- Email: [email protected]
Novel Hairless RET-Transgenic Mouse Line with Melanocytic Nevi and Anagen Hair Follicles
M Kato et al. Novel Hairless RET-Transgenic Mouse Line
lipid barrier in the stratum corneum (Akiyama, 2006). As illustrated here, mutation analysis of the ALDH3A2 gene is a highly sensitive method to confirm a diagnosis of SLS, which does not require a skin biopsy and can complement or replace FALDH enzymatic assays or analysis in SLS. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS We thank Professor James R. McMillan for his critical reading of the manuscript; Ms Maki Goto, Ms Akari Nagasaki, and Ms Megumi Sato for their fine technical assistance on this project. This work was supported in part by Grant-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan to M. Akiyama (Kiban B 16390312 and Kiban B 18390310).
Kaori Sakai1, Masashi Akiyama1, Tomoyuki Watanabe2, Kazunori Sanayama2, Katsuo Sugita3, Mari Takahashi4, Keisuke Suehiro4, Kazuhiko Yorifuji4, Akihiko Shibaki1 and Hiroshi Shimizu1 1
Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan; 2Department of Pediatrics, Japanese Red Cross Narita Hospital, Narita, Japan; 3Division of Child Health, Faculty of Education, Chiba University, Chiba, Japan and 4Department of Dermatology, Japanese Red Cross Narita Hospital, Narita, Japan. E-mail: [email protected]
REFERENCES Akiyama M (2006) Harlequin ichthyosis and other autosomal recessive congenital ichthyoses: the underlying genetic defects and pathomechanisms. J Dermatol Sci 42:83–9 Aoki N, Suzuki H, Ito K, Ito M (2000) A novel point mutation of the FALDH gene in a Japanese family with Sjo¨gren–Larsson syndrome. J Invest Dermatol 114:1065–6 Chang C, Yoshida A (1997) Human fatty aldehyde dehydrogenase gene (ALDH10): organization and tissue-dependent expression. Genomics 40:80–5 De Laurenzi V, Rogers GR, Hamrock DJ, Marekov LN, Steinert PM, Compton JG et al. (1996) Sjo¨gren–Larsson syndrome is caused by mutations in the fatty aldehyde dehydrogenase gene. Nat Genet 12:52–7 Freshney NW, Rawlinson L, Guesdon F, Jones E, Cowley S, Hsuan J et al. (1994) Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27. Cell 78:1039–49 Ito M, Oguro K, Sato Y (1991) Ultrastructural study of the skin in Sjo¨gren–Larsson syndrome. Arch Dermatol Res 283:141–8 Kelson TL, Secor McVoy JR, Rizzo WB (1997) Human liver fatty aldehyde dehydrogenase: microsomal localization, purification, and biochemical characterization. Biochim Biophys Acta 1335:99–110 Kraus C, Braun-Quentin C, Ballhausen WG, Pfeiffer RA (2000) RNA-based mutation screening in German families with Sjogren–Larsson syndrome. Eur J Hum Genet 8: 299–306 Liu Z-J, Sun Y-J, Rose J, Chung YJ, Hsiao CD, Chang WR et al. (1997) The first structure of an aldehyde dehydrogenase reveals novel
interactions between NAD and the Rossmann fold. Nat Struct Biol 4:317–26 Paige DG, Morse-Fisher N, Harper JI (1994) Quantification of stratum corneum ceramides and lipid envelope ceramides in the hereditary ichthyoses. Br J Dermatol 131: 23–7 Perozich J, Nicholas H, Wang B-C, Lindahl R, Hempel J (1999) Relationships within the aldehyde dehydrogenase extended family. Protein Sci 8:137–46 Rizzo WB (1993) Sjo¨gren–Larsson syndrome. Semin Dermatol 2:210–8 Rizzo WB, Carney G (2005) Sjo¨gren–Larsson syndrome: diversity of mutations and polymorphisms in the fatty aldehyde dehydrogenase gene (ALDH3A2). Hum Mutat 26: 1–10 Rizzo WB, Carney G, Lin Z (1999) The molecular basis of Sjo¨gren–Larsson syndrome: mutation analysis of the fatty aldehyde dehydrogenase gene. Am J Hum Genet 65: 1547–60 Rizzo WB, Dammann AL, Craft DA (1988) Sjo¨gren–Larsson syndrome. Impaired fatty alcohol oxidation in cultured fibroblasts due to deficient fatty alcohol: nicotinamide adenine dinucleotide oxidoreductase activity. J Clin Invest 81:737–44 Rogers GR, Markova NG, De Laurenzi V, Rizzo WB, Compton JG (1997) Genomic organization and expression of the human fatty aldehyde dehydrogenase gene (FALDH). Genomics 39:127–35 Shibaki A, Akiyama M, Shimizu H (2004) Novel ALDH3A2 heterozygous mutations are associated with defective lamellar granule formation in a Japanese family of Sjo¨gren–Larsson syndrome. J Invest Dermatol 123:1197–9
Novel Hairless RET-Transgenic Mouse Line with Melanocytic Nevi and Anagen Hair Follicles Journal of Investigative Dermatology (2006) 126, 2547–2550. doi:10.1038/sj.jid.5700444; published online 15 June 2006
TO THE EDITOR The c-RET proto-oncogene encodes a receptor-tyrosine kinase and glial cell line-derived neurotrophic factor ligands, including glial cell line-derived neurotrophic factor, neurturin, artemin, and persephin, have been reported to be ligands of RET (Takahashi, 2001). RFP/ RET is a hybrid oncogene between cRET and RFP that was isolated by NIH3T3 transfection assays (Takahashi et al., 1985). Previously, we established
a metallothionein-I/RFP-RET (RET)-transgenic mouse line (242) that spontaneously develops systemic skin melanosis without macroscopic tumors (Iwamoto et al., 1991; Kato et al., 1999). Generally, most hair follicles in adult wild-type mice are in telogen (Kato et al., 2001). It is basically impossible to induce continuous anagen hair follicles in adult wild-type mice, although temporal anagen hair follicles are inducible by shaving hairs (Kato et al.,
2001). Interestingly, adult RET-transgenic mice have continuous anagen hair follicles with hyper melanin production (Kato et al., 2001, 2004). Moreover, hair growth of adult transgenic mice was promoted compared with that of control C57BL/6 mice (Kato et al., 2001). These results suggest that a continuous anagen phase of hair follicles plays an important role in hair growth. We also established another RETtransgenic mouse line (304/B6) (Kato www.jidonline.org 2547
M Kato et al. Novel Hairless RET-Transgenic Mouse Line
a
e
f b
c
d
g
h
Figure 1. Macroscopic and microscopic characteristics of HL/RET-transgenic mice. Macroscopic (a, g) and microscopic (b–f, h) appearances of control skin (b, c), hyperpigmented skin (d, black arrowheads in e and f), congenital melanocytic nevus (white arrows in e and f), and benign tumor (white arrow in g and h) from a representative 12-week-old HL/RET-transgenic mouse of line 242-hr/hr (bottom mouse of a, d, e, f), a control 12-week-old hairless non-transgenic albino mouse (top mouse of a, b), a control 12-week-old hairless non-transgenic conventionally pigmented (non-albino) mouse (c), and an original haired 12-week-old RET-transgenic mouse of line 304/B6 (g, h) are presented. Tissues were stained with hematoxylin and eosin (b–e, h) or immunohistochemically with anti-S100 antibody (f). There were basically no differences between an albino non-transgenic hairless mouse and a pigmented hairless mouse in hair cycle (b, c) and macroscopic appearance (data not shown). The nevus was composed of S100-positive round or spindle-shaped cells with round nuclei and without a mitotic profile (e, f). Melanophages were also observed in the nevus (e, f). The benign tumor in the original RET-transgenic mice of line 304/B6 consisted of S-100-positive cells as reported previously (data not shown) (Iwamoto et al., 1991; Kato et al., 1998a, b). Immunohistochemistry was performed by a previously reported method (Kato et al., 2004). Bar ¼ 100 mm (b, c) and 200 mm (d, e, f, h).
et al., 1998a, b, 1999). The process of benign tumor development and its malignant transformation in the 304/ B6 line macroscopically resembles that of the human giant congenital melanocytic nevus, which is present at birth and frequently gives rise to malignant melanoma. However, we could not notice a melanocytic nevus in the dermis when we reported the mouse line (Kato et al., 1998a, b, 2000). In fact, very limited numbers of mouse lines with spontaneously developed nevi have been reported. Alternatively, nevi are inducible in animals by treatment with 7,12-dimethylbenzanthracene and/ or UV light irradiation (Elmets et al., 2004; Menzies et al., 2004). In this study, we crossed the original RET-transgenic mice of line 242 with hairless HRS/J mice and newly prepared a hairless RET (HL/RET)-transgenic mouse of line 242-hr/hr in the
Animal Center of Nagoya University, which approved the experiment. As expected, skin of the HL/RET-transgenic mouse (Figure 1a bottom, d) was highly pigmented compared with that of the control albino non-transgenic hairless mouse (Figure 1a top, b) or the control conventionally pigmented (nonalbino) non-transgenic hairless mouse (Figure 1c). Using this new mouse line, we for the first time investigated RET-associated hair growth in mice bearing an hr/hr genotype. Although no anagen is usually observed in hair follicles from adult wild-type hairless mice (Figure 1b and c), most hair follicles in the HL/ RET-transgenic mice were anagen (Figure 1d). These results suggest that the activated RET gene partially complemented the hairless defect. As shown in Figure 1a (bottom), however, no hairs were macroscopically observed
2548 Journal of Investigative Dermatology (2006), Volume 126
in the HL/RET-transgenic mice. Other factors in addition to anagen may be necessary for hair development. Unexpectedly, melanocytic nevi with slightly enlarged lymph nodes with hyperpigmentation in the dermis were microscopically found to develop up to 10 days after birth in 90% (18/20) of the HL/RET-transgenic mice (Figure 1d and e), suggesting that the nevi are of genetic origin rather than environmental origin. The histopathological characteristics of the nevi in the HL/RET-transgenic mice were compatible with those of human congenital melanocytic nevi, for which an important criterion for diagnosis is the presence of melanocytes (S-100-positive cells) along epithelial structures of adnexa and their angiocentric distribution (white arrows in Figure 1e and f) (Kerl et al., 2006). On the other hand, there is histopathologically a slight increase in the number of melanocytes in hyperpigmented skin of the HL/RETtransgenic mice (black arrowheads in Figure 1d, e, and f) (Kerl et al., 2006). Benign melanocytic tumors in the original haired RET-transgenic mice of line 304/B6 (white arrow in Figure 1g) are histologically in the subcutaneous lesion (white arrow in Figure 1h). Based on these findings and the difference in macroscopic appearance (Figure 1a and g), we finally concluded that the nevi in the dermis in HL/RET-transgenic mice of line 242-hr/hr are different from hyperpigmented skin and benign melanocytic tumors, and we finally diagnosed them as congenital melanocytic nevi. Recently, hyperpigmented skin has been reported in steel factor (SLF)-, Nras-, and Ha-ras-transgenic mouse lines (Powell et al., 1995; Kunisada et al., 1998; Ackermann et al., 2005). Nevi were found in the Ha-ras-transgenic mouse line (Powell et al., 1995) but not in SLF- and N-ras-transgenic mouse lines (Kunisada et al., 1998; Ackermann et al., 2005). The reason why the nevi developed in the HL/RET-transgenic mice is unknown. However, the hairless mice have a significant depression of T-cell, but not B-cell, immune response (Johnson et al., 1982). Therefore, T-cell-mediated immune response may
M Kato et al. Novel Hairless RET-Transgenic Mouse Line
be involved in the development of nevi. On the other hand, it was reported that congenital melanocytic nevi have a significantly higher risk for development of a malignant melanoma in humans (Zaal et al., 2005). No malignant melanomas developed from the nevi in the HL/RET-transgenic mice of line 242 (n4200) throughout their life (41.5 years). However, melanomas may be inducible in the HL/RET-transgenic mice by treatment of the nevi with 7,12-dimethylbenzanthracene or injection of cultured melanocytes from the nevi for immunodeficient mice. Moreover, the HL/RET-transgenic mice might be useful for analyzing the mechanisms of nevus development and the recently suggested linkage between nevus development and onset of some popular diseases. For example, two study groups reported that children with atopic dermatitis had few melanocytic nevi (Broberg and Augustsson, 2000; Synnerstad et al., 2004). One of those groups also revealed that there was a significant negative correlation between serum IgE and number of nevi in patients with atopic dermatitis. Another group reported that allergic rhinitis easily developed in individuals with few nevi (Awaya et al., 2003). Unfortunately, however, the mechanisms underlying these apparently important observations have not been clarified because these studies were performed only in humans. There was no clear difference between serum IgE levels in young aged (less than 6-month old) HL/RET-transgenic mice and control wild-type mice in our preliminary examination (Kato et al., unpublished observation). However, it might be possible to determine the basic mechanisms underlying the correlation between nevus development and occurrence of atopic dermatitis by crossing the nevi-developing HL/RETtransgenic mouse with a previously reported hairless model mouse with atopic dermatitis (Matsumoto et al., 2005). On the other hand, allergic rhinitis is easily inducible by treatment with ovalbumin (Hellings et al., 2001). Therefore, the mechanism underlying the negative correlation between nevus development and occurrence of allergic rhinitis might be
clarified through comparison of the sensitivity for ovalbumin-induced allergic rhinitis in HL/RET-transgenic mice and that in control hairless mice. In summary, we have newly established a hairless mouse line with hyperpigmented skin and continuous anagen hair follicles. Using this mouse model, we demonstrated that a continuous anagen phase of follicles is not a sufficient condition for hair growth. More interestingly, we found that melanocytic nevi develop spontaneously in the transgenic mouse line. This novel mouse model may open a new window in research on biology and related pathogenic events of melanocytic nevi. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS This study was supported in part by the Grants-inAid for Scientific Research (B) and the Grant-inAid for Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), the Grant-in Aid for JSPS Fellows from Japan Society for the Promotion of Science (JSPS), a Grant for the Basic Dermatological Research from Shiseido Co. Ltd, and the Cosmetology Research Foundation, the Tokyo Biochemical Research Foundation (TBRF) Postdoctoral fellowship for Asian Researcher, the Uehara Memorial Foundation Research Grant, and the research fund of the Institute of Kampo Medicine.
Masashi Kato1, Kozue Takeda1, Yoshiyuki Kawamoto2, Toyonori Tsuzuki3, Yoko Kato1, Tamio Ohno4, Khaled Hossain1, Imtiaz IftakharE-Khuda1, Nobutaka Ohgami1, Ken-ichi Isobe5, Masahide Takahashi3 and Izumi Nakashima2 1 Unit of Environmental Health Sciences, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai-shi, Aichi, Japan; 2Unit of Immunology, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai-shi, Aichi, Japan; 3 Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan; 4Division of Experimental Animals, Center for Promotion of Medical Research and Education, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan and 5 Department of Immunology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan. E-mail: [email protected]
REFERENCES Ackermann J, Frutschi M, Kaloulis K, McKee T, Trumpp A, Beermann F (2005) Metastasizing
melanoma formation caused by expression of activated N-RasQ61K on an INK4adeficient background. Cancer Res 65: 4005–11 Awaya A, Watanabe K, Kato S (2003) Individuals exhibiting conspicuous nevi (lentigo simplex) are resistant to allergic rhinitis/conjunctivitis (pollinosis), but those who do not show increased susceptibility to pollinosis. Microbiol Immunol 47:101–3 Broberg A, Augustsson A (2000) Atopic dermatitis and melanocytic naevi. Br J Dermatol 142:306–9 Elmets CA, Yusuf N, Hamza S, Iranikakh N, Smith J, Volk AL et al. (2004) Topical application of dimethylbenz[a]anthracene results in the generation of multiple melanocytic nevi in C3H/HeN mice. Toxicol Appl Pharmacol 195:355–60 Hellings PW, Hessel EM, Van Den Oord JJ, Kasran A, Van Hecke P, Ceuppens JL (2001) Eosinophilic rhinitis accompanies the development of lower airway inflammation and hyper-reactivity in sensitized mice exposed to aerosolized allergen. Clin Exp Allergy 31:782–90 Iwamoto T, Takahashi M, Ito M, Hamatani K, Ohbayashi M, Wajjwalku W et al. (1991) Aberrant melanogenesis and melanocytic tumour development in transgenic mice that carry a metallothionein/ret fusion gene. EMBO J 10:3167–75 Johnson DA, Shultz LD, Bedigian HG (1982) Immunodeficiency and reticulum cell sarcoma in mice segregating for HRS/J and SJL/J genes. Leukemia Res 6:711–20 Kato M, Liu W, Akhand AA, Dai Y, Ohbayashi M, Tuzuki T et al. (1999) Linkage between melanocytic tumor development and early burst of Ret protein expression for tolerance induction in metallothionein-I/ret transgenic mouse lines. Oncogene 18: 837–42 Kato M, Liu W, Akhand AA, Hossain K, Takeda K, Takahashi M et al. (2000) Ultraviolet radiation induces both full activation of ret kinase and malignant melanocytic tumor promotion in RFP-RETtransgenic mice. J Invest Dermatol 115: 1157–8 Kato M, Liu W, Yi H, Asai N, Hayakawa A, Kozaki K et al. (1998a) The herbal medicine Sho-saiko-to inhibits growth and metastasis of malignant melanoma primarily developed in ret-transgenic mice. J Invest Dermatol 111:640–4 Kato M, Takeda K, Kawamoto Y, Tsuzuki T, Hossain K, Tamakoshi A et al. (2004) c-Kit-targeting immunotherapy for hereditary melanoma in a mouse model. Cancer Res 64:801–6 Kato M, Takahashi M, Akhand AA, Liu W, Dai Y, Shimizu S et al. (1998b) Transgenic mouse model for skin malignant melanoma. Oncogene 17:1885–8 Kato M, Takeda K, Kawamoto Y, Tsuzuki T, Dai Y, Nakayama S et al. (2001) RET tyrosine kinase enhances hair growth in association with promotion of melanogenesis. Oncogene 20:7536–41
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Kerl H, Massi D, LeBoit PE, Bastin BC (2006) Melanocytic tumours. In: Pathology & genetics skin tumours. (LeBoit PE, Burg G, Weedon D, Sarasin A, eds), Lyon: IARC Press, 49–120 Kunisada T, Yoshida H, Yamazaki H, Miyamoto A, Hemmi H, Nishimura E et al. (1998) Transgene expression of steel factor in the basal layer of epidermis promotes survival, proliferation, differentiation and migration of melanocyte precursors. Development 125:2915–23 Matsumoto K, Mizukoshi K, Oyobikawa M, Ohshima H, Sakai Y, Tagami H (2005) Objective evaluation of the efficacy of daily topical applications of cosmetics bases using
the hairless mouse model of atopic dermatitis. Skin Res Technol 11:209–17 Menzies SW, Greenoak GE, Abeywardana CM, Crotty KA, O’Neill ME (2004) UV light from 290 to 325 nm, but not broadband UVA or visible light, augments the formation of melanocytic nevi in a guineapig model for human nevi. J Invest Dermatol 123:354–60 Powell MB, Hyman P, Bell OD, Balmain A, Brown K, Alberts D et al. (1995) Hyperpigmentation and melanocytic hyperplasia in transgenic mice expressing the human T24 Ha-ras gene regulated by a mouse tyrosinase promoter. Mol Carcinog 12:82–90
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Synnerstad I, Nilsson L, Fredrikson M, Rosdahl I (2004) Fewer melanocytic nevi found in children with active atopic dermatitis than in children without dermatitis. Arch Dermatol 140:1471–5 Takahashi M (2001) The GDNF/RET signaling pathway and human diseases. Cytokine Growth Factor Rev 12:361–73 Takahashi M, Ritz J, Cooper GM (1985) Activation of a novel human transforming gene, ret, by DNA rearrangement. Cell 42:581–8 Zaal LH, Mooi WJ, Klip H, van der Horst CM (2005) Risk of malignant transformation of congenital melanocytic nevi: a retrospective nationwide study from The Netherlands. Plast Reconstr Surg 116:1902–9
lipid barrier in the stratum corneum (Akiyama, 2006). As illustrated here, mutation analysis of the ALDH3A2 gene is a highly sensitive method to confirm a diagnosis of SLS, which does not require a skin biopsy and can complement or replace FALDH enzymatic assays or analysis in SLS. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS We thank Professor James R. McMillan for his critical reading of the manuscript; Ms Maki Goto, Ms Akari Nagasaki, and Ms Megumi Sato for their fine technical assistance on this project. This work was supported in part by Grant-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan to M. Akiyama (Kiban B 16390312 and Kiban B 18390310).
Kaori Sakai1, Masashi Akiyama1, Tomoyuki Watanabe2, Kazunori Sanayama2, Katsuo Sugita3, Mari Takahashi4, Keisuke Suehiro4, Kazuhiko Yorifuji4, Akihiko Shibaki1 and Hiroshi Shimizu1 1
Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan; 2Department of Pediatrics, Japanese Red Cross Narita Hospital, Narita, Japan; 3Division of Child Health, Faculty of Education, Chiba University, Chiba, Japan and 4Department of Dermatology, Japanese Red Cross Narita Hospital, Narita, Japan. E-mail: [email protected]
REFERENCES Akiyama M (2006) Harlequin ichthyosis and other autosomal recessive congenital ichthyoses: the underlying genetic defects and pathomechanisms. J Dermatol Sci 42:83–9 Aoki N, Suzuki H, Ito K, Ito M (2000) A novel point mutation of the FALDH gene in a Japanese family with Sjo¨gren–Larsson syndrome. J Invest Dermatol 114:1065–6 Chang C, Yoshida A (1997) Human fatty aldehyde dehydrogenase gene (ALDH10): organization and tissue-dependent expression. Genomics 40:80–5 De Laurenzi V, Rogers GR, Hamrock DJ, Marekov LN, Steinert PM, Compton JG et al. (1996) Sjo¨gren–Larsson syndrome is caused by mutations in the fatty aldehyde dehydrogenase gene. Nat Genet 12:52–7 Freshney NW, Rawlinson L, Guesdon F, Jones E, Cowley S, Hsuan J et al. (1994) Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27. Cell 78:1039–49 Ito M, Oguro K, Sato Y (1991) Ultrastructural study of the skin in Sjo¨gren–Larsson syndrome. Arch Dermatol Res 283:141–8 Kelson TL, Secor McVoy JR, Rizzo WB (1997) Human liver fatty aldehyde dehydrogenase: microsomal localization, purification, and biochemical characterization. Biochim Biophys Acta 1335:99–110 Kraus C, Braun-Quentin C, Ballhausen WG, Pfeiffer RA (2000) RNA-based mutation screening in German families with Sjogren–Larsson syndrome. Eur J Hum Genet 8: 299–306 Liu Z-J, Sun Y-J, Rose J, Chung YJ, Hsiao CD, Chang WR et al. (1997) The first structure of an aldehyde dehydrogenase reveals novel
interactions between NAD and the Rossmann fold. Nat Struct Biol 4:317–26 Paige DG, Morse-Fisher N, Harper JI (1994) Quantification of stratum corneum ceramides and lipid envelope ceramides in the hereditary ichthyoses. Br J Dermatol 131: 23–7 Perozich J, Nicholas H, Wang B-C, Lindahl R, Hempel J (1999) Relationships within the aldehyde dehydrogenase extended family. Protein Sci 8:137–46 Rizzo WB (1993) Sjo¨gren–Larsson syndrome. Semin Dermatol 2:210–8 Rizzo WB, Carney G (2005) Sjo¨gren–Larsson syndrome: diversity of mutations and polymorphisms in the fatty aldehyde dehydrogenase gene (ALDH3A2). Hum Mutat 26: 1–10 Rizzo WB, Carney G, Lin Z (1999) The molecular basis of Sjo¨gren–Larsson syndrome: mutation analysis of the fatty aldehyde dehydrogenase gene. Am J Hum Genet 65: 1547–60 Rizzo WB, Dammann AL, Craft DA (1988) Sjo¨gren–Larsson syndrome. Impaired fatty alcohol oxidation in cultured fibroblasts due to deficient fatty alcohol: nicotinamide adenine dinucleotide oxidoreductase activity. J Clin Invest 81:737–44 Rogers GR, Markova NG, De Laurenzi V, Rizzo WB, Compton JG (1997) Genomic organization and expression of the human fatty aldehyde dehydrogenase gene (FALDH). Genomics 39:127–35 Shibaki A, Akiyama M, Shimizu H (2004) Novel ALDH3A2 heterozygous mutations are associated with defective lamellar granule formation in a Japanese family of Sjo¨gren–Larsson syndrome. J Invest Dermatol 123:1197–9
Novel Hairless RET-Transgenic Mouse Line with Melanocytic Nevi and Anagen Hair Follicles Journal of Investigative Dermatology (2006) 126, 2547–2550. doi:10.1038/sj.jid.5700444; published online 15 June 2006
TO THE EDITOR The c-RET proto-oncogene encodes a receptor-tyrosine kinase and glial cell line-derived neurotrophic factor ligands, including glial cell line-derived neurotrophic factor, neurturin, artemin, and persephin, have been reported to be ligands of RET (Takahashi, 2001). RFP/ RET is a hybrid oncogene between cRET and RFP that was isolated by NIH3T3 transfection assays (Takahashi et al., 1985). Previously, we established
a metallothionein-I/RFP-RET (RET)-transgenic mouse line (242) that spontaneously develops systemic skin melanosis without macroscopic tumors (Iwamoto et al., 1991; Kato et al., 1999). Generally, most hair follicles in adult wild-type mice are in telogen (Kato et al., 2001). It is basically impossible to induce continuous anagen hair follicles in adult wild-type mice, although temporal anagen hair follicles are inducible by shaving hairs (Kato et al.,
2001). Interestingly, adult RET-transgenic mice have continuous anagen hair follicles with hyper melanin production (Kato et al., 2001, 2004). Moreover, hair growth of adult transgenic mice was promoted compared with that of control C57BL/6 mice (Kato et al., 2001). These results suggest that a continuous anagen phase of hair follicles plays an important role in hair growth. We also established another RETtransgenic mouse line (304/B6) (Kato www.jidonline.org 2547
M Kato et al. Novel Hairless RET-Transgenic Mouse Line
a
e
f b
c
d
g
h
Figure 1. Macroscopic and microscopic characteristics of HL/RET-transgenic mice. Macroscopic (a, g) and microscopic (b–f, h) appearances of control skin (b, c), hyperpigmented skin (d, black arrowheads in e and f), congenital melanocytic nevus (white arrows in e and f), and benign tumor (white arrow in g and h) from a representative 12-week-old HL/RET-transgenic mouse of line 242-hr/hr (bottom mouse of a, d, e, f), a control 12-week-old hairless non-transgenic albino mouse (top mouse of a, b), a control 12-week-old hairless non-transgenic conventionally pigmented (non-albino) mouse (c), and an original haired 12-week-old RET-transgenic mouse of line 304/B6 (g, h) are presented. Tissues were stained with hematoxylin and eosin (b–e, h) or immunohistochemically with anti-S100 antibody (f). There were basically no differences between an albino non-transgenic hairless mouse and a pigmented hairless mouse in hair cycle (b, c) and macroscopic appearance (data not shown). The nevus was composed of S100-positive round or spindle-shaped cells with round nuclei and without a mitotic profile (e, f). Melanophages were also observed in the nevus (e, f). The benign tumor in the original RET-transgenic mice of line 304/B6 consisted of S-100-positive cells as reported previously (data not shown) (Iwamoto et al., 1991; Kato et al., 1998a, b). Immunohistochemistry was performed by a previously reported method (Kato et al., 2004). Bar ¼ 100 mm (b, c) and 200 mm (d, e, f, h).
et al., 1998a, b, 1999). The process of benign tumor development and its malignant transformation in the 304/ B6 line macroscopically resembles that of the human giant congenital melanocytic nevus, which is present at birth and frequently gives rise to malignant melanoma. However, we could not notice a melanocytic nevus in the dermis when we reported the mouse line (Kato et al., 1998a, b, 2000). In fact, very limited numbers of mouse lines with spontaneously developed nevi have been reported. Alternatively, nevi are inducible in animals by treatment with 7,12-dimethylbenzanthracene and/ or UV light irradiation (Elmets et al., 2004; Menzies et al., 2004). In this study, we crossed the original RET-transgenic mice of line 242 with hairless HRS/J mice and newly prepared a hairless RET (HL/RET)-transgenic mouse of line 242-hr/hr in the
Animal Center of Nagoya University, which approved the experiment. As expected, skin of the HL/RET-transgenic mouse (Figure 1a bottom, d) was highly pigmented compared with that of the control albino non-transgenic hairless mouse (Figure 1a top, b) or the control conventionally pigmented (nonalbino) non-transgenic hairless mouse (Figure 1c). Using this new mouse line, we for the first time investigated RET-associated hair growth in mice bearing an hr/hr genotype. Although no anagen is usually observed in hair follicles from adult wild-type hairless mice (Figure 1b and c), most hair follicles in the HL/ RET-transgenic mice were anagen (Figure 1d). These results suggest that the activated RET gene partially complemented the hairless defect. As shown in Figure 1a (bottom), however, no hairs were macroscopically observed
2548 Journal of Investigative Dermatology (2006), Volume 126
in the HL/RET-transgenic mice. Other factors in addition to anagen may be necessary for hair development. Unexpectedly, melanocytic nevi with slightly enlarged lymph nodes with hyperpigmentation in the dermis were microscopically found to develop up to 10 days after birth in 90% (18/20) of the HL/RET-transgenic mice (Figure 1d and e), suggesting that the nevi are of genetic origin rather than environmental origin. The histopathological characteristics of the nevi in the HL/RET-transgenic mice were compatible with those of human congenital melanocytic nevi, for which an important criterion for diagnosis is the presence of melanocytes (S-100-positive cells) along epithelial structures of adnexa and their angiocentric distribution (white arrows in Figure 1e and f) (Kerl et al., 2006). On the other hand, there is histopathologically a slight increase in the number of melanocytes in hyperpigmented skin of the HL/RETtransgenic mice (black arrowheads in Figure 1d, e, and f) (Kerl et al., 2006). Benign melanocytic tumors in the original haired RET-transgenic mice of line 304/B6 (white arrow in Figure 1g) are histologically in the subcutaneous lesion (white arrow in Figure 1h). Based on these findings and the difference in macroscopic appearance (Figure 1a and g), we finally concluded that the nevi in the dermis in HL/RET-transgenic mice of line 242-hr/hr are different from hyperpigmented skin and benign melanocytic tumors, and we finally diagnosed them as congenital melanocytic nevi. Recently, hyperpigmented skin has been reported in steel factor (SLF)-, Nras-, and Ha-ras-transgenic mouse lines (Powell et al., 1995; Kunisada et al., 1998; Ackermann et al., 2005). Nevi were found in the Ha-ras-transgenic mouse line (Powell et al., 1995) but not in SLF- and N-ras-transgenic mouse lines (Kunisada et al., 1998; Ackermann et al., 2005). The reason why the nevi developed in the HL/RET-transgenic mice is unknown. However, the hairless mice have a significant depression of T-cell, but not B-cell, immune response (Johnson et al., 1982). Therefore, T-cell-mediated immune response may
M Kato et al. Novel Hairless RET-Transgenic Mouse Line
be involved in the development of nevi. On the other hand, it was reported that congenital melanocytic nevi have a significantly higher risk for development of a malignant melanoma in humans (Zaal et al., 2005). No malignant melanomas developed from the nevi in the HL/RET-transgenic mice of line 242 (n4200) throughout their life (41.5 years). However, melanomas may be inducible in the HL/RET-transgenic mice by treatment of the nevi with 7,12-dimethylbenzanthracene or injection of cultured melanocytes from the nevi for immunodeficient mice. Moreover, the HL/RET-transgenic mice might be useful for analyzing the mechanisms of nevus development and the recently suggested linkage between nevus development and onset of some popular diseases. For example, two study groups reported that children with atopic dermatitis had few melanocytic nevi (Broberg and Augustsson, 2000; Synnerstad et al., 2004). One of those groups also revealed that there was a significant negative correlation between serum IgE and number of nevi in patients with atopic dermatitis. Another group reported that allergic rhinitis easily developed in individuals with few nevi (Awaya et al., 2003). Unfortunately, however, the mechanisms underlying these apparently important observations have not been clarified because these studies were performed only in humans. There was no clear difference between serum IgE levels in young aged (less than 6-month old) HL/RET-transgenic mice and control wild-type mice in our preliminary examination (Kato et al., unpublished observation). However, it might be possible to determine the basic mechanisms underlying the correlation between nevus development and occurrence of atopic dermatitis by crossing the nevi-developing HL/RETtransgenic mouse with a previously reported hairless model mouse with atopic dermatitis (Matsumoto et al., 2005). On the other hand, allergic rhinitis is easily inducible by treatment with ovalbumin (Hellings et al., 2001). Therefore, the mechanism underlying the negative correlation between nevus development and occurrence of allergic rhinitis might be
clarified through comparison of the sensitivity for ovalbumin-induced allergic rhinitis in HL/RET-transgenic mice and that in control hairless mice. In summary, we have newly established a hairless mouse line with hyperpigmented skin and continuous anagen hair follicles. Using this mouse model, we demonstrated that a continuous anagen phase of follicles is not a sufficient condition for hair growth. More interestingly, we found that melanocytic nevi develop spontaneously in the transgenic mouse line. This novel mouse model may open a new window in research on biology and related pathogenic events of melanocytic nevi. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS This study was supported in part by the Grants-inAid for Scientific Research (B) and the Grant-inAid for Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), the Grant-in Aid for JSPS Fellows from Japan Society for the Promotion of Science (JSPS), a Grant for the Basic Dermatological Research from Shiseido Co. Ltd, and the Cosmetology Research Foundation, the Tokyo Biochemical Research Foundation (TBRF) Postdoctoral fellowship for Asian Researcher, the Uehara Memorial Foundation Research Grant, and the research fund of the Institute of Kampo Medicine.
Masashi Kato1, Kozue Takeda1, Yoshiyuki Kawamoto2, Toyonori Tsuzuki3, Yoko Kato1, Tamio Ohno4, Khaled Hossain1, Imtiaz IftakharE-Khuda1, Nobutaka Ohgami1, Ken-ichi Isobe5, Masahide Takahashi3 and Izumi Nakashima2 1 Unit of Environmental Health Sciences, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai-shi, Aichi, Japan; 2Unit of Immunology, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai-shi, Aichi, Japan; 3 Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan; 4Division of Experimental Animals, Center for Promotion of Medical Research and Education, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan and 5 Department of Immunology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan. E-mail: [email protected]
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