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-h (TGF-h), 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-h. Irradiation of HepG2 cells led to a high increase in PAI-1mRNA levels and a mild increase in Timp-1mRNA levels. In contrast,TGF-h1and Smad7 were not increased. Radiation and TGF-h showed strong cooperative effects in transcription of the PAI-1 gene. The TGF-b1 gene showed a mild cooperative activation, whereas Timp-1and Smad7 were not cooperatively activated by radiation and TGF-h. Analysis using the proximal 800 bp of the human PAI-1 promoter revealed a dose-dependent increase of PAI-1levels between 2 and 32 Gy g-rays that was independent of latent TGF-h 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-1activation and the cooperativity with theTGF-h/Smad pathway. Together, these data show that radiation and TGF-h 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.