RelB Forms Transcriptionally Inactive Complexes with RelA/p65

Abstract

RelB is an unusual member of the NF-κB transcription factor family that acts as both a transcriptional activator as well as a repressor of NF-κB-dependent gene expression. Although RelB promotes gene expression when it associates with p50/NF-κB1 or p52/NF-κB2, the precise molecular mechanisms through which it represses NF-κB remain unclear. To examine this inhibitory function in more detail, we employed reporter gene assays and found that RelB represses at the level of RelA. Furthermore, electrophoretic mobility shift analysis revealed that in vitro translated RelB impaired the DNA binding activity of RelA and that overexpressed RelB significantly reduced tumor necrosis factor-α-induced RelA activity in murine embryonic fibroblasts. Intriguingly, this inhibitory effect was due to the formation of RelA·RelB heterodimers that were unable to bind to κB sites in vitro strongly suggesting that these newly described NF-κB dimers cannot bind DNA. Expression pattern analysis revealed that RelA·RelB heterodimers appeared at relatively low levels in both lymphoid and non-lymphoid cells. However, the presence of these complexes increased following stimulation with phorbolesters or lipopolysaccharide or by overexpression of constitutively active IKKβ. Functional characterization of RelA·RelB heterodimers in NIH3T3 murine embryonic fibroblasts revealed that they are not regulated by IκB proteins and are located in both the cytoplasm and the nucleus. Taken together, our findings demonstrate that sequestration of RelA in transcriptionally inactive RelA·RelB complexes provides a molecular mechanism that may explain the repressive role of RelB on NF-κB-dependent gene expression. RelB is an unusual member of the NF-κB transcription factor family that acts as both a transcriptional activator as well as a repressor of NF-κB-dependent gene expression. Although RelB promotes gene expression when it associates with p50/NF-κB1 or p52/NF-κB2, the precise molecular mechanisms through which it represses NF-κB remain unclear. To examine this inhibitory function in more detail, we employed reporter gene assays and found that RelB represses at the level of RelA. Furthermore, electrophoretic mobility shift analysis revealed that in vitro translated RelB impaired the DNA binding activity of RelA and that overexpressed RelB significantly reduced tumor necrosis factor-α-induced RelA activity in murine embryonic fibroblasts. Intriguingly, this inhibitory effect was due to the formation of RelA·RelB heterodimers that were unable to bind to κB sites in vitro strongly suggesting that these newly described NF-κB dimers cannot bind DNA. Expression pattern analysis revealed that RelA·RelB heterodimers appeared at relatively low levels in both lymphoid and non-lymphoid cells. However, the presence of these complexes increased following stimulation with phorbolesters or lipopolysaccharide or by overexpression of constitutively active IKKβ. Functional characterization of RelA·RelB heterodimers in NIH3T3 murine embryonic fibroblasts revealed that they are not regulated by IκB proteins and are located in both the cytoplasm and the nucleus. Taken together, our findings demonstrate that sequestration of RelA in transcriptionally inactive RelA·RelB complexes provides a molecular mechanism that may explain the repressive role of RelB on NF-κB-dependent gene expression. NF-κB describes a family of ubiquitously expressed transcription factors that play crucial roles in regulating innate and adaptive immunity, cell differentiation, and apoptosis (1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Abstract Full Text Full Text PDF PubMed Scopus (3329) Google Scholar). NF-κB is composed of a dimer of distinct members of the Rel protein family (RelA, c-Rel, RelB, p50, and p52), and in the majority of non-stimulated cells it is maintained inactive in the cytoplasm via association with members of a family of small inhibitory proteins named the IκBs. Following stimulation of cells with a wide variety of agents, including TNFα, 1The abbreviations used are: TNFα, tumor necrosis factor α; EMSA, electrophoretic mobility shift assay; IKK, IκB kinase, IκB; inhibitor of NF-κB; PMA, phorbol 12-mystrate 13-acetate; IB, immunoblot; IP, immunoprecipitation; LPS, lipopolysaccharide; RHD, Rel homology domain; MEF, murine embryonic fibroblast; Ab, antibody; PVDF, polyvi-nylidene difluoride; HA, hemagglutinin; IL-7, interleukin-7. LPS, or phorbol esters, IκB proteins become phosphorylated on two amino-terminal serine residues through the activity of a specific upstream kinase complex named the IκB kinase (IKK). This phosphorylation targets the IκB proteins for ubiquitination and degradation by the proteasomal pathway enabling the free NF-κB to translocate to the nucleus where it binds to specific DNA elements in the control regions of various genes and promotes their expression (2Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4657) Google Scholar). The generation of mice deficient for each Rel protein has revealed that the individual NF-κB family members have distinct, non-redundant functions. Furthermore, the separate combinations of NF-κB dimers recognize distinctive variants of the κB DNA binding site (3Kunsch C. Rosen C.A. Mol. Cell. Biol. 1993; 13: 6137-6146Crossref PubMed Google Scholar, 4Kunsch C. Ruben S.M. Rosen C.A. Mol. Cell. Biol. 1992; 12: 4412-4421Crossref PubMed Google Scholar). This modulatory design therefore contributes to the differential patterns of target gene expression induced by the NF-κB family of proteins. Classic NF-κB is considered to be a heterodimer of RelA and p50 that is efficiently complexed by IκB proteins and is thereby highly inducible by stimulation with agents such as TNFα, which transduce signals via the IKK complex. The importance of RelA is highlighted by the fact that it supports the expression of a wide variety of genes that include various leukocyte adhesion molecules, chemokines, effector enzymes, and certain IκB and NF-κB members (5Pahl H.L. Oncogene. 1999; 18: 6853-6866Crossref PubMed Scopus (3495) Google Scholar, 6Bren G.D. Solan N.J. Miyoshi H. Pennington K.N. Pobst L.J. Paya C.V. Oncogene. 2001; 20: 7722-7733Crossref PubMed Scopus (171) Google Scholar). Due to this widespread action RelA has been demonstrated to be of exceptional importance for the anti-apoptotic and pro-inflammatory roles of NF-κB. In contrast to RelA and the other NF-κB family members, RelB has been demonstrated to act as both a positive promoter of NF-κB-dependent gene expression and a repressor of NF-κB activity (7Ruben S.M. Klement J.F. Coleman T.A. Maher M. Chen C.H. Rosen C.A. Genes Dev. 1992; 6: 745-760Crossref PubMed Scopus (128) Google Scholar, 8Suhasini M. Pilz R.B. Oncogene. 1999; 18: 7360-7369Crossref PubMed Scopus (37) Google Scholar, 9Bours V. Azarenko V. Dejardin E. Siebenlist U. Oncogene. 1994; 9: 1699-1702PubMed Google Scholar). Moreover, RelB is only weakly controlled by IκB proteins and is the major contributor toward the constitutive NF-κB activity observed in lymphoid cells (10Lernbecher T. Kistler B. Wirth T. EMBO J. 1994; 13: 4060-4069Crossref PubMed Scopus (83) Google Scholar). Another specific feature of RelB is its limited ability to form complexes with the other Rel proteins such that it is unable to homodimerize and only heterodimerizes with p50 and p52, but not with RelA or c-Rel (36Dobrzanzki P. Ryseck R.P. Bravo R. EMBO J. 1994; 13: 4608-4616Crossref PubMed Scopus (106) Google Scholar). Although only a few RelB target genes have been reported, its positive transcriptional role is clearly manifested in studies using RelB-deficient mice. These mice are not only deficient for a thymic medulla and a subset of dendritic cells, but they also display defects in the development of secondary lymphatic organs suggesting a crucial role of RelB for these developmental processes (12Weih F. Carrasco D. Durham S.K. Barton D.S. Rizzo C.A. Ryseck R.P. Lira Bravo R. Cell. Full Text PDF PubMed Scopus Google Scholar). it has been demonstrated that heterodimers function in an NF-κB pathway that play a role in the of RelB-deficient mice. This pathway in the regulated through the action of the IKK of to form p52, which associates with RelB U. Chen Karin M. 2001; PubMed Scopus Google Scholar, M. EMBO J. 2001; 20: PubMed Scopus Google Scholar). However, the of mice also a repressive role for in RelB-deficient the expression of various and is and through a mechanism that is at in NF-κB Furthermore, the gene is active in but not in it has been that RelB an role in the gene to the of the in cells D. J. Google Scholar, Chen S. D. Mol. Cell. Biol. 1999; PubMed Google Scholar). Another for the repressive function of RelB is its ability to the and NF-κB activity in cells (7Ruben S.M. Klement J.F. Coleman T.A. Maher M. Chen C.H. Rosen C.A. Genes Dev. 1992; 6: 745-760Crossref PubMed Scopus (128) Google Scholar). we a of RelB in cells, a for of the or the positive RelB function for a cell R. F. E. M. Oncogene. 2001; 20: PubMed Scopus Google Scholar). In our we in the repressive effect of RelB on NF-κB found that RelB NF-κB activity by the activity of RelA and that the molecular mechanism this effect was of DNA binding by RelA through its complex formation with of the demonstrated that only the Rel homology are for this to other NF-κB dimers R.P. J. Bravo R. Mol. Cell. Biol. PubMed Scopus Google Scholar). we were unable to binding of the RelA·RelB heterodimer to κB sites the that RelA·RelB complexes are unable to bind to DNA. observed that RelA·RelB heterodimers in lymphoid as well as in non-lymphoid cells and are at in the non-lymphoid cells, controlled by or the gene is a target for the in RelB expression is by an in levels of RelA·RelB we using murine embryonic fibroblasts as a that levels of RelB protein to a in active RelA and that this is due to an in the formation of RelA·RelB and Expression following were used for and and and and were and the for the and and were RelB were by using the murine RelB as a The were in the and sites of the The and the p50 expression were as described H. S. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The is described M.J. R.B. S. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus (128) Google and the is control of the promoter in the reporter of and murine embryonic fibroblasts and and cells as well as cells were in cells, murine cells, and murine cells were in mice were to mice or mice with a constitutively active were as described E. M. S. 13: Full Text Full Text PDF PubMed Scopus Google Scholar). were by using a cell and the cells were with a to cells and In stimulation was used at a of and assays were using the and the reporter to the were in and at with In and in vitro of the expression was with of the in vitro transcription and and of The was for at and expression of the proteins of was by were to and DNA the of to of lymphoid and non-lymphoid cells were with in of with inhibitor The were by at for at and protein were R. M. S. C. B. E. J. Immunol. PubMed Scopus Google and assays and were as described M. T. S. B. M. E. EMBO J. PubMed Scopus Google Scholar). for were to protein and in the as a were and the were on for to the of a The were an on a with with and and in of were using to a and with the were also as described R. M. S. C. B. E. J. Immunol. PubMed Scopus Google using the κB which for RelB or the κB site the that RelA binding U. Chen Karin M. 2001; PubMed Scopus Google Scholar, H. S. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the assays of a specific was to each on The were for an to the RelB a on NF-κB-dependent is the only member of the family that both and NF-κB-dependent gene expression. is that RelB promotes NF-κB-dependent gene expression when complexed with p50 or p52, but the molecular mechanisms that its function remain unclear. To this function in detail, we cells with a reporter in the or presence of of an expression murine RelB the cells with or for in overexpression of RelB reduced NF-κB-dependent gene expression to of both the and and To this was due to with the function of other NF-κB we cells with expression for p50, or RelB, or in RelB not NF-κB activity when with p50 to a in activity that induced by p50 RelB overexpression reduced gene expression To that this effect was not due to reduced levels of expressed we these by using specific for the or the in the the levels of overexpressed RelA in the presence of of Furthermore, the fact that of RelB and p50 significantly increased the NF-κB activity the that the reduced RelA activity is on the sequestration of its complex p50 in RelB a with of RelA activity through with other transcription including or has been described Rev. 1999; 20: PubMed Google Scholar, J. 2002; PubMed Scopus Google Scholar, R. T. PubMed Scopus Google Scholar). association with the the DNA binding activity of it has been demonstrated that the ability of RelA to bind to DNA. To the molecular mechanism through which RelB RelA we an using a with in vitro translated of RelB and the Rel homology of RelA used this of RelA the protein binds to the κB binding site due to an H. S. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In by this in vitro we binding of NF-κB complexes to the κB In our both proteins were in vitro translated with the to with the κB RelB was unable to bind to DNA the DNA binding activity of the of was reduced by the of RelB of in vitro translated RelB protein reduced the DNA binding of but not to binding of an we that the RelA·RelB complex is unable to bind to the used in this To this impaired DNA binding of RelA was due to complex formation with RelB, we cells with expression RelA or RelB or in and analysis using cell these cells. in RelA was with the only in the presence of that RelA with RelB to form a complex. a reduced protein of both Rel proteins in this which is due to a of the expression of DNA in the various RelA and RelB we the for RelA·RelB complex formation and found that the of both proteins are for the association that this mechanism strongly the other Rel proteins. RelB RelA in that complex formation with RelB the DNA binding of RelA in we to this mechanism the of the proteins in therefore a murine embryonic cell as a as in the levels of p50, p52, and their and in these cells with control cells. therefore the DNA binding activity in control cells or the cells that were or with for and analysis revealed that overexpression of RelB the and and and and binding binding activity was low in control cells but increased strongly stimulation with for and In in cells we a constitutive activity of complexes that we as complexes and stimulation to a in the binding activity of only complex the complexes and and To the of these distinct κB we used control and the cells with for for analysis specific for RelB, p50, and in specific for both p50 and RelA reduced binding of the complex in control cells specific for RelB or effect In cells we a in complex binding using for RelA and more strongly with specific for RelB, p50, and The RelB also the binding of both complex and Furthermore, complex binding was by the These findings to that the inducible binding activity in control cells of in cells, complex is of a of and and complex a complex to a we were unable to other proteins in this complex. it that this a RelB RelB cannot bind to κB Taken together, the studies described that RelB overexpression the inducible binding activity of RelA through the formation of IκB and of RelA in studies using fibroblasts RelB-deficient mice that RelB IKK activity in cells with Chen S. D. Mol. Cell. Biol. 1999; PubMed Google Scholar). the that the in RelA activity we observed via degradation of IκB proteins due to reduced activity of the IKK complex. therefore analysis for IκB proteins using control cells or cells that were or for and with in we an inducible degradation of and in the control as well as the cells with Moreover, the protein levels of were control cells and RelB cells and we observed that was not as efficiently in cells and In to IκB of the Rel proteins is also for their transcriptional we analysis for RelA using control and cells. In control cells we a low level of which was increased following of stimulation and The of this the activity observed in overexpression of RelB to an in both the as well as the inducible levels of RelA levels of RelB were in RelB cells in to the control cells. In we a of the induced by in both the control cells as well as the cells The inducible degradation of and that IKK activity induced by is by RelB we not of inducible IKK activity by in vitro kinase assays not In the in RelA in to in RelB cells also that the of RelA is not by RelB therefore RelB with RelA and and RelB overexpression RelA activity and this through formation of RelA·RelB To this we cell of control and cells using an specific for RelA. were using to the of the not in analysis of the revealed that RelA·RelB heterodimers are in both control cells and at increased levels in RelB cells that the overexpressed RelB is and therefore in the of RelA·RelB in that RelA·RelB heterodimers in the control cell at low levels to a more characterization of these complexes using these cells. by the of RelB that was complexed with RelA with the levels with p50 or by analysis using specific for p50, and by with demonstrated in we a with each the of RelB that with p50 and was we RelB with RelA. To control for the level of we the using and as we a RelA RelB were in using not the p50 and with the in our we the level of each protein in with the to the of the and In of the fact that of RelA is efficiently controlled by and we to RelA·RelB heterodimers are by of these IκB proteins. with the we complex formation RelA and or In RelB not with IκB protein suggesting at in RelA·RelB heterodimers are not to control by or This is with the that the only IκB protein regulating RelB in fibroblasts is the protein N.J. Miyoshi H. G.D. Paya C.V. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus (171) Google Scholar). The of NF-κB proteins is controlled by their with such that NF-κB heterodimers that are not to are to the nucleus. To the of RelA·RelB heterodimers in we and and using an as not we found that the majority of RelA·RelB heterodimers were located in the a small was also located in the nucleus suggesting that the of RelA·RelB heterodimers is a regulated of IκB proteins. we also a small of heterodimers in the nucleus and a control for the of the we used an to and and NF-κB-dependent in RelA·RelB in mice display various defects in the development and of the F. H. Bravo R. J. Immunol. Google Scholar, P. Bravo R. F. J. PubMed Scopus Google Scholar, F. Durham S.K. Barton D.S. D. Bravo R. J. Immunol. Google and RelB an role in lymphoid cells where it is constitutively located in the nucleus (10Lernbecher T. Kistler B. Wirth T. EMBO J. 1994; 13: 4060-4069Crossref PubMed Scopus (83) Google Scholar). this importance of RelB in lymphoid cells, we and and and cell for the expression of RelA·RelB heterodimers and in each of these we were to RelA with RelB using a we also c-Rel association with RelB that in the To RelB expression in lymphoid cells, we used cell in a using and we were to RelA and RelB, that RelA·RelB dimers in lymphoid cells. RelB is constitutively located in the of cells, but its in cells with or or M. S. Kistler B. Wirth T. E. J. Immunol. Google Scholar, R. Immunol. 1998; PubMed Scopus Google Scholar). Furthermore, the of cells with a in RelB expression B. R. M. Wirth T. J. Immunol. 1998; Google Scholar). To an in the level of RelB expression with an in RelA·RelB heterodimer formation we cells of the cell with or for various with for to a of RelB protein levels in the by an and in RelB expression was also stimulation with for and in we a in the expression of RelA by analysis To the of RelB complexed with we with to a in the RelA·RelB heterodimer formation and In control with RelA RelB was not RelB is a NF-κB target gene and its expression is by cell stimulation in a NF-κB-dependent we the in RelA·RelB is NF-κB we RelB expression in mice with a overexpression of a constitutively active of in the that were to NF-κB activity and low levels of protein E. M. S. 13: Full Text Full Text PDF PubMed Scopus Google Scholar). in and RelB protein expression levels are in cells Moreover, in an using an Ab, we a in the RelA·RelB heterodimer formation with control mice and This strongly that increased RelA·RelB heterodimer formation in lymphoid cells is the induced activity of the IKK complex and the of NF-κB. The limited ability of RelB to form heterodimers with p50 or p52, but not with RelA or c-Rel, with its to homodimerize RelB as an unusual member of the NF-κB However, when with p50 or p52, RelB promotes the expression of a limited of NF-κB-dependent genes G.D. Solan N.J. Miyoshi H. Pennington K.N. Pobst L.J. Paya C.V. Oncogene. 2001; 20: 7722-7733Crossref PubMed Scopus (171) Google Scholar, R. U. S. PubMed Scopus Google Scholar). the importance of RelB when complexed with or its as the of an NF-κB which is on the activity of but not on the activity of the IKK pathway and U. Chen Karin M. 2001; PubMed Scopus Google Scholar). The role of RelB, is and the of this was to more the molecular mechanism this and described as an inhibitory Rel RelB NF-κB when overexpressed in cells. also an of NF-κB in reporter gene assays stimulation with or using cells. However, the level of the role of RelB as an inhibitor and positive effector of NF-κB. NF-κB activity overexpression of low of RelB but at the level when we used RelB This be due to the fact that RelB heterodimers with newly p50 and thereby to the reporter gene the inhibitory function is due to binding of RelA. was of individual proteins such that reporter gene expression was impaired by of RelB, of p50 and RelB a positive effect were observed in a the differential control of the promoter and the κB site in NIH3T3 cells by overexpression of the protein C. J. 2002; PubMed Scopus Google Scholar). was that protein which expression of the reporter but not the promoter controlled Furthermore, overexpression of RelB the expression of the gene induced by expressed RelA. Another molecular mechanism by which RelB the inducible NF-κB activity was in studies using RelB-deficient fibroblasts. In the of RelB the as well as the IKK activity and degradation were Chen S. D. Mol. Cell. Biol. 1999; PubMed Google Scholar). However, we were not to a role of RelB on by overexpression of RelB in murine embryonic fibroblasts Furthermore, the our reporter gene assays with NF-κB factors using cells with the DNA binding studies using in vitro translated RelA and RelB proteins or cells strongly that RelB the activity of RelA by its Moreover, our that of RelA activity through its sequestration in RelA·RelB To our this is the that RelB is a complex of RelA. may for the to RelA·RelB These are RelA·RelB heterodimers not bind efficiently to κB sites and the of RelA·RelB heterodimers is low with and However, of by a constitutively active or stimulation with an NF-κB a or LPS, to a in the RelA·RelB complex formation in cells and with these are the findings that the gene is regulated by NF-κB and its expression is induced by of the In a in RelB protein levels has been in cells and cells by or M. S. Kistler B. Wirth T. E. J. Immunol. Google Scholar, B. R. M. Wirth T. J. Immunol. 1998; Google Scholar). the function of a RelA·RelB heterodimer but we were unable to DNA binding of these complexes in we that they are transcriptionally inactive through a to with DNA. we described a degradation of RelB in cells with or and This degradation therefore RelA·RelB heterodimers and RelA p50 and as active NF-κB the that the function of heterodimer formation RelB and RelA is the of the RelA activity R. F. E. M. Oncogene. 2001; 20: PubMed Scopus Google Scholar). the low of RelA·RelB heterodimers and the of the IκB proteins to RelA this in the of binding sites for the distinct combinations of NF-κB family members have been described (3Kunsch C. Rosen C.A. Mol. Cell. Biol. 1993; 13: 6137-6146Crossref PubMed Google Scholar, 4Kunsch C. Ruben S.M. Rosen C.A. Mol. Cell. Biol. 1992; 12: 4412-4421Crossref PubMed Google it is that RelA·RelB heterodimers bind to a DNA separate the κB in the of the NF-κB factors with various such as the stimulation of cells with or R. Immunol. 1998; PubMed Scopus Google Scholar). in cells, active NF-κB is composed of RelB and c-Rel stimulation with both for RelA heterodimers are induced by a of active RelB in the of the only the DNA but not the of RelB, to be R. Immunol. 1998; PubMed Scopus Google Scholar). RelA·RelB heterodimers not to be controlled by the NF-κB or in fibroblasts This is with the that and to a are the only IκB proteins complexed with RelB in this cell N.J. Miyoshi H. G.D. Paya C.V. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus (171) Google Scholar). we found only a of RelA·RelB heterodimers in the to the of the of the of RelA·RelB heterodimers is The control of dimers in has been a of heterodimers are only weakly at controlled by IκB but the majority of heterodimers was found in the is RelB is complexed with of p50 in this or mechanism is for this The molecular mechanisms the RelB are and highly cell was therefore to both lymphoid as well as non-lymphoid cells. we RelA·RelB heterodimers in both cell However, RelB has been demonstrated to bind to IκB proteins in lymphoid cells, the control of RelA·RelB heterodimers be in lymphoid non-lymphoid cells. the expression of RelB is controlled by NF-κB G.D. Solan N.J. Miyoshi H. Pennington K.N. Pobst L.J. Paya C.V. Oncogene. 2001; 20: 7722-7733Crossref PubMed Scopus (171) Google and therefore an in the protein levels of RelB in a constitutively active of is not In increased RelB levels have been cell development B. R. M. Wirth T. J. Immunol. 1998; Google Scholar, M. H. C. P. E. E. E. PubMed Google Scholar). Taken together, our a feature of the family member RelB, which explain its repressive role on NF-κB other molecular mechanism such as of κB of p50 or in as well as through the to transcriptional also for the repressive role of our findings clearly demonstrate that complex formation RelB and RelA RelA the of NF-κB-dependent gene expression is such a of RelA in NF-κB to a of genes and therefore a mechanism to gene expression patterns an NF-κB-dependent