Tomoyuki Nemoto

Interferon alfa down-regulates collagen gene transcription and suppresses experimental hepatic fibrosis in mice.

The equilibrium between the production and degradation of collagen is rigorously controlled by a number of growth factors and cytokines. Interferon alfa (IFN‐α) is now widely used for the treatment of chronic hepatitis C, which can improve serum levels of fibrotic markers and the degree of hepatic fibrosis, not only in patients who responded to therapy but also in those in whom it is ineffective. These findings may suggest that IFN‐α possesses direct antifibrotic effects in addition to its antiviral activity. However, in contrast to IFN‐γ, which has been shown to suppress collagen gene transcription, little is known about the mechanisms responsible for the antifibrotic effects of IFN‐α. Here, we report that IFN‐α, when administered into transgenic mice harboring the α2(I) collagen gene (COL1A2) promoter sequence, significantly repressed promoter activation and prevented the progression of hepatic fibrosis induced by carbon tetrachloride injection. Transient transfection assays indicated that IFN‐α decreased the steady‐state levels of COL1A2 messenger RNA (mRNA) and inhibited basal and TGF‐β/Smad3‐stimulated COL1A2 transcription in activated hepatic stellate cells (HSC). These inhibitory effects of IFN‐α on COL1A2 transcription were exerted through the interaction between phosphorylated Stat1 and p300. Blocking of the IFN‐α signal by overexpressing the intracellular domain‐deleted IFN receptor increased basal COL1A2 transcription and abolished the inhibitory effects of IFN‐α. In conclusion, our results indicate that IFN‐α antagonizes the TGF‐β/Smad3‐stimulated COL1A2 transcription in vitro and suppresses COL1A2 promoter activation in vivo, providing a molecular basis for antifibrotic effects of IFN‐α. (Hepatology 2003;38:890–899).

Constitutive phosphorylation and nuclear localization of Smad3 are correlated with increased collagen gene transcription in activated hepatic stellate cells

Hepatic stellate cells (HSC) are the main producers of type I collagen in fibrotic liver, and transforming growth factor‐β (TGF‐β) plays critical roles in stimulating collagen gene expression in the cells mainly at the level of transcription. We have previously identified an upstream sequence of α2(I) collagen gene (COL1A2) that is essential for its basal and TGF‐β‐stimulated transcription in skin fibroblasts and HSC. We designated this region the TGF‐β‐responsive element (TbRE). Recently Smad3, an intracellular mediator of TGF‐β signal transduction, has been shown to bind to the TbRE and stimulate COL1A2 transcription when overexpressed in skin fibroblasts. In the present study, we demonstrate increased transcription of COL1A2 and plasminogen activator inhibitor‐1 (PAI‐1) genes and low response to TGF‐β in an activated HSC clone derived from a cirrhotic liver. Western blot analyses indicated constitutive phosphorylation of Smad3 in the cells. Immunofluorescence studies revealed that, in contrast to Smad2 that translocated from the cytoplasm to the nucleus upon TGF‐β treatment, Smad3 and Smad4 were present in the nucleus irrespective of ligand stimulation. Increased COL1A2 and PAI‐1 gene transcription in the cells was not affected by overexpression of inhibitory Smad7. Altogether, the results correlate abnormality in TGF‐β/Smad signaling with pathologically accelerated collagen gene transcription in activated HSC.

RFX1 mediates the serum-induced immediate early response of Id2 gene expression.

Id2, a negative regulator of basic helix-loop-helix transcription factors, is involved in regulating cell differentiation and proliferation. To obtain insight into the role of Id2 in cell cycle control, we investigated the mechanisms underlying the immediate early response of Id2 expression to serum stimulation in NIH3T3 cells. Luciferase reporter analysis with deletion and point mutants demonstrated the serum response element of Id2 (Id2-SRE) to be a consensus binding site for RFX1 (regulatory factor for X-box 1) present 3.0 kb upstream of the transcription initiation site of Id2. Gel shift and chromatin immunoprecipitation assays confirmed the binding of RFX1 to Id2-SRE in vitro and in vivo, respectively. In both assays, RFX1 binding was observed not only in serum-stimulated cells, but also in serum-starved cells. Knockdown of RFX1 by RNA interference disturbed the immediate early response of Id2 expression in cells and abrogated the Id2-SRE-mediated induction of luciferase activity by serum. These alterations were rescued by the introduction of RNA interference-resistant RFX1 into cells. On the other hand, in the Id2-SRE-mediated reporter assay, RFX1 with an N-terminal deletion abrogated the serum response, whereas RFX1 with a C-terminal deletion enhanced the reporter activity in serum-starved cells. Furthermore, HDAC1 was recruited to Id2-SRE in serum-starved cells. These results demonstrate that RFX1 mediates the immediate early response of the Id2 gene by serum stimulation and suggest that the function of RFX1 is regulated intramolecularly in its suppression in growth-arrested cells. Our results unveil a novel transcriptional control of immediate early gene expression.

Gene expression profiling of hepatocarcinogenesis in a mouse model of chronic hepatitis B

Background Hepatocellular carcinoma (HCC) is a common complication of chronic viral hepatitis. In support of this notion, we have reported that hepatitis B surface antigen (HBsAg)-specific CD8+ T lymphocytes critically contribute to inducing chronic liver cell injury that exerts high carcinogenic potential in a hepatitis B virus (HBV) transgenic mouse model. The dynamics of the molecular signatures responsible for hepatocellular carcinogenesis are not fully understood. The current study was designed to determine the serial changes in gene expression profiles in a model of chronic immune-mediated hepatitis. Methods Three-month-old HBV transgenic mice were immunologically reconstituted with bone marrow cells and splenocytes from syngeneic nontransgenic donors. Liver tissues were obtained every three months until 18 months at which time all mice developed multiple liver tumors. Nitrative DNA lesions and hepatocyte turnover were assessed immunohistochemically. Gene expression profiles were generated by extracting total RNA from the tissues and analyzing by microarray. Results The nitrative DNA lesions and the regenerative proliferation of hepatocytes were increased during the progression of chronic liver disease. In a gene expression profile analysis of liver samples, the chemokine- and T cell receptor (TCR)-mediated pathways were enhanced during chronic hepatitis, and the EGF- and VEGF-mediated pathways were induced in HCC. Among these molecules, the protein levels of STAT3 were greatly enhanced in all hepatocyte nuclei and further elevated in the cytoplasm in HCC tissue samples at 18 months, and the levels of phosphorylated TP53 (p-p53-Ser 6 and -Ser 15) were increased in liver tissues. Conclusions HBV-specific immune responses caused unique molecular signatures in the liver tissues of chronic hepatitis and triggered subsequent carcinogenic gene expression profiles in a mouse model. The results suggest a plausible molecular basis responsible for HBV-induced immune pathogenesis of HCC.

Transcriptional Activation of Type I Collagen Gene during Hepatic Fibrogenesis

Publisher Summary Collagens represent a family of proteins involved not only in the maintenance of organ architecture and tissue integrity, but also in various physiological conditions, such as developmental program, tissue repair process, and wound healing. There is a dynamic balance between production and degradation of collagen, and a disruption of this equilibrium results in either excessive collagen deposition or inadequate tissue integrity. Irrespective of the initial stimuli, fibrosis in different organs is caused commonly by a chronic and uncontrolled inflammatory and repair process, leading to excessive deposition of collagen and other components of ECM. The liver is one of those organs undergoing progressive fibrosis as a result of chronic repeating inflammation. Type I collagen, the major component of ECM in fibrotic tissues, is a heterotrimer composed of two α1 chains and one α2 chain. They are coordinately expressed but encoded by the distinct genes, COL1A1 and COLIA2, respectively. Transforming growth factor-β1 plays a critical role in stimulating type I collagen gene expression mainly at the levels of transcription. This chapter provides a summary of the cell type-specific activation of the COL1A2 promoter during hepatic fibrogenesis and reveals molecular mechanisms responsible for pathologically accelerated collagen gene transcription in activated hepatic stellate cells.

Long-term prognosis after biliary stenting for common bile duct stones in high-risk elderly patients: Biliary stenting in high-risk elderly patients

To evaluate the long‐term outcomes of complete common bile duct (CBD) stone removal and biliary stenting in elderly patients (≥85 years) with CBD stones.

Duplication cyst of the ileum presenting with severe anemia detected by double-balloon endoscopy

Background and study aims  Duplication cysts of the ileum are rare and present with non-specific clinical manifestations such as abdominal pain, vomiting, melena, and intussusception. Therefore, preoperative diagnosis is difficult. Here, we report a case of duplication cyst of the small intestine that was diagnosed preoperatively using double-balloon enteroscopy. A 19-year-old man presented with severe iron deficiency anemia, abdominal pain, and exertional dyspnea. Gastroscopy and colonoscopy revealed no remarkable findings. Abdominal computed tomography revealed a cystic structure in the ileum. Therefore, we performed double-balloon enteroscopy via the anal route. The intestinal tract was bifurcated, with one segment ending in a blind sac containing normal villi and an ulceration. Tc-99 m pertechnetate scintigraphy showed no accumulation in the lesion. Accordingly, we diagnosed a duplication cyst and suspected that this was the cause of severe anemia. Following small bowel resection with cyst excision and anastomosis, the anemia and presenting symptoms resolved. This report highlights the usefulness of double-balloon enteroscopy of the small intestine for preoperative diagnosis of the obscure gastrointestinal bleeding, including duplication cysts .

Transcriptional Regulation of Type I Collagen Gene Expression by Transforming Growth Factor-ß and Smad Proteins

Increased production of type I collagen is a common hallmark of fibrotic diseases in various organs including the liver. This increase is exerted mainly at the level of transcription, and transforming growth factor-β (TGFβ) is the key factor in stimulating gene transcription. We have previously shown that the -313 to -183 upstream sequence of α2(I) collagen gene (C0L1A2) is essential for basal and TGFs-stimulated transcription in skin fibroblasts and hepatic stellate cells. We designated this region the TGFs-responsive element (TbRE) and revealed that a ubiquitous trans-activator Spl and unknown nuclear factor(s) bind to this region to mediate the stimulatory effect of TGFs. Experiments using transgenic mice harboring the -313 C0L1A2 promoter have revealed that the promoter is activated in a cell type-specific manner during hepatic fibrogenesis in vivo. Smad proteins have been recently identified as mediators of intracellular signal transduction of TGFs superfamily members. One of them, Smad3 is shown to bind to the TbRE and is implicated in mediating TGFβ-elicited stimulation of C0L1A2 transcription.

A case of erythropoietic protoporphyria with early liver cirrhosis improved by chenodeoxycholic acid administration.

症例は35歳男性.1965年より露光部に紅斑・浮腫が出現し,1987年,赤血球中プロトポルフィジン(PP)の高値より造血性プロトポルフィリン症(EPP)と診断された.1994年,肝障害にて当科入院.入院時の血液生化学検査で,AST 63IU/l, ALT 92IU/l, γGTP 303IU/lの肝障害と赤血球中PP値の増加(7,606μg/l)を認めた.肝組織像では小葉構造は乱れ,一部に再生結節が認められた.偏光顕微鏡で重屈折性を示す,褐色色業沈着を肝細胞,Kupffer細胞に認め,以上よりEPPによる早期肝硬変と診断した.ケノデオキシコール酸900mg/日の投与により,ALTは30IU/lと正常化し,γGTP 63IU/l,赤血球中PP5, 748μg/lと低下した.EPPにおいては時に肝不全を呈することがあり,早期診断と肝機能の厳重な経過観察が必要である.本例ではケノデオキシコール酸療法が赤血球中PPの低下と肝機能の改善に有効であったと考えられた.