Nuclear localization of epidermal growth factor and epidermal growth factor receptors in human thyroid tissues

Epidermal growth factor (EGF) has widespread growth effects, and in some tissues proliferation is associated with the nuclear localization of EGF and epidermal growth factor receptor (EGFR). In the thyroid, EGF promotes growth but differs from thyrotropin (TSH) in inhibiting rather than stimulating functional parameters. We have therefore studied the occurrence and cellular distribution of EGF and EGFR in normal thyroid, in Graves' disease, where growth is mediated through the thyrotropin receptor (TSHR), and in a variety of human thyroid tumors. In the normal gland the staining was variable, but largely cytoplasmic, for both EGF and EGFR. In Graves' disease there was strong cytoplasmic staining for both EGF and EGFR, with frequent positive nuclei. Nuclear positivity for EGF and particularly for EGFR was also a feature of both follicular adenomas and follicular carcinomas. Interestingly, nuclear staining was almost absent in papillary carcinomas. These findings document for the first time the presence of nuclear EGF and EGFR in thyroid. Their predominant occurrence in tissues with increased growth (Graves' disease, follicular adenoma, and carcinoma) may indicate that nuclear EGF and EGFR play a role in growth regulation in these conditions. The absence of nuclear EGF and EGFR in papillary carcinomas would suggest that the role played by EGF in growth control differs between papillary carcinoma and follicular adenomas/carcinomas of the thyroid.

Experimental arthritis: effect on growth parameters and total skeletal calcium

Aim of the study was to investigate the possible mechanisms leading to stunted growth and osteoporosis in experimental arthritis. Fourty-two female rats of 7-8 weeks of age were randomly assigned to three groups of 14 animals each: (a) controls; (b) adjuvant-inoculated (AA); and (c) adjuvant-inoculated rats receiving 10 mg cyclosporin A (CsA) orally for 30 days. Biological parameters studied were: hindpaw swelling; vertebral length progression expressed as Delta increments between days 1 and 30 as a parameter of skeletal growth, and estimation of total skeletal mineral content by dual energy X-ray absorptiometry (n=10 each group) on day 30. Endocrine parameters measured were pulsatile release of growth hormone (rGH) on day 30 following jugular cannulation and measurement of insulin-like growth factor (IGF-1) in pooled plasma from rGH profiles. Results can be summarized as follows: Untreated AA rats exhibited local signs of inflammation in comparison with controls (hindpaw diameter 8.1-8.9 mm vs. 5.3-5.6 mm in controls). Treatment with CsA normalized this parameter (4.9-5.6 mm). Vertebral growth was significantly retarded in AA rats in comparison with controls (214+/-32 vs. 473+/-33 microm; p<0.001). Administration of CsA normalized vertebral size increment with a clear tendency to overgrowth (523+/-43 microm, n.s.). There was also a marked reduction in total skeletal mineral content in diseased (AA) rats as compared to controls (5.8+/-0.1 vs. 7.5+/-0.1g [OH-apatite]; p<0.001), and a moderate but significant increment above controls in the group receiving CsA (8.0+/-0.1 vs. 7.5+/-0.1g [OH-appatite]; p<0.04). Integrated rGH profiles exhibited a significant fall in arthritic rats and were completely restored to normal under CsA treatment. A trend toward higher rGH values was observed in the latter group (2908+/-554 in AA vs. 8317+/-1492 ng/ml/240 min in controls; p<0.001, and 10940+/-222 ng/ml/240 min, n.s. in the CsA group). There was a good correlation between skeletal growth and rGH pulsatility (r=0.81; p<0.001). IGF-1 followed a similar pattern (630+/-44 in AA vs. 752+/-30 ng/ml in controls; p<0.04, and 769+/-59 ng/ml in the CsA group, n.s. vs. controls). Thus, a clear tendency to skeletal overgrowth following treatment was observed in agreement with the hormonal data. It can therefore be concluded that, in experimental arthritis, attenuated GH-spiking and reduced circulating IGF-1 appear to be causally related to growth retardation, probably mimicking signs and symptoms observed in juvenile arthritis. Therapy with CsA is followed by normalization of hormonal and biological parameters accompanied by a catch up phenomenon in skeletal growth which is also observed clinically in juvenile arthritis. Generalized osteopenia is a prominent feature seemingly connected with the growth abnormalities as they parallel each other during the evolution of the disease and respond equally to therapy.

Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway

Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFalpha and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NFkappaB was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production.

Elevated levels of placental growth factor represent an adaptive host response in sepsis

Recently, we demonstrated that circulating levels of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are increased in sepsis (Yano, K., P.C. Liaw, J.M. Mullington, S.C. Shih, H. Okada, N. Bodyak, P.M. Kang, L. Toltl, B. Belikoff, J. Buras, et al. 2006. J. Exp. Med. 203:1447-1458). Moreover, enhanced VEGF/Flk-1 signaling was shown to contribute to sepsis morbidity and mortality. We tested the hypothesis that PlGF also contributes to sepsis outcome. In mouse models of endotoxemia and cecal ligation puncture, the genetic absence of PlGF or the systemic administration of neutralizing anti-PlGF antibodies resulted in higher mortality compared with wild-type or immunoglobulin G-injected controls, respectively. The increased mortality associated with genetic deficiency of PlGF was reversed by adenovirus (Ad)-mediated overexpression of PlGF. In the endotoxemia model, PlGF deficiency was associated with elevated circulating levels of VEGF, induction of VEGF expression in the liver, impaired cardiac function, and organ-specific accentuation of barrier dysfunction and inflammation. Mortality of endotoxemic PlGF-deficient mice was increased by Ad-mediated overexpression of VEGF and was blocked by expression of soluble Flt-1. Collectively, these data suggest that up-regulation of PlGF in sepsis is an adaptive host response that exerts its benefit, at least in part, by attenuating VEGF signaling.

The vascular ectonucleotidase CD39 is permissive for sinusoidal endothelial cell proliferation during liver regeneration: evidence for synergism between growth factors and extracellular nucleotides

Crosstalk between elements of the sinusoidal vasculature, platelets and hepatic parenchymal cells influences regenerative responses to liver injury and/or resection. Such paracrine interactions include hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), IL-6 and small molecules such as serotonin and nucleotides. CD39 (nucleoside triphosphate diphosphohydrolase-1) is the dominant vascular ectonucleotidase expressed on the luminal surface of endothelial cells and modulates extracellular nucleotide signaling. We have previously shown that integrity of P2-receptors, as maintained by CD39, is required for angiogenesis in Matrigel plugs in vivo and that there is synergism between nucleotide P2-receptor- and growth factor-mediated cell proliferation in vitro. We have now explored effects of CD39 on liver regeneration and vascular endothelial growth factor responses in a standard small animal model of partial hepatectomy. The expression of CD39 on liver sinusoidal endothelial cells (LSEC) is substantially boosted during liver regeneration. This transcriptional upregulation precedes maximal sinusoidal endothelial cell proliferation, noted at day 5-8 in C57BL6 wild type mice. In matched mutant mice null for CD39 (n=14), overall survival is decreased to 71% by day 10. Increased lethality occurs as a consequence of extensive LSEC apoptosis, decreased endothelial proliferation and failure of angiogenesis leading to hepatic infarcts and regenerative failure in mutant mice. This aberrant vascular remodeling is associated with biochemical liver injury, elevated serum levels of VEGF (113.9 vs. 65.5pg/ml, p=0.013), and decreased circulating HGF (0.89 vs. 1.43 ng/ml, p=0.001) in mice null for CD39. In agreement with these observations, wild type LSEC but not CD39 null cultures upregulate HGF expression and secretion in response to exogenous VEGF in vitro. CD39 null LSEC cultures show poor proliferation responses and heightened levels of apoptosis when contrasted to wild type LSEC where agonists of P2Y receptors augment cell proliferation in the presence of growth factors. These observations are associated with features of P2Y-desensitization, normal levels of the receptor tyrosine kinase VEGFR-1 (Flt-1) and decreased expression of VEGFR-2 (FLK/KDR) in CD39 null LSEC cultures. We provide evidence that CD39 and extracellular nucleotides impact upon growth factor responses and tyrosine receptor kinases during LSEC proliferation. We propose that CD39 expression by LSEC might co-ordinate angiogenesis-independent liver protection by facilitating VEGF-induced paracrine release of HGF to promote vascular remodeling in liver regeneration.

Adenovirus-mediated transforming growth factor-beta ameliorates ischemic necrosis of epigastric skin flaps in a rat model

BACKGROUND: Gene therapy has been recently introduced as a novel approach to treat ischemic tissues by using the angiogenic potential of certain growth factors. We investigated the effect of adenovirus-mediated gene therapy with transforming growth factor-beta (TGF-beta) delivered into the subdermal space to treat ischemically challenged epigastric skin flaps in a rat model. MATERIAL AND METHODS: A pilot study was conducted in a group of 5 animals pretreated with Ad-GFP and expression of green fluorescent protein in the skin flap sections was demonstrated under fluorescence microscopy at 2, 4, and 7 days after the treatment, indicating a successful transfection of the skin flaps following subdermal gene therapy. Next, 30 male Sprague Dawley rats were divided into 3 groups of 10 rats each. An epigastric skin flap model, based solely on the right inferior epigastric vessels, was used as the model in this study. Rats received subdermal injections of adenovirus encoding TGF-beta (Ad-TGF-beta) or green fluorescent protein (Ad-GFP) as treatment control. The third group (n = 10) received saline and served as a control group. A flap measuring 8 x 8 cm was outlined on the abdominal skin extending from the xiphoid process proximally and the pubic region distally, to the anterior axillary lines bilaterally. Just prior to flap elevation, the injections were given subdermally in the left upper corner of the flap. The flap was then sutured back to its bed. Flap viability was evaluated seven days after the initial operation. Digital images of the epigastric flaps were taken and areas of necrotic zones relative to total flap surface area were measured and expressed as percentages by using a software program. RESULTS: There was a significant increase in mean percent surviving area between the Ad-TGF-beta group and the two other control groups (P < 0.05). (Ad-TGF-beta: 90.3 +/- 4.0% versus Ad-GFP: 82.2 +/- 8.7% and saline group: 82.6 +/- 4.3%.) CONCLUSIONS: In this study, the authors were able to demonstrate that adenovirus-mediated gene therapy using TGF-beta ameliorated ischemic necrosis in an epigastric skin flap model, as confirmed by significant reduction in the necrotic zones of the flap. The results of this study raise the possibility of using adenovirus-mediated TGF-beta gene therapy to promote perfusion in random portion of skin flaps, especially in high-risk patients.

Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma

PURPOSE: Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS: Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS: An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION: This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

Inhibition of integrin alphavbeta6 on cholangiocytes blocks transforming growth factor-beta activation and retards biliary fibrosis progression

BACKGROUND ; AIMS: Integrin alphavbeta6 is highly expressed on certain activated epithelia, where it mediates attachment to fibronectin and serves as coreceptor for the activation of latent transforming growth factor (TGF)-beta1. Because its role in liver fibrosis is unknown, we studied alphavbeta6 function in vitro and explored the antifibrotic potential of the specific alphavbeta6 antagonist EMD527040. METHODS: Experimental liver fibrosis was studied in rats after bile duct ligation (BDL) and in Mdr2(abcb4)(-/-) mice. Different doses of EMD527040 were given to rats from week 2 to 6 after BDL and to Mdr2(-/-) mice from week 4 to 8. Liver collagen was quantified, and expression of alphavbeta6 and fibrosis-related transcripts was determined by quantitative reverse-transcription polymerase chain reaction. alphavbeta6-expressing cells, bile duct proliferation, and apoptosis were assessed histologically. The effect of EMD527040 on cholangiocyte adhesion, proliferation, apoptosis, and TGF-beta1 activation was studied in vitro. RESULTS: alphavbeta6 was highly expressed on proliferating bile duct epithelia in fibrosis, with 100-fold increased transcript levels in advanced fibrosis. EMD527040 attenuated bile ductular proliferation and peribiliary collagen deposition by 40%-50%, induced down-regulation of fibrogenic and up-regulation of fibrolytic genes, and improved liver architecture and function. In vitro alphavbeta6 inhibition reduced activated cholangiocyte proliferation, their adhesion to fibronectin, and endogenous activation of TGF-beta1 by 50% but did not affect bile duct apoptosis. CONCLUSIONS: Integrin alphavbeta6 is strongly up-regulated in proliferating bile duct epithelia and drives fibrogenesis via adhesion to fibronectin and auto/paracrine TGF-beta1 activation. Pharmacologic inhibition of alphavbeta6 potently inhibits the progression of primary and secondary biliary fibrosis.

Evaluation of the transforming growth factor beta1 codon 25 (Arg-->Pro) polymorphism in alcoholic liver disease

INTRODUCTION: Liver cirrhosis develops only in a minority of heavy drinkers. Genetic factors may account for some variation in the progression of fibrosis in alcoholic liver disease (ALD). Transforming growth factor beta 1 (TGFbeta1) is a key profibrogenic cytokine in fibrosis and its gene contains several polymorphic sites. A single nucleotide polymorphism at codon 25 has been suggested to affect fibrosis progression in patients with chronic hepatitis C virus infection, fatty liver disease, and hereditary hemochromatosis. Its contribution to the progression of ALD has not been investigated sufficiently so far. PATIENTS AND METHODS: One-hundred-and-fifty-one heavy drinkers without apparent ALD, 149 individuals with alcoholic cirrhosis, and 220 alcoholic cirrhotics who underwent liver transplantation (LTX) were genotyped for TGFbeta1 codon 25 variants. RESULTS: Univariate analysis suggested that genotypes Arg/Pro or Pro/Pro are associated with decompensated liver cirrhosis requiring LTX. However, after adjusting for patients' age these genotypes did not confer a significant risk for cirrhosis requiring LTX. CONCLUSION: TGFbeta1 codon 25 genotypes Arg/Pro or Pro/Pro are not associated with alcoholic liver cirrhosis. Our study emphasizes the need for adequate statistical methods and accurate study design when evaluating the contribution of genetic variants to the course of chronic liver diseases.

Connective tissue growth factor, steatosis and fibrosis in patients with chronic hepatitis C

BACKGROUND/AIM: Both steatosis and insulin resistance have been linked to accelerated fibrosis in chronic hepatitis C. Connective tissue growth factor (CTGF) plays a major role in extracellular matrix production in fibrotic disorders including cirrhosis, and its expression is stimulated in vitro by insulin and glucose. We hypothesized that CTGF may link steatosis, insulin resistance and fibrosis. METHODS: We included 153 chronic hepatitis C patients enrolled in the Swiss Hepatitis C Cohort Study and for whom a liver biopsy and plasma samples were available. CTGF expression was assessed quantitatively by immunohistochemistry. In 94 patients (57 with genotypes non-3), plasma levels of glucose, insulin and leptin were also measured. CTGF synthesis was investigated by immunoblotting on LX-2 stellate cells. RESULTS: Connective tissue growth factor expression was higher in patients with steatosis (P=0.039) and in patients with fibrosis (P=0.008) than those without these features. CTGF levels were neither associated with insulinaemia or with glycaemia, nor with inflammation. By multiple regression analysis, CTGF levels were independently associated with steatosis, a past history of alcohol abuse, plasma leptin and HCV RNA levels; when only patients with genotypes non-3 were considered, CTGF levels were independently associated with a past history of alcohol abuse, plasma leptin levels and steatosis. Leptin stimulated CTGF synthesis in LX-2 cells. CONCLUSIONS: In patients with chronic hepatitis C and steatosis, CTGF may promote fibrosis independently of inflammation. CTGF may link steatosis and fibrosis via increased leptin levels.

Insulin-like growth factor I improves aspects of mycophenolate mofetil-impaired anastomotic healing in an experimental model

BACKGROUND: Patients taking immunosuppressants after transplantation may require intestinal surgery. Mycophenolate mofetil (MMF) has been found to impair the healing of colonic anastomoses in rats. This study examined whether insulin-like growth factor (IGF) I prevents MMF impairment of anastomotic healing. METHODS: Sixty-three rats were divided into three groups (MMF, MMF/IGF and control). Animals underwent a sigmoid colon anastomosis with a 6/0 suture, and were killed on days 2, 4 and 6 after surgery. Investigations included bursting pressure measurement, morphometric analysis, and assessment of mucosal proliferation by 5-bromo-2'-deoxyuridine and Ki67 immunohistochemistry of the anastomoses. RESULTS: The leak rate was three of 21, one of 20 and two of 20 in the MMF, MMF/IGF-I and control groups respectively. Anastomotic bursting pressures were significantly lower in the MMF group than in the control group on days 2 and 4, but there was no significant difference by day 6. Values in the MMF/IGF-I and control groups were similar. Colonic crypt depth was significantly reduced in MMF-treated animals on days 2 and 4, but this impairment was attenuated by IGF-I on day 4. Similarly, IGF-I reduced the negative impact of MMF on mucosal proliferation on days 2 and 6. CONCLUSION: Exogenous IGF-I improves some aspects of MMF-impaired anastomotic healing.

Subcellular distribution of the insulin-like growth factor (IGF) binding proteins (IGFBPs) 2 and 3 in articular chondrocytes

The insulin-like growth factor (IGF) is a major anabolic regulator in articular cartilage. The IGF-binding proteins (IGFBPs) are increased during osteoarthritis (OA), but the function of the later proteins remains unknown. In general, the IGFBPs are pluripotential effectors capable of IGF regulation and of acting on their own to control key cell functions, including survival and proliferation. The independent functions are often associated with their cell location, and therefore this study explores the distribution of IGFBP-2 and IGFBP-3 in articular chondrocytes. Immunohistochemistry was used to localize IGFBP-2 in normal human articular cartilage. Bovine chondrocytes were used for subcellular fractionation (hypotonic cell lysis) under nonreducing conditions and nuclear purification (centrifugation on sucrose cushions). Cell fraction markers and IGFBPs were assayed in the subcellular fractions by Western immunoblot. The IHC results showed association of IGFBP-2 with chondrocytes, but not with the nuclei. Subcellular fractionation of isolated chondrocytes yielded intact nuclei as assessed at the light microscopic level; the nuclear marker histone H1 was exclusively associated with this fraction. More than 90% of the cytoplasmic marker GAPDH and all the detectable IGFBP-2 were in the cytoplasmic fraction. Immunoreactive IGFBP-3 was found in the cytoplasmic and peri-nuclear/nuclear fractions. Chondrocytes contain intracellular IGFBP-2 and IGFBP-3 but only IGFBP-3 is associated with nuclei. This suggests the hypothesis that the actions of these IGFBPs in articular cartilage extend beyond the classic modulation of IGF receptor action.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is closely associated with the chondrocyte nucleus in human articular cartilage

OBJECTIVE: Insulin-like growth factor-I (IGF-I) is critically involved in the control of cartilage matrix metabolism. It is well known that IGF-binding protein-3 (IGFBP-3) is increased during osteoarthritis (OA), but its function(s) is not known. In other cells, IGFBP-3 can regulate IGF-I action in the extracellular environment and can also act independently inside the cell; this includes transcriptional gene control in the nucleus. These studies were undertaken to localize IGFBP-3 in human articular cartilage, particularly within cells. DESIGN: Cartilage was dissected from human femoral heads derived from arthroplasty for OA, and OA grade assessed by histology. Tissue slices were further characterized by extraction and assay of IGFBPs by IGF ligand blot (LB) and by enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry (IHC) for IGF-I and IGFBP-3 was performed on cartilage from donors with mild, moderate and severe OA. Indirect fluorescence and immunogold-labeling IHC studies were included. RESULTS: LBs of chondrocyte lysates showed a strong signal for IGFBP-3. IHC of femoral cartilage sections at all OA stages showed IGF-I and IGFBP-3 matrix stain particularly in the top zones, and closely associated with most cells. A prominent perinuclear/nuclear IGFBP-3 signal was seen. Controls using non-immune sera or antigen-blocked antibody showed negative or strongly reduced stain. In frozen sections of human ankle cartilage, immunofluorescent IGFBP-3 stain co-localized with the nuclear 4',6-diamidino-2-phenyl indole (DAPI) stain in greater than 90% of the cells. Immunogold IHC of thin sections and transmission electron immunogold microscopy of ultra-thin sections showed distinct intra-nuclear staining. CONCLUSIONS: IGFBP-3 in human cartilage is located in the matrix and within chondrocytes in the cytoplasm and nuclei. This new finding indicates that the range of IGFBP-3 actions in articular cartilage is likely to include IGF-independent roles and opens the door to studies of its nuclear actions, including the possible regulation of hormone receptors or transcriptional complexes to control gene action.

The etiology of liver damage imparts cytokines transforming growth factor beta1 or interleukin-13 as driving forces in fibrogenesis

It is unknown whether transforming growth factor beta1 (TGF-beta1) signaling uniformly participates in fibrogenic chronic liver diseases, irrespective of the underlying origin, or if other cytokines such as interleukin (IL)-13 share in fibrogenesis (e.g., due to regulatory effects on type I pro-collagen expression). TGF-beta1 signaling events were scored in 396 liver tissue samples from patients with diverse chronic liver diseases, including hepatitis B virus (HBV), hepatitis C virus (HCV), Schistosoma japonicum infection, and steatosis/steatohepatitis. Phospho-Smad2 staining correlated significantly with fibrotic stage in patients with HBV infection (n = 112, P < 0.001) and steatosis/steatohepatitis (n = 120, P < 0.01), but not in patients with HCV infection (n = 77, P > 0.05). In tissue with HBx protein expression, phospho-Smad2 was detectable, suggesting a functional link between viral protein expression and TGF-beta1 signaling. For IL-13, immunostaining correlated with fibrotic stage in patients with HCV infection and steatosis/steatohepatitis. IL-13 protein was more abundant in liver tissue lysates from three HCV patients compared with controls, as were IL-13 serum levels in 68 patients with chronic HCV infection compared with 20 healthy volunteers (72.87 +/- 26.38 versus 45.41 +/- 3.73, P < 0.001). Immunohistochemistry results suggest that IL-13-mediated liver fibrogenesis may take place in the absence of phospho-signal transducer and activator of transcription protein 6 signaling. In a subgroup of patients with advanced liver fibrosis (stage > or =3), neither TGF-beta nor IL-13 signaling was detectable. Conclusion: Depending on the cause of liver damage, a predominance of TGF-beta or IL-13 signaling is found. TGF-beta1 predominance is detected in HBV-related liver fibrogenesis and IL-13 predominance in chronic HCV infection. In some instances, the underlying fibrogenic mediator remains enigmatic.