* Department of Pharmacology and Toxicology,
Department of Biochemistry and Molecular Biology,
Institute for Environmental Toxicology, and
National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824; and
¶ Department of Biology, Kyonggi University, Paldal-gu, Suwon-Si, Korea
Received August 26, 2003; accepted October 2, 2003
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ABSTRACT |
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Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin; Pax5; X-box-binding-protein-1; immunoglobulin heavy chain; immunoglobulin kappa light chain; B cell.
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INTRODUCTION |
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The gene product of Pax5, also termed B-cell-lineagespecific activator protein, is a bifunctional transcription factor capable of activating and repressing transcription. In hematopoietic cells, Pax5 is only present within the B-cell lineage and functions as an essential regulator of B-cell development and differentiation (Adams et al., 1992). Pax5 is present in pro-B, pre-B, and mature B cells but is down-regulated in plasma cells (Horcher et al., 2001
; Nutt et al., 1997
, 2001
). In mice lacking Pax5, B-cell development is arrested at the pro-Bcell stage identifying Pax5 as an essential B-celllineage commitment factor (Urbanek et al., 1997
). In mature B cells, Pax5 positively regulates the expression of CD19 (Kozmik et al., 1992
) while strongly repressing genes associated with the plasma cell phenotype including immunoglobulin heavy chain (IgH), kappa light chain (Ig
), J chain, and XBP-1 (Neurath et al., 1994
; Reimold et al., 1996
; Rinkenberger et al., 1996
; Roque et al., 1996
). Pax5 DNA binding motifs are present within the IgH 3'
enhancer, Ig
3' enhancer, J chain promoter, and XBP-1 promoter to which Pax5 is recruited to exert transcriptional repression (Maitra and Atchison 2000
; Neurath et al., 1994
, 1995
; Rinkenberger et al., 1996
; Roque et al., 1996
). The specific mechanisms by which Pax5 represses transcription have been partially elucidated, revealing its ability to inhibit the transcriptional activating properties of other transactivating factors, including PU.1 and c-jun/AP-1 through direct protein:protein interaction (Maitra and Atchison, 2000
). Based on the well-established suppression produced by TCDD on B-cell differentiation and immunoglobulin secretion, the objective of the present study was to investigate the effect of TCDD on Pax5 and its downstream targets, IgH, Ig
, J chain, and XBP-1. Our studies show that B cells activated in the presence of TCDD exhibited elevated Pax5 mRNA levels, protein, and DNA binding activity compared to B cells activated in the absence of TCDD. Concomitant with elevated cellular Pax5, the TCDD-treated B cells also displayed marked suppression of IgH, Ig
, and J chain mRNA levels and cellular XBP-1.
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MATERIALS AND METHODS |
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Cell line.
The CH12.LX B-cell line derived from the murine CH12 B-cell lymphoma (Arnold et al., 1983) has been previously characterized by Bishop and coworkers (Bishop and Haughton, 1986
) and was a generous gift from Dr. Geoffrey Haughton (University of North Carolina). CH12.LX cells were grown in RPMI-1640 (Gibco BRL, Grand Island, NY) supplemented with 10% bovine calf serum (BCS; Hyclone, Logan, UT), 13.5 mM HEPES, 23.8 mM sodium bicarbonate, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, and 50 µM ß-mercaptoethanol. The cells were maintained at 37°C in an atmosphere of 5% CO2. The day before treatment, the CH12.LX cells (2.5 x 104 cells/ml) were cultured in treatment media (growth media as stated above but with 5% heat-inactivated BCS) overnight at 37°C in an atmosphere of 5% CO2.
Enzyme-linked immunosorbant assay.
The supernatants were harvested at the indicated times from naive or LPS (5 µg/ml)-activated CH12.LX cells that were treated with 10 nM TCDD or the vehicle (0.01% DMSO). The supernatants were analyzed for IgM by sandwich ELISA as described by Sulentic et al.(1998). Briefly, 100 µl of supernatant or standard (mouse IgM
light chain) were added to wells of a 96-well microtiter plate (Immulon 4, Dynex Technologies Inc., Chantilly, VA) previously coated with anti-mouse Ig capture antibody (Roche Molecular Biochemicals, Indianapolis, IN) and then incubated at 37°C for 1.5 h. After the incubation period, the plate was washed with 0.05% Tween-20 PBS and H2O. A horseradish peroxidaselinked anti-mouse IgM detection antibody was added to the plate and incubated for 1.5 h at 37°C. Unbound detection antibody was washed from the plate with 0.05% Tween-20 PBS and H2O. ABTS substrate (Roche Molecular Biochemicals, Indianapolis, IN) was added, and colorimetric detection was performed over a 1-h period using an EL808 automated microplate reader with a 405-nm filter (Bio-Tek, Winooski, VT). The concentration of total IgM in the supernatants was calculated using a standard curve generated from the absorbance readings of known IgM
concentrations.
Western blot analysis.
Western blot analysis was performed on cell lysates from CH12.LX cells. The cell lysates were prepared in HEG (25 mM HEPES, 2 mM EDTA, and 10% glycerol) containing protease inhibitors (complete mini tablets, Roche Molecular Biochemicals), sonicated three times for 5 s to break open the nuclei, and centrifuged at 100,000 x g for 1 h at 4°C. Protein concentrations were determined by the Bradford protein assay (Bio-Rad, Hercules, CA). The cell lysate proteins were resolved by denaturing SDSPAGE (Life Science Products Inc., Denver, CO). The percent acrylamide is indicated in the figure legends. The proteins were transferred to nitrocellulose following electrophoresis (Amersham Pharmacia Biotech, Arlington Heights, IL). Protein blots were blocked in BLOTTO buffer (4% low-fat dry milk/1% BSA in 0.1% Tween-20 TBS) for 12 h at room temperature. Rabbit anti-mouse Pax5 and XBP-1 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Donkey HRPconjugated anti-rabbit IgG was purchased from Amersham Pharmacia Biotech. Immunochemical staining was performed as described in Williams et al.(1996). Detection was performed using the enhanced chemical luminescence method (Amersham Pharmacia Biotech). All blots were stripped and normalized by reprobing for ß-actin using an anti-mouse ß-actin antibody (Sigma-Aldrich). Stripping was performed by submerging the membrane in stripping buffer [100 mM 2-ME, 2% SDS, and 62.5 mM Tris (pH 6.7)] for 30 min at 50°C. The protein blots were then washed, blocked, and reprobed as stated above. Optical density for the protein of interest was measured by densitometry using a model 700 imaging system (Bio Rad).
Real-time reverse transcriptase polymerase chain reaction (RT-PCR).
Real-time RT-PCR was performed on a PE Applied Biosystems PRISM 7000 Sequence Detection System (Foster City, CA). Total RNA was isolated from naive or LPS (5 µg/ml)-activated CH12.LX cells that were treated with 10 nM TCDD and/or the vehicle (0.01% DMSO) at the indicated times using the SV Total RNA Isolation kit (Promega, Madison, WI). To synthesize cDNA, total RNA (500 ng/sample) was used as the template for a reverse transcriptase reaction in 20 µl 1X First Strand Synthesis buffer (Life Technologies, Inc.) containing 500 ng oligo d(T18A/C/GN), 0.2 mM dNTPs, 10 mM dithiothreitol, and 200 U SuperScript II reverse transcriptase (Life Technologies, Inc.). The reaction mixture was incubated at 42°C for 60 min and was stopped by incubation at 75°C for 15 min. Amplification of cDNA (1/20th) was performed using the SYBR Green PCR Core Reagents (PE Applied Biosystems) as suggested by the manufacturers instructions. Primer pairs for each gene were designed using PrimerExpress (PE Applied Biosystems). Gene names, locus link numbers, the forward and reverse primer sequences, and amplicon size are listed in Table 1. The PCR cycling conditions were as follows: initial denaturation and enzyme activation for 10 min at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Each plate contained duplicate standards of purified PCR products with a known template concentration covering at least six orders of magnitude to interpolate the relative template concentration of the experimental samples from standard curves of log copy numbers vs. threshold cycle (Ct). No template controls (NTC) were also included on each plate. The relative level of mRNA was standardized to the housekeeping gene ß-actin in order to control for differences in RNA loading, quality, and cDNA synthesis.
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Statistical analysis of data.
The mean ± standard deviation was generated for each treatment group. The statistical differences between treatment groups and the appropriate controls were determined by first performing a one-way ANOVA that was followed by a Dunnetts two-tailed t test.
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RESULTS |
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DISCUSSION |
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In mature resting B cells, Pax5 represses the transcription of a number of genes that are directly involved in immunoglobulin production and characteristically expressed by plasma cells, including IgH, Ig, J chain, and XBP-1. Based on this fact, the effect of TCDD on IgH, Ig
, J chain, and XBP-1 were investigated in the CH12.LX cells. Consistent with the attenuation of Pax5 down-regulation produced by TCDD in LPS-activated CH12.LX cells, modest IgH, Ig
, J chain transcript levels, and XBP-1 protein were observed. The effect of TCDD in this respect was striking on IgH, Ig
, and J chain mRNA expression as the magnitude of expression for all three genes was comparable to control cells that had not been activated with LPS. Moreover, the inhibition of IgH, Ig
, and J chain mRNA levels in TCDD-treated cells was persistent, lasting throughout the entire 72-h culture period. The repression of J chain by Pax5 has been previously shown to be mediated directly through its binding to a negative regulatory element located on base pairs -113 to -97 within the J chain promoter (Rinkenberger et al., 1996
). Repression of IgH expression by Pax5 is mediated through its DNA binding within at least two distinct regulatory domains of the IgH 3'
enhancer, the hs1,2 and the hs4, thus decreasing IgH 3'
enhancer activity (Neurath et al., 1994
, 1995
). Interestingly, we have previously demonstrated strong inhibition of IgH mRNA levels (Sulentic et al., 1998
, 2000
) by TCDD treatment in LPS-activated CH12.LX cells that was closely correlated with a decrease in IgH 3'
enhancer activity (manuscript submitted for publication). In addition to the previously identified Pax5 binding sites, analysis of IgH 3'
enhancer led to the identification of several TCDD-inducible AhR binding motifs (i.e., DRE-like sites) within the IgH 3'
enhancer, one in the hs4 and one in the hs1,2 domain (Sulentic et al., 2000
). Analysis of the hs4 domain alone (i.e., removed from the context of other regulatory IgH 3'
enhancer domains) showed enhanced activity with TCDD treatment that was only partially attenuated by the mutation of the DRE site. Although further investigation is needed, the dysregulation of the hs4 regulatory domain of the 3'
enhancer in activated B cells treated with TCDD appears to be mediated by multiple mechanisms that, in addition to the induction of AhR DNA binding, may include persistent Pax5 DNA binding.
The influence of Pax5 on Ig has also been recently investigated but remains poorly understood. Similar to the IgH 3'
enhancer, Ig
also possesses a 3' enhancer that is crucial to the regulation of Ig
expression. Within the Ig
3' enhancer, several Pax5 DNA binding sites have been identified that have been shown to repress enhancer activity (Maitra and Atchison 2000
). Interestingly, the mechanism for Pax5 repression of Ig
3' enhancer involves targeting of the transcriptional function of the transcription factor, PU.1, which, like Pax5, is essential for B-cell development (McKercher et al., 1996
; Scott et al., 1994
). The repression of PU.1 transcriptional function by Pax5 is not mediated through the displacement of PU.1 DNA binding but does involve physical interactions between the two factors. In light of these findings by Maitra and Atchison (2000)
, our observation that TCDD treatment interferes with Pax5 down-regulation is consistent with the suppression of Ig
mRNA levels and previously reported Ig
protein (Crawford et al., 2003
) in LPS-activated B cells treated with TCDD.
As observed with IgH, Ig, and J chain mRNA levels, cellular XBP-1 was also strongly inhibited in LPS-activated CH12.LX cells treated with TCDD. Although XBP-1 cellular protein was assayed in this investigation, its inhibition by TCDD most likely also occurs through transcriptional repression by Pax5 rather than at the protein level or at the level of XBP-1 mRNA splicing within the ER. This is suggested by the fact that both p54XBP-1 and p30XBP were decreased similarly in magnitude and that Pax5 is also well established as a potent transcriptional repressor of XBP-1 expression (Reimold et al., 1996
). Current evidence supporting a critical role for XBP-1 in immunoglobulin secretion and the development of plasma cells is based on several important observations. First, when introduced into B-lineage cells, XBP-1 initiated plasma cell differentiation (Reimold et al., 2001
). Second, mouse lymphoid chimeras deficient in XBP-1 possessed a normal number of activated B cells that were able to proliferate, secrete normal amounts of cytokines, and form germinal centers but possessed very little immunoglobulin and were devoid of plasma cells (Reimold et al., 2001
). The role of XBP-1, a basic-region leucine zipper protein in the ATF/CREB family of transcription factors, in B-cell differentiation and immunoglobulin secretion has been linked to its involvement in triggering the assembly of the secretory apparatus necessary for IgM secretion by plasma cells (Calfon et al., 2002
). The very low level of cellular XBP-1 in TCDD-treated CH12.LX cells after LPS activation is again concordant with the marked inhibition of IgM secretion and previously reported inhibition of antibody forming cells by TCDD in a variety of model systems.
An important question remaining to be answered concerns the mechanism by which TCDD interferes with the down-regulation of Pax5, which is known to disrupt the execution of the B-cell differentiation program. At least two putative mechanisms could account for the altered regulation of Pax5 identified in activated B cells treated with TCDD. The first is through positive and direct regulation of the Pax5 promoter by the AhR. In fact, examination of the mouse and human Pax5 promoter revealed at least three core DRE sites within the first 3000 bp 5' of the transcriptional start site. Therefore, it is conceivable that the ligand-activated AhR may positively regulate Pax5 at a time when Pax5 is normally being actively repressed to promote B-cell differentiation. A second putative mechanism that could account for the inadequate down-regulation of Pax5 in TCDD-treated B cells is through the disruption of critical mediators that regulate Pax5 expression. It is important to emphasize that the two putative mechanisms are not mutually exclusive.
In summary, the present investigation demonstrates that TCDD treatment alters the regulation of Pax5, a critical repressor of B-cell differentiation. Typically, as activated B cells differentiate into the plasma cells, Pax5 is down-regulated, leading to an up-regulation of four well-established Pax5 downstream targets, IgH, Ig, J chain, and XBP-1, which are strongly repressed by Pax5 prior to the initiation of the B-cell differentiation program. Using CH12.LX cells as a model, we show that Pax5 mRNA levels, protein, and DNA binding activity are not down-regulated in activated B cells treated with TCDD, which in turn results in concomitant modest levels of IgH, Ig
, J chain mRNA, cellular XBP-1, and, ultimately, secreted IgM.
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ACKNOWLEDGMENTS |
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NOTES |
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