RESUMO
Two homologs of the tumor suppressor p53, named p63 and p73, are each expressed from at least two start sites of mRNA synthesis, yielding full-length, transactivating (TA) isoforms, and also aminoterminally truncated (DeltaN) isoforms that act as antagonists to p53. The expression of TAp73-transcripts is induced by E2F and negatively regulated by transforming growth factor beta (TGFbeta). The DeltaNp73 promoter is induced by p53, resulting in negative feedback to control p53 activity. Here, we have analysed the expression of p63 in comparison with p73. In contrast to the induction of DeltaNp73, the expression of DeltaNp63 was reduced by p53 particularly in human keratinocytes, at the mRNA and protein levels. Accordingly, the 3' promoter of p73, but not that of p63, was activated by p53 in reporter assays. DeltaNp73 mRNA and DeltaNp73 protein, but not the p63 gene products, also accumulated when HaCat cells (lacking functional p53) were grown to high density. TAp73, but not TAp63, expression was suppressed by TGFbeta in these cells, and the TAp73, but not the TAp63, promoter was induced by E2F-1. Thus, in contrast to the functional similarities of their respective products, the expression levels of p63 and p73 are regulated by different mechanisms. This might be responsible for the discordant biological roles of p63 and p73 in development, as well as their deviant expression characteristics in cancer.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Proteínas de Membrana , Proteínas Nucleares/genética , Fosfoproteínas/genética , Transativadores/genética , Contagem de Células , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Genes Supressores de Tumor , Humanos , Regiões Promotoras Genéticas , Fatores de Transcrição/fisiologia , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/fisiologia , Proteínas Supressoras de TumorRESUMO
The p53 tumor suppressor protein activates transcription and induces cell death. A close homologue of p53, termed p73, is expressed in transactivating (TA) forms that induce growth arrest and apoptosis much like p53. However, the p73 gene contains a second promoter, giving rise to the expression of p73 Delta N, a species of p73 proteins that lack the N-terminal transactivation domain. We show here that the expression of p73 Delta N is induced by p53 on the mRNA and protein level. The promoter that regulates p73 Delta N expression in human cells was cloned and found to be activated by p53, as well as by p73TA, directly through a specific DNA element. The p73 Delta N proteins, that are thereby expressed, bound to p53-responsive promoter DNA, competed with p53 for DNA binding, antagonized the activation of transcription by p53, and prevented p53-induced cell death. In addition, a transcriptional repressor domain was identified within the splicing variant p73 Delta Nalpha. The combination of p73DeltaNalpha and mdm2 antagonized p53 more strongly than either p73Nalpha or mdm2 alone. Blocking endogenous p73 Delta N by a trans dominant fragment, or its removal by siRNA, increased the activity of a p53-responsive promoter in cells that contain a wild type p53 gene. Thus, the induction of p73 Delta N expression by p53 establishes an autoregulatory feedback loop that keeps the trigger of cell death under tight control.
Assuntos
Proteínas Nucleares/biossíntese , Ativação Transcricional , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/fisiologia , Apoptose , Sequência de Bases , Linhagem Celular , Proteínas de Ligação a DNA , Retroalimentação Fisiológica , Genes Supressores de Tumor , Homeostase , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Células Tumorais Cultivadas , Proteína Tumoral p73 , Proteínas Supressoras de TumorRESUMO
DeltaNp73alpha is an isoform of the p53 homologue p73 that lacks an amino-terminal transactivation domain and antagonizes the induction of gene expression by p53. Here, we examined whether DeltaNp73alpha might also modulate cellular transcription in the absence of p53. The expression of DeltaNp73alpha in the p53-/- cell line H1299 reduced the mRNA levels of p21/CDKN1A, but did not affect other p53-responsive genes. Correspondingly, the p21/CDKN1A promoter was downregulated by DeltaNp73alpha in reporter assays, whereas other p53-responsive promoters were not inhibited. To identify additional genes that respond to DeltaNp73alpha in the absence of p53, microarrays carrying 4600 cDNA clones were hybridized. The expression of 30 genes was found to be altered more than threefold by overexpressed DeltaNp73alpha. For instance, DeltaNp73alpha increased the expression of EGR1 and CDC6, whereas it decreased the mRNA levels of c-MYC, cyclin A2/CCNA2, NF-kappaB1, ODC1, and RET finger protein/RFP. Semiquantitative reverse transcription-PCR confirmed these results and further revealed that the influence of DeltaNp73alpha on the regulation of these genes differs from other p73 isoforms and p53. We conclude that the impact of DeltaNp73alpha on gene expression is not limited to p53-responsive genes. Rather, DeltaNp73alpha can regulate the expression of a variety of genes independently of p53.