Tissue specific induction of p62/sqstm1 by farnesoid X receptor

Background: Farnesoid X Receptor (FXR) is a member of the nuclear receptor superfamily and is a ligand-activated transcription factor essential for maintaining liver and intestinal homeostasis. FXR is protective against carcinogenesis and inflammation in liver and intestine as demonstrated by the development of inflammation and tumors in the liver and intestine of FXR knock-out mice. However, mechanisms for the protective effects of FXR are not completely understood. This study reports a novel role of FXR in regulating expression of Sqstm1, which encodes for p62 protein. p62 plays an important role in maintaining cellular homeostasis through selective autophagy and activating signal transduction pathways, such as NF-κB to support cell survival and caspase-8 to initiate apoptosis. FXR regulation of Sqstm1 may serve as a protective mechanism. Methods and Results: This study showed that FXR bound to the Sqstm1 gene in both mouse livers and ileums as determined by chromatin immunoprecipitation. In addition, FXR activation enhanced transcriptional activation of Sqstm1 in vitro. However, wild-type mice treated with GW4064, a synthetic FXR ligand, showed that FXR activation induced mRNA and protein expression of Sqstm1/p62 in ileum, but not in liver. Interestingly, FXR-transgenic mice showed induced mRNA expression of Sqstm1 in both liver and ileum compared to wild-type mice. Conclusions: Our current study has identified a novel role of FXR in regulating the expression of p62, a key factor in protein degradation and cell signaling. Regulation of p62 by FXR indicates tissue-specific and gene-dosage effects. Furthermore, FXR-mediated induction of p62 may implicate a protective mechanism of FXR. © 2012 Williams et al

Heterozygous B beta-chain C-terminal 12 amino acid elongation variant, B beta X462W (Kyoto VI), showed dysfibrinogenemia

A heterozygous patient with dysfibrinogenemia with slight bleeding and no thrombotic complications was diagnosed with fibrinogen Kyoto VI (K-VI). To elucidate the genetic mutation(s) and characterize the variant protein, we performed the following experiments and compared with identical and similar variants that have already been reported. The proposita's PCR-amplified DNA was analyzed by sequencing and her purified plasma fibrinogen underwent SDS-PAGE followed by immunoblotting, fibrin polymerization, and scanning electron microscopic observation of fibrin clot and fibers. Sequence analyses showed that K-VI fibrinogen substituted W (TGG) for terminal codon (TAG), resulting in 12 amino acid elongation 462-473 (WSPIRRFLLFCM) in the B beta-chain. Protein analyses indicated that the presence of some albumin-binding variant fibrinogens and a dimeric molecule of variant fibrinogens reduced fibrin polymerization, with a thinner fiber and aberrant fibrin network. These results are almost the same as for the identical variant of Magdeburg, however, different from the similar variant of Osaka VI [ 12 amino acid elongation 462-473 (KSPIRRFLLFCM) in the B beta-chain] in the presence of variant forms and clot structure. We speculate the side-chain difference at 462 residues, W in K-VI, K in Osaka VI, and/or the difference in the presence of disulfide bridged forms of variant fibrinogens, led to the notable difference in the fibrin bundle network. Although a strong evolutional and structural association between B beta-chain and gamma-chain molecules is established, the corresponding recombinant 15 residue elongation variants of the fibrinogen gamma-chain showed reduced assembly and secretion.ArticleBLOOD COAGULATION & FIBRINOLYSIS. 23(1):87-90 (2012)journal articl

Nuclear abnormalities in aspirated thyroid cells and chromosome aberrations in lymphocytes of residents near the Semipalatinsk Nuclear Test Site

Chromosomal studies in peripheral lymphocytes from 63 residents near the Semipalatinsk nuclear test site, at ages of 52–63 years old, were performed in 2001–2002. A higher rate of chromosome aberrations was observed in the two contaminated villages, Dolon and Sarjal, compared with the control village, Kokpekti. Moreover, a relationship of frequency of cells with radiation induced chromosome aberrations and the previously estimated exposure dose was observed. Furthermore, apparent nuclear abnormalities (ANA) of thyroid follicular cells were studied in 30 out of 63 residents, who were examined for chromosome aberrations. A higher rate of ANA was also found in the residents in the exposed villages compared with those in the control village. These results suggest radiation effects both on the chromosomes in peripheral lymphocytes and on the follicular cells in the thyroid

Nuclear abnormalities in aspirated thyroid cells and chromosome aberrations in lymphocytes of residents near the Semipalatinsk Nuclear Test Site

Chromosomal studies in peripheral lymphocytes from 63 residents near the Semipalatinsk nuclear test site, at ages of 52–63 years old, were performed in 2001–2002. A higher rate of chromosome aberrations was observed in the two contaminated villages, Dolon and Sarjal, compared with the control village, Kokpekti. Moreover, a relationship of frequency of cells with radiation induced chromosome aberrations and the previously estimated exposure dose was observed. Furthermore, apparent nuclear abnormalities (ANA) of thyroid follicular cells were studied in 30 out of 63 residents, who were examined for chromosome aberrations. A higher rate of ANA was also found in the residents in the exposed villages compared with those in the control village. These results suggest radiation effects both on the chromosomes in peripheral lymphocytes and on the follicular cells in the thyroid

The antiparasitic drug ivermectin is a novel FXR ligand that regulates metabolism

Farnesoid X receptor (FXR) has important roles in maintaining bile acid and cholesterol homeostasis. Here we report that the antiparasitic drug ivermectin is a ligand for nuclear FXR. We identify ivermectin using a high-throughput compound library screening and show that it induces the transcriptional activity of the FXR with distinctive properties in modulating coregulator recruitment. The crystal structure of ivermectin complexed with the ligand-binding domain of FXR reveals a unique binding mode of ivermectin in the FXR ligand-binding pocket, including the highly dynamic AF-2 helix and an expanded ligand-binding pocket. Treatment of wild-type mice, but not of FXR-null mice, with ivermectin decreases serum glucose and cholesterol levels, suggesting that ivermectin regulates metabolism through FXR. Our results establish FXR as the first mammalian protein targeted by ivermectin with high selectivity. Considering that ivermectin is a widely used clinical drug, our findings reveal a safe template for the design of novel FXR ligands

Farnesoid X Receptor Induces Murine Scavenger Receptor Class B Type I via Intron Binding

Farnesoid X receptor (FXR) is a nuclear receptor and a key regulator of liver cholesterol and triglyceride homeostasis. Scavenger receptor class B type I (SR-BI) is critical for reverse cholesterol transport (RCT) by transporting high-density lipoprotein (HDL) into liver. FXR induces SR-BI, however, the underlying molecular mechanism of this induction is not known. The current study confirmed induction of SR-BI mRNA by activated FXR in mouse livers, a human hepatoma cell line, and primary human hepatocytes. Genome-wide FXR binding analysis in mouse livers identified 4 putative FXR response elements in the form of inverse repeat separated by one nucleotide (IR1) at the first intron and 1 IR1 at the downstream of the mouse Sr-bi gene. ChIP-qPCR analysis revealed FXR binding to only the intronic IR1s, but not the downstream one. Luciferase assays and site-directed mutagenesis further showed that 3 out of 4 IR1s were able to activate gene transcription. A 16-week high-fat diet (HFD) feeding in mice increased hepatic Sr-bi gene expression in a FXR-dependent manner. In addition, FXR bound to the 3 bona fide IR1s in vivo, which was increased following HFD feeding. Serum total and HDL cholesterol levels were increased in FXR knockout mice fed the HFD, compared to wild-type mice. In conclusion, the Sr-bi/SR-BI gene is confirmed as a FXR target gene in both mice and humans, and at least in mice, induction of Sr-bi by FXR is via binding to intronic IR1s. This study suggests that FXR may serve as a promising molecular target for increasing reverse cholesterol transport

統合的ストレス応答の制御によるNAFLDでの肝切除後肝再生障害の新規治療法の解明

肝臓の持つ強靭な再生能は、脂肪肝では障害され、肝切除術後の肝不全など合併症を引き起こす。脂肪肝での再生障害の危険性が広く知られる一方で、その病因・治療法は、十分に解明されていなかった。代表者は、脂肪肝での再生障害に、多様な細胞内ストレスに共通して誘導される「統合的ストレス応答」が、主要な役割を果たす事を見出している。本研究では、脂肪肝再生障害メカニズムとして、ストレス誘導性転写因子ATF3が重要な役割を担うことを明らかにした。The liver has robust regenerative potential in response to damage, but hepatic steatosis weakens this potential. That is known the impaired liver regeneration causes complications after surgery, such as liver failure. However the detail of mechanisms was unclear. The enhanced integrated stress response (ISR) is commonly induced by various cellular stress. We found that ISR plays a key role in impaired regeneration of hepatic steatosis. In this study, we revealed that stress induced transcription factor has an important role as the mechanism of liver regeneration disorder in fatty liver disease.研究課題/領域番号:15H05678, 研究期間(年度):2015-04-01 – 2019-03-3

高度脂肪肝再生過程で生じる広汎細胞死の発症メカニズムの解明

金沢大学新学術創成研究機構The liver has robust regenerative potential in response to damage, but this potential is impaired in hepatic steatosis. In particular, impaired liver regeneration in severe fatty liver is a trigger for complications after hepatectomy. We have found that solitary cell death and focal cell death (necrosis) occur in regenerating severe fatty liver after hepatectomy, and that this focal cell death is an important trigger for impaired fatty liver regeneration. In this study, we found that the stress response gene activating transcription factor 3 (ATF3) is important for regulation of focal cell death during liver regeneration in severe fatty liver.肝臓は強靭な再生力を持つ臓器であるが、脂肪肝ではその再生力が障害される。特に高度脂肪肝における肝再生障害は、肝切除術後合併症の誘因となっている。我々は、高度脂肪肝での切除後再生過程では孤発性の細胞死と広汎な細胞死（壊死）が発症すること、この広汎細胞死が肝再生障害の誘因として重要であることを明らかにしている。本研究課題では、高度脂肪肝再生過程における広汎細胞死の発症メカニズムの解明を行い、ストレス誘導性転写因子ATF3が重要な役割を担うことを明らかにした。研究課題/領域番号:19K09192, 研究期間(年度):2019-04-01 – 2022-03-31出典：研究課題「高度脂肪肝再生過程で生じる広汎細胞死の発症メカニズムの解明」課題番号19K09192（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-19K09192/19K09192seika/）を加工して作