S Dooley

Smad7 prevents activation of hepatic stellate cells and liver fibrosis in rats

BACKGROUND & AIMS Numerous studies implicate transforming growth factor (TGF)-beta signaling in liver fibrogenesis. To perturb the TGF-beta pathway during this process, we overexpressed Smad7, an intracellular antagonist of TGF-beta signaling, in vivo and in primary-cultured hepatic stellate cells (HSCs). METHODS Ligation of the common bile duct (BDL) was used to induce liver fibrosis in rats. Animals received injections of an adenovirus carrying Smad7 cDNA into the portal vein during surgery and via the tail vein at later stages. The effect of Smad7 on TGF-beta signaling and activation of HSC was further analyzed in primary-cultured cells. RESULTS Smad7-overexpressing BDL rats displayed reduced collagen and alpha-SMA expression and reduced hydroxyproline content in the liver, when compared with animals administered AdLacZ. Such a beneficial effect was also observed when Smad7 was expressed in animals with established fibrosis. Accordingly, Smad7 arrested transdifferentiation of primary-cultured HSCs. AdSmad7 infected cells remained in a quiescent stage and retained storage of vitamin A droplets. Smad7 expression totally blocked TGF-beta signal transduction, shown by inhibiting Smad2/3 phosphorylation, nuclear translocation of activated Smad complexes, and activation of (CAGA)(9)-MLP-Luc, resulting in decreased collagen I expression. Smad7 also abrogated TGF-beta-dependent proliferation inhibition of HSC. Smad7 did not decrease expression of alpha-SMA, but immunofluorescent staining with anti alpha-SMA antibodies displayed destruction of the fibrillar organization of the actin cytoskeleton. CONCLUSIONS In summary, gene transfer of Smad7 inhibits experimental fibrogenesis in vivo. Studies with isolated HSC suggest that the underlying mechanisms involve inhibition of TGF-beta signaling and HSC transdifferentiation.

Participation of Smad2, Smad3, and Smad4 in transforming growth factor beta (TGF-beta)-induced activation of Smad7. THE TGF-beta response element of the promoter requires functional Smad binding element and E-box sequences for transcriptional regulation.

Smad7 has recently been identified as a player that antagonizes transforming growth factor β (TGF-β) signals by acting downstream of TGF-β receptors. TGF-β rapidly induces expression of Smad7 mRNA in a variety of cell types, suggesting participation in a negative feedback loop to control TGF-β responses. We have previously described the genomic locus of rat Smad7 including the promoter region. Here we report polymerase chain reaction cloning of the corresponding promoter regions of human and murine Smad7 genes and functional characterization of the rat Smad7 promoter. Using transient transfection experiments of HepG2 cells, we identified the TGF-β response element within a strongly conserved region, containing a perfect Smad binding element (SBE; GTCTAGAC). Performing electrophoretic mobility shift assay and cotransfection experiments, we were able to delineate DNA-binding complexes and identified Smad3, Smad4, and Smad2. Mutation of the SBE completely abolished TGF-β inducibility of Smad7 in HepG2 cells, indicating that this sequence is necessary for TGF-β-induced transcription. Furthermore, a 3-base pair adjacent E-box is additionally essential for TGF-β-dependent promoter activation and an overlapping AP1 site is also involved. We conclude that regulation of Smad7 transcription by TGF-β is mediated via a specific constellation of recognition motifs localized around the SBE, which is conserved in human, rat, and murine genes.

Y-box Protein-1 Is the Crucial Mediator of Antifibrotic Interferon-γ Effects

Y-box protein-1 (YB-1) is a known negative regulator of collagen (Col) expression by two different mechanisms, acting directly through binding to an interferon-γ response element within the col1A2 promoter and/or by physically interacting with p300/Smad3, thereby abrogating the stimulatory effect of transforming growth factor-β (TGF-β). Here, we report that YB-1 activation via the Jak1 signaling pathway is required and sufficient to confer interferon-γ-dependent activation of the smad7 gene. By binding to a bona fide recognition site within the smad7 promoter, YB-1 up-regulates smad7 transcription, which was additively enhanced by autoinhibitory TGF-β signaling. Importantly, the anti-TGF-β effect was not only supplied by induced Smad7 expression but was recapitulated in the context of the col1A2 promoter, where YB-1 overexpression abolished the trans-stimulatory TGF-β effect in a dominant fashion. In conclusion, YB-1 is the main target of interferon-γ signaling via Jak1 that exerts antifibrotic action by both interference with TGF-β signaling and direct down-regulation of collagen expression.

Genomic locus and promoter region of rat Smad7, an important antagonist of TGFβ signaling

Abstract. SMAD proteins are essential components of the intracellular signaling pathways utilized by members of the transforming growth factor β (TGFβ) superfamily of growth factors. Certain SMAD proteins (Smad1, 2, 3, and 5) can act as regulated transcriptional activators. This process involves phosphorylation of these proteins by activated TGFβ receptors. Recently, Smad6 and Smad7 were identified; they antagonize TGFβ signaling by preventing the activation of signal-transducing SMAD complexes. TGFβ rapidly induces the expression of Smad7 mRNA, suggesting participation of Smad7 in a negative feedback loop to control TGFβ responses. Similarly, epidermal growth factor (EGF) and interferon γ (IFNγ) have been reported to induce Smad7 expression. In a rat model system of liver fibrosis, TGFβ inducibility of Smad7 is abrogated during transformation of hepatic stellate cells (HSC), indicating an important switch in transcriptional regulation of the gene. With the detailed characterization of the rat Smad7 genomic organization including the promoter region, we present the first identified Smad7 gene so far. The gene is composed of four exons separated by three introns covering a DNA region of about 30 kilobases (kb) in total. The major transcription start site is conserved between rat and mouse, and two polyadenylation signals were detected. In the promoter region, a potential CAGA box, a signal transducer and activator of transcription (STAT) factor-related recognition site, and different AP1 sites were identified, which could be the targets of TGFβ, IFNγ, and EGF-dependent Smad7 transcription initiation.

Hepatocyte-specific Smad7 deletion accelerates DEN-induced HCC via activation of STAT3 signaling in mice

TGF-β signaling in liver cells has variant roles in the dynamics of liver diseases, including hepatocellular carcinoma (HCC). We previously found a correlation of high levels of the important endogenous negative TGF-β signaling regulator SMAD7 with better clinical outcome in HCC patients. However, the underlying tumor-suppressive molecular mechanisms are still unclear. Here, we show that conditional (TTR-Cre) hepatocyte-specific SMAD7 knockout (KO) mice develop more tumors than wild-type and corresponding SMAD7 transgenic mice 9 months after diethylnitrosamine (DEN) challenge, verifying SMAD7 as a tumor suppressor in HCC. In line with our findings in patients, Smad7 levels in both tumor tissue as well as surrounding tissue show a significant inverse correlation with tumor numbers. SMAD7 KO mice presented with increased pSMAD2/3 levels and decreased apoptosis in the tumor tissue. Higher tumor incidence was accompanied by reduced P21 and upregulated c-MYC expression in the tumors. Activation of signal transducer and activator of transcription factor 3 signaling was found in Smad7-deficient mouse tumors and in patients with low tumoral SMAD7 expression as compared with surrounding tissue. Together, our results provide new mechanistic insights into the tumor-suppressive functions of SMAD7 in hepatocarcinogenesis.

Cytokine secretion profile of culture-activated mouse hepatic stellate cells upon stimulation with LPS

Background: Hepatic stellate cells (HSC) are considered the primary fibrogenic cell population. They are activated by various damage-associated molecular patterns (DAMPs) and transdifferentiate to myofibroblasts. Upon activation, they proliferate and produce extracellular matrix, cytokines, growth factors and chemokines. These secreted factors contribute to intermediary metabolism changes, xenobiotic responses, wound healing processes, immunoregulation, fibrogenesis and regeneration. HSC are the main source of extracellular matrix and therefore are considered a main target when aiming at fibrosis reduction and resolution. Their involvement in inflammatory responses is still under investigation. Methodology: HSC were isolated from mouse livers and cultivated for 48 hours before adding LPS (50 ng/ml) to the culture medium. Tissue culture medium supernatants were collected after 24 hours of LPS stimulation. The release of altogether 23 cytokines and chemokines into the medium was analysed by Luminex cytokine array technology and secretion of TGF-β and HGF were investigated by ELISA. Untreated cells and tissue culture medium were used as controls. Results: Secretion levels of most of the 23 measured factors as well as TGF-β and HGF were increased upon treatment with LPS. Of the 23, particularly G-CSF, KC (Cxcl1), IL-12 (p70), MCP-1a (Ccl2), IL-1b, TNFα and MIP1a (Ccl3) showed significant upregulation compared to untreated HSCs. Conclusion: The increase in released cytokines and chemokines points to the conclusion that HSC play a central role not only in the production of extracellular matrix, but also in the orchestration of the livers inflammatory response to damage and infection. Besides feedback loops between HSC and Kupffer cells which may function as “signal amplifiers” HSC most likely also enhance inflammation in the liver by endogenously producing relevant amounts of inflammatory mediators as direct response to LPS.

IFN-gamma inhibits hepatic progenitor cell activation in chronic liver damage

Background & Aims: Hepatic progenitor cells (HPC) are thought to promote a periportal ductular reaction (DR) and contribute to periportal fibrosis in HCV- and non-alcholic steatohepatitis (NASH)-associated liver diseases. IFN-gamma is a well recognized fibrotic inhibitor in vitro and in vivo. The present study investigate the role of IFN-gamma in HPC activation in chronic HBV infected patients and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) treated mouse. Methods: Hepatic progenitor cells were quantified by Cytokeratin-19 (CK19) immunostaining in 110 HBV, as were oval cells (HPCs in rodents) in DDC treated wild-type and three kinds of IFN-gamma-associated gene knockout mice, including IFN-gamma-, IFN-gamma receptor- and interferon regulator factor (IRF)-1 knockout mice. Results: The CK19 staining score significantly correlates with inflammatory grade (r=0.61, P<0.001) and fibrotic stage (r=0.61, P<0.001) in 110 chronic HBV infected patients. 9-months IFN-gamma treatment resulted in decreased HPC numbers concomitant with reduced inflammatory and fibrotic degree in chronic HBV infection (13/18 patients). In line with HBV patients, the number of oval cells and DRs significantly increased in IFN-gamma knockout mice compare with wild-type mice after 4 weeks DDC treatment. Furthermore, elevated oval cells and DR also demonstrated in interferon regulator factor (IRF)-1, downstream of IFN-gamma signalling, knockout mice with 4 weeks DDC administration. Either IFN-gamma- or IRF-1 knockout mice showed serious fibrotic degree compare with wild-type mice in DDC model. Conclusions: Besides a role in anti-fibrosis, IFN-gamma inhibits HPC activation in chronic liver damage.

IL-13 induces connective tissue growth factor in rat hepatic stellate cells via TGF- independent Smad signaling

Aims: Connective tissue growth factor (CTGF) plays a central role in stimulating extracellular matrix deposition in the liver, and hence is considered as a critical mediator of TGF-β dependent fibrogenesis. Hepatic stellate cells (HSCs) are known as the major source of CTGF. However, previous studies revealed that IL-13, rather than TGF-beta, represents the main inducer of CTGF expression in HSCs. Methods: We evaluate the effect of IL-13 on CTGF expression in primary cultured HSCs. We also investigated how IL-13 downstream signaling modulates CTGF expression in HSCs. Results: IL-13 induces a time- and dosage-dependent increase of CTGF in a TGF-beta independent manner. This process involves different Smad proteins and their upstream receptor kinases (ALKs). Smad1 and Smad2 were identified as key mediators for IL-13 dependent CTGF expression. Furthermore, IL-13 induces Stat6 phosphorylation in HSCs, but Stat6 was not involved in CTGF induction. Instead, the Erk1/2-MAPK pathway was responsible for IL-13 induced early Smad phosphorylation and CTGF production. Conclusion: We demonstrate that IL-13 induces CTGF expression in HSCs by activating TGF-beta independent ALK/Smad signaling via the Erk-MAPK pathway rather than via its canonical JAK/Stat6 pathway. These results may provide an improved new insight into the molecular mechanisms of pro-fibrotic IL-13 activity in the liver.

120 IFN-G INHIBITS HEPATIC PROGENITOR CELL ACTIVATION IN CHRONIC LIVER DAMAGE

Hepatic fibrosis represents a significant health problem worldwide of which no acceptable therapy exists. It is associated with the accumulation of extracellular matrix in response to acute or chronic liver injury and may ultimately lead to cirrhosis. Combined with our recent findings that showed upregulation of Interleukin (IL)-33 in different models of experimental fibrosis, we were endorsed to further investigate the role of IL-33 in liver fibrosis. IL-33, a novel member of IL-1 family of cytokines signalling via IL-1 receptor-related protein ST2, is known to be involved in several autoimmune and inflammatory disorders; however, its function in liver fibrosis still remains ambiguous. To address this question, we analyzed IL-33 transgenic, expression vector-based overexpressing, knockout and receptor (ST2) knockout mice. Different experimental fibrosis models were assessed, at which point IL-33 knockout mice showed protection ascertained by collagen-specific staining, hydroxyproline assay and realtime PCR. In order to analyze profibrotic cellular targets, bone marrow cells from IL-33 receptor knockout (ST2) and wildtype mice were transplanted into lethally radiated wildtype recipients, followed by IL-33 injection after transfer recovery. ST2 mice revealed protection against fibrosis indicating to the relevance of hematopoietic cells in the setting. Gene chip analysis of liver from IL-33 overexpressing mice identified several gene candidates that are differentially regulated during fibrosis, including type 2 IL-4 receptor (IL-4R) signaling molecules. IL-33 overexpression in IL-4Raand IL-13 knockout mice exhibited predominant protection in fibrosis. Similarly, mast cell deficiency is also likely to protect mice, demonstrating the importance of these cells in fibrosis. Based on these results we conclude that IL-33 signal-associated pathways have a considerable role in the development of liver fibrosis. Therefore, our findings may have important implications for therapeutic interventions across a range of fibrosis-based diseases.

105 NOTCH SIGNALING PLAYS A CRITICAL ROLE IN EXPERIMENTAL AND HUMAN LIVER FIBROGENSIS

105 NOTCH SIGNALING PLAYS A CRITICAL ROLE IN EXPERIMENTAL AND HUMAN LIVER FIBROGENSIS S. Dooley, Y. Liu, I. Ilkavets, H. Shen, V. Zimmer, R.M. Bohle, C. Zhu, C. Xu, C. Yu, C. Meyer, L. Ciuclan, C. Stump, A. Muller, T. Huang, Y. Li, F. Lammert, M.V. Singer, P.R. Mertens, H.-L. Weng. Molecular Hepatology – Alcohol Dependent Diseases, II. Medical Clinic Faculty of Medicine at Mannheim, University of Heidelberg, Mannheim, Department of Internal Medicine II, Institute of Pathology, University of the Saarland, Homburg, Department of Nephrology and Hypertention, Otto-von-Guericke-University, Magdeburg, Germany; Department of Gastroenterology, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Department of Cardiac Vascular Medicine, Affiliated Hospital, Medical School, Ningbo University, Ningbo, China E-mail: honglei.weng@medma.uni-heidelberg.de