Okio Hino

Tsc tumour suppressor proteins antagonize amino-acid–TOR signalling

Target of Rapamycin (TOR) mediates a signalling pathway that couples amino acid availability to S6 kinase (S6K) activation, translational initiation and cell growth. Here, we show that tuberous sclerosis 1 (Tsc1) and Tsc2, tumour suppressors that are responsible for the tuberous sclerosis syndrome, antagonize this amino acid–TOR signalling pathway. We show that Tsc1 and Tsc2 can physically associate with TOR and function upstream of TOR genetically. In Drosophila melanogaster and mammalian cells, loss of Tsc1 and Tsc2 results in a TOR-dependent increase of S6K activity. Furthermore, although S6K is normally inactivated in animal cells in response to amino acid starvation, loss of Tsc1–Tsc2 renders cells resistant to amino acid starvation. We propose that the Tsc1–Tsc2 complex antagonizes the TOR-mediated response to amino acid availability. Our studies identify Tsc1 and Tsc2 as regulators of the amino acid–TOR pathway and provide a new paradigm for how proteins involved in nutrient sensing function as tumour suppressors.

Autophagy-deficient mice develop multiple liver tumors

Autophagy is a major pathway for degradation of cytoplasmic proteins and organelles, and has been implicated in tumor suppression. Here, we report that mice with systemic mosaic deletion of Atg5 and liver-specific Atg7⁻/⁻ mice develop benign liver adenomas. These tumor cells originate autophagy-deficient hepatocytes and show mitochondrial swelling, p62 accumulation, and oxidative stress and genomic damage responses. The size of the Atg7⁻/⁻ liver tumors is reduced by simultaneous deletion of p62. These results suggest that autophagy is important for the suppression of spontaneous tumorigenesis through a cell-intrinsic mechanism, particularly in the liver, and that p62 accumulation contributes to tumor progression.

A germ-line Tsc1 mutation causes tumor development and embryonic lethality that are similar, but not identical to, those caused by Tsc2 mutation in mice

Tuberous sclerosis (TS) is characterized by the development of hamartomas in various organs and is caused by a germ-line mutation in either TSC1 or TSC2 tumor suppressor genes. From the symptomatic resemblance among TS patients, involvement of TSC1 and TSC2 products in a common pathway has been suggested. Here, to analyze the function of the Tsc1 product, we established a line of Tsc1 (TSC1 homologue) knockout mouse by gene targeting. Heterozygous Tsc1 mutant (Tsc1+/−) mice developed renal and extra-renal tumors such as hepatic hemangiomas. In these tumors, loss of wild-type Tsc1 allele was observed. Homozygous Tsc1 mutants died around embryonic days 10.5–11.5, frequently associated with neural tube unclosure. As a whole, phenotypes of Tsc1 knockout mice resembled those of Tsc2 knockout mice previously reported, suggesting that the presumptive common pathway for Tsc1 and Tsc2 products may also exist in mice. Notably, however, development of renal tumors in Tsc1+/− mice was apparently slower than that in Tsc2+/− mice. The Tsc1 knockout mouse described here will be a useful model to elucidate the function of Tsc1 and Tsc2 products as well as pathogenesis of TS.

Hepatitis C Virus Core Protein Interacts with 14-3-3 Protein and Activates the Kinase Raf-1

ABSTRACT Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver dysfunction in humans and is epidemiologically closely associated with the development of human hepatocellular carcinoma. Among HCV components, core protein has been reported to be implicated in cell growth regulation both in vitro and in vivo, although mechanisms explaining those effects are still unclear. In the present study, we identified that members of the 14-3-3 protein family associate with HCV core protein. 14-3-3 protein bound to HCV core protein in a phosphoserine-dependent manner. Introduction of HCV core protein caused a substantial increase in Raf-1 kinase activity in HepG2 cells and in a yeast genetic assay. Furthermore, the HCV core–14-3-3 interaction was essential for Raf-1 kinase activation by HCV core protein. These results suggest that HCV core protein may represent a novel type of Raf-1 kinase-activating protein through its interaction with 14-3-3 protein and may contribute to hepatocyte growth regulation.

Mutation analysis of the TSC1 and TSC2 genes in Japanese patients with pulmonary lymphangioleiomyomatosis

AbstractPulmonary lymphangioleiomyomatosis (LAM) is a destructive lung disease characterized by a diffuse hamartomatous proliferation of smooth muscle cells (LAM cells) in the lungs. Pulmonary LAM can occur as an isolated form (sporadic LAM) or in association with tuberous sclerosis complex (TSC) (TSC-LAM), a genetic disorder with autosomal dominant inheritance with various expressivity resulting from mutations of either the TSC1 or TSC2 gene. We examined mutations of both TSC genes in 6 Japanese patients with TSC-LAM and 22 patients with sporadic LAM and identified six unique and novel mutations. TSC2 germline mutations were detected in 2 (33.3%) of 6 patients with TSC-LAM and TSC1 germline mutation in 1 (4.5%) of 22 sporadic LAM patients. In accordance with the tumor-suppressor model, loss of heterozygosity (LOH) was detected in LAM cells from 3 of 4 patients with TSC-LAM and from 4 of 8 patients with sporadic LAM. Furthermore, an identical LOH or two identical somatic mutations were demonstrated in LAM cells microdissected from several tissues, suggesting LAM cells can spread from one lesion to another. Our results from Japanese patients with LAM confirmed the current concept of pathogenesis of LAM: TSC-LAM has a germline mutation but sporadic LAM does not; sporadic LAM is a TSC2 disease with two somatic mutations; and a variety of TSC mutations causes LAM. However, our study indicates that a fraction of sporadic LAM can be a TSC1 disease; therefore, both TSC genes should be examined, even for patients with sporadic LAM.

Biphasic Response of Pancreatic β-Cell Mass to Ablation of Tuberous Sclerosis Complex 2 in Mice

ABSTRACT Recent studies have demonstrated the importance of insulin or insulin-like growth factor 1 (IGF-1) for regulation of pancreatic β-cell mass. Given the role of tuberous sclerosis complex 2 (TSC2) as an upstream molecule of mTOR (mammalian target of rapamycin), we examined the effect of TSC2 deficiency on β-cell function. Here, we show that mice deficient in TSC2, specifically in pancreatic β cells (βTSC2−/− mice), manifest increased IGF-1-dependent phosphorylation of p70 S6 kinase and 4E-BP1 in islets as well as an initial increased islet mass attributable in large part to increases in the sizes of individual β cells. These mice also exhibit hypoglycemia and hyperinsulinemia at young ages (4 to 28 weeks). After 40 weeks of age, however, the βTSC2−/− mice develop progressive hyperglycemia and hypoinsulinemia accompanied by a reduction in islet mass due predominantly to a decrease in the number of β cells. These results thus indicate that TSC2 regulates pancreatic β-cell mass in a biphasic manner.

Interaction of folliculin (Birt-Hogg-Dubé gene product) with a novel Fnip1-like (FnipL/Fnip2) protein.

Birt-Hogg-Dubé (BHD) syndrome is characterized by the development of pneumothorax, hair folliculomas and renal tumors and the responsible BHD gene is thought to be a tumor suppressor. The function of folliculin (Flcn), encoded by BHD, is totally unknown, although its interaction with Fnip1 has been reported. In this study, we identified a novel protein binding to Flcn, which is highly homologous to Fnip1, and which we named FnipL (recently reported in an independent study as Fnip2). The interaction between FnipL/Fnip2 and Flcn may be mediated mainly by the C-terminal domains of each protein as is the case for the Flcn-Fnip1 interaction. FnipL/Fnip2 and Flcn were located together in the cytoplasm in a reticular pattern, although solely expressed Flcn was found mainly in the nucleus. Cytoplasmic retention of Flcn was canceled with C-terminal truncation of FnipL/Fnip2, suggesting that FnipL/Fnip2 regulates Flcn distribution through their complex formation. By the employment of siRNA, we observed a decrease in S6K1 phosphorylation in the BHD-suppressed cell. We also observed a decrease in S6K1 phosphorylation in FNIP1- and, to a lesser extent, in FNIPL/FNIP2-suppressed cells. These results suggest that Flcn-FnipL/Fnip2 and Flcn-Fnip1 complexes positively regulate S6K1 phosphorylation and that FnipL/Fnip2 provides an important clue to elucidating the function of Flcn and the pathogenesis of BHD.

Rapamycin reverses impaired social interaction in mouse models of tuberous sclerosis complex

Impairment of reciprocal social interaction is a core symptom of autism spectrum disorder. Genetic disorders frequently accompany autism spectrum disorder, such as tuberous sclerosis complex caused by haploinsufficiency of the TSC1 and TSC2 genes. Accumulating evidence implicates a relationship between autism spectrum disorder and signal transduction that involves tuberous sclerosis complex 1, tuberous sclerosis complex 2 and mammalian target of rapamycin. Here we show behavioural abnormalities relevant to autism spectrum disorder and their recovery by the mammalian target of rapamycin inhibitor rapamycin in mouse models of tuberous sclerosis complex. In Tsc2+/− mice, we find enhanced transcription of multiple genes involved in mammalian target of rapamycin signalling, which is dependent on activated mammalian target of rapamycin signalling with a minimal influence of Akt. The findings indicate a crucial role of mammalian target of rapamycin signalling in deficient social behaviour in mouse models of tuberous sclerosis complex, supporting the notion that mammalian target of rapamycin inhibitors may be useful for the pharmacological treatment of autism spectrum disorder associated with tuberous sclerosis complex and other conditions that result from dysregulated mammalian target of rapamycin signalling.

Clinical and genetic spectrum of Birt–Hogg–Dubé syndrome patients in whom pneumothorax and/or multiple lung cysts are the presenting feature

Background Birt–Hogg–Dubé syndrome (BHDS) is an inherited autosomal genodermatosis characterised by fibrofolliculomas of the skin, renal tumours and multiple lung cysts. Genetic studies have disclosed that the clinical picture as well as responsible germline FLCN mutations are diverse. Objectives BHDS may be caused by a germline deletion which cannot be detected by a conventional genetic approach. Real-time quantitative polymerase chain reaction (qPCR) may be able to identify such a mutation and thus provide us with a more accurate clinical picture of BHDS. Methods This study analysed 36 patients with multiple lung cysts of undetermined causes. Denaturing high performance liquid chromatography (DHPLC) was applied for mutation screening. If no abnormality was detected by DHPLC, the amount of each FLCN exon in genome was quantified by qPCR. Results An FLCN germline mutation was found in 23 (63.9%) of the 36 patients by DHPLC and direct sequencing (13 unique small nucleotide alterations which included 11 novel mutations). A large genomic deletion was identified in two of the remaining 13 patients by qPCR (one patient with exon 14 deletion and one patient with a deletion encompassing exons 9 to 14). Mutations including genomic deletions were most frequently identified in the 3′-end of the FLCN gene including exons 12 and 13 (13/25=52.0%). The BHDS patients whose multiple cysts prompted the diagnosis in this study showed a very low incidence of skin and renal involvement. Conclusions BHDS is due to large deletions as well as small nucleotide alterations. Racial differences may occur between Japanese and patients of European decent in terms of FLCN mutations and clinical manifestations.

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.