Identification of a Novel Bone Marrow Cell-Derived Accelerator of Fibrotic Liver Regeneration Through Mobilization of Hepatic Progenitor Cells in Mice.

Granulocyte colony stimulating factor (G-CSF) has been reported to ameliorate impaired liver function in patients with advanced liver diseases through mobilization and proliferation of hepatic progenitor cells (HPCs). However, the underlying mechanisms remain unknown. We previously showed that G-CSF treatment increased the number of bone marrow (BM)-derived cells migrating to the fibrotic liver following repeated carbon tetrachloride (CCl4 ) injections into mice. In this study, we identified opioid growth factor receptor-like 1 (OGFRL1) as a novel BM cell-derived accelerator of fibrotic liver regeneration in response to G-CSF treatment. Endogenous Ogfrl1 was highly expressed in the hematopoietic organs such as the BM and spleen, whereas the liver contained a relatively small amount of Ogfrl1 mRNA. Among the peripheral blood cells, monocytes were the major sources of OGFRL1. Endogenous Ogfrl1 expression in both the peripheral blood monocytes and the liver was decreased following repeated CCl4 injections. An intrasplenic injection of cells overexpressing OGFRL1 into CCl4 -treated fibrotic mice increased the number of HPC and stimulated proliferation of hepatic parenchymal cells after partial resection of the fibrotic liver. Furthermore, overexpression of OGFRL1 in cultured HPC accelerated their differentiation as estimated by increased expression of liver-specific genes such as hepatocyte nuclear factor 4α, cytochrome P450, and fatty acid binding protein 1, although it did not affect the colony forming ability of HPC. These results indicate a critical role of OGFRL1 in the mobilization and differentiation of HPC in the fibrotic liver, and administration of OGFRL1-expressing cells may serve as a potential regenerative therapy for advanced liver fibrosis. Stem Cells 2019;37:89-101.

Stearoyl-CoA Desaturase Promotes Liver Fibrosis and Tumor Development in Mice via a Wnt Positive-Signaling Loop by Stabilization of Low-Density Lipoprotein-Receptor-Related Proteins 5 and 6.

BACKGROUND & AIMS: Stearoyl-CoA desaturase (SCD) synthesizes monounsaturated fatty acids (MUFAs) and has been associated with the development of metabolic syndrome, tumorigenesis, and stem cell characteristics. We investigated whether and how SCD promotes liver fibrosis and tumor development in mice. METHODS: Rodent primary hepatic stellate cells (HSCs), mouse liver tumor-initiating stem cell-like cells (TICs), and human hepatocellular carcinoma (HCC) cell lines were exposed to Wnt signaling inhibitors and changes in gene expression patterns were analyzed. We assessed the functions of SCD by pharmacologic and conditional genetic manipulation in mice with hepatotoxic or cholestatic induction of liver fibrosis, orthotopic transplants of TICs, or liver tumors induced by administration of diethyl nitrosamine. We performed bioinformatic analyses of SCD expression in HCC vs nontumor liver samples collected from patients, and correlated levels with HCC stage and patient mortality. We performed nano-bead pull-down assays, liquid chromatography-mass spectrometry, computational modeling, and ribonucleoprotein immunoprecipitation analyses to identify MUFA-interacting proteins. We examined the effects of SCD inhibition on Wnt signaling, including the expression and stability of low-density lipoprotein-receptor-related proteins 5 and 6 (LRP5 and LRP6), by immunoblot and quantitative polymerase chain reaction analyses. RESULTS: SCD was overexpressed in activated HSC and HCC cells from patients; levels of SCD messenger RNA (mRNA) correlated with HCC stage and patient survival time. In rodent HSCs and TICs, the Wnt effector ß-catenin increased sterol regulatory element binding protein 1-dependent transcription of Scd, and ß-catenin in return was stabilized by MUFAs generated by SCD. This loop required MUFA inhibition of binding of Ras-related nuclear protein 1 (Ran1) to transportin 1 and reduced nuclear import of elav-like protein 1 (HuR), increasing cytosolic levels of HuR and HuR-mediated stabilization of mRNAs encoding LRP5 and LRP6. Genetic disruption of Scd and pharmacologic inhibitors of SCD reduced HSC activation and TIC self-renewal and attenuated liver fibrosis and tumorigenesis in mice. Conditional disruption of Scd2 in activated HSCs prevented growth of tumors from TICs and reduced the formation of diethyl nitrosamine-induced liver tumors in mice. CONCLUSIONS: In rodent HSCs and TICs, we found SCD expression to be regulated by Wnt-ß-catenin signaling, and MUFAs produced by SCD provided a forward loop to amplify Wnt signaling via stabilization of Lrp5 and Lrp6 mRNAs, contributing to liver fibrosis and tumor growth. SCD expressed by HSCs promoted liver tumor development in mice. Components of the identified loop linking HSCs and TICs might be therapeutic targets for liver fibrosis and tumors.

Rosmarinic acid and baicalin epigenetically derepress peroxisomal proliferator-activated receptor γ in hepatic stellate cells for their antifibrotic effect.

UNLABELLED: Hepatic stellate cells (HSCs) undergo myofibroblastic transdifferentiation (activation) to participate in liver fibrosis and identification of molecular targets for this cell fate regulation is essential for development of efficacious therapeutic modalities for the disease. Peroxisomal proliferator-activated receptor γ (PPARγ) is required for differentiation of HSCs and its epigenetic repression underlies HSC activation. The herbal prescription Yang-Gan-Wan (YGW) prevents liver fibrosis, but its active ingredients and molecular mechanisms are unknown. Here we demonstrate YGW prevents and reverses HSC activation by way of epigenetic derepression of Pparγ involving reductions in MeCP2 expression and its recruitment to Pparγ promoter, suppressed expression of PRC2 methyltransferase EZH2, and consequent reduction of H2K27di-methylation at the 3' exon. High-performance liquid chromatography / mass spectrometry (HPLC/MS) and nuclear magnetic resonance (NMR) analyses identify polyphenolic rosmarinic acid (RA) and baicalin (BC) as active phytocompounds. RA and BC suppress the expression and signaling by canonical Wnts, which are implicated in the aforementioned Pparγ epigenetic repression. RA treatment in mice with existing cholestatic liver fibrosis inhibits HSC activation and progression of liver fibrosis. CONCLUSION: These results demonstrate a therapeutic potential of YGW and its active component RA and BC for liver fibrosis by way of Pparγ derepression mediated by suppression of canonical Wnt signaling in HSCs.

Novel anti-fibrotic modalities for liver fibrosis: molecular targeting and regenerative medicine in fibrosis therapy.

Based on the cellular and molecular mechanisms underlying hepatic fibrogenesis, several kinds of approaches have been proposed to treat liver fibrosis. Among a number of growth factors and cytokines that regulate collagen metabolism, transforming growth factor (TGF)-ß is the most potent factor to accelerate liver fibrosis by activating hepatic stellate cells, stimulating collagen gene transcription, and suppressing matrix metalloproteinases expression. Thus, TGF-ß as well as its intracellular mediators, Smad proteins, can be potential therapeutic targets for liver fibrosis. Constitutive phosphorylation and nuclear accumulation of Smad3 is the common feature of activated stellate cells. We have synthesized a novel small compound that inhibits Smad3-dependent collagen gene transcription by promoting nuclear import of a transcriptional repressor, YB-1. Another insight into anti-fibrotic strategies is the contribution of bone marrow-derived cells to the regression of liver fibrosis. Administration of granulocyte-colony stimulating factor enhanced the migration of bone marrow-derived cells into fibrotic liver tissue and accelerated the regression of experimental liver fibrosis. We have recently identified novel unknown factors expressed by bone marrow-derived cells that not only ameliorate liver fibrosis but also accelerate regeneration of fibrotic liver.

Negligible contribution of bone marrow-derived cells to collagen production during hepatic fibrogenesis in mice.

BACKGROUND & AIMS: Recent studies have reported that bone marrow (BM)-derived cells migrating into fibrotic liver tissue exhibit a myofibroblast-like phenotype and may participate in the progression of liver fibrosis. However, their contribution to collagen production has not been fully verified yet. We revisited this issue by using 2 mechanistically distinct liver fibrosis models introduced into transgenic collagen reporter mice and their BM recipients. METHODS: BM of wild-type mice was replaced by cells obtained from transgenic animals harboring tissue-specific enhancer/promoter sequences of alpha2(I) collagen gene (COL1A2) linked to enhanced green fluorescent protein (EGFP) or firefly luciferase (LUC) gene. Liver fibrosis was introduced into those mice by repeated carbon tetrachloride injections or ligation of the common bile duct. Activation of COL1A2 promoter was assessed by confocal microscopic examination detecting EGFP signals and luciferase assays of liver homogenates. RESULTS: The tissue-specific COL1A2 enhancer/promoter was activated in hepatic stellate cells following a single carbon tetrachloride injection or during primary culture on plastic. A large number of EGFP-positive collagen-expressing cells were observed in liver tissue of transgenic COL1A2/EGFP mice in both liver fibrosis models. In contrast, there were few EGFP-positive BM-derived collagen-producing cells detected in fibrotic liver tissue of COL1A2/EGFP recipients. Luciferase assays of liver tissues from COL1A2/LUC-recipient mice further indicated that BM-derived cells produced little collagen in response to fibrogenic stimuli. CONCLUSIONS: By using a specific and sensitive experimental system, which detects exclusively BM-derived collagen-producing cells, we conclude an unexpectedly limited role of BM-derived cells in collagen production during hepatic fibrogenesis.

Hepatocyte growth factor suppresses profibrogenic signal transduction via nuclear export of Smad3 with galectin-7.

BACKGROUND & AIMS: Hepatocyte growth factor (HGF) and transforming growth factor-beta (TGF-beta) regulate diversified cellular functions and often act antagonistically against each other. For example, TGF-beta is the most potent factor accelerating liver fibrosis, whereas HGF treatment prevents its progression. Here, we propose a novel molecular mechanism by which HGF counter represses TGF-beta-stimulated profibrogenic signal transduction. METHODS: Effects of HGF on TGF-beta-responsive gene transcription of type I collagen, the major matrix component of fibrotic liver, were examined by using cultured hepatic stellate cells (HSC) and transgenic mice harboring alpha2(I) collagen gene (COL1A2) promoter. Expression and subcellular localization of Smad3 were determined by Western blot analyses and immunofluorescence staining, respectively. A mass spectrometric analysis was employed to identify immunoprecipitated proteins with antiphospho-Smad2/3 antibodies. RESULTS: Over expression of HGF inhibited COL1A2 transcription in cultured HSC and suppressed activation of COL1A2 promoter in liver tissue induced by carbon tetrachloride administration. A mass spectrometric analysis identified galectin-7 as one of the immunoprecipitated proteins with antiphospho-Smad2/3 antibodies following HGF treatment. HGF accelerated nuclear export of Smad3 by enhancing its interaction with galectin-7. Transfection of cells with galectin-7 small interfering RNA inhibited nuclear export of Smad3 and abolished suppressive effect of HGF on expression of TGF-beta-responsive genes such as COL1A2 and plasminogen activator inhibitor-1. On the other hand, over expression of galectin-7 suppressed TGF-beta-stimulated expression of those target genes. CONCLUSIONS: These results reveal a novel function of intracellular galectin-7 as a transcriptional regulator via its interaction with Smad3 and provide a molecular basis for the antifibrotic effect of HGF.

Glycyrrhizin and its metabolite inhibit Smad3-mediated type I collagen gene transcription and suppress experimental murine liver fibrosis.

AIMS: Glycyrrhizin has been widely used for the treatment of chronic hepatitis C. It decreases the serum levels of aminotransferases, and suppresses progression of liver fibrosis as well as subsequent occurrence of hepatocellular carcinoma. Although previous studies have shown that glycyrrhizin and its metabolite inhibit collagen gene expression, its underlying mechanisms are virtually unknown. This study was aimed to explore molecular mechanisms responsible for the inhibitory effect of glycyrrhizin on type I collagen gene transcription. MAIN METHODS: Effects of glycyrrhizin and its metabolite, glycyrrhetinic acid, on collagen promoter activity were examined by using transgenic reporter mice harboring alpha2(I) collagen gene (COL1A2) promoter. Their effects on the TGF-beta/Smad signaling pathway were studied by cell transfection assays and immunofluorescence studies using cultured hepatic stellate cells. KEY FINDINGS: Administration of glycyrrhizin or its metabolite, glycyrrhetinic acid, significantly suppressed COL1A2 promoter activation and progression of liver fibrosis induced by repeated carbon tetrachloride injections. In cultured hepatic stellate cells, glycyrrhetinic acid, but not glycyrrhizin, inhibited type I collagen synthesis mostly at the level of gene transcription. This inhibitory effect of glycyrrhetinic acid was abolished by a mutation introduced into a Smad3-binding region within the COL1A2 promoter. Glycyrrhetinic acid did not affect gene expression of TGF-beta receptors or Smad proteins, but inhibited nuclear accumulation of Smad3 in activated hepatic stellate cells. In addition to those direct inhibitory effects on COL1A2 transcription, glycyrrhetinic acid also suppressed activation of quiescent hepatic stellate cells in primary culture. SIGNIFICANCE: The results provide a molecular basis for the anti-fibrotic effect of glycyrrhizin treatment.

Wnt Pathway Stabilizes MeCP2 Protein to Repress PPAR-γ in Activation of Hepatic Stellate Cells.

PPAR-γ is essential for differentiation of hepatic stellate cells (HSC), and its loss due to epigenetic repression by methyl-CpG binding protein 2 (MeCP2) causes HSC myofibroblastic activation mediated in part via Wnt pathway, the key cellular event in liver fibrosis. Decreased miR-132 was previously proposed to promote MeCP2 protein translation for Ppar-γ repression in activated HSC (aHSC). The present study aimed to test this notion and to better understand the mechanisms of MeCP2 upregulation in aHSC. MeCP2 protein is increased on day 3 to 7 as HSC become activated in primary culture on plastic, but this is accompanied by increased but not reduced miR-132 or miR-212 which is also expected to target MeCP2 due to its similar sequence with miR-132. The levels of these mRNAs are decreased 40~50% in aHSCs isolated from experimental cholestatic liver fibrosis but increased 6-8 fold in aHSC from hepatotoxic liver fibrosis in rats. Suppression of either or both of miR132 and miR212 with specific anti-miRNA oligonucleotides (anti-oligo), does not affect MeCP2 protein levels in aHSCs. The Wnt antagonist FJ9 which inhibits HSC activation, increases miR-132/miR-212, reduces MeCP2 and its enrichment at 5' Ppar-γ promoter, and restores Ppar-γ expression but the anti-oligo do not prevent Ppar-γ upregulation. The pan-NADPH oxidase (NOX) inhibitor diphenyleneiodonium (DPI) also reduces both MeCP2 and stabilized non-(S33/S37/Thr41)-phospho ß-catenin and reverts aHSC to quiescent cells but do not affect miR-132/miR-212 levels. Wnt antagonism with FJ9 increases MeCP2 protein degradation in cultured HSC, and FJ9-mediated loss of MeCP2 is rescued by leupeptin but not by proteasome and lysozome inhibitors. In conclusion, canonical Wnt pathway increases MeCP2 protein due to protein stability which in turn represses Ppar-γ and activates HSC.

Association of change in the type of job with prevalence of components of the metabolic syndrome-special reference to job stress.

OBJECTIVE: It is well established that job stress is a leading cause of cardiovascular disease. The relationship with the metabolic syndrome, however, has received only limited attention. The present study was designed to investigate associations between change of the type of job and the prevalence of metabolic syndrome components from the aspect of on-the-job stress and alteration in life style. METHODS: Thirty-six male workers of the manufacturing department were transferred to the carsales department at the same automobile company in 1992 to 1993. These same workers were transferred back to the manufacturing department after two years. We compared the first health-check data before the transfer in 1992 (Term A), a second set of data two years after transfer in 1994-95 (Term B) and a third set of data two years following transfer back to the manufacturing department in 1996-1998 (Term C). The workers were requested to provide information about drinking and smoking habits, and answer Karasek's questionnaire and a simple stress questionnaire in order to clarify the possibility of job stress in occurrence of the metabolic syndrome, defined in terms of obesity, hypertension, dyslipidemia, and impaired glucose tolerance as components. RESULTS: Five workers had two or more components of the metabolic syndrome before the transfer to the car-sales department (Group I). One demonstrated improvement, three no change, and one increase in symptoms from A to B. Seven workers had more than two components after the transfer to car-sales department (Group II), and six of them exhibited decrease two years following transfer back to the manufacturing department. Five of them also showed elevated liver enzymes in serum with the appearance of the components, and three of them demonstrated recover. Three workers had two components of the metabolic syndrome only at time point C (Group III), while the remaining 21 workers had 0 to one component throughout the observation period (Group IV). Amount of drinking and smoking increased significantly when working in the sales department but these items returned to the previous values after rejoining manufacturing, though differences were not observed between workers with (Group II) and without (Group IV) components of the metabolic syndrome. Body mass index (BMI) and alanine aminotransferase (ALT) increased significantly when workers moved to the sales department and that was significant in Group II as compared to Group IV. Three components of Karasek's JCQ changed significantly during job transfer, though differences were not observed between the workers with (Group II) and without (Group IV) components of the metabolic syndrome. Logistic regression analysis with age, lifestyle, Karasek's JCQ, and ALT revealed that elevation of ALT value was associated with having two or more components of metabolic syndrome, while hours of sleep demonstrated an inverse association. CONCLUSION: Elevated ALT and reduction of sleep hours may be associated with development of the metabolic syndrome in workers who change their type of job.

Change of components of the metabolic syndrome in a workers' health checkup after five years--relation with elevated liver enzymes, gene polymorphisms for ALDH 2, beta3-AR and lifestyle.

We previously reported that the prevalence of elevated alanine aminotransferase (ALT) increases with accumulation of metabolic syndrome components, and a greater degree of involvement of aldehyde dehydrogenase 2 (ALDH2) than beta3-adrenergic receptor gene (beta3-AR) polymorphisms. The present study was designed to clarify the effect of aging, lifestyle and the two gene polymorphisms on the relationship between 4 components of the metabolic syndrome (obesity, hypertension, dyslipidemia and impaired glucose tolerance) and elevated ALT values in a subset of 73 out of 148 male workers who were 35 years of age in the baseline study and 40 years old in the present study. Study subjects completed questionnaires about drinking and smoking habits, and underwent urinalysis, physical examination and peripheral blood tests, blood chemistry, electrocardiogram and chest X-rays each year as required by Japanese law. Information from the questionnaires and physical examinations, including liver function tests, were compared with previously reported ALDH2 and beta3-AR genotypes for the 73 workers. Of the 73 workers studied, 14 (19%) demonstrated decrease in metabolic syndrome components, 39 (53%) demonstrated no change, and 20 (27%) demonstrated an increase. Ten workers (14%) showed liver dysfunction at age 35 and 20 workers (27%) at age 40. Fourteen workers were newly diagnosed as having liver dysfunction at their 40-year checkup, thus being associated with the BMI and an active ALDH2 genotype. Accumulation of components of the metabolic syndrome were associated with the presence of liver dysfunction at 35 years. In conclusion, these findings indicate that ALDH2 genotyping as well as lifestyle habits may be important factors in causing metabolic syndrome with liver dysfunction.

Interplay between bone marrow and liver in the pathogenesis of hepatic fibrosis.

Recent advances in the technologies of both molecular biology and regenerative medicine have made it possible to identify bone marrow (BM)-derived cells migrating into various fibrotic organs including the liver. A number of studies have reported that BM-derived cells migrating into fibrotic liver tissue exhibit a myofibroblast-like phenotype and may participate in the progression of liver fibrosis. On the other hand, it has also been shown that BM-derived cells express matrix metalloproteinases and contribute to the regression of experimental liver fibrosis. These contradictory results may arise, at least in part, from the uncertainty of various different methods that have been used in those studies. In this review article, we describe the interplay between BM and liver in the progression and regression of liver fibrosis, with an emphasis on the necessity of qualified methods with high specificity and sensitivity to evaluate the role of BM-derived cells in collagen production.

Mouse intragastric infusion (iG) model.

Direct intragastric delivery of a diet, nutrient or test substance can be achieved in rodents (mice and rats) on a long-term (2-3 months) basis using a chronically implanted gastrostomy catheter and a flow-through swivel system. This rodent intragastric infusion (iG) model has broad applications in research on food intake, gastrointestinal (GI) physiology, GI neuroendocrinology, drug metabolism and toxicity, obesity and liver disease. It achieves maximal control over the rate and pattern of delivery and it can be combined with normal ad libitum feeding of solid diet if so desired. It may be adopted to achieve infusion at other sites of the GI system to test the role of a bypassed GI segment in neuroendocrine physiology, and its use in genetic mouse models facilitates the genetic analysis of a central question under investigation.

Differential contribution of dermal resident and bone marrow-derived cells to collagen production during wound healing and fibrogenesis in mice.

Recent studies show that bone marrow (BM)-derived cells migrating into a dermal wound promote healing by producing collagen type I. However, their contribution to the repair process has not been fully verified yet. It is also unclear whether BM-derived cells participate in dermal fibrogenesis. We have addressed these issues using transgenic mice that harbor tissue-specific enhancer/promoter sequences of α2(I) collagen gene linked to either enhanced green fluorescent protein (COL/EGFP) or the luciferase (COL/LUC) reporter gene. Following dermal excision or subcutaneous bleomycin administration, a large number of EGFP-positive collagen-producing cells appeared in the dermis of COL/EGFP reporter mice. When wild-type mice were transplanted with BM cells from transgenic COL/EGFP animals and subjected to dermal excision, no EGFP-positive BM-derived collagen-producing cells were detected throughout the repair process. Luciferase assays of dermal tissues from COL/LUC recipient mice also excluded collagen production by BM-derived cells during dermal excision healing. In contrast, a limited but significant number of CD45-positive collagen-producing cells migrated from BM following bleomycin injection. These results indicate that resident cells in the skin are the major source of de novo collagen deposition in both physiological and pathological conditions, whereas BM-derived cells participate, in part, in collagen production during dermal fibrogenesis.

Bone marrow-derived cells express matrix metalloproteinases and contribute to regression of liver fibrosis in mice.

UNLABELLED: Liver fibrosis is usually progressive, but it can occasionally be reversible if the causative agents are adequately removed or if patients are treated effectively. However, molecular mechanisms responsible for this reversibility of liver fibrosis have been poorly understood. To reveal the contribution of bone marrow (BM)-derived cells to the spontaneous regression of liver fibrosis, mice were treated with repeated carbon tetrachloride injections after hematopoietic reconstitution with enhanced green fluorescent protein (EGFP)-expressing BM cells. The distribution and characteristics of EGFP-positive (EGFP(+)) cells present in fibrotic liver tissue were examined at different time points after cessation of carbon tetrachloride intoxication. A large number of EGFP(+) cells were observed in liver tissue at peak fibrosis, which decreased during the recovery from liver fibrosis. Some of them, as well as EGFP-negative (EGFP(-)) liver resident cells, expressed matrix metalloproteinase (MMP)-13 and MMP-9. Whereas MMP-13 was transiently expressed mainly in the cells clustering in the periportal areas, MMP-9 expression and enzymatic activity were detected over the resolution process in several different kinds of cells located in the portal areas and along the fibrous septa. Therapeutic recruitment of BM cells by granulocyte colony-stimulating factor (G-CSF) treatment significantly enhanced migration of BM-derived cells into fibrotic liver and accelerated the regression of liver fibrosis. Experiments using transgenic mice overexpressing hepatocyte growth factor (HGF) indicated that G-CSF and HGF synergistically increased MMP-9 expression along the fibrous septa. CONCLUSION: Autologous BM cells contribute to the spontaneous regression of liver fibrosis, and their therapeutic derivation could be a new treatment strategy for intractable liver fibrosis.

Detection of individual over-smoking using conventional laboratory tests.

OBJECTIVE: The present study was designed to find useful markers for detecting the severity of smoking effects on the human body from conventional laboratory tests used in community health check-ups. METHODS: The subjects were 18,576 persons who visited Tokai University Hospital Health Check Clinic between January 5, 2000, and December 28, 2000. The data on their life-style information and laboratory tests were analyzed by multiple regression analysis and logistic analysis. RESULTS: White blood cell counts (WBC), the numbers of lymphocytes (Ly), hemoglobin concentration (Hb), mean corpuscular volume of red blood cells (MCV), and carcinoembriogenic antigen (CEA) were positively correlated with numbers of cigarettes consumed, while high density lipoprotein cholesterol (HDL-C) was negatively correlated, with statistically significant difference. Although the relationship between the grade of smoking habit and MCV, HDL-C and CEA was affected by alcohol intake, the WBC, MCV, HDL-C and CEA were shown to be the grading index for smoking effect regardless of drinking habit. Logistic analysis indicated that an accumulation of these marker abnormalities was not useful for differentiating smokers from non-smokers as compared with a single marker abnormality. CONCLUSION: WBC, MCV, HDL-C and CEA can be the grading index for smoking effect. Abnormalities in one or some of those values observed in healthy smokers indicate some effects of smoking on their bodies.

Cell type-specific intervention of transforming growth factor beta/Smad signaling suppresses collagen gene expression and hepatic fibrosis in mice.

BACKGROUND & AIMS: Transforming growth factor beta and its intracellular mediators, Smad proteins, play important roles in stimulating collagen gene transcription and, thus, could be the targets for treating hepatic fibrosis. However, intervention of transforming growth factor beta/Smad signaling affects physiological signal transduction as well and may cause serious adverse effects on clinical application. Here we have attempted to suppress hepatic fibrosis by expressing a transforming growth factor beta/Smad antagonist selectively in collagen-producing cells only in the fibrotic liver. METHODS: Recombinant adenoviruses expressing either green fluorescent protein or a transforming growth factor beta/Smad signal repressor, YB-1, were injected into mice untreated or treated with carbon tetrachloride. Green fluorescent protein expression was analyzed under a confocal laser scanning microscope. Antifibrotic effects of YB-1 overexpression were examined by luciferase assays and histological examination with transgenic reporter mice. RESULTS: When the CAG expression unit was used as a control, green fluorescent protein was strongly expressed in a large number of hepatocytes in both normal and carbon tetrachloride-treated liver. In contrast, green fluorescent protein expression driven by a tissue-specific enhancer of the mouse alpha2(I) collagen gene ( COL1A2 ) was detected in activated hepatic stellate cells in carbon tetrachloride-induced fibrotic liver, but not in untreated normal liver. No green fluorescent protein fluorescence was observed in any other organs when the COL1A2 enhancer was used. Adenovirus-mediated YB-1 expression under the control of the COL1A2 enhancer significantly decreased COL1A2 promoter activity after carbon tetrachloride injection and subsequently suppressed the progression of hepatic fibrosis. CONCLUSIONS: These results validate a new concept of the therapy for hepatic fibrosis to achieve cell type-specific gene expression only in the fibrotic liver, with little damage to other organs.

c-Jun NH2-terminal kinase pathway is involved in constitutive matrix metalloproteinase-1 expression in a hepatocellular carcinoma-derived cell line.

Transcription factor c-Jun serves for cellular proliferation, survival, differentiation and transformation and is recognized as an important factor in cancer development, including hepatocellular carcinoma (HCC). The purpose of present study is to determine the involvement of c-Jun in matrix metalloproteinase-1 (MMP-1) expression, which is previously reported by us to be expressed only in the early stage of human HCC showing stromal invasion. Of 5 human HCC cell lines examined, only HLE cells revealed mRNA and protein expression as well as enzymatic activity of MMP-1. Transient transfection of an MMP-1 promoter/luciferase construct (including 4.4 kb full promoter region) into HLE and HCC-T cells (MMP-1 nonproducer) showed that high promoter activity was observed only in HLE cells without inducers, and that this promoter activity was still observed when a shorter 0.6 kb proximal promoter construct was transfected. The 0.6 kb promoter region contained 3 AP-1 sites, and c-jun mRNA was constitutively expressed in HLE cells without inducers. Furthermore, phosphorylated c-Jun and c-Jun NH2-terminal kinase (JNK) were detected in HLE cells. Promoter activity of the 0.6 kb construct was suppressed with SP600125, a potent inhibitor of JNK, but not with PD98059 and SB203580, potent inhibitors of MEK1/2 and p38, respectively. The inhibitory effect of SP600125 was also observed at protein expression level and in enzymatic activity of MMP-1. Taken together, this study suggests that the JNK pathway is involved in the expression of MMP-1 in HCC cells and may represent a new functional role of c-Jun for HCC development.