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β-catenin Correlation with Hair Follicle Activity in Cashmere Goat
Journal of Integrative Agriculture
July 2012
2012, 11(7): 1159-1166
RESEARCH ARTICLE
Hoxc13/β-catenin Correlation with Hair Follicle Activity in Cashmere Goat WU Jiang-hong1, 2*, ZHANG Yan-jun2*, ZHANG Jia-xin2, CHANG Zi-li1, LI Jin-quan1, YAN Zu-wei3, Husile 1 and ZHANG Wen-guang2 Key Laboratory of Animal Genetics, Breeding and Reproduction, Inner Mongolian Autonomous Region, Hohhot 010018, P.R.China College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, P.R.China 3 College of Science, Inner Mongolia Agricultural University, Huhhot 010018, P.R.China 1 2
Abstract Seasonal hair follicle activity and fibre growth in some Cashmere-bearing goats (Caprus hircus) is a cyclic process that is well characterized morphologically but understood incompletely at the molecular level. As an initial step in discovering regulators in hair-follicle activity and cycling, we used qPCR to investigate 19 genes expression in Cashmere goat side skin from 12 mon. Many of these genes may be associated with the hair follicle development-relevant genes (HFDRGs) in the literature. Here we show that Hoxc13/β-catenin gene associated with the follicle activity. In addition, Hoxc13 was found to be expressed with an drastic increase between July and November for melatonin treatments. To further investigate the role of Hoxc13 on HFDRGs, fibroblasts and keratinocytes from Cashmere goat skin were transfected with p-ECFPHoxc13. The result suggested that overexpression of Hoxc13 gene decreased HFDRGs with negative role for hair follicle development and increase HFDRGs with positive role for hair follicle development in vitro. These findings provide data on the Hoxc13 expression profile of normal Cashmere goat skin and Cashmere goat skin with melatonin treatment, and demonstrate hair-follicle-activity dependent regulation of Hoxc13 expression. Key words: Hoxc13, β-catenin, Cashmere goat, hair follicle activity, cycle
INTRODUCTION The hair cycle could be divided into quiescent and active states at the points of rapid transition (early proanagen and mid catagen) (Nixon 1993). Many mammalian species including goats exhibit seasonal changes in their pelages, characterized by a dense coat in winter and annual visible molting. These seasonal changes are in response to cyclical activation of the hair follicles (HFs), in which melatonin may modulates the seasonality of cashmere growth (Fischer et al. 2008). Melatonin, the chief secretory product of the pineal
gland, has long been known to modulate hair growth and/or molting in many species, presumably as a key neuroendocrine regulator that couples coat phenotype and function to photoperiod-dependent on environmental and reproductive changes (Fischer et al. 2008). The administration of melatonin to New Zealand goats resulted in increased melatonin blood levels, and this was associated with the transition of HFs from telogen (resting phase) into the growing pro-anagen phase; HFs of the untreated goats remained in the telogen stage (Nixon et al. 1993). Moreover, the secondary active follicle ratio was greater for treatments with melatonin administration, compared to the control in female Span-
Received 19 January, 2011 Accepted 14 June, 2011 WU Jiang-hong, E-mail: [email protected]; Correspondence ZHANG Wen-guang, E-mail: [email protected]; LI Jin-quan, Mobile: 15047996076, E-mail: [email protected] * These authors contributed equally to this study. © 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
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ish goats (Wuliji et al. 2006). However, the detailed effects and mechanisms of melatonin on the hair follicles regarding growth control have not yet been completely understood (Fischer et al. 2008). Hoxc13 belongs to the Abd-B class of Hox gene family, which participated in the hair follicle formation and hair growth regulation process (Wu et al. 2010). The first Hox gene shown to play a universal role in hair follicle development is Hoxc13, as both Hoxc13-deficient and overexpressing mice exhibit severe hair growth and patterning defects (Awgulewitsch 2003). Hoxc13 plays an important role in controlling transcription of hair keratins protein (KP) and hair keratin-associated protein (KAP) gene, and may have other roles in skin function (Pruett et al. 2004; Sander and Powell 2004; Jave-Suarez and Schweizer 2006). Our prior study indicated that changes of the Hoxc13 gene expression and thickness of skin have a similar trend during hair follicle morphogenesis in Cashmere goat (Wu et al. 2009). Previous studies have implicated members of the βcatenin signaling pathway as vital regulators of the epithelial-mesenchymal interactions that specify the development of HFs (Fuchs 2007). Documents show that those studies using embryos have revealed that embryonic HF fate change, HF differentiation and its excessive induction are induced by stabilized β-catenin (Narhi et al. 2008; Zhang et al. 2008). However, the role involved in the effects of β-catenin signaling to regulate HF fate are poorly understood in Cashmere goat. In this article, we identified genes showing hair follicle activity associated changes in expression within the skin. We first identified hair cycle-related genes
based on published papers in Cashmere goat. These hair follicle activity related genes were monitoring by quantitative real-time PCR. The result show that Hoxc13/β-catenin correlated with secondary hair follcile activity. Furthermore, we investigated the role of Hoxc13 gene and melatonin to hair follicle developmentrelevant genes (HFDRGs) in vitro.
RESULTS Hoxc13/β-catenin expression correlates with hair follicle activity Hair follicle activity was studied using histological sections from the skin of 30 adult doelings sampled every 4 wk for 12 mon in previous studies from our laboratory (Fig. 1-A) (Yin 2004). To identify genes showing expression changes related to the hair follicle activity, we used quantitative real-time PCR to profile mRNA expression in Cashmere goat skin of control from 12 mon during hair follicle cycle (Appendix A). Furthermore, we calculated the correlation coefficient between the expression level of HFDRGs and hair follicle activity (Appendix B). The result showed that Hoxc13 and β-catenin associated with the follicle activity of control (Fig. 1-B). The correlation coefficient between the expression level of Hoxc13/β-catenin and the second hair follicle is 0.84 (P<0.001), while 0.62 (P<0.05) for Hoxc13 and primary hair follicle. The correlation coefficient between the expression level of Hoxc13 and β-catenin is 0.86 (P<0.001).
Fig. 1 The hair follicle activity and the expression level of Hoxc13/β-catenin in Cashmere goat skin. A, hair follicle activity. Pf, primary hair follicle; Sf, second hair follicle. B, the expression level of Hoxc13/β-catenin. C, control. Data are mean±SD. The same as below.
© 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
Hoxc13/β -catenin Correlation with Hair Follicle Activity in Cashmere Goat
Melatonin increase Hoxc13 expression level during anagen We first examined the effects of melatonin on Hoxc13 expression level to understand the mechanism melatonin on the hair follicle. Hoxc13 was expressed weakly during telogen, with an increase in early anagen and mid and late anagen in control. However, Hoxc13 was found to be expressed with an drastic increase between July and November for melatonin treatments (Fig. 2). On the other hand, melatonin also changed the expression pattern of β-catenin gene in Cashmere goat skin.
Melatonin diversity affect to Hoxc13 expression in keratinocytes and fibroblasts To analyze Hoxc13 gene expression for fibroblasts and keratinocytes in skin of Cashmere goat. We examined the effects of melatonin pre-treatment on fibroblasts
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and keratinocytes to increase Hoxc13 expression level. Treatment of cells with 10-6 M melatonin last 48 h (Fig. 3). The result show that melatonin significantly increase the expression level of Hoxc13 in keratinocytes, while relatively stable in fibroblasts.
The gene expression level induced by Hoxc13 To further investigate the role of Hoxc13 on HFDRGs, fibroblasts and keratinocytes from Cashmere goat skin were transfected with p-ECFP-Hoxc13 or p-ECFP-c1 vector as control (Fig. 4). We found that overexpression Hoxc13 in keratinocytes of skin of Cashmere goat can cause apoptosis (data not shown). So we can not constructe the cell line of keratinocytes with transgene. qPCR analysis of HFDRGs, 10 HFDRGs, including Pdgfra, Fgf5, Pdgfa, Wnt10b, FRZB, TgfβrII, Rorα2, Rorc, Nanog, and KPII.1, were upregulated in the transgenic fibroblasts and 4 HFDRGs, including Msx2,
Fig. 2 The expression change of Hoxc13/β-catenin in Cashmere goat skin during hair follicle cycle. MI, melatonin implant. The same as below.
Delta, Bmp2, and Ntrk3, were downregulated in the transgenic fibroblasts (Fig. 5). In addition, several other HFDRGs, including β-catenin, BmprIb, Notch1, and Hr, were almost no significantly change in the transgenic fibroblasts.
DISCUSSION
Fig. 3 The effect of melatonin to Hoxc13 in skin cell. MT, melatonin treatment. * means P<0.05.
The cashmere produced by the secondary hair follicles of Inner Mongolia Cashmere goats is one of the softest,
© 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
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WU Jiang-hong et al.
Fig. 4 Overexpression vectors and detection of GFP/RT-PCR analysis. FT, transgenic fibroblasts; FC, control fibroblasts. The same as below.
finest and warmest materials for textile manufacturing. The fineness and length of the hair are crucial in determining its quality. It is significant to find key molecules involved in the development and cycling of skin hair follicles. Hair follicle activity was studied using histological sections from the skin of 30 doelings sampled every month for one year. The main period of secondary follicles grew fine, long, nonmedullated fibres (cashmere) from July to November. Shedding of these fibres from secondary follicles had commenced by December and cashmere was absent from the fleece by June. Annual pelage changes were therefore achieved with one main growth period, although many secondary follicles underwent another brief hair cycle in autumn (Yin 2004). We wanted to know the molecular mechanisms that constitute this process. Using qPCR, we searched for cyclic molecular expressions that correlate with hair cycle. Previous studies have found that Hoxc13 play an important role in controlling transcription of hair keratins protein (KP) and hair keratin-associated protein (KAP) gene in skin and appendage. However, the pattern of the Hoxc13 gene expression in skin of Cashmere goat during secondary hair cycle has not been ascertained until this report. In this paper, the expression of Hoxc13 gene significantly correlated with secondary hair follicle activity. Studies have already indicated that exogenous melatonin has a positive role on cashmere growth, resulting in increased cashmere production in some breeds (Teh et al. 1991; Jia 1996); the consistent con-
clusion was obtained in Chinese Inner Mongolia Cashmere goats (Wang et al. 2007). Our experiment supports the viewpoint that melatonin has an effect on Hoxc13 in skin of Cashmere goat, especially during anagen. The essential role of Wnt/β-catenin signaling during HF morphogenesis has been suggested by transgenic and knockout mouse studies. In this study, data show that β-catenin gene also correlated with hair follicle activity in Cashmere goat skin. Transient activation of β-catenin signalling in adult mouse epidermis is sufficient to induce new hair follicles (Lo Celso et al. 2004). Overexpression of β-catenin led to the upregulation of hair keratin 16 and 17 in vitro (Sohn et al. 2009). In this paper, expression levels of β-catenin show a declining trend in FT. The analysis of correlation coefficients in gene expressions suggested that the strength of the relationship among these genes. β-catenin is required genetically upstream of Bmp and Shh in placode formation and essential for fate decisions of skin stem cells (Huelsken et al. 2001). Here we show Hoxc13 downregulates Bmp2 expression in fibroblast cells in vitro. Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration, with high levels of BMP signaling being associated with the resting non-proliferative phase of HF growth cycle (Plikus et al. 2008). Altogether, both Hoxc13 and β-catenin can regulated hair keratin to responsed to the cycle of hair follicle cells. Additionally, in the hair follicle cycle, melatonin can affect the expression pattern of Hoxc13
© 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
Hoxc13/β -catenin Correlation with Hair Follicle Activity in Cashmere Goat
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Fig. 5 The expression of HFDRGs in transgenic fibroblasts and wild fibroblasts.
and β-catenin in skin. These results show that Hoxc13 and β-catenin may play an important role in modulating of melatonin on the hair follicle growth cycle. The molecular mechanisms that drive hair follicle cycling remain obscure in Cashmere goat. We wanted to know the molecular mechanisms that constitute this pattern. Using transgeic fibroblasts of skin in Cashmere goat with Hoxc13 and qPCR technology, we investigated the role of Hoxc13 gene to HFDRGs. We observed Msx2, Bmp2 and Ntrk3 to be downregulated by Hoxc13 in transgenic fibroblasts. Periodically expressed Bmp2 coordinate the function of hair in re-
sponse to the external environment. Mouse mutants have demonstrated that BMP antagonists (e.g., Noggin) act as anagen-inducing signals (Plikus et al. 2008). Bmp2 expression was also found to be higher in late telogen and early anagen phases and lower in late anagen in the goat’s skin and hair follicle (Su et al. 2009). When Msx2 is overexpressed in transgenic mice under the control of the CMV promoter, hairs are shorter and the matrix region is shrunken (Wang et al. 1999). ETV6NTRK3 chimeric tyrosine kinase could suppresses TGF-β signaling by directly binding to the type II TGFβ receptor, thereby preventing it from interacting with © 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
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the type I TGF-β receptor (Jin et al. 2005). At same time, we observed the TgfβrII gene to be upregulated by Hoxc13. We suggest that Hoxc13 gene may be an activator to TGF-β signaling. On the other hand, Pdgfra, Fgf5, Pdgfa, and Wnt10b to be upregulated by Hoxc13 in transgenic fibroblasts in our study. Pdgfra, Fgf5 and Pdgfa belong to MAPK pathway, and be activated simultaneously. In addition, TGF-β signaling is necessary to activate the MAPK pathway in the hair bud (Jamora et al. 2005). Furthermore, Wnt10b has an important role in the initiation of hair follicle development and following trichogenesis (Ouji et al. 2006). In sum, overexpression of Hoxc13 gene decreased HFDRGs with negative role for hair follicle development and increase HFDRGs with positive role for hair follicle development in vitro.
CONCLUSION In summary, we have investigated the role of Hoxc13 gene and melatonin to HFDRGs in vitro and in vivo for Cashmere goat. We find that Hoxc13/β-catenin gene associated with the follicle activity. In addition, Hoxc13 was found to be expressed with an drastic increase between July and November for melatonin treatments. Furthermore, the result suggested that overexpression of Hoxc13 gene decreased HFDRGs with negative role for hair follicle development and increase HFDRGs with positive role for hair follicle development in vitro. These findings provide data on the Hoxc13/β-catenin expression profile of normal Cashmere goat skin and Cashmere goat skin with melatonin treatment, and demonstrate hair-follicle-activity dependent regulation of Hoxc13/β-catenin expression.
(C); melatonin implant (MI). Silastic capsules (10 mm long) were filled with crystalline melatonin. Capsules were implanted under the skin on the ear. Control experiments consisted of implanting empty silastic capsules in place of the melatonin filled capsules. Prior to sample collection, procedures for skin sampling from adult animals were approved by the National Animal Care Standard (GB149252001). All samples were taken under supervision. Skin samples of 1 cm 2 were harvested from the side of the body per month in a year long, frozen in liquid nitrogen and stored at -80°C for analysis.
Cell culture The side of the body of adult female goat was shaved and sterilized with 70% alcohol before a piece of skin was excised. The skin tissue was rinsed three times in phosphate-buffered saline (PBS) containing antibiotics (penicillin G 100 U mL-1 and streptomycin 100 mg mL-1) and was minced. Explants were cultured in DMEM (Hyclone, Beijing, China) supplemented with 15% fetal bovine serum (FBS) and antibiotics (penicillin G 100 U mL-1 and streptomycin 100 mg mL-1) at 37°C in 5% CO2. While the explant cultures contained a mixed population of cells, fibroblasts were predominant. When the cells from the explants reached 70% confluency, they were removed with 0.05% trypsin-EDTA treatment, counted, and frozen into aliquots in 10% DMSO+90% FBS. Frozen aliquots of cells with a normal chromosome number were thawed and was transfected with the DNA construct to generate stable lines. Experiments with melatonin were performed under subdued light due to their sensitivity to light.
p-ECFP-Hoxc13 vector construction
MATERIALS AND METHODS
For expression of Hoxc13, the Cashmere goat complementary DNA was cloned into the p-ECFP-c1 cytomegalovirus (CMV)-based expression vector. Full-length Hoxc13 were obtained by overlap extension PCR. The full-length product amplified by flanking primers that can include restriction enzyme sites (Xba I and EcoR I) for inserting the product into p-ECFP-c1 for cloning purposes and was confirmed by DNA sequencing.
Animals
Gene transduction and expression
Inner Mongolia Cashmere goats from a goat stud farm (39.2°N and 107.2°E), Inner Mongolia, Erdos) were used for this study. Six doelings (1 yr old) were selected, shorn and randomly allotted to the two treatments (3 doelings per treatment), which were maintained together. Mean initial body weights were (24.5±1.2) kg. Treatments commenced on 22 June, 2009, and were as follows: control
Dermal fibroblasts cells were plated in six-well plates and cultured until reaching a subconfluent state. Cells were transfected with 1 µg of p-ECFP-Hoxc13 using Lipofectamine TM2000 (Invitrogen, Carlsbad, CA, USA) in normal growth medium. 3 d later, cells were plated on 10cm dishes and cultured until reaching a confluent state. After G418 selection, clones were subjected to RT-PCR.
© 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
Hoxc13/β -catenin Correlation with Hair Follicle Activity in Cashmere Goat
RNA isolation and cDNA synthesis Total RNA was extracted from skin (100 mg) or harvest cells (106) using RNAiso reagent (TaKaRa, Dalian, China). RNA concentration and purity were determined using the OD260:OD280 absorbance and ratios. Total RNA was used for cDNA synthesis. The reverse transcription (DRR037S, TaKaRa, Dalian, China) was carried out at 37°C for 15 min in a 20 µL reaction volume with 1 µL PrimeScriptTM RT Enzyme Mix I, 4 µL PrimeScriptTM buffer, 25 pmol of oligo(dT)18, 200 pmol of random 6 mers, and RNase-free H2O. The resulting reaction mix was incubated for 5 s at 85°C and then used as template in qRT-PCR.
Quantitative real-time PCR RNA levels were analysed using real-time rtPCR carried out on a Bio-Rad iQ5 Gradient Real-Time PCR (Bio-Rad Laboratories, Hercules, CA) using the PrimeScript ® RT Master Mix (TaKaRa, Dalian, China). Primers were designed according to Primer Premier 5 guidelines and all reactions were performed using Power SYBR Green PCR Master Mix (TaKaRa, Dalian, China), 250 nmol L-1 primers (TaKaRa, Dalian, China) and 100 ng cDNA in a 25 µL reaction volume. The qPCR conditions were: 95°C for 30 s, 40 cycles of 95°C for 5 s, 59°C for 15 s and 72°C for 20 s. Here, we analyzed the expression levels of 19 HFDRGs, which include Hoxc13, Msx2, Pdgfa, Pdgfra, Fgf5, Bmp2, Ntrk3, Wnt10b, β -catenin, BmprIb, Notch1, Tgf β rII, Hr, Rorα 2, Rorc, FRZB, Nanog, KPII.1, and Delta. The sequences of the primers used for amplification are listed in Appendix C. Three measurement replicates was performed to determine the expression level (critical threshold value) per sample, and the expression for each sample is normalized to the endogenous control gene, Gapdh. Relative expression levels were calculated with 2 -ΔΔCT method (Schmittgen and Livak 2008).
Statistical analysis The unpaired t-test and one-way ANOVA were used for statistical comparison. Calculations were made with the help of Microsoft Excel computer software (Microsoft, Redmond, WA, USA). P<0.05 was considered statistically significant.
Acknowledgements We thank Wu Ping and Fu Shaoyin, College of Animal Science, Inner Mongolia Agricultural University, China, for RNA extraction, Prof. Cao Jinshan, College of Animal Science, Inner Mongolia Agricultural University, for providing a lab to qPCR, the Inner Mongolia Cashmere goat stud farm staff for the provisions and help with sample
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collection. This research was conducted with financial support from the National Natural Science Foundation of China (30960246), the Key Project of National Science and Technology Pillar Program of China (2011BAD28B05), the National High Technology Research and Development Program of China (2007AA10Z151), the Specialized Research Fund for the Doctoral Program of Higher Education (20091515120010), the Inner Mongolia Natural Science Foundation, China (20080404ZD04), and the China Agriculture Research System (CARS-40). Appendix associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm
References Awgulewitsch A. 2003. Hox in hair growth and development. Naturwissenschaften, 90, 193-211. Fischer T W, Slominski A, Tobin D J, Paus R. 2008. Melatonin and the hair follicle. Journal of Pineal Research, 44, 1-15. Fuchs E. 2007. Scratching the surface of skin development. Nature, 445, 834-842. Huelsken J, Vogel R, Erdmann B, Cotsarelis G, Birchmeier W. 2001. β-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin. Cell, 105, 533545. Jamora C, Lee P, Kocieniewski P, Azhar M, Hosokawa R, Chai Y, Fuchs E. 2005. A signaling pathway involving TGF-β2 and snail in hair follicle morphogenesis. PLoS Biology, 3, e11. Jave-Suarez L F, Schweizer J, 2006. The HOXC13-controlled expression of early hair keratin genes in the human hair follicle does not involve TALE proteins MEIS and PREP as cofactors. Archives of Dermatological Research, 297, 372-376. Jia Z H, The T H, An M. 1996. Effects of exogenous melatonin on the productivity of cashmere goats. In: VI International Conference on Goats. International Academic Publishers, China. pp. 890-893. Jin W, Kim B C, Tognon C, Lee H J, Patel S, Lannon C L, Maris J M, Triche T J, Sorensen P H, Kim S J. 2005. The ETV6-NTRK3 chimeric tyrosine kinase suppresses TGFβ signaling by inactivating the TGF-β type II receptor. Proceedings of the National Academy of Sciences of the United States of America, 102, 16239-16244. Lo Celso C, Prowse D M, Watt F M. 2004. Transient activation of β-catenin signalling in adult mouse epidermis is sufficient to induce new hair follicles but continuous activation is required to maintain hair follicle tumours. Development, 131, 1787-1799. Narhi K, Jarvinen E, Birchmeier W, Taketo M M, Mikkola M L, Thesleff I. 2008. Sustained epithelial β-catenin activity induces precocious hair development but disrupts hair follicle down-growth and hair shaft formation. Development, 135, 1019-1028.
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Nixon A J. 1993. A method for determining the activity state of hair follicles. Biotechnic and Histochemistry, 68, 316325. Nixon A J, Choy V J, Parry A L, Pearson A J. 1993. Fiber growth initiation in hair follicles of goats treated with melatonin. Journal of Experimental Zoology, 267, 4756. Ouji Y, Yoshikawa M, Shiroi A, Ishizaka S. 2006. Promotion of hair follicle development and trichogenesis by Wnt10b in cultured embryonic skin and in reconstituted skin. Biochemical and Biophysical Research Communications, 345, 581-587. Plikus M V, Mayer J A, de la Cruz D, Baker R E, Maini P K, Maxson R, Chuong C M. 2008. Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration. Nature, 451, 340-344. Pruett N D, Tkatchenko T V, Jave-Suarez L, Jacobs D F, Potter C S, Tkatchenko A V, Schweizer J, Awgulewitsch A. 2004. Krtap16, characterization of a new hair keratinassociated protein (KAP) gene complex on mouse chromosome 16 and evidence for regulation by Hoxc13. Journal of Biological Chemistry, 279, 51524-51533. Sander G R, Powell B C. 2004. Structure and expression of the ovine Hoxc-13 gene. Gene, 327, 107-116. Schmittgen T D, Livak K J. 2008. Analyzing real-time PCR data by the comparative C(T) method. Nature Protocols, 3, 1101-1108. Sohn K C, Shi G, Jang S, Choi D K, Lee Y, Yoon T J, Park H, Hwang C, Kim H J, Seo Y J, et al. 2009. Pitx2, a β-cateninregulated transcription factor, regulates the differentiation of outer root sheath cells cultured in vitro. Journal of Dermatological Science, 54, 6-11. Su R, Zhang W G, Sharma R, Chang Z L, Yin J, Li J Q. 2009. Characterization of BMP2 gene expression in embryonic and adult Inner mongolia cashmere goat (Capra hircus) hair follicles. Canadian Journal of Animal Science,
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89, 457-462. Teh T H, Jia Z H, Odgen K D, Newtona G I. 1991. The effects of photoperiod and melatonin implant on cashmere production. Journal of Animal Science, 69, 496. Wang L, Lu D, Sun H, Zhao X, Shan D, Yang G, Fang L, 2007. Effects of photoperiod and melatonin on nitrogen partitioning and production performance of Inner mongolia white cashmere goats. Frontiers of Agriculture in China, 1, 229-236. Wang W P, Widelitz R B, Jiang T X, Chuong C M. 1999. Msx-2 and the regulation of organ size: epidermal thickness and hair length. Journal of Investigative Dermatology Symposium Proceedings, 4, 278-281. Wu J H, Yan Z W, Husile, Zhang W G, Li J Q. 2010. Hoxc13 and the development of hair follicle. Hereditas, 32, 656662. (in Chinese) Wu J H, Zhang W G, Li J G, Yin J, Zhang Y J. 2009. Hoxc13 expression pattern in cashmere goat skin during hair follicle development. Agricultural Sciences in China, 8, 491-496. Wuliji T, Litherland A, Goetsch A L, Sahlu T, Puchala R, Dawson L J, Gipson T. 2006. Evaluation of melatonin and bromocryptine administration in Spanish goats: III. Effects on hair follicle activity, density and relationships between follicle characteristics. Small Ruminant Research, 66, 11-21. Yin J. 2004. The research of hair follicle development, growth cycle and related genes in Inner Mongolia cashmere goat. Ph D thesis, Inner Mongolia University, Hohhot. (in Chinese) Zhang Y, Andl T, Yang S H, Teta M, Liu F, Seykora J T, Tobias J W, Piccolo S, Schmidt-Ullrich R, Nagy A, et al. 2008. Activation of β-catenin signaling programs embryonic epidermis to hair follicle fate. Development 135, 2161-2172. (Managing editor ZHANG Juan)
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July 2012
2012, 11(7): 1159-1166
RESEARCH ARTICLE
Hoxc13/β-catenin Correlation with Hair Follicle Activity in Cashmere Goat WU Jiang-hong1, 2*, ZHANG Yan-jun2*, ZHANG Jia-xin2, CHANG Zi-li1, LI Jin-quan1, YAN Zu-wei3, Husile 1 and ZHANG Wen-guang2 Key Laboratory of Animal Genetics, Breeding and Reproduction, Inner Mongolian Autonomous Region, Hohhot 010018, P.R.China College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, P.R.China 3 College of Science, Inner Mongolia Agricultural University, Huhhot 010018, P.R.China 1 2
Abstract Seasonal hair follicle activity and fibre growth in some Cashmere-bearing goats (Caprus hircus) is a cyclic process that is well characterized morphologically but understood incompletely at the molecular level. As an initial step in discovering regulators in hair-follicle activity and cycling, we used qPCR to investigate 19 genes expression in Cashmere goat side skin from 12 mon. Many of these genes may be associated with the hair follicle development-relevant genes (HFDRGs) in the literature. Here we show that Hoxc13/β-catenin gene associated with the follicle activity. In addition, Hoxc13 was found to be expressed with an drastic increase between July and November for melatonin treatments. To further investigate the role of Hoxc13 on HFDRGs, fibroblasts and keratinocytes from Cashmere goat skin were transfected with p-ECFPHoxc13. The result suggested that overexpression of Hoxc13 gene decreased HFDRGs with negative role for hair follicle development and increase HFDRGs with positive role for hair follicle development in vitro. These findings provide data on the Hoxc13 expression profile of normal Cashmere goat skin and Cashmere goat skin with melatonin treatment, and demonstrate hair-follicle-activity dependent regulation of Hoxc13 expression. Key words: Hoxc13, β-catenin, Cashmere goat, hair follicle activity, cycle
INTRODUCTION The hair cycle could be divided into quiescent and active states at the points of rapid transition (early proanagen and mid catagen) (Nixon 1993). Many mammalian species including goats exhibit seasonal changes in their pelages, characterized by a dense coat in winter and annual visible molting. These seasonal changes are in response to cyclical activation of the hair follicles (HFs), in which melatonin may modulates the seasonality of cashmere growth (Fischer et al. 2008). Melatonin, the chief secretory product of the pineal
gland, has long been known to modulate hair growth and/or molting in many species, presumably as a key neuroendocrine regulator that couples coat phenotype and function to photoperiod-dependent on environmental and reproductive changes (Fischer et al. 2008). The administration of melatonin to New Zealand goats resulted in increased melatonin blood levels, and this was associated with the transition of HFs from telogen (resting phase) into the growing pro-anagen phase; HFs of the untreated goats remained in the telogen stage (Nixon et al. 1993). Moreover, the secondary active follicle ratio was greater for treatments with melatonin administration, compared to the control in female Span-
Received 19 January, 2011 Accepted 14 June, 2011 WU Jiang-hong, E-mail: [email protected]; Correspondence ZHANG Wen-guang, E-mail: [email protected]; LI Jin-quan, Mobile: 15047996076, E-mail: [email protected] * These authors contributed equally to this study. © 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
WU Jiang-hong et al.
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ish goats (Wuliji et al. 2006). However, the detailed effects and mechanisms of melatonin on the hair follicles regarding growth control have not yet been completely understood (Fischer et al. 2008). Hoxc13 belongs to the Abd-B class of Hox gene family, which participated in the hair follicle formation and hair growth regulation process (Wu et al. 2010). The first Hox gene shown to play a universal role in hair follicle development is Hoxc13, as both Hoxc13-deficient and overexpressing mice exhibit severe hair growth and patterning defects (Awgulewitsch 2003). Hoxc13 plays an important role in controlling transcription of hair keratins protein (KP) and hair keratin-associated protein (KAP) gene, and may have other roles in skin function (Pruett et al. 2004; Sander and Powell 2004; Jave-Suarez and Schweizer 2006). Our prior study indicated that changes of the Hoxc13 gene expression and thickness of skin have a similar trend during hair follicle morphogenesis in Cashmere goat (Wu et al. 2009). Previous studies have implicated members of the βcatenin signaling pathway as vital regulators of the epithelial-mesenchymal interactions that specify the development of HFs (Fuchs 2007). Documents show that those studies using embryos have revealed that embryonic HF fate change, HF differentiation and its excessive induction are induced by stabilized β-catenin (Narhi et al. 2008; Zhang et al. 2008). However, the role involved in the effects of β-catenin signaling to regulate HF fate are poorly understood in Cashmere goat. In this article, we identified genes showing hair follicle activity associated changes in expression within the skin. We first identified hair cycle-related genes
based on published papers in Cashmere goat. These hair follicle activity related genes were monitoring by quantitative real-time PCR. The result show that Hoxc13/β-catenin correlated with secondary hair follcile activity. Furthermore, we investigated the role of Hoxc13 gene and melatonin to hair follicle developmentrelevant genes (HFDRGs) in vitro.
RESULTS Hoxc13/β-catenin expression correlates with hair follicle activity Hair follicle activity was studied using histological sections from the skin of 30 adult doelings sampled every 4 wk for 12 mon in previous studies from our laboratory (Fig. 1-A) (Yin 2004). To identify genes showing expression changes related to the hair follicle activity, we used quantitative real-time PCR to profile mRNA expression in Cashmere goat skin of control from 12 mon during hair follicle cycle (Appendix A). Furthermore, we calculated the correlation coefficient between the expression level of HFDRGs and hair follicle activity (Appendix B). The result showed that Hoxc13 and β-catenin associated with the follicle activity of control (Fig. 1-B). The correlation coefficient between the expression level of Hoxc13/β-catenin and the second hair follicle is 0.84 (P<0.001), while 0.62 (P<0.05) for Hoxc13 and primary hair follicle. The correlation coefficient between the expression level of Hoxc13 and β-catenin is 0.86 (P<0.001).
Fig. 1 The hair follicle activity and the expression level of Hoxc13/β-catenin in Cashmere goat skin. A, hair follicle activity. Pf, primary hair follicle; Sf, second hair follicle. B, the expression level of Hoxc13/β-catenin. C, control. Data are mean±SD. The same as below.
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Hoxc13/β -catenin Correlation with Hair Follicle Activity in Cashmere Goat
Melatonin increase Hoxc13 expression level during anagen We first examined the effects of melatonin on Hoxc13 expression level to understand the mechanism melatonin on the hair follicle. Hoxc13 was expressed weakly during telogen, with an increase in early anagen and mid and late anagen in control. However, Hoxc13 was found to be expressed with an drastic increase between July and November for melatonin treatments (Fig. 2). On the other hand, melatonin also changed the expression pattern of β-catenin gene in Cashmere goat skin.
Melatonin diversity affect to Hoxc13 expression in keratinocytes and fibroblasts To analyze Hoxc13 gene expression for fibroblasts and keratinocytes in skin of Cashmere goat. We examined the effects of melatonin pre-treatment on fibroblasts
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and keratinocytes to increase Hoxc13 expression level. Treatment of cells with 10-6 M melatonin last 48 h (Fig. 3). The result show that melatonin significantly increase the expression level of Hoxc13 in keratinocytes, while relatively stable in fibroblasts.
The gene expression level induced by Hoxc13 To further investigate the role of Hoxc13 on HFDRGs, fibroblasts and keratinocytes from Cashmere goat skin were transfected with p-ECFP-Hoxc13 or p-ECFP-c1 vector as control (Fig. 4). We found that overexpression Hoxc13 in keratinocytes of skin of Cashmere goat can cause apoptosis (data not shown). So we can not constructe the cell line of keratinocytes with transgene. qPCR analysis of HFDRGs, 10 HFDRGs, including Pdgfra, Fgf5, Pdgfa, Wnt10b, FRZB, TgfβrII, Rorα2, Rorc, Nanog, and KPII.1, were upregulated in the transgenic fibroblasts and 4 HFDRGs, including Msx2,
Fig. 2 The expression change of Hoxc13/β-catenin in Cashmere goat skin during hair follicle cycle. MI, melatonin implant. The same as below.
Delta, Bmp2, and Ntrk3, were downregulated in the transgenic fibroblasts (Fig. 5). In addition, several other HFDRGs, including β-catenin, BmprIb, Notch1, and Hr, were almost no significantly change in the transgenic fibroblasts.
DISCUSSION
Fig. 3 The effect of melatonin to Hoxc13 in skin cell. MT, melatonin treatment. * means P<0.05.
The cashmere produced by the secondary hair follicles of Inner Mongolia Cashmere goats is one of the softest,
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Fig. 4 Overexpression vectors and detection of GFP/RT-PCR analysis. FT, transgenic fibroblasts; FC, control fibroblasts. The same as below.
finest and warmest materials for textile manufacturing. The fineness and length of the hair are crucial in determining its quality. It is significant to find key molecules involved in the development and cycling of skin hair follicles. Hair follicle activity was studied using histological sections from the skin of 30 doelings sampled every month for one year. The main period of secondary follicles grew fine, long, nonmedullated fibres (cashmere) from July to November. Shedding of these fibres from secondary follicles had commenced by December and cashmere was absent from the fleece by June. Annual pelage changes were therefore achieved with one main growth period, although many secondary follicles underwent another brief hair cycle in autumn (Yin 2004). We wanted to know the molecular mechanisms that constitute this process. Using qPCR, we searched for cyclic molecular expressions that correlate with hair cycle. Previous studies have found that Hoxc13 play an important role in controlling transcription of hair keratins protein (KP) and hair keratin-associated protein (KAP) gene in skin and appendage. However, the pattern of the Hoxc13 gene expression in skin of Cashmere goat during secondary hair cycle has not been ascertained until this report. In this paper, the expression of Hoxc13 gene significantly correlated with secondary hair follicle activity. Studies have already indicated that exogenous melatonin has a positive role on cashmere growth, resulting in increased cashmere production in some breeds (Teh et al. 1991; Jia 1996); the consistent con-
clusion was obtained in Chinese Inner Mongolia Cashmere goats (Wang et al. 2007). Our experiment supports the viewpoint that melatonin has an effect on Hoxc13 in skin of Cashmere goat, especially during anagen. The essential role of Wnt/β-catenin signaling during HF morphogenesis has been suggested by transgenic and knockout mouse studies. In this study, data show that β-catenin gene also correlated with hair follicle activity in Cashmere goat skin. Transient activation of β-catenin signalling in adult mouse epidermis is sufficient to induce new hair follicles (Lo Celso et al. 2004). Overexpression of β-catenin led to the upregulation of hair keratin 16 and 17 in vitro (Sohn et al. 2009). In this paper, expression levels of β-catenin show a declining trend in FT. The analysis of correlation coefficients in gene expressions suggested that the strength of the relationship among these genes. β-catenin is required genetically upstream of Bmp and Shh in placode formation and essential for fate decisions of skin stem cells (Huelsken et al. 2001). Here we show Hoxc13 downregulates Bmp2 expression in fibroblast cells in vitro. Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration, with high levels of BMP signaling being associated with the resting non-proliferative phase of HF growth cycle (Plikus et al. 2008). Altogether, both Hoxc13 and β-catenin can regulated hair keratin to responsed to the cycle of hair follicle cells. Additionally, in the hair follicle cycle, melatonin can affect the expression pattern of Hoxc13
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Hoxc13/β -catenin Correlation with Hair Follicle Activity in Cashmere Goat
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Fig. 5 The expression of HFDRGs in transgenic fibroblasts and wild fibroblasts.
and β-catenin in skin. These results show that Hoxc13 and β-catenin may play an important role in modulating of melatonin on the hair follicle growth cycle. The molecular mechanisms that drive hair follicle cycling remain obscure in Cashmere goat. We wanted to know the molecular mechanisms that constitute this pattern. Using transgeic fibroblasts of skin in Cashmere goat with Hoxc13 and qPCR technology, we investigated the role of Hoxc13 gene to HFDRGs. We observed Msx2, Bmp2 and Ntrk3 to be downregulated by Hoxc13 in transgenic fibroblasts. Periodically expressed Bmp2 coordinate the function of hair in re-
sponse to the external environment. Mouse mutants have demonstrated that BMP antagonists (e.g., Noggin) act as anagen-inducing signals (Plikus et al. 2008). Bmp2 expression was also found to be higher in late telogen and early anagen phases and lower in late anagen in the goat’s skin and hair follicle (Su et al. 2009). When Msx2 is overexpressed in transgenic mice under the control of the CMV promoter, hairs are shorter and the matrix region is shrunken (Wang et al. 1999). ETV6NTRK3 chimeric tyrosine kinase could suppresses TGF-β signaling by directly binding to the type II TGFβ receptor, thereby preventing it from interacting with © 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
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the type I TGF-β receptor (Jin et al. 2005). At same time, we observed the TgfβrII gene to be upregulated by Hoxc13. We suggest that Hoxc13 gene may be an activator to TGF-β signaling. On the other hand, Pdgfra, Fgf5, Pdgfa, and Wnt10b to be upregulated by Hoxc13 in transgenic fibroblasts in our study. Pdgfra, Fgf5 and Pdgfa belong to MAPK pathway, and be activated simultaneously. In addition, TGF-β signaling is necessary to activate the MAPK pathway in the hair bud (Jamora et al. 2005). Furthermore, Wnt10b has an important role in the initiation of hair follicle development and following trichogenesis (Ouji et al. 2006). In sum, overexpression of Hoxc13 gene decreased HFDRGs with negative role for hair follicle development and increase HFDRGs with positive role for hair follicle development in vitro.
CONCLUSION In summary, we have investigated the role of Hoxc13 gene and melatonin to HFDRGs in vitro and in vivo for Cashmere goat. We find that Hoxc13/β-catenin gene associated with the follicle activity. In addition, Hoxc13 was found to be expressed with an drastic increase between July and November for melatonin treatments. Furthermore, the result suggested that overexpression of Hoxc13 gene decreased HFDRGs with negative role for hair follicle development and increase HFDRGs with positive role for hair follicle development in vitro. These findings provide data on the Hoxc13/β-catenin expression profile of normal Cashmere goat skin and Cashmere goat skin with melatonin treatment, and demonstrate hair-follicle-activity dependent regulation of Hoxc13/β-catenin expression.
(C); melatonin implant (MI). Silastic capsules (10 mm long) were filled with crystalline melatonin. Capsules were implanted under the skin on the ear. Control experiments consisted of implanting empty silastic capsules in place of the melatonin filled capsules. Prior to sample collection, procedures for skin sampling from adult animals were approved by the National Animal Care Standard (GB149252001). All samples were taken under supervision. Skin samples of 1 cm 2 were harvested from the side of the body per month in a year long, frozen in liquid nitrogen and stored at -80°C for analysis.
Cell culture The side of the body of adult female goat was shaved and sterilized with 70% alcohol before a piece of skin was excised. The skin tissue was rinsed three times in phosphate-buffered saline (PBS) containing antibiotics (penicillin G 100 U mL-1 and streptomycin 100 mg mL-1) and was minced. Explants were cultured in DMEM (Hyclone, Beijing, China) supplemented with 15% fetal bovine serum (FBS) and antibiotics (penicillin G 100 U mL-1 and streptomycin 100 mg mL-1) at 37°C in 5% CO2. While the explant cultures contained a mixed population of cells, fibroblasts were predominant. When the cells from the explants reached 70% confluency, they were removed with 0.05% trypsin-EDTA treatment, counted, and frozen into aliquots in 10% DMSO+90% FBS. Frozen aliquots of cells with a normal chromosome number were thawed and was transfected with the DNA construct to generate stable lines. Experiments with melatonin were performed under subdued light due to their sensitivity to light.
p-ECFP-Hoxc13 vector construction
MATERIALS AND METHODS
For expression of Hoxc13, the Cashmere goat complementary DNA was cloned into the p-ECFP-c1 cytomegalovirus (CMV)-based expression vector. Full-length Hoxc13 were obtained by overlap extension PCR. The full-length product amplified by flanking primers that can include restriction enzyme sites (Xba I and EcoR I) for inserting the product into p-ECFP-c1 for cloning purposes and was confirmed by DNA sequencing.
Animals
Gene transduction and expression
Inner Mongolia Cashmere goats from a goat stud farm (39.2°N and 107.2°E), Inner Mongolia, Erdos) were used for this study. Six doelings (1 yr old) were selected, shorn and randomly allotted to the two treatments (3 doelings per treatment), which were maintained together. Mean initial body weights were (24.5±1.2) kg. Treatments commenced on 22 June, 2009, and were as follows: control
Dermal fibroblasts cells were plated in six-well plates and cultured until reaching a subconfluent state. Cells were transfected with 1 µg of p-ECFP-Hoxc13 using Lipofectamine TM2000 (Invitrogen, Carlsbad, CA, USA) in normal growth medium. 3 d later, cells were plated on 10cm dishes and cultured until reaching a confluent state. After G418 selection, clones were subjected to RT-PCR.
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Hoxc13/β -catenin Correlation with Hair Follicle Activity in Cashmere Goat
RNA isolation and cDNA synthesis Total RNA was extracted from skin (100 mg) or harvest cells (106) using RNAiso reagent (TaKaRa, Dalian, China). RNA concentration and purity were determined using the OD260:OD280 absorbance and ratios. Total RNA was used for cDNA synthesis. The reverse transcription (DRR037S, TaKaRa, Dalian, China) was carried out at 37°C for 15 min in a 20 µL reaction volume with 1 µL PrimeScriptTM RT Enzyme Mix I, 4 µL PrimeScriptTM buffer, 25 pmol of oligo(dT)18, 200 pmol of random 6 mers, and RNase-free H2O. The resulting reaction mix was incubated for 5 s at 85°C and then used as template in qRT-PCR.
Quantitative real-time PCR RNA levels were analysed using real-time rtPCR carried out on a Bio-Rad iQ5 Gradient Real-Time PCR (Bio-Rad Laboratories, Hercules, CA) using the PrimeScript ® RT Master Mix (TaKaRa, Dalian, China). Primers were designed according to Primer Premier 5 guidelines and all reactions were performed using Power SYBR Green PCR Master Mix (TaKaRa, Dalian, China), 250 nmol L-1 primers (TaKaRa, Dalian, China) and 100 ng cDNA in a 25 µL reaction volume. The qPCR conditions were: 95°C for 30 s, 40 cycles of 95°C for 5 s, 59°C for 15 s and 72°C for 20 s. Here, we analyzed the expression levels of 19 HFDRGs, which include Hoxc13, Msx2, Pdgfa, Pdgfra, Fgf5, Bmp2, Ntrk3, Wnt10b, β -catenin, BmprIb, Notch1, Tgf β rII, Hr, Rorα 2, Rorc, FRZB, Nanog, KPII.1, and Delta. The sequences of the primers used for amplification are listed in Appendix C. Three measurement replicates was performed to determine the expression level (critical threshold value) per sample, and the expression for each sample is normalized to the endogenous control gene, Gapdh. Relative expression levels were calculated with 2 -ΔΔCT method (Schmittgen and Livak 2008).
Statistical analysis The unpaired t-test and one-way ANOVA were used for statistical comparison. Calculations were made with the help of Microsoft Excel computer software (Microsoft, Redmond, WA, USA). P<0.05 was considered statistically significant.
Acknowledgements We thank Wu Ping and Fu Shaoyin, College of Animal Science, Inner Mongolia Agricultural University, China, for RNA extraction, Prof. Cao Jinshan, College of Animal Science, Inner Mongolia Agricultural University, for providing a lab to qPCR, the Inner Mongolia Cashmere goat stud farm staff for the provisions and help with sample
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collection. This research was conducted with financial support from the National Natural Science Foundation of China (30960246), the Key Project of National Science and Technology Pillar Program of China (2011BAD28B05), the National High Technology Research and Development Program of China (2007AA10Z151), the Specialized Research Fund for the Doctoral Program of Higher Education (20091515120010), the Inner Mongolia Natural Science Foundation, China (20080404ZD04), and the China Agriculture Research System (CARS-40). Appendix associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm
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