- Email: [email protected]
beta-catenin signalling promotes dermal adipocyte differentiation during hair follicle morphogenesis and regeneration
JSID Abstracts / Journal of Dermatological Science 69 (2013) e47–e93
is only partly understood. Here we show that fibroblast growth factor (Fgf)18 is expressed in a hair stem cell niche throughout telogen, and that it regulates the hair cycle through the non-growth phases. When the Fgf18 gene is conditionally knocked out in keratin 5-positive epithelial cells in mice, telogen becomes very short, giving rise to a strikingly rapid succession of hair cycles. In wildtype mice, hair follicle growth during anagen is strongly suppressed by local delivery of FGF18 protein. Our results demonstrate that epithelial FGF18 signaling and its reduction in the milieu of hair stem cells are crucial for the maintenance of telogen and anagen phase, respectively. http://dx.doi.org/10.1016/j.jdermsci.2012.11.499 P09-02[C03-02] Human induced pluripotent stem cell line with hair forming capacity is ectodermal/epithelial lineage-prone and in WNTactivated state Ophelia K. Veraitch 1 , Tetsuro Kobayashi 1 , Yoichi Imaizumi 2 , Wado Akamatsu 2 , Masayuki Amagai 1 , Hideyuki Okano 2 , Manabu Ohyama 1,∗ 1 2
Department of Dermatology, Keio University School of Medicine Department of Physiology, Keio University School of Medicine
Recently, we demonstrated that human induced pluripotent stem cell-derived keratinocyte precursors (hiPS-KCPs) contributed to hair follicle (HF) regeneration. Among three hiPS cell lines tested, 201B7, a hiPS line generated with 4 Yamanaka factors, better communicated with hair inductive mesenchyme than WD39 and WDT2 (4 and 3 factor) lines and provided optimal material for HF formation experiments. The aim of the current study is to elucidate underlying mechanisms that endowed 201B7 hiPS line such functional advantage enabling HF reconstitution. To probe natural fate, embryo bodies were generated from each hiPS line and allowed to differentiate without any induction. 201B7 line more readily expressed MAP2, an ectoderm/neuronal marker, and epithelial markers KRT8 and KRT18 than other lines. In contrast, WD39 and WDT2 lines were prone to express endoderm and mesoderm markers. Thus, 201B7 line innately differentiated towards ectodermal/epithelial lineages than other lines. Activation of WNT signalling has been shown to dispose keratinocytes to HF fate. Intriguingly, all hiPS-KCP lines, especially 201B7 hiPSC-KCPs, expressed WNT signalling genes CTNNB and LEF1 more intensely than normal human adult keratinocytes. WNT signal inhibition abrogated the interactions between 201B7 hiPS-KCPs and human dermal papilla cells in co-culture, further supporting the importance of WNT-activated state for participating in HF regeneration. Accordingly, 201B7 hiPS and its descendant were distinguished by follicular epithelium prone nature, which may be essential to achieve efficient HF regeneration using hiPS-KCPs. In addition, this study emphasizes the necessity of pre-assessing functional aspect of individual hiPS lines for downstream applications, including tissue engineering. http://dx.doi.org/10.1016/j.jdermsci.2012.11.500
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P09-03[C03-03] Indispensable roles of BNIP3, an inducer of autophagy, in both differentiation and maintenance of epidermal keratinocytes Mariko Moriyama 1,∗ , Junki Uda 1 , Akifumi Matsuyama 2 , Hiroyuki Moriyama 1 1
Pharmaceutical Research and Technology Institute, Kinki University, Osaka, Japan 2 Department of Somatic Stem Cell Therapy and Health Policy, Foundation for Biomedical Research and Innovation, Kobe, Japan Recent studies have revealed that autophagy, a lysosomal degradation pathway, is involved in differentiation of erythrocytes, lymphocytes, and adipocytes. Keratinocyte differentiation is also going along with an activation of lysosomal enzymes and organelle clearance, expecting the contribution of autophagy in this process. We found that BNIP3, a pro-apoptotic member of Bcl-2 family, was expressed in the suprabasal layer of mouse epidermis and reconstituted human epidermal equivalent. Forced expression of BNIP3 in human primary epidermal keratinocytes (HPEK) by adenoviral infection resulted in upregulation of involucrin, whereas knockdown of BNIP3 had an opposite effect. Intriguingly, addition of 3-methyladenine, an inhibitor of autophagy, significantly suppressed BNIP3-stimulated differentiation of keratinocytes, suggesting that autophagy is involved in the process. Moreover, we also found that overexpression of BNIP3 induced autophagy in HPEK. These data clearly suggest that BNIP3 plays a crucial role in keratinocytes differentiation by inducing autophagy. Furthermore, dead cells were increased in human epidermal equivalent reconstituted from BNIP3 knockdown keratinocytes, which gave us the idea that BNIP3 is also indispensable for maintenance of skin epidermis. To test the hypothesis, HEPK were irradiated with UVB. UVB irradiation stimulated BNIP3 expression and cleavage of caspase3. Surprisingly, suppression of BNIP3 expression induced by UVB irradiation using RNAi caused a further increase of the cleaved caspase3 protein level, suggesting that BNIP3 has a protective effect against UVB-induced apoptosis in keratinocytes. Overall, our data shed light on functions of BNIP3, an inducer of autophagy, in both differentiation and maintenance of epidermal keratinocytes. http://dx.doi.org/10.1016/j.jdermsci.2012.11.501 P09-04[C03-04] Epidermal Wnt/beta-catenin signalling promotes dermal adipocyte differentiation during hair follicle morphogenesis and regeneration Hironobu Fujiwara 1,∗ , Giacomo Donati 2 , Valentina Proserpio 3 , Ken Natsuga 2 , Charlotte Collins 4 , Fiona Watt 2,4 1
RIKEN Center for Developmental Biology Cancer Research UK Cambridge Research Institute 3 MRC Laboratory of Molecular Biology 4 Wellcome Trust Centre for Stem Cell Research 2
Reciprocal interactions between different lineages of cells within mammalian skin are essential for skin morphogenesis, maintenance and function. Although it has long been known that morphogenesis and cyclic expansion and regression of subcutaneous/dermal adipocytes occur in parallel with the hair regeneration cycle, the mechanisms involved in this correlation remain largely unknown. Here we show that the activation of epidermal Wnt/beta-catenin signalling in growing hair follicles not only controls hair follicle stem cell (HFSC) activation but also regulates dermal lineages to promote adipocyte differen-
e66
JSID Abstracts / Journal of Dermatological Science 69 (2013) e47–e93
tiation. Inhibition of Wnt/beta-catenin signalling in the mouse epidermis reduces the adipocyte maturation in the developing and adult dermis. Conversely, the activation of Wnt/beta-catenin signalling in the embryonic epidermis dramatically increase the adipocyte contents even without the hair follicle morphogenesis, indicating that the activation of epidermal Wnt/beta-catenin signalling is sufficient to induce adipocyte generation. In vitro model of pre-adipocyte differentiation reveals that Wnt/betacatenin signalling-dependent soluble factors from keratinocytes regulate adipocyte differentiation. Our results demonstrate that the cyclic activation of Wnt/beta-catenin signalling in the hair follicle regulates dermal lineage decision. http://dx.doi.org/10.1016/j.jdermsci.2012.11.502 P09-05[C03-05] Transcription factors Runx1 and Runx3 regulate differentiation and proliferation of keratinocyte Ryuhei Uchiyama 1,∗ , Eisaku Ogawa 1 , Yukiko Kiniwa 1 , Hisashi Uhara 1 , Won F. Wong 2 , Kazuyoshi Kofu 3 , Shuntaro Ikawa 4 , Masanobu Satake 2 , Ryuhei Okuyama 1 1
The Department of Dermatology, Shinshu University School of Medicine, Matsumoto, Japan 2 Institute of Development, Aging and Cancer, Tohoku University, Sendai 3 National University Singapore, Singapore 4 Center for Interdisciplinary Research, Tohoku University, Sendai Runt-related transcription factor (Runx) family is implicated in stem cell regulation, tissue development, and oncogenesis. Runx1 and Runx3 play important roles in several organs, including blood, muscle, gastric mucosa, the nervous system, and hair follicles by affecting cell survival, proliferation, and differentiation. However, it is unknown whether and how Runx1 and Runx3 regulate in the epidermis. We investigated the functions of Runx1 and Runx3 in the epidermis. First, we overexpressed Runx1 and Runx3 in primary mouse keratinocyte by using adenovirus vectors. The amounts of keratin1 (K1) and keratin10 (K10) were decreased by Runx overexpression. Next, we decreased the expression of Runx1 and Runx3 by using sequence-specific siRNAs. The amounts of K1 and K10 were increased by the Runx knockdown. These results suggested that both Runx molecules inhibit the induction of early differentiation markers in the keratinocytes. We further elucidated how Runx regulate expression of K1 and K10. Chromatin immunoprecipitation assay showed that Runx1 and Runx3 bound to Runx consensus sequences located in 5’regions of K1 and K10 genome. While Runx1 and Runx3 bound to the 5’regions in undifferentiated state, they come off in association with differentiation induced by increasing calcium concentration within the medium. These results show that Runx negatively regulates transcription of K1 and K10. Further, Runx regulates keratinocyte growth. Overexpression of Runx1 and Runx3 induced keratinocytes growth arrest as well as apoptosis. However, the knockdown of Runx did not advance proliferation. Thus, our findings show that Runx inhibits both differentiation and proliferation in keratinocyte. http://dx.doi.org/10.1016/j.jdermsci.2012.11.503
P09-06[C03-06] CXCL10 induces infiltrates of Th1/Tc1 cells in the acute alopecia areata followed by sustained Tc1 cell-accumulation in the chronic phase Taisuke Ito 1,∗ , Hideo Hashizume 2 , Takatoshi Shimauchi 1 , Atsuko Funakoshi 1 , Natsuho Ito 1 , Masahiro Takigawa 1 , Yoshiki Tokura 1 1 2
Hamamatsu University School of Medicine Shimada Municipal Hospital
Alopecia areata (AA) is an organ-specific autoimmune disease with cell-mediated autoimmune reactions. T lymphocytes densely surround hair bulbs in the lesion of acute AA, which is referred to as “swarm of bees”. We investigated the pathological mechanisms of “swarm of bees”, focusing on T-cell chemokine and chemokine receptors. In the PBMCs, the frequency of CXCR3+ CD4+ T cells (Th1) was significantly higher in acute AA than in chronic AA or healthy control, while CXCR3+ CD8+ T cells (Tc1) were significantly increased in chronic AA. In the skin lesions of acute AA, CXCR3+ CD4+ and CXCR3+ CD8+ T cells infiltrated in the juxtafollicular area, but CCR4+ CD4+ and CCR4+ CD8+ T cells were scarce. In chronic-phase AA, CXCR3+ CD8+ T cells dominated the infiltrate around hair bulbs, contributing to the prolonged state of hair loss. Lymphocytes obtained by culturing a lesional skin specimen of acute AA contained CXCR3+ CD4+ and CXCR3+ CD8+ T cells at higher percentages than those of PBMC, suggesting preferential emigration from the blood. Immunohistochemical and real-time RT-PCR studies demonstrated that hair follicles of acute AA expressed a high level of Th1-associated chemokine CXCL10 compared to Th2 chemokine CCL17/TARC. In addition, plasmacytoid DCs (pDCs) were found around hair bulbs in AA lesion, that may indicate the source of IFN-␣. The number of pDCs were also increased in PBMCs obtained from acute AA patients. By chemotaxis assay, freshly isolated PBMC from acute AA patients had a strong velocity of chemotaxis toward CXCL10 with increased expression of F-actin. These results suggest that the increased production of CXCL10 induces preferential infiltrates of Th1 and Tc1 cells in the acute AA, and Tc1 cell infiltration remains prolonged in the chronic phase. http://dx.doi.org/10.1016/j.jdermsci.2012.11.504 P09-07 Estrogen induce VEGF expression in dermal papilla cells Yuuki Sakurai 1 , Ichitaro Niibe 2 , Satoshi Takeda 1 , Takashi Matsuzaki 2,∗ 1 2
Ogawa 1 , Yasufumi
Central Research Institute, Milbon Co., Ltd. Faculty of Life and Environmental Science, Shimane University
It is required to reveal the mechanisms of female pattern hair loss (FPHL) augmenting in Japanese women with age. Estrogen could have a pivotal role on the hair cycle control, because anagen extends during pregnancy. Estrogen levels in blood considerably drop in women who are in ages suffering FPHL. Therefore, we analyzed expressions of several genes associated with hair cycles using real time RT-PCR in human follicular dermal papilla cells (DPCs) after treatment with a various extent of estradiol. The cells were culture with serum-free medium without phenol red. We found that vascular endothelial growth factor (VEGF) expression increased in DPCs with estradiol at 1 nM, but showed no difference at higher concentration compared to controls without estradiol. ELISA assays revealed that VEGF protein expression was also elevated in DPCs treated with estradiol at concentrations similar to the above experiments. These concentrations are close to the upper limit of physiological level in blood in women, i.e. in pregnancy.
is only partly understood. Here we show that fibroblast growth factor (Fgf)18 is expressed in a hair stem cell niche throughout telogen, and that it regulates the hair cycle through the non-growth phases. When the Fgf18 gene is conditionally knocked out in keratin 5-positive epithelial cells in mice, telogen becomes very short, giving rise to a strikingly rapid succession of hair cycles. In wildtype mice, hair follicle growth during anagen is strongly suppressed by local delivery of FGF18 protein. Our results demonstrate that epithelial FGF18 signaling and its reduction in the milieu of hair stem cells are crucial for the maintenance of telogen and anagen phase, respectively. http://dx.doi.org/10.1016/j.jdermsci.2012.11.499 P09-02[C03-02] Human induced pluripotent stem cell line with hair forming capacity is ectodermal/epithelial lineage-prone and in WNTactivated state Ophelia K. Veraitch 1 , Tetsuro Kobayashi 1 , Yoichi Imaizumi 2 , Wado Akamatsu 2 , Masayuki Amagai 1 , Hideyuki Okano 2 , Manabu Ohyama 1,∗ 1 2
Department of Dermatology, Keio University School of Medicine Department of Physiology, Keio University School of Medicine
Recently, we demonstrated that human induced pluripotent stem cell-derived keratinocyte precursors (hiPS-KCPs) contributed to hair follicle (HF) regeneration. Among three hiPS cell lines tested, 201B7, a hiPS line generated with 4 Yamanaka factors, better communicated with hair inductive mesenchyme than WD39 and WDT2 (4 and 3 factor) lines and provided optimal material for HF formation experiments. The aim of the current study is to elucidate underlying mechanisms that endowed 201B7 hiPS line such functional advantage enabling HF reconstitution. To probe natural fate, embryo bodies were generated from each hiPS line and allowed to differentiate without any induction. 201B7 line more readily expressed MAP2, an ectoderm/neuronal marker, and epithelial markers KRT8 and KRT18 than other lines. In contrast, WD39 and WDT2 lines were prone to express endoderm and mesoderm markers. Thus, 201B7 line innately differentiated towards ectodermal/epithelial lineages than other lines. Activation of WNT signalling has been shown to dispose keratinocytes to HF fate. Intriguingly, all hiPS-KCP lines, especially 201B7 hiPSC-KCPs, expressed WNT signalling genes CTNNB and LEF1 more intensely than normal human adult keratinocytes. WNT signal inhibition abrogated the interactions between 201B7 hiPS-KCPs and human dermal papilla cells in co-culture, further supporting the importance of WNT-activated state for participating in HF regeneration. Accordingly, 201B7 hiPS and its descendant were distinguished by follicular epithelium prone nature, which may be essential to achieve efficient HF regeneration using hiPS-KCPs. In addition, this study emphasizes the necessity of pre-assessing functional aspect of individual hiPS lines for downstream applications, including tissue engineering. http://dx.doi.org/10.1016/j.jdermsci.2012.11.500
e65
P09-03[C03-03] Indispensable roles of BNIP3, an inducer of autophagy, in both differentiation and maintenance of epidermal keratinocytes Mariko Moriyama 1,∗ , Junki Uda 1 , Akifumi Matsuyama 2 , Hiroyuki Moriyama 1 1
Pharmaceutical Research and Technology Institute, Kinki University, Osaka, Japan 2 Department of Somatic Stem Cell Therapy and Health Policy, Foundation for Biomedical Research and Innovation, Kobe, Japan Recent studies have revealed that autophagy, a lysosomal degradation pathway, is involved in differentiation of erythrocytes, lymphocytes, and adipocytes. Keratinocyte differentiation is also going along with an activation of lysosomal enzymes and organelle clearance, expecting the contribution of autophagy in this process. We found that BNIP3, a pro-apoptotic member of Bcl-2 family, was expressed in the suprabasal layer of mouse epidermis and reconstituted human epidermal equivalent. Forced expression of BNIP3 in human primary epidermal keratinocytes (HPEK) by adenoviral infection resulted in upregulation of involucrin, whereas knockdown of BNIP3 had an opposite effect. Intriguingly, addition of 3-methyladenine, an inhibitor of autophagy, significantly suppressed BNIP3-stimulated differentiation of keratinocytes, suggesting that autophagy is involved in the process. Moreover, we also found that overexpression of BNIP3 induced autophagy in HPEK. These data clearly suggest that BNIP3 plays a crucial role in keratinocytes differentiation by inducing autophagy. Furthermore, dead cells were increased in human epidermal equivalent reconstituted from BNIP3 knockdown keratinocytes, which gave us the idea that BNIP3 is also indispensable for maintenance of skin epidermis. To test the hypothesis, HEPK were irradiated with UVB. UVB irradiation stimulated BNIP3 expression and cleavage of caspase3. Surprisingly, suppression of BNIP3 expression induced by UVB irradiation using RNAi caused a further increase of the cleaved caspase3 protein level, suggesting that BNIP3 has a protective effect against UVB-induced apoptosis in keratinocytes. Overall, our data shed light on functions of BNIP3, an inducer of autophagy, in both differentiation and maintenance of epidermal keratinocytes. http://dx.doi.org/10.1016/j.jdermsci.2012.11.501 P09-04[C03-04] Epidermal Wnt/beta-catenin signalling promotes dermal adipocyte differentiation during hair follicle morphogenesis and regeneration Hironobu Fujiwara 1,∗ , Giacomo Donati 2 , Valentina Proserpio 3 , Ken Natsuga 2 , Charlotte Collins 4 , Fiona Watt 2,4 1
RIKEN Center for Developmental Biology Cancer Research UK Cambridge Research Institute 3 MRC Laboratory of Molecular Biology 4 Wellcome Trust Centre for Stem Cell Research 2
Reciprocal interactions between different lineages of cells within mammalian skin are essential for skin morphogenesis, maintenance and function. Although it has long been known that morphogenesis and cyclic expansion and regression of subcutaneous/dermal adipocytes occur in parallel with the hair regeneration cycle, the mechanisms involved in this correlation remain largely unknown. Here we show that the activation of epidermal Wnt/beta-catenin signalling in growing hair follicles not only controls hair follicle stem cell (HFSC) activation but also regulates dermal lineages to promote adipocyte differen-
e66
JSID Abstracts / Journal of Dermatological Science 69 (2013) e47–e93
tiation. Inhibition of Wnt/beta-catenin signalling in the mouse epidermis reduces the adipocyte maturation in the developing and adult dermis. Conversely, the activation of Wnt/beta-catenin signalling in the embryonic epidermis dramatically increase the adipocyte contents even without the hair follicle morphogenesis, indicating that the activation of epidermal Wnt/beta-catenin signalling is sufficient to induce adipocyte generation. In vitro model of pre-adipocyte differentiation reveals that Wnt/betacatenin signalling-dependent soluble factors from keratinocytes regulate adipocyte differentiation. Our results demonstrate that the cyclic activation of Wnt/beta-catenin signalling in the hair follicle regulates dermal lineage decision. http://dx.doi.org/10.1016/j.jdermsci.2012.11.502 P09-05[C03-05] Transcription factors Runx1 and Runx3 regulate differentiation and proliferation of keratinocyte Ryuhei Uchiyama 1,∗ , Eisaku Ogawa 1 , Yukiko Kiniwa 1 , Hisashi Uhara 1 , Won F. Wong 2 , Kazuyoshi Kofu 3 , Shuntaro Ikawa 4 , Masanobu Satake 2 , Ryuhei Okuyama 1 1
The Department of Dermatology, Shinshu University School of Medicine, Matsumoto, Japan 2 Institute of Development, Aging and Cancer, Tohoku University, Sendai 3 National University Singapore, Singapore 4 Center for Interdisciplinary Research, Tohoku University, Sendai Runt-related transcription factor (Runx) family is implicated in stem cell regulation, tissue development, and oncogenesis. Runx1 and Runx3 play important roles in several organs, including blood, muscle, gastric mucosa, the nervous system, and hair follicles by affecting cell survival, proliferation, and differentiation. However, it is unknown whether and how Runx1 and Runx3 regulate in the epidermis. We investigated the functions of Runx1 and Runx3 in the epidermis. First, we overexpressed Runx1 and Runx3 in primary mouse keratinocyte by using adenovirus vectors. The amounts of keratin1 (K1) and keratin10 (K10) were decreased by Runx overexpression. Next, we decreased the expression of Runx1 and Runx3 by using sequence-specific siRNAs. The amounts of K1 and K10 were increased by the Runx knockdown. These results suggested that both Runx molecules inhibit the induction of early differentiation markers in the keratinocytes. We further elucidated how Runx regulate expression of K1 and K10. Chromatin immunoprecipitation assay showed that Runx1 and Runx3 bound to Runx consensus sequences located in 5’regions of K1 and K10 genome. While Runx1 and Runx3 bound to the 5’regions in undifferentiated state, they come off in association with differentiation induced by increasing calcium concentration within the medium. These results show that Runx negatively regulates transcription of K1 and K10. Further, Runx regulates keratinocyte growth. Overexpression of Runx1 and Runx3 induced keratinocytes growth arrest as well as apoptosis. However, the knockdown of Runx did not advance proliferation. Thus, our findings show that Runx inhibits both differentiation and proliferation in keratinocyte. http://dx.doi.org/10.1016/j.jdermsci.2012.11.503
P09-06[C03-06] CXCL10 induces infiltrates of Th1/Tc1 cells in the acute alopecia areata followed by sustained Tc1 cell-accumulation in the chronic phase Taisuke Ito 1,∗ , Hideo Hashizume 2 , Takatoshi Shimauchi 1 , Atsuko Funakoshi 1 , Natsuho Ito 1 , Masahiro Takigawa 1 , Yoshiki Tokura 1 1 2
Hamamatsu University School of Medicine Shimada Municipal Hospital
Alopecia areata (AA) is an organ-specific autoimmune disease with cell-mediated autoimmune reactions. T lymphocytes densely surround hair bulbs in the lesion of acute AA, which is referred to as “swarm of bees”. We investigated the pathological mechanisms of “swarm of bees”, focusing on T-cell chemokine and chemokine receptors. In the PBMCs, the frequency of CXCR3+ CD4+ T cells (Th1) was significantly higher in acute AA than in chronic AA or healthy control, while CXCR3+ CD8+ T cells (Tc1) were significantly increased in chronic AA. In the skin lesions of acute AA, CXCR3+ CD4+ and CXCR3+ CD8+ T cells infiltrated in the juxtafollicular area, but CCR4+ CD4+ and CCR4+ CD8+ T cells were scarce. In chronic-phase AA, CXCR3+ CD8+ T cells dominated the infiltrate around hair bulbs, contributing to the prolonged state of hair loss. Lymphocytes obtained by culturing a lesional skin specimen of acute AA contained CXCR3+ CD4+ and CXCR3+ CD8+ T cells at higher percentages than those of PBMC, suggesting preferential emigration from the blood. Immunohistochemical and real-time RT-PCR studies demonstrated that hair follicles of acute AA expressed a high level of Th1-associated chemokine CXCL10 compared to Th2 chemokine CCL17/TARC. In addition, plasmacytoid DCs (pDCs) were found around hair bulbs in AA lesion, that may indicate the source of IFN-␣. The number of pDCs were also increased in PBMCs obtained from acute AA patients. By chemotaxis assay, freshly isolated PBMC from acute AA patients had a strong velocity of chemotaxis toward CXCL10 with increased expression of F-actin. These results suggest that the increased production of CXCL10 induces preferential infiltrates of Th1 and Tc1 cells in the acute AA, and Tc1 cell infiltration remains prolonged in the chronic phase. http://dx.doi.org/10.1016/j.jdermsci.2012.11.504 P09-07 Estrogen induce VEGF expression in dermal papilla cells Yuuki Sakurai 1 , Ichitaro Niibe 2 , Satoshi Takeda 1 , Takashi Matsuzaki 2,∗ 1 2
Ogawa 1 , Yasufumi
Central Research Institute, Milbon Co., Ltd. Faculty of Life and Environmental Science, Shimane University
It is required to reveal the mechanisms of female pattern hair loss (FPHL) augmenting in Japanese women with age. Estrogen could have a pivotal role on the hair cycle control, because anagen extends during pregnancy. Estrogen levels in blood considerably drop in women who are in ages suffering FPHL. Therefore, we analyzed expressions of several genes associated with hair cycles using real time RT-PCR in human follicular dermal papilla cells (DPCs) after treatment with a various extent of estradiol. The cells were culture with serum-free medium without phenol red. We found that vascular endothelial growth factor (VEGF) expression increased in DPCs with estradiol at 1 nM, but showed no difference at higher concentration compared to controls without estradiol. ELISA assays revealed that VEGF protein expression was also elevated in DPCs treated with estradiol at concentrations similar to the above experiments. These concentrations are close to the upper limit of physiological level in blood in women, i.e. in pregnancy.