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The Journal of Immunology, 2007, 178: 2631-2635.
Copyright © 2007 by The American Association of Immunologists, Inc.

CUTTING EDGE

Cutting Edge: TFII-I Controls B Cell Proliferation via Regulating NF-{kappa}B1

Todd Ashworth* and Ananda L. Roy2,*,{dagger},{ddagger}

* Program in Immmunology and {dagger} Program in Genetics, {ddagger} Department of Pathology, Tufts University School of Medicine, Boston, MA 02111


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
 References
 
The multifunctional transcription factor TFII-I physically and functionally interacts with Bruton’s tyrosine kinase in murine B cells. However, the downstream functions of TFII-I in B cells are unknown. Toward achieving this goal, we established stable posttranscriptional silencing of TFII-I in WEHI-231 immature murine B cells, which undergoes growth arrest and apoptosis either upon anti-IgM or TGF-beta signaling. In this study, we show that TFII-I promotes growth arrest of cells in a signal-dependent manner. Unlike control cells, B cells exhibiting loss of TFII-I function fail to undergo arrest upon signaling due to up-regulation of c-Myc expression and concomitant down-regulation of both p21 and p27. Loss of TFII-I is also associated with simultaneous increase in nuclear c-rel and decrease in p50 homodimer binding. Thus, besides controlling c-myc transcription, TFII-I controls B cell proliferation by regulating both nuclear translocation of c-rel and DNA-binding activity of p50 NF-{kappa}B.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Disclosures
 References
 
Autoreactive cells are eliminated during B cell development by clonal deletion, which is important for maintenance of immune tolerance (1). Clonal deletion of B cells occurs primarily at the immature stage. A failure to destroy immature B cells that are not necessary results in uncontrolled proliferation and survival, which may trigger development of autoimmune disease and cancer. Signal transduction via the B cell Ag receptor (Bcr)3 is important in regulating activation, clonal deletion, and expansion of B lymphocytes (1). Unlike the immature B cells, which undergo apoptosis or anergy upon stimulation via the Bcr, engagement of Bcr in mature B cells promotes growth and survival. The murine B lymphoma cell line WEHI-231 provides a model to study clonal deletion of immature B cells because treatment with anti-IgM (or TGF-beta) induces the WEHI-231 cells to arrest at the G0/G1 phase of the cell cycle before eventually undergoing cell death by apoptosis (2, 3). However, cell cycle arrest and apoptosis in WEHI-231 cells can be rescued by signals derived through the CD40 receptor (4).

The transcriptional pathways that control anti-IgM-induced growth regulation in WEHI-231 cells are unclear but involve both NF-{kappa}B-dependent and -independent events. Because Bcr-signaling induces activation of transcription factor TFII-I (reviewed in Ref. 5), we sought to determine the precise role of TFII-I in Bcr-mediated growth regulation in WEHI-231 cells. TFII-I is a ubiquitously expressed, signal-induced multifunctional transcription factor that facilitates communication between upstream regulatory factors and the basal machinery (5, 6, 7, 8). In resting B cells, TFII-I associates with Bruton’s tyrosine kinase (Btk) (9, 10). Mutations in Btk lead to Xid in humans (X-linked agammaglobulinemia) and mice (Xid) (reviewed in Ref. 11). Although TFII-I associates in vivo with Btk in the resting state, membrane IgM cross-linking in B cells leads to tyrosine phosphorylation of TFII-I, dissociation from Btk, and its subsequent nuclear localization. Importantly, wild-type but not Xid mutant Btk associates with TFII-I (10). These results suggest that association between TFII-I and Btk is functionally important and that impairment in this association may result in diminished TFII-I-dependent transcription and contribute to defective B cell development or function (10). Despite these observations, the precise role of TFII-I in Bcr-induced cellular proliferation is not known.

Apart from the TFII-I pathway, activation of Btk leads to the activation of the NF-{kappa}B pathway, which is necessary for cell survival (12, 13, 14, 15). It is shown that activation of Btk leads to the activation of protein kinase Cbeta, which phosphorylates the I{kappa}B kinase-{alpha}, resulting in phosphorylation of the I{kappa}B (14). This subsequently leads to nuclear translocation of the active NF-{kappa}B required for survival (15). Whether (and how) the TFII-I pathway and the NF-{kappa}B pathway intersect in B cells is unknown. In this regard, it is interesting to note that although a physical link between TFII-I and NF-{kappa}B has been shown (16), whether or not the two pathways functionally converge in B cells is unclear. Consistent with this intriguing possibility, we show that short hairpin (sh)RNA-induced silencing of TFII-I in WEHI-231 cells results in deregulation of NF-{kappa}B. Moreover, deregulation of NF-{kappa}B leads to up-regulation of c-myc and down-regulation in both p21 and p27, both of which are necessary for anti-IgM-induced growth arrest in WEHI-231 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Disclosures
 References
 
Abs and reagents

WEHI-231 cells (a gift from P. Brodeur, Boston, MA) were grown in complete RPMI 1640 with 10% FBS and cultured at a density of 0.5–1 x 106 cells/ml. Abs specific for c-Myc (sc-788), p50 (sc-114), c-Rel (sc-71), p21 (sc-471), p27 (sc-528), cyclin D2 (sc-593), and cyclin E (sc-481) were obtained from Santa Cruz Biotechnology. TGF-beta1 (240-B-002) was obtained from R&D Systems. Real-time primers and probes for c-myc were from obtained from Applied Biosystems, and designed using the TaqMan 1.5 express software.

Generation of shs and infection of the WEHI-231 cell line

The shRNA against TFII-I was generated as described previously (17). The lentiviral system consists of three distinct plasmids, the envelope (pVSVG), the helper (pH82{Delta}R’), and pLLP3.7. Briefly, 2 µg of total plasmid was used for production of lentivirus using 0.5 µg of pVSVG, 0.5 µg of p82{Delta}R’, and a total of 1 µg of pLLP3.7 constructs containing TFII-I hairpins 1 and 3. The exact protocol was followed for the nonsilencing hairpin 2 (control). Lentivirus was generated for 44–48 h in the BL3 facility, followed by infection, with the addition of viral supernatant to 0.5 x 106 WEHI-231 cells. After 24 h, single cells were sorted in 96-well round-bottom plates. Optimal infection of WEHI-231 cells yielded 85% GFP expression. Clones were grown for 1 wk followed by transfer to 6-well plates.

Cell cycle analysis of WEHI-231 cells stimulated with anti-IgM or TGF-beta

WEHI-231 cells were plated in 6-well plates at a density of 1 x 106cells/ml before stimulation, equilibrated for 0.5–1 h at 37°C, and stimulated with goat anti-mouse IgM (5 µg/ml) or TGF-beta1 (5 ng/ml) for 24 h at a final density of 0.5 x 106 cells/ml. The cells were fixed overnight in 70% ethanol at 4°C, washed, RNase A treated (100 µg) for 30 min at 37°C, and stained with propidium iodide (50 µg/ml). Cell cycle events were acquired using a FACSCalibur (BD Biosciences) and analyzed with ModFit software.

Thymidine incorporation assay

A total of 1 x 104 cells were plated in 96-well plates at 100 µl/well and stimulated at a final concentration of 5 µg/ml goat anti-mouse IgM for 24 h followed by the addition of 1 µCi of [3H]thymidine 20 h before measuring incorporation levels. Thymidine uptake was measured using the Trilux software in conjunction with the Trilux 1450 microbeta scintillation counter.

Quantitative PCR analysis

Cells were stimulated in triplicate with goat anti-mouse IgM for 0, 4, 8, and 24 h, harvested, and total RNA was isolated using the Qiagen RNeasy Mini kit. A total of 250–500 ng of total RNA was reverse transcribed using the Applied Biosystems RT-PCR kit in a final volume of 25 µl/reaction. The RT-PCR conditions were as follows: 24°C for 10 min, 42°C for 60 min, and 99°C for 5 min. As a template control, quantitative analysis was performed using probes specific for the 18S ribosomal cDNA. The ratio of c-myc:18S was determined and relative values between groups were compared.

Nuclear/cytoplasmic extraction

Following stimulation, cells were washed in PBS, spun down at 1,000 rpm for 5 min, and resuspended in 5 vol of hypotonic lysis buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 1 mM MgCl2, 1 mM DTT, and 0.05% Triton X-100), then incubated for 10 min on ice. Cells were then spun at 1,400 rpm for 5 min, and the pellet was resuspended in 2 vol of hypotonic lysis buffer (supplemented with 1x complete protease inhibitor tablet; Roche), lysed by repeated passage through a 27-gauge syringe (5–10 times), and centrifuged for 20 min at 9,200 rpm. The supernatant was removed and stored as the cytoplasmic fraction. The remaining pellet was then resuspended in a high salt nuclear extraction buffer (20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM DTT, 10% glycerol, and 1% Igepal-300) and rotated for 1 h at 4°C. The pellet was centrifuged at 13,000 rpm for 5 min, and the supernatant was transferred to a new tube.

EMSA

Nuclear extracts were generated from WEHI-231 control and TFII-I KD cells as described above. Extracts (5 µg) were then incubated with a radiolabeled NF-{kappa}B-specific probe (derived from the c-myc promoter sequence) for 30 min. Approximately 1 µg of poly(dI:dC), as a nonspecific DNA, was included during the probe incubation. Reaction mixtures were then loaded onto a 6% polyacrylamide native gel, ran for 2 h at 175 V, dried for 1 h, and exposed.

Retroviral transduction of WEHI-231 cells

293 FT cells were transfected with 4 µg of pCL-Eco and 4 µg of human pSFG TFII-I{Delta} (18). Virus was harvested 48 h posttransfection, filtered (0.45 µM), and added to 0.2 x 106 WEHI-231 cells stably expressing shRNA against murine TFII-I in the presence of polybrene (8 µg/ml). Cells were infected for 24 h in a 6-well plate, washed, replated, and analyzed for TFII-I{Delta} expression 48 h postinfection by Western blot.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Disclosures
 References
 
Loss of TFII-I leads to continued proliferation of WEHI-231 cells

Because genetic ablation of TFII-I results in early embryonic lethality (D. Bayarsaihan and F. Ruddle, personal communication), we established stable posttranscriptional silencing of TFII-I to determine its role in B cells. WEHI-231 cells were infected with a lentiviral vector carrying shRNA against TFII-I according to published protocols (19). GFP-bright cells were sorted (Fig. 1A), and selected clones were analyzed for reduction in TFII-I protein by Western blot (Fig. 1B). Greater than 80% "knockdown" (KD) of TFII-I (TFII-I KD) was achieved in multiple clones, a representative clone is shown in Fig. 1B. A noninhibitory clone was selected as a control KD for all subsequent experiments. Because TFII-I has been implicated in cellular proliferation (17), we tested the proliferative potential of the control and TFII-I KD WEHI-231 clones upon anti-IgM stimulation. Although anti-IgM stimulation of both the parental WEHI-231 and control KD cells resulted in a significant but expected proliferative block, surprisingly loss of TFII-I resulted in continued cellular proliferation under the same conditions (Fig. 1C). Anti-IgM or TGF-beta stimulation of WEHI-231 cells is known to induce G0/G1 arrest. Hence, the TFII-I KD cells compared with the control cells were analyzed for their G0/G1 content. Both TGF-beta and anti-IgM stimulation resulted in significant arrest in G0/G1 phase in control KD cells, but neither induced similar arrest in TFII-I KD cell clones (Fig. 1D). The effect was more pronounced upon TGF-beta stimulation. Thus, compared with the control KD cells, TFII-I KD cells exhibited a nearly 50% reduction in G0/G1 cells, indicating that these cells are still proliferating (Fig. 1D). Taken together, these results demonstrate that loss of TFII-I leads to a failure of anti-IgM or TGF-beta-induced cell cycle arrest and proliferation of WEHI-231 B cells. That the effect is specifically due to silencing of TFII-I was demonstrated by reconstitution experiments (Fig. 2). WEHI-231 cells exhibiting silencing of TFII-I was reconstituted with retrovirally expressed ectopic human TFII-I{Delta} isoform (Fig. 2A) because the human TFII-I is not silenced by the shRNA against the murine TFII-I (18). The migration of the ectopic protein is slightly higher than the endogenous protein because it is fused to a 6x histidine moiety. Importantly, the cell cycle arrest upon silencing of TFII-I is nearly reversed upon TFII-I reconstitution (Fig. 2B).


Figure 1
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  		FIGURE 1. TFII-I KD results in decreased growth arrest of WEHI-231 cells. A, FACS analysis of WEHI-231 cells infected with shRNA against TFII-I. Approximately 85% of the cells were infected following the procedure. B, Western blot analysis was performed to assess the level of KD 1 wk after the infection. C, Thymidine incorporation assays were performed in the absence and presence of anti-IgM stimulation after 24 h in culture. D, Cell cycle profiles were assessed in the presence and absence of anti-IgM and TGF-beta. Two different TFII-I KD clones were compared with the KD control. These results are the average of five independent experiments.

 

Figure 2
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  		FIGURE 2. Reconstitution of TFII-KD WEHI-231 cells with human TFII-I{Delta} restores growth arrest. A, TFII-I KD cells were transduced with the human pSFG TFII-I{Delta} retroviral vector. Western blot analysis was performed on lysates from control KD, TFII-I KD, and TFII-I KD reconstituted with pSFG TFII-I{Delta}. Endogenous levels of TFII-I{Delta} were restored upon retroviral transduction. B, Control KD, TFII-I KD, and TFII-I KD+ pSFG TFII-I{Delta} cells were stimulated with either medium or goat anti-mouse IgM (5 µg/ml) for 24 h and analyzed for growth arrest (%G0/G1) by cell cycle analysis. Results are the average of four independent experiments.

 
Derepression of c-Myc upon silencing of TFII-I

Stimulation of WEHI-231 cells with anti-IgM results in down-regulation of c-myc, which could account for the block in proliferation (20, 21). We thus analyzed the levels of c-Myc protein (Fig. 3A) and RNA in TFII-I KD cells compared with the control KD cells upon anti-IgM stimulation. Although the c-Myc levels dropped significantly in control cells upon anti-IgM stimulation between 4 and 8 h and continued to be low until 24 h (Fig. 3A), the c-Myc levels did not drop significantly in TFII-I KD cells. Consistent with the constant levels of c-Myc protein in TFII-I KD cells, quantitative RT-PCR analysis confirmed that the myc RNA levels also remained significantly higher in these cells compared with the control cells (Fig. 3B), suggesting that TFII-I directly or indirectly controls c-myc transcription.


Figure 3
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  		FIGURE 3. Lack of c-myc repression upon anti-IgM stimulation of WEHI-231 cells in the absence of TFII-I. A, WEHI-231 control KD and TFII-I KD cells were stimulated for 4, 8, and 24 h with anti-IgM and analyzed for c-Myc protein levels by Western blot. B, c-myc RNA levels were monitored by quantitative PCR between 0-24 h poststimulation, in TFII-I and control KD cells. Results are representative of three independent experiments.

 
Down-regulation of p21 and p27 in the absence of TFII-I

It has been demonstrated that c-Myc transcriptionally down-regulates p27 and p21 in WEHI-231 cells (22, 23). Compared with the control KD cells, both p27 (Fig. 4A) and p21 (Fig. 4B) protein levels were down in TFII-I KD WEHI-231 cells. In contrast, neither the cyclin D2 nor the cyclin E levels were reduced in TFII-I KD cells (Fig. 4C). Thus, silencing of TFII-I does not cause general inhibition of all cell cycle regulators but results in selective inhibition of p21 and p27 gene products. However, because the basal p21 or p27 levels are down in TFII-I KD cells compared with the control cells, these genes may also directly require TFII-I for basal level transcription. Given the fact that these genes have Inr-containing promoters (22), basal action of TFII-I on these genes is likely. Thus, inhibition of these genes could likely be due to a combined effect of reduced TFII-I levels (basal) as well as persistent c-Myc expression (induced).


Figure 4
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  		FIGURE 4. Reduced p27 and p21 expression in the absence of TFII-I. A, Western Blot analysis was performed on whole cell extracts isolated from control and TFII-I KD cells and analyzed for the expression of p27, p21 (B), and cyclins D2 and E (C). The induction of p27 and p21, but not cyclins D2 and E, were abrogated upon TFII-I KD. In each case, the cells were stimulated for 0-24 h with anti-IgM.

 
Deregulation of NF-{kappa}B pathway upon silencing of TFII-I

Transformation of WEHI-231 cells is largely dependent on the instability of the NF-{kappa}B inhibitory protein, I{kappa}B (24). Loss of I{kappa}B{alpha} stability results in increased nuclear localization of NF-{kappa}B heterodimers (p50/c-rel). Moreover, ectopic expression of c-Rel rescued anti-IgM-stimulated WEHI-231 cells from apoptosis (25). We thus tested whether the lack of anti-IgM-induced growth arrest observed upon silencing of TFII-I is a consequence of increased levels of nuclear NF-{kappa}B. Surprisingly, a significant increase in constitutive nuclear c-Rel was observed upon TFII-I KD compared with the control cells (Fig. 5A, upper panel, compare lanes 1 and 7), which persisted until 8 h after anti-IgM stimulation in TFII-I KD cells compared with the control cells (compare lanes 4 and 6 with 10 and 11). Western blot analysis showed equivalent levels of total c-Rel in control as well as TFII-I KD cells (data not shown). Moreover, a direct interaction of TFII-I with c-Rel was also not observed (data not shown). Thus, an increased nuclear c-Rel upon loss of TFII-I is not due to direct sequestration of cytoplasmic c-Rel by TFII-I. Next, we tested the levels of cytoplasmic and nuclear p50 and found that the levels of p50 remains virtually unchanged in both control and TFII-I KD cells (Fig. 4A, lower panel). Because I{kappa}B{alpha} up-regulation and stabilization was shown to promote down-regulation of NF-{kappa}B activity in WEHI-231 cells (25, 26, 27), we tested whether I{kappa}B{alpha} levels are altered in TFII-I KD cells upon anti-IgM stimulation. However, no significant defect in I{kappa}B{alpha} was observed upon loss of TFII-I (Fig. 5B), suggesting that enhanced nuclear c-Rel and maintenance of c-Myc levels in TFII-I KD are independent of I{kappa}B{alpha}.


Figure 5
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  		FIGURE 5. TFII-I KD is associated with increased nuclear c-Rel, and decreased NF-{kappa}B p50 homodimer binding. A, Nuclear and cytoplasmic extracts were generated from Ag-stimulated control and TFII-I KD WEHI-231 cells and analyzed for c-Rel localization. Densitometric scan revealed a 2-fold increase in nuclear c-Rel upon TFII-I KD before stimulation and nearly 1.5-fold after anti-IgM stimulation. B, Increased nuclear c-Rel is not associated with a decrease in I{kappa}B{alpha} expression. C, TFII-I KD results in decreased NF-{kappa}B homodimer binding upon anti-IgM stimulation. EMSA was performed using nuclear extracts generated from control, and TFII-KD WEHI-231 cells were stimulated for the indicated times. D, Ectopically expressed p50 interacts with TFII-I{Delta}. COS7 cells were transfected with GST-p50 and GFP-TFII-I{Delta} expression constructs and subjected to coimmunoprecipitation with GST Ab. E, TFII-I can regulate both p50 DNA binding and nuclear translocation of c-Rel. Silencing of TFII-I may result in decreased p50 homodimer binding to the c-myc promoter as well as enhanced formation of p50-c-Rel heterodimer formation, both of which can contribute to c-myc activation. Sustained c-Myc levels would in turn result in repression of p21 and p27, leading to cell cycle progression and enhanced S phase entry.

 
Because the p50 subunit of NF-{kappa}B lacks a classical transactivation domain, but contains a functional DNA-binding domain, p50 homodimers have been shown to repress the expression of NF-{kappa}B-dependent genes (25). Formation of the p50 homodimer was observed as a late event (12–24 h) following anti-IgM stimulation of WEHI-231 cells and has been shown to repress c-myc transcription (25). To test whether loss of TFII-I results in decreased p50 DNA binding to the c-myc promoter, which could explain continued expression of c-myc following anti-IgM stimulation, we performed an EMSA. Although the DNA binding of p50 homodimer was enhanced in control cells following anti-IgM stimulation, no such increase was observed in TFII-I KD cells (Fig. 5C). Moreover, consistent with an increase in nuclear c-Rel in TFII-I KD cells, an increase in c-rel containing DNA-binding complexes was also observed under these conditions. We next tested whether TFII-I might physically interact with p50. When these proteins were ectopically coexpressed in COS cells, an interaction between p50 and TFII-I was observed because GST-tagged p50 was capable of pulling down GFP-tagged TFII-I (Fig. 5D). Together with the fact that TFII-I and p50 has been shown previously to augment each other’s DNA-binding capabilities (16), our current data further suggest that TFII-I physically interacts with p50 and might serve (directly or indirectly) as a cofactor for p50 DNA binding. These observations might provide a rationale for the diminished DNA binding by p50 observed in the absence of TFII-I.

Although our data indicates a novel pathway of Bcr-mediated gene regulation via TFII-I to control B cell proliferation, the precise mechanistic basis for this pathway at present is less clear. Because transient transfection experiments failed to show a significant direct effect of TFII-I on the c-myc promoter (data not shown), it is likely that the transcriptional effects of TFII-I on c-myc are indirectly mediated via NF-{kappa}B. We speculate that this could be in part due to a lack of recruitment of the p50 homodimer to the c-myc promoter (Fig. 5E). Whether this is due to alteration of p50 interaction with histone deacetylases in the absence of TFII-I is, however, not known yet. Nevertheless, it remains to be tested whether TFII-I might, under some conditions, have a more direct effect on c-myc transcription. Besides controlling the DNA-binding activity of the p50 homodimer, TFII-I appears to control the nuclear translocation of c-Rel in an I{kappa}B{alpha}-independent fashion (Fig. 5E). One possible scenario is that lack of p50 DNA binding and concomitant increase in nuclear c-Rel might lead to a more efficient p50-c-Rel heterodimer formation, which would result in an activation of the c-myc gene. Future experiments are designed to address many of these questions.


   Acknowledgments
 
We thank Carl Novina for the lentilox plasmid system, Peter Brodeur for WEHI-231 cells, Sanker Ghosh for the p50 construct.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results and 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 in part by a grant from National Institutes of Health (AI 45150; to A.L.R.). Back

2 Address correspondence and reprint requests to Dr. Ananda L. Roy, Department of Pathology, Tufts University School of Medicine, 150 Harrison Avenue, Boston, MA 02111. E-mail address: ananda.roy{at}tufts.edu Back

3 Abbreviations used in this paper: Bcr, B cell Ag receptor; Btk, Bruton’s tyrosine kinase; sh, short hairpin; KD, knockdown. Back

Received for publication April 26, 2006. Accepted for publication December 21, 2006.


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

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