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Background/aims The activation of hepatic stellate cells (HSC) to extracellular matrix (ECM) producing myofibroblasts (MFB) is the key pathogenetic event in human liver fibrogenesis. Latent transforming growth factor beta binding protein (LTBP), a component of the profibrogenic large latent transforming growth factor (TGF)-beta complex, is suggested to be important for secretion, latency, storage and activation of TGF-beta in the ECM. This study was performed to identify the expression profile of all hitherto known LTBP isoforms and LTBP splice variants in conjunction with that of TGF-beta isoforms in cultured human liver MFB. Methods Cultured human MFB were analyzed for TGF-beta and LTBP using reverse-transcription polymerase chain reaction (RT-PCR), sequence analysis, immunofluorescence staining, metabolic labeling, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). Results Transcripts of all three TGF-beta isoforms, of all four LTBP isoforms and of nearly all splice variants of LTBP-1 and LTBP-4 so far known were detected. Metabolic labeling followed by immunoprecipitation with anti-LTBP-1 antibody revealed the synthesis of LTBP proteins. Secretion of free LTBP and LTBP integrated into the large latent TGF-beta complex was demonstrated by size-exclusion chromatography. Co-localization of LTBP-1 and -2 with fibronectin and collagen type I was observed by double immunofluorescence staining. Conclusion The expression of a complete profile of hitherto known LTBP proteins by cultured human MFB suggests a role in modulating the bioactivity of TGF-beta in the diseased liver.

Activation of hepatic stellate cells (HSCs) is the key step in liver fibrogenesis. Increased transforming growth factor beta (TGF-beta) expression and extracellular matrix production in patients with hepatic fibrosis and experimental models of liver fibrogenesis support implication of TGF-beta in the pathogenesis of this disease. However, a causative role for TGF-beta during transdifferentiation of HSCs has not been delineated in molecular detail. Using a rat cell culture model of HSC transdifferentiation, we analyzed TGF-beta signal transduction and identified changes between stellate cells and their transdifferentiated phenotype. Fully transdifferentiated myofibroblasts, opposed to HSCs, were not inhibited in proliferation activity on treatment with TGF-beta1. Furthermore, stimulation of alpha2 (I) collagen and Smad7 messenger RNA (mRNA) expression by TGF-beta1 was achieved in stellate cells but not in myofibroblasts. Northern and Western blot analyses indicated significant expression of TGF-beta receptors I and II in both cell types. In contrast, [(125)I]-TGF-beta1 receptor affinity labeling displayed strongly reduced types I, II, and III receptor presentation at the cell surface of myofibroblasts. Moreover, myofibroblasts did not display DNA-binding SMAD proteins in electrophoretic mobility shift assays with a CAGA box. These data indicate that stellate cells are responsive to TGF-beta1 treatment and transduce a signal that may play an important role in liver fibrogenesis. Myofibroblasts display decreased availability of surface receptors for TGF-beta, which could be based on autocrine stimulation. However, lack of activated SMAD complexes with DNA-binding activity and absence of alpha2 (I) collagen transcription inhibition by latency-associated peptide (LAP)/anti-TGF-beta antibody raise the possibility of TGF-beta signaling independent receptor down-regulation in myofibroblasts.

Two point mutations in the hemochromatosis gene (HFE) are considered responsible for the development of hereditary hemochromatosis (HH), an autosomal recessive iron overload disease. One of these mutations produces a cysteine-to-tyrosine amino acid substitution at position 282 of the HFE protein (Cys282Tyr), caused by a G-to-A transition at nucleotide position 845. A homozygous Cys282Tyr mutation is present in 80–100% of hemochromatosis patients (1)(2). This mutation prevents interaction of HFE protein with β2-microglobulin, leading to an inability to bind to the transferrin receptor, a process that is necessary for iron resorption (3)(4). A second mutation, which changes histidine at position 63 to aspartic acid (His63Asp), caused by a C187G transversion does not affect this binding but possibly blocks the interaction with other ligands (3)(5). In contrast to the Cys282Tyr mutation, the role of the His63Asp mutation in the pathogenesis of the disease is uncertain. A heterozygous His63Asp mutation is often observed in Cys282Tyr heterozygous patients. This genotype combination is called compound heterozygosity and is observed in 4–5% of HH cases. Therefore, it must be considered, like the homozygous Cys282Tyr mutation, as a genetic disposition for HH. However, it seems to play a subordinate role and seems to be important only in combination with the Cys282Tyr mutation (6). Approximately 3% of the population is homozygous for the His63Asp mutation, and thus is not considered in the risk group for HH (6)(7). In rare HH cases, none of these known HFE mutations are present. Other mutations, possibly not associated with HFE , might contribute to the development of hemochromatosis (2). There are many current methods available for genotyping the two known hemochromatosis-causing mutations, including oligonucleotide ligation assay (8), single-strand conformation polymorphism (9), allele-specific PCR (10)(11), and PCR-restriction fragment length …

Latent transforming growth factor-beta (TGFbeta) binding protein (LTBP), a component of the high-molecular-weight latent TGFbeta complex, is found in various cell and tissue types. Originally described as a TGFbeta-masking protein, recent detections of four isoforms and numerous splice variants provide new aspects of its putative functional role. Regulation and sequestration of TGFbeta activity and structural remodeling of the extracellular matrix (ECM) seem to be the main tasks, but other possible functions might exist. The mechanism by which LTBP interacts with cell surface molecules or cellular receptors and ECM components remains unclear. Cellular, molecular and functional aspects will be discussed.

Rheumatoid arthritis (RA) is characterised by chronic inflammation of synovial tissue with aggressive proliferation of synovial cells causing destruction of cartilage and bone. Immunopathological mechanisms, infectious causes and genetic factors have been discussed, but the etiology of the disease has not been understood until now. Especially, the mechanisms driving tumourlike growth and invasive behaviour of fibroblastoid synovial cells have not been identified yet. Our aim is to find cellular factors which are mediators for such pathways. One possibility to approach this, is searching for disease-relevant genes. We applied the mRNA-differential display technique to compare mRNA expression patterns of normal and rheumatic synovial fibroblasts. We identified an upregulation of the human semaphorin E gene in rheumatoid synovial fibroblastoid cells. Interestingly semaphorin E is a member of a protein-family described to play an immunosuppressive role via inhibition cytokines. A relevance of this finding towards the pathogenesis of RA is discussed.

Background: The objective of the present study was to elucidate possible relationships between four polymorphisms of the TGF-β1 gene (−800G>A; −509C>T; Leu10Pro; Arg25Pro) and stage, histological activity grade and progression rate of liver fibrosis, classified according to the METAVIR-score. Methods: Three study groups, i.e. 48 patients with hepatic fibrosis (26 with known duration of hepatitis C virus infection), 47 patients with non-fibrotic diseases and 50 healthy blood donors, were analyzed for TGF-β1 polymorphisms using ARMS-PCR and sequence analysis. The concentrations of total TGF-β1 in plasma and of hyaluronan, P-III-NP and activities of transminases in serum were measured. Results: The presence of proline at codons 10 and/or 25 was associated with a faster progression of fibrosis than other polymorphisms. Patients with the genotype 25ArgPro developed fibrosis significantly faster (0.23 units/year) than those having 25ArgArg (0.08 units/year). Similarly, the fibrosis progression rate of patients with genotypes 10LeuPro and 10ProPro was almost three times as fast as of those having genotype 10LeuLeu. Stage and histological activity grade of fibrosis in 25ArgPro in comparison to 25ArgArg were higher. Also 10LeuPro showed a higher average stage of fibrosis than 10LeuLeu. The TGF-β1 plasma concentrations of patients with hepatic fibrosis were not significantly different between carriers of 25ArgArg and 25ArgPro genotypes. The frequency of the genotype 25ArgPro in liver fibrotic patients was about three times that of the control group whereas the frequency distribution of the genotype 25ArgArg tended to lower frequency in the fibrosis group. TGF-β1-promoter polymorphisms did not show any correlation with stage, grade or progression of liver fibrosis. Conclusion: Our results indicate that the heterozygous ArgPro of codon 25 predicts significantly faster fibrotic progression of chronic hepatitis C than the homozygous 25ArgArg genotype. The homozygous LeuLeu genotype of codon 10 showed a slow progression of fibrosis.