Kazuo Okimoto

A germ-line insertion in the Birt-Hogg-Dubé (BHD) gene gives rise to the Nihon rat model of inherited renal cancer

A rat model of hereditary renal carcinoma (RC) was found in a rat colony of the Sprague–Dawley strain in Japan and named the “Nihon” rat. In heterozygotes, RCs, predominantly the clear cell type, develop from early preneoplastic lesions, which began to appear as early as 3 weeks of age, to adenocarcinomas by the age of 6 months. The Nihon rat is an example of a Mendelian dominantly inherited predisposition for development of RCs like the Eker (Tsc2 gene mutant) rat. We have previously shown that the Nihon mutation was tightly linked to genes that are located on the distal part of rat chromosome 10. The order of the genes is the Eker (Tsc2 gene (human 16p13.3)–Il3 gene–Nihon gene–Llgl1 locus– Myhse gene. We now describe a germ-line mutation in the Birt–Hogg–Dubé gene (Bhd) (human 17p11.2) caused by the insertion of a single nucleotide in the Nihon rat, resulting in a frameshift and producing a stop codon 26 aa downstream. We found that the homozygous mutant condition was lethal at an early stage of fetal life in the rat. We detected a high frequency of loss of heterozygosity (LOH) in primary RCs (10/11) at the Bhd locus and found a point mutation (nonsense) in one LOH-negative case, fitting Knudsons “two-hit” model. The Nihon rat may therefore provide insights into a tumor-suppressor gene that is related to renal carcinogenesis and an animal model of human BHD syndrome.

A Novel Renal Carcinoma Predisposing Gene of the Nihon Rat Maps on Chromosome 10

A novel rat model of hereditary renal cell carcinoma (RC) was found in a rat colony of the Spra‐gue‐Dawley (SD) strain in Japan, and named the “Nihon” rat in 2000. This study was designed to map the RC susceptibility gene in the Nihon rat using 113 backcross annuals. Our present data clearly show that the Nihon gene is genetically linked to interleukin‐3 (IL3) gene (χ2=93.6, Lod score=25.16), lethal (2) giant larvae (LLGL1) locus (χ2=109.0, Lod score=31.56) and myosin heavy chain, embryonic skeletal muscle (MYHSE) gene (χ2=90.6, Lod score=23.87), which are located on the distal part of rat chromosome 10. The order of the genes is the Eker (Tsc2) gene (located on the proximal part of rat chromosome 10; human chromosome 16p 13.3)–21.3 cM–IL3 gene (human 5q23‐31)–4.4 cM–Nihon gene–0.9 cM–LLGL1 locus (human 17p11.2)‐4.4 cM–MYHSE gene (human 17pl3.1). We also detected loss of the wild‐type allele at the MYHSE locus, fitting Knudsons “two hit” model. Thus, the Nihon rat should have a mutation of a novel tumor suppressor gene related to renal carcinogenesis.

Natural history of the Nihon rat model of BHD.

Hereditary cancer was first described in the rat by Eker and Mossige in 1954 in Oslo. The Eker rat model of hereditary renal carcinoma (RC) was the first example of a Mendelian dominantly inherited predisposition to a specific cancer in an experimental animal, and has been contributing to the elucidation of renal carcinogenesis. Recently, we found a second hereditary RC model in the Sprague-Dawley (SD) rat, in Japan in 2000, which was named the Nihon rat. The Nihon rat is also an example of a Mendelian dominantly inherited predisposition for development of RCs like the Eker rat, which are predominantly of the clear cell type (this type represents approximately 75 % of human RCC), and develop from earlier preneoplastic lesions than the Eker rat. We performed a genetic linkage analysis of the Nihon rat using 113 backcross animals, and found that the Nihon mutation was tightly linked to genes, which are located on the distal part of rat chromosome 10. Finally, we identified a germline mutation in the Birt-Hogg-Dubé gene (Bhd) (rat chromosome 10, human chromosome 17p11.2) caused by the insertion of a single nucleotide in the Nihon rat gene sequence, resulting in a frame shift and producing a stop codon 26 amino acids downstream. Thus, the Nihon rat will contribute to understanding the BHD gene function and renal carcinogenesis.

Serine 62 is a phosphorylation site in folliculin, the Birt-Hogg-Dubé gene product

MINT‐7298229: FNIPL (uniprotkb:Q9P278) physically interacts (MI:0915) with Flcn (uniprotkb:Q76JQ2) by anti tag coimmunoprecipitation (MI:0007)

Regulation of folliculin (the BHD gene product) phosphorylation by Tsc2-mTOR pathway.

The Birt-Hogg-Dubé gene (BHD) encodes the tumor suppressor protein folliculin (FLCN). The function of FLCN has recently been implicated in the regulation of rapamycin-sensitive mTOR complex (mTORC1). Reciprocally, the mTORC1-dependent phosphorylation of FLCN was reported. However, precise mechanism of FLCN phosphorylation and functional interaction of FLCN with tuberin, the product of tuberous sclerosis 2 gene (TSC2) which is a negative regulator of mTORC1, are unclear. Here we report that multiple phosphorylation in FLCN are evoked by downregulation of tuberin as well as by Rheb expression. We found that phosphorylation at Ser62 and Ser302 are differently regulated by mTORC1-dependent pathway. Some unknown kinases downstream of tuberin-mTORC1 are thought to directly phosphorylate FLCN. Interestingly, our results also suggest that the complex formation of FLCN with AMPK is modulated by FLCN phosphorylation. These results suggest that FLCN is involved in a novel mechanism of signal transduction downstream of tuberin.

Transgenic rescue from embryonic lethality and renal carcinogenesis in the Nihon rat model by introduction of a wild-type Bhd gene

We recently reported that a germline insertion of a single nucleotide in the rat homologue of the human Birt–Hogg–Dubé gene (BHD) gives rise to dominantly inherited cancer in the Nihon rat model. In this study, we constructed transgenic Nihon rats with introduction of a wild-type Bhd gene to ascertain whether suppression of the Nihon phenotype is possible. Rescue from embryonic lethality of mutant homozygotes (Nihon/Nihon) and suppression of renal carcinogenesis in heterozygotes (Nihon/+) were both observed, defining the germline Bhd mutation in the Nihon rat as an embryonal lethal and tumor predisposing mutation. This transgenic rescue system will be useful to analyse Bhd gene function, its relation to tumorigenesis in vivo, and genetic–environmental interactions in carcinogenesis.

Di(2-ethylhexyl)phthalate Induces Hepatocellular Adenoma in Transgenic Mice Carrying a Human Prototype c-Ha-ras Gene in a 26-Week Carcinogenicity Study

To evaluate the transgenic mouse carrying a human prototype c-Ha-ras gene (rasH2 mouse) as a model for 26-week carcinogenicity tests, Di(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, was administered to 15 rasH2 mice/sex/group at concentrations of 1,500, 3,000 or 6,000 ppm, and to 15 wild-type (non-Tg) mice/sex/group at a concentration of 6,000 ppm in their diets for 26 weeks. Survival rates and food consumption in the groups treated with DEHP and in the control group were similar. Body weight gain in rasH2 and non-Tg mice at 6,000 ppm in the terminal week decreased about 10% as compared to the control group. Common findings related to treatment with DEHP in rasH2 and non-Tg mice included hypertrophy with coarse granules and deposit of pigment in the liver, hydronephrosi s and tubular regeneration in the kidney, focal atrophy in the testis, and increased eosinophilic body in the nasal cavity. Hepatocellular adenoma was induced by treatment with DEHP, and was confined to male rasH2; mice the incidence being 7%(1/15), 13%(2/15), and 27%(4/15) in the 1,500-, 3,000-, and 6,000-ppm group, respectively. Point mutation was not detected in codon 12 and 61 of human c-Ha-ras transgene upon DNA analyses on frozen samples taken from these hepatocellular adenomas. From the results obtained in this 26-week carcinogenicity study, it is concluded that DEHP is a hepato-carcinogen for transgenic mouse carrying a human prototype c-Ha-ras gene.

The First Case of Feline Infectious Peritonitis-like Pyogranuloma in a Ferret Infected by Coronavirus in Japan.

A male ferret, which was purchased from abroad at 9 months of age, had shown significant weight loss starting at 13 months of age. The ferret subsequently showed decreasing motor activity and recumbency and was euthanized at 14 months of age. At necropsy, a white, quail egg-sized mass was found in the mesentery. Histopathologically, multifocal granulomas consisting of necrotic foci, macrophages, fibroblasts and plentiful fibrous connective tissues were observed in the mesenteric mass. Surrounding the granulomas, inflammatory cell infiltration consisting of neutrophils, lymphocytes and plasmacytes was observed diffusely and significantly. Immunohistochemistry revealed small numbers of macrophages around necrotic foci that were positively stained for anti-mouse feline coronavirus. Electron microscopically, the cytoplasm of the macrophages contained viral particles, which were identified as coronavirus. The histopathological features in this ferret were similar to those in cats with feline infectious peritonitis (FIP). This was the first case in ferrets in Japan.

Histopathology of spontaneous lesions in beagles used for toxicity studies.

A histopathological study was carried out on spontaneous subclinical lesions in 86 beagle dogs employed in toxicity studies. The lesions found in a young adult group were compared with those in an older age group. Lesions detected more frequently in the older age group included acidophilic crystalline intranuclear inclusions in the liver and kidney, cell infiltration in the submaxillary gland, a tubular fatty change and glomerular lipid deposition in the kidney, atrophy and cyst formation in the thymus, siderofibrotic nodules in the spleen, and parafollicular cell hyperplasia in the thyroid. Focal mesothelial proliferation in the epicardium of the heart, intranuclear inclusions in the epididymis, focal granulomatous inflammation in the kidney, and aberrant pancreas in the gallbladder were noted each in 1 case. Granulomas possibly caused by parasitic infection were found in 21 cases (24.4%).

Hyaline Glomerulopathy with Tubulo-Fibrillary Deposits in Young ddY Mice

Hyaline glomerulopathy with tubulo-fibrillary deposits was observed in two young female ddY mice. One of the mice showed gross systemic edema and bilateral enlargement and pale color of the kidneys, whereas no significant gross findings were noted in the other mouse. Microscopically, a large number of the glomeruli in both mice were enlarged because of diffuse and global deposition of amorphous eosinophilic materials. The deposits were negatively stained with Congo red and positively stained with IgG, IgM, IgA, C3, and periodic acid–Schiff. Electron microscopic examination revealed microtubular and fibrillary deposits with diameters of 80–100 and 9–16 nm, respectively, in the subendothelial space of the glomeruli. These features are histopathologically similar to immunotactoid glomerulopathy or fibrillary glomerulonephritis according to the classification of human glomerular lesions. Understanding of these characteristics of hyaline glomerulopathy in ddY mice is essential when evaluating pharmacological, pharmacokinetic, and toxicological studies using this mouse strain.