p53 transcription factors control apoptosis susceptibility by regulation of Mcl1

Background: Hepatocellular carcinoma (HCC) represents a complication of liver cirrhosis limiting the curing option by liver transplantation. Depending on their splice variants – with transactivation (TA) domain or dominant negative (DN) – p53-family transcription factors (p53, p63, p73) exert tumorsuppressive or oncogenic functions in cell cycle by transcriptional regulation of a specific set of genes. In previous studies we identified the MCL1 gene (Myeloid cell leukemia sequence 1, Mcl-1) as potential target gene of the p53 family and confirmed its prognostic relevance in HCC. Mcl-1 is a member of the Bcl-2 protein family being involved in the control of mitochondrial integrity. Mcl-1 represents an anti-apoptotic member of the Bcl-2 family, supporting cell survival by binding and inhibition of pro-apoptotic Bcl-2 proteins. Aim of this study was to elucidate the impact of p53 family members on MCL1 gene regulation.

Methods: Hep3B cells were transfected with rAd-GFP, -p53, -TAp63α, -TAp73β, -DNp63α, and DNp73β. MCL1 expression was measured by real time qPCR. Western Blot analyses determined intracellular levels of Mcl-1 after specific siRNA interference. Potential binding sites of p53 family members in the MCL1 locus were identified by database analyses (pDraw32, Husar, MatchTM) and verified by luciferase reporter assays. Direct protein interactions of Mcl-1 and p53 proteins were evaluated by LUminescence-based Mammalian IntERactome mapping (LUMIER) technology.

Results: p53 and the isoforms TAp63 and TAp73 inhibited MCL1 expression, whereas ΔNp63α and ΔNp73β induced transcription of the MCL1 gene. These effects were confirmed for Mcl-1 protein levels. Reduction of Mcl-1 protein was abrogated after silencing of p53, TAp63 and TAp73. Database analyses identified two potential p53 binding sites and one potential p63 binding site each in promoter, intron 1 and 2 of the MCL1 gene. Luciferase reporter assays of these cloned putative binding sites confirmed a regulation of the MCL1 gene by p53 family members. Furthermore, a direct protein-protein interaction of Mcl-1 with p53 and TAp63, respectively, was detected.

Conclusion: p53 family members directly regulate the expression of the MCL1 gene. p53, TAp63 and TAp73 are potent repressors, whereas DNp63 and DNp73 act as inducers of MCL1. On posttranslational level, Mcl-1 interacts with p53 and TAp63. Since Mcl-1 has anti-apoptotic functions, a downregulation of Mcl-1 by TA-isoforms of p53 proteins results in an increase of apoptosis susceptibility. HCC is often associated with chemotherapeutic resistance, which could be overcome by reconstitution with p53, TAp63 and TAp73 or a direct inhibition of Mcl1.