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 r egeneration. 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 th at endogenous ATF3 mRNA expression is rapidly induced up to 4-fold upon mit ogenic stimulation of quiescent Hepa 1-6 mouse hepatoma cells. Overexpressi on of exogenous ATF3 results in a significant, dose-dependent increase in D NA synthesis of up to 140% over control cells. ATF3; transfected cells also display significantly higher rates of [H-3]thymidine incorporation in comp arison with nontransfected controls in the presence of serum, Northern blot analysis and co transfection experiments demonstrate that overexpression o f ATF3 enhances cyclin D1 mRNA expression and activates the cyclin D1 promo ter 2.5-fold when activating protein-1 (AP-1) and cyclic AMP response eleme nt (CRE) sites within the promoter are intact. ATF3-mediated promoter activ ation 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 studie s demonstrate that ATF3 binds directly to the AP-I site within the cyclin D I promoter. These results indicate that ATF3 expression stimulates hepatoce llular proliferation, suggesting that this effect is mediated, at least in part, by the ATF3-dependent activation of cyclin D1 transcription.