Novel hairless RET-transgenic mouse line with melanocytic nevi and anagen hair follicles.

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 c-RET 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 RET-transgenic mouse line (304/B6) (Kato et al., 1998a, 1998b, 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, 1998b, 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 (non-albino) 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 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/RET-transgenic 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)-, N-ras-, 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 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 (n>200) throughout their life (>1.5 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/RET-transgenic 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. The authors state no conflict of interest. This study was supported in part by the Grants-in-Aid for Scientific Research (B) and the Grant-in-Aid 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.