Radiation and transforming growth factor-beta cooperate in transcriptional activation of the profibrotic plasminogen activator inhibitor-1 gene.
Radiation-induced fibrosis is an important side effect in the treatment of cancer. Profibrotic proteins, such as plasminogen activator inhibitor-1 (PAI-1), transforming growth factor-beta (TGF-beta), and tissue type inhibitor of metalloproteinases-1 (Timp-1), are thought to play major roles in the development of fibrosis via the modulation of extracellular matrix integrity. We did a detailed analysis of transcriptional activation of these profibrotic genes by radiation and TGF-beta. Irradiation of HepG2 cells led to a high increase in PAI-1 mRNA levels and a mild increase in Timp-1 mRNA levels. In contrast, TGF-beta1 and Smad7 were not increased. Radiation and TGF-beta showed strong cooperative effects in transcription of the PAI-1 gene. The TGF-beta1 gene showed a mild cooperative activation, whereas Timp-1 and Smad7 were not cooperatively activated by radiation and TGF-beta. Analysis using the proximal 800 bp of the human PAI-1 promoter revealed a dose-dependent increase of PAI-1 levels between 2 and 32 Gy gamma-rays that was independent of latent TGF-beta activation. Subsequent site-directed mutagenesis of the PAI-1 promoter revealed that mutation of a p53-binding element abolished radiation-induced PAI-1 transcription. In line with this, PAI-1 was not activated in p53-null Hep3B cells, indicating that p53 underlies the radiation-induced PAI-1 activation and the cooperativity with the TGF-beta/Smad pathway. Together, these data show that radiation and TGF-beta activate PAI-1 via partially nonoverlapping signaling cascades that in concert synergize on PAI-1 transcription. This may play a role in patient-to-patient variations in susceptibility toward fibrosis after radiotherapy.
Hageman
Jurre
J
Department of Radiation and Stress Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.
Eggen
Bart J
BJ
Rozema
Tom
T
Damman
Kevin
K
Kampinga
Harm H
HH
Coppes
Robert P
RP
D015153:Blotting, Western; D045744:Cell Line, Tumor; D004307:Dose-Response Relationship, Radiation; D005720:Gamma Rays; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051906:Smad7 Protein / Q000235:genetics; D013997:Time Factors; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D051906:Smad7 Protein; C508960:TGFB1 protein, human; D019715:Tissue Inhibitor of Metalloproteinase-1; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D016159:Tumor Suppressor Protein p53; D008156:Luciferases