From the
Section of Immunobiology and Department of Molecular Biophysics and Biochemistry, Yale University Medical School, New Haven, Connecticut 06520, the ¶Institute of Virology and Immunobiology and ||Department of Molecular Pathology, Institute of Pathology, University of Würzburg, Josef Schneider Strasse 2, Würzburg 97080, Germany, and the **Institute for Experimental Internal Medicine, University of Magdeburg, Leipziger Strasse 44, Magdeburg 39120, Germany
Received for publication, February 24, 2003 , and in revised form, March 25, 2003.
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ABSTRACT |
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INTRODUCTION |
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The generation of mice deficient for each Rel protein has revealed that the individual NF-B family members have distinct, non-redundant functions. Furthermore, the separate combinations of NF-
B dimers recognize distinctive variants of the
B DNA binding site (3, 4). This modulatory design therefore contributes to the differential patterns of target gene expression induced by the NF-
B family of proteins. Classic NF-
B is considered to be a heterodimer of RelA and p50 that is efficiently complexed by I
B proteins and is thereby highly inducible by stimulation with agents such as TNF
, 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 I
B and NF-
B members (5, 6). Due to this widespread action RelA has been demonstrated to be of exceptional importance for the anti-apoptotic and pro-inflammatory roles of NF-
B.
In contrast to RelA and the other NF-B family members, RelB has been demonstrated to act as both a positive promoter of NF-
B-dependent gene expression and a repressor of NF-
B activity (7, 8, 9). Moreover, RelB is only weakly controlled by I
B proteins and is the major contributor toward the constitutive NF-
B activity observed in lymphoid cells (10). 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 (36). 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 (12).
Recently, it has been demonstrated that RelB·p52 heterodimers function in an alternative NF-B activation pathway that might play a central role in the phenotype of RelB-deficient mice. This pathway culminates in the regulated processing through the action of the IKK subunit IKK
of p100 to form p52, which then associates predominantly with RelB (13, 14). However, the phenotype of RelB/ mice also indicates a repressive role for RelB. Thus, in RelB-deficient fibroblasts, the expression of various chemokines and cytokines is augmented and prolonged through a mechanism that is at least in part, dependent upon IKK-induced NF-
B activity. Furthermore, because the TNF
gene is active in RelB/ but not in wild type fibroblasts, it has been suggested that RelB plays an epigenetic role in silencing the TNF
gene similar to the reported RelB-specific demethylation of the Ig
locus in B cells (15, 16). Another example for the repressive function of RelB is its ability to impair the combined phorbolester and ionomycin-induced NF-
B activity in T cells (7). Recently, we reported a signal-induced proteolysis of RelB in activated T cells, implying a necessity for removal of either the negative or the positive RelB function for a proper T cell activation response (17).
In our current study we analyzed in greater detail the repressive effect of RelB on NF-B activity. We found that RelB reduces NF-
B activity by directly influencing the activity of RelA and that the molecular mechanism underlying this negative effect was inhibition of DNA binding by RelA through its complex formation with RelB. Mapping of the interaction domains demonstrated that only the Rel homology domains (RHDs) are necessary for this interaction similar to all other NF-
B dimers (11). More importantly, we were unable to detect binding of the RelA·RelB heterodimer to known
B sites raising the possibility that endogenous RelA·RelB complexes are unable to bind to DNA. We further observed that RelA·RelB heterodimers exist in lymphoid as well as in non-lymphoid cells and are not, at least in the non-lymphoid cells, controlled by I
B
or I
B
. Because the relb gene itself is a target for NF-
B, the increase in RelB expression is paralleled by an increase in levels of RelA·RelB heterodimers. Finally, we show using murine embryonic fibroblasts (MEFs) as a model system, that high levels of RelB protein lead to a drastic reduction in active RelA and that this reduction is due to an increase in the formation of RelA·RelB heterodimers.
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EXPERIMENTAL PROCEDURES |
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Preparation of Lymphocytes, Cell Culture, and Reporter Gene AssaysWild type murine embryonic fibroblasts and human Raji and Daudi B cells as well as human 293 HEK cells were cultured in Dulbecco's modified Eagle's medium plus 10% fetal calf serum. Human Jurkat T leukemic cells, murine 70Z/3 pre B cells, and murine EL-4 thymoma cells were cultured in RPMI medium containing 5% fetal calf serum. Murine splenocytes from Balb/c mice were prepared according to standard protocols. Thymocytes from wild type mice or mice with a tissue-specific constitutively active IKK transgene were prepared as previously described (20). Briefly, prepared thymocytes were disrupted by using a cell strainer (Falcon), and the resulting cells were pelleted, washed twice with cold phosphate-buffered saline then loaded onto a Ficoll gradient to remove dead cells and erythrocytes. In all cytokine stimulation experiments, recombinant hTNF
(R&D systems) was used at a concentration of 10 ng/ml. Transient transfections and luciferase assays were performed using the Fugen6 transfection reagent (Roche Diagnostics) and the dual luciferase reporter assay system (Promega), respectively, according to the manufacturer's protocols. All experiments were performed in parallel and repeated at least three times with similar results.
In Vitro Transcription and TranslationFor in vitro translation, 1 µg of the appropriate expression vector was mixed with 20 µl of rabbit reticulocyte lysate from the TNTT7 Quick in vitro transcription and translation system (Promega) and 1 µl of 35S-labeled methionine. The reaction was incubated for 30 min at 30 °C, and successful expression of the proteins of interest was analyzed by SDS-PAGE, then exposures were made to x-ray film.
Immunoblots, Immunoprecipitations, and DNA Binding StudiesFor the preparation of whole cellular extracts 5 x 106 to 1 x 107 of lymphoid and non-lymphoid cells were pelleted, washed once with ice-cold phosphate-buffered saline, then resuspended in 200 µl of TNT buffer (200 mM Tris-HCl, pH 8.0, 20 mM NaCl, 1% Triton X-100, 1 mM dithiothreitol) supplemented with protease inhibitors (Complete protease inhibitor mixture, Roche Applied Science). The extracts were cleared by centrifugation at 14,000 rpm for 10 min at 4 °C. Nuclear and cytoplasmic protein extracts were prepared (21), and immunoblot (IB) assays and immunoprecipitations (IPs) were performed as previously described (22). Briefly, for IP experiments samples were equalized to contain identical protein concentrations (between 500 and 1000 µg/sample) in the TNT buffer. Specific antibodies (2 µg per sample) or, as a control, non-immune serum were added, and the samples were incubated on ice for 1 h prior to the addition of 30 µl a 50% Protein-A-Sepharose slurry. The samples were then incubated an additional 412 h on a rotor, washed extensively with TNT lysis buffer supplemented with protease and phosphatase inhibitors, and then boiled in 25 µl of SDS-sample buffer. Immunocomplexes were separated using SDS-PAGE (8%), transferred to a PVDF membrane, and then blotted with the appropriate antibodies. EMSAs were also performed as previously described (21) using either the palindromic B site, which allows for RelB binding, or the
B site from the Ig
enhancer that favors RelA binding (13, 18). For the supershift assays 1 µg of a specific antibody was added to each protein-DNA mixture after 10-min incubation on ice. The samples were incubated for an additional 10 min prior to loading onto the gels.
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RESULTS |
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RelB Forms a Complex with RelAInhibition of RelA activity through interaction with other transcription factors, including glucocorticoid receptors, p53, or c-Myc has been previously described (23, 24, 25). Whereas association with the glucocorticoid receptors maintains the DNA binding activity of RelA, it has been demonstrated that p53 hinders the ability of RelA to bind to DNA. To analyze the molecular mechanism through which RelB inhibits RelA activity, we performed an EMSA experiment using a radiolabeled B-oligonucleotide together with in vitro translated versions of full-length RelB and the Rel homology domain (RHD) of RelA (RelA
313). We used this deletion mutant of RelA because the full-length protein binds poorly to the
B binding site due to an intramolecular interaction (18). In addition, by employing this in vitro assay system we avoided additional binding of other, endogenous NF-
B complexes to the
B site. In our experiments, both proteins were in vitro translated separately then mixed together with the indicated relative amounts prior to incubation with the radiolabeled
B probe. As expected, RelB alone was unable to bind to DNA (Fig. 2A; lane 6), whereas the robust DNA binding activity of the homodimer of RelA
313 (RelA
313(2)) was dose-dependently reduced by the addition of RelB (lanes 25). Because addition of in vitro translated RelB protein reduced the DNA binding of RelA
313 but did not lead to binding of an additional, slower migrating band (Fig. 2A), we conclude that the RelA·RelB complex is unable to bind to the
B-oligonucleotide used in this assay.
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To determine whether this impaired DNA binding of RelA was due to complex formation with RelB, we transiently transfected 293 HEK cells with expression vectors encoding HA-tagged RelA or FLAG-tagged RelB either individually or in combination and performed immunoprecipitation (IP) analysis using whole cell extracts prepared from these cells. As shown in Fig. 2B, HA-tagged RelA was immunoprecipitated with the FLAG-specific antibody only in the presence of cotransfected FLAG-RelB (lane 4) demonstrating that RelA interacts with RelB to form a complex. We noticed a reduced protein concentration of both ectopic-expressed Rel proteins in this particular experiment, which is probably due to a mutual repression of the expression caused high amounts of plasmid DNA in the transfection. Using various RelA and RelB deletion mutants, we mapped the interaction domains responsible for RelA·RelB complex formation and found that the RHDs of both proteins are required for the association (Fig. 2C), hence, indicating that this interaction mechanism strongly resembles the classic heterodimerization between other Rel proteins.
Overexpressed RelB Impairs TNF-induced RelA Activity in FibroblastsHaving established that complex formation with RelB blocks the DNA binding capacity of RelA in vitro, we wished to determine whether this mechanism regulates the activities of the endogenous proteins in vivo. We therefore established a stable RelB-overexpressing murine embryonic fibroblast cell line as a model system, and, as shown in Fig. 3A, the levels of RelA, p50, p52, and their precursors p100 and p105, respectively, remained unaltered in these cells compared with control cells. We therefore compared the
B-specific DNA binding activity in nuclear extracts from either control cells or the RelB-overexpressing cells that were either untreated or stimulated with TNF
for 20 and 60 min. EMSA analysis revealed that overexpression of RelB dramatically altered the basal (Fig. 3B, compare lanes 1 and 4) and TNF
-induced (lanes 2 and 3 versus lanes 5 and 6)
B-specific binding complexes. Thus, basal binding activity was very low in untreated control cells but increased strongly after stimulation with TNF
for 20 min (complex C1') and decreased thereafter (Fig.3B, lanes 13). In contrast, in RelB-overexpressing cells we detected a strong constitutive
B-binding activity of multiple complexes that we designated as complexes C1, C2, and C3 (Fig. 3B, lane 4). Furthermore TNF
stimulation led to a slight increase in the binding activity of only complex C1, whereas the complexes C2 and C3 remained unaltered (Fig. 3B, lanes 5 and 6). To determine the composition of these distinct
B complexes, we used nuclear extracts from control and the RelB-overexpressing cells stimulated with TNF
for 20 min for supershift analysis employing antibodies specific for RelA, RelB, p50, and p52. As shown in Fig. 3C, antibodies specific for both p50 and RelA reduced binding of the complex C1' in control cells extracts, whereas antibodies specific for RelB or p52 had no effect (Fig. 3C, lanes 15). In extracts from RelB-overexpressing cells we detected a partial reduction in complex C1 binding using antibodies for RelA and more strongly with antibodies specific for RelB, p50, and p52 (Fig. 3C, lanes 610). The RelB antibody also affected the binding of both complex C2 and C3. Furthermore, complex C2 binding was blocked by the p52 antibody. These findings led us to conclude that the strong inducible binding activity (complex C1') in control cells consists of RelA·p50 heterodimers, whereas in RelB-overexpressing cells, complex C1 is comprised of a mixture of RelA·p50 and RelB·p50 heterodimers, and complex C2 represents a RelB·p52 heterodimer. Interestingly, complex C3 appears to represent a slower migrating RelB-containing complex, although we were unable to precisely identify any other proteins in this complex. Nevertheless, it seems unlikely that this represents a RelB homodimer, because RelB alone cannot bind to
B sites. Taken together, the studies described above reveal that RelB overexpression dramatically reduces the inducible binding activity of RelA through the formation of heterodimeric complexes.
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Efficient IB Degradation and Nuclear Translocation of RelA in RelB-overexpressing FibroblastsPrevious studies using fibroblasts from RelB-deficient mice implied that RelB might negatively regulate IKK activity in cells stimulated with LPS (16). Hence, the possibility remained that the reduction in RelA activity we observed occurs via inefficient degradation of I
B proteins due to reduced activity of the IKK complex. We therefore performed immunoblot analysis for I
B proteins using cytoplasmic extracts from control cells or RelB-overexpressing cells that were either left untreated or stimulated for 20 and 60 min with TNF
. As shown in Fig. 4A, we detected an effective inducible degradation of I
B
and I
B
in the control as well as the RelB-overexpressing cells (compare lanes 13 with lanes 46). Moreover, although the basal protein levels of I
B
were comparable between control cells and RelB cells (Fig. 4A, lanes 1 and 4) we consistently observed that I
B
was not resynthesized as efficiently in RelB-overexpressing cells (compare lanes 3 and 6).
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In addition to IB degradation, nuclear translocation of the Rel proteins is also critical for their transcriptional activity. Therefore, we performed immunoblot analysis for RelA using nuclear extracts from control and RelB-overexpressing cells. In control cells we detected a low basal level of nuclear RelA, which was increased following 20 min of TNF
stimulation and declined thereafter (Fig. 4B, lanes 13). The time course of this nuclear localization reflected the DNA-binding activity observed in Fig. 3B. Stable overexpression of RelB led to an increase in both the basal as well as the inducible levels of nuclear RelA (Fig. 4B, lanes 46). Not surprisingly, higher levels of nuclear RelB were detected in RelB cells in comparison to the control cells. In addition, we detected a slight increase of the nuclear localization induced by TNF
in both the control cells as well as the RelB-overexpressing cells (Fig. 4B, lower panel). The effective inducible degradation of I
B
and I
B
(Fig. 4A) suggests that IKK activity induced by TNF
is unaffected by RelB overexpression; indeed, we did not detect significant reduction of inducible IKK activity by in vitro kinase assays (data not shown). In addition, the augmented nuclear translocation in RelA in response to TNF
seen in RelB cells also suggests that the mobilization of RelA is not affected by RelB overexpression. We therefore reasoned that, because RelB can associate with RelA (Fig. 2, B and C) and RelB overexpression inhibits RelA activity (Figs. 1 and 2C), this might occur through formation of RelA·RelB heterodimers. To investigate this possibility we performed immunoprecipitations from whole cell extracts of control and RelB-overexpressing cells using an antibody specific for RelA. As control, similar IP experiments were performed using non-immune serum to ensure the specificity of the interaction (data not shown). As shown in Fig. 4C, immunoblot analysis of the resulting immunoprecipitated material revealed that RelA·RelB heterodimers are present in both control cells and at substantially increased levels in RelB overexpressing cells (Fig. 4C). Note that the ectopic overexpressed RelB is FLAG-tagged and therefore migrates slower in the polyacrylamide gel (upper arrow).
Characterization of RelA·RelB Heterodimers in FibroblastsThe finding that endogenous RelA·RelB heterodimers exist in the control cell line albeit at low levels (Fig. 4C, lane 3), prompted us to perform a more detailed characterization of these complexes using these cells. We began by determining the relative amount of RelB that was complexed with RelA compared with the levels associated with either p50 or p52 by performing coimmunoprecipitation analysis using antibodies specific for RelA, p50, and p52 followed by immunoblotting with anti-RelB. As demonstrated in Fig. 5A we detected a RelB-specific signal after immunoprecipitation with each antibody. Nevertheless, although the amount of RelB that coimmunoprecipitated with p50 and p52 antibodies was equally strong, we consistently detected less RelB associated with RelA. To control for the level of immunoprecipitated RelA, we reprobed the membrane using anti-RelA, and as expected, we detected a strong RelA-specific signal (Fig. 5A, lower panel). Neither RelA nor RelB were detected in similar experiments using non-immune serum (data not shown). Because the p50 and p52 bands overlapped with the Ig heavy chain signal in our immunoblots, we compared the level of each protein in pre-IP samples with the immunodepleted extracts to ensure the success of the IP (lanes 4 and 5).
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In light of the fact that activation of RelA is efficiently controlled by IB
and I
B
, we sought to determine whether RelA·RelB heterodimers are bound by either of these I
B proteins. Consistent with the previous reports we detected efficient complex formation between RelA and either I
B
or I
B
(Fig. 5B). In contrast, RelB did not coimmunoprecipitate with antibodies against either I
B protein suggesting that, at least in fibroblasts, RelA·RelB heterodimers are not subject to control by I
B
or I
B
. This finding is consistent with the previous observations that the only I
B protein regulating RelB in fibroblasts is the NF-
B2/p100 protein (26). The subcellular localization of NF-
B proteins is mainly controlled by their interaction with I
Bs such that NF-
B heterodimers that are not bound to I
Bs are able to enter the nucleus. To analyze the subcellular distribution of RelA·RelB heterodimers in unstimulated fibroblasts, we prepared nuclear and cytoplasmic extracts from MEFs and performed IP experiments using an anti-RelA antibody or, as control, non-immune serum (data not shown). Surprisingly, we found that the majority of RelA·RelB heterodimers were located in the cytoplasm, although a small fraction was also located in the nucleus (Fig. 5C) suggesting that the nuclear translocation of RelA·RelB heterodimers is a regulated process, possibly independent of I
B proteins. Interestingly, we also detected a small amount of RelA·p50 heterodimers in the nucleus (Fig. 5C, lanes 1 and 2). As a control for the purity of the nuclear extracts, we used an I
B
-specific antibody to immunoblot nuclear and cytoplasmic extracts (lanes 3 and 4).
NF-B-dependent Increase in RelA·RelB Heterodimer Formation in Lymphoid CellsRelB/ mice display various defects in the development and regulation of the immune system (27, 28, 29), and RelB plays an important role in lymphoid cells where it is constitutively located in the nucleus (10). Given this importance of RelB in lymphoid cells, we analyzed different B (Daudi and Raji) and T (Jurkat and EL-4) cell lines for the expression of RelA·RelB heterodimers and in each of these we were able to coimmunoprecipitate RelA with RelB using a RelB-antibody. Interestingly, we also detected c-Rel association with RelB that occurred predominantly in the B-cell lines (Fig. 6A, middle panel). To investigate RelB expression in primary lymphoid cells, we used whole cell extracts from splenocytes in a similar IP experiment using RelA- and RelB-specific Abs (Fig. 6B). Once again we were able to coimmunoprecipitate RelA and RelB, demonstrating that RelA·RelB dimers exist in primary lymphoid cells.
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RelB is constitutively located in the nuclei of mature B cells, but its nuclear concentration increases in B cells stimulated with CD40 or LPS or anti-Ig (32, 35). Furthermore, the differentiation of pre B cells after IL-7 withdrawal correlates with a strong increase in RelB expression (33). To determine whether an increase in the level of RelB expression correlates with an increase in RelA·RelB heterodimer formation we stimulated cells of the pre B cell line 70Z/3 with PMA or LPS for various times. Stimulation with PMA for 1 h led to a temporary drop of RelB protein levels in the pre-immunoprecipitation samples followed by an increase after 8 and 24 h (Fig. 7A, lower panels). An increase in RelB expression was also detected after stimulation with LPS for 8 and 24 h and, in addition, we detected a slight increase in the expression of RelA by immunoblot analysis (Fig. 7A, lower panel). To determine the amount of RelB complexed with RelA, we performed immunoprecipitation experiments with anti-RelA-specific antibodies (Fig. 7A, upper part). Both stimuli led to a significant increase in the RelA·RelB heterodimer formation after 8 and 24 h. In control IP experiments with non-immune serum neither RelA nor RelB was detected (data not shown). Because RelB is a NF-B target gene and its expression is up-regulated by cell stimulation in a NF-
B-dependent fashion, we directly tested whether the increase in RelA·RelB heterodimerization is dependent upon NF-
B activation. Hence, we examined RelB expression in transgenic mice with a tissue-specific overexpression of a constitutively active isoform of IKK
(IKK
CA) in the thymus that were reported to exhibit high basal NF-
B activity and low levels of I
B
protein (20). As shown in Fig. 7B (lanes 1 and 2), RelB protein expression levels are higher in IKK
CA transgenic cells than wild type thymocytes. Moreover, in an IP experiment using an RelA-specific Ab, we detected a significant increase in the RelA·RelB heterodimer formation compared with thymocytes from control mice (Fig. 6D, compare lanes 3 and 4). This finding strongly suggests, therefore, that increased RelA·RelB heterodimer formation in lymphoid cells is dependent upon the induced activity of the IKK complex and the subsequent activation of NF-
B.
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DISCUSSION |
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Initially cloned and described as human I-Rel, an inhibitory Rel protein, RelB inhibits phorbolester-induced NF-B activation when overexpressed in Jurkat T cells. We also detected an inhibition of NF-
B in reporter gene assays after stimulation with TNF
or phorbolester using 293 HEK cells. However, the level of inhibition reflected the dual role of RelB as an inhibitor and positive effector of NF-
B. Thus, NF-
B activity declined after overexpression of low amounts of RelB but stayed at the same level when we used higher RelB concentrations (Fig. 1A). This might be due to the fact that RelB formed heterodimers with newly synthesized p50 and thereby contributed positively to the reporter gene expression, whereas the inhibitory function is due to binding of RelA. A clearer result was obtained upon transient transfection of individual NF-
B/Rel proteins such that high RelA-induced reporter gene expression was impaired by addition of RelB, whereas cotransfection of p50 and RelB had a positive effect (Fig. 1B). Similar results were observed in a previous study analyzing the differential control of the Bcl-x(L) promoter and the c-myc
B site in NIH3T3 cells by overexpression of the cytomegalovirus immediate-early protein 1 (31). It was reported that immediate-early protein 1 specifically activated RelB·p50 heterodimers, which promoted expression of the c-myc
B-site-driven reporter construct but not the Bcl-x(L) promoter controlled reporter. Furthermore, overexpression of RelB inhibited the expression of the Bcl-x(L) gene induced by exogenously expressed RelA.
Another potential molecular mechanism by which RelB might affect the inducible NF-B activity was identified in studies using RelB-deficient fibroblasts. In the absence of RelB the basal as well as the LPS-induced IKK activity and consequently I
B
degradation were augmented (16). However, we were not able to detect a negative role of RelB on TNF
-induced I
B-degradation by overexpression of RelB in murine embryonic fibroblasts (Fig. 4). Furthermore, the results from our reporter gene assays with cotransfected NF-
B factors using 293 HEK cells together with the DNA binding studies using either in vitro translated RelA and RelB proteins or nuclear extracts from RelB-overexpressing cells strongly suggest that RelB inhibits the activity of RelA by affecting its DNA-binding activity. Moreover, our results imply that inhibition of RelA activity occurs through its sequestration in RelA·RelB heterodimers. To our knowledge this is the first demonstration that RelB is a complex partner of RelA.
Two possible explanations may exist for the previous failure to detect RelA·RelB heterodimers. These are (a) RelA·RelB heterodimers do not bind efficiently to conventional B sites and (b) the amount of RelA·RelB heterodimers is low compared with RelB·p52 and RelB·p50 heterodimers. However, activation of NF-
B, by either a constitutively active IKK
or stimulation with an NF-
B agonists like a phorbolester or LPS, led to a significant increase in the RelA·RelB complex formation in 70Z/3 pre B cells (Fig. 7, A and B). Consistent with these observations are the recent findings that the relb gene itself is regulated by NF-
B and its expression is induced by most of the known NF-
B-inducing agonists. In addition, a substantial increase in cellular RelB protein levels has been reported in B cells and pre-B cells stimulated by CD40 or IL-7 withdrawal, respectively (32, 33). Currently, the biological function of a RelA·RelB heterodimer remains unclear, but because we were unable to detect DNA binding of these complexes in vitro, we conclude that they are transcriptionally inactive through a failure to associate with DNA. Recently, we described a signal-induced proteolytic degradation of RelB in T cells stimulated with anti-CD3/CD28 antibodies or phorbolester and calcium ionophore. This degradation process would therefore remove RelA·RelB heterodimers and would release RelA (or p50 and p52) as active NF-
B factor(s), thus supporting the possibility that the biological function of heterodimer formation between RelB and RelA is the inhibition of the RelA activity (17). Nevertheless, the low amounts of RelA·RelB heterodimers and the much higher efficiency of the classic I
B proteins to inhibit RelA argue against this possibility. Alternatively, because variations in the composition of binding sites for the distinct combinations of NF-
B family members have been described previously (3, 4), it is possible that RelA·RelB heterodimers bind to a DNA sequence separate from the known
B sites. A change in the composition of the NF-
B factors after time occurs after treatment with various inducers such as the stimulation of resting mature B cells with anti-Ig antibodies or CD40 ligand (35). Thus in unstimulated B cells, active NF-
B is composed of RelB and c-Rel heterodimers, whereas, during stimulation with both agonists for 2 h, RelA heterodimers are induced followed by a signal-specific loss of active RelB after 24 h in the case of the CD40 stimulation. Interestingly, only the DNA binding, but not the nuclear localization of RelB, seemed to be affected (35). Interestingly RelA·RelB heterodimers do not seem to be controlled by the NF-
B inhibitors I
B
or I
B
in fibroblasts (Fig. 5B). This is consistent with the recent finding that p100 and to a lesser extent p105 are the only I
B proteins complexed with RelB in this cell type (26). Because we found only a portion of RelA·RelB heterodimers in the nucleus, comparable to the amount of nuclear RelA·p50 heterodimers, the question of how the cytoplasmic retention of RelA·RelB heterodimers is achieved still remains. The control of RelB-containing dimers in general has always been a matter of discussion. For example, RelB·p50 heterodimers are only weakly if at all controlled by I
B proteins, but the majority of RelB·p50 heterodimers was found in the cytoplasm. It is still unclear if RelB is complexed with p105 instead of p50 in this compartment or if another unidentified mechanism is responsible for this effect. The molecular mechanisms underlying the different RelB functions are versatile and highly cell type-dependent. It was therefore important to analyze both lymphoid as well as non-lymphoid cells. Interestingly, we detected RelA·RelB heterodimers in both cell types. However, because RelB has been demonstrated to bind to I
B proteins in lymphoid cells, the control of RelA·RelB heterodimers might be different in lymphoid versus non-lymphoid cells.
As mentioned earlier, the expression of RelB is controlled by NF-B (6), and therefore an increase in the protein levels of RelB in thymocytes overexpressing a constitutively active isoform of IKK
is not surprising. In addition, increased cellular RelB levels have been reported during B cell development (33, 34). Taken together, our data describe a new feature of the NF-
B/Rel family member RelB, which helps explain its repressive role on NF-
B activity. Clearly, other molecular mechanism such as blocking of
B sites, sequestering of p50 or p52 in heterodimers, as well as epigenetic effects through the inability to recruit transcriptional cofactors might also contribute for the repressive role of RelB. Nevertheless, our findings clearly demonstrate that complex formation between RelB and RelA inhibits RelA activity. Because the regulation of NF-
B-dependent gene expression is so versatile, such a repression of RelA in one context might redirect NF-
B to induce a different set of genes and therefore potentially represents a mechanism to switch gene expression patterns during an NF-
B-dependent cellular response.
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FOOTNOTES |
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To whom correspondence should be addressed: Section of Immunobiology and Dept. of Molecular Biophysics and Biochemistry, Yale University Medical School, 300 Cedar St., New Haven, CT 06520. Tel.: 203-737-4424; Fax: 203-785-3855; E-mail: rmarienfeld{at}hotmail.com.
1 The abbreviations used are: TNF, tumor necrosis factor
; EMSA, electrophoretic mobility shift assay; IKK, I
B kinase, I
B; inhibitor of NF-
B; 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.
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REFERENCES |
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