Transforming growth factor beta-1 enhances Smad transcriptional activity through activation of p8 gene expression.
We report that exposure of mouse embryonic fibroblasts to transforming growth factor beta-1 (TGFbeta-1) (5 ng/ml) results in a strong activation of p8 mRNA expression that precedes the induction of cell growth. Involvement of the p8 promoter in the regulation was demonstrated by using a p8-chloramphenicol acetyltransferase construct. We therefore speculated that p8 might be a mediator of TGFbeta-1 in these cells. The incorporation of [(3)H]thymidine on treatment with TGFbeta-1 was indeed significantly higher in p8(+/+) fibroblasts than in p8(-/-) fibroblasts. Smad transcriptional activity was used as marker of the TGFbeta-1 signalling pathway, to probe the lower p8(-/-) response to TGFbeta-1. Two Smad-binding elements (SBEs)-luciferase constructs were transfected into p8(-/-) and p8(+/+) embryonic fibroblasts before treatment with TGFbeta-1. A lower level of Smad transactivation was observed in p8(-/-) embryonic fibroblasts, under basal conditions and after stimulation with TGFbeta-1. To test whether Smad underexpression in p8(-/-) cells was actually due to p8 depletion, p8(-/-) embryonic fibroblasts were transfected with a human p8 expression plasmid together with an SBE-luciferase construct. The expression of p8 restored Smad transactivation in unstimulated and TGFbeta-1-treated cells to the level found in p8(+/+) cells. We concluded that TGFbeta-1 activates p8 expression, which in turn enhances the Smad-transactivating function responsible for TGFbeta-1 activity.
García-Montero
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INSERM U.315, 46 boulevard de la Gaye, F-13009 Marseille, France.
Vasseur
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Giono
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LE
Canepa
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Moreno
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Dagorn
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Iovanna
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D016475:3T3 Cells; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology*; D017353:Gene Deletion; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D017930:Genes, Reporter; D006133:Growth Substances / Q000235:genetics* / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000235:genetics; D006720:Homozygote; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009363:Neoplasm Proteins; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology*; D015533:Transcriptional Activation; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D051792:Basic Helix-Loop-Helix Proteins; D015500:Chloramphenicol O-Acetyltransferase; D004247:DNA; D004268:DNA-Binding Proteins; D006133:Growth Substances; D006609:High Mobility Group Proteins; D008156:Luciferases; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D013936:Thymidine; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; C491643:NUPR1 protein, human; C491644:Nupr1 protein, mouse