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β–catenin signaling
Biochemical and Biophysical Research Communications xxx (2017) 1e4
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Hairless controls hair fate decision via Wnt/becatenin signaling Kuicheng Zhu a, b, Cunshuan Xu a, Mengduan Liu c, Jintao Zhang c, *, 1 a
College of Life Science and Key Laboratory for Bioengineering, Henan Normal University, Xinxiang 453007, China Laboratory Animal Center of Zhengzhou University, Zhengzhou 450052, China c Institute of Medical and Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450052, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 July 2017 Accepted 29 July 2017 Available online xxx
The hairless (Hr) gene plays a central role in the hair cycle, considering that mutations in the gene result in hair loss with the exception of a few vibrissae after the first hair growth cycle in both mice and humans. This study examinedthe uncommon phenotype and using microarray analyses and functional studies, we found that b-catenin was mediated by Hr. Progenitor keratinocytes from the bulge region differentiate into both epidermis and sebaceous glands, and fail to adopt the hair keratinocytes fate in the mutant scalp, due to the decreased Wnt/b-catenin signaling in the absence of the hairless protein. This may be attributed to the dysfunction of normal epithelial-mesenchymal interactions in the hair follicle (HF). © 2017 Elsevier Inc. All rights reserved.
Keywords: Hairless Wnt/becatenin signaling Hair follicle Alopecia
1. Introduction Alopecia is a group of common dermatologic diseases that tend to lead to hair loss. This includes diseases such as androgenetic alopecia (AGA) and alopecia areata (AA) both of which involve the alopecia moving outward in a circular pattern on the scalp, which is similar to a beard where hair growth frequently starts above the mouth and on the chin until it extends across the face. A beard typically develops normally in men with AGA or AA; however, the molecular basis of this largely remained unexplored [1,2]. Unidentified gene mutations may play a role in the development of this disorder as it is frequent in people with a family history of alopecia [3,4]. Hairless (Hr), is a potentially important regulatory gene for the hair cycle control since an autosomal recessive mutation of Hr in mice and humans has been reported to evoke severe, irreversible HF abnormalities except for a few vibrissae [5e7]. In our previous study, a similar phenotype was observed in mice that harbored a mutation of the Hr gene named rhinocerotic (symbol: hrrhsl) [8]. To identify the differentially expressed genes that are responsible for this unusual phenotype, we performed a microarray analysis on body sites-specific skins around the corners of the mouth and the scalp at postnatal day 12 (P12) in hrrhsl/hrrhsl mouse. Our findings demonstrate that the Wnt
* Corresponding author. E-mail address: [email protected] (J. Zhang). 1 The authors contributed equally to this work.
pathway gene (b-catenin) regulated by Hr could play a central role in hair regeneration. 2. Materials and methods 2.1. Animals and tissue collection Hrrhsl/Hrrhslmice were maintained as previously reported [8]. Skin samples were collected from 12-day-old mutant mice (n ¼ 3). The animal experiments comply with the ARRIVE guidelines and approved by the Animal Ethics Committee of the Zhengzhou University (Zhengzhou, China). Animals were maintained on a 12-h light: 12-h dark circadian schedule in a 20e24 C housing room, and provided water and mouse chow ad libitum. 2.2. Histology and transmission electron microscopy (TEM) Skin samples from the scalp and chin were collected for both histological and ultrastuctural studies. Skin tissues were cut into pieces, fixed in 10% neutral buffered formalin overnight, embedded in paraffin, sectioned to 5 mm, and stained with hematoxylin and eosin (H&E) for histological studies. The samples were washed in 5% sucrose cacodylate buffer, postfixed with 1% osmium tetroxide, dehydrated, and embedded in epoxy resin after 4 h in Karnovsky's fixative for the transmission electron microscopy (TEM). Ultrathin sections were cut, collected on formvar coated grids, and examined via electron microscopy.
http://dx.doi.org/10.1016/j.bbrc.2017.07.164 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: K. Zhu, et al., Hairless controls hair fate decision via Wnt/becatenin signaling, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.164
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K. Zhu et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e4
Fig. 1. Phenotypic and structural changes in mutant mice at P12. (A, B) Note the hair loss in the scalp except for vibrissae at P12 and P21, respectively. The dilatation of the pilary and utricle formation in HFs of the scalp was present (C) versus the vibrissae (D) via histology at P12. The central part of the cyst was characterized by an accumulation of lipid droplets in the scalp (E) versus the vibrissae (F) by TEM at P12. Lipids were detected in the cyst contents via Oil Red O staining at P12 (G). Scale bars: 20 mm (C, D and G); 1 mm (E); 0.5 mm (F).
2.3. Oil Red O staining
3. Results
Samples were embedded in Tissue-TeK® (Sakura, Finetek, Inc., USA) and 6 mm vertical cryosections were stained with Oil Red O solution (Sigma).
3.1. Histological changes in scalp and chin
2.4. Microarray and real-time PCR Total RNA was extracted from skin samples extracted from the scalp and chin using the TRIzol® reagent (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Microarray analysis was performed with an Agilent MouseWG-6 v2 Gene Expression BeadChip (Santa Clara, CA, USA) to identify differentially expressed genes. Quantitative PCR (qRT-PCR) was performed with the SYBR Premix Ex Taq (Takara) using aLightCycler480 Software Setup (Roche, USA) to validate the differences. Differences between the scalp and the chin were calculated based on the 2eDDCt method. 2.5. Immunostaining Skin samples were taken from both the scalp and the chin of three mutant mice for immunohistochemistry. The tissue sections were deparaffinized, and blocked with 10% goat serum. All tissue sections were then incubated overnight at 4 C with the following primary antibodies: HR (1: 200, Santa Cruz Biotechnology, Inc), bcatenin (1:500, Cell Signaling, Abcam), Caspase 14 (Casp14, 1:100, epidermal differentiation, Abcam), and Keratin 86 (KRT86, 1:200, Keratin, Abcam). The primary antibodies were detected with biotinylated goat anti-rabbit secondary antibodies, and visualized using a DAB Peroxidase Substrate Kit (Sigma). Counterstaining of the sections was performed using hematoxylin. 2.6. Statistical analysis The data obtained from each mouse were averaged per group, and the standard deviation of the mean values was calculated. An unpaired student's t-test was used to determine the significance between two groups: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.
The most prominent effect observed in mutant mice is hair loss from P12 (Fig. 1A). Progressive hair loss is initiated around the eyes and progresses caudally, resulting in a completely hairless condition by postnatal day 21 (P21) with the exception of the vibrissae (Fig. 1B). Histology revealed that there was an excessively keratinized layer in the epidermis, with accompanied loss of the hair shaft, prominent dilatation of the pilary canal (infundibulum), and numerous intradermal follicular cysts filled with keratinized material in the scalp (Fig. 1C) versus the chin (Fig. 1D). The ultrastructure of the scalp was also compared with that of the chin. The results showed that in the peripheral part of the cysts, the cytoplasm of the wall epithelial cells that occupied the same position as the initial follicular bulb was filled with lipid droplets at P12 (Fig. 1E). This pattern was not seen in the chin samples (Fig. 1F). The cysts also contain lipids, a component of sebum (Fig. 1G). 3.2. Regional differences in Wnt/becatenin signaling pathway genes between scalp and chin To clarify which regional differences from within the signaling mechanisms were unique to the uncommon phenotype of the mutant mouse, we conducted a microarray analysis to investigate the genes with specifically affected expressions by the loss of Hr on a body site, specifically on the scalp and the chin. We determined the time point where hair loss (P12) first occurred to clarify the initial events that caused these morphological changes. The results revealed that the WNT signaling pathway gene- b-catenin underwent significant changes (2, P < 0.05) in the scalp versus the chin of the same mouse(Fig. 2A). There was a 0.32-fold decrease in the expression of b-catenin in the scalp versus the chin. In addition, the Casp14, the Keratin (KRT)83, and the KRT86 (that were associated with keratinocyte differentiation and hair-shaft structure) were more highly transcribed in the scalp than in the chin. QRT-PCR confirmed that the above expressed genes in the P12 scalp were higher than in the chin (Fig. 2B).
Please cite this article in press as: K. Zhu, et al., Hairless controls hair fate decision via Wnt/becatenin signaling, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.164
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vibrissa (Fig. 3D). The high expression of Casp14 was located in the center of the cyst and the epidermis of the mutant scalp (Fig. 3E) versus the chin (Fig. 3F). This indicated that the progenitor keratinocytes that would normally be destined to become follicular structures ultimately populated the strata corneum of the epidermis and the sebaceous glands due to an increase in terminal differentiation. KRT86 were highly expressed in the scalp (Fig. 3G) but not in the chin (Fig. 3H), indicating that the elevated level of these proteins may disturb the balance of their normal array in follicular keratinocytes. 4. Discussion
Fig. 2. Specific changes in gene expression in the scalp versus the chin. (A) Scatter plot of microarray analyses of the P12 scalp and the chin skin. Relative expression was assessed via qRT-PCR using the same RNA source as was used in the microarray analysis for the differentially expressed genes. (B) Relative amounts of mRNA were normalized against b-actin mRNA.All experiments were independently performed in triplicate, and unpaired student's t-test was used to determine the significance between the two groups:*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
3.3. Loss of Hr blocks hair cycling, leading to dermal and epidermal changes in fate determinants of epidermis and sebaceous glands To test whether regional differences in expressed genes are critical to the unusual phenotype, we examined their expression via immunohistochemical analysis. We found that the structures of HF in scalp and chin were determined through regional skin differences in the epithelial expression of the genes associated with Wnt/ b-catenin (WNT) signaling. The hair follicles in the scalp of mutant mice were Hr negative (Fig. 3A). In contrast, the upper part of hair follicles was Hr positive in vibrissa (Fig. 3B). In the scalp HFs, bcatenin that is the effector of canonical WNT signaling was merely expressed in the mesenchymal dermal papilla (DP), but not in the epithelial cells (Fig. 3C). The b-catenin, however, was strongly expressed in epithelial and mesenchymal dermal papilla cells in the
There appears to be a spreading of changes in AGA and/or AA in humans, which resembles the waves seen in the hrrhsl/hrrhsl mice. First, beard growth starts above the mouth and on the chin and extends across the face, similar to the pattern of receding hairline that forms a characteristic “M” shape in men [9]. Second, the beard develops normally in both humans and mice. Third, the hair usually appears normal on the scalp at birth but never regrows after alopecia [10]. However, it has been difficult to visualize these changes and to decipher the underlying mechanisms in vivo, especially at disease onset because the development of alopecia is currently not possible to predict. Given the above facts, a mutant mouse system was used to visualize and analyze the disease in the present study. Histologically, the follicles in the scalp showed characteristics of cysts near the follicular infundibulum in contrast to that of the chin. Since the cysts closely resembled unopened sebaceous glands based on morphological examination and oil scalp in human with AGA and/or AA, we conducted a lipid analysis and ultrastructural study. The results showed that the utricle organized in acini and contain numerous signs of sebocyte differentiation, which is consistent with the result that the overgrowth (multilobulation) of the sebaceous gland could contribute to the formation of AGA that usually has a bald oily scalp [11]. We conducted a microarray analysis to understand the regional differences in the pathway genes that are regulated by a loss of the Hr gene that encodes the HR functional protein as a transcriptional repressor. Comparative bioinformatics revealed levels of WNTe (bcatenin) in the scalp compared to the chin, which was further confirmed via qRT-PCR and immunohistochemical analyses. Mice without b-catenin displayed the same phenotype as Hr mutant animals. However, Hr expression was not affected in the b-cateninnegative skin, indicating that genetically Hr acts upstream of bcatenin during the hair cycle [12]. Based on histological and ultrastructural observations, there must be specific changes in expression of genes that are associated with terminal keratinocyte differentiation. Casp14 was found to be increased beyond 8-fold in the scalp compared to the chin. This increase in terminal differentiation of keratinocytes induced the formation of cystic-like structures, accompanied by high expression of KRT83 and KRT86 that are major building blocks of hair keratins, leading to the formation of dysfunctional intermediate filaments, and resulting in easily breaking fragile hair fibres [13]. These results, combined with the function of HR as a transcriptional co-repressor, suggest that Casp14, KRT83, and KRT86 may depend on HR, since the Casp14/ mice failed to grow hairs after the first hair shedding, and the expression of keratin genes was decreased in mutant skin that over-expressed Hr [14]. Whether the genes are directly or indirectly regulated by Hr and/or b-catenin requires further study, as Hr could be upstream of b-catenin. Normally, multipotent progenitors originating from the bulge region can adopt various cell fates by responding to cues provided by multiple signaling molecules [15]. The present results showed that progenitors are unable to adopt the fate of hair keratinocytes
Please cite this article in press as: K. Zhu, et al., Hairless controls hair fate decision via Wnt/becatenin signaling, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.164
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K. Zhu et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e4
Fig. 3. Lack of hair cycling in Hr-mutant scalp Skin. Histological sections of scalp(A) and chin skins(B), stained with an HR antibody (brown staining). (C, D) Expression of bcatenin and (E, F) Casp14 expression was determined in the scalp HFs versus vibrissa. Immunohistochemistry of KRT86 in the scalp HFs (G) and the vibrissa (H). Antibody binding was visualized via brown DAB staining, followed by hematoxylin counterstaining. One sample section from three mutant mice is shown. Scale bars: 20 mm (AeH).
and instead, differentiate into epidermis and sebaceous glands in the absence of HR. This supports the notion that the function of Wnt/becatenin signaling may play a key role in both maintenance and differentiation of stem cells [16]. Especially, the fact that bcatenin is expressed in the DP, but not in the epithelial cells in the HFs of scalp, indicates that signals from DP could not directly interact with stem cells, although a potent inductive ability of DP to promote follicle formation has been recognized for decades [17]. Our findings may pave the way for therapeutic advances in the treatment of AGA and/or AA. Conflict of interest None. Acknowledgements This work was supported by the grants from the National Natural Science Foundation of China (No. 39970119 and 31372270). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.07.164. References [1] C.M. Chuong, V.A. Randall, R.B. Widelitz, P. Wu, T.X. Jiang, Physiological regeneration of skin appendages and implications for regenerative medicine, Physiology 27 (2012) 61e72. [2] C.H. Pratt, L.E. King Jr., A.G. Messenger, et al., Alopecia areata, Nat. Rev. Dis. Prim. 3 (2017) 1e17.
€ter, H. Hamm, et al., [3] P. van der Steen, H. Traupe, R. Happle, J. Boezeman, R. Stra The genetic risk for alopecia areata in first degree relatives of severely affected patients, An Estim. Acta Derm Venereol 72 (1992) 373e375. [4] L. Petukhova, M. Duvic, M. Hordinsky, et al., Genome-wide association study in alopecia areata implicates both innate and adaptive immunity, Nature 466 (2010) 113e117. [5] F. Benavides, T.M. Oberyszyn, A.M. Vanbuskirk, V.E. Reeve, D.F. Kusewitt, The hairless mouse in skin research, J. Dermatological Sci. 53 (2009) 10e18. [6] C.C. Thompson, Hairless is a nuclear receptor corepressor essential for skin function, Nucl. Recept. Signal 7 (2009) 1e11. [7] Y. Wen, Y. Liu, Y. Xu, et al., Loss-of-function mutations of an inhibitory upstream ORF in the human Hairless transcript cause Marie Unna hereditary hypotrichosis, Nat. Genet. 41 (2009) 228e233. [8] J.T. Zhang, S.G. Fang, C.Y. Wang, A novel nonsense mutation and polymorphisms in the mouse hairless gene, J. Invest. Dermatol 124 (2005) 1200e1205. [9] J.B. Hamilton, Patterned loss of hair in man; types and incidence, Ann. N.Y. Acad. Sci. 53 (1951) 708e728. [10] W. Ahmad, M. Faiyaz ul Haque, V. Brancolini, H.C. Tsou, S. ul Haque, et al., Alopecia universalis associated with a mutation in the human hairless gene, Science 279 (1998) 720e724. [11] K. Kure, T. Isago, T. Hirayama, Changes in the sebaceous gland in patients with male pattern hair loss (androgenic alopecia), J. Cosmet. Dermatol 14 (2015) 178e184. [12] J. Huelsken, R. Vogel, B. Erdmann, et al., b-catenin controls hair follicle morphogenesis and stem cell differentiation in the skin, Cell 105 (2001) 533e545. [13] N. Nanashima, K. Ito, T. Ishikawa, M. Nakano, T. Nakamura, Damage of hair follicle stem cells and alteration of keratin expression inexternal radiationinduced acute alopecia, Int. J. Mol. Med. 30 (2012) 579e584. [14] M. Devos, P. De Groote, B. Gilbert, I. Bruggeman, K. Leurs, S. Lippens, P. Vandenabeele, W. Declercq, Caspase-14 overexpression in hairless mice is not involved in utricle formation, Exp. Dermatol 22 (2013) 484e486. [15] C.P. Lu, L. Polak, B.E. Keyes, E. Fuchs, Spatiotemporal antagonism in mesenchymal-epithelial signaling in sweatversus hair fate decision, Science 354 (2016) 1551e1563. [16] C.C. Thompson, J.M. Sisk, G.M. Beaudoin 3rd, Hairless and Wnt signaling: allies in epithelial stem cell differentiation, Cell. Cycle 5 (2006) 1913e1917. [17] M.H. Hardy, The secret life of the hair follicle, Trends. Genet. 8 (1992) 55e61.
Please cite this article in press as: K. Zhu, et al., Hairless controls hair fate decision via Wnt/becatenin signaling, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.164
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Hairless controls hair fate decision via Wnt/becatenin signaling Kuicheng Zhu a, b, Cunshuan Xu a, Mengduan Liu c, Jintao Zhang c, *, 1 a
College of Life Science and Key Laboratory for Bioengineering, Henan Normal University, Xinxiang 453007, China Laboratory Animal Center of Zhengzhou University, Zhengzhou 450052, China c Institute of Medical and Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450052, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 July 2017 Accepted 29 July 2017 Available online xxx
The hairless (Hr) gene plays a central role in the hair cycle, considering that mutations in the gene result in hair loss with the exception of a few vibrissae after the first hair growth cycle in both mice and humans. This study examinedthe uncommon phenotype and using microarray analyses and functional studies, we found that b-catenin was mediated by Hr. Progenitor keratinocytes from the bulge region differentiate into both epidermis and sebaceous glands, and fail to adopt the hair keratinocytes fate in the mutant scalp, due to the decreased Wnt/b-catenin signaling in the absence of the hairless protein. This may be attributed to the dysfunction of normal epithelial-mesenchymal interactions in the hair follicle (HF). © 2017 Elsevier Inc. All rights reserved.
Keywords: Hairless Wnt/becatenin signaling Hair follicle Alopecia
1. Introduction Alopecia is a group of common dermatologic diseases that tend to lead to hair loss. This includes diseases such as androgenetic alopecia (AGA) and alopecia areata (AA) both of which involve the alopecia moving outward in a circular pattern on the scalp, which is similar to a beard where hair growth frequently starts above the mouth and on the chin until it extends across the face. A beard typically develops normally in men with AGA or AA; however, the molecular basis of this largely remained unexplored [1,2]. Unidentified gene mutations may play a role in the development of this disorder as it is frequent in people with a family history of alopecia [3,4]. Hairless (Hr), is a potentially important regulatory gene for the hair cycle control since an autosomal recessive mutation of Hr in mice and humans has been reported to evoke severe, irreversible HF abnormalities except for a few vibrissae [5e7]. In our previous study, a similar phenotype was observed in mice that harbored a mutation of the Hr gene named rhinocerotic (symbol: hrrhsl) [8]. To identify the differentially expressed genes that are responsible for this unusual phenotype, we performed a microarray analysis on body sites-specific skins around the corners of the mouth and the scalp at postnatal day 12 (P12) in hrrhsl/hrrhsl mouse. Our findings demonstrate that the Wnt
* Corresponding author. E-mail address: [email protected] (J. Zhang). 1 The authors contributed equally to this work.
pathway gene (b-catenin) regulated by Hr could play a central role in hair regeneration. 2. Materials and methods 2.1. Animals and tissue collection Hrrhsl/Hrrhslmice were maintained as previously reported [8]. Skin samples were collected from 12-day-old mutant mice (n ¼ 3). The animal experiments comply with the ARRIVE guidelines and approved by the Animal Ethics Committee of the Zhengzhou University (Zhengzhou, China). Animals were maintained on a 12-h light: 12-h dark circadian schedule in a 20e24 C housing room, and provided water and mouse chow ad libitum. 2.2. Histology and transmission electron microscopy (TEM) Skin samples from the scalp and chin were collected for both histological and ultrastuctural studies. Skin tissues were cut into pieces, fixed in 10% neutral buffered formalin overnight, embedded in paraffin, sectioned to 5 mm, and stained with hematoxylin and eosin (H&E) for histological studies. The samples were washed in 5% sucrose cacodylate buffer, postfixed with 1% osmium tetroxide, dehydrated, and embedded in epoxy resin after 4 h in Karnovsky's fixative for the transmission electron microscopy (TEM). Ultrathin sections were cut, collected on formvar coated grids, and examined via electron microscopy.
http://dx.doi.org/10.1016/j.bbrc.2017.07.164 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: K. Zhu, et al., Hairless controls hair fate decision via Wnt/becatenin signaling, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.164
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K. Zhu et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e4
Fig. 1. Phenotypic and structural changes in mutant mice at P12. (A, B) Note the hair loss in the scalp except for vibrissae at P12 and P21, respectively. The dilatation of the pilary and utricle formation in HFs of the scalp was present (C) versus the vibrissae (D) via histology at P12. The central part of the cyst was characterized by an accumulation of lipid droplets in the scalp (E) versus the vibrissae (F) by TEM at P12. Lipids were detected in the cyst contents via Oil Red O staining at P12 (G). Scale bars: 20 mm (C, D and G); 1 mm (E); 0.5 mm (F).
2.3. Oil Red O staining
3. Results
Samples were embedded in Tissue-TeK® (Sakura, Finetek, Inc., USA) and 6 mm vertical cryosections were stained with Oil Red O solution (Sigma).
3.1. Histological changes in scalp and chin
2.4. Microarray and real-time PCR Total RNA was extracted from skin samples extracted from the scalp and chin using the TRIzol® reagent (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Microarray analysis was performed with an Agilent MouseWG-6 v2 Gene Expression BeadChip (Santa Clara, CA, USA) to identify differentially expressed genes. Quantitative PCR (qRT-PCR) was performed with the SYBR Premix Ex Taq (Takara) using aLightCycler480 Software Setup (Roche, USA) to validate the differences. Differences between the scalp and the chin were calculated based on the 2eDDCt method. 2.5. Immunostaining Skin samples were taken from both the scalp and the chin of three mutant mice for immunohistochemistry. The tissue sections were deparaffinized, and blocked with 10% goat serum. All tissue sections were then incubated overnight at 4 C with the following primary antibodies: HR (1: 200, Santa Cruz Biotechnology, Inc), bcatenin (1:500, Cell Signaling, Abcam), Caspase 14 (Casp14, 1:100, epidermal differentiation, Abcam), and Keratin 86 (KRT86, 1:200, Keratin, Abcam). The primary antibodies were detected with biotinylated goat anti-rabbit secondary antibodies, and visualized using a DAB Peroxidase Substrate Kit (Sigma). Counterstaining of the sections was performed using hematoxylin. 2.6. Statistical analysis The data obtained from each mouse were averaged per group, and the standard deviation of the mean values was calculated. An unpaired student's t-test was used to determine the significance between two groups: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.
The most prominent effect observed in mutant mice is hair loss from P12 (Fig. 1A). Progressive hair loss is initiated around the eyes and progresses caudally, resulting in a completely hairless condition by postnatal day 21 (P21) with the exception of the vibrissae (Fig. 1B). Histology revealed that there was an excessively keratinized layer in the epidermis, with accompanied loss of the hair shaft, prominent dilatation of the pilary canal (infundibulum), and numerous intradermal follicular cysts filled with keratinized material in the scalp (Fig. 1C) versus the chin (Fig. 1D). The ultrastructure of the scalp was also compared with that of the chin. The results showed that in the peripheral part of the cysts, the cytoplasm of the wall epithelial cells that occupied the same position as the initial follicular bulb was filled with lipid droplets at P12 (Fig. 1E). This pattern was not seen in the chin samples (Fig. 1F). The cysts also contain lipids, a component of sebum (Fig. 1G). 3.2. Regional differences in Wnt/becatenin signaling pathway genes between scalp and chin To clarify which regional differences from within the signaling mechanisms were unique to the uncommon phenotype of the mutant mouse, we conducted a microarray analysis to investigate the genes with specifically affected expressions by the loss of Hr on a body site, specifically on the scalp and the chin. We determined the time point where hair loss (P12) first occurred to clarify the initial events that caused these morphological changes. The results revealed that the WNT signaling pathway gene- b-catenin underwent significant changes (2, P < 0.05) in the scalp versus the chin of the same mouse(Fig. 2A). There was a 0.32-fold decrease in the expression of b-catenin in the scalp versus the chin. In addition, the Casp14, the Keratin (KRT)83, and the KRT86 (that were associated with keratinocyte differentiation and hair-shaft structure) were more highly transcribed in the scalp than in the chin. QRT-PCR confirmed that the above expressed genes in the P12 scalp were higher than in the chin (Fig. 2B).
Please cite this article in press as: K. Zhu, et al., Hairless controls hair fate decision via Wnt/becatenin signaling, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.164
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vibrissa (Fig. 3D). The high expression of Casp14 was located in the center of the cyst and the epidermis of the mutant scalp (Fig. 3E) versus the chin (Fig. 3F). This indicated that the progenitor keratinocytes that would normally be destined to become follicular structures ultimately populated the strata corneum of the epidermis and the sebaceous glands due to an increase in terminal differentiation. KRT86 were highly expressed in the scalp (Fig. 3G) but not in the chin (Fig. 3H), indicating that the elevated level of these proteins may disturb the balance of their normal array in follicular keratinocytes. 4. Discussion
Fig. 2. Specific changes in gene expression in the scalp versus the chin. (A) Scatter plot of microarray analyses of the P12 scalp and the chin skin. Relative expression was assessed via qRT-PCR using the same RNA source as was used in the microarray analysis for the differentially expressed genes. (B) Relative amounts of mRNA were normalized against b-actin mRNA.All experiments were independently performed in triplicate, and unpaired student's t-test was used to determine the significance between the two groups:*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
3.3. Loss of Hr blocks hair cycling, leading to dermal and epidermal changes in fate determinants of epidermis and sebaceous glands To test whether regional differences in expressed genes are critical to the unusual phenotype, we examined their expression via immunohistochemical analysis. We found that the structures of HF in scalp and chin were determined through regional skin differences in the epithelial expression of the genes associated with Wnt/ b-catenin (WNT) signaling. The hair follicles in the scalp of mutant mice were Hr negative (Fig. 3A). In contrast, the upper part of hair follicles was Hr positive in vibrissa (Fig. 3B). In the scalp HFs, bcatenin that is the effector of canonical WNT signaling was merely expressed in the mesenchymal dermal papilla (DP), but not in the epithelial cells (Fig. 3C). The b-catenin, however, was strongly expressed in epithelial and mesenchymal dermal papilla cells in the
There appears to be a spreading of changes in AGA and/or AA in humans, which resembles the waves seen in the hrrhsl/hrrhsl mice. First, beard growth starts above the mouth and on the chin and extends across the face, similar to the pattern of receding hairline that forms a characteristic “M” shape in men [9]. Second, the beard develops normally in both humans and mice. Third, the hair usually appears normal on the scalp at birth but never regrows after alopecia [10]. However, it has been difficult to visualize these changes and to decipher the underlying mechanisms in vivo, especially at disease onset because the development of alopecia is currently not possible to predict. Given the above facts, a mutant mouse system was used to visualize and analyze the disease in the present study. Histologically, the follicles in the scalp showed characteristics of cysts near the follicular infundibulum in contrast to that of the chin. Since the cysts closely resembled unopened sebaceous glands based on morphological examination and oil scalp in human with AGA and/or AA, we conducted a lipid analysis and ultrastructural study. The results showed that the utricle organized in acini and contain numerous signs of sebocyte differentiation, which is consistent with the result that the overgrowth (multilobulation) of the sebaceous gland could contribute to the formation of AGA that usually has a bald oily scalp [11]. We conducted a microarray analysis to understand the regional differences in the pathway genes that are regulated by a loss of the Hr gene that encodes the HR functional protein as a transcriptional repressor. Comparative bioinformatics revealed levels of WNTe (bcatenin) in the scalp compared to the chin, which was further confirmed via qRT-PCR and immunohistochemical analyses. Mice without b-catenin displayed the same phenotype as Hr mutant animals. However, Hr expression was not affected in the b-cateninnegative skin, indicating that genetically Hr acts upstream of bcatenin during the hair cycle [12]. Based on histological and ultrastructural observations, there must be specific changes in expression of genes that are associated with terminal keratinocyte differentiation. Casp14 was found to be increased beyond 8-fold in the scalp compared to the chin. This increase in terminal differentiation of keratinocytes induced the formation of cystic-like structures, accompanied by high expression of KRT83 and KRT86 that are major building blocks of hair keratins, leading to the formation of dysfunctional intermediate filaments, and resulting in easily breaking fragile hair fibres [13]. These results, combined with the function of HR as a transcriptional co-repressor, suggest that Casp14, KRT83, and KRT86 may depend on HR, since the Casp14/ mice failed to grow hairs after the first hair shedding, and the expression of keratin genes was decreased in mutant skin that over-expressed Hr [14]. Whether the genes are directly or indirectly regulated by Hr and/or b-catenin requires further study, as Hr could be upstream of b-catenin. Normally, multipotent progenitors originating from the bulge region can adopt various cell fates by responding to cues provided by multiple signaling molecules [15]. The present results showed that progenitors are unable to adopt the fate of hair keratinocytes
Please cite this article in press as: K. Zhu, et al., Hairless controls hair fate decision via Wnt/becatenin signaling, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.164
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K. Zhu et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e4
Fig. 3. Lack of hair cycling in Hr-mutant scalp Skin. Histological sections of scalp(A) and chin skins(B), stained with an HR antibody (brown staining). (C, D) Expression of bcatenin and (E, F) Casp14 expression was determined in the scalp HFs versus vibrissa. Immunohistochemistry of KRT86 in the scalp HFs (G) and the vibrissa (H). Antibody binding was visualized via brown DAB staining, followed by hematoxylin counterstaining. One sample section from three mutant mice is shown. Scale bars: 20 mm (AeH).
and instead, differentiate into epidermis and sebaceous glands in the absence of HR. This supports the notion that the function of Wnt/becatenin signaling may play a key role in both maintenance and differentiation of stem cells [16]. Especially, the fact that bcatenin is expressed in the DP, but not in the epithelial cells in the HFs of scalp, indicates that signals from DP could not directly interact with stem cells, although a potent inductive ability of DP to promote follicle formation has been recognized for decades [17]. Our findings may pave the way for therapeutic advances in the treatment of AGA and/or AA. Conflict of interest None. Acknowledgements This work was supported by the grants from the National Natural Science Foundation of China (No. 39970119 and 31372270). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.07.164. References [1] C.M. Chuong, V.A. Randall, R.B. Widelitz, P. Wu, T.X. Jiang, Physiological regeneration of skin appendages and implications for regenerative medicine, Physiology 27 (2012) 61e72. [2] C.H. Pratt, L.E. King Jr., A.G. Messenger, et al., Alopecia areata, Nat. Rev. Dis. Prim. 3 (2017) 1e17.
€ter, H. Hamm, et al., [3] P. van der Steen, H. Traupe, R. Happle, J. Boezeman, R. Stra The genetic risk for alopecia areata in first degree relatives of severely affected patients, An Estim. Acta Derm Venereol 72 (1992) 373e375. [4] L. Petukhova, M. Duvic, M. Hordinsky, et al., Genome-wide association study in alopecia areata implicates both innate and adaptive immunity, Nature 466 (2010) 113e117. [5] F. Benavides, T.M. Oberyszyn, A.M. Vanbuskirk, V.E. Reeve, D.F. Kusewitt, The hairless mouse in skin research, J. Dermatological Sci. 53 (2009) 10e18. [6] C.C. Thompson, Hairless is a nuclear receptor corepressor essential for skin function, Nucl. Recept. Signal 7 (2009) 1e11. [7] Y. Wen, Y. Liu, Y. Xu, et al., Loss-of-function mutations of an inhibitory upstream ORF in the human Hairless transcript cause Marie Unna hereditary hypotrichosis, Nat. Genet. 41 (2009) 228e233. [8] J.T. Zhang, S.G. Fang, C.Y. Wang, A novel nonsense mutation and polymorphisms in the mouse hairless gene, J. Invest. Dermatol 124 (2005) 1200e1205. [9] J.B. Hamilton, Patterned loss of hair in man; types and incidence, Ann. N.Y. Acad. Sci. 53 (1951) 708e728. [10] W. Ahmad, M. Faiyaz ul Haque, V. Brancolini, H.C. Tsou, S. ul Haque, et al., Alopecia universalis associated with a mutation in the human hairless gene, Science 279 (1998) 720e724. [11] K. Kure, T. Isago, T. Hirayama, Changes in the sebaceous gland in patients with male pattern hair loss (androgenic alopecia), J. Cosmet. Dermatol 14 (2015) 178e184. [12] J. Huelsken, R. Vogel, B. Erdmann, et al., b-catenin controls hair follicle morphogenesis and stem cell differentiation in the skin, Cell 105 (2001) 533e545. [13] N. Nanashima, K. Ito, T. Ishikawa, M. Nakano, T. Nakamura, Damage of hair follicle stem cells and alteration of keratin expression inexternal radiationinduced acute alopecia, Int. J. Mol. Med. 30 (2012) 579e584. [14] M. Devos, P. De Groote, B. Gilbert, I. Bruggeman, K. Leurs, S. Lippens, P. Vandenabeele, W. Declercq, Caspase-14 overexpression in hairless mice is not involved in utricle formation, Exp. Dermatol 22 (2013) 484e486. [15] C.P. Lu, L. Polak, B.E. Keyes, E. Fuchs, Spatiotemporal antagonism in mesenchymal-epithelial signaling in sweatversus hair fate decision, Science 354 (2016) 1551e1563. [16] C.C. Thompson, J.M. Sisk, G.M. Beaudoin 3rd, Hairless and Wnt signaling: allies in epithelial stem cell differentiation, Cell. Cycle 5 (2006) 1913e1917. [17] M.H. Hardy, The secret life of the hair follicle, Trends. Genet. 8 (1992) 55e61.
Please cite this article in press as: K. Zhu, et al., Hairless controls hair fate decision via Wnt/becatenin signaling, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.164