High-incidence spontaneous tumors in JF1/Ms mice: relevance of hypomorphic germline mutation and subsequent promoter methylation of Ednrb.

PURPOSE: JF1/Ms mice, an inbred strain derived from Japanese wild mice, carry a germline hypomorphic mutation in the endothelin receptor type B gene (Ednrb). We observed that the JF1/Ms mice develop various spontaneous tumors at a high incidence late in life. The aim of this study was to elucidate the mechanism responsible for spontaneous tumors in these mice. Possible relevance of milk-borne mammary tumor virus and gene alterations in Ednrb to tumorigenesis was explored. METHODS: Expression and methylation status of Ednrb were quantitatively analyzed in normal and cancer tissues of mammary gland, liver, submandibular gland as well as in a cultured cell line, MW1, established from a submandibular gland adenocarcinoma. The biological effects of EDNRB were examined in the MW1 cells transfected with wild-type Ednrb. RESULTS: Transcripts of Ednrb were barely detectable, and the promoter region of Ednrb was hypermethylated in tumor tissues and the MW1 cells. In contrast, normal counterpart tissues showed positive expression of Ednrb transcripts and had unmethylated promoter regions. Treatment of the MW1 cells with 5-Aza-dC restored transcription of Ednrb to normal levels. Transfection of the MW1 cells with Ednrb1 (MW1-Ednrb1) resulted in lower growth rates and morphological changes compared with the mock-transfected MW1 cells (MW1-mock1). Furthermore, the MW1-Ednrb1 cells transplanted in syngeneic mice showed a lower proliferation rate than the MW1-mock1 cells. CONCLUSIONS: Germline mutation and subsequent promoter methylation of Ednrb may be relevant to cancer susceptibility in the JF1/Ms mice. These data indicate that Ednrb acts as a tumor suppressor, as reported in human prostate, bladder, and clear cell renal carcinomas.

A mouse model of Waardenburg syndrome type 4 with a new spontaneous mutation of the endothelin-B receptor gene.

Waardenburg syndrome (WS) is a hereditary auditory-pigmentary syndrome with hearing impairment and pigmentation anomaly of the skin and iris. In addition to these major symptoms, WS type 4 is associated with Hirschsprung disease. To date, three genes responsible for WS4 have been cloned: genes for a transcription factor SOX10, endothelin 3 (EDN3), and endothelin B receptor (EDNRB). We here describe a novel mutant mouse with a mutation of the Ednrb gene, and propose the mouse as an animal model of WS4. These mutants are with mixed genetic background of BALB/c and MSM (an inbred strain of Japanese wild mice) and have extensive white spotting. They died between 2 and 7 weeks after birth owing to megacolon: their colon distal to the megacolon lacked Auerbach's plexus cells. Interestingly, these mutants did not respond to sound, and the stria vascularis of their cochlea lacked intermediate cells, i.e., neural crest-derived melanocytes. Since these symptoms resembled those of human WS4 and were transmitted in autosomal recessive hereditary manner, the mutants were named WS4 mice. Breeding analysis revealed that WS4 mice are allelic with piebald-lethal and JF1 mice, which are also mutated in the Ednrb gene. Mutation analysis revealed that their Ednrb lacked 318 nucleotides encoding Ednrb transmembrane domains owing to deletion of exons 2 and 3. Interaction between endothelin 3 and its receptor is required for normal differentiation and development of melanocytes and Auerbach's plexus cells. We concluded that a missing interaction here led to a lack of these cells, which caused pigmentation anomaly, deafness, and megacolon in WS4 mice.

Hereditary keratoconus-like keratopathy in Japanese wild mice mapped to mouse Chromosome 13.

Human keratoconus is a common corneal disease with non-inflammatory corneal ectasia, and a subset of this disease is heritable. In an effort to establish animal models for this disease, we discovered Japanese keratoconus (JKC) mice among Mishima molosinus (MSM) mice, an inbred strain of Japanese wild mice (Mus musculus molossinus). Typical phenotypic corneas of JKC mice are, like human keratoconus, conical in shape, although the corneas were often associated with a red punctum at the tip. In contrast to human keratoconus, histological examination revealed the inflammatory changes such as infiltration of capillaries and hematocytes in JKC mouse corneas. Although JKC mouse corneal change is probably secondary to keratitis and is a mouse-specific keratopathy, its pathogenesis may be relevant to that of human keratoconus. Linkage analysis mapped the responsible gene at the markers D13Mit21, D13Mit252, D13Mit279, and D13Mit39, which are located between 21.9 and 34.0 cm of the mouse Chr 13. Candidate genes in this region include genes for cathepsins, interleukin, and chemotaxin. Further study of JKC mice may shed light on pathogenesis of human keratoconus.