Gastroenterology

Gastroenterology

Volume 126, Issue 7, June 2004, Pages 1828-1843
Gastroenterology

Basic-liver, pancreas, and biliary tract
Activin A stimulates vascular endothelial growth factor gene transcription in human hepatocellular carcinoma cells

https://doi.org/10.1053/j.gastro.2004.03.011Get rights and content

Abstract

Background & Aims: Up-regulation of vascular endothelial growth factor is known to play a critical role in hepatocellular tumor biology. In an attempt to identify factors responsible for vascular endothelial growth factor induction in human hepatocellular carcinoma, we evaluated the effects of activin A, a member of the transforming growth factor-β cytokine superfamily, on vascular endothelial growth factor gene expression. Methods: Expression of vascular endothelial growth factor, activin A, and its receptors was analyzed by immunohistochemistry, polymerase chain reaction, and enzyme-linked immunosorbent assay. Functional vascular endothelial growth factor promoter analysis and gel shift assays were performed to define minimal promoter requirements and potential transcription factors. Nuclear expression and biochemical modifications of Sp1, as well as subcellular distribution, expression, and physical interaction of Smad proteins with Sp1, were assessed with immunoprecipitation and Western blot analysis. Results: Hepatocellular carcinoma tumors and cell lines expressed activin A and its receptors. Activin A stimulated vascular endothelial growth factor gene transcription through Sp1-dependent induction of vascular endothelial growth factor promoter activity. Furthermore, activin A stimulated the DNA-binding and transactivation potential of Sp1. Immunoprecipitation showed activin A-dependent nuclear translocation of Smad2 and induction of Sp1/Smad2 interaction. The functional relevance of Sp1/Smad2 interaction was confirmed by transient transfection experiments, which showed that overexpression of Smad2 increased vascular endothelial growth factor promoter activity and endogenous vascular endothelial growth factor protein expression, whereas dominant negative Smad2 blocked activin A responsiveness. Conclusions: This study identifies activin A as a novel stimulus of vascular endothelial growth factor gene expression in hepatocellular carcinoma and delineates physical and functional cooperation of Sp1 and Smad2 as the underlying mechanism.

Section snippets

Materials

The following were purchased: Dulbecco’s modified Eagle medium and phosphate-buffered saline (Gibco, Berlin, Germany); UltraCulture medium (BioWhittaker, Verviers, Belgium); fetal calf serum, trypsin/ethylenediaminetetraacetic acid, penicillin, and streptomycin (Biochrom, Berlin, Germany); RNAzol B (Wak-Chemie Medical, Bad Soden, Germany); Thermus aquaticus DNA polymerase and PCR Nucleotide Mix (Roche Diagnostics, Indianapolis, IN); DNA molecular size markers, oligodeoxythymidylic acid residues

Expression of activin A and activin receptors in hepatocellular carcinoma

To evaluate the postulated role of activin A in regulation of VEGF gene expression, we initially analyzed the expression of VEGF in 9 surgically resected human HCC samples. Using a polyclonal antibody specific for hVEGF, we consistently observed strong VEGF expression over HCC cells in all tumor specimens tested (Figure 1A). To investigate the potential interaction of VEGF with activin A, we next characterized the expression pattern of activin A and its receptors. Immunohistochemical analysis

Discussion

The pivotal role of VEGF in tumor angiogenesis and, thus, the propagation and metastasis of HCC is supported by several lines of evidence: (1) expression of the VEGF/VEGF-R system is up-regulated in HCC,13, 14 (2) hepatocellular overexpression of VEGF is inversely correlated with the prognosis and survival of HCC patients,43, 44 and (3) inhibition of VEGF has successfully been used to block angiogenesis and restrain HCC cell growth and metastases in a variety of preclinical models.45, 46

Acknowledgements

The authors thank G. Finkenzeller (GeneScan AG, Freiburg, Germany), G. Suske (Institut für Molekularbiologie und Tumorforschung, Marburg, Germany), and R. Derynck (Department of Growth and Development and Anatomy, and Programs in Cell Biology and Developmental Biology, University of California, San Francisco) for generous gifts of plasmids.

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    S.R. was supported by grants from the Deutsche Krebshilfe, Wilhelm Sander-Stiftung, Deutsche Forschungsgemeinschaft, Berliner Krebsgesellschaft, Else Kröner Fresenius Stiftung, and Sonnenfeld Stiftung.

    1

    K.W. and M.P. contributed equally to this work.

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