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Expression and tissue distribution of hepatocyte growth factor (HGF) and its receptor (c-Met) in alpacas (Vicugna pacos) skins associated with white and brown coat colors
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Expression and tissue distribution of hepatocyte growth factor (HGF) and its receptor (c-Met) in alpacas (Vicugna pacos) skins associated with white and brown coat colors Xiuju Yu 1 , Xiaoyan He 1 , Junbing Jiang, Junping He, Ruiwen Fan, Haidong Wang, Jianjun Geng, Changsheng Dong ∗ College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi, 030801, PR China
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Article history: Received 19 March 2015 Received in revised form 28 May 2015 Accepted 4 June 2015 Available online xxx Keywords: HGF c-Met Alpaca Coat color Skin
a b s t r a c t Hepatocyte growth factor (HGF)/c-Met signaling has been considered as a key pathway in both melanocyte development and melanogenesis. To understand better the expression patterns and tissue distribution characterization of HGF and its receptor c-Met in skin of white versus brown alpaca (Vicugna pacos), we detected the tissue distribution of HGF and c-Met using immunohistochemistry and analyzed the expression patterns by using Western blot and quantitative real time PCR (qPCR). Immunohistochemistry analysis demonstrated that HGF staining robustly increased in the dermal papilla and mesenchymal cells of white alpaca skin compared with that of brown. However, c-Met staining showed strongly positive result, particularly inhair matrix and root sheath in brown alpaca skin. Western blot and qPCR results suggested that HGF and c-Met were expressed at significantly high levels in white and brown alpaca skins, respectively, and protein and transcripts possessed the same expression pattern in white and brown alpaca skins. The results suggested that HGF/c-Met signaling functions in alpaca coat color formation offer essential theoretical basis for further exploration of the role of HGF/c-Met signaling in pigment formation. © 2015 Elsevier GmbH. All rights reserved.
Introduction In adult animals, coat color is dependent on pigment-producing melanocytes that localize at the dermal border and transfer melanin-containing organelles to adjacent keratinocytes that are pushed upward as they proliferate (Barsh, 2007). Melanocyte generates two distinct types of melanin, namely, the yellow to reddish pheomelanins and the black to brown eumelanins (Ito and Wakamatsu, 2008, Pape et al., 2008). Melanin synthesis is regulated by more than 350 genes in mammals, including melanogenic enzymes, transcription factors, hormones, neurotransmitters, cytokines, and growth factors (Slominski et al., 2004, Yamaguchi and Hearing, 2009, Montoliu et al., 2015). However, the molecular and cellular mechanisms regulating coat color in woolproducing animal have not been completely elucidated. Alpacas have more than 22 natural coat colors; thus, the species is therefore suited for studies on the mechanism of
∗ Corresponding author. Tel.: +86 354 628 9208; fax: +86 354 628 9208. E-mail address: [email protected] (C. Dong). 1 The authors contributed equally to the work.
natural coat color formation (Lupton et al., 2006, Mcgregor, 2006). Previous studies, including those we conducted, demonstrated that the three pigmentation critical enzymes, namely, tyrosinase (Tyr), tyrosinase-related protein 1 (Tyrp1), tyrosinaserelated protein 2 (DCT), and the critical factors, which include melanocortin-1 receptor protein (Mc1r), inducible nitric oxide synthase, micropthalmia-associated transcription factor (MITF), solute carrier family 7 member 11(Slc7a11), and -catenin, were stimulators of melanogenesis in alpaca and agouti signaling proteins (Asip), miR-25 and miR-137 were inhibitors of melanogenesis in alpaca (Feeley and Munyard, 2009, Zhu, 2010, Chandramohan, 2013, Dong, 2012, Xue et al., 2014). Although the role of numerous genes in regulating the coat color of mice, dog, cat, and sheep has been verified (Kaelin and Barsh, 2013, Fan et al., 2013, Sturm, 2009, Montoliu et al., 2015), minimal knowledge on the potential role of hepatocyte growth factor (HGF)/c-Met signaling in the forming of alpaca coat color is available. HGF is a polypeptide growth factor consisting of heavy ␣ and light  chains linked by a disulfide bond; the receptor for HGF is a 145 kDa protein with tyrosine kinase activity that is the product of the c-Met proto-oncogene (Hirobe et al., 2004). In normal skin, c-Met present on epithelial cells and melanocytes
http://dx.doi.org/10.1016/j.acthis.2015.06.002 0065-1281/© 2015 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Yu X, et al. Expression and tissue distribution of hepatocyte growth factor (HGF) and its receptor (c-Met) in alpacas (Vicugna pacos) skins associated with white and brown coat colors. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acthis.2015.06.002
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control multiple aspects of melanocyte function in response to HGF, which is upregulated by ultraviolet radiation (UVR) exposure in keratinocytes and fibroblasts (Mildner, 2007, Soong, 2012). In normal melanocytes, HGF/c-Met signaling has been observed to play a role not only for survival, proliferation, and differentiation of melanocyte precursors, but also for melanogenesis by regulating Tyr and MITF (Kos, 1999, Mcgill, 2006, Cheli et al., 2010). Although previous evidence has indicated that HGF/c-Met has a function in melanocyte development and melanogenesis regulation, the expression and localization of HGF/c-Met in alpacas have not been fully elucidated. In this study, we observed the localization of HGF/c-Met protein within the skin tissue using immunohistochemistry and analyzed its transcript and protein expression level in the skin of white and brown alpacas. Results indicated that HGF/c-Met signaling may have a function in coat-color regulation and melanogenesis in alpacas.
polymerized HRP-conjugated goat anti-rabbit secondary antibody (Cat. No. bs-0295G; 1:100 in PBS, Beijing Biosynthesis Biotechnology Co., Beijing, China) at 37 ◦ C for 30 min, and then rinsed three times in PBS (5 min each). Immunoreactivity was visualized by incubating sections in the presence of 3,3 -diaminobenzidine (DAB, Beijing Biosynthesis Biotechnology Co., Beijing, China) substrate at RT for 5 min. The sections were then counterstained with hemotoxylin, and coverslips were sealed with neutral balsam. After the completion of immunostaining, sections were examined using a Leica DMIRB computerized microscope (Leica, Wetzlar, Germany) with a Leica digital camera DFC 320 attachment (Leica Microsystems Imaging Solutions Ltd., Cambridge, UK). Images were captured and stored for analysis. The SPSS statistical package version 17.0 (SPSS Inc., USA) was used to analyze all data. ANOVA testing was used to determine statistical differences in the data. All results were expressed as mean ± SD; P-values below 0.05 were considered statistically significant.
Materials and methods Animals and tissue collection Housing and care of alpacas and collection of skin samples that were used in the experiments were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals (http://www.cioms. ch/frame 1985 texts of guidelines.htm) and approved by the Animal Experimentation Ethics Committee of Shanxi Agricultural University, Taigu, China. Three pieces of skin were harvested from the hindquarters of each alpaca (3 white and 3 brown) by 8 mm (biopsy punches Miltex, Japan). Two biopsies were immediately stored in liquid nitrogen for RNA and protein extractions, and the third biopsy was fixed in Bouin’s solution for 24 h at 4 ◦ C and then extensively washed in 70% ethanol. Subsequently, the fixed samples were dehydrated in a graded series of ethanol (85%, 95%, and 100%), cleared in xylene, and embedded in paraffin wax. Sections of skin tissue (5 m) were prepared and mounted onto 2% 3-aminopropyltriethoxysilane-coated slides for immunohistochemical localization of HGF and c-Met. Immunohistochemistry of HGF and c-Met in the skin A polyclonal rabbit anti-HGF (or c-Met) antibody was utilized for the localization studies. All incubations were performed in a humidified chamber. Sections of paraffin-embedded skin were deparaffinized and rehydrated in a graded series of ethanol (100%, 95%, 90%, 85%, 80%, and 70%). Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 10 min. After three rinses (5 min each) in phosphate-buffered saline (PBS, pH 7.4), sections were incubated with 5% bovine serum albumin (BSA) in PBS at room temperature for 20 min to block the non-specific binding, then incubated overnight at 4 ◦ C in the presence of polyclonal rabbit anti-HGF (Cat. No. bs-1025R; 1:50 in PBS, Beijing Biosynthesis Biotechnology Co., Beijing, China) or anti-c-Met (Cat. No. bs-0668R; 1:50 in PBS, Beijing Biosynthesis Biotechnology Co., Beijing, China). For determination of non-specific staining, the primary antibody was replaced by non-immune bovine serum. After rinsing, HGF and c-Met staining was carried out according to the HGF and c-Met programs. In the HGF program, sections were incubated in biotinylated goat anti-rabbit second antibody (Boster, Wuhan, China) at 37 ◦ C for 30 min, and then rinsed three times in PBS (5 min each). Immunoreactivity was visualized by incubating sections in the presence of 5-Bromo-4-Chloro-3-Indolyl Phosphate/Nitroblue Tetrazolium (BCIP/NBT, 1:20, Boster, Wuhan, China) substrate at RT for 20 min. The sections were then rinsed and counterstained with nuclear fast red (Boster, Wuhan, China), and coverslips were sealed with neutral balsam. In the c-Met program, sections were incubated with
Western blot analysis of HGF and c-Met proteins Total protein was extracted from six alpaca full-thickness buttock skin samples (3 white and 3 brown) using a protein extraction kit (Beyotime, Beijing, China). The protein concentration was measured using the BCA protein assay kit (CWBIO, Beijing, China). Twenty five g of denatured protein from each sample were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% skimmed milk in TBST (150 mM NaCl, 10 mMTris pH 7.0, 0.05% Tween-20), and incubated with primary antibodies (HGF, 1:200 in TBST, bioss, China; c-Met, 1:200 in TBST, bioss, China; -actin, 1:1000 in TBST, CWBIO, and China) in 5% skimmed milk blocking buffer overnight at 4 ◦ C. Membranes were washed three times for 15 min with TBST, and incubated with goat anti-rabbit secondary antibody (1:10,000, CWBIO, China) for 2 h at RT. The membranes were again washed for 15 min with TBST. The ECL Western Blot Kit (CWBIO, Beijing, China) was used to detect the signal. The intensity of each protein was analyzed using ImageJ Software (National Institutes of Health, USA) and normalized to values obtained for -actin (Suppl. Table 1). All experiments were performed in triplicates.
qPCR analysis of HGF and c-Met mRNA Total RNA was isolated from six alpaca full-thickness buttock skin samples (3 white and 3 brown) using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA integrity was evaluated using 1% gel electrophoresis, and the concentration of total RNA was determined using the ND100 (NanoDrop Technologies, Wilmington, DE, USA). Subsequently, 1 g DNase-treated RNA of each sample was converted to cDNA using PrimeScript® RT Master Mix (Perfect Real Time) (TaKaRa, Dalian, China). The cDNA was used for the qPCR analysis of mRNA abundance using the gene specific forward and reverse primers (HGF-F, HGF-R, c-Met-F, c-Met-R). -actin served as a reference gene (primers are listed in Table 1). All reactions were performed in triplicates on the Stratagene Mx3005P qPCR system (Stratagene Agilent, USA). The 25 l PCR reactions used 12.5 l SYBR Premix Ex TaqTM II, 0.5 l forward primer (10 pM), 0.5 l reverse primer (10 pM), 0.5 l ROX reference dye, 2.0 l template, and 9 l water. The reactions were incubated in a 96-well plate at 95 ◦ C for 10 s, followed by 40 cycles at 95 ◦ C, 52 ◦ C, and 72 ◦ C for 5, 20, and 15 s. Abundance of HGF and c-Met mRNA was normalized relative to the abundance of -actin mRNA using the comparative threshold cycle method established by Livak (2001).
Please cite this article in press as: Yu X, et al. Expression and tissue distribution of hepatocyte growth factor (HGF) and its receptor (c-Met) in alpacas (Vicugna pacos) skins associated with white and brown coat colors. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acthis.2015.06.002
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Table 1 Primer sequences used in this study. Primer name
Sequence(5 –3 )
HGF
F: 5 -ACGCTACGAAGTCTGTGA-3 R: 5 -AAGAATTTGTGCCGGTGT-3
c-Met
F: 5 -TGAGAAGGCTAAAGGAAA-3 R: 5 -TCAAAGGCGTGGACATAC-3
-actin
F: 5 -ACCCTCATAGATGGGCACAG-3 R: 5 -AGCCATGTACGTAGCCATCC-3
Tm (◦ C)
Size(bp)
48.2 53.1 47.8 51.5 57.1 56.6
150 115 148
F: Sense primers; R: Antisense primers.
Results Localization of HGF and c-Met proteins in white and brown alpacas To investigate the distribution of HGF and c-Met protein, immunohistochemistry analysis was used on white and brown alpaca skins. As shown in Fig. 1, signals of HGF were detected only in the hair follicle mesenchyme (dermal papilla) and root sheath, and c-Met was localized in the epithelial cells, hair follicle matrix, and root sheath. No specific staining was found in the negative controls, where primary antibodies were replaced by nonimmune bovine serum (Figs. 1B, D, F, and H). The expression level of HGF/c-Met was robustly different between brown and white alpaca skins. HGF staining was significantly more positive in the dermal papilla of white alpaca skin compared with that of brown skin
(Fig. 1A). In brown alpaca skin, c-Met staining showed strongly positive result, particularly in the hair follicle matrix and root sheath (Fig. 1G).
Western blot assay of HGF and c-Met proteins between brown and white alpaca skins Protein levels of HGF and c-Met were determined using immunoblot analysis. As shown in Fig. 2A, HGF and c-Met protein were detected at 82 and 147 kDa in the total protein extracted from the white and brown alpaca skins, respectively. HGF protein in the brown alpaca skin significantly increased to as much as 2.00 folds of that detected in brown alpaca skin (Fig. 2B), whereas c-Met protein in the brown alpaca skin significantly increased to as much as 1.83 folds of that detected in white alpaca skin (Fig. 2C).
Fig. 1. Immunohistochemistry analysis of HGF and c-Met protein expression level in skin samples collected from the white and brown alpacas. (A) and (C): Expression of HGF in white and brown alpaca skin; (E) and (G): Expression of c-Met in white and brown alpaca skin; (B), (D), (F) and (H): Negative control group in white and brown alpaca skin. (1). hair shaft; (2). root sheath; (3). dermal papilla; (4): hair follicle matrix.
Please cite this article in press as: Yu X, et al. Expression and tissue distribution of hepatocyte growth factor (HGF) and its receptor (c-Met) in alpacas (Vicugna pacos) skins associated with white and brown coat colors. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acthis.2015.06.002
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Fig. 2. Western blot analysis of HGF and c-Met protein in skin samples collected from the white and brown alpacas. (A): Immunoblot results of HGF and c-Met in white and brown alpaca; (B): protein relative expression level of HGF in white and brown alpaca; (C): protein relative expression level of c-Met in white and brown alpaca; Bars in panels (B) and (C) represent the mean ± standard error (n — 3 each), * P < 0.05,** P < 0.01.
Expression profile of HGF and c-Met mRNA in white and brown alpaca skins Expression profiles of HGF and c-Met in alpaca skin with white and brown coat colors are shown in Fig. 3. Abundance of HGF tended to be higher in skin samples collected from alpacas with white coat color. However, abundance of c-Met was higher in skin samples collected from alpacas with brown coat color. The mRNA levels of HGF and c-Met were consistent with the protein expression levels determined from the skin samples of alpacas with white and brown coat colors. Discussion Coat color is of particular importance in alpacas because of the effect of color on fiber value (Cransberg et al., 2013). Coat color is defined by the number and melanogenic activity of follicular melanocytes, and the transfer of melanin to keratinocytes (Hoekstra and Genetics, 2006). Recently, progress has been attained in relation to the understanding of pigmentation in alpaca. However, the role of HGF/c-Met signaling in the formation of alpaca coat color has not been defined. HGF/c-Met signaling might also be required not only for normal melanocyte development (Kos, 1999), but also for maintenance of melanocyte-specific genes, such as Tyr in mice (Omoteno et al., 2000). We have obtained the partial sequence of HGF and c-Met in our previous study (Fan, 2011), and demonstrated that HGF/c-Met signaling might play some physiological roles in the alpaca skin. In this report, we investigated c-Met present on the hair follicle matrix and root sheath and revealed that c-Met staining in brown alpaca hair follicle matrix showed stronger positive result than that in the white hair follicle matrix. Consequently, the dependence of animal coat color on the pigment-producing melanocytes located in the hair follicle matrix and root sheath was confirmed. Moreover, the melanocytes
in the hair follicle matrix was found to contribute mainly to coat color formation, compared with the hair follicle matrix, the root sheath has extremely fewer melanocytes (Slominski et al., 2005, Tobin, 2011). Previous research demonstrated that HGF is one of the fibroblast-derived factors secreted from keratinocytes that stimulates pigmentation via their receptors on melanocytes (Yamaguchi and Hearing, 2009). The current study detected HGF in dermal papilla and mesenchymal cells, and found that HGF staining was significantly more positive in the dermal papilla of white alpaca skin compared with that of brown skin. Interestingly, Western blot and qPCR results showed that the protein and RNA levels of HGF and c-Met in white and brown alpaca skins were different. The protein level of HGF was higher in white than brown skin, whereas c-Met was higher in the skin of brown alpacas compared with those of white alpacas. qPCR results also demonstrated the same difference between white and brown alpaca skins. Abundance of HGF was higher in skin samples collected from alpacas with white coat color. However, abundance of c-Met was higher in skin samples collected from alpacas with brown coat color. Two reasons could account for this phenomenon. On the one hand, the HGF expression in white alpaca skin is more affected by ultraviolet (Wolnicka-Glubisz et al., 2015); thus, HGF content is high in white skin. On the other hand, the protein expression of c-Met is not induced by HGF (Beuret, 2007); thus, higher amount of melanin is not synthesized in white alpaca skin. In transgenic mice overexpression HGF, the number of hair follicles and the number of extrafollicular melanocytes in the dermis increase, but melanogenesis is not affected (WolnickaGlubisz, 2013). In summary, the expression of c-Met may be related with the brown coat color formation in alpaca. Thus, we speculate that the increased expression of c-Met in melanocytes may produce more pigments in alpacas, leading to the brown coat color formation. However, further research is needed to prove this speculation.
Fig. 3. Relative abundance of HGF and c-Met mRNA in skin samples collected from the white and brown alpacas. Abundance of HGF and c-Met were normalized relative to abundance of -actin. Bars in each panel represent the mean ± standard error(n — 3 each), ** P < 0.01, *** P < 0.001.
Please cite this article in press as: Yu X, et al. Expression and tissue distribution of hepatocyte growth factor (HGF) and its receptor (c-Met) in alpacas (Vicugna pacos) skins associated with white and brown coat colors. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acthis.2015.06.002
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Conclusions We detected the tissue distribution of HGF and c-Met through immunohistochemistry and analyzed the expression patterns using Western blot and qPCR. The results showed that signals of HGF are localized in dermal papilla and root sheath, and c-Met is localized in epithelial cells, hair follicle matrix, and root sheath. HGF has significantly high expression at the mRNA and protein levels in white alpaca skins. By contrast, c-Met has significantly high expression at the mRNA and protein levels in brown alpaca skins. Our results indicate that c-Met might have an important function in the regulation of coat color formation. Acknowledgments This research was sponsored by the National Natural Science Foundation of China (31172283), the Science and Technology Innovation Foundation of SXAU (201204) and the National High Technology Research and Development Program of China (863 Program) Project (2013AA102506). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.acthis.2015.06. 002 References Barsh G. How hair gets its pigment. Cell 2007;130:779–81. Beuret L. Up-regulation of MET expression by alpha-melanocyte-stimulating hormone and MITF allows hepatocyte growth factor to protect melanocytes and melanoma cells from apoptosis. J Biol Chem 2007;282:14140–7. Chandramohan B. The alpaca agouti gene: genomic locus, transcripts and causative mutations of eumelanic and pheomelanic coat color. Gene 2013;521:303–10. Cheli Y, Ohanna M, Ballotti R, Bertolotto C. Fifteen-year quest for microphthalmia-associated transcription factor target genes. Pigment Cell Melanoma Res 2010;23:27–40. Cransberg R, Wakamatsu K, Munyard K. Melanin characterisation suggests that the “brown” phenotype in alpaca (Vicugna pacos) is predominantly pheomelanic. Small Rumin Res 2013;114:240–6. Dong C. Coat color determination by mir-137 mediated down-regulation of microphthalmia-associated transcription factor in a mouse model. RNA Publ RNA Soc 2012;18:1679–86. Fan R. Gene expression profile in white alpaca (Vicugna pacos) skin. Animal 2011:5. Fan R, Xie J, Bai J, Wang H, Xue T, Rui B, et al. Skin transcriptome profiles associated with coat color in sheep. BMC Genomics 2013;14:3001–8. Feeley NL, Munyard KA. Characterisation of the melanocortin-1 receptor gene in alpaca and identification of possible markers associated with phenotypic variations in colour. Anim Prod Sci 2009;49:675–81.
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Hirobe T, Osawa M, Nishikawa S. Hepatocyte growth factor controls the proliferation of cultured epidermal melanoblasts and melanocytes from newborn mice. Pigment Cell Res 2004;17:51–61. Hoekstra HE. Genetics development and evolution of adaptive pigmentation in vertebrates. Heredity 2006;97:222–34, http://dx.doi.org/10.1038/sj.hdy.6800861. Ito S, Wakamatsu K. Chemistry of mixed melanogenesis-pivotal roles of dopaquinone 69. Photochem Photobiol 2008;84:582–92. Kaelin CB, Barsh GS. Genetics of pigmentation in dogs and cats. Annu Rev Anim Biosci 2013;1:125–56. Kos L. Hepatocyte growth factor/scatter factor-MET signaling in neural crest-derived melanocyte development. Pigment Cell Res 1999;12:13–21. Livak KJ. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402–8. Lupton CJ, Mccoll A, Stobart RH. Fiber characteristics of the Huacaya Alpaca. Small Rumin Res 2006;64:211–24. Mcgill GG. c-Met expression is regulated by Mitf in the melanocyte lineage. J Biol Chem 2006;281:10,365–73. Mcgregor BA. Production, attributes and relative value of alpaca fleeces in southern Australia and implications for industry development. Small Rumin Res 2006;61:93–111. Mildner M. Hepatocyte growth factor establishes autocrine and paracrine feedback loops for the protection of skin cells after UV irradiation. J Investig Dermatol 2007;127:2637–44. Montoliu L, Oetting W, Bennett D. Color Genes. 2015. European Society for Pigment Cell Research. World Wide Web (URL: http://wwwespcrorg/micemut). Omoteno M, Tagaya H, Hemmi H. Keratinocyte expression of transgenic hepatocyte growth factor affects melanocyte development, leading to dermal melanocytosis. Mech Dev 2000;94(12):67–78. Pape EL, Wakamatsu K, Ito S, Wolber R, Hearing VJ. Regulation of eumelanin/pheomelanin synthesis and visible pigmentation in melanocytes by ligands of the melanocortin 1 receptor. Pigment Cell Melanoma Res 2008;21:477–86. Slominski A, Tobin DJ, Shibahara S, Wortsman J. Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev 2004;84: 1155–228. Slominski A, Wortsman J, Plonka PM, Schallreuter KU, Paus R, Dj. T. Hair follicle pigmentation. J Investig Dermatol 2005;124:13–21. Soong J. Sema4D, the ligand for Plexin B1, suppresses c-Met activation and migration and promotes melanocyte survival and growth. J Investig Dermatol 2012;132:1230–8. Sturm RA. Molecular genetics of human pigmentation diversity. Hum Mol Genet 2009;18:2920–3020. Tobin DJ. The cell biology of human hair follicle pigmentation. Pigment Cell Melanoma Res 2011;24:75–88. Wolnicka-Glubisz A. HGF/SF increases number of skin melanocytes but does not alter quality or quantity of follicular melanogenesis. Plos One 2013;8:e74883. Wolnicka-Glubisz A, Strickland FM, Wielgus A, Anver M, Merlino G, Ec DF, et al. A melanin-independent interaction between Mc1r and Met signaling pathways is required for HGF-dependent melanoma. Int J Cancer J Int Cancer 2015;136:752–60. Xue T, Meng X, Wang L, Song Y, Zhang D, Ji Y, et al. Molecular cloning, mRNA expression and tissue distribution analysis of Slc7a11 gene in alpaca (Lama paco) skins associated with different coat colors. Gene 2014;555:88–94. Yamaguchi Y, Hearing VJ. Physiological factors that regulate skin pigmentation. Biofactors 2009;35:193–9. Zhu Z. MicroRNA-25 functions in regulation of pigmentation by targeting the transcription factor MITF in Alpaca (Lama pacos) skin melanocytes. Domest Anim Endocrinol 2010;38:200–9.
Please cite this article in press as: Yu X, et al. Expression and tissue distribution of hepatocyte growth factor (HGF) and its receptor (c-Met) in alpacas (Vicugna pacos) skins associated with white and brown coat colors. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acthis.2015.06.002
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Contents lists available at ScienceDirect
Acta Histochemica journal homepage: www.elsevier.de/acthis
Expression and tissue distribution of hepatocyte growth factor (HGF) and its receptor (c-Met) in alpacas (Vicugna pacos) skins associated with white and brown coat colors Xiuju Yu 1 , Xiaoyan He 1 , Junbing Jiang, Junping He, Ruiwen Fan, Haidong Wang, Jianjun Geng, Changsheng Dong ∗ College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi, 030801, PR China
a r t i c l e
i n f o
Article history: Received 19 March 2015 Received in revised form 28 May 2015 Accepted 4 June 2015 Available online xxx Keywords: HGF c-Met Alpaca Coat color Skin
a b s t r a c t Hepatocyte growth factor (HGF)/c-Met signaling has been considered as a key pathway in both melanocyte development and melanogenesis. To understand better the expression patterns and tissue distribution characterization of HGF and its receptor c-Met in skin of white versus brown alpaca (Vicugna pacos), we detected the tissue distribution of HGF and c-Met using immunohistochemistry and analyzed the expression patterns by using Western blot and quantitative real time PCR (qPCR). Immunohistochemistry analysis demonstrated that HGF staining robustly increased in the dermal papilla and mesenchymal cells of white alpaca skin compared with that of brown. However, c-Met staining showed strongly positive result, particularly inhair matrix and root sheath in brown alpaca skin. Western blot and qPCR results suggested that HGF and c-Met were expressed at significantly high levels in white and brown alpaca skins, respectively, and protein and transcripts possessed the same expression pattern in white and brown alpaca skins. The results suggested that HGF/c-Met signaling functions in alpaca coat color formation offer essential theoretical basis for further exploration of the role of HGF/c-Met signaling in pigment formation. © 2015 Elsevier GmbH. All rights reserved.
Introduction In adult animals, coat color is dependent on pigment-producing melanocytes that localize at the dermal border and transfer melanin-containing organelles to adjacent keratinocytes that are pushed upward as they proliferate (Barsh, 2007). Melanocyte generates two distinct types of melanin, namely, the yellow to reddish pheomelanins and the black to brown eumelanins (Ito and Wakamatsu, 2008, Pape et al., 2008). Melanin synthesis is regulated by more than 350 genes in mammals, including melanogenic enzymes, transcription factors, hormones, neurotransmitters, cytokines, and growth factors (Slominski et al., 2004, Yamaguchi and Hearing, 2009, Montoliu et al., 2015). However, the molecular and cellular mechanisms regulating coat color in woolproducing animal have not been completely elucidated. Alpacas have more than 22 natural coat colors; thus, the species is therefore suited for studies on the mechanism of
∗ Corresponding author. Tel.: +86 354 628 9208; fax: +86 354 628 9208. E-mail address: [email protected] (C. Dong). 1 The authors contributed equally to the work.
natural coat color formation (Lupton et al., 2006, Mcgregor, 2006). Previous studies, including those we conducted, demonstrated that the three pigmentation critical enzymes, namely, tyrosinase (Tyr), tyrosinase-related protein 1 (Tyrp1), tyrosinaserelated protein 2 (DCT), and the critical factors, which include melanocortin-1 receptor protein (Mc1r), inducible nitric oxide synthase, micropthalmia-associated transcription factor (MITF), solute carrier family 7 member 11(Slc7a11), and -catenin, were stimulators of melanogenesis in alpaca and agouti signaling proteins (Asip), miR-25 and miR-137 were inhibitors of melanogenesis in alpaca (Feeley and Munyard, 2009, Zhu, 2010, Chandramohan, 2013, Dong, 2012, Xue et al., 2014). Although the role of numerous genes in regulating the coat color of mice, dog, cat, and sheep has been verified (Kaelin and Barsh, 2013, Fan et al., 2013, Sturm, 2009, Montoliu et al., 2015), minimal knowledge on the potential role of hepatocyte growth factor (HGF)/c-Met signaling in the forming of alpaca coat color is available. HGF is a polypeptide growth factor consisting of heavy ␣ and light  chains linked by a disulfide bond; the receptor for HGF is a 145 kDa protein with tyrosine kinase activity that is the product of the c-Met proto-oncogene (Hirobe et al., 2004). In normal skin, c-Met present on epithelial cells and melanocytes
http://dx.doi.org/10.1016/j.acthis.2015.06.002 0065-1281/© 2015 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Yu X, et al. Expression and tissue distribution of hepatocyte growth factor (HGF) and its receptor (c-Met) in alpacas (Vicugna pacos) skins associated with white and brown coat colors. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acthis.2015.06.002
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control multiple aspects of melanocyte function in response to HGF, which is upregulated by ultraviolet radiation (UVR) exposure in keratinocytes and fibroblasts (Mildner, 2007, Soong, 2012). In normal melanocytes, HGF/c-Met signaling has been observed to play a role not only for survival, proliferation, and differentiation of melanocyte precursors, but also for melanogenesis by regulating Tyr and MITF (Kos, 1999, Mcgill, 2006, Cheli et al., 2010). Although previous evidence has indicated that HGF/c-Met has a function in melanocyte development and melanogenesis regulation, the expression and localization of HGF/c-Met in alpacas have not been fully elucidated. In this study, we observed the localization of HGF/c-Met protein within the skin tissue using immunohistochemistry and analyzed its transcript and protein expression level in the skin of white and brown alpacas. Results indicated that HGF/c-Met signaling may have a function in coat-color regulation and melanogenesis in alpacas.
polymerized HRP-conjugated goat anti-rabbit secondary antibody (Cat. No. bs-0295G; 1:100 in PBS, Beijing Biosynthesis Biotechnology Co., Beijing, China) at 37 ◦ C for 30 min, and then rinsed three times in PBS (5 min each). Immunoreactivity was visualized by incubating sections in the presence of 3,3 -diaminobenzidine (DAB, Beijing Biosynthesis Biotechnology Co., Beijing, China) substrate at RT for 5 min. The sections were then counterstained with hemotoxylin, and coverslips were sealed with neutral balsam. After the completion of immunostaining, sections were examined using a Leica DMIRB computerized microscope (Leica, Wetzlar, Germany) with a Leica digital camera DFC 320 attachment (Leica Microsystems Imaging Solutions Ltd., Cambridge, UK). Images were captured and stored for analysis. The SPSS statistical package version 17.0 (SPSS Inc., USA) was used to analyze all data. ANOVA testing was used to determine statistical differences in the data. All results were expressed as mean ± SD; P-values below 0.05 were considered statistically significant.
Materials and methods Animals and tissue collection Housing and care of alpacas and collection of skin samples that were used in the experiments were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals (http://www.cioms. ch/frame 1985 texts of guidelines.htm) and approved by the Animal Experimentation Ethics Committee of Shanxi Agricultural University, Taigu, China. Three pieces of skin were harvested from the hindquarters of each alpaca (3 white and 3 brown) by 8 mm (biopsy punches Miltex, Japan). Two biopsies were immediately stored in liquid nitrogen for RNA and protein extractions, and the third biopsy was fixed in Bouin’s solution for 24 h at 4 ◦ C and then extensively washed in 70% ethanol. Subsequently, the fixed samples were dehydrated in a graded series of ethanol (85%, 95%, and 100%), cleared in xylene, and embedded in paraffin wax. Sections of skin tissue (5 m) were prepared and mounted onto 2% 3-aminopropyltriethoxysilane-coated slides for immunohistochemical localization of HGF and c-Met. Immunohistochemistry of HGF and c-Met in the skin A polyclonal rabbit anti-HGF (or c-Met) antibody was utilized for the localization studies. All incubations were performed in a humidified chamber. Sections of paraffin-embedded skin were deparaffinized and rehydrated in a graded series of ethanol (100%, 95%, 90%, 85%, 80%, and 70%). Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 10 min. After three rinses (5 min each) in phosphate-buffered saline (PBS, pH 7.4), sections were incubated with 5% bovine serum albumin (BSA) in PBS at room temperature for 20 min to block the non-specific binding, then incubated overnight at 4 ◦ C in the presence of polyclonal rabbit anti-HGF (Cat. No. bs-1025R; 1:50 in PBS, Beijing Biosynthesis Biotechnology Co., Beijing, China) or anti-c-Met (Cat. No. bs-0668R; 1:50 in PBS, Beijing Biosynthesis Biotechnology Co., Beijing, China). For determination of non-specific staining, the primary antibody was replaced by non-immune bovine serum. After rinsing, HGF and c-Met staining was carried out according to the HGF and c-Met programs. In the HGF program, sections were incubated in biotinylated goat anti-rabbit second antibody (Boster, Wuhan, China) at 37 ◦ C for 30 min, and then rinsed three times in PBS (5 min each). Immunoreactivity was visualized by incubating sections in the presence of 5-Bromo-4-Chloro-3-Indolyl Phosphate/Nitroblue Tetrazolium (BCIP/NBT, 1:20, Boster, Wuhan, China) substrate at RT for 20 min. The sections were then rinsed and counterstained with nuclear fast red (Boster, Wuhan, China), and coverslips were sealed with neutral balsam. In the c-Met program, sections were incubated with
Western blot analysis of HGF and c-Met proteins Total protein was extracted from six alpaca full-thickness buttock skin samples (3 white and 3 brown) using a protein extraction kit (Beyotime, Beijing, China). The protein concentration was measured using the BCA protein assay kit (CWBIO, Beijing, China). Twenty five g of denatured protein from each sample were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% skimmed milk in TBST (150 mM NaCl, 10 mMTris pH 7.0, 0.05% Tween-20), and incubated with primary antibodies (HGF, 1:200 in TBST, bioss, China; c-Met, 1:200 in TBST, bioss, China; -actin, 1:1000 in TBST, CWBIO, and China) in 5% skimmed milk blocking buffer overnight at 4 ◦ C. Membranes were washed three times for 15 min with TBST, and incubated with goat anti-rabbit secondary antibody (1:10,000, CWBIO, China) for 2 h at RT. The membranes were again washed for 15 min with TBST. The ECL Western Blot Kit (CWBIO, Beijing, China) was used to detect the signal. The intensity of each protein was analyzed using ImageJ Software (National Institutes of Health, USA) and normalized to values obtained for -actin (Suppl. Table 1). All experiments were performed in triplicates.
qPCR analysis of HGF and c-Met mRNA Total RNA was isolated from six alpaca full-thickness buttock skin samples (3 white and 3 brown) using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA integrity was evaluated using 1% gel electrophoresis, and the concentration of total RNA was determined using the ND100 (NanoDrop Technologies, Wilmington, DE, USA). Subsequently, 1 g DNase-treated RNA of each sample was converted to cDNA using PrimeScript® RT Master Mix (Perfect Real Time) (TaKaRa, Dalian, China). The cDNA was used for the qPCR analysis of mRNA abundance using the gene specific forward and reverse primers (HGF-F, HGF-R, c-Met-F, c-Met-R). -actin served as a reference gene (primers are listed in Table 1). All reactions were performed in triplicates on the Stratagene Mx3005P qPCR system (Stratagene Agilent, USA). The 25 l PCR reactions used 12.5 l SYBR Premix Ex TaqTM II, 0.5 l forward primer (10 pM), 0.5 l reverse primer (10 pM), 0.5 l ROX reference dye, 2.0 l template, and 9 l water. The reactions were incubated in a 96-well plate at 95 ◦ C for 10 s, followed by 40 cycles at 95 ◦ C, 52 ◦ C, and 72 ◦ C for 5, 20, and 15 s. Abundance of HGF and c-Met mRNA was normalized relative to the abundance of -actin mRNA using the comparative threshold cycle method established by Livak (2001).
Please cite this article in press as: Yu X, et al. Expression and tissue distribution of hepatocyte growth factor (HGF) and its receptor (c-Met) in alpacas (Vicugna pacos) skins associated with white and brown coat colors. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acthis.2015.06.002
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Table 1 Primer sequences used in this study. Primer name
Sequence(5 –3 )
HGF
F: 5 -ACGCTACGAAGTCTGTGA-3 R: 5 -AAGAATTTGTGCCGGTGT-3
c-Met
F: 5 -TGAGAAGGCTAAAGGAAA-3 R: 5 -TCAAAGGCGTGGACATAC-3
-actin
F: 5 -ACCCTCATAGATGGGCACAG-3 R: 5 -AGCCATGTACGTAGCCATCC-3
Tm (◦ C)
Size(bp)
48.2 53.1 47.8 51.5 57.1 56.6
150 115 148
F: Sense primers; R: Antisense primers.
Results Localization of HGF and c-Met proteins in white and brown alpacas To investigate the distribution of HGF and c-Met protein, immunohistochemistry analysis was used on white and brown alpaca skins. As shown in Fig. 1, signals of HGF were detected only in the hair follicle mesenchyme (dermal papilla) and root sheath, and c-Met was localized in the epithelial cells, hair follicle matrix, and root sheath. No specific staining was found in the negative controls, where primary antibodies were replaced by nonimmune bovine serum (Figs. 1B, D, F, and H). The expression level of HGF/c-Met was robustly different between brown and white alpaca skins. HGF staining was significantly more positive in the dermal papilla of white alpaca skin compared with that of brown skin
(Fig. 1A). In brown alpaca skin, c-Met staining showed strongly positive result, particularly in the hair follicle matrix and root sheath (Fig. 1G).
Western blot assay of HGF and c-Met proteins between brown and white alpaca skins Protein levels of HGF and c-Met were determined using immunoblot analysis. As shown in Fig. 2A, HGF and c-Met protein were detected at 82 and 147 kDa in the total protein extracted from the white and brown alpaca skins, respectively. HGF protein in the brown alpaca skin significantly increased to as much as 2.00 folds of that detected in brown alpaca skin (Fig. 2B), whereas c-Met protein in the brown alpaca skin significantly increased to as much as 1.83 folds of that detected in white alpaca skin (Fig. 2C).
Fig. 1. Immunohistochemistry analysis of HGF and c-Met protein expression level in skin samples collected from the white and brown alpacas. (A) and (C): Expression of HGF in white and brown alpaca skin; (E) and (G): Expression of c-Met in white and brown alpaca skin; (B), (D), (F) and (H): Negative control group in white and brown alpaca skin. (1). hair shaft; (2). root sheath; (3). dermal papilla; (4): hair follicle matrix.
Please cite this article in press as: Yu X, et al. Expression and tissue distribution of hepatocyte growth factor (HGF) and its receptor (c-Met) in alpacas (Vicugna pacos) skins associated with white and brown coat colors. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acthis.2015.06.002
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Fig. 2. Western blot analysis of HGF and c-Met protein in skin samples collected from the white and brown alpacas. (A): Immunoblot results of HGF and c-Met in white and brown alpaca; (B): protein relative expression level of HGF in white and brown alpaca; (C): protein relative expression level of c-Met in white and brown alpaca; Bars in panels (B) and (C) represent the mean ± standard error (n — 3 each), * P < 0.05,** P < 0.01.
Expression profile of HGF and c-Met mRNA in white and brown alpaca skins Expression profiles of HGF and c-Met in alpaca skin with white and brown coat colors are shown in Fig. 3. Abundance of HGF tended to be higher in skin samples collected from alpacas with white coat color. However, abundance of c-Met was higher in skin samples collected from alpacas with brown coat color. The mRNA levels of HGF and c-Met were consistent with the protein expression levels determined from the skin samples of alpacas with white and brown coat colors. Discussion Coat color is of particular importance in alpacas because of the effect of color on fiber value (Cransberg et al., 2013). Coat color is defined by the number and melanogenic activity of follicular melanocytes, and the transfer of melanin to keratinocytes (Hoekstra and Genetics, 2006). Recently, progress has been attained in relation to the understanding of pigmentation in alpaca. However, the role of HGF/c-Met signaling in the formation of alpaca coat color has not been defined. HGF/c-Met signaling might also be required not only for normal melanocyte development (Kos, 1999), but also for maintenance of melanocyte-specific genes, such as Tyr in mice (Omoteno et al., 2000). We have obtained the partial sequence of HGF and c-Met in our previous study (Fan, 2011), and demonstrated that HGF/c-Met signaling might play some physiological roles in the alpaca skin. In this report, we investigated c-Met present on the hair follicle matrix and root sheath and revealed that c-Met staining in brown alpaca hair follicle matrix showed stronger positive result than that in the white hair follicle matrix. Consequently, the dependence of animal coat color on the pigment-producing melanocytes located in the hair follicle matrix and root sheath was confirmed. Moreover, the melanocytes
in the hair follicle matrix was found to contribute mainly to coat color formation, compared with the hair follicle matrix, the root sheath has extremely fewer melanocytes (Slominski et al., 2005, Tobin, 2011). Previous research demonstrated that HGF is one of the fibroblast-derived factors secreted from keratinocytes that stimulates pigmentation via their receptors on melanocytes (Yamaguchi and Hearing, 2009). The current study detected HGF in dermal papilla and mesenchymal cells, and found that HGF staining was significantly more positive in the dermal papilla of white alpaca skin compared with that of brown skin. Interestingly, Western blot and qPCR results showed that the protein and RNA levels of HGF and c-Met in white and brown alpaca skins were different. The protein level of HGF was higher in white than brown skin, whereas c-Met was higher in the skin of brown alpacas compared with those of white alpacas. qPCR results also demonstrated the same difference between white and brown alpaca skins. Abundance of HGF was higher in skin samples collected from alpacas with white coat color. However, abundance of c-Met was higher in skin samples collected from alpacas with brown coat color. Two reasons could account for this phenomenon. On the one hand, the HGF expression in white alpaca skin is more affected by ultraviolet (Wolnicka-Glubisz et al., 2015); thus, HGF content is high in white skin. On the other hand, the protein expression of c-Met is not induced by HGF (Beuret, 2007); thus, higher amount of melanin is not synthesized in white alpaca skin. In transgenic mice overexpression HGF, the number of hair follicles and the number of extrafollicular melanocytes in the dermis increase, but melanogenesis is not affected (WolnickaGlubisz, 2013). In summary, the expression of c-Met may be related with the brown coat color formation in alpaca. Thus, we speculate that the increased expression of c-Met in melanocytes may produce more pigments in alpacas, leading to the brown coat color formation. However, further research is needed to prove this speculation.
Fig. 3. Relative abundance of HGF and c-Met mRNA in skin samples collected from the white and brown alpacas. Abundance of HGF and c-Met were normalized relative to abundance of -actin. Bars in each panel represent the mean ± standard error(n — 3 each), ** P < 0.01, *** P < 0.001.
Please cite this article in press as: Yu X, et al. Expression and tissue distribution of hepatocyte growth factor (HGF) and its receptor (c-Met) in alpacas (Vicugna pacos) skins associated with white and brown coat colors. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acthis.2015.06.002
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Conclusions We detected the tissue distribution of HGF and c-Met through immunohistochemistry and analyzed the expression patterns using Western blot and qPCR. The results showed that signals of HGF are localized in dermal papilla and root sheath, and c-Met is localized in epithelial cells, hair follicle matrix, and root sheath. HGF has significantly high expression at the mRNA and protein levels in white alpaca skins. By contrast, c-Met has significantly high expression at the mRNA and protein levels in brown alpaca skins. Our results indicate that c-Met might have an important function in the regulation of coat color formation. Acknowledgments This research was sponsored by the National Natural Science Foundation of China (31172283), the Science and Technology Innovation Foundation of SXAU (201204) and the National High Technology Research and Development Program of China (863 Program) Project (2013AA102506). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.acthis.2015.06. 002 References Barsh G. How hair gets its pigment. Cell 2007;130:779–81. Beuret L. Up-regulation of MET expression by alpha-melanocyte-stimulating hormone and MITF allows hepatocyte growth factor to protect melanocytes and melanoma cells from apoptosis. J Biol Chem 2007;282:14140–7. Chandramohan B. The alpaca agouti gene: genomic locus, transcripts and causative mutations of eumelanic and pheomelanic coat color. Gene 2013;521:303–10. Cheli Y, Ohanna M, Ballotti R, Bertolotto C. Fifteen-year quest for microphthalmia-associated transcription factor target genes. Pigment Cell Melanoma Res 2010;23:27–40. Cransberg R, Wakamatsu K, Munyard K. Melanin characterisation suggests that the “brown” phenotype in alpaca (Vicugna pacos) is predominantly pheomelanic. Small Rumin Res 2013;114:240–6. Dong C. Coat color determination by mir-137 mediated down-regulation of microphthalmia-associated transcription factor in a mouse model. RNA Publ RNA Soc 2012;18:1679–86. Fan R. Gene expression profile in white alpaca (Vicugna pacos) skin. Animal 2011:5. Fan R, Xie J, Bai J, Wang H, Xue T, Rui B, et al. Skin transcriptome profiles associated with coat color in sheep. BMC Genomics 2013;14:3001–8. Feeley NL, Munyard KA. Characterisation of the melanocortin-1 receptor gene in alpaca and identification of possible markers associated with phenotypic variations in colour. Anim Prod Sci 2009;49:675–81.
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Please cite this article in press as: Yu X, et al. Expression and tissue distribution of hepatocyte growth factor (HGF) and its receptor (c-Met) in alpacas (Vicugna pacos) skins associated with white and brown coat colors. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acthis.2015.06.002