Mdm2 inhibition of p53 induces E2F1 transactivation via p21

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Abstract
The transcription factor E2F1 functions as a key regulator for both cell-cycle progression and apoptosis. Mdm2, a major cellular regulator of the p53 tumor suppressor protein, is also closely involved in cell cycle and apoptosis. In addition to regulation of p53, Mdm2 has been reported to stimulate E2F1 transactivation by a mechanism that remains unclear. Here we examined how overexpression of Mdm2 alters E2F1/DP1 transactivation. Using a set of cell lines with differing p53 and Rb status we determined that Mdm2 induction of E2F1 transactivation was p53-dependent, resulting from release of repression by p53. While Mdm2 association with p53 was required to increase E2F1 transactivation, Mdm2 mediated degradation of p53 was not. p53 repression of E2F1 transactivation required a functional DNA binding and transactivation domain. Consistent with Mdm2 activation of E2F1 via an inhibition of p53 transactivation we demonstrate a concomitant reduction in p21 protein levels with Mdm2 overexpression. Furthermore, E2F1 repression by an Rb-phosphorylation mutant could not be reversed by Mdm2 overexpression. Mdm2 was also unable to enhance E2F1 transactivation in Mouse embryo fibroblasts lacking p21. Taken together, these results suggest that Mdm2 activation of E2F1 occurs through the repression of p53-dependent transcription of p21, a p53-target gene and cyclin dependent kinase inhibitor.