ABSTRACT
The E2F1 transcription factor is a critical downstream target of the tumor suppressor RB. When activated, E2F1 induces cell proliferation. In addition, E2F1 can induce apoptosis via both p53-dependent and p53-independent pathways. A number of E2F-regulated genes, including ARF, ATM and Chk2, contribute to E2F-induced p53 stabilization. However, it is not known how E2F directs p53 activity towards apoptosis rather than growth arrest. We show that E2F1 upregulates the expression of four proapoptotic cofactors of p53--ASPP1, ASPP2, JMY and TP53INP1--through a direct transcriptional mechanism. Adenovirus E1A protein also induces upregulation of these genes, implicating endogenous E2F in this effect. TP53INP1 was shown to mediate phosphorylation of p53 on serine 46. We demonstrate that activation of E2F1 leads to phosphorylation of p53 on serine 46 and this modification is important for E2F1-p53 cooperation in apoptosis. Overall, these data provide novel functional links between RB/E2F pathway and p53-induced apoptosis.
Subject(s)
Apoptosis/physiology , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Transcription Factors/metabolism , Tumor Suppressor Protein p53/metabolism , Adaptor Proteins, Signal Transducing , Apoptosis Regulatory Proteins , Carrier Proteins/metabolism , E2F Transcription Factors , E2F1 Transcription Factor , Gene Expression Regulation/physiology , Heat-Shock Proteins/metabolism , Humans , Nuclear Proteins/metabolism , Phosphorylation , Trans-Activators/metabolism , Up-RegulationABSTRACT
Protein kinase C encodes a family of enzymes implicated in cellular differentiation, growth control and tumor promotion. The generation and characterization of NIH-3T3 cells which stably overexpress the PKCeta isoform has been previously described by us. In these cells, overexpression of PKCeta altered the expression of specific cell cycle regulators and promoted differentiation [20]. Since PKC has been implicated in the regulation of gene expression, including that of various cytokines, we examined the production of several cytokines in these cells. We report here that out of the major pro-inflammatory cytokines examined, IL-1alpha, IL-1beta, TNF-alpha and IL-6, only IL-6 was generated and secreted in PKCeta -expressing cells without any additional inducer in serum-supplemented cultures (10% FCS). IL-6 was not detected in the control cell line, transfected with the same vector, but lacking the cDNA coding for PKCeta. Moreover, the production of IL-6 on serum stimulation correlated with the levels of PKCeta expressed in these cells. This implies that factors in the serum activate PKCeta and induce IL-6 production. We have examined several growth factors and cytokines for their ability to induce IL-6 production in our PKCeta-expressing cells. Among the growth factors tested (EGF, PDGF, FGF, insulin, IGF-1 and IL-1), PDGF and FGF were the most potent IL-6 inducers. The effects of FGF and PDGF on IL-6 production were blocked in the presence of PKC inhibitors. We also examined the signaling pathways that mediate production of IL-6 in PKCeta-expressing cells. Using specific inhibitors of the MAPK pathway, we have shown a role for ERK and p38 MAPK in FGF- and serum-stimulated IL-6 production, but only for p38 MAPK in PDGF-stimulated IL-6 production. Our studies provide evidence that PDGF and FGF can serve as upstream regulators of PKCeta and that PKCeta is involved in the expression of IL-6. This suggests that inhibition of PKC may provide a basis for the development of drugs for the treatment of disorders in which IL-6 is pathologically involved.