Smad proteins play a key role in the intracellular signalling of transforming growth factor β (TGFβ), which elicits a large variety of cellular responses. Upon TGFβ receptor activation, Smad2 and Smad3 become phosphorylated and form heteromeric complexes with Smad4. These complexes translocate to the nucleus where they control expression of target genes. However, the mechanism by which Smads mediate transcriptional regulation is largely unknown. Human plasminogen activator inhibitor-1 (PAI-1) is a gene that is potently induced by TGFβ. Here we report the identification of Smad3/Smad4 binding sequences, termed CAGA boxes, within the promoter of the human PAI-1 gene. The CAGA boxes confer TGFβ and activin, but not bone morphogenetic protein (BMP) stimulation to a heterologous promoter reporter construct. Importantly, mutation of the three CAGA boxes present in the PAI-1 promoter was found to abolish TGFβ responsiveness. Thus, CAGA elements are essential and sufficient for the induction by TGFβ. In addition, TGFβ induces the binding of a Smad3/Smad4-containing nuclear complex to CAGA boxes. Furthermore, bacterially expressed Smad3 and Smad4 proteins, but not Smad1 nor Smad2 protein, bind directly to this sequence in vitro. The presence of this box in TGFβ-responsive regions of several other genes suggests that this may be a widely used motif in TGFβ-regulated transcription.
Connective tissue growth factor (CTGF) stimulates in vitro fibroblast proliferation and extracellular matrix synthesis. The aim of this study was to assess the role of CTGF in liver fibrogenesis. CTGF expression was investigated both at the protein and mRNA level in biopsies of chronic liver diseases, in experimental models of liver fibrosis, and in hepatic stellate cells in culture. CTGF immunostaining was observed in most human liver biopsies with significant fibrosis. An increase of CTGF immunostaining was associated with a higher score of fibrosis both in the group of chronic hepatitis C ( 2 ؍ 9.3; P F .01) and in the non-hepatitis C group ( 2 ؍ 7.2; P F .02). In situ hybridization showed CTGF mRNA expression in spindle cells in both the fibrous septa and sinusoidal lining. In experimental models of liver fibrosis, CTGF accumulated in parallel with the development of septal fibrosis and cirrhosis. Quantification of CTGF mRNA by a real-time reversetranscription polymerase chain reaction (RT-PCR) assay showed a significant increase of CTGF mRNA in both CCl 4 -induced and bile duct-ligated rat models of liver fibrosis. Expression of CTGF protein and mRNA was definitively assigned to hepatic stellate cells, because CTGF was detected by Western blot both in lysate and supernatant of a hepatic stellate cell line derived from rats. These cells also displayed CTGF protein and mRNA as shown by immunohistochemistry and in situ hybridization. In conclusion, this study shows that CTGF is strongly expressed during liver fibrogenesis, and hepatic stellate cells seem to be the major cellular sources of CTGF in the liver. (HEPATOL-OGY 1999;30:968-976.)
Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor β (TGFβ) superfamily. We used two-hybrid screening to map Smad signaling protein–protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.
1 Chronic liver disease is characterized by an exacerbated accumulation of matrix, causing progressive fibrosis, which may lead to cirrhosis. Transforming growth factor beta (TGF-b), a wellknown profibrotic cytokine, transduces its signal through the ALK5 ser/thr kinase receptor, and increases transcription of different genes including PAI-1 and collagens. The identification of GW6604 (2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine), an ALK5 inhibitor, allowed us to evaluate the therapeutic potential of inhibiting TGF-b pathway in different models of liver disease. 2 A cellular assay was used to identify GW6604 as a TGF-b signaling pathway inhibitor. This ALK5 inhibitor was then tested in a model of liver hepatectomy in TGF-b-overexpressing transgenic mice, in an acute model of liver disease and in a chronic model of dimethylnitrosamine (DMN)-induced liver fibrosis. 3 In vitro, GW6604 inhibited autophosphorylation of ALK5 with an IC 50 of 140 nM and in a cellular assay inhibited TGF-b-induced transcription of PAI-1 (IC 50 : 500 nM). In vivo, GW6604 (40 mg kg À1 p.o.) increased liver regeneration in TGF-b-overexpressing mice, which had undergone partial hepatectomy. In an acute model of liver disease, GW6604 reduced by 80% the expression of collagen IA1. In a chronic model of DMN-induced fibrosis where DMN was administered for 6 weeks and GW6604 dosed for the last 3 weeks (80 mg kg À1 p.o., b.i.d.), mortality was prevented and DMNinduced elevations of mRNA encoding for collagen IA1, IA2, III, TIMP-1 and TGF-b were reduced by 50-75%. Inhibition of matrix genes overexpression was accompanied by reduced matrix deposition and reduction in liver function deterioration, as assessed by bilirubin and liver enzyme levels. 4 Our results suggest that inhibition of ALK5 could be an attractive new approach to treatment of liver fibrotic diseases by both preventing matrix deposition and promoting hepatocyte regeneration.
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