Activin A stimulates vascular endothelial growth factor gene transcription in human hepatocellular carcinoma cells.
OBJECTIVE
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-beta 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.
Wagner
Karola
K
Department of Hepatology and Gastroenterology, Charité, Campus Virchow-Klinikum, Humboldt University, Augustenburger Platz 1, 13353 Berlin, Germany.
Peters
Michael
M
Scholz
Arne
A
Benckert
Christoph
C
Ruderisch
Hugo Sanchez
HS
Wiedenmann
Bertram
B
Rosewicz
Stefan
S
D029404:Activin Receptors / Q000235:genetics / Q000378:metabolism; D028341:Activins / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D028322:Inhibin-beta Subunits / Q000494:pharmacology*; D008113:Liver Neoplasms; D009389:Neovascularization, Pathologic / Q000503:physiopathology; D011401:Promoter Regions, Genetic / Q000502:physiology; D051899:Smad2 Protein; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
D004268:DNA-Binding Proteins; C497782:SMAD2 protein, human; D051899:Smad2 Protein; D016329:Sp1 Transcription Factor; D015534:Trans-Activators; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; C422405:activin A; D028341:Activins; D051705:Sp3 Transcription Factor; D028322:Inhibin-beta Subunits; D029404:Activin Receptors