eCB synthesizing and degrading enzymes; and ii) explore the effects of modulation of the eCB tone on human SZ95 sebocytes by using inhibitors of the eCB membrane transporter (EMT), which has been hypothesized to facilitate the transmembrane transport (and thereby degradation) of eCBs. We found that the major eCB synthesizing (NAPE-PLD, DAGLa and -b) and degrading (FAAH, MAGL) enzymes are expressed in SZ95 sebocytes as well as in SGs in situ, and that the EMT is also functionally active in these cells. Without influencing viability, the EMT-inhibitor VDM11 induced a small, but significant increase in SLP. Intriguingly, co-administration of the most efficient pro-lipogenic concentrations of VDM11 and AEA appeared to decrease the pro-lipogenic effect of the latter. This effect might have been due to the increased cytotoxic potential of the combined treatment. Finally, we found that VDM11 was able to prevent the pro-inflammatory action of the Toll-like receptor 4 activator lipopolysaccharide. Our data suggest that the increase of eCB tone exerts remarkable anti-inflammatory actions and slightly elevates SLP; thus its modulation might be beneficial in cutaneous inflammatory conditions accompanied by dry skin.
S194 Journal of Investigative Dermatology (2016), Volume 136
A novel splice site mutation in LIPH identified in a Japanese patient with autosomal recessive woolly hair A Tanaka1, Y Matsuo1, Y Shimomura2 and M Hide1 1 Department of Dermatology, Hiroshima University, Hiroshima, Japan and 2 Laboratory of Genetic Skin Diseases, Niigata University, Niigata, Japan Autosomal recessive woolly hair/hypotrichosis (ARWH) is a rare hereditary hair disease characterized by patients having short and tightly curled scalp hair with hypotrichosis. Mutations in two genes, the lipase gene (LIPH) and lysophosphatidic acid receptor 6 (LPAR6), are reported to cause ARWH. The patient was a 4-year-old Japanese boy. He had had short and tightly curled scalp hair since birth. His hair loss had gradually progressed with aging without any other manifestations. The patient’s parents and sister did not show these manifestations. Based on the clinical features, we diagnosed the patient as having ARWH and conducted genetic studies by using extracted genomic DNA from a peripheral blood sample. We performed direct sequencing analysis of LIPH and identified a heterozygous missense mutation c.736T>A (p.Cys246Ser) in exon 6 of LIPH, which is known to be a prevalent founder mutation in the Japanese population. Additionally, we identified a novel heterozygous mutation (c.417+1G>C) in intron 2 of LIPH. To investigate the expression of the mutant LIPH transcript, total RNA was extracted from the blood samples. The LIPH cDNA were amplified from exon 1 to exon 7 by RT-PCR. The PCR product using LIPH cDNA from the patient showed two fragments. These two mutations were located on different alleles; a normal splicing event occurred from the c.736T>A mutant allele, while an aberrant splicing event was induced from the c.417+1G>C mutant allele. Sequencing analysis also identified a heterozygous 144-bp deletion in exon 2, caused by the c.417+1G>C. The protein is expected to comprise a specific amino acid sequence in which 48 amino acid residues are deleted. Most of the deleted amino acid residues contribute to form an N-terminal a-helix of PA-PLA1a. Before our finding, two splice site mutations in LIPH have been reported. The c.417+1G>C is the third splice site mutation identified in LIPH, thereby expanding the spectrum of LIPH mutations causing ARWH.
193
194
195
MicroRNAs and their regulatory interactions in the human hair follicle LM Hochfeld, T Anhalt, A Hofmann, N Fricker, MM No¨then and S Heilmann-Heimbach Institute of Human Genetics, Dept. Genomics, University of Bonn, Bonn, Germany Human hair follicle (HF) cycling involves numerous signaling cascades and cellular processes that have been reported to be controlled by micro(mi)RNAs. However, our current knowledge on the expression and function of these miRNAs in hair biology is largely based on murine data or cell culture experiments and little is known about their exact role in the human HF. We therefore sought to gain a deeper understanding of the role of 9 previously reported miRNAs in human hair biology by systematically investigating their expression and regulatory interactions in HF samples from 25 healthy male donors. After nucleic acid extraction, we performed a genome-wide miRNA and mRNA expression profiling and subsequently correlated miRNA expression levels with mRNA-expression levels from the same samples to identify potential target genes. Moreover, we tested for an enrichment of target genes in biological pathways and searched for protein-protein interactions (PPIs). Our analysis confirmed the expression of 7 of the 9 candidate miRNAs. For 3 of them (miR-24, miR-31, miR-106a), significant miRNA/mRNA correlations were detected. The identified target genes included genes with known functions in hair biology (e.g. ITGB1, SOX9), genes whose described function matched the described function of their corresponding miRNA (e.g. RXRA) and numerous genes that had not previously been implicated in hair biology. The pathwayand PPI-based analysis revealed an enrichment of target genes in relevant pathways such as integrin and GnRH signaling and confirmed the interaction of target gene products in PPI-networks. In summary, our data provide novel insights into the role of miRNAs and their regulatory interactions in the human HF and will eventually contribute to a deeper understanding of healthy hair growth and the pathobiology of hair loss disorders.
Unveiling the role of estradiol in the pathogenesis of female pattern hair loss M Takahashi1, Y Endo1, Y Obayashi1, T Serizawa1, M Murakoshi1, R Ueki2 and M Ohyama3 1 Life Science Reserch Laboratories, Lion Corporation, Odawara, Japan, 2 Department of Dermatology, Juntendo University School of Medicine, Koto-ku, Japan and 3 Department of Dermatology, Kyorin University School of Medicine, Mitaka, Japan Female pattern hair loss (FPHL) shares major features with male androgenetic alopecia (AGA). Despite that major progress has been made in the investigation and management of AGA, our understanding of FPHL has been limited. As the incidence of FPHL is relatively high in postmenopausal females, we attempted to dissect the mechanism responsible for increased hair shedding in FPHL in the context of estradiol (E2) decrease. Thirty 25 to 58-year-old healthy women were evaluated for serum E2 levels, the frequency and the anchoring strength of telogen hairs. Interestingly, the decrease in E2 levels correlated with the increase in the proportion of telogen hairs. The extraction force required to pluck telogen hairs was significantly weaker in those with low E2 levels, suggesting that the reduction in E2 not only altered hair cycles but also affected hair shaft anchoring with resultant increase in hair shedding in low E2 individuals. PCR array analysis of human outer root sheath cells cultured with or without E2 detected up-regulation of adherence junction genes including desmocollin 3 and E-cadherin and down-regulation of metallopeptidase 10 and 15, implying that E2 may enhance hair follicle epithelia cell adhesion to suppress hair follicle shedding in homeostasis. Forced hair pluck experiments using C57BL/6 mice demonstrated marked decrease in hair anchoring strength in later telogen phase accompanied with altered expression of the genes detected in the experiment using human outer root sheath cells. Importantly, ovariectomized mice without E2 showed weaker telogen hair anchoring when compared to control mice. These findings suggested a previously unrecognized role of E2 in the pathophysiology of FPHL and may provide strategies to better manage postmenopausal FPHL by supplementing E2.
An underlying mechanism of hair loss in acrodermatitis enteropathica Y Ogawa, T Kawamura and S Shimada Dermatology, University of Yamanashi, Yamanashi, Japan The triad of acrodermatitis enteropathica (AE) is dermatitis, diarrhea and hair loss. We previously elucidated an underlying mechanism of characteristic dermatitis in AE using skin samples of AE patients and dietary Zinc (Zn)-deficient mice. We herein assessed underlying mechanisms of hair loss in AE using dietary Zn-adequate (ZA) and -deficient (ZD) mice. Fiveweek-old female Balb/c mice were continued ZA diet or switched to ZD diet for ZA or ZD mice, respectively. Although ZD mice did not exhibit obvious hair loss, the hair coat of ZD mice was sparse when compared to that of ZA mice. Hair morphology of ZD mice was stuck in telogen stage even after one week and thereafter of initiation of ZD diet, implying disruption of hair cycle. Moreover, depilation of telogen hair of ZA mice induced anagen hair, but not of ZD mice, in which hair did not regrow after depilation. Strikingly, loss of hair cycle and depilation-induced anagen initiation in ZD mice was completely restored by supplementation of Zn to ZD mice, suggesting that functions of some molecules related to hair cycle and regeneration was reversibly impaired by Zn deficiency. Immunofluorescence study revealed that lack of positivity of tissue-nonspecific alkaline phosphatase (TNAP) in dermal papilla (DP) and of P-cadherin in hair germ cells in ZD mice. TNAP, a zinc dependent enzyme, has been reported to be critical for hair regeneration by DP cells. Thus, the lack of TNAP activity in DP cells due to Zn deficiency appeared to be the major cause of loss of hair cycle and depilation-induced anagen initiation in ZD mice. Additionally, qPCR with dermis from ZA and ZD mice revealed downregulation of mRNA expression of pdgfa, pdgfb, fgf7 and fgf10. These data suggest that Zn deficiency decreases the production of PDGF and FGF in dermal cells including intra-dermal adipocytes, followed by downregulation of TNAP activity in DP cells, thereby resulting in loss of hair cycle and depilation-induced anagen initiation.
Androgen action and 3D culture conditions influence dermal papilla cells inductive properties: the role of Wnt agonists/antagonists balance JM Ceruti, GJ Leiro´s, AG Kusinsky and ME Balan˜a´ Fundacion Cassara´, ICT-MIlstein CONICET, Buenos Aires, Argentina In androgenetic alopecia, circulating androgens act on dermal papilla cells (DPC) and alter paracrine factors that influence hair epithelial cells leading to hair follicle (HF) miniaturization. We showed that androgens deregulate DPC-secreted factors involved in normal HF stem cell (HFSC) differentiation inhibiting the canonical Wnt signalling pathway reported as important for hair morphogenesis and for the maintenance of DPC inductive properties. In this work, we studied the effect of dihydrotestosterone (DHT) and culture conditions on Wnt agonists/antagonists balance in DPC and evaluated the role of the Wnt antagonist DKK-1 in dermal papilla-induced HFSC differentiation. To assess the effect that culture conditions could have on androgen-driven signals, monolayer and spheroids DPC cultures were established. The presence of DHT in both types of DPC cultures downregulated the expression of the agonists Wnt 5a and Wnt 10b, and upregulated the expression of Dkk1. Notably, in basal conditions, a higher expression of Wnt agonists was observed in DPC-spheroids culture system. HFSC differentiation was evaluated in presence of media conditioned by DPC after androgen treatment and in presence of DKK-1. DKK-1 presence in DPC culture abrogated the inductive ability of its conditioned medium mimicking the androgen effect previously reported. Furthermore, DPC-conditioned medium supplemented with Dkk-1 lost this HFSCdifferentiation inductive ability failing in the Wnt-b catenin pathway activation, evidenced by failure of accumulation of b catenin in the cytoplasm of these cells. We conclude that threedimensional-tissue architecture of DPC-spheroids appears to contribute in maintaining Wnt pathway activation, and probably DPC inductive properties. DHT can alter the balance of Wnt agonist/antagonist in DPC, reducing its ability to induce HFSC differentiation to hair lineage. The paracrine action of DKK-1 would be enough to abrogate HFSC differentiation, suggesting that DHT-inducible DKK-1 is involved in DHT-driven balding.
196
197
The endocannabinoid tone regulates human sebocyte biology A Ola´h1, N Za´ka´ny1, A Markovics1, S Nicolussi2, J Gertsch2, F Piscitelli3, V Di Marzo3, A´ Po´r6, C Zouboulis4 and T Bı´ro´5 1 Department of Physiology, University of Debrecen, Debrecen, Hungary, 2 Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland, 3 Consiglio Nazionale delle Ricerche, Pozzuoli, Italy, 4 Dessau Medical Center, Dessau, Germany, 5 Department of Immunology, University of Debrecen, Debrecen, Hungary and 6 Gyula Kene´zy Hospital, Debrecen, Hungary We have previously shown that i) endocannabinoids (eCB; e.g. anandamide [AEA]) are key players in the maintenance of homeostatic sebaceous lipid production (SLP) in sebaceous glands (SG); and ii) eCB treatment of sebocytes dramatically increases their lipid synthesis. Thus, here, we aimed to i) investigate the expression of the major eCB synthesizing and degrading enzymes; and ii) explore the effects of modulation of the eCB tone on human SZ95 sebocytes by using inhibitors of the eCB membrane transporter (EMT), which has been hypothesized to facilitate the transmembrane transport (and thereby degradation) of eCBs. We found that the major eCB synthesizing (NAPE-PLD, DAGLa and -b) and degrading (FAAH, MAGL) enzymes are expressed in SZ95 sebocytes as well as in SGs in situ, and that the EMT is also functionally active in these cells. Without influencing viability, the EMT-inhibitor VDM11 induced a small, but significant increase in SLP. Intriguingly, co-administration of the most efficient pro-lipogenic concentrations of VDM11 and AEA appeared to decrease the pro-lipogenic effect of the latter. This effect might have been due to the increased cytotoxic potential of the combined treatment. Finally, we found that VDM11 was able to prevent the pro-inflammatory action of the Toll-like receptor 4 activator lipopolysaccharide. Our data suggest that the increase of eCB tone exerts remarkable anti-inflammatory actions and slightly elevates SLP; thus its modulation might be beneficial in cutaneous inflammatory conditions accompanied by dry skin.
S194 Journal of Investigative Dermatology (2016), Volume 136
A novel splice site mutation in LIPH identified in a Japanese patient with autosomal recessive woolly hair A Tanaka1, Y Matsuo1, Y Shimomura2 and M Hide1 1 Department of Dermatology, Hiroshima University, Hiroshima, Japan and 2 Laboratory of Genetic Skin Diseases, Niigata University, Niigata, Japan Autosomal recessive woolly hair/hypotrichosis (ARWH) is a rare hereditary hair disease characterized by patients having short and tightly curled scalp hair with hypotrichosis. Mutations in two genes, the lipase gene (LIPH) and lysophosphatidic acid receptor 6 (LPAR6), are reported to cause ARWH. The patient was a 4-year-old Japanese boy. He had had short and tightly curled scalp hair since birth. His hair loss had gradually progressed with aging without any other manifestations. The patient’s parents and sister did not show these manifestations. Based on the clinical features, we diagnosed the patient as having ARWH and conducted genetic studies by using extracted genomic DNA from a peripheral blood sample. We performed direct sequencing analysis of LIPH and identified a heterozygous missense mutation c.736T>A (p.Cys246Ser) in exon 6 of LIPH, which is known to be a prevalent founder mutation in the Japanese population. Additionally, we identified a novel heterozygous mutation (c.417+1G>C) in intron 2 of LIPH. To investigate the expression of the mutant LIPH transcript, total RNA was extracted from the blood samples. The LIPH cDNA were amplified from exon 1 to exon 7 by RT-PCR. The PCR product using LIPH cDNA from the patient showed two fragments. These two mutations were located on different alleles; a normal splicing event occurred from the c.736T>A mutant allele, while an aberrant splicing event was induced from the c.417+1G>C mutant allele. Sequencing analysis also identified a heterozygous 144-bp deletion in exon 2, caused by the c.417+1G>C. The protein is expected to comprise a specific amino acid sequence in which 48 amino acid residues are deleted. Most of the deleted amino acid residues contribute to form an N-terminal a-helix of PA-PLA1a. Before our finding, two splice site mutations in LIPH have been reported. The c.417+1G>C is the third splice site mutation identified in LIPH, thereby expanding the spectrum of LIPH mutations causing ARWH.