Birgit Lahme

Pharmacological application of caffeine inhibits TGF-β-stimulated connective tissue growth factor expression in hepatocytes via PPARγ and SMAD2/3-dependent pathways ☆

BACKGROUND/AIMS Epidemiological studies suggest that coffee drinking is inversely correlated with the risk of development of liver fibrosis but the molecular basis is unknown. METHODS We investigated the pharmacological mechanisms involved in caffeine-dependent regulation of CTGF expression, an important modulator protein of fibrogenic TGF-beta, in rat hepatocytes using Western-blot, co-immunoprecipitations, reporter-gene-assays and ELISAs. RESULTS It is demonstrated that caffeine, similar to 8-Br-cAMP, suppresses CTGF expression, decreases SMAD2 protein levels and inhibits SMAD1/3-phosphorylation. The SMAD2 level can be restored by a proteasome inhibitor. Additionally, caffeine leads to an up-regulation of PPARgamma expression, that enhances the inhibitory effect of the natural PPARgamma agonist 15-PGJ(2) on CTGF expression by inducing a dissociation of the SMAD2/3-CBP/p300-transcriptional complex. CONCLUSIONS We show that caffeine strongly down-modulates TGF-beta-induced CTGF expression in hepatocytes by stimulation of degradation of the TGF-beta effector SMAD 2, inhibition of SMAD3 phosphorylation and up-regulation of the PPARgamma-receptor. Long-term caffeinization might be an option for anti-fibrotic trials in chronic liver diseases.

TGF-β-mediated hepatocellular apoptosis by rat and human hepatoma cells and primary rat hepatocytes

Abstract Background/Aims: Primary cultures of rat hepatocytes, rat (FAO) and human (HepG2) hepatoma cells were studied by immunocytochemistry for expression of transforming growth factor (TGF)-β, for the release of TGF-β into the medium, and generation of hepatocellular apoptosis by the respective cell-conditioned media. Methods/Results: Using the alkaline-phosphatase anti-alkaline-phosphatase technique, intense TGF-β immunostaining was shown in all cell types. The cytokine is released almost entirely in the latent form into the culture medium; only the FAO-cells had a substantial fraction of bioactive TGF-β in the native (unacidified) culture fluid. Exposure of hepatocytes with the respective cell-conditioned media in the activated, but not in the native form (except for FAO-cell media), induced severe detrimental effects as evidenced by: (i) gross morphological alterations, (ii) functional impairment (reduction of WST-1 test, detachment of cells, lactate dehydrogenase increase in the medium), and (iii) generation of apoptosis. The latter phenomenon was confirmed by an increase of internucleosomal DNA fragments, positive TUNEL reaction, and intense binding of the fluorochrome Hoechst 33342 to fragmented nuclei. All these effects, which were mimicked by addition of recombinant human TGF-β 1 , were almost entirely antagonized by pre-incubation of the conditioned media with latency associated peptide. In contrast to hepatocytes, both types of hepatoma cells were completely resistant to the multiple actions of TGF-β and activated conditioned media. Conclusions: It is concluded that hepatocytes might have the ability to induce autocrine, TGF-β-mediated apoptosis, whereas hepatoma cells, because of their TGF-β resistance, might generate TGF-β-mediated peritumorous apoptosis of hepatocytes in a paracrine way, which could facilitate their expansion in situ . Both mechanisms, however, are critically dependent on extracellular TGF-β activation.

Induction of neutrophil-attracting chemokines in transforming rat hepatic stellate cells

BACKGROUND & AIMS Hepatic stellate cells (HSCs) play a key role in the pathogenesis of liver fibrosis. Immigrating leukocytes can potentiate the progression of liver fibrosis by release of fibrogenic mediators and cytotoxic actions. The inducible production of neutrophil chemotactic activities in HSCs was investigated to understand the underlying mechanisms responsible for the attraction of leukocytes in the pathogenesis of liver fibrosis. METHODS Cultured HSCs of different transformation grades and after transformation to myofibroblasts (MFBs) were stimulated with tumor necrosis factor (TNF)-alpha and lipopolysaccharide (LPS), respectively. Induced leukocyte chemotactic activities were evaluated by chemotaxis assays, enzyme-linked immunosorbent assay, and Northern blot analysis. RESULTS A transformation grade-dependent differential responsiveness of HSCs and MFBs was observed. TNF-alpha-inducible production of chemotactic mediators increased substantially with advancing transformation. Only transformed MFBs were LPS responsive. Macrophage inflammatory protein 2 was identified as one of the inducible chemokines. CONCLUSIONS The results suggest that chemokines play an important role in the pathogenesis of liver fibrosis. Proinflammatory cytokines can initiate the production of chemotactic activities. The more HSCs are transformed to MFBs, e.g., by chronic injury, the more sensitive the cells become to LPS, which may lead to a vicious circle of enhanced fibrogenic and chemotactic mediator production.

Activation of TGF‐β within cultured hepatocytes and in liver injury leads to intracrine signaling with expression of connective tissue growth factor

Recently, synthesis and secretion of connective tissue growth factor (CTGF)/CYR61/CTGF/NOV‐family member 2 (CCN2) in cultures of hepatocytes were shown, which are sensitively up‐regulated by exogenous TGF‐β. In this study TGF‐β‐dependent CTGF/CCN2 expression in hepatocytes cultured under completely TGF‐β‐free conditions was analysed by Western‐blots, metabolic labelling, and CTGF‐reporter gene assays. In alkaline phosphatase monoclonal anti‐alkaline phosphatase complex (APAAP)‐staining of cultured hepatocytes it was demonstrated that latent TGF‐β within the hepatocytes becomes rapidly detectable during culture indicating an intracellular demasking of the mature TGF‐β antigen. Subsequent signaling to theCTGF/CCN2 promoter occurs via p‐Smad2, whereas p‐Smad3 does not seem to be involved. Cycloheximide did not abolish the rapid immunocytochemical appearance of mature TGF‐β, but calpain inhibitors partially suppressed intracellular TGF‐β activation and subsequently CTGF up‐regulation. Calpain treatment had the reverse effect. None of the inhibitors of extracellular TGF‐β signalling was effective in the reduction of spontaneous CTGF synthesis, but intracellularly acting Alk 4‐/Alk 5‐specific inhibitor SB‐431542 was able to diminish CTGF expression. The assumption that latent intracellular TGF‐β is activated by calpains during culture‐induced stress or injurious conditions in the liver in vivo was further validated by a direct effect of calpains on the activation of recombinant latent TGF‐β. In conclusion, these data are the first to suggest the possibility of intracrine TGF‐β signalling due to calpain‐dependent intracellular proteolytic activation leading to transcriptional activation of CTGF/CCN2 as a TGF‐β‐sensitive reporter gene. This mechanism might be deleterious for keeping long‐term hepatocyte cultures due to TGF‐β‐induced apoptosis and, further, might be of relevance for induction of apoptosis or epithelial‐mesenchymal transition of hepatocytes in injured liver.

Differential expression of monocyte chemotactic protein-1 (MCP-1) in transforming rat hepatic stellate cells.

BACKGROUNDS/AIMS Hepatic stellate cells and infiltrating leukocytes play a key role in the pathogenesis of liver fibrosis. The chronic phase of liver inflammation is characterized by immigrating mononuclear cells. To understand the underlying mechanisms responsible for the attraction of mononuclear cells in the pathogenesis of liver fibrosis, we investigated the inducible production of chemotactic activities in hepatic stellate cells. METHODS Cultured hepatic stellate cells of different transformation grades and after in vitro transformation to myofibroblast-like cells were stimulated with tumor necrosis factor-a or bacterial lipopolysaccharide. Mononuclear cell attracting chemotactic activities were evaluated by chemotaxis assays, ELISA, and Northern blot analysis. RESULTS We observed a transformation grade-dependent differential responsiveness of hepatic stellate cells and myofibroblast-like cells. Monocyte chemotactic protein-1 was inducible by tumor necrosis factor-alpha in non-transformed hepatic stellate cells. In contrast, monocyte chemotactic protein-1 was not inducible by bacterial lipopolysaccharide until the cells were fully transformed into myofibroblast-like cells. Despite a delayed onset, the bacterial lipopolysaccharide-inducible monocyte chemotactic protein-1 expression did not depend on an endogenous production of tumor necrosis factor-alpha. CONCLUSIONS Our results indicate that the tumor necrosis factor-alpha and bacterial lipopolysaccharide-inducible production of chemokines plays a central role in the pathogenesis of liver fibrosis. These data suggest that when hepatic stellate cells have been transformed to a myofibroblast-like cells phenotype, e.g. by chronic injury, the cells become more sensitive to bacterial lipopolysaccharide, which may potentiate the production of chemotactic and fibrogenic mediators. A strong secretion of monocyte chemotactic protein-1 may contribute to the maintenance of an inflammatory infiltrate dominated by mononuclear cells.

Validation of connective tissue growth factor (CTGF/CCN2) and its gene polymorphisms as noninvasive biomarkers for the assessment of liver fibrosis

Summary.  Clinical and experimental studies have demonstrated that connective‐tissue growth factor (CTGF) expression is increased in fibrotic human liver and experimental animal models of liver fibrogenesis. CTGF has been linked to transforming growth factor‐beta (TGF‐β) pathways in fibroproliferative diseases and specific polymorphisms within the CTGF gene may predispose for fibrosis in systemic sclerosis. As CTGF is detectable in various human fluids (serum, plasma and urine), it may provide information about fibrotic remodelling processes and reflect hepatic TGF‐β bioactivity. We established a novel ELISA for the measurement of serum CTGF and tested its clinical value in patients with chronic hepatitis C virus (HCV) infection and chronic liver disease (CLD). HCV infected patients (n = 138) had significantly higher serum CTGF levels than healthy controls. CTGF was linked to the histological degree of liver fibrosis. To expand the results to other aetiologies, a separate cohort of CLD patients (n = 129) was evaluated, showing higher serum CTGF than healthy controls and again an association with advanced stages of liver cirrhosis (Child B and C). Although independent of the underlying aetiology, serum CTGF was most powerful in indicating fibrosis/advanced disease states in HCV‐related disorders. The genotyping of six polymorphisms (rs6917644, rs9399005, rs6918698, rs9493150, rs2151532 and rs11966728) covering the CTGF locus in 365 patients suffering from chronic hepatitis C revealed that none of these polymorphisms showed a genotypic or allelic association with the severity of hepatic fibrosis. Taken together, serum CTGF is suitable for determination of hepatic fibrosis and most powerful in patients with chronic HCV infection.

Connective tissue growth factor is a Smad2 regulated amplifier of transforming growth factor β actions in hepatocytes—But without modulating bone morphogenetic protein 7 signaling†

In vivo knockdown of connective tissue growth factor (CTGF/CCN2) was recently shown to attenuate the formation of experimental liver fibrosis. The secreted, cysteine‐rich growth factor is proposed to adversely modulate the binding of profibrogenic transforming growth factor β (TGF‐β) and its natural antagonist bone morphogenetic protein (BMP) to their cognate receptors in several cellular systems, but the functionality of CTGF in modulation of the TGF‐β/BMP signaling pathways is still unknown. This study aims at characterizing a potentially differential modulating role of CTGF on TGF‐β– and BMP7‐dependent transactivation of reporter gene [Ad‐(CAGA)12‐MLP‐luc, Ad‐hCTGF‐luc, and Ad‐(BRE)2‐luc reporter gene] expression in rat hepatocytes. In this context, emphasis is also placed on the differential roles of Smad2 and Smad3 in the TGF‐β–dependent transactivation of the endogenous CTGF gene and the CTGF gene reporter, as investigated following adenoviral infection of wild‐type and dominant negative Smad2/3 or treatment with the specific inhibitor of Smad3 or ALK5‐specific (SB‐431542) inhibitor. In this analysis, we found (1) a selective transcriptional activation of the CTGF promoter by Smad2 (but not Smad3); (2) the failure of BMP7 to inhibit the transcriptional activation of the Smad3‐selective (CAGA)12‐luc reporter by TGF‐β, as well as the failure of TGF‐β to inhibit the transcriptional activation of the Smad5‐selective (BRE)2‐luc reporter by BMP7; and (3) the sensitization of hepatocytes toward TGF‐β type I receptor (ALK5)/Smad2 and Smad3‐mediated TGF‐β signaling by CTGF, whereas BMP type I receptor (ALK1)/Smad5‐mediated BMP7 signaling is not modulated. Conclusion: CTGF acts as a Smad2‐dependent sensitizer of TGF‐β actions that does not influence BMP7 signaling in hepatocytes. (HEPATOLOGY 2009.)

Intracrine signalling of activin A in hepatocytes upregulates connective tissue growth factor (CTGF/CCN2) expression

Background/Aims: Up to now, the effect of activin A on the expression of the important transforming growth factor (TGF)‐β downstream modulator connective tissue growth factor (CTGF) is not known, but might be of relevance for the functional effects of this cytokine on several liver cell types.

Gc-globulin (vitamin D binding protein) is synthesized and secreted by hepatocytes and internalized by hepatic stellate cells through Ca2+-dependent interaction with the megalin/gp330 receptor

BACKGROUND Gc-globulin or vitamin D binding protein is a highly expressed, multifunctional and polymorphic serum protein, which also serves as the major transporter for vitamin D metabolites in the circulation. The present study was performed to analyze the interaction between gc-globulin of hepatocytes and hepatic stellate cells, the most important fat-/retinol-storing cell type in the liver, which spontaneously transdifferentiates to myofibroblasts in culture. METHODS Hepatic stellate cells and hepatocytes were isolated by the pronase/collagenase reperfusion method, hepatocytes by collagenase reperfusion of the organ. Gc-globulin expression was monitored by immunocytochemistry, immunoblotting, RT-PCR, metabolic labelling with [(35)S]-methionine, and its intracellular binding to alpha-smooth-muscle actin was investigated by co-immunoprecipitation. Cytoskeletal stainings of gc-globulin and alpha-smooth-muscle actin in hepatic stellate cells and the identification of the receptors megalin/gp330, HCAM/CD44, cubilin and annexin A2 were performed with confocal immunocytochemistry, immunoblotting and/or FACS-analysis. RESULTS Hepatocytes synthesize and secrete gc-globulin as shown by RT-PCR and [(35)S]-methionine labelling, which could be suppressed by cycloheximide. Also, a strong signal for gc-globulin was detected in the immunoblot of native hepatic stellate cell lysates. However, no mRNA for gc-globulin was found in this cell type, which suggests no active synthesis by hepatic stellate cells. Hepatic stellate cells were tested positively for the presence of known gc-globulin interacting receptors megalin/gp330, HCAM/CD44, cubilin and annexin A2. Inhibition of the megalin/gp330 receptor by a competitive, neutralizing antibody resulted in decreased intracellular availability of gc-globulin in hepatic stellate cells. The latter effect was enhanced by additional incubation of hepatic stellate cells with EDTA for complexing Ca(2+), suggesting a Ca(2+)-dependent internalization of gc-globulin into hepatic stellate cells via the megalin/gp300 receptor. This was supported by confocal microscopy which showed a co-localization of gc-globulin with the multifunctional megalin/gp330 receptor on this cell type. Inside hepatic stellate cells, a linkage between gc-globulin and alpha-smooth muscle actin filaments of hepatic stellate cells was detected by immunocytochemistry. Intracellular binding of gc-globulin to alpha-smooth-muscle actin filaments was confirmed by co-immunoprecipitation. CONCLUSION We give evidence to the expression of the megalin/gp330 receptor on hepatic stellate cells and that this receptor is involved in the Ca(2+)-dependent internalization of gc-globulin into hepatic stellate cells, a protein synthesized and secreted into the extracellular space and circulation by hepatocytes. Inside hepatic stellate cells, it co-localizes with and binds to alpha-smooth muscle actin filaments. Under consideration of the available literature, these findings propose a participation of gc-globulin in hepatic vitamin D metabolism as well as in hepatic stellate cell stability and apoptosis as important mechanisms of liver regeneration.

Identification of paraxanthine as the most potent caffeine-derived inhibitor of connective tissue growth factor expression in liver parenchymal cells.

Background: Recently, we identified hepatocytes as the major cellular source of profibrogenic connective tissue growth factor (CTGF/CCN2) in the liver. Based on reports of a hepatoprotective effect of coffee consumption, we were the first to provide evidence that caffeine suppresses transforming growth factor (TGF)‐β dependent and ‐independent CTGF expression in hepatocytes in vitro and in vivo, thus suggesting this xanthine‐alkaloid as a potential therapeutic agent.