Rag-1 and Rag-2 are essential for the construction of the BCR repertoire. Regulation of Rag gene expression is tightly linked with BCR expression and signaling during B cell development. Earlier studies have shown a major role of the PI(3)K/Akt pathway in regulating the transcription of Rag genes. In this study, by using the 38c13 murine B cell lymphoma we show that transcription of Rag genes is also regulated by the MEK/ERK pathways, and that both pathways additively coordinate in this regulation. The additive effect is observed for both ligand-dependent (upon BCR ligation) and ligand independent (tonic) signals. However, whereas the PI(3)K/Akt regulation of Rag transcription is mediated by Foxo1, we show in this study that the MEK/ERK pathway coordinates with the regulation of Rag by controlling the phosphorylation and turnover of E47 and its consequential binding to the Rag enhancer regions. Our results suggest that the PI(3)K and MEK/ERK pathways additively coordinate in the regulation of Rag transcription in an independent manner.
The products of Rag-1 and Rag-2 are essential for rearrangement of Ig genes during B cell development (1–3). Maintenance or reinduction of Rag during bone marrow lymphopoiesis occurs when B lymphocytes attempt to replace an inappropriate receptor with a new one, either in the case of self reactivity (negative selection) (4–6) or in the case of inappropriate BCR-mediated tonic signal transduction (positive selection) (7, 8). This mechanism is called receptor editing and is strongly correlated with elevated Rag-1 and Rag-2 mRNA levels (9, 10). The expression of Rag and occurrence of DNA rearrangements have also been shown in a subset of germinal center B cells, a process referred to as receptor revision (11, 12). In these cells, however, BCR ligation effectively suppresses the expression of Rag mRNA (13, 14).
The expression of a signaling-competent BCR is essential for the developmental progression in B lymphopoiesis and for the survival of mature B cells (15–18). Studies have shown that the generation of BCR-proximal tonic signals is required to promote developmental progression and survival (reviewed in Refs. 19 and 20), as well as to establish allelic exclusion at the heavy and L chain loci (21). Thus, B cell development and Rag expression are linked together under stringent and coordinated BCR-mediated control.
The signaling pathways regulating Rag gene expression at various stages of B cell development are currently under investigation. In a previous study we showed that BCR tonic signals regulate Rag gene expression and establishment of allelic exclusion (7, 22). More recently, it has been shown that this tonic signaling activates PI(3)K and Akt kinases (23, 24), which phosphorylate and lead to the cytoplasmic sequestration of FOXO proteins, the major transcriptional activators of Rag gene expression (25–27). In contrast, inappropriate tonic signals, such as those in B cells deficient of CD19, impose a developmental arrest and maintain Rag gene expression and activation of receptor editing (7, 8, 28). In earlier studies we showed that ongoing Rag expression in immature CD19−/− B cells is associated with reduced ERK phosphorylation (7, 8), which is in agreement with studies showing that efficient ERK phosphorylation depends on CD19 (29). It has also been shown that tonic signaling in T cells activates Abl and ERK kinases to inactivate Rag gene expression (30). More recently, an ERK inhibitor has been shown to modify the receptor editing process in self-reactive B cells in vivo (31). These studies suggest that in addition to the PI(3)K/Akt pathway, the regulation of the Rag gene expression by BCR signals is also mediated through the ERK pathway. However, the mechanism of this regulation and the interrelationship with the PI(3)K/Akt pathway are not known.
To study the role of ERK kinase in regulating Rag gene expression we used the 38c13 murine B cell lymphoma cell line (32), which has been shown to express Rag and to undergo L chain gene replacement (33, 34). This cell line is a carcinogen-induced lymphoid tumor (35) that effectively responds to BCR signals by activation of main signaling pathways (36). Ligation of the BCR in 38c13 cells effectively downregulates Rag expression, implicating that Rag genes in these cells are under stringent regulation of BCR signaling (34). In this study, we show in 38c13 cells that BCR-mediated regulation of the Rag gene expression is additively controlled by the MEK/ERK and PI(3)K pathways, in both ligand-dependent and ligand-independent manners. However, whereas PI(3)K function is mediated through phosphorylation of FOXO proteins (26), the regulation of Rag genes by the MEK/ERK pathway is independently mediated by phosphorylation and inactivation of E47 and its consequential binding to the Rag enhancer region. Hence, our data suggest that the MEK/ERK and the PI(3)K pathways additively coordinate to regulate the expression of Rag genes in B cells.
Materials and Methods
Cell line growth, maintenance, and stimulation
All experiments were carried out using the 38c13 murine B cell lymphoma (32), grown in RPMI 1640 culture medium (10% FCS). For stimulations, cells were adjusted to 2–5 × 106 cells/ml in culture medium at 37°C and stimulated with goat anti-mouse IgM Abs (SouthernBiotech, Birmingham, AL) or with the F(ab′)2 of goat anti-mouse IgM (Jackson ImmunoResearch Laboratories, West Grove, PA) at 0.1–20 μg/ml. For the experiments conducted, we used the MEK/ERK inhibitor UO126 (10 μM), the PI(3)K inhibitor LY294002 (10 μM), the JNK inhibitor sb202190 (10 μM) or the p38 inhibitor sp600125 (5 μM) (all from Sigma-Aldrich, St. Louis, MO). Stimulations were allowed to proceed for the indicated intervals and cells were lysed for Western blot analysis or for the preparation of total RNA.
Generation of expression cassettes and viral vectors
In some experiments cells were transfected with a constitutively active Ras-expressing vector (HRas). The HRas (G12V onco-RAS mutant) sequence described in Frankel et al. (37) was cloned into EcoRI and HINDIII sites of the retroviral expression vector pBABE. To achieve inducible activation of ERK, 38c13 cells were transfected with the pBABE vector encoding the estradiol-inducible (DD)Raf1:ER fusion protein as described (38). A pBABE vector encoding an inactive form of a fusion protein (301) was used for control (38). To activate ERK, β-estradiol (Sigma-Aldrich) was added to a final concentration of 10−7 μM, as described (38).
Viral packaging and infection
Retroviral vectors were transfected into Phoenix cells as described (39). Transfected cells were cultured for 48 h at 37°C in a humidified incubator, and the viral-rich supernatant was collected and filtered with a 0.45-μm-diameter pore filter. For infection, 1 × 106 38c13 cells were resuspended in 1 ml viral-rich supernatant with 10 μg/ml polybrene (American Bioanalytical, Natick, MA) and placed in 24-well plates. Next, cells were centrifuged at 140 × g for 60 min, resuspended in culture medium, and incubated for 16 h at 37°C. Cells were then transferred to petri dishes for an additional 24 h followed by addition of puromycin (Sigma-Aldrich) at 3 μg/ml for 10 d to select stably transfected clones.
Immunoprecipitation and Western blot analysis
Immunoprecipitations and Western blotting were performed as we have previously described (7). Briefly, cells were lysed and proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membrane (Millipore, Bedford, MA). Blots were probed with anti-phosphotyrosine (4G10), rabbit anti–phospho-ERK1/2, rabbit anti-ERK1/2, rabbit anti–phosph-Akt, rabbit anti-Akt (all from Cell Signaling Technology, Beverly, MA) and goat anti-mouse actin (Santa Cruz Biotechnology, Santa Cruz, CA). For immunoprecipitations of E47 we used mouse monoclonal anti-E47 (BD Biosciences, Mountain View, CA). Immune complexes were isolated by incubation with protein G–agarose beads and then analyzed by Western blotting using anti–phospho-threonine–proline mouse monoclonal Abs (Cell Signaling Technology) and by anti-E47 Abs. To reveal bound Abs we used HRP- conjugated secondary Abs and the blot was developed with ECL reagent. To obtain semiquantitative estimates for the total E47 and for E47 phosphorylation, bands were quantified and densitometry analysis was performed using Tina 2.0 software (Raytest, Straubenhardt, Germany). Values were normalized to respective control bands.
Flow cytometry and cell cycle analysis
Abs used for cell staining were anti-mouse IgM-PE and anti-mouse CD19 (clone 1D3) FITC (BD Pharmingen, San Diego, CA). For cell cycle and apoptosis analysis, cells were incubated for 1 h at room temperature with propidium iodide solution (a buffer containing 0.3% [w/v] saponin, 50 μg/ml RNAse, 5 mM EDTA [pH 8], and 5 μg/ml propidium iodide [all from Sigma-Aldrich]) and were analyzed by FACS. The data collection was performed using a FACSCalibur (BD Biosciences) and FlowJo software (Tree Star, Ashland, OR).
Total RNA was prepared using TRI Reagent (Sigma-Aldrich), according to the manufacturer’s protocol, and reverse transcribed to cDNA as described earlier (22). Real-time quantitative PCR for Rag-1, Rag-2, E2A, germline λ transcripts, and hypoxanthine phosphoribosyltransferase (HPRT) expression was performed using ABsolute Blue SYBR Green ROX mix (Thermo Scientific/ABgene, Epsom, U.K.) according to the manufacturer’s instructions on a Rotor-Gene 6000 system (Corbett Research, Sydney, Australia) using its software, version 1.7. Melting curves were determined to ensure the amplification of a single product. The primer sequences used for Rag-1 and Rag-2 and HPRT amplifications were described in Wang et al. (40) and primer sequences for germline λ transcripts were described in Xu and Feeney (41). Primer sequences for E2A were: forward, 5′-cctggatactcagccgaaga-3′, and reverse, 5′-agcatccctgctgtagctgt-3′.
Chromatin immunoprecipitation (ChIP) analysis was performed as previously described (42). Briefly, cells (10–24 × 107) were first cross-linked on ice, lysed, and sonicated at 4°C. Samples were centrifuged and supernatants were precleared with slurry of salmon sperm DNA-coated protein A– or protein G–Sepharose beads (ssDNA beads). Cleared samples were incubated overnight at 4°C with 10 μg anti-Foxo1 (H128; Santa Cruz Biotechnology), anti-E47 (554077; BD Pharmingen), or anti-IgG (sc-1703; Santa Cruz Biotechnology), followed by incubation with ssDNA beads. After immunoprecipitation, washes, and reverse cross-linking, the samples were further extracted with phenol/chloroform and ethanol precipitated in the presence of 30 μg glycogen. Samples were then subjected to real-time quantitative PCR for ERag1 and ERag2 enhancer regions using the primers described in Hu et al. (43). Dissociation curves after amplification showed that all primer pairs generated single products, and the amount of PCR product amplified was calculated relative to a standard curve of the input. The binding activity of E47 or Foxo1 is presented as the ratio between values obtained by a specific immunoprecipitation and the value of control (IgG) immunoprecipitation.
The statistical significance of the difference between the experimental groups was determined using an unpaired two-tailed Student t test with differences considered significant at p < 0.05.
BCR ligation rapidly suppresses Rag gene expression in 38c13
In 38c13 B lymphoma cells Rag genes are constitutively expressed, but they are rapidly suppressed upon cross-linking of the BCR (34). To study the intracellular mechanisms regulating Rag gene expression in this cell line, we first conducted kinetic experiments to quantify the suppression effect. As shown in Fig. 1, the cross-linking of the BCR in 38c13 cells stimulated total tyrosine and ERK phosphorylations that were detected within 5 min and lasted for at least 2 h (Fig. 1A), with no effect on the apoptosis rate or the cell cycle (Fig. 1B). Quantification analysis of Rag gene expression revealed a significant reduction of 1.7-fold in Rag1 and 1.25-fold in Rag2 mRNA levels as early as 10 min following BCR ligation, reaching 2-fold reduction within 30 min relative to the untreated cells (Fig. 1C). The suppression reached maximal level (>10-fold reduction) after 1–2 h of BCR ligation, as compared with WEHI 231 cells that do not express Rag (Fig. 1C). The suppression degree of Rag was also dependent on the concentration of the stimulating Abs (Supplemental Fig. 1), indicating that BCR signals can tune the level of Rag gene expression in 38c13 cells. However, to carefully study the mechanism by which BCR signals regulate Rag gene expression in 38c13 cells, we chose to carry out the experiments at a time point where Rag expression in BCR-stimulated cells is suppressed by 2-fold (30 min, Fig. 1C).
Inhibition of PI(3)K partially blocks BCR-mediated suppression of Rag gene expression in 38c13 cells
Earlier studies have shown that regulation of Rag expression is mediated through the activation of PI(3)K/Akt (23–27). To determine whether the PI(3)K/Akt pathway is the exclusive regulator of Rag expression, we tested whether inhibition of PI(3)K would block the BCR-mediated suppression in Rag expression. Ligation of the BCR in 38c13 cells for 30 min stimulated the phosphorylation of Akt (Fig. 2A) and suppressed Rag-1 and Rag-2 expression by 2-fold (Fig. 2B). However, despite the effective inhibition of the PI(3)K pathway (by the specific PI(3)K inhibitor LY294002, as revealed by inhibition of Akt phosphorylation; Fig. 2A), the expression levels of Rag-1 and Rag-2 were only partially restored (still reduced by 1.25-fold relative to those of the untreated cells; Fig. 2B). Hence, we concluded that BCR-mediated suppression in Rag gene expression in 38c13 cells is not exclusively regulated by the PI(3)K/Akt pathway.
MEK/ERK and PI(3)K additively regulate BCR-mediated suppression of Rag gene expression in 38c13 cells
We next assessed the role of the MEK/ERK pathway in regulating Rag gene expression in 38c13 cells. To do so, 38c13 cells were co-treated with the MEK specific inhibitor UO126 and with stimulating anti-BCR Abs for 30 min. Upon ligation of the BCR, ERK phosphorylation was detected (Fig. 3A) and expression levels of Rag-1 and Rag-2 were suppressed by 2-fold (Fig. 3B). However, we found that this BCR-mediated suppression of Rag gene expression in 38c13 cells was partially blocked by inhibition of the MEK/ERK pathway. Quantitative analysis for Rag gene expression revealed that in cells co-treated with anti-BCR and MEK inhibitor, the expression levels of Rag-1 and Rag-2 were only partially restored (still reduced by 1.45-fold relative to those of the untreated cells; Fig. 3B). The inhibition of JNK or p38 had no effect on BCR-mediated suppression of Rag (Supplemental Fig. 2), indicating that the MEK/ERK and PI(3)K pathways are specifically involved in the regulation of Rag gene expression in 38c13 cells.
Because each of the PI(3)K and MEK inhibitors alone blocked only partially the BCR-mediated suppression of Rag expression in 38c13 cells, we next tested whether the two pathways may cooperate in the regulation of Rag expression in these cells. Quantitative analysis revealed that only in cells treated with both inhibitors was the anti-BCR mediated suppression of Rag gene expression completely blocked and levels of Rag-1 and Rag-2 mRNA were not different from those expressed in the unstimulated control cells (Fig. 3C). Similar results were obtained upon extended duration of the treatments (up to 2 h; see Supplemental Fig. 3). Thus, we concluded that upon BCR ligation, MEK/ERK and PI(3)K additively coordinate to downregulate Rag gene expression in 38c13 cells.
Specific activation of ERK downregulates Rag gene expression in 38c13 cells
Our results implicate that BCR-mediated phosphorylation of ERK is necessary to downregulate Rag gene expression in 38c13 cells. To exclude a potential contribution of other signaling pathways and/or intracellular interactions that are activated upon BCR ligation, we next assessed whether specific activation of ERK is capable of suppressing Rag gene expression in 38c13 cells. To do so, we transfected 38c13 cells with a retroviral vector encoding the oncogenic activated form of HRas (G12V onco-RAS mutant; see Ref. 37), and stable clones were selected and confirmed for constitutive activation of ERK (Fig. 4A). Transfected and control cells were then analyzed for Rag-1 and Rag-2 gene expression by quantitative PCR. The results in Fig. 4B reveal that in HRas-transfected 38c13 cells the levels of Rag-1 and Rag-2 mRNA expression were reduced by 5- and 2.5-fold, respectively, relative to the control cells.
However, we considered the possibility that the long-term antibiotic selection promoted growth and survival of clones where the Rag genes were suppressed due to intracellular compensatory mechanisms, rather than representing a net effect of the activation of ERK. To directly resolve this issue we used an inducible system to activate the ERK pathway. For this propose, we stably transfected 38c13 cells with a retroviral vector (P-Babe) encoding the estradiol-inducible (DD)Raf1:ER fusion protein. Importantly, RAF is a downstream signaling molecule to RAS and therefore actiactivatesvates ERK more specifically. As described previously (38), the (DD)Raf1:ER molecule is expressed in stably transfected cells in its inactive form but can be activated by the addition of estradiol to the growth medium with a subsequent increase in ERK phosphorylation (38) (Fig. 4C). In contrast, control cells transfected with the nonactive (301)Raf1:ER did not exhibit an increased phosphorylation of ERK upon the addition of estradiol (Fig. 4C). Real-time PCR analysis of mRNA from the estradiol-treated (DD)Raf1:ER 38c13 cells revealed a significant reduction of 2-folds in Rag-1 and 1.55-fold in Rag-2 expression relative to the control untreated (DD)Raf1:ER 38c13 cells (Fig. 4D), with no effect on cell cycle and/or apoptosis rate (Supplemental Fig. 4). Thus, specific activation of the ERK pathway downregulates Rag gene expression in 38c13 cells.
Tonic activation of MEK/ERK and PI(3)K additively cooperate in regulating Rag gene expression in 38c13 cells
Because the specific activation of ERK downregulated Rag expression, we next assessed in a reciprocal experiment whether the specific inhibition of ERK would result in an increased Rag expression. To do so, 38c13 cells were cultured in the presence of the MEK specific inhibitor UO126, which effectively blocked the tonic ERK phosphorylation for at least 8 h (Fig. 5A), with no effect on cell cycle or apoptosis rate (Supplemental Fig. 5). Quantitative analysis for Rag gene expression revealed that the levels of Rag-1 and Rag-2 gradually increased with time, reaching to significant enhanced values in cells treated with UO126 for at least 6 h. Thus, a 2-fold increase in Rag-1 and a 3-fold increase in Rag-2 were found 8 h after UO126 treatment (Fig. 5A), suggesting that tonic activation of ERK is necessary to regulate Rag gene expression in 38c13 cells.
To find whether tonic ERK and PI(3)K activation cooperate in regulating Rag expression, 38c13 cells were cultured for 8 h in the presence of MEK inhibitor or PI(3)K inhibitor or with both. Quantitative analysis revealed that inhibition of PI(3)K resulted in a 20-fold increase in Rag-1 and a 4-fold increase in Rag-2, whereas inhibition of ERK resulted in a 2-fold increase in both Rag-1 and Rag-2. All differences were statistically significant (p < 0.05). However, in cells that were treated with both inhibitors, we obtained a significant additive 25-fold increase in Rag-1 and a 10-fold increase in Rag-2 (Fig. 5B). We concluded that tonic activation of both ERK and PI(3)K cooperate in regulating Rag expression levels in 38c13 cells.
The effect of MEK/ERK in regulating Rag expression is mediated by the E47 transcription factor
We next studied the mechanism by which the MEK/ERK pathway exerts the additive effect in regulating Rag gene expression. Earlier studies have shown that the PI(3)K/Akt pathway regulates Rag gene expression by phosphorylation and consequent inactivation of Foxo1 protein (25–27). Hence, we first assessed whether this additive effect is mediated by Foxo1. In agreement with earlier studies (27), ChIP assays performed using 38c13 cells revealed that treatment with the PI(3)K inhibitor resulted in a profound 15- to 20-fold increase in the binding of Foxo1 to the Rag enhancer (ERag) regions (Fig. 6A). Although we found that treatment with the MEK inhibitor increased Foxo1 binding to ERag regions by 2- to 3-fold, the inhibition of both PI(3)K and MEK did not result in an additive increase in the binding of Foxo1 beyond that obtained upon inhibition of PI(3)K alone (Fig. 6A). This finding suggests that the additive effect in regulating Rag gene expression by the MEK/ERK pathway is not mediated by Foxo1.
It has been shown that binding of the E2A gene product E47 to the ERag region is necessary to activate transcription of Rag in the B lineage (44, 45), and that binding of this transcription factor is regulated by the ERK/MAPK pathway (46, 47). Thus, we next tested whether the additive effect of MEK/ERK in regulating Rag expression is mediated by E47. ChIP analysis revealed that inhibition of the MEK/ERK pathway in 38c13 cells resulted in a 1.5- and 2-fold increase in binding of E47 to ERag1 and ERag2, respectively (Fig. 6B). No significant increase in binding of E47 to ERag regions was obtained upon treatment with PI(3)K inhibitor. Also, the inhibition of both MEK and PI(3)K did not result in an additive increase in the binding of E47 beyond that obtained upon inhibition of MEK alone (Fig. 6B). Amplification of DNA fragments that are >23 kb downstream to the ERag regions reveal no changes in relative E47 binding, indicating the specificity of the ChIP assay (Supplemental Fig. 6). These results suggest that the MEK/ERK pathway additively regulates Rag gene expression through modification of E47 binding to the ERag regions.
Inhibition of the MEK/ERK pathway reduces phosphorylated E47 in 38c13 cells
Activation of the ERK/MAPK pathway in T and B lymphocytes has been shown to modulate the turnover of E2A proteins by phosphorylation of Thr355 of the E47/E12 that is followed by ubiquitination and subsequent degradation (46–48). Hence, we next tested whether inhibition of MEK reduces the phosphorylation of E47. To do so, E47 proteins were immune-precipitated from lysates of 38c13 cells, which were treated or untreated with UO126 for 6 h and analyzed for the relative level of threonine phosphorylation by Western blotting. The results in Fig. 7 show that treatment of 38c13 cells with UO126 reduces the level of phosphorylated E47 by 1.7-fold (Fig. 7A). Because phosphorylation reduces E47 stability and increases its degradation (46, 47), we tested whether E47 accumulates in 38c13 cells treated with MEK/ERK inhibitor. To do so, we analyzed total E47 in cell lysates of 38c13 cells treated with UO126 for 6–12 h. Fig. 7B shows that treatment with UO126 resulted in a significant 1.7- to 1.75-fold increase in the total amount of E47, which was evident upon extended culturing duration (12 h) . Importantly, E2A mRNA levels were not altered in the UO126-treated cells, thus indicating that transcription of E2A gene is not perturbed (Fig. 7C). We concluded that upon inhibition of MEK/ERK, phosphorylation and turnover of E47 are reduced, leading to accumulation of E47 and increased binding and expression of Rag genes in 38c13 cells. In support of these findings, we found that germline λ transcription, which is also regulated by E2A proteins (49), is increased in UO126-treated cells (Supplemental Fig. 7).
BCR signals are integrated through multiple signaling pathways into cellular differentiation, activation proliferation, and survival. One critical outcome of these signals is the regulation of Rag expression during early development of B lineage cells (23, 24). Earlier studies have shown that the PI(3)K/Akt pathway is a major regulator of Rag expression in B cell development and that this regulation is mediated by Foxo1 (23, 26, 27). However, there are many examples in which multiple signaling pathways and transcriptional regulators cooperate in tuning the expression of genes. The cooperation may reflect synergism, additive effect, complementary effect, and, in many cases, redundancy (50). In agreement with this, we show in this study that the MEK/ERK pathway coordinates with the PI(3)K/Akt pathway in regulating Rag expression in 38c13 B lineage cells, and that this coordination is mediated through E47 transcription factor.
The conclusions drawn in this study are based on the use of the B cell lymphoma cell line 38c13. Although this fact may limit the scope of the findings, we think that the use of the 38c13 cell line confers a significant advantage by allowing us to analyze the regulation of Rag expression independently of cellular differentiation. This is particularly important since in normal B cell development the regulation of Rag and cellular differentiation are linked together (1). The uniqueness of Rag expression in 38c13 cells has been used in the past to study secondary rearrangements and receptor revision at the L chain locus (33, 51). It is unclear why Rag is continuously expressed in these cells, and one possible explanation for this is the inappropriate BCR-mediated suppressing signals. Indeed, elevation of these signals, imposed by BCR ligation, suppresses Rag expression (this study and Ref. 34), whereas inhibition of these signals enhances the expression of Rag (this study). Overall, these findings indicate that BCR-mediated regulation of Rag gene expression in 38c13 cells is intact.
The present study has revealed an important role of “tonic” ligand-independent activation of MEK/ERK pathway in the regulation of Rag expression, as inhibition of these signals significantly increased the levels of Rag in 38c13 cells. There are several studies supporting these findings. First, we have previously shown that in normal and Ig transgenic B cells that fail to suppress Rag expression, the level of ERK phosphorylation is reduced (7, 8). Second, in T lymphocytes, suppression of Rag expression is mediated through tonic activation of ERK and Abl kinases (30). In contrast, it has been shown that activation of RAS-MEK-ERK is necessary to induce RAG transcription and to initiate Ig gene rearrangements. Thus, activated Ras expression induces developmental progression and Ig-κ gene rearrangements in the absence of pre-BCR signals (52), and inhibition of either MEK or ERK upon IL-7 withdrawal attenuated the transcription of both Rag-1 and Rag-2 (53). These findings may suggest that activation of MEK/ERK is necessary to initiate the transcription of Rag genes, but appropriate tonic signals are also necessary to turn it off once the BCR is expressed. These tonic signals may also coordinate with other cellular changes associated with developmental progression.
We show in this study that ligation of the BCR suppresses RAG expression in 38c13 cells and that this suppression is also mediated by MEK/ERK pathway. These findings are in apparent contradiction with receptor editing in negative selection of B cells. Thus, immature B cells that encounter self Ag upregulate Rag expression and undergo secondary L chain rearrangements (4, 10). In these cells the MEK/ERK pathway is activated upon BCR ligation (54), and treatment with the MEK/ERK inhibitor partially abolishes receptor editing (31). In contrast, some peripheral B cell populations have also been shown to express Rag and to revise the BCR (receptor revision; 11, 12), but upon BCR ligation Rag expression is effectively suppressed (13, 14). This suggests that the outcome effects of BCR signaling and MEK/ERK activation on Rag gene expression may be developmentally regulated. Thus, whereas in immature B cells BCR signaling stimulates Rag expression, in peripheral B cells it turns it off. In the case of the 38c13 cells, it is unclear why Rag is continuously expressed. However, as has been shown earlier, the response of the cells to BCR ligation by suppressing Rag expression resembles that of peripheral B cells undergoing L chain gene rearrangement, rather than that of immature B cells in the bone marrow (34). We show in this study that this response is mediated through the MEK/ERK pathway.
Earlier studies suggested that PI(3)K/Akt is a key regulator of Rag transcription in primary B cells (26), and that this function depends on the PI(3)K catalytic domain p110δ (24). The data shown in this study are in agreement with studies demonstrating that inhibition of PI(3)K resulted in the most profound increase in Rag expression in 38c13 cells. However, our data indicate that PI3K is not the exclusive pathway in the regulation of Rag. This finding is supported by the fact that κ-chain rearrangement or Rag transcription were significantly reduced, but not completely abolished, upon inhibition of PI(3)K pathway or suppressing Foxo1 expression in immature B cells (25, 26). To this end, we show that specific inhibition or activation of the MEK/ERK pathway effectively modulates Rag expression in 38c13 cells. Moreover, we show that the MEK/ERK and PI(3)K pathways coordinate in regulating Rag, and an additive effect in the induction or suppression of Rag is obtained when both pathways are specifically inhibited. There are many examples in which expression of genes is regulated through multiple pathways, including NF-κB (55), BCL-2 family proteins (56), and activation-induced cytidine deaminase (57), which in many cases function for tuning expression levels and/or balancing functional properties of the target genes. Thus, the BCR activates two independent pathways that coordinate for induction of Rag and for tuning its expression level in B lineage cells.
An important issue that is raised here is how the two pathways coordinate the regulation of Rag. Whereas the PI(3)K/Akt regulation of Rag transcription is mediated by Foxo1, we show in this study that the MEK/ERK pathway coordinates with the regulation of Rag through modification of E47 turnover and binding to the ERag regions. There are several studies supporting the existence of this regulatory pathway. First, the E2A proteins are essential regulators of V(D)J recombination by binding to the heavy and L chain enhancers (58, 59), as well as to the ERag region (44, 45), to drive transcription. Second, expression of the transcription factor E2A with the recombinases Rag-1 and Rag-2 is sufficient to induce Ig-κ recombination in a nonlymphoid cell line (60). Third, in E47-defficinet mice, germline transcripts of Rag at the common lymphoid progenitors are profoundly reduced (61). In accordance with this, Rag expression and receptor editing in E47+/− immature B cells undergoing negative selection are suppressed (62). More recent studies have shown directly that E47 is essential for promoting developmental progression at the proper-B stage, for Ig-λ genes rearrangements, and for the induction of receptor editing (49). Fourth, studies have shown that activation of the ERK/MAPK pathway in T and B lymphocytes leads to E2A degradation, thus suggesting that the turnover of E2A proteins is regulated by MAPK activities (46, 47). Hence, in addition to their essential role for differentiation and commitment in the B lineage (63), E2A may also regulate transcription of Rag.
Inhibition of MEK/ERK resulted in a small increase in the binding of Foxo1 to the ERag region, which is in agreement with earlier studies showing that the MEK/ERK pathway also regulates transcription and turnover of Foxo gene products (64, 65). However, when both PI(3)K and MEK/ERK pathways were inhibited we did not find an increased binding of Foxo1 to ERag region beyond that obtained upon inhibition of PI(3)K alone. This is in agreement with earlier studies proposing that PI(3)K/Foxo1 is a major pathway regulating the transcription of Rag gene in B lineage cells (25–27). Instead, we found that inhibition of MEK/ERK, but not PI(3)K, resulted in a significant increase in the binding of E47 to the ERag region. The MEK/ERK pathway regulates the turnover of E2A proteins, possibly by phosphorylating E2A-encoded proteins on serine/threonine residues as a prelude to ubiquitin modification (46, 47). Consistent with this, we found that threonine phosphorylation of E47 and its consequential turnover rate were reduced in 38c13 cells treated with MEK/ERK inhibitor, which led to the accumulation of E47. This may explain the increased binding of E47 to the ERag regions and the increased Rag expression. The possibility that inhibition of MEK/ERK increases E47 synthesis is unlikely since activation of the MEK/ERK pathway does not increase E2A mRNA above that of the steady-state level (47), and since E2A mRNA levels are not altered in UO126-treated cells (shown in this study). Additionally, the accumulation of E47 was only evident upon extended duration of the culture, suggesting that under steady-state conditions E47 protein is produced at a relatively slow rate. The finding that tonic phosphorylation of ERK was abolished within 2 h after treatment with MEK/ERK inhibitor, but that a significant elevation of Rag mRNA was detected only after 6 h, supports this suggestion. Hence, our study suggests that the PI(3)K and MEK/ERK pathways coordinate in the regulation of Rag transcription in independent manners. A similar coordination between the MAPK and PI(3)K pathways has been shown in the growth regulation of tumor cells (64, 66). Also, in pre-B cells, IL-7R and pre-BCR signaling coordinate expansion and subsequent L chain recombination, and this function is mediated through the activation of MEK/ERK and PI(3)K pathways (53).
In this study, we show changes in RAG expression as measured on mRNA levels. Importantly, these changes can be regulated by differential synthesis, differential degradation, or both. Earlier studies with B cell lines have shown that both processes can be regulated by BCR signals (67–69). In primary B cells undergoing receptor editing, however, RAG mRNA expression is regulated at the level of RAG transcription, rather than mRNA stability (70). The changes in the binding of E47 (shown in this study) and Foxo1 (Refs. 25–27 and shown in this study) to the ERag regions may suggest that the effects on RAG mRNA levels we found in this study are regulated at the transcriptional level, but that they do not support or exclude changes in Rag mRNA stability.
Finally, B lymphocytes make crucial fate decisions based on the Ag receptor signals. Appropriate tonic signals are necessary for immature B cells to suppress Rag expression and to progress along the developmental pathway. Downregulation of Rag is also important for the establishment of allelic exclusion. As we have shown in this study, the BCR-mediated regulation of Rag is coordinated by the PI(3)K/Foxo1 and the MEK/ERK/E47 pathways. Such coordination may facilitate the fine tuning of Rag expression during B lymphopoiesis, receptor editing, and reactivation in the periphery, rather than an all-or-none expression pattern. Although this coordination is shown in this study using a B cell lymphoma line, it is important now to recapitulate and extend these findings in normal B cells.
We thank Drs. D. Nemazee (The Scripps Research Institute, La Jolla, CA) and A. Aharonheim (Technion, Haifa, Israel) for reviewing the manuscript.
Disclosures The authors have no financial conflicts of interest.
This work was supported in part by the National Council for Research and Development, Israel, jointly with the Deutsches Krebsforschungszentrum (Heidelberg, Germany) and the Colleck Research Fund.
The online version of this article contains supplemental material.
Abbreviations used in this paper:
G1 phase of cell cycle