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Constitutive Nuclear Translocation of NF-B in B Cells in the Absence of IB Degradation¹

Department of Microbiology, Boston University School of Medicine, Boston, MA 02118

	Abstract

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Members of the NF-B/Rel family of transcription factors are involved in many aspects of B lymphocyte development and function. NF-B is constitutively active in these cells, in contrast with most other cell types. In the inactive form, NF-B/Rel proteins are sequestered in the cytoplasm by members of the IB family of NF-B inhibitors. When activated, NF-B is translocated to the nucleus, a process that involves the phosphorylation and proteasomal degradation of IB proteins. Thus, NF-B activation is accompanied by the rapid turnover of IB proteins. We show that while this "classical" mode of NF-B activation is a uniform feature of IgM⁺ B cell lines, all IgG⁺ B cells analyzed contain nuclear NF-B yet have stable IB, IBß, and IB. Furthermore, Iß levels are at least 10 times lower in IgG⁺ B cells than in IgM⁺ B cells, an additional indication that the regulation of constitutive NF-B activity in these two types of B cells is fundamentally different. These data imply the existence of a novel mechanism of NF-B activation in IgG⁺ B cells that operates independently of IB degradation. They further suggest that different isoforms of the B cell receptor may have distinct roles in regulating NF-B activity.

	Introduction

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Nuclear factor-B/Rel transcription factors are involved in many processes that are important for B cell development, differentiation, and function (1, 2, 3, 4). NF-B was originally described as a constitutive nuclear factor in B cells and an inducible factor in pre-B cells that is required for the expression of the Ig light chain ()³ gene (2, 5, 6). In addition to the regulation of expression, NF-B also plays a role in the rearrangement of the Ig locus (7), suggesting a central role of NF-B in the initial stages of B cell receptor expression. NF-B is important in the rescue of B cells from apoptosis and in the maintenance of cell cycling, events important in the survival, selection, and maturation of B cells (8, 9, 10). NF-B has also been implicated in the initiation of Ig isotype switching (11, 12), suggesting a role in adaptive humoral immune responses.

NF-B is now known to be ubiquitously expressed and to play a major role in controlling the expression of proteins involved in immune, inflammatory, and acute phase responses (4, 13). NF-B activity is primarily regulated through nuclear translocation (5, 6, 13, 14, 15, 16, 17). In its inactive form, NF-B is sequestered in the cytoplasm, bound by members of the IB family of inhibitor proteins, which include IB, IBß, IB, and the recently identified IB (16, 18, 19, 20). Activation occurs when NF-B is released from IB and translocated to the nucleus. Nuclear translocation of NF-B can be induced by a variety of stimuli, including TNF-, IL-1, LPS, and phorbol esters such as PMA (1, 3). These stimuli trigger the phosphorylation of IB, which is followed by the ubiquitination of IB and its subsequent degradation through the proteasome (17, 21, 22). This sequence of events has been called the classical pathway of NF-B activation, and most signals that activate NF-B converge into this pathway (17, 23). The IB proteins identified to date that are the targets of this pathway include IB, IBß, and IB. Each of these IB proteins contains two highly conserved serine residues in their N-terminal domain that serve as substrates for IB kinase and are necessary for NF-B activation (20, 21, 22). A key feature of the classical pathway is that IB proteins are unstable and exhibit shortened half-lives during NF-B activation.

In most cells, NF-B activation is transient because NF-B induces the transcription of IB, among other target genes (13, 15). Consequently, NF-B activation normally results in a negative feedback loop, in which newly synthesized IB leads to the resequestration of NF-B in the cytoplasm (15). However, in the B lineage, NF-B activation becomes constitutive at the transition from the pre-B to the B cell stage (5, 24). Although much of the NF-B remains sequestered in the cytoplasm of primary B cells and B cell lines, these cells contain constitutive levels of nuclear NF-B that can be increased upon stimulation (2, 5, 6, 25, 26, 27, 28, 29). Using the immature B cell line, WEHI231, as a model system, several groups have shown that both IB and IBß undergo rapid degradation (30, 31, 32, 33). The degradation of these IB proteins has been shown to be required for the presence of constitutive nuclear NF-B, suggesting that NF-B activation in WEHI231 cells occurs via the classical pathway (33). Whether this cell line serves as a general model for the constitutive activation of NF-B in B cells is not known.

In the present study we evaluated the stability of IB, IBß, and IB in a number of B cell lines to determine whether the rapid turnover of IB is a general feature of constitutive NF-B activation in the B lineage. Although several B cell lines, including WEHI231, exhibited enhanced degradation of IB and IBß, other B cell lines showed no evidence of rapid IB turnover despite the presence of nuclear NF-B. The differences in IB turnover correlated with the isotype of the surface Ig receptor. Although all B cells exhibiting unstable IB and IBß were IgM⁺, the B cells with stable IB expressed surface IgG. These data suggest the presence of an alternative mechanism of constitutive NF-B activation in IgG⁺ B cells that is independent of the degradation of IB and IBß. Interestingly, while IB was stable in all B cell lines, IgG⁺ B cells expressed much lower levels of IB than the IgM⁺ B cells, suggesting that reduced levels of IB may play a role in NF-B activation in IgG⁺ B cells. We also suggest that the B cell receptor plays a role in regulating the different mechanisms of constitutive NF-B activation in B cells.

	Materials and Methods

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Cell lines and cell cultures

A panel of murine B cell lymphoma lines representing different stages of development and differentiation was used in this study; many of these have been previously described (34, 35, 36). The IgM⁺ B cell lines WEHI231 and CH31 are representative of immature B cells that are susceptible to tolerance induction (37). CH27 (38) and CH12-LBK (39) are both representative of mature IgM⁺ B cell lines. A20 and M12 (40) (provided by L. Glimcher, Harvard School of Public Health, Boston, MA) are IgG⁺ B cell lymphoma cells that are representative of mature B cells. LK (41) (provided by A. Marshak-Rothstein (Boston University, Boston, MA)) is a B cell hybridoma derived by fusion of splenic B cells from B10.BR mice with the B cell lymphoma, L10.A. We found that the LK cell line expresses surface IgG but not IgM. CH12.LX2.1A7 (42) (provided by Dr. L. Arnold, University of North Carolina, Chapel Hill, NC) is a subclone of the CH12 B cell lymphoma that has undergone spontaneous class switching and expresses surface IgG but lacks IgM. All Ig isotypes were confirmed by Western blot and/or FACS analysis. The pre-B cell lines 70Z/3 and Bine4.8 (the latter provided by H.-M. Jäck, University of Erlangen-Nurnberg, Erlangen-Nurnberg, Germany) and the T cell hybridoma BW5147 (43) (provided by D. Woodland, St. Jude Children’s Research Hospital, Memphis, TN) served as controls. All cell lines were maintained in DMEM containing 7% FBS (Life Technologies, Gaithersburg, MD) and 50 µM 2-ME (Sigma, St. Louis, MO) and supplemented as previously described (44). In some experiments cells were treated with cycloheximide (CHX; 50 µg/ml, Sigma), LPS (50 µg/ml; Escherichia coli 055:B5, Difco, Detroit, MI), or PMA (50 nM; Sigma) for various periods of time as described in Results.

Immunoblotting

Cells were washed in ice-cold BSS and lysed at 1 x 10⁵ cells/µl in ice-cold whole cell lysis buffer (150 mM NaCl, 10 mM Tris (pH 7.50, and 1% Nonidet P-40) containing 1 mM DTT, 1 mM PMSF, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 0.5 µg/ml pepstatin, 50 mM NaF, and 1 mM Na₃VO₄. Cell lysates were incubated for 30 min on ice and centrifuged at 13,000 x g at 4°C for 15 min, and supernatants were snap-frozen in liquid nitrogen. For the lysates used to determine IB expression levels, protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA). Lysates from equivalent cell numbers or equivalent amounts of protein were fractionated on 10% SDS-PAGE. Gels were transferred to nitrocellulose membranes (Micron Separations, Westboro, MA), and membranes were stained with Ponceau S (Sigma) to ensure equivalent loading and transfer. Membranes were probed with the appropriate Abs and developed with the ECL system (Amersham, Arlington Heights, IL). Autoradiographs were quantified using a Molecular Dynamics densitometer with ImageQuant software (Molecular Dynamics, Sunnyvale, CA). All primary Abs were developed in rabbit and were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). They included Abs against IB (sc-371), IBß (sc-945), IB (sc-7155), RelA (sc-109), RelB (sc-226), c-Rel (sc-71), and NF-B1 (sc-114). In some experiments the anti-IB Ab detected an additional protein that migrated slightly more slowly than IB. This protein was stable after PMA or LPS activation (see Fig. 5). It is unclear whether this protein represents a stable phosphoisoform of IB or is an unrelated cross-reacting protein.

View larger version (45K): [in this window] [in a new window]   FIGURE 5. Rapid IB degradation is inducible in B cell lines with constitutively stable IB. A20 or M12 cells were pretreated for 45 min with LPS or for 15 min with PMA before CHX addition. Whole cell lysates were prepared at the indicated times after CHX addition and analyzed by Western blot. IB is identified by an arrow (see Materials and Methods).

Cells were washed in BSS, resuspended at 4–5 x 10⁶ cells/ml in complete DMEM lacking cysteine and methionine, and incubated at 37°C for 1 h. Cells were labeled for 1 h (or indicated times) by addition of 60–100 µCi/ml of [³⁵S]cysteine and [³⁵S]methionine (New England Nuclear, Boston, MA). Cells were washed in BSS and chased with complete DMEM for the times indicated in the respective experiment. Cells were washed twice in BSS, and whole cell lysates were prepared as described above. Lysates were precleared using normal rabbit serum and protein A-Sepharose (Sigma). IB protein was immunoprecipitated with Ab sc-371 and protein A-Sepharose. Precipitated material from the preclearing step and from the IB immunoprecipitations was separated by 10% SDS-PAGE, and gels were analyzed by autoradiography or phosphorimaging. The m.w. standards were ¹⁴C-labeled High Range standards (Life Technologies). We found that the Ab to IBß (sc-945) did not immunoprecipitate IBß (not shown).

Preparation of nuclear and cytoplasmic extracts

Nuclear extracts were prepared according to standard procedures (45). Cells (1–4 x 10⁸) were washed once with BSS and once with hypotonic buffer (10 mM HEPES (pH 7.9), 1.5 mM MgCl₂, 10 mM KCl, 1 mM PMSF, and 1 mM DTT), resuspended in hypotonic buffer, and incubated on ice for 10 min. Cells were homogenized using a Dounce homogenizer (Kontes, Vineland, NJ) until >90% lysis was detected as determined by trypan blue exclusion. Nuclei were collected by centrifugation for 5 min at 3000 x g. Supernatants were saved for the preparation of cytoplasmic extracts. Nuclei were washed once in hypotonic buffer and resuspended in low salt buffer (20 mM HEPES (pH 7.9), 0.02 mM KCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 25% glycerol, 1 mM PMSF, and 1 mM DTT). An equal volume of high salt buffer (same composition as low salt buffer, but containing 800 mM KCl) was added dropwise with occasional vortex mixing. The nuclei were incubated for 30 min at 4°C and centrifuged for 30 min at 13,000 x g, and supernatants were snap-frozen in liquid nitrogen. All nuclear extracts were free from cytoplasmic contamination as determined by Western blot with an Ab against the cytoplasmic protein NF-B p105, with the exception of the WEHI231 extracts, which contained about 3–5% of cytoplasmic contamination.

Cytoplasmic extracts from above were supplemented with cytoplasmic extract buffer to a final concentration of 40 mM HEPES (pH 7.9) and 150 mM KCl, incubated for 30 min on ice, and centrifuged for 30 min at 13,000 x g, and supernatants were snap-frozen. The protein concentration of all extracts was determined by Bradford assay and further controlled by separating equal aliquots of the samples via SDS-PAGE, transfer to a nitrocellulose membrane, and staining of the membrane with Ponceau S.

DNA-protein binding studies

A photoreactive, radiolabeled B-specific oligonucleotide probe (B-pd) was prepared by primer extension labeling with [³²P]dCTP (New England Nuclear) and bromodeoxyuridine (Sigma) as previously described (46). For gel-shift assays, 20 µg of nuclear extracts were added to a reaction mix containing final concentrations of 20 mM HEPES (pH 7.9), 50 mM KCl, 1 mM EDTA, 5% glycerol, 5 mM DTT, 250 µg/ml BSA, and the nonspecific competitors pd(N)₆ (250 µg/ml; Amersham) and poly(dI-dC) (250 µg/ml, Amersham) in a total volume of 20 µl. Radiolabeled probe (5 x 10⁴ cpm) was added, and the reactions were incubated at 20°C for 20 min. DNA-protein complexes were analyzed by gel retardation on native 5% acrylamide gels as previously described (47) and were visualized by phosphorimaging.

For UV cross-linking analyses, 50 µg of nuclear extracts were incubated for 20 min in a total volume of 50 µl in the same reaction mix as that described above, except containing 5 x 10⁵ cpm of the B-pd probe, and were irradiated for 20 min with a UV light source (300 nm; Fotodyne, Hartland, WI). The reaction was diluted in lysis buffer, and equal aliquots were subjected to immunoprecipitation with Abs against either RelA or c-Rel. Precipitated material was analyzed by SDS-PAGE and autoradiography together with an aliquot of each cross-linking reaction. RelB was not detectable by this analysis due to the failure of RelB to be efficiently cross-linked or to the inability of the anti-RelB Ab to precipitate RelB-DNA adducts.

	Results

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IgM⁺, but not IgG⁺, B cell lines show accelerated IB degradation

We first sought to determine whether the paradigm established using WEHI231 cells was generally applicable and whether other B cell lines showed accelerated degradation of IB proteins. We therefore analyzed the stability of IB proteins in a panel of murine B cell lines using two different approaches. In the first, the stability of IB proteins was determined by treatment of cells with the protein synthesis inhibitor CHX. Degradation of the respective IB proteins was monitored by Western blot analysis of protein extracts prepared at different times following CHX treatment.

As expected, IB and IBß were rapidly degraded in WEHI231 cells (Fig. 1). The half-lives of these proteins were calculated to be 50 and 140 min, respectively. Similar results were obtained using another IgM⁺ B cell line, CH27. In striking contrast, IB and IBß were significantly more stable in two IgG⁺ B cell lines, A20 and M12, and one IgG⁺ B cell hybridoma, LK, with half-lives of >4 h (Fig. 1). These half-lives were similar to or even longer than those found in a pre-B cell line (Bine4.8) and a T cell line (BW5147; Fig. 1). Pre-B cells and T cells generally do not contain significant levels of constitutive nuclear NF-B, and their IB proteins are stable in the absence of stimuli (5, 32, 48, 49, 50, 51, 52) (see also Fig. 3). In contrast to IB and IBß, IB was stable in all cell lines analyzed, including WEHI231. This suggests that IB degradation in WEHI231 cells is controlled independently of IB and IBß.

View larger version (62K): [in this window] [in a new window]   FIGURE 1. Turnover of IB, -ß, and - pools in different cell lines. A, Western blot analysis of IB proteins in whole cell extracts from CHX-treated cells. B cell lines included the IgM+ cell lines WEHI231 (WEHI) and CH27, the IgG+ cell lines A20, M12, and LK. Control cells included the pre-B cell line Bine4.8 (BINE) and the T cell line BW5147 (BW). Note that the signal intensities for the IB proteins cannot be used to compare their relative levels of expression. Different exposures were chosen to match the respective signals for better comparison of the half-lives. A comparison of the steady state levels of IB, -ß, and - in these cell lines is shown in Fig. 6. B, IB levels were quantitated by densitometry, and the half-lives were calculated by linear regression. C, Half-life determination of IBß was conducted as described in B.

View larger version (58K): [in this window] [in a new window]   FIGURE 3. Presence of nuclear NF-B in cell lines exhibiting stable IB. A, Gel-shift assay. Nuclear extracts from the indicated cell lines were incubated with a radiolabeled B-specific probe and resolved on native PAGE gels, and the DNA-protein complexes were visualized by autoradiography. The bands corresponding to NF-B/Rel heterodimers and NF-B homodimers are indicated. B and C, UV cross-linking analysis of nuclear NF-B complexes. Nuclear extracts were incubated with a radiolabeled B-specific probe and exposed to UV light. Aliquots of the reaction mixture were separated by SDS-PAGE (top panel in B, lane 1 in C) or immunoprecipitated with Abs to RelA or c-Rel (middle and lower panels in B, lanes 2 and 3, respectively, in C) before SDS-PAGE analysis. D, Comparison of nuclear and cytoplasmic levels of c-Rel and RelB. Nuclear (N; 10 µg) and cytoplasmic (C; 40 µg) extracts were subjected to Western blot analysis; autoradiographs were quantified by densitometry. The quantity of nuclear and cytoplasmic extracts used for the analysis corresponded to equivalent numbers of cells, with the exception of WEHI231, where the amount of nuclear extract used was from twice as many cells as the cytoplasmic extract.

View larger version (43K): [in this window] [in a new window]   FIGURE 2. Pulse-chase analysis of IB in different B cell lines. A, Cells were metabolically labeled for 1 h and chased for the times indicated. Lysates were prepared from equal cell numbers and analyzed by immunoprecipitation and SDS-PAGE. The B cell lines CH31 and CH12-LBK cells are IgM+; the other cell lines are as described in Fig. 1. B, Half-life determination of IB was conducted as in Fig. 1B.

NF-B is efficiently translocated to the nucleus in B cells with stable IB

B cells normally express constitutive levels of nuclear NF-B and express all known mammalian members of the NF-B/Rel transcription factor family, including NF-B1, NF-B2, RelA, RelB, and c-Rel (2, 25, 26, 27, 28, 31). These proteins exist as homodimers or heterodimers; the latter are predominantly formed between either NF-B1 or NF-B2 and RelA, RelB, or c-Rel (2, 13). RelA and c-Rel serve as receptors for IB, IBß, and IB, and consequently, complexes containing these Rel proteins are normally retained in the cytoplasm in the absence of stimuli (16, 18, 19, 20, 53). Because the vast majority of the known pathways of NF-B activation converge in the degradation of IB or IBß, we were especially interested in determining whether the B cell lines with stable IB also contained nuclear RelA and c-Rel. We also included RelB in this analysis, since it is expressed at high levels in B cells and has a functional trans-activation domain as do RelA and c-Rel. However, it should be noted that not all NF-B complexes containing RelB can be inhibited by IB (54).

We first analyzed the B cell lines for the presence of nuclear DNA binding NF-B by gel-shift assay. This assay can distinguish between different NF-B complexes, with complexes comprised of NF-B1 or NF-B2 homodimers migrating faster than NF-B/Rel heterodimers (47). As controls, we used the pre-B cell lines 70Z/3 and Bine4.8, which, as expected, contained little nuclear NF-B (Fig. 3A).

All the B cell lines contained nuclear NF-B, including the B cell lines that exhibited stable IB (Fig. 3A). The complexes detected were consistent with the presence of Rel-containing heterodimers, suggesting that these NFB complexes are efficiently translocated to the nucleus in B cells with stable IB.

To directly demonstrate the presence of RelA, RelB, and c-Rel in the nucleus of B cells with stable IB, two types of experiments were performed. In the first, nuclear extracts from IgM⁺ and IgG⁺ B cell lines were cross-linked to a B probe by UV irradiation, and RelA- and c-Rel-containing DNA-protein adducts were identified by immunoprecipitation and SDS-PAGE (Fig. 3, B and C). Importantly, in all cell lines nuclear DNA binding RelA and c-Rel could be detected by this method, albeit at different concentrations. The IgM⁺ cell lines WEHI231, CH27, and CH12-LBK had consistently higher levels of c-Rel than the IgG⁺ B cell lines A20 and M12. In contrast, Rel A levels did not correlate with Ig phenotype or IB half-life. WEHI231 and M12 cells had the highest RelA levels, followed by CH27 and CH12 cells, while RelA was barely detectable in A20 cells. A direct comparison between the cell line with the highest (WEHI231) and that with the lowest (A20) content of nuclear RelA and c-Rel is shown in an independent UV cross-link experiment, in which the respective bands are separated at a higher resolution (Fig. 3C). This experiment clearly shows the presence of both RelA and cRel in the nuclei of A20 cells. RelB could not be detected by this method, but its presence in nuclear extracts could be confirmed by Western blot analysis (Fig. 3D).

To obtain an independent measure of the relative efficiency of NF-B translocation in the two groups of B cell lines, we estimated the nuclear fraction of total RelB and c-Rel by Western blot of nuclear and cytoplasmic extracts (Fig. 3D). Nuclear RelA levels were too low to be detected by this method. From the four cell lines used in these experiments, two were IgM⁺ and had unstable IB and IBß proteins (WEHI231 and CH12), and two were IgG⁺ and exhibited stable IB (A20 and M12). All these B cells contained nuclear RelB and c-Rel, although some differences in the proportions of these NF-B constituents were observed. The cell lines with unstable IB contained a higher proportion of c-Rel in the nucleus (20% of the total) than cell lines with stable IB (6–7%). This is consistent with the results of the UV cross-linking experiments (Fig. 3B), which suggested the presence of higher levels of nuclear c-Rel in WEHI231 than in A20 cells. Both of the IgG⁺ cell lines contained nuclear RelB in somewhat higher proportions (30–70% of the total) than cells with unstable IB (<25%). Taken together, these data confirm that RelA, RelB, and c-Rel, the three NF-B members that are transcriptional activators, are constitutively present in the nucleus of IgG⁺ B cells. They also demonstrate that nuclear translocation of RelA and c-Rel, which are the targets for cytoplasmic sequestration by IB, IBß, and IB, can occur without the accelerated degradation of these IB proteins.

B cells with stable IB have a slower rate of IB synthesis

For a cell to maintain homeostatic levels of a protein, the protein must be synthesized at the same rate as it is being degraded. We therefore reasoned that the differences in IB turnover in the various B cell lines would be accompanied by different IB synthesis rates. The relative rates of IB biosynthesis in WEHI231 and A20 cells were therefore determined by following the incorporation of radiolabeled amino acids into IB, which was quantified by immunoprecipitation and phosphorimaging. As predicted, WEHI231 cells synthesized IB at a much faster rate than A20 cells (Fig. 4). Two types of control experiments showed that this was not true for all proteins. First, we fractionated labeled proteins by SDS-PAGE and determined the incorporation of radioactivity into an abundant random protein of 55 kDa. As shown in Fig. 4, this protein was synthesized more rapidly in A20 cells than in WEHI231 cells. In addition, we determined the total incorporation of radiolabel into proteins in these two cell lines using a beta counter and again found that A20 cells incorporated label faster than WEHI231 cells (data not shown). We conclude that the reduced rate of IB synthesis in A20 cells is not due to a lower general translation efficiency, but, rather, is specific for IB and reflects the requirement for a lower rate of synthesis of IB due to its stability in these cells.

View larger version (66K): [in this window] [in a new window]   FIGURE 4. IB synthesis rates in different B cell lines correlate with IB half-lives. Cells were metabolically labeled for the indicated times. A, Lysates from equivalent cell numbers were immunoprecipitated with an anti-IB Ab, and the precipitated material was analyzed by SDS-PAGE and phosphorimaging. B, Aliquots of the total cell lysates were analyzed by SDS-PAGE and phosphorimaging. C, The IB signals from A were quantitated, and the relative signal intensities are graphically displayed. D, The signals corresponding to an unknown 55-kDa protein (indicated by the arrow in B) were quantitated and analyzed as described in C.

LPS and PMA can induce IB degradation in B cells with stable IB

We next wanted to determine whether IB turnover could be stimulated in the B cell lines, A20 and M12, that otherwise contained stable IB. We used LPS and PMA, since these agents are known to induce IB degradation and NF-B activation in pre-B cells and T cells (5, 51, 52). A20 and M12 cells were treated with CHX in the presence or the absence of LPS or PMA, and IB levels were assessed by Western blot analysis. As shown above (Fig. 1), IB levels remained unchanged for >4 h in the absence of stimuli. In contrast, IB was almost completely degraded after 4 h in the LPS-treated cells (Fig. 5). Consistent with previous reports (51, 52), PMA induced an even more rapid turnover of IB than LPS, resulting in complete degradation of the total IB pool of M12 cells in <1 h (Fig. 5). The stability of IB in A20 cells was unaffected by PMA treatment, possibly because these cells are refractory to its effects (data not shown). These results demonstrate that the ability to signal IB degradation is not defective in A20 and M12 cells and suggest that the classical pathway of NF-B translocation can be activated.

IgG⁺ and IgM⁺ B cells express comparable levels of IB and IBß, but strikingly different levels of IB

We next compared the steady state levels of IB, IBß, and IB in the various B cell lines. We were particularly interested in determining whether the different half-lives of IB and IBß detected in IgM⁺ and IgG⁺ B cell lines would result in different expression levels of these proteins. As shown in Fig. 6, all cell lines tested expressed high steady state levels of IB and IBß. Although the expression levels varied somewhat between the different cell lines, they did not correlate with the respective half-lives. For example, one IgG⁺ cell line, A20, expressed the highest levels of IB, but the other IgG⁺ cell line, M12, expressed equal or lower levels of IB than IgM⁺ cells.

View larger version (84K): [in this window] [in a new window]   FIGURE 6. Comparison of steady state levels of IB, IBß, and IB in various B cell lines. Cytoplasmic extracts (50 µg) of the indicated cell lines were separated by SDS-PAGE and analyzed by Western blot analysis with Abs specific for the IB proteins. Membranes were stained with Ponceau S before probing to verify equivalent loading of proteins. Two different exposures of the anti-IB blot are shown to illustrate the large differences in IB expression in IgM+ and IgG+ cell lines.

View larger version (68K): [in this window] [in a new window]   FIGURE 7. Analysis of IB proteins in the IgG+ class switch variant of CH12, CH12-LX2.1A7 (1A7). A, The half-life of IB in the IgG+ 1A7 cells was compared with the half-life in the IgM+ CH12-LBK (CH12) cells. Half-life analysis was conducted as described in Fig. 1. B, Comparison of steady state levels of IB proteins in 1A7 (IgG+), CH12 (IgM+), A20, and Lk (both IgG+). Whole cell extracts (50 µg) of the indicated cell lines were resolved by SDS-PAGE and analyzed by Western blot analysis with the respective Abs.

A class-switched variant of CH12 cells shows altered IB stability and IB levels

To better establish the correlation among Ig isotype, IB stability, and IB expression levels, we analyzed a spontaneous class-switched variant of the IgM⁺ CH12 B cell lymphoma, CH12.LX2.1A7, which expresses surface IgG and not IgM (42). In these cells we found the stability of IB to be significantly increased in the IgG⁺ variant compared with that in IgM⁺ CH12 cells (Fig. 7A). In addition, the levels of IB were considerably lower in the IgG⁺ variant than in the IgM⁺ cell line and were similar to the levels found in other IgG⁺ B cells (Fig. 7B). Thus, the differences in IB stability and IB expression levels detected between several IgM⁺ and IgG⁺ B cell lines are also observed in two CH12 subclones expressing these different Ig isotypes. These results strongly suggest that the Ig isotype plays a role, either direct or indirect, in the differential regulation of IB proteins in IgM⁺ and IgG⁺ B cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
A central tenet of the NF-B signaling pathway is the release of NF-B from its inhibitor, IB, and its subsequent translocation to the nucleus (13, 16, 17). The release of IB is accompanied by its degradation, a process that is the hallmark of NF-B activation. This pathway has been well documented during the transient activation of NF-B, but it has also been proposed to function in the constitutive activation of NF-B in B cells, based on studies of the immature B cell line, WEHI231. In these cells, IB and IBß are rapidly degraded (30, 31, 32, 33). Although the precise mechanism of NF-B translocation in these cells remains unknown, several studies find that the degradation of IB, IBß, or both is required for nuclear translocation of NF-B (33, 55, 56). However, despite the central role that NF-B plays in regulating many aspects of B cell differentiation and function, detailed studies of other B cell lines have not been conducted, and the mechanism for constitutive NF-B activation in B cells remains largely unknown. Here we provide evidence that different B cell lines use distinct strategies for the nuclear translocation of NF-B, and that this difference may be associated with the isotype of the B cell receptor.

Like WEHI231, we found that IB and IBß were rapidly degraded in several other B cell lines, including CH31, CH12-LBK, and CH27, all of which are IgM⁺. In striking contrast, IB and IBß were very stable in the three B cell lines tested, A20, M12, and LK (Fig. 1), all of which express surface IgG. In these cells, IB and IBß were at least as stable as in pre-B and T cell lines, which do not express significant quantities of nuclear NF-B (32, 51, 52) (see also Fig. 1). At the very least, these results demonstrate that the rapid degradation of IB proteins is not a general feature of all B cell lines. They also suggest that the stability of IB and IBß may be a general feature of IgG⁺ B cells, or at least of B cells that are not IgM⁺.

Despite the stability of IB, IBß, and IB in the IgG⁺ B cell lines, they contained constitutive nuclear NF-B, including complexes containing RelA and c-Rel (Fig. 3) that are normally susceptible to cytoplasmic retention by IB (16, 18, 19, 20, 53). The presence of these complexes is in striking contrast to other cells, such as pre-B cells and T cells, that have stable IB proteins but do not contain activated NF-B (32, 51, 52). To our knowledge, this is the first demonstration that constitutive translocation of NF-B complexes containing RelA and c-Rel can occur without accelerated degradation of IB, IBß, or IB. We therefore conclude that these IgG⁺ B cell lines use a novel mechanism of nuclear NF-B translocation that is independent of the degradation of these IB proteins.

The presence of nuclear RelB in IgG⁺ B cells is perhaps less surprising than the presence of RelA and c-Rel, since the nuclear translocation of RelB-containing complexes is regulated very differently. NF-B p52/RelB heterodimers are not inhibited by IB, while NF-B p50/RelB heterodimers are only poorly retained by IB and are not bound by IBß (54). Thus, the effect that the novel mechanism of NF-B activation in IgG⁺ B cells has on modulating levels of nuclear RelB, if any, cannot be determined at this time.

Although IB and IBß were degraded at different rates in IgM⁺ and IgG⁺ B cell lines, they were expressed at comparable levels in these two groups of cells. In contrast, IB was very stable in both types of B cell lines, suggesting that the turnover of IB is regulated independently of that of IB and IBß, at least in IgM⁺ B cells. Despite the similarity in stability of IB in the different cell lines, it was expressed at high levels only in the IgM⁺ B cells. All IgG⁺ B cells expressed very low levels of IB, estimated to be at most 10% of the levels in IgM⁺ cells. These data indicate that IB expression is regulated independently of IB and IBß and is also regulated differently in the IgM⁺ and IgG⁺ B cells. We therefore propose that the newly identified member of the IB family, IB, may play a distinct role in regulating NF-B activity.

The nature of the novel mechanism of NF-B translocation in the IgG⁺ B cell lines from this study remains to be determined. Several possibilities exist to account for the presence of Rel-containing complexes in the nucleus of these cells. For example, NF-B may dissociate from one or more IB proteins without their degradation. Such a possibility has been suggested in studies of T cell activation after pervanadate treatment (57). It is also possible that a portion of the cytoplasmic NF-B is associated with an as yet unidentified IB protein, one that is being degraded and thus releases NF-B for nuclear translocation. This would imply that the degradation of this putative IB protein occurs independently of IB, IBß, and IB. A third possibility is that the efficient retention of NF-B requires a minimal level of total IB proteins in the cytoplasm. Thus, a significant reduction in the level of any IB protein would allow the translocation of NF-B complexes to the nucleus after the assembly and processing of their precursors. IB has been shown to be similar to IB and IBß in its specificity, in that it binds complexes containing RelA or c-Rel (19, 20). Thus, it is possible that the levels of IB and IBß are insufficient to compensate for the low levels of IB in IgG⁺ B cells. On the other hand, IB has been reported to preferentially bind homodimers of RelA and c-Rel, rather than heterodimers formed with NF-B1 (19, 20). Thus, even the presence of high levels of IB and IBß might be unable to sequester these complexes if levels of IB are too low. In either case, NF-B translocation may be permitted in IgG⁺ cells without prior engagement of IB. At this point, none of these proposed mechanisms can be ruled out, and further studies are required to distinguish between them.

What might the consequences be of using two different mechanisms of NF-B translocation? It is possible that they provide a tool to differentially regulate the composition of nuclear NF-B and thus the pattern of NF-B dependent target genes. While our analysis has not revealed any dramatic differences in nuclear NF-B composition between the two groups of B cells, it remains possible that their different mechanisms of NF-B activation may lead to subtle changes that are not immediately obvious from the current results. The various NF-B complexes differ in their preference for B sites from different promoters and also in their trans-activation potentials (reviewed in Ref. 58). Thus, even small changes in NF-B composition might alter the expression pattern of NF-B-dependent genes. Such changes might explain why the IgG⁺ B cells had slower rates of IB synthesis and lower steady state levels of IB. The expression of both these IB proteins is regulated at least in part by NF-B (19, 20, 59, 60, 61).

What leads to the different mechanisms of NF-B activation? It is striking that the only apparent difference between cells with unstable IB and cells with stable IB is reflected in their surface Ig receptors. All IgM⁺ cells had unstable IB and IBß regardless of their stage of differentiation. In these studies we used representatives of both immature (WEHI231, CH31) and mature (CH27, CH12-LBK) IgM⁺ B cells, which we found had similar rates of IB degradation. In sharp contrast, all IgG⁺ cells analyzed contained stable IB and IBß. Consequently, it is unlikely that the differences observed between IgM⁺ and IgG⁺ B cells simply reflect their states of differentiation, but rather correlate with the surface Ig phenotype. This idea is further supported by the finding that IB stability and IB levels are different in a subclone that has undergone spontaneous class switching from IgM to IgG compared with the parental cell line.

These findings strongly suggest that the Ig receptor itself may play a direct or an indirect role in regulating IB and IBß stability and IB expression levels. For example, surface IgM, but not IgG, may have the ability to constitutively stimulate IB turnover, possibly by activating IB kinase (reviewed in Ref. 21). Such IgM-mediated IB turnover might occur in the absence of ligand binding. Indeed, Rajewsky and colleagues (62) have recently shown that surface IgM expression is required for the survival of B cells in the periphery. Their studies imply that surface IgM delivers a constitutive signal that cannot be provided by other mechanisms in the absence of B cell stimulation. Given the important role that NF-B plays in cell survival and protection from apoptosis (63), we predict that this survival signal leads to IB degradation, and thus NF-B activation, providing a testable hypothesis for the role of IgM in B cell survival in the periphery.

	Acknowledgments

 
We thank Drs. David Woodland (St. Jude Children’s Research Hospital, Memphis, TN), Hans-Martin Jäck (University of Erlangen-Nurnberg, Erlangen-Nurnberg, Germany), Laurie Glimcher (Harvard School of Public Health, Boston, MA), Ann Marshak-Rothstein (Boston University, Boston, MA), and Larry Arnold (University of North Carolina, Chapel Hill, NC) for cell lines, and Dr. Greg Viglianti, Sarah Olken, and Michele Youd for critical reading of the manuscript.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants AI31209 and CA36642 (to R.B.C.) and by Grant IRG-97S from the American Cancer Society (to S.D.).

² Address correspondence and reprint requests to Dr. Stefan Doerre, Department of Microbiology, R509, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118. E-mail address:

³ Abbreviations used in this paper: , Ig light chain; CHX, cycloheximide.

Received for publication November 13, 1998. Accepted for publication April 13, 1999.

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