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The Journal of Immunology, 2004, 173: 818-827.
Copyright © 2004 by The American Association of Immunologists

Decreased E47 in Senescent B Cell Precursors Is Stage Specific and Regulated Posttranslationally by Protein Turnover1

Elaine Van der Put, Daniela Frasca, Anne M. King, Bonnie B. Blomberg2 and Richard L. Riley2,3

Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33101


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The E2A-encoded transcription factor E47 is crucial to B lymphopoiesis. Senescent BALB/c mice (~2 years old) had reduced pre-B cells ex vivo. Pro-B/early pre-B cells from these aged mice, both ex vivo and in vitro, were deficient in E47 protein. In vitro, IL-7 expanded pro-B/early pre-B cells from young BALB/c mice expressed E47 protein that was relatively stable over a 5-h period. Cultured senescent pro-B/early pre-B cells exhibited reduced E47 protein stability with ~50–90% loss of E47 over the same time period. Degradation of E47 was effectively blocked by the proteasome inhibitor lactacystin as well as calpain I and II inhibitors; E2A proteins were also shown to undergo ubiquitination. Although senescent B cell precursors expressed less E47 protein, E47 mRNA levels and turnover were normal. Therefore, E47 protein levels are reduced relatively early in B lineage differentiation in senescence and the decline in E47 protein occurs via increased protein degradation by proteasome and, possibly, calpain pathways. In contrast, normal E47 protein levels were observed within the highly reduced pre-B cell pool in aged mice. This suggests that pre-B cells in senescence undergo selection based on E47 expression. Increased degradation rates and lower steady-state levels were also observed for the transcription factors Pax-5/BSAP, Bob-1, and Ikaros, but this was not a general property of all proteins in aged B cell precursors. Therefore, altered turnover of multiple, select proteins crucial to B cell development may contribute to diminished B lymphopoiesis in old age.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The E2A gene encodes two transcription factors, E12 and E47, which are essential in the genetic program for B cell differentiation. These two RNA splice variants encode class I basic helix-loop-helix (bHLH)4 proteins and are involved in a large variety of developmental processes, characterized by their broad tissue distribution and by their ability to form homodimers or heterodimers with tissue-restricted class II bHLH. The two proteins share identical N-terminal transactivation domains, but have distinct dimerization and DNA-binding motifs (1, 2, 3, 4).

In B lymphocytes, the active DNA-binding complex consists of E47 homodimers (5, 6, 7), whereas in non-B cells, E47 binds DNA only as a heterodimer with cell-restricted bHLH proteins, such as MyoD or NeuroD (8, 9). E2A-null mutant mice fail to generate precursors and mature B cells (10, 11, 12, 13). The arrest of B cell development occurs at an early stage, since neither Ig DJ rearrangements nor expression of B lineage-specific markers can be detected in homozygous mutant mice (10, 11). Although either E12 or E47 expression can promote reconstitution of B cell precursors in transgenic mice, E47 appears to be considerably more efficient in promoting pre-B cell generation (12, 13).

E2A transcription factors have been shown to regulate the expression of several B lineage genes, such as {lambda}5, early B cell factor (EBF), TdT, and RAG-1 (12, 13, 14, 15, 16, 17). They also regulate Ig rearrangements (18, 19) and promote Ig class switch recombination (20). Studies in our laboratory (21) and others (22, 23) have shown that the levels of E2A proteins increase during the pro-B to pre-B cell transition. Notably, E2A mRNA levels differ little with stage of B lymphopoiesis (21, 23). Therefore, changes in E2A protein levels appear to be regulated, at least in part, via posttranslational mechanisms (e.g., phosphorylation; ubiquitination) and protein degradation (21).

Senescence is associated with decreased numbers of late-stage pre-B cells in the bone marrow (24, 25, 26, 27, 28). This has been shown to correlate with reduced responsiveness to the growth factor IL-7 as well as increased susceptibility to apoptosis (26, 27, 29, 30, 31). Pre-B cell generation is critically dependent on expression of the pre-BCR, comprised of the µ H chain along with the surrogate L chain proteins {lambda}5 and VpreB (32). We have previously demonstrated that aged B cell precursors show reduced expression of surrogate L chain along with decreased pre-B cell numbers (25, 33, 34). Notably, reduced surrogate L chain in aged B cell precursors results, in part, from decreased surrogate L chain transcription (25, 33). Since E47 functions to regulate surrogate L chain transcription, it is possible that decreased E47 expression and/or function may affect surrogate L chain levels. Our previous studies have shown decreased E2A (E47/E12) protein expression and DNA binding capacity in B cell precursors from aged mice after in vitro expansion with recombinant murine (rm) IL-7 (33, 34); this correlated with a decline in surrogate L chain expression (33). However, whether differences in E2A protein expression also occur in vivo during senescence and, if so, the mechanisms responsible for altering E2A protein levels in aged B cell precursors have not been addressed.

Our studies indicate that, like cultured B cell precursors, ex vivo isolated pro-B/early pre-B cells from senescent BALB/c mice have reduced E47 protein. However, although few in number, the residual late-stage pre-B cells in senescent mice maintain normal, relatively high E47 protein expression. The loss of E47 protein in aged B cell precursors mainly results from increased turnover of E47 protein. Although E47 protein levels at the pro-B cell stage in aged mice are reduced via increased degradation, the maintenance of normal E47 protein levels in the small residual late-stage pre-B cell pool of old mice presumably reflects selection for relatively high E47 expression and the necessity for increased E47 for pre-B cell development. Our findings indicate that alterations in the turnover of E2A proteins, as well as other proteins involved in regulating B cell development (Pax-5/BSAP; Bob-1/OCA-B, OBF-1; Ikaros), may contribute to the dysregulation of B lymphopoiesis seen in murine senescence.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Male and female young (2–4 mo of age) and old (21–27 mo of age) BALB/c mice were obtained from the National Institute on Aging.

Fluorescence-activated cell sorting

Bone marrow cells isolated from tibia and femur pairs of young and aged mice were sorted for pro-B/early pre-B cells and late-stage pre-B cells. Cells were stained for surface IgM (FITC), CD43 (PE, clone S7), and B220 (allophycocyanin, clone RA3-6B2; BD PharMingen, San Diego, CA) and sorted for lymphoid cells by forward light scatter, for IgM– cells and for either CD43+B220+ pro-B/early pre-B cells or CD43–B220+ late-stage pre-B cells by fluorescence flow cytometry using a FACSVantage SE (BD Immunocytometry Systems, San Jose, CA) with purity ranging between 80 and 95% for pro-B/early pre-B and 93–98% for late-stage pre-B cells. In some experiments, bone marrow cells were first enriched for B220+ cells by magnetic sorting according to the MiniMacs protocol (Miltenyi Biotec, Auburn, CA) and then sorted for pro-B/early pre-B and late-stage pre-B cell populations by fluorescence flow cytometry. Additional analysis for B lineage Hardy fractions (Fr.) was performed with an LSR flow cytometer (BD Immunocytometry Systems).

Bone marrow IL-7 pro-B/pre-B cultures

IL-7 expanded pro-B/pre-B cell cultures were performed as previously described (24, 25, 33). Bone marrow cells were resuspended at 1 x 106/ml in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 5% FCS (Sigma-Aldrich, St. Louis, MO), 3 x 10–5 M 2-ME (Sigma-Aldrich) plus penicillin-streptomycin (Life Technologies). rIL-7 (rmIL-7; BioSource International, Camarillo, CA) was added at 5 ng/ml at the initiation of culture. Nonadherent cells were harvested after 7 days and lysates were derived for EMSA or Western blot analysis. Data were analyzed for each individual experiment, which consisted of paired young and aged pro-B/pre-B cell cultures.

Preparation of nuclear and cytoplasmic extracts and total cell lysates

Nuclear and cytoplasmic extracts were prepared from cultured bone marrow cells essentially as previously described (34). Briefly, cells were harvested and centrifuged, and the pellet was resuspended in 30 µl of solution A containing 10 mM HEPES buffer with 10 mM KCl/1 mM EDTA/1 mM DTT/1.5 mM MgCl2/1 mM PMSF (pH 7.9) plus protease inhibitor mixture (Boehringer Mannheim, Mannheim, Germany) and Nonidet P-40 (0.1%). The supernatant containing the cytoplasmic extract was removed and the pellet containing the nuclei was resuspended in solution B containing 20 mM HEPES (pH 7.9) supplemented with 0.1 mM EDTA/1 mM DTT/1.5 mM MgCl2/2 mM PMSF plus protease inhibitor mixture and 10% glycerol. The lysate was incubated on ice for 20 min and protein was sonicated for a few seconds and centrifuged. Aliquots of the nuclear extract were stored at –80°C. Lysates were briefly vortexed and centrifuged. Protein content was determined by the Bradford assay.

For total cell lysates, cells were harvested and immediately lysed with 10 µl/106cells of Mammalian Protein Extraction reagent (Pierce, Rockford, IL) supplemented with Halt Protease Inhibitor Cocktail (Pierce) at 20 µl/ml to avoid any further protein degradation. For immunoprecipitation assays, lysates were incubated with agitation overnight at 4°C with 4 µg of affinity-purified rabbit pan-E2A Ab (E2A.E12, clone V-18; Santa Cruz Biotechnology, Santa Cruz, CA). The immune complexes were isolated by incubation with protein G agarose beads (Sigma-Aldrich) for 3 h at 4°C with agitation, followed by several washes (three times with PBS-Tween 20 and twice with PBS) and analyzed by Western blotting.

Assay of protein degradation rates

In vitro IL-7-expanded B cell precursors from young and aged BALB/c mice were harvested and cultured for up to 8 h with 200 µg/ml cycloheximide. Briefly, nonadherent B cell precursors were harvested at day 7 of culture, washed, and resuspended at 1 x 106/ml in RPMI 1640 supplemented with 5% FCS, 3 x 10–5 M 2-ME, and rIL-7 at 5 ng/ml. At 1-h intervals, equivalent numbers of cells were harvested and total cell lysates were prepared as described. E47 and actin proteins were detected by Western blot analysis as previously described (25, 33, 34) with identical cell equivalents of lysate loaded per lane at each time point. In some experiments, inhibitors of degradation pathways dissolved in DMSO were added or DMSO alone was added along with cycloheximide to cultured pro-B cells. Inhibitors used were lactacystin (Alexis Biochemicals, Carlsbad, CA), calpain I inhibitor (LLnL; Sigma-Aldrich), or calpain II inhibitor (LLM; Sigma-Aldrich).

DNA probes and EMSA

The µE5 probe was prepared as previously described (34) and labeled with [{gamma}-32P]ATP. A gel mobility shift assay to determine DNA binding of E47/E12 was performed as follows. The radiolabeled DNA probe was incubated with 10 µg of nuclear extract in the presence of 0.5 µg of poly(dI:dC) as nonspecific competitors for µE5. The reaction was performed at room temperature in 15 µl of running buffer (10x Tris-borate-EDTA,1 mM DTT, and 1 mM PMSF). The samples were electrophoresed in a 5% polyacrylamide gel at 175 V for 3 h at room temperature. The gels were dried on Whatman 3M and exposed to Kodak x-ray films (Rochester, NY) at –80°C overnight.

Western blotting

Reduced SDS-PAGE protein separation and Western blot analysis was performed as described elsewhere (25). Extracts of either sorted bone marrow cells or IL-7-expanded pro-B/pre-B cells were denatured by boiling for 4 min in sample buffer and subjected to SDS-PAGE using a 4–12% polyacrylamide gel under reducing conditions. Proteins were then electrotransferred onto nitrocellulose filters. Nonspecific sites were blocked by incubation of the membranes with PBS-Tween 20 (1x PBS/0.05% Tween 20) containing 10% milk for 1 h at room temperature (blocking solution). Filters were incubated with purified anti-human monoclonal anti-E47 (clone G127-32), purified mouse monoclonal anti-Pax-5 (BSAP), and purified hamster anti-mouse Bcl-2 Abs (BD PharMingen, San Diego, CA); mouse monoclonal IgG1 anti-actin Ab, rabbit polyclonal anti-Bob-1, goat polyclonal anti-UBC9 (N15), goat polyclonal anti-Ikaros (E-20), and rabbit polyclonal anti-Tyk 2 (C-20) (Santa Cruz Biotechnology); rabbit anti-Ku (80 kDa; Serotec, Raleigh, NC); HRP-labeled goat anti-mouse IgM µ-chain specific (Jackson ImmunoResearch, West Grove, PA); hamster anti-{lambda}5 mAb FS1 (25); or mouse anti-ubiquitin (clone Ubi-1; Zymed Laboratories, San Francisco, CA). Following overnight incubation with the primary Ab, immunoblots were incubated with the appropriate HRP-labeled secondary Abs for 2 h at room temperature, developed by enzyme chemiluminescence, and exposed to CL-XPosure film (Pierce).

RNA extraction and reverse transcription

Total RNA was isolated from 107 IL-7-expanded bone marrow pro-B/early pre-B cells using TRIzol reagent (Life Technologies) according to the manufacturer’s protocol, eluted into 100 µl of distilled water, and stored at –20°C until use. RT-PCR was performed using 2 µl of RNA at 0.5 µg/µl as template for cDNA synthesis. After an initial 4-min denaturation at 95°C, the cDNA was amplified for 30 cycles. Each cycle was programmed for denaturation at 95°C for 2 min, with annealing at 60°C for 2 min, with elongation at 72°C for 2 min, followed by a final extension phase of 3 min at 72°C. Primers for PCR amplification were: E2A forward, GCC-TGA-GCA-AGA-TGG-AGG-ACC-GCT-TG; E2A reverse, CAG-GGA-CAG-CAC-CTC-ATC-TGT-AC; GAPDH forward, ACC-ACA-GTC-CAT-GCC-ATC-AC; and GAPDH reverse, TCC-ACC-ACC-CTG-TTG-CTG-TA. Sizes of the detected PCR products were 452 and 454 bp for GAPDH and E2A, respectively. The PCR products were separated on 1.5% agarose gels. Gels were photographed using the Bio-Rad Gel-doc system (Hercules, CA) and images were quantitated using the Scion Image for Windows (Scion, Frederick, MD).

To evaluate RNA stability, RNA transcription was blocked in cultures of IL-7-expanded pro-B/early pre-B cells by actinomycin D (10 µg/ml; Sigma-Aldrich). After 10, 45, and 90 min, RNA was extracted and processed as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vivo senescent pro-B/early pre-B cells have low E47 protein expression

E2A proteins in general and E47 in particular have been shown to be crucial for generation of both pro-B and pre-B cells (10, 11, 12, 13). We have previously shown that levels of E2A proteins, both E47 and E12, are reduced in cultured B cell precursors derived from aged BALB/c mice (33, 34). To confirm that the reduced E47 protein levels seen in vitro in aged B cell precursors were also seen in vivo, E47 protein levels were estimated via Western blot analysis in B cell precursors freshly isolated from bone marrow of young (2–3 mo.) and aged (24–25 mo.) BALB/c mice. Table I shows the late-stage pre-B and pro-B/early pre-B cell composition of the young and aged BALB/c mice analyzed. All aged mice showed substantial decline (>75%) in late-stage pre-B cells (Hardy Fr. D (35, 36)) and mild (27–40%, O1, O3, O4, and O5) to severe (95%, O2) reductions in pro-B/early pre-B cells (Hardy Frs. A–C' (35, 36)) compared with young adult controls. Pro-B/early pre-B cells were gated as surface IgM–CD43+B220+ (Hardy Frs. A–C' (35, 36)) and late-stage pre-B cells as IgM–CD43–B220+ (Hardy Fr. D (35)) and isolated by fluorescence-activated cell sorting (Fig. 1). As shown in Fig. 2, pro-B/early pre-B cells isolated from four bone marrow preparations from senescent BALB/c mice all showed a decline in detectable E47 protein compared with that of the young controls via Western blot analysis.


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  		Table I. B Lineage precursors are decreased in aged BALB/c micea

 


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  		FIGURE 1. Isolation of late-stage pre-B cells and pro-B/early pre-B cells from young and aged BALB/c mice. Bone marrow cells from young and aged BALB/c mice were stained for surface B220, CD43, and IgM. Bone marrow cells were sorted by four parameters: forward light scatter as lymphoid cells, as IgM– cells, and as either CD43+B220+ cells (Frs. A–C'; pro-B/early pre-B cells) or CD43– B220+ cells (Fr. D; late-stage pre-B cells). Representative pre-sort histograms for bone marrow cells from young and aged mice are shown as well as post-sort histograms for isolated surface IgM–CD43+B220+ Frs. A–C' and IgM–CD43–B220+ Fr. D cells. Percentages refer to the proportions of Frs. A–C' and Fr. D cells in the bone marrow before sorting and in the isolated populations.

 


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  		FIGURE 2. Decreased E47 protein in senescent pro-B/early pre-B cells in vivo. Pro-B/early pre-B cells from the femurs/tibiae of young and aged mice (see Table I) were detected as surface IgM– CD43+B220+ (Hardy Frs. A–C') and were isolated by fluorescence flow cytometry. Pro-B/early pre-B cell lysates were electrophoresed and Western blotted for detection of E47 and actin proteins. A, E47 protein expression for BALB/c pro-B/early pre-B cells from young preparations Y1, Y2, Y3, and Y5 and old preparations O1, O2, O3, and O5. Because of poor recovery of pro-B/early pre-B cells from O4, the Y4/O4 pair was not analyzed. B, Summary of E47 protein levels relative to actin determined via densitometry for the young and aged pro-B/early pre-B cells shown in A.

 
In contrast to the reduced E47 protein seen in aged pro-B/early pre-B cells, senescent late-stage Fr. D pre-B cells expressed E47 protein that was equal to or greater than that seen in young pre-B cells (Fig. 3). Importantly, late-stage pre-B cells in these aged mice were markedly reduced compared with young controls (Table I). These results suggest that, while E47 expression is reduced early in B cell development in senescence, the limited, residual small pool of pre-B cells generated in aged mice maintains relatively high levels of E47 protein.



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  		FIGURE 3. Late-stage pre-B cells from senescent mice retain high levels of E47 protein in vivo. Late-stage pre-B cells (Hardy Fr. D) were detected as surface IgM–CD43–B220+ cells in bone marrow of young and aged BALB/c mice (Table I) and were isolated by fluorescence cell sorting. E47 and actin proteins from pre-B cell lysates were detected by Western blotting. A, E47 protein expression in pre-B cells isolated from BALB/c young bone marrow preparations Y1, Y3, Y4, and Y5 and aged preparations O1, O3, O4, and O5. O2 had extremely few pre-B cells (Table I), precluding their isolation and analysis of the Y2/O2 pair. B, Summary of the expression of E47 protein relative to actin determined via densitometry for the pre-B cell preparations in A. C, Comparison of E47 and actin protein expression in both pro-B/early pre-B cells and late-stage pre-B cells from young and aged bone marrow preparations Y1/O1, Y3/O3, and Y5/O5. B lineage cell fractions were detected and isolated, and E47 and actin proteins were measured as in Figs. 1 and 2.

 
In vitro senescent pro-B/early pre-B cells have low E47 protein expression

The low numbers of pro-B/early pre-B cells that could be isolated directly from individual mice precluded examination of the mechanisms responsible for reduced E47 expression in senescence. However, previously we have reported that E2A protein levels were similarly reduced in B cell precursors from aged mice upon expansion and culture with the growth cytokine IL-7 (33, 34). These IL-7-expanded B cell precursors were used for further analysis of the mechanisms of E47 decrease in aged mice. Typically, B cell precursors derived in vitro from either young or aged mice consisted predominantly of pro-B cells (80–90%, cytoplasmic µ negative) with ≤20% pre-B cells (cytoplasmic µ positive) (31, 33). As shown in Fig. 4, A and C, decreased E47 protein was observed in IL-7-expanded B cell precursors from aged mice relative to young controls as expected and consistent with the decline in E47 protein seen in pro-B/early pre-B cells ex vivo.



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  		FIGURE 4. In vitro IL-7-expanded B cell precursors from aged mice have reduced E47 protein levels and show decreased E2A binding to DNA. B cell precursors from both young and aged mice were expanded in vitro with rmIL-7 as described in Materials and Methods. Whole-cell or nuclear lysates were prepared from cultured B cell precursors and used in Western blots or EMSA analysis. A, Western blot for individual young and aged BALB/c mice (representative of five to six individual mice of each group). Cultured pro-B cells were derived from both aged mice with moderate depletion of late stage (Fr. D) pre-B cells (30–70%) and no loss of pro-B/early pre-B cells (Frs. A-C/C') or from aged mice with severe depletion of pre-B cells (>80%) and partial (~50%) loss of pro-B cells as described previously (27 ). B, EMSA results indicating binding of nuclear E2A proteins from cultured B cell precursors from aged mice to the µE5 probe. Inclusions of either anti-E47 or anti-E12 mAbs were used to establish specificity. Results are for the same mice depicted in A. C, Summary of Western blot analyses showing relative E47 protein to actin ratios for IL-7-expanded pro-B cells from five young BALB/c mice, six aged BALB/c mice with moderate pre-B/pro-B depletion, and six aged BALB/c mice with severe pre-B/pro-B depletion. *, p < 0.05 by Student’s t test vs young controls.

 
Moreover, decreased E2A proteins in cultured senescent B cell precursors also led to reduced E47 and E12 transcription factor-binding activity to the µE5 sequence (Ref.34 and Fig. 4B). Both E12 and E47 participate in the DNA-binding complexes in B cell precursors, as indicated by the depletion of protein-DNA complexes seen in EMSA with both anti-E12 and anti-E47 Abs (Ref.34 and Fig. 4B). Both E47-E47 and E47-E12 complexes with µE5 sequences were observed (Ref.34 and Fig. 4B). However, since E12 has a lower binding affinity for target DNA sequences as compared with E47 (1), our studies herein have focused on determining the levels of E47 protein in aged and young B cell precursors.

We have recently shown that the reduction in B cell precursors, which characterizes murine senescence, is highly variable (27, 37). This is exemplified by the range of pro-B/early pre-B and late-stage pre-B cells seen in the aged mice in Table I. We have categorized those senescent mice with ~30–70% loss of Fr. D pre-B cells in vivo as "moderately pre-B depleted" (27, 37). Those with more extensive loss of Fr. D pre-B cells in vivo (>80%) are termed "severely pre-B depleted." These categories of senescent mice differ not only in their numbers of B cell precursors, but also in the capacity of their B cell precursors to undergo proliferation and survival (27). The incidences of mice with "severe pre-B depletion" increases with old age, suggesting a progression from the moderate to severe pre-B cell phenotype (27).

Previous results indicated that the E47 protein levels seen in cultured senescent B cell precursors were associated with the extent of pre-B cell decline observed in aged mice in vivo (33). As shown in Fig. 4, A and C, cultured B cell precursors from aged mice with severe depletion of pre-B cells showed extensive (~70%) reduction in relative levels of E47 protein and highly reduced DNA-binding activity by EMSA (Fig. 4B). Cultured B cell precursors from aged mice with more moderate pre-B cell depletion showed variable and intermediate decline in E47 protein levels.

E2A mRNA levels and mRNA stability are not altered in aged mice

The decline in E47 proteins seen among aged pro-B cells could reflect alterations in transcriptional regulation or, alternatively, increased protein turnover. E2A mRNA levels were analyzed from cultured B cell precursors from aged and young BALB/c mice by semiquantitative RT-PCR. E2A mRNA levels from in vitro expanded pro-B cells from aged mice either moderately depleted or severely depleted of pre-B cells in vivo were comparable to those seen in young controls (Fig. 5). A possible role for altered RNA stability of these transcripts was evaluated by blocking mRNA transcription with actinomycin D and following mRNA loss over time. E2A mRNA transcripts from both young and aged mice showed similar stability (Fig. 5), suggesting that alterations in transcription are not responsible for the reduced levels of E47 protein seen in senescent B cell precursors.



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  		FIGURE 5. IL-7-expanded pro-B cells from aged mice maintain normal expression and stability of E2A mRNA. A, RNA was extracted from the same number (1 x 106) of cultured pro-B cells from young, severely depleted, or moderately depleted aged mice, untreated or treated with actinomycin D (10 µg/ml) for 10, 45, and 90 min, and RT-PCR was performed. B, Means of the densitometric estimates (normalized to GAPDH) ± SE of three independent RT-PCR experiments (1/4 titration). Differences between young and severely or moderately depleted old mice were not significant.

 
E47 protein degradation is increased in aged B cell precursors in vitro

The possibility that lower E47 protein levels in pro-B cells from aged mice resulted from changes in protein turnover was tested with cultured B cell precursors from individual young and aged mice. Upon blockage of protein synthesis with cycloheximide, E47 proteins from cultured B cell precursors from young mice were relatively stable over a 5-h period (Fig. 6). In contrast, aged B cell precursors showed accelerated E47 protein degradation. Over a 5-h period, aged B cell precursors derived from mice with moderate pre-B depletion exhibited ~50% reduction in E47 protein (Fig. 6). The kinetics of E47 protein loss in these aged B cell precursors appeared to be biphasic with about one-half of E47 protein being stable over the 5-h period. In contrast, the extent of E47 protein degradation was greater and more complete in aged B cell precursors cultured from the bone marrow of mice with severe pre-B loss as compared with those with more moderate pre-B reduction (Fig. 6). For these severely pre-B-depleted mice, ~90% of E47 protein was degraded within 5 h (Fig. 6). Note that actin was stable for 5 h after cycloheximide treatment for both young and aged mice (Fig. 6).



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  		FIGURE 6. IL-7-expanded B cell precursors from aged mice exhibit increased turnover of E47 protein. In vitro IL-7-expanded B cell precursors from young and aged BALB/c mice were harvested and cultured for up to 5 h with 200 µg/ml cycloheximide (CHX). Cultured B cell precursors were lysed and E47 and actin proteins were detected by Western blot analysis with time after initiation of the cycloheximide protein synthesis block. A, Representative results for E47 protein turnover for B cell precursors from young and aged mice that were either moderately or severely deficient in ex vivo late-stage (Fr. D) pre-B cells. Lysates equivalent to ~4 x 105 cells for young and to ~6 x 105 cells for aged mice were loaded per lane. B, Summary of E47 protein turnover for young and aged mice shown in Fig. 4C: five young mice as well as six aged mice with moderate and six aged mice with severe deficits in B lymphopoiesis as defined previously (27 ). *, p < 0.05 by Student’s t test.

 
IL-7-expanded B cell precursors from both young and aged mice, up to 5 h after cycloheximide treatment, did not show differences in apoptosis as measured by annexin V staining. Before cycloheximide treatment, young B cell precursors exhibited 8.9 ± 1.4% annexin V+ cells compared with 9.3 ± 1.1% for aged B cell precursors (averages ± 1 SD; n = 3). After 5 h of cycloheximide treatment, annexin V staining was 23.4 ± 5.2% for young and 21.6 ± 1.7% for aged B cell precursors. Cell viability at 5 h after cycloheximide, as measured by trypan blue exclusion, was routinely ~90% for both young and aged B cell precursors. Therefore, it is unlikely that differences in apoptosis between young and aged B cell precursors contribute to the altered turnover of E47 protein in our studies.

As shown above, E47 protein steady-state levels were generally decreased in aged B cell precursors. Decreased levels of several other proteins were seen in cultured B cell precursors from aged mice, including Pax-5/BSAP, {lambda}5, and Bob-1/OCA-B/OBF-1 (Table II, Fig. 7, and Ref.33). The declines in Pax-5/BSAP and Bob-1 proteins, like E47, also were associated with accelerated degradation (Fig. 7 and Table III). Although {lambda}5 was decreased in aged B cell precursors, the turnover of {lambda}5 protein was not significantly different from that seen in young B cell precursors (Fig. 7 and Table III). Therefore, the reduced steady-state levels of {lambda}5 protein likely result from declines in {lambda}5 mRNA expression in aged B cell precursors as we have previously reported (33). For Ikaros, variable levels were observed in aged compared with young B cell precursors from individual mice (range, 35–94% of young levels; Fig. 7 and Table II) and this was reflected in the degradation seen over time (Fig. 7 and Table III). More limited or no changes in steady-state levels and protein degradation were observed for Ku80, Bcl-2, Tyk-2, and UBC9 (Fig. 7 and Tables II and III).


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  		Table II. Steady-state levels of proteins differ in aged B cell precursorsa

 


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  		FIGURE 7. Altered protein turnover is selective in aged B cell precursors. Whole-cell lysates (~5 x 104 cells) from either young or aged BALB/c IL-7-expanded bone marrow B cell precursors were assayed for a panel of proteins by Western blot. Lysates were obtained either at time 0 or after a 5-h.treatment with cycloheximide as in Fig. 6. Data are representative of at least three experiments.

 

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  		Table III. Altered protein degradation is selective in aged B cell precursorsa

 
E47 protein is degraded by proteasome and calpain pathways

Previous reports indicated that E2A proteins might undergo ubiquitin addition and degradation via the proteasome (38, 39). Alternatively, E2A proteins could undergo cleavage via calpain or other protease recognition of a PEST domain proximal to the DNA-binding region (39). Upon treatment with cycloheximide, IL-7-expanded cultured B cell precursors from aged mice showed almost complete degradation of E47 protein within 8 h (Fig. 8). Addition of lactacystin, a proteasome-specific inhibitor (40), led to significant increases in E47 protein levels above those seen in untreated controls (Fig. 8). Inhibition of calpain, an intracellular nonlysosomal cysteine protease (41, 42), with either the aldehyde peptides LLnL (calpain inhibitor I) or LLM (calpain inhibitor II) also partially increased E47 protein levels in aged B cell precursors (Fig. 8A). Similar results were seen in five experiments using IL-7-expanded B cell precursors from aged mice with moderate to severe deficits in B lymphopoiesis. These experiments clearly indicate that the low levels of E47 protein typical of pro-B/early pre-B cells in senescence derive predominantly from extensive degradation mediated by proteasome and possibly nonproteasome pathways.



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  		FIGURE 8. The rapid turnover of E47 protein in aged B cell precursors is prevented by proteasome and calpain inhibitors. A, In vitro IL-7-expanded B cell precursors from aged BALB/c mice were treated with cycloheximide (CHX) as in Fig. 6, but for 8 h and in the presence or absence of either 10 µM lactacystin, 100 µM calpain I inhibitor (LLnL), or 100 µM calpain II inhibitor (LLM). E47 and actin protein levels in whole-cell lysates were measured by Western blotting. Data are for one of five representative experiments using aged individual mice that were either moderately depleted or severely depleted of Fr. D late-stage pre-B cells as described elsewhere (27 ). Levels of E47 protein were determined via densitometry and are shown as a percentage of initial E47 protein present at time 0. B, Cell lysates were obtained from IL-7-expanded B cell precursors from either two young BALB/c mice (Y1 and Y2) or two aged BALB/c mice (O1 and O2) that exhibited the moderate pre-B cell depletion phenotype. E2A proteins were immunoprecipitated from these cell lysates with rabbit pan-anti-E2A Ab. Lysate equivalent to either 5 or 8 x 106 IL-7-expanded B cell precursors from young or aged mice, respectively, were immunoprecipitated as described in Materials and Methods. Approximately 2-fold more lysate from aged B cell precursors was immunoprecipitated since aged B cell precursors typically exhibited less E2A proteins (see Fig. 4 and Ref.33 ). In either case, total immunoprecipitated material was loaded into the lanes indicated. E47 protein was detected with mAb G127-32 and Ub protein was detected with anti-Ub mAb by Western blotting. Two mock immunoprecipitation controls were performed, C1 and C2. C1 consisted of saline mixed with anti-E2A capture Ab and protein G beads (without cell lysate) while C2 consisted of cell lysate mock immunoprecipitated with protein G beads in the absence of specific E2A capture Ab.

 
That E2A proteins undergo ubiquitination was directly shown with immunoprecipitates of E2A derived from both young and aged IL-7-expanded B cell precursors. We have examined ubiquitin modification of E2A proteins produced by both young and aged B cell precursors. Note that the latter were derived in vitro from aged mice with the "moderate depletion" phenotype which typically show a milder increase in E47 degradation (Fig. 6). B cell precursors cultured from aged mice with "severe depletion" phenotypes yielded little or no E2A in immunoprecipitation, as discussed above, and were not amenable to analysis. As shown in Fig. 8B, E2A proteins immunoprecipitated from either young or aged B cell precursors showed ubiquitin (Ub) modification. A range of Ub-labeled species was observed; this was reproducible for three independent experiments. Although minimal Ub modification was seen for full-length E2A molecules (~72 kDa.), Ub modification was particularly evident for smaller immunoprecipitated proteins that presumably represent partially degraded E2A fragments (33). Ub-labeled species were also seen at molecular masses above 72 kDa; these may represent polyubiquitinated E2A molecules and were seen in lysates from both young and aged B cell precursors. The observation that E2A proteins are Ub modified in B cell precursors cultured from aged mice is consistent with their degradation via the proteasome and partial inhibition of degradation upon treatment with lactacystin (43).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The expression of E2A transcription factors signifies an early, critical step along the B lymphopoietic pathway (1). E2A proteins promote the commitment to the B lineage via regulation of more distal transcription factors including EBF and Pax-5/BSAP (1). E2A, often in tandem with these other transcription factors, facilitates expression of a variety of B lineage-associated molecules including TdT, RAG-1, mb-1 (Ig{alpha}), IL-7R{alpha}, and surrogate L chain ({lambda}5, VpreB) (1, 12, 13, 14, 15, 16, 17). In addition, E2A proteins participate in regulation of Ig H and L chain rearrangement (18, 19) as well as proliferation (23) and survival (44) of B cell precursors. Although both E47 and E12 E2A encoded splice variants, when expressed as transgenes, promote partial recovery of B cell precursors in mice lacking endogenous E2A, E47 appears critical in progression to the late pre-B cell stage (12, 13). In the adaptive immune responses of mature B cells, E2A proteins are required for efficient isotype switching (20) and somatic hypermutation (45). Given the numerous molecules and functions affected by E2A proteins, it is likely that levels of E2A proteins are subject to stringent controls.

In old age, B lymphopoiesis is compromised in murine models and is generally characterized by progressive loss of pre-B cells and, in more severe instances, partial loss of pro-B cells within the bone marrow (24, 25, 26, 27, 28). The B lymphopoietic deficiencies in senescence are predominantly manifest at the pro-B to pre-B cell transition (24, 25, 26, 27, 28). Decline in pre-B cells in old age has been associated with several mechanisms including poor responses to the growth promoting cytokine IL-7 (26, 27, 30), reduced Ig rearrangement (46), and/or increased apoptosis (27, 29). We have shown a decline in surrogate L chain expression during B lymphopoiesis in senescence and correlation between reduction in surrogate L chain and loss of pre-B cells (25, 33). Presumably, reduced surrogate L chain affects expression of the pre-BCR required for early pre-B cell proliferation and selection at the pro-B to pre-B cell transition (32). E47, in combination with EBF, regulates expression of surrogate L chain during pro-B cell development (16). Initial studies using IL-7-expanded, cultured B cell precursors from aged mice indicated that E2A protein levels were significantly decreased in senescence and this was associated with both reduced surrogate L chain and pre-B cell generation in vivo (33).

Our current studies indicated that, similar to in vitro observations, E47 protein levels are also decreased in senescent pro-B/early pre-B cells in vivo. As shown in cultured B cell precursors, the reduced E47 protein levels seen in senescence are not due to changes in E2A mRNA expression or stability. Rather the decreased levels of E47 protein observed in aged B cell precursors are due to increased protein turnover. Blocking degradation of E47 via either proteasome-mediated or calpain-mediated pathways effectively restored E47 protein levels in aged B cell precursors. Although lactacystin is regarded as highly specific for inhibition of the proteasome (40), calpain I inhibitor may affect both calpain and the proteasome (41, 42). Calpain II inhibitor is regarded as more specific for the calpain pathway (41, 42); therefore, while proteasome-mediated degradation of E2A proteins is likely, based on our results and previous observations (38, 39), the role of the calpain pathway in E47 degradation remains to be firmly established. A role for the proteasome in the degradation of E2A molecules has also been indicated by previous findings, in vitro, in cell lines and activated splenocytes, where E2A is ubiquitinated (38, 39, 43). Herein, we now show that E2A molecules are also Ub-modified in pro-B/early pre-B cells and our data suggest that the Ub-proteasome pathway contributes to the decreased E47 protein stability and steady-state levels seen in senescent B cell precursors. Increased E47 protein turnover was associated with the extent of pre-B cell reduction in old age. Cultured B cell precursors from aged mice with "severe" pre-B loss had more extensive reduction of E47 protein and increases in E47 turnover than did those from aged mice with "moderate" pre-B depletion. Therefore, decline in early B cell precursor E2A protein, in part due to alterations in protein turnover, may be progressive during old age.

The increased degradation seen in E47 in aged B cell precursors is not a "global" phenomenon since turnover of a variety of other diverse proteins is not altered in aged B cell precursors. However, several other transcription factors, including Pax-5/BSAP, Bob-1/OCA-B/OBF-1, and, to a lesser extent, Ikaros, do show increased turnover in aged B cell precursors. Notably, like E2A, these transcription factors play important roles in B lineage differentiation (1, 47, 48, 49). Of interest, aged B cell precursors show a decline in capacity to proliferate in response to IL-7 (26, 27, 30, 31, 50). We have previously shown that E47 protein levels are adversely affected by stem cell factor (SCF)/c-kit ligand stimulation of B cell precursors via increased E47 protein degradation (21). Aged B cell precursors respond poorly to IL-7 (26, 27, 30, 31, 50), but normally to SCF (50). Conceivably, imbalances among cytokine signals (IL-7 vs SCF) and/or other microenvironmental signals may affect the stability of E47 in aged pro-B cells.

Aged pro-B/early pre-B cells, both in vivo and in vitro, showed reduced E47 protein when compared with young controls; in contrast the remaining low numbers of late-stage pre-B cells from aged mice retained relatively high E47 protein expression. Previous reports indicate that the levels of E47 protein may increase with transition from early pro-B to late-stage pre-B cells (21, 22, 23). Since E47 expression appears to be involved in progression to the pre-B stage (12, 13), conceivably up-regulation of E47 is an important step in the pro-B to pre-B transition. The relatively poor expression of E47 protein seen in aged pro-B/early pre-B cells may contribute to decreased pre-B cell generation in senescence. This may be related to the importance of E47 for surrogate L chain, and preBCR, expression (25, 33, 34) or other functions related to proliferation and/or survival (24, 25, 26, 27, 28, 29, 30, 31). However, the limited number of pre-B cells produced in aged mice do retain normal, relatively high, levels of E47 protein. Conceivably, pre-B cells in senescent mice are subject to extensive selection, which may be affected by compromised surrogate L chain expression (25, 33, 34) and increased susceptibility to apoptosis (27, 29). Presumably only those pre-B cells in aged mice that retain capacity for sufficient E47 expression continue further maturation. This underscores the necessity for relatively high E2A expression as a likely prerequisite for pre-B formation.

Our studies establish that 1) E47 protein levels are reduced in pro-B/early pre-B cells of aged BALB/c mice; 2) E47 protein levels are maintained normally in the small late-stage pre-B cell pool of aged mice; and 3) decreased E47 protein levels in aged B cell precursors are a result of increased proteasome-mediated protein degradation. Two molecules which serve, directly or indirectly, as E47 target genes, the {lambda}5 surrogate L chain and Pax-5/BSAP transcription factor, are also reduced in aged B cell precursors. In the case of Pax-5/BSAP, the reduction is, in part, due to accelerated degradation. For {lambda}5, reduced steady-state levels do not correlate with altered turnover, but rather decreased mRNA expression (25, 33). This, along with the association of reduced {lambda}5 protein levels with E2A loss in aged B cell precursors, suggests diminished E2A levels may affect {lambda}5 transcription.

E2A molecules have multiple roles in promoting such diverse functions as Ig rearrangement, preBCR selection, proliferation, and survival. Although the spectrum of target genes that may be abnormally influenced by partial E2A reduction in aged B cell precursors has yet to be fully explored, it is notable that partial E2A decreases even in young wild-type mice substantially affects pro-B and/or pre-B cell development as well as functions of mature B cells (11, 23, 51, 52, 53). That reduced E2A expression in senescent B lymphopoiesis occurs via increased degradation and altered turnover also extends to other proteins crucial for B lineage development (e.g., Pax-5/BSAP; Bob-1) suggests that dysregulation of protein turnover likely constitutes a novel and important defect in B lymphopoiesis during senescence.


   Acknowledgments
 
We thank James Phillips and the Sylvester Comprehensive Cancer Center Flow Cytometry Core Resource for assistance with cell sorting.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AG 15474 (to R.L.R.) and AG 17618 (to B.B.B.). Back

2 B.B.B. and R.L.R. contributed equally as senior authors. Back

3 Address correspondence and reprint requests to Dr. Richard L. Riley, Department of Microbiology and Immunology, University of Miami School of Medicine, P.O. Box 016960 (R-138), Miami, FL 33101. E-mail addressed: rriley{at}med.miami.edu Back

4 Abbreviations used in this paper: bHLH, basic helix-loop-helix; EBF, early B cell factor; rm, recombinant murine; Fr., fraction; Ub, ubiquitin; SCF, stem cell factor. Back

Received for publication October 22, 2003. Accepted for publication April 19, 2004.


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