Activating transcription factor 3 induces DNA synthesis and expression of cyclin D1 in hepatocytes

Activating transcription factor 3 (ATF3) is an early response gene that is induced rapidly during in vivosituations of cellular growth such as liver regeneration. However, neither the physiological function nor the potential target genes of this transcription factor related to cellular proliferation have been identified in the liver or other tissues. We demonstrate here that endogenous ATF3 mRNA expression is rapidly induced up to 4-fold upon mitogenic stimulation of quiescent Hepa 1-6 mouse hepatoma cells. Overexpression of exogenous ATF3 results in a significant, dose-dependent increase in DNA synthesis of up to 140% over control cells. ATF3; transfected cells also display significantly higher rates of [H-3]thymidine incorporation in comparison with nontransfected controls in the presence of serum, Northern blot analysis and co transfection experiments demonstrate that overexpression of ATF3 enhances cyclin D1 mRNA expression and activates the cyclin D1 promoter 2.5-fold when activating protein-1 (AP-1) and cyclic AMP response element (CRE) sites within the promoter are intact. ATF3-mediated promoter activation is reduced to 1.3-fold and 1.6-fold respectively when the AP-1 or CRE sites are mutated, and mutation of both sites simultaneously leads to the complete abrogation of promoter activation. Furthermore, DNA-binding studies demonstrate that ATF3 binds directly to the AP-I site within the cyclin DI promoter. These results indicate that ATF3 expression stimulates hepatocellular proliferation, suggesting that this effect is mediated, at least in part, by the ATF3-dependent activation of cyclin D1 transcription.