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The Journal of Immunology, 2007, 178: 3521-3529.
Copyright © 2007 by The American Association of Immunologists, Inc.

Accelerated Notch-Dependent Degradation of E47 Proteins in Aged B Cell Precursors Is Associated with Increased ERK MAPK Activation1

Anne M. King, Elaine Van der Put, Bonnie B. Blomberg and Richard L. Riley2

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The transcriptional regulator E47, encoded by the E2A gene, is crucial to B lymphopoiesis. In BALB/c senescent mice (~2 years old), the incidence of E47-expressing pro-B cells in vivo and E47 protein steady state levels in B cell precursors in vitro were reduced. Poor expression of E47 protein was a consequence of accelerated proteasome-mediated turnover and was associated with heightened ubiquitin modification of E2A-encoded proteins in aged B cell precursors. Both MAPK and Notch activity have been previously associated with E2A-encoded protein stability in lymphocytes. Aged B cell precursors exhibited heightened levels of MAPK activity reflected in increased levels of phospho-ERK proteins. Phosphorylation of E2A-encoded proteins was also increased in aged B cell precursors and pharmacologic inhibition of MEK-1 resulted in a partial restoration of their E47 protein. Both Notch proteins and their Delta-like ligands were detected comparably in young and aged B cell precursors. Either inhibition of Notch activation via gamma-secretase or Ab blockade of Notch-Delta-like ligand interactions partially restored E47 expression in aged B cell precursors. We hypothesize that increased MAPK activity promotes phosphorylation of E2A-encoded protein in aged B cell precursors. Subsequently, E2A-encoded proteins undergo ubiquitination and accelerated degradation in a Notch-dependent process. The dysregulation of E2A-encoded protein expression may contribute to the reductions seen in early B lymphopoiesis during murine senescence.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
As mice age, the process of B lymphopoiesis diminishes substantially (1, 2, 3, 4, 5, 6, 7, 8). By 2 years of age, marked losses are apparent in the late pre-B cell compartment and, in a subset of aged mice, within the pro-B cell compartments as well (3, 4, 6, 8). The decline in B lymphopoiesis in senescent mice coincides with reduced expression of RAG-2 and capacity to rearrange genes at the VH loci (5, 6, 7). Furthermore, diminished expression of the surrogate L chain components {lambda}5 and VpreB in aged B cell precursors suggests that signaling may be compromised at the pre-BCR checkpoint (3, 8, 9). Both transcription of the RAG enzymes and the surrogate L chain proteins in B cell precursors are, in part, mediated by the E2A-encoded transcription factors E47 and E12 (10). Previously, we have shown that E2A-encoded protein levels are reduced in B cell precursors from aged mice after their expansion with IL-7 in vitro (9, 11, 12). This was associated with reduction in the expression of {lambda}5 protein and reflected in decreased {lambda}5 and VpreB mRNAs (9). The reduced steady-state levels of E47 proteins seen in cultured aged B cell precursors were demonstrated to result primarily from increased proteasome-mediated protein turnover (12).

The mechanisms that regulate E2A gene expression are of importance to an understanding of B lineage commitment and development. We have previously shown that, in senescence, posttranscriptional mechanisms in B cell precursors generate reduced E47 protein expression (12); however, in activated splenic B cells reduced E47 mRNA stability is responsible for lower levels of E47 protein (13). In a recent report (14), the degradation of E47 proteins in lymphocytes was shown to be promoted by serine/threonine MAPKs and to be highly dependent upon activation of the Notch pathway. It is likely that phosphorylation is an early step in the degradation pathway for E47.

At present, the mechanisms responsible for the accelerated turnover of E47 protein in aged B cell precursors have not been established. In this study, we have examined the expression of E47 proteins in vivo and in vitro by B lineage precursors from young and aged mice and addressed the hypothesis that alterations in the MAPK and/or Notch pathways promote the ubiquitination and degradation of E47 protein in aged B cell precursors. Dysregulation of E2A-encoded protein expression in old age may curtail effective generation of B cell precursors and contribute to the deficiencies in B lymphopoiesis typical of murine senescence.


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

Young (2–4 mo) and aged (21–26 mo) BALB/c mice were purchased from the National Institutes of Aging colony at Harlan Sprague Dawley. Mice with obvious abdominal tumors and/or splenomegaly were eliminated from the studies. RAG-2 knock-out mice were purchased from Taconic Laboratories and were 2–4 mo of age. E2A heterozygous mice (15, 16) on the C57BL/6 background were provided by Drs. M. O’ Riordan and R. Grosschedl (University of California, San Francisco, CA) originally from Dr. Y. Zhuang (Duke University Medical Center, Durham, NC) and bred in our colony.

Expansion of B cell precursors with IL-7 in vitro

Femur and tibia pairs were flushed to harvest cells from the bone marrow. RBC were removed by treatment with ACK (0.15 M NH4Cl, 1 mM KHCO3, and 0.1 mM EDTA) for 5 min at room temperature followed by centrifugation to remove red cell debris. Bone marrow cells were counted and resuspended at 1 x 106/ml in RPMI 1640 (Invitrogen Life Technologies), supplemented with 10% FCS (Sigma-Aldrich) plus 1% penicillin-streptomycin, 1% L-glutamine, and 2-ME at 2 x 10–5 M. Purified recombinant mouse IL-7 (BioSource International) was added at 5 ng/ml and remained in culture for 4–8 days after which nonadherent cells were harvested and used for RT-PCR, Western blot, or immunoprecipitation analysis. In some experiments, CD19+ bone marrow cells were isolated by magnetic sorting (>96% purity) before culture with IL-7.

Immunoprecipitation and Western blot analysis

For total cell lysates, cells were harvested, counted, and lysed with Mammalian Protein Extraction Reagent (Pierce) at 10 µl/106 cells. Protein extraction reagent was supplemented with Halt Protease Inhibitor Cocktail (Pierce) at 10 µl/ml. For phospho-ERK (P-ERK)3 analysis, total cell lysates also contained 2 mM Na3VO4. Extracts of IL-7-expanded pro-B/pre-B cells were denatured by boiling for 4 min in sample buffer, subjected to reducing conditions, and electrophoresed using SDS-PAGE 4–12% polyacrylamide gels for 50 min at 200 V. Proteins were run out on gels, then transferred onto nitrocellulose membranes for 90 min at 100 V. Nonspecific sites were blocked by incubation of the membranes with PBS-Tween 20 (1x PBS/0.05% Tween 20) containing 10% milk for 2 h at room temperature. Membranes were incubated as required with purified mouse monoclonal anti-E47 (G127-32) (BD Biosciences); mouse monoclonal anti-actin (C-2), goat polyclonal anti-UBC9 (N-15); goat polyclonal anti-Notch 1 (M-20); rabbit polyclonal anti-Jagged 1 (H-66); rabbit polyclonal anti-Delta (H-265) (Santa Cruz Biotechnology); rabbit polyclonal Ab specific for phosphorylated (Thr355) on either E47 or E12 proteins (BL 1029; Bethyl Laboratories); rabbit polyclonal Ab anti-phospho-p44/42 MAPK (197G2) (Cell Signaling Technology); and/or mouse anti-ubiquitin (Ubi-1; Zymed Laboratories). 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) or analyzed via an Alpha Innotech FluorChem Gel-doc system.

For immunoprecipitation assays, lysates were incubated with mixing overnight at 4°C with 8 µg of affinity-purified rabbit anti-E2A (E2A.E12,V-18; Santa Cruz Biotechnology) or, in some instances, a mixture of monoclonal anti-E47/anti-E12 Abs (BD Biosciences). Immune complexes were isolated by incubation with protein G agarose beads (Sigma-Aldrich) for 2 h at 4°C with agitation, followed by three washes with 1x PBS-Tween 20, two washes with 1x PBS, and then analyzed by Western blotting. For quantitation, in each experiment (comprised of both aged cell lysate and young controls), individual blots were scanned and analyzed for desired proteins using densitometry. Densitometry values for desired proteins were normalized to that of the loading controls. In some experiments, normalized values of experimental proteins in old lysates (e.g., E47) were expressed relative to young controls (9, 12).

Flow cytometry and magnetic cell sorting

Mouse bone marrow cells harvested ex vivo were stained fluorescently for surface IgM (II/41), CD43 (S7), B220 (RA3-6B2), CD19 (1D3) (BD Biosciences), and/or AA4.1 (eBioscience). Cells were cytoplasmically stained for E47 with PE-anti-E47 per BD Biosciences (G127-32). In brief, cells were resuspended in 200 µl of Cytofix for 20 min on ice following the primary stain. Cells were then washed with PBS plus 5% FCS and permeabilized with 200 µl of PBS/0.2% Tween 20 for 15 min at 37°C. Cells were finally washed with PBS plus 5% FCS and stained cytoplasmically for E47. Cells were analyzed within a half-hour of staining.

Analysis was either on an LSR I or LSR II flow cytometer (BD Biosciences). IL-7-expanded B cell precursors were surface stained for CD19 (1D3; BD Biosciences) and CD2 (LFA-2; eBioscience). In other studies, IL-7-cultured B cell precursors were sorted for either CD2+ or CD2– cells by magnetic bead separation before analysis. Cells were first surface stained with anti-CD2 PE and then secondary stained using anti-PE microbeads according to the MiniMacs protocol (Miltenyi Biotec). Purity of CD2+ cells was >96% and for CD2– cells was >98%. CD19-positive cells were similarly magnetically sorted by first surface staining with anti-CD19 PE (1D3) followed by secondary incubation with anti-PE microbeads. CD19-positive cells were separated from CD19-negative cells on a magnetic column and post sorts revealed 90–95% purity.

RNA extraction and RT-PCR

Total RNA was isolated from 2 x 106 IL-7-expanded bone marrow pro-B/early pre-B cells using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s protocol. Following isolation, RNA was eluted into 10 µl of diethyl pyrocarbonate-treated water and stored at –80°C. Reverse transcription was performed using 2 µl of RNA at 0.5 µg/µl as template for cDNA synthesis. The cDNA was amplified for 35 cycles using the following program: denaturation at 94°C for 30 s, with annealing at 55°C for 1 min, with elongation at 72°C for 45 s, followed by a final extension phase of 3 min at 72°C. Primers for PCR amplification were for Notch-1 through Notch-4 and Hes-1 and Hes-5 (17): Notch-1 forward, 5'-TGCCTGTGCACACCATTCTGC-3'; Notch 1 reverse, 5'-CAATCAGAGATGTTGGAATGC-3'; Notch 2 forward, 5'-ATGCACCATGACATCGTTCG-3'; Notch 2 reverse, 5'-GATAGAGTCACTGAGCTCTCG-3'; Notch 3 forward, 5'-TTGGTCTGCTCAATCCTGTAGC-3'; Notch 3 reverse, 5'-TGGCATTGGTAGCAGTTGCTG-3'; Notch 4 forward, 5'-AAGCGACACGTACGAGTCTGG-3'; Notch 4 reverse, 5'-ATAGTTGCCAGCTACTTGTGG-3'; Hes-1 forward, 5'-TCTACACCAGCAACAGTGG-3'; Hes-1 reverse, 5'-TCAAACATCTTTGGCATCAC-3'; Hes-5 forward, 5'-AAGTGACTTCTGCGAAGTTCC-3'; Hes-5 reverse, 5'-AAGGCCATGTGGACCTTGAGG-3'. For Deltex primers, the cDNA was amplified for 30 cycles using the following program: 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 (18): Deltex forward, 5'-CACTGGCCCTGTCCACCCAGCCTTGGCAGG-3'; Deltex reverse, 5'-GGGAAGGCGGGCAACTCAGGCCTCAGG-3'. Housekeeping gene primers were as follows: hypoxanthine-guanine phosphoribosyl transferase (HGPRT) forward, 5'-CACAGGACTAGAACACCTGC-3', HGPRT reverse, 5'-GCTGGTGAAAAGGACCTCT-3'; GAPDH forward, 5'-ACCACAGTCCATGCCATCAC-3', GAPDH reverse, 5'-TCCACCACCCTGTTGCTGTA-3'. Either HGPRT or GAPDH were used as controls depending on the desired sizes of products in each experiment. The PCR products were separated on 1.5% agarose gels. Ethidium-stained gels were photographed using the Alpha Innotech Gel-doc system.

Gamma-secretase, Mek-1 inhibition, and Delta-like ligand blocking assays

IL-7-expanded B cell precursors were harvested, counted, and diluted to 1 x 106 cell/ml. Gamma-secretase inhibitor XII (Z-IL-CHO; Calbiochem) 50 µM in DMSO or DMSO alone was added to cells in complete medium for 1–2 h. For MEK-1 inhibition assays, PD 98059 (50 µM in DMSO) was added to B cell precursors for 6 h. Cells were harvested, lysed, and Western blots were performed. Similar assays were performed using goat anti-Delta-like Ab (H-20; Santa Cruz Biotechnology). Anti-Delta-like Ab was added to cultures at a concentration of 50 µg/ml at culture inception and again 24 h before harvest at day 6. Normal goat IgG (Jackson ImmunoResearch Laboratories) was used at 50 µg/ml as a control.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Aged B cell precursors exhibit reduced E47 protein in vivo and in vitro

BALB/c mice by ~2 years of age generally show a variable loss in late-stage pre-B cells (1, 2, 3, 4, 5, 6, 7, 8). Furthermore, some losses in pro-B cell fractions have also been observed (1, 3, 4, 6, 8, 19). Pro-B/early pre-B cells express the CD43 surface Ag together with the B lineage Ags CD45R (B220) and CD19 as well as CD95 (AA4.1) and are negative for surface IgM (19). Using these markers, we observed that aged BALB/c mice showed reductions in late-stage pre-B/immature B cells and in pro-B/early pre-B cells (Fig. 1A).


Figure 1
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  		FIGURE 1. Aged B cell precursors are reduced in aged mice and exhibit reduced E47 protein in vivo. A, Young (2–4 mo) and aged (21–26 mo) mouse bone marrow cells were stained ex vivo for CD43, B220, CD19, and AA4.1. Late-stage pre-B/immature B cells were characterized as B220+ CD43– (moderate B220 stain) and pro-B/early pre-B cells were characterized as B220+CD43+CD19+AA4.1+. Cells as a percent of total bone marrow are shown for a single young/aged mouse pair where the aged mouse showed substantial loss (>90%) of both pre-B/immature B cells and pro-B cells. Cumulative numbers of these cell types in individual young and aged mice (n = 8) present in combined femurs/tibias were calculated and averaged. Significance was determined by Student’s t test (*) with p < 0.05. B, Ex vivo cytoplasmic stain for E47 protein in young and aged B cell precursors is shown for a 2-mo-old BALB/c and a 23-mo-old BALB/c mouse. Similar staining patterns were observed in three other experiments. Bone marrow cells were first depleted of IgM-bearing cells, followed by staining for CD19, CD43, and E47. Staining with an IgG-PE isotype control (C) is also shown.

 
We then assessed the expression of the E47 protein in young and aged pro-B/early pre-B cells using cytoplasmic fluorescent staining as described by Quong et al. (20). Typically, as shown in Fig. 1B, most cells in the pro-B/early pre-B subset from young adult BALB/c mice expressed E47 protein. E47 protein levels were heterogeneous in young adult (2–4 mo) pro-B/early pre-B cells. In aged BALB/c mice, E47 protein expression was also seen among pro-B/early pre-B cells (Fig. 1B). However, as shown in this representative experiment, the spectrum of E47 protein levels was often skewed in favor of lower E47 expression. This pattern was seen in experiments with four individual aged mice each of which exhibited impairment of B lymphopoiesis. Therefore, those pro-B/early pre-B cells capable of relatively high E47 protein levels appear to be preferentially reduced in aged bone marrow. The deficits seen in B cell precursors are variable in aged mice (4). Altered expression of E47 protein was particularly evident in those aged mice with severe losses in pro-B and pre-B cell compartments.

E2A-encoded protein expression may change during progressive stages of B lymphopoiesis (20, 21). Because B cell precursors previously examined (9, 12) and above are a mixture of pro-B and early CD43+ pre-B cells, these above results do not distinguish whether reduced E47 protein occurs in both pro-B and early pre-B cell compartments in aged mice.

To address whether E2A-encoded proteins are reduced in both pro-B and early CD43+ pre-B cells, we expanded pro-B/early pre-B cells from young and aged mice with IL-7 in vitro and then separated pro-B from pre-B cells based on expression of surface CD2 (22). CD2 is found on pre-B and B cells, but not pro-B cells (22). CD2 was expressed by ~40–60% of IL-7-expanded B cell precursors from young and aged mice (Fig. 2A). As a control, pro-B cells expanded in vitro from RAG-2 knockout bone marrow failed to express CD2 (Fig. 2A), confirming that CD2 expression coincides with capacity for V gene recombination. Furthermore, the CD2– fraction did not stain for cytoplasmic µ H chain; cµ+ cells were exclusively contained within the CD2+ fraction obtained from young and aged cultures as shown by fluorescence flow cytometry (data not shown). However, only about one-third of CD2+ pre-B cells had cµ H chain sufficient for detection.


Figure 2
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  		FIGURE 2. E47 protein levels are decreased in both aged pre-B and pro-B cells in vitro. A, IL-7-expanded B cell precursors from young and aged BALB/c mice and RAG-2 KO mice on the BALB/c background were stained for surface CD2 and CD19 following 5–7 days in culture; percentages shown are of total CD19+ cells. CD2–CD19+ cells were considered pro-B cells and CD2+CD19+ as pre-B cells. B cell contamination in the CD2+CD19+ pre-B cells was <10%. B, Magnetic bead-sorted CD2+ and CD2– IL-7-expanded B cell precursors were harvested, lysed, and E47 proteins were detected by Western blot with Ubc9 protein as loading control. Similar data were obtained in six experiments. C, B cell precursors from young and aged mice were derived in vitro after IL-7 stimulation for 7 days of unfractionated (total) bone marrow or sorted CD19+ B lineage cells. Western blot was performed for E47 protein levels with Ubc9 as loading control. Data are representative of three experiments.

 
In B cell precursors cultured from aged mice, reduced levels of E47 protein (2–4-fold) were observed in both CD2+ pre-B and CD2– pro-B cells compared with that in young controls (Fig. 2B). Therefore, rather than diminished E47 protein being restricted to only one early B precursor compartment, both pro-B and early pre-B cells from aged mice exhibited lower steady-state levels of E47 protein.

Typically, when unfractionated bone marrow cells are used to establish expansion of B cell precursors in response to exogenous IL-7, a mixed monolayer of bone marrow-derived adherent cells is also established and is comprised principally of macrophages and fibroblastic stromal cells. To determine whether contact with adherent bone marrow cells, or their secreted products, is required for the continued poor expression of E47 in aged B cell precursors in vitro, we isolated B lineage cells from the bone marrow of young and aged mice as CD19+ cells and cultured these with exogenous IL-7 in the absence of non-B lineage cells. As shown in Fig. 2C, ~85% reduction in E47 protein was similarly observed in B cell precursors cultured from unfractionated bone marrow and from B lineage-enriched cell preparations cultured in the absence of a bone marrow adherent cell environment.

Partial reduction in E2A expression impairs bone marrow B lineage development

We hypothesize that decreased E47 expression at the pro-B/early pre-B cell stages in aged mice contributes to reduced B lymphopoiesis. Therefore, we tested whether partial loss of E2A-encoded protein expression in young mice was sufficient to affect B cell development at pro-B and pre-B stages as seen in aged mice. Previously, Quong et al. (20) showed that E2A heterozygous mice (E2A+/–) have ~2-fold reductions in late stage pre-B cells. This was confirmed in our analysis of young (2–3 mo.) E2A+/– mice compared with their wild-type controls (Fig. 3), where CD43– B220+ pre-B/immature cells were reduced by an average of 2.5-fold. We also observed an ~2-fold loss in pro-B/early pre-B cells defined as CD43+B220+AA4.1+CD19+ cells. These results indicate that B lymphopoiesis, at both the pro-B and pre-B cell stages, is sensitive to even relatively modest alterations in E2A-encoded protein expression. Aged B cell precursors often show ≥2-fold reduction in E2A-encoded proteins, particularly in those aged mice where numbers of pre-B cells are severely depleted (>80%) (9, 12). Therefore, even in the absence of senescence-associated effects, this level of E2A-encoded protein loss can contribute to depletion of B cell precursors.


Figure 3
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  		FIGURE 3. E2A+/– young adult mice are depleted in B cell precursors. Mouse bone marrow cells from wild-type C57BL/6 (WT) and E2A heterozygous (E2A+/–) mice were stained for CD43, B220, CD19, and AA4.1. Pre-B/immature B cells and pro-B/early pre-B cells were characterized as in Fig. 1. The percent of B cell precursors for a representative WT and E2A+/– pair are shown and cumulative numbers for three WT and four E2A heterozygous mice were calculated and averaged. Significance was determined by Student’s t test (*) with p < 0.05.

 
B cell precursors derived from senescent mice exhibit increased phosphorylation and ubiquitin modification of E2A-encoded proteins

Previously, we demonstrated that E2A-encoded proteins were reduced in B cell precursors derived from aged BALB/c bone marrow upon culture with recombinant mouse IL-7 (9, 11, 12). This reflected increased protein turnover rather than alterations in expression of E2A-encoded mRNA (12). In particular, turnover of E47 protein from both B cell precursors derived in vitro from young adult (~3–4 mo) and ~2-year-old BALB/c mice was proteasome-mediated and prevented by incubation with the irreversible proteosome inhibitor lactacystin as we have previously described (Ref. 12 and data not shown).

Degradation of E2A-encoded proteins has been associated with their ubiquitination (14, 23). Previously, we showed that E2A-encoded proteins were ubiquitinated in B cell precursors from both young and aged mice (12). This included high molecular mass, presumably polyubiquitinated, E2A-encoded proteins and smaller mass fragments (12). Herein, we have quantitated ubiquitin modification of E2A-encoded proteins present in both young and aged B cell precursors expanded in vitro with IL-7. As shown in Fig. 4A and Ref. 12 , immunoprecipitated E2A-encoded proteins from both young and aged mice exhibited ubiquitin modification. Although the full-length (~72 kDa) E2A-encoded proteins showed little ubiquitination, at higher molecular masses ubiquitination was demonstrable (Fig. 4A) and likely represents polyubiquitinated protein (14). The levels of ubiquitin-modified relative to unmodified full-length E2A-encoded proteins were ~2.5-fold greater in aged B cell precursors compared with young controls (Fig. 4D). Hence, higher proportions of E2A-encoded proteins in aged B cell precursors are ubiquitin modified.


Figure 4
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  		FIGURE 4. IL-7-expanded B cell precursors from aged mice show increased phosphorylation and ubiquitination of E2A-encoded proteins. A, Lysates from IL-7-expanded B cell precursors from young (Yg) and aged mice were immunoprecipitated with anti-E2A (E47/E12) Ab and Western blots probed with either anti-E47 or anti-ubiquitin (Ub) Abs. B, Lysates from IL-7-expanded B cell precursors were analyzed by Western blot for total ubiquitinated proteins. Ubc9 protein was used as a loading control. C, E2A-encoded proteins from IL-7-expanded B cell precursors were immunoprecipitated from young and aged mice using anti-E2A (E47/E12) Ab. Western blots were probed with either a mixture of anti-E47/E12 mAbs or rabbit anti-phosphoE2A (Thr355) (pE2A). The box on the right shows controls consisting of either eluted products from 1) B cell precursor lysate incubated with protein G beads alone with no capture Ab or 2) anti-E2A (E47/E12) Ab incubated with protein G beads in the absence of cell lysate. Blots were developed with anti-pE2A and anti-rabbit HRP. D, Ubiquitinated E2A-encoded proteins (Ub-E2A), total ubiquitinated proteins (Ub-proteins), and phosphorylated (Thr355) (pE2A) E2A-encoded proteins for young and aged B cell precursor lysates were determined by densitometry, expressed relative to levels of full-length E2A proteins (Ub-E2A; pE2A) or Ubc9 (Ub-protein) and then normalized to their respective young controls and averaged for three separate experiments.

 
Although E2A-encoded proteins in aged B cell precursors showed increased ubiquitination (Fig. 4, A and D), this was not a "global" phenomenon. Total levels of ubiquitin-modified proteins were not different in young vs aged B cell precursors (Fig. 4B).

Phosphorylation of E2A-encoded protein occurs as a prelude to ubiquitin modification. In particular, phosphorylation at threonine 355 of E47/E12 has been implicated; mutation of Thr355 results in decreased degradation and stabilization of the E2A-encoded proteins (14). Therefore, we tested whether phosphorylation of Thr355 (pT355) on E2A-encoded proteins was detected in cultured B cell precursors and whether this was increased in aged vs young mice. As shown in Fig. 4C, immunoprecipitates of E47/E12 proteins from both young and aged mice exhibited reactivity with a polyclonal Ab specific for pT355 (14). The relative expression of pT355 E2A-encoded proteins, compared with that of full-length E47/E12, was higher in aged cultured B cell precursors (Fig. 4, C and D). In aged B cell precursors, a greater proportion of E2A-encoded proteins appear to have undergone phosphorylation and ubiquitin modification consistent with their increased rates of proteasome-mediated degradation.

ERK MAPK activity is increased in aged B cell precursors and its inhibition restores E47 protein expression

Nie et al. (14), have indicated that activity of MAPKs, in particular the Ras-MEK-ERK pathway, promotes the degradation of E2A-encoded proteins by mediating their phosphorylation before ubiquitination. Because increased phosphorylation, as well as ubiquitination, of E2A-encoded proteins was seen in aged B cell precursors, we tested whether Ras-MEK-ERK MAPK activity coincided with reduced E47 protein levels in aged B cell precursors.

As indicated in Fig. 5A, aged B cell precursors, upon culture with IL-7, often exhibited increased levels of phosphorylated ERK proteins (P-ERK1/2). This was observed at several time points assessed during culture (Fig. 5B). Levels of total ERK1/2 protein were similar in young and aged B cell precursors (data not shown). Increased levels of P-ERK proteins were also observed when P-ERK was assessed in freshly isolated CD19+ bone marrow B lineage cells from aged compared with young mice, albeit the differences seen were lower than after activation (Fig. 5B, inset). The mechanisms responsible for increased P-ERK levels in aged B cell precursors are unclear. Although IL-7 signaling contributes to activation of the Ras-MEK-ERK pathway, levels of IL-7R{alpha} and the common {gamma}-chain are similar in young vs aged cultured B cell precursors (9, 24) as well as freshly obtained bone marrow B cell precursors (data not shown).


Figure 5
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  		FIGURE 5. MEK 1 inhibition increases E47 protein in aged B cell precursors and aged B cell precursors express increased P-ERK. A, CD19+ B cell precursors from young and aged IL-7-supplemented cultures were magnetic bead-sorted and P-ERK1/2 determined by Western blot with Ubc9 as loading control. B, IL-7-supplemented cultures were established with either sorted CD19+ B lineage cells or unfractionated bone marrow cells from young and aged mice in three independent experiments. B cell precursors from young and aged cultures were harvested on days 4, 6, and 8. Protein levels for P-ERK1/2 and the loading controls, Ubc9 or actin, were determined by Western blot. For comparison among time points and experiments, P-ERK levels were normalized to the loading controls. In addition, P-ERK values are expressed relative to that of young controls at day 8 in each experiment. Averages shown are +1 SEM. Inset, CD19+ cells were freshly isolated from bone marrow of three young and three aged mice. After normalization for differences in loading controls, levels of P-ERK proteins for aged mice are shown as a ratio relative to that of young controls for each experiment. C, P-ERK and E47 proteins were normalized to Ubc9 loading controls from five individual young and aged pairs. Closed symbols represent IL-7-expanded bone marrow cells (≥75% CD19+ B cell precursors) from young and aged mice. Open symbols represent CD19+ B cell precursors isolated from IL-7-stimulated bone marrow cultures (≥90% CD19+) by magnetic bead sorting. D, IL-7-expanded B cell precursors from young and aged mice were cultured with the MEK1 inhibitor PD098059 (50 µM) (MEK1 Inh) for 6 h. E47 protein was analyzed by Western blot with actin as loading control. Data are representative of three experiments.

 
In a more extensive survey, aged B cell precursors from IL-7-supplemented bone marrow cultures not only exhibited high P-ERK protein levels, but also reduced levels of E47 protein compared with young controls (Fig. 5C). Culture of IL-7-expanded B cell precursors from aged mice with the MEK-1 inhibitor PD 98059 resulted in a 2-fold increase in E47 protein levels after 6 h (Fig. 5D). Increases in E47 protein levels were also seen in young B cell precursors after an extended time period (e.g., 24 h) (data not shown). No discernable changes in E47 protein levels were seen in young B cell precursors at earlier time points presumably due to the already relatively high E47 expression. Therefore, an increase in the Ras-MEK-ERK pathway is associated with the reduction of E47 protein levels seen in aged B cell precursors in vitro and inhibition of this pathway enhances E47 protein expression.

Both young and aged B cell precursors express Notch in vitro

MAPK activity is not sufficient for degradation of E2A; Notch activity has been reported as obligatory for the degradation of E2A-encoded proteins in lymphocytes (14). Notch expression, at low levels, has been shown in pro-B and pre-B cells as have expression of known Notch target genes, e.g., Hes-1, Hes-5, and Deltex (25, 26). We therefore determined the expression of each of the four Notch genes in young and aged cultured B cell precursors. As shown in Fig. 6A, RT-PCR analysis indicated expression of transcripts for all four Notch genes in cultured nonadherent B cell precursors from both young and aged mice. Moreover, Notch activity was apparent in both young and aged cultured B cell precursors as reflected in comparable levels of detected transcripts for Hes-1, Hes-5, and Deltex (Figs. 6A and 7A). As shown in Fig. 7A, expression of these Notch target genes was completely inhibited by inclusion of the gamma-secretase inhibitor Z-IL-CHO which interferes with cleavage of Notch to yield its intracellular activation fragment.


Figure 6
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  		FIGURE 6. Notch expression in young and aged cultured B cell precursors. A, RNA was extracted from young (Y) and aged (A) B cell precursors after culture with IL-7, reverse transcribed to yield cDNA, and PCRs were performed with Notch 1, 2, 3, 4 primers as well as Hes-1 and Hes-5 primers with GAPDH primers as control. No reverse transcriptase, –RT, in the cDNA reaction. PCR products were visualized in gels with ethidium bromide stain. Data are representative of four experiments. B, Whole cell lysates from cultured B cell precursors were prepared and Notch-1 protein detected by Western blot with Ubc9 as a loading control. C, The majority CD19+ and minority CD19– cells from bone marrow IL-7-stimulated cultures were sorted and Notch-1 protein assayed by Western blot with actin as loading control. Similar data were obtained for both young and aged cells in three experiments.

 

Figure 7
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  		FIGURE 7. Gamma-secretase inhibition increases E47 protein in cultured aged B cell precursors. A, RNA was extracted from IL-7-cultured B cell precursors from young and aged mice, reverse transcribed, and PCRs were performed using Hes-1 and Deltex primers with HGPRT primers as control. PCR products were detected on gels by ethidium bromide stain. B cell precursors were treated with DMSO or with the gamma-secretase inhibitor Z-IL-CHO ({gamma} sec, 50 µM) in DMSO as shown for 1 h before assay. B, Young and aged B cell precursors were harvested from IL-7-supplemented bone marrow cultures and cultured for an additional 1 h with either DMSO or 50 µM gamma-secretase inhibitor Z-IL-CHO in DMSO ({gamma} sec Inh). Whole cell lysates were prepared from each and used in Western blot analysis to detect E47 protein with actin protein as a loading control. C, Results for four experiments with cultured B cell precursors from young and aged mice were analyzed with E47 levels normalized to that of DMSO-treated young B cell precursors. The increase in E47 protein seen in gamma-secretase inhibited aged B cell precursors was statistically different (p < 0.05) compared with that in aged B cell precursors treated with only DMSO. However, there was no statistical difference between gamma-secretase inhibited aged B cell precursors and gamma-secretase inhibited young B cell precursors. D, IL-7-expanded B cell precursors from aged bone marrow were harvested and cultured for 5 h with DMSO (as control), cycloheximide (CHX) (12 ), or with CHX plus gamma-secretase inhibitor (50 µM) for 5 h. Ubc9 serves as a loading control.

 
Notch-1, in particular, has been shown to impact the T cell vs B cell differentiation choice (27); therefore, we assessed Notch-1 protein in young and aged B cell precursors. Notch-1 protein in cultured B cell precursors from aged mice was detectable and, although B cell precursors from some individual aged mice had increased Notch-1 levels, overall, no significant differences in Notch-1 levels from young vs aged B cell precursors were apparent (Fig. 6B). To insure that Notch-1 protein expression was limited to B lineage cells, both the majority CD19+ and minority CD19– (<15%) cells present in IL-7 supplemented bone marrow cultures were isolated. Although lysates from CD19+ B cell precursors expressed Notch-1 protein, little or no Notch-1 protein was seen in CD19– cells from the bone marrow cultures (Fig. 6C).

Notch/Delta-like ligand signaling is necessary for maintaining low E47 protein levels in aged B cell precursors

Notch activity was required to maintain accelerated E47 protein turnover and reduced E47 protein steady-state levels in aged B cell precursors. Aged cultured B cell precursors, when exposed to the gamma-secretase inhibitor Z-IL-CHO for limited time periods (1–2 h), showed markedly increased levels of E47 protein (~3-fold) (Fig. 7, B and C). This coincided with altered E47 protein turnover; upon cycloheximide-induced blockade of new protein synthesis, the rapid loss of E47 protein over time (5 h) was not observed in aged B cell precursors in the presence of gamma-secretase inhibitors (Fig. 7D). In contrast, young B cell precursors, already expressing relatively high E47 protein levels with slow turnover kinetics, showed no increase in E47 protein at up to 2 h after gamma-secretase inhibition; however, longer time periods (e.g., ~9 h) did result in significant increases in E47 protein (data not shown). Inhibition of Notch activity did not affect the levels of E2A-encoded mRNA in either young or aged B cell precursors as determined by RT-PCR (data not shown).

IL-7-expanded B cell precursors (CD19+) from both young and aged BALB/c bone marrow expressed Notch ligands of the Delta-like family (DLL) in comparable amounts (Fig. 8A). Notch ligands of the Jagged family were not detected in either young or aged cultured B cell precursors (data not shown), but Jagged proteins (and not DLL) were expressed by non-B lineage adherent cells in cultures of young and aged bone marrow (Fig. 8B).


Figure 8
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  		FIGURE 8. Delta-like proteins are expressed by cultured B cell precursors from young and aged mice. A, IL-7-expanded B cell precursors from young and aged mice were grown for 5–7 days after which CD19+ cells were sorted from the cultures. Cells were lysed and Delta-like ligand proteins (DLL), with actin as a loading control, were detected by Western blot. Data are representative of three experiments. B, Adherent cells from IL-7-supplemented bone marrow cultures from young and aged mice were trypsinized for cell removal and cell lysates used to detect DLL and Jagged proteins by Western blot. Data are representative of two experiments.

 
To assess whether interference with Notch/DLL interactions would alter E47 expression in aged B cell precursors, polyclonal Ab reactive with DLL 1, 3, and 4 was added to cultures of young and aged bone marrow and E47 protein levels in IL-7-expanded B cell precursors were determined. In contrast to the gamma-secretase inhibition studies where effects were determined over several hours, anti-DLL Ab was continuously present throughout the 7-day culture. When anti-DLL Ab was added in culture to block DLL/Notch interactions, increased levels of E47 protein in cultures of aged, as well as young, B cell precursors were observed (Fig. 9). Although increases in E47 protein levels of ~40% were seen in young B cell precursors, more marked increases (~150%) were seen in aged B cell precursors. Therefore, limiting Notch activity consequent to either inhibition of gamma-secretase or interference with Notch/DLL interactions led to increased E47 protein levels and these effects were particularly apparent within aged B cell precursors.


Figure 9
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  		FIGURE 9. Ab to DLL increases E47 protein in cultured aged B cell precursors. Anti-Delta-like Ab (DLL; 50 µg/ml) or IgG control Ab was added to B cell precursor cultures established from young and aged bone marrow. Ab was added at culture inception and again at day 5 of culture. After 6 days, cultured cells were harvested and lysed for detection of E47 and Ubc9 proteins, as control, by Western blot. Summary of three experiments is shown with E47 proteins normalized to that of the young controls. Significance between aged anti-DLL-treated and control cultures was determined by Student’s t test (*) with p < 0.05. Although anti-DLL treated aged B cell precursors and control IgG-treated aged B cell precursors differed significantly in E47 levels, there was no significant difference between the levels of E47 in young anti-DLL-treated and aged anti-DLL Ab-treated B cell precursors.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It is well established that senescence in mice results in impaired B lymphopoiesis (1, 2, 3, 4, 5, 6, 7, 8). This is primarily seen as a loss of pre-B cells, but also affects both the development of immature B cells and the readout of the B cell specificity repertoire (3, 28, 29, 30). As reported previously (4, 6, 19) and confirmed herein, deficits are also observed at the pro-B cell stage in aged mice; this may result from even earlier developmental defects affecting the common lymphoid/early B cell precursors (CLPs/EBPs) (19). Loss of RAG expressing early B cell precursors with accompanying deficient Ig H chain recombination has been shown in aged mice and may directly result in diminished production of new pre-B cells (5, 6, 7). However, it is likely that additional mechanisms, operative at the pre-B cell stage, also contribute to pre-B cell reduction (3). This may involve diminished survival (31, 32) or limited expansion at the pre-BCR checkpoint (8, 9).

E2A-encoded proteins function as transcription factors early in B lymphopoiesis that regulate, directly or indirectly, the expression of other transcription factors including EBF and Pax-5 (10, 33) and multiple B lineage genes including the surrogate L chain proteins {lambda}5 and VpreB, Ig{alpha} and Igbeta, and TdT (10). Transcription of the RAG-1 and RAG-2 enzymes are highly dependent upon presence of the Erag enhancer recognized by E2A-encoded proteins (34). Therefore, any dysregulation in the expression and function of the E2A gene products during senescence likely would have a significant effect on B lymphopoiesis both at early stages characterized by V gene recombination and, later, at the pre-BCR checkpoint.

In aged mice, as we have shown herein and elsewhere (3, 9, 11, 12), pro-B/early pre-B cells have reduced overall levels of E47 protein. Although our previous studies have relied on Western blotting of lysates from pooled B cell precursors, the current study using fluorescence flow cytometry provides for determination of E47 protein expression by individual pro-B/early pre-B cells in aged mice. Young adult pro-B/early pre-B cells from BALB/c mice show heterogeneity in E47 protein expression. It is likely that relatively high E47 protein expression in a subset of pro-B cells facilitates RAG expression and Ig H chain rearrangement. In aged pro-B/early pre-B cells, expression of E47 protein is more limited. Whether these pro-B cells undergo normal RAG expression, V gene rearrangements, and further differentiation is not known. However, given the dependency of RAG expression on E2A, it may be that these pro-B cells in aged mice are compromised in their further development. As shown herein and by others (20), modest alterations in E2A-encoded protein expression, as seen in young E2A heterozygous mice, are capable of limiting B lymphopoiesis. Aged mice do express, as shown here, a smaller pool of early B cell precursors capable of high E47 protein levels. These, like their young counterparts, would be expected to undergo normal H chain rearrangement, but would lead to formation of a reduced pre-B cell population in aged mice.

Pre-B cells in aged mice may be subject to additional negative regulatory influences. Population of the late-stage pre-B cell compartment requires expression of the pre-BCR, effective synergistic signaling via the pre-BCR and IL-7R (35), proliferation, and survival. E2A, together with EBF, regulates expression of the surrogate L chains and consequently, the pre-BCR (10). We have shown decreased surrogate L chain expression in aged B cell precursors after culture and expansion with IL-7 (8, 9). Notably, this coincides with decline in E47 protein levels (9). Hence, it is likely that reduced expression of E2A gene products in aged B cell precursors also affects pre-BCR expression and signaling at this checkpoint.

We have investigated the mechanisms responsible for losses in E47 protein expression in aged B cell precursors using in vitro IL-7-expanded populations of pro-B/early pre-B cells. We have previously reported that, while E2A mRNA levels are similar in cultured B cell precursors from young and aged mice, their lower levels of E47 protein coincide with increased protein turnover by the proteasome pathway (12).

Increased targeting of E47 for degradation in aged B cell precursors is suggested by the more extensive ubiquitin modification of E47 observed in vitro. We have previously shown that not all proteins are rendered less stable in aged B cell precursors and that the rapid turnover of E47 protein observed is not a "global" phenomenon (12). The levels of total ubiquitin modified proteins was estimated to be similar in young vs aged B cell precursors; this also suggests that the increased protein degradation by the ubiquitin/proteasome pathway may apply to only a subset of proteins (including E47) in aged B cell precursors.

Nie et al. (14), have reported that both MAPK and Notch activity regulate the degradation of E2A proteins in lymphocytes. Consistent with a role for Notch in B cell precursors, both Notch family members and Notch ligands of the Delta-like family were detected in cultured B cell precursors from young and aged mice. Notch expression and/or signaling by pro-B and pre-B cells have previously been suggested (25, 26) and Delta-like expression may occur in B cell precursors (26, 36). In addition, Notch ligands of the Jagged family were also demonstrated in the adherent cell fractions present in young and aged B cell precursor cultures. Therefore, opportunities for Notch activation via Delta-like and/or Jagged ligands are available in bone marrow cultures and presumably in vivo as well. The presence of Notch and DLL in B cell precursors also suggests the capacity for interactions among B cell precursors in addition to B cell precursor/stromal cell interactions.

Notch signaling was apparent in both young and aged B cell precursors as evidenced by Hes-1 and Deltex transcripts that were eliminated by gamma-secretase treatment. Notch-1 protein levels were generally comparable in young and aged B cell precursors as were Hes-1, Hes-5, and Deltex transcripts, suggesting roughly comparable Notch signaling in young and aged B cell precursors. However, inhibition of either gamma-secretase, and hence Notch activation, or interference with Notch-DLL interactions had lesser effects on levels of E47 protein in young B cell precursors, but markedly increased E47 protein levels in aged B cell precursors. Although the precise mechanisms by which Notch affects E2A degradation remain to be elucidated, these data point to involvement of the Notch pathway in promoting the loss of E47 protein in aged B cell precursors.

Notch activity is key to blocking B lineage development and promoting commitment of emerging lymphoid precursors to the T cell (27, 37), and possibly plasmacytoid dendritic cell (38), pathways. Whether reduced E2A expression consequent to Notch activation in aged B cell precursors biases lymphocyte lineage choice or commitment in old mice remains to be determined. B lineage development is clearly sensitive to the levels of available E2A. In aged mice, reduced E2A expression would be expected to adversely affect function and development of B lineage cells. E2A is expressed before the pro-B cell stage, e.g., in CLP/EBP (39). CLPs/EBPs are also reduced in aged mice (19). Conceivably, low E2A protein expression at such early developmental stages may compromise CLP/EBP generation and that of subsequent pro-B cells. Additional studies on E2A expression at such early steps in B lymphopoiesis in senescence need to be performed for further insight.

In addition to the importance of Notch activity in regulating E2A protein levels, MAPK activity also promotes the turnover of E2A proteins (14). In particular, activation of the Ras-MEK-ERK pathway appears to be involved, possibly by phosphorylating E2A-encoded proteins on serine/threonine residues as a prelude to ubiquitin modification (14). Therefore, it is of considerable importance that, in aged B cell precursors, activation of the Ras-MEK-ERK pathway appears elevated as evidenced by substantially increased levels of phosphorylated ERK proteins. Moreover, inhibition of this pathway resulted in recovery of E47 protein levels in aged B cell precursors. Consistent with actions of the Ras-MEK-ERK pathway, phosphorylation of E2A proteins from aged mice on threonine 355 was increased in aged B cell precursors. Phosphorylation of Thr355 via the Ras-MEK-ERK pathway has been implicated in the degradation of E2A proteins (14). Increased activation of the Ras-MEK-ERK pathway, resulting in heightened phosphorylation of E2A in aged B cell precursors, may provide an initial spur that promotes loss of E2A-encoded proteins in a Notch and ubiquitin/proteasome-dependent manner.

Previous studies have implicated the senescent bone marrow microenvironment in down-regulating RAG expression in aged B cell precursors (5, 6). Whether the bone marrow microenvironment regulates E2A expression is not known. In our studies, reduced E47 protein was seen in isolated CD19+ aged B cell precursors cultured in the absence of a bone marrow microenvironment. Although this may suggest an intrinsic B cell precursor defect in E2A expression in aged mice, it is also conceivable that B cell precursors developing in an aged microenvironment in vivo are already programmed for low E47 expression following subsequent in vitro culture. Further studies to define the role of the senescent bone marrow microenvironment on E2A regulation are in progress.

In summary, dysregulation of E2A-encoded protein expression may contribute significantly to the poor production of new B cell precursors during senescence. This may result from accelerated turnover of E2A-encoded proteins as a consequence of heightened activities of the ERK/MAPK pathway leading to accelerated Notch-dependent, proteasome-mediated degradation. Altered E2A-encoded protein expression in aged B cell precursors likely affects B lymphopoiesis at multiple stages and contributes to reduced pre-B cell pools and altered further B cell development in aged mice.


   Acknowledgments
 
We thank Jim Phillips and the Sylvester Cancer Center Flow Cytometry Core Facility for assistance with cell sorting and analysis. We acknowledge Anjali Prabhu for technical assistance. We also thank all members of the Riley and Blomberg laboratories for their support.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AG025256 and AI064591 (to R.L.R.) and AG17618 and AG23717 (to B.B.B.). Back

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

3 Abbreviations used in this paper: P-ERK, phospho-ERK; DLL, Delta-like ligand protein; CLP, common lymphoid precursor; EBP, early B cell precursor; HGPRT, hypoxanthine-guanine phosphoribosyl transferase. Back

Received for publication June 23, 2006. Accepted for publication December 21, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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