Background & aims: Normal intestinal mucosal growth requires cellular polyamines that regulate expression of various genes involved in cell proliferation, growth arrest, and apoptosis. We have recently shown that growth inhibition after polyamine depletion is associated with an increase in JunD/AP-1 activity in normal intestinal epithelial cells (IEC-6 line). The current study tests the hypothesis that polyamine depletion-induced JunD/activator protein 1 (AP-1) activity results from the activation of junD gene expression and plays a critical role in regulation of intestinal epithelial cell growth.
Methods: The junD gene transcription was examined by nuclear run-on assays, and messenger RNA (mRNA) stability was measured by determination of JunD mRNA half-life. Functions of JunD were investigated by using JunD antisense oligodeoxyribonucleotides and transient transfection with the junD-expressing vector.
Results: Depletion of cellular polyamines by DL-alpha-difluoromethylornithine (DFMO) induced levels of JunD mRNA and protein, which was associated with an increase in G(1) phase growth arrest. Polyamine depletion did not increase the rate of junD gene transcription but significantly increased the stability of JunD mRNA. Decreasing JunD protein by using JunD antisense oligomers promoted cell growth in polyamine-deficient cells. Growth arrest following polyamine depletion also was accompanied by increases in both p21 expression and its promoter activity. Treatment with JunD antisense oligomers inhibited the p21 promoter and prevented the increase in p21 expression in the presence of DFMO. Ectopic expression of the wild-type junD increased p21-promoter activity and inhibited epithelial cell growth.
Conclusions: Polyamines negatively regulate junD gene expression posttranscriptionally, and increased JunD/AP-1 inhibits intestinal epithelial cell proliferation at least partially through the activation of p21 promoter.