Radiation and Transforming Growth Factor-β Cooperate in Transcriptional Activation of the Profibrotic Plasminogen Activator Inhibitor-1 Gene

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Abstract
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-β (TGF-β), 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-β. 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-β1 and Smad7 were not increased. Radiation and TGF-β showed strong cooperative effects in transcription of the PAI-1 gene. The TGF-β1 gene showed a mild cooperative activation, whereas Timp-1 and Smad7 were not cooperatively activated by radiation and TGF-β. 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 γ-rays that was independent of latent TGF-β 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-β/Smad pathway. Together, these data show that radiation and TGF-β 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.