CREB-binding Protein Is a Nuclear Integrator of Nuclear Factor-κB and p53 Signaling
Transcriptional coactivators may function as nuclear integrators by coordinating diverse signaling events. Here we show that the p65 (RelA) component of nuclear factor-κB (NF-κB) and p53 mutually repress each other's ability to activate transcription. Additionally, tumor necrosis factor-activated NF-κB is inhibited by UV light-induced p53. Both p65 and p53 depend upon the coactivator CREB-binding protein (CBP) for maximal activity. Increased levels of the coactivator relieve p53-mediated repression of NF-κB activity and p65-mediated repression of p53-dependent gene expression. Nuclear competition for limiting amounts of CBP provides a novel mechanism for altering the balance between the expression of NF-κB-dependent proliferation or survival genes and p53-dependent genes involved in cell cycle arrest and apoptosis. Transcriptional coactivators may function as nuclear integrators by coordinating diverse signaling events. Here we show that the p65 (RelA) component of nuclear factor-κB (NF-κB) and p53 mutually repress each other's ability to activate transcription. Additionally, tumor necrosis factor-activated NF-κB is inhibited by UV light-induced p53. Both p65 and p53 depend upon the coactivator CREB-binding protein (CBP) for maximal activity. Increased levels of the coactivator relieve p53-mediated repression of NF-κB activity and p65-mediated repression of p53-dependent gene expression. Nuclear competition for limiting amounts of CBP provides a novel mechanism for altering the balance between the expression of NF-κB-dependent proliferation or survival genes and p53-dependent genes involved in cell cycle arrest and apoptosis. nuclear factor-κB chloramphenicol acetyltransferase CREB-binding protein cAMP-response element-binding protein tumor necrosis factor. Nuclear factor-κB (NF-κB)1 is an inducible transcription factor that plays an essential role in the regulation of gene expression in response to inflammatory stimuli (1Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4631) Google Scholar). It is composed of members of the Rel family (p50, p52, p65 (RelA), c-Rel, and RelB), which share a region of homology known as the Rel homology domain capable of directing DNA binding and mediating dimerization. In most cells, NF-κB is found in an inactive form in the cytoplasm bound to an inhibitory protein, IκB. In response to multiple activating signals, the inhibitor is degraded by the ubiquitin-proteasome complex, and NF-κB translocates to the nucleus and induces gene expression. NF-κB components can interact with other DNA binding proteins, as well as with a series of non-DNA-binding coactivator proteins. Among these interactions, the p65 component of NF-κB, like a variety of signal-dependent transcriptional activators, can associate with CREB-binding protein (CBP) or its structural homolog p300 (2Gerritsen M.E. Williams A.J. Neish A.S. Moore S. Shi Y. Collins T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2927-2932Crossref PubMed Scopus (717) Google Scholar,3Perkins N.D. Felzien L.K. Betts J.C. Leung K. Beach D.H. Nabel G.J. Science. 1997; 275: 523-527Crossref PubMed Scopus (666) Google Scholar). Activation of NF-κB is associated with resistance to programmed cell death (4Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2940) Google Scholar, 5Liu Z.G. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1784) Google Scholar, 6Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2452) Google Scholar, 7Wang C.Y. Mayo M.W. Baldwin A.S. 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The biological consequences of p53-mediated repression of gene expression are not fully understood. The divergent roles played by NF-κB and p53 suggest that there might be mechanisms that integrate the activities of these regulatory factors. Some of this control may be provided by the common dependence of both NF-κB and p53-dependent gene expression on limiting levels of transcriptional coactivators. Since transactivation by both p53 and p65 involves CBP, we investigated the role of this coactivator in the p53 and NF-κB signaling pathways. We find that mutual transrepression of these diverse signaling systems results, at least in part, from competition for a limiting amount of this versatile transcriptional coactivator. Interactions between the p65 and p53 signaling pathways mediated by CBP may be an important aspect of regulating the diverse changes in gene expression associated with cell survival. SaoS2 and COS-7 were obtained from the American Type Culture Collection (ATCC). ECV-304 cells were obtained from the European Collection of Animal Cell Cultures (ECACC, Salisbury, Wiltshire, United Kingdom). The cells were cultivated in Dulbecco's modified Eagle's medium from Life Technologies, Inc. supplemented with 10% fetal calf serum, 2 mml-glutamine, and antibiotics. Cells were grown on 10-cm2 dishes and cultured at 37 °C in a 5% CO2 incubator. The COS cells were cotransfected with −578 E-selectin promoter-CAT (26Read M.A. Whitley M.Z. Gupta S. Pierce J.W. Best J. Davis R.J. Collins T. J. Biol. Chem. 1997; 272: 2753-2761Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar) or a p21-promoter-luciferase reporter construct (p21-luciferase), along with expression vectors for human p53 (pC53-SN3), p65 (pcDNA p65), MDM2 (pCHDMIA), or CBP (pRc/RSV-mCBP-HA), as described in the figure legends. Whole cell extracts were prepared from the transfected cells and CAT or luciferase activity determined as described previously (2Gerritsen M.E. Williams A.J. Neish A.S. Moore S. Shi Y. Collins T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2927-2932Crossref PubMed Scopus (717) Google Scholar, 27Sheppard K.A. Phelps K.M. Williams A.J. Thanos D. Glass C.K. Rosenfeld M.G. Gerritsen M.E. Collins T. J. Biol. Chem. 1998; 273: 29291-29294Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). Transfected cells were treated with 100 units/ml TNFα (Endogene Sciences) for 30 min and then exposed to increasing amounts of ultraviolet light (10, 20, and 40 J/m2) using a Stratalinker (Stratagene) at 254 nm UV. Cells were harvested 4 h after exposure to UV light and whole cell extracts prepared. Aliquots from the cotransfection studies were saved prior to the freeze/thaw step in the CAT assay harvesting procedure. Cells were lysed in 5 × cell lysis buffer (Promega, Madison, WI) for 20 min on ice. Samples were centrifuged briefly, and equivalent amounts of protein were resuspended in SDS sample buffer, boiled for 2 min, and analyzed on 10% SDS-polyacrylamide gels. Proteins were electrophoretically transferred to nitrocellulose (Schleicher and Schuell), the membranes blocked with 5% non-fat dry milk in TBST buffer (containing 20 mm Tris-HCl, pH 7.6, 137 mm NaCl, 0.5% Tween 20), and incubated with either p65 or p53 (Rockland, Gilbertsville, PA and PharMingen, San Diego, CA, respectively) antisera for 16 h at 4 °C. Blots were washed three times with TBST buffer, incubated for 1 h with a secondary antibody conjugated to horseradish peroxidase (Amersham Pharmacia Biotech), and then washed three times in TBST. The antigen antibody interactions were visualized by incubation with ECL chemiluminescence reagent (Amersham Pharmacia Biotech). Blots were exposed to x-ray film for 10 s to 10 min. If CBP functions as a signal integrator for the NF-κB and p53 pathways, there might be mutual transcriptional interference between these two signal-dependent activators. To determine whether the p65 component of NF-κB alters p53 function, a p53 reporter plasmid containing the p21 promoter, a known p53 target gene associated with arrest of the cell cycle, was cotransfected with a fixed amount of p53 and increasing amounts of a p65 expression plasmid. As expected, p53 strongly activates the p21 promoter (Fig.1 A, lanes 2 and3). Cotransfection of p65 resulted in a dose-dependent suppression of this reporter plasmid in SaoS2 cells (Fig. 1 A, lanes 5–8) or in COS cells (Fig. 1 B, lanes 3–5). p65-mediated suppression was also seen with a Bax promoter-reporter, a gene that is associated with the induction of apoptosis, as well as an artificial promoter containing only multiple p53 binding sites (data not shown). In contrast to the p53-dependent genes, no repression of a Gal4-dependent promoter-reporter gene was seen (data not shown). Control studies also demonstrate that overexpressed p65 did not decrease production of p53 from the corresponding expression construct (Fig. 1, A and B, insets). To determine whether p53 could alter NF-κB-dependent gene expression, similar studies were done with a NF-κB-dependent E-selectin promoter-reporter construct (2Gerritsen M.E. Williams A.J. Neish A.S. Moore S. Shi Y. Collins T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2927-2932Crossref PubMed Scopus (717) Google Scholar, 28Whitley M.Z. Thanos D. Read M.A. Maniatis T. Collins T. Mol. Cell. Biol. 1994; 14: 6464-6467Crossref PubMed Scopus (177) Google Scholar), a fixed amount of p65, and increasing amounts of p53. As predicted, p65 results in an induction of the NF-κB-dependent reporter gene (Fig. 1 C,lane 2). Increasing amounts of p53 resulted in a dose-dependent suppression of p65-mediated transactivation (Fig. 1 C, lanes 3–6). This effect was reversed by MDM2 (Fig. 1 C, lanes 7–9), an inhibitor of p53 transactivation. Control studies demonstrated that overexpression of MDM2 did not alter levels of p65 (Fig. 1 C,insets), although in some experiments it modestly increased p65-dependent gene expression (Fig. 1 C,lanes 7–9). MDM2 does not physically interact with p65 (data not shown). The presence of p53 had no effect on nuclear accumulation of NF-κB or DNA binding activity, and it did not physically interact with p65 (data not shown). Additionally, p53 did not increase expression of an inhibitor of NF-κB, IκB-α (data not shown). Thus p65-mediated transcriptional activation is repressed by p53, and p53-dependent gene expression is repressed by p65. One possibility suggested by the preceding findings is that the formation of complexes between either p65 or p53 and specific coactivators would reduce the amount of coactivator available for transcriptional activation. If competition for limiting amounts of CBP accounts for the inhibitory effect of p53, then increased levels of the coactivators should restore, or rescue, p65-dependent gene expression. Indeed, the inhibitory effect of p65 on p53-dependent gene expression was completely abolished by cotransfection of a vector expressing CBP (Fig.1 B, lane 6). In a similar manner, the suppression of p53 on p65-dependent gene expression was significantly decreased by CBP (Fig. 1 C, lanes 10–13). Control studies demonstrated that CBP overexpression did not alter levels of either p53 (Fig. 1, B and C, insets) or p65 (Fig. 1 C, inset). Additionally, overexpression of an irrelevant transcriptional activator, or mutated forms of CBP, did not result in rescue (data not shown). Collectively, these functional studies demonstrate that CBP is limiting for both p65- and p53-dependent transactivation and suggest that CBP can rescue the mutually repressive interaction between the two activators. The mutual transrepression of p53- and p65-dependent gene expression described above was observed with over expressed activators and might not reflect the situation with authentic levels of these transcription factors. To address this important issue, we determined whether TNFα-activated NF-κB was capable of inhibiting endogenous p53-mediated gene expression. Endothelial cells transfected with an E-selectin promoter-reporter were treated with TNFα and exposed to increasing amounts of UV irradiation. As expected, TNFα activated expression of the E-selectin promoter-reporter construct (Fig.2 A, lane 2). UV illumination resulted in a dose-dependent suppression of this activity (Fig. 2 A, lanes 3–5). Cotransfection of an MDM2 expression plasmid, while significantly inhibiting a p53 transcriptional response and not increased expression from the NF-κB-dependent reporter gene (Fig.2 A, lane 6). This that the transcriptional activating of endogenous p53 is for the suppression of the E-selectin promoter-reporter The inhibitory effect of endogenous p53 on NF-κB-dependent gene expression was completely abolished by cotransfection of a vector expressing CBP (Fig. 2 A, lane In control levels of activity were in with the activity of the reporter In contrast to the results with the NF-κB-dependent the UV cotransfection with either MDM2 or CBP the of activity (Fig. 2 These results are with the possibility that the coactivator endogenous NF-κB-dependent gene NF-κB activity is following irradiation. A, UV exposure induces expression of a p53-dependent reporter ECV-304 cells were cotransfected with 2 of a p21-promoter-luciferase construct in the presence or of 1 of an MDM2 expression cells were exposed to 40 UV ultraviolet light and cells were harvested at the times following B, NF-κB activity by UV is by MDM2. ECV-304 cells were transfected with 2 of an E-selectin promoter-CAT reporter construct in the presence or of 1 of an MDM2 expression Transfected cells were treated with UV as described above and harvested at the times The results are of three The findings described above that endogenous NF-κB and p53 are would each other's transcriptional activity. UV activates both transcription K. M. S. EMBO J. 1998; PubMed Scopus Google Scholar), we it as a to the effect of the signaling pathways on each UV exposure strongly induces expression of a p21 promoter-reporter construct (Fig. in a In UV also activated expression of an E-selectin promoter-reporter gene Cotransfection of an MDM2 expression plasmid blocked the p53 transcriptional response (Fig. while increasing expression of the NF-κB-dependent reporter plasmid These results are with the that the transcriptional activity of endogenous NF-κB is regulated by p53. these studies we suggest that between the p53 and NF-κB signaling is mediated by These findings are with that the levels of p300 are limiting to of p65 Felzien L.K. Nabel G.J. EMBO J. 1998; PubMed Scopus Google Scholar) and that are to the gene of either CBP or its p300 M. D. Bronson R.T. Livingston D.M. R. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The common dependence of both p65 and p53 on that the coactivators may integrate multiple signaling pathways that on these transcription factors. p53 inhibits transcription, and overexpression of p300 abolished the ability of p53 to activity (18Avantaggiati M.L. Ogryzko V. Garner K. Giordano A. Levine A.S. Kelly K. Cell. 1997; 89: 1175-1184Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar). was observed between p53 and through the coactivator p300 1998; Scopus Google Scholar). NF-κB-dependent gene expression is also regulated by coactivator The p65 component of NF-κB and the mutually repress each other's ability to activate transcription A. J.A. Baldwin Jr., A.S. Mol. Cell. Biol. 1995; 15: PubMed Google Scholar). Increased levels of CBP relieve the inhibition of repression of NF-κB activity and the NF-κB mediated repression of activity K.A. Phelps K.M. Williams A.J. Thanos D. Glass C.K. Rosenfeld M.G. Gerritsen M.E. Collins T. J. Biol. Chem. 1998; 273: 29291-29294Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). The results described that NF-κB and p53 mutually repress each other's transcription, not only in also in cells, and suggest that these signaling systems on the coactivators. Collectively, these studies are with that coactivator can function as a mechanism to determine of gene expression Y. T. J. R. B. Glass C.K. Rosenfeld M.G. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). This control may be important in the of cells in which both NF-κB and p53 are In NF-κB is as a survival NF-κB would induce genes that promote resistance to apoptosis and cell through coactivator NF-κB would also suppress ability to stimulate genes involved in cell such as Bax T. J.C. Cell. 1998; Scholar), which might the survival In the of cell p53 would activate expression of genes involved in apoptosis and suppress the survival signals by NF-κB-dependent gene expression. This control could be formation of Trends Genet. 1998; 14: Full Text Full Text PDF PubMed Scopus Google Scholar). to this regulation is CBP can integrate the functions of these diverse transcriptional activators. The in which the transcription are in the various target gene may determine ability to for the coactivators and activate transcription M. Williams A.J. G. Collins T. Thanos D. Mol. Cell. 1998; 1: Full Text Full Text PDF Scopus Google Scholar). Additionally, proteins, such as may a series of coactivator complexes Genet. Dev. 1998; PubMed Scopus Google Scholar). These could be by specific of transcription limiting between signaling pathways. The p53 expression vector and a p21 reporter construct were provided by B. Vogelstein The human MDM2 expression vector was provided by A. Levine a CBP expression vector was provided by R. and a Bax promoter-reporter construct was provided by J.