2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Alters the Regulation of Pax5 in Lipopolysaccharide-Activated B Cells

Byung S. Yoo*, Darrell R. Boverhof{dagger},{ddagger}, Dina Shnaider*,{ddagger}, Robert B. Crawford*, Timothy R. Zacharewski{dagger},{ddagger},§ and Norbert E. Kaminski*,{ddagger},§,1

* Department of Pharmacology and Toxicology, {dagger} Department of Biochemistry and Molecular Biology, {ddagger} 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces a profound suppression of the primary immunoglobulin-M (IgM) antibody response. The suppression of IgM production by TCDD can occur through direct interactions with the B cell, is aryl hydrocarbon receptor–dependent, and is mediated through alterations in the differentiation of B cells into plasma cells. The objective of the present investigation was to characterize the effects of TCDD on the regulation of Pax5, a crucial repressor of B-cell differentiation, and four downstream targets that are directly regulated by Pax5 and involved in immunoglobulin regulation, immunoglobulin heavy chain (IgH), kappa light chain (Ig{kappa}), J chain, and X box protein-1 (XBP-1). Lipopolysaccharide (LPS) activation of aryl hydrocarbon receptor–expressing CH12.LX cells induced B cell differentiation and robust immunoglobulin secretion that was markedly (~50%) suppressed in the presence of 10 nM TCDD. Kinetic studies show that LPS-activation induced a time-dependent decrease in Pax5 mRNA levels, protein, and DNA binding activity during a 72-h culture period that was almost completely blocked in the presence of TCDD. Concomitant with the time-dependent down-regulation of Pax5 in LPS-activated control CH12.LX cells, a reciprocal induction of IgH, Ig{kappa}, J chain mRNA levels, and cellular XBP-1 was observed. Conversely, and consistent with the absence of Pax5 down-regulation associated with TCDD treatment, IgH, Ig{kappa}, J chain mRNA, and XBP-1 protein were persistently repressed in LPS-activated CH12.LX cells. Collectively, these studies demonstrate the involvement of altered Pax5 regulation in the suppression of the primary IgM antibody response by TCDD.

Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin; Pax5; X-box-binding-protein-1; immunoglobulin heavy chain; immunoglobulin kappa light chain; B cell.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most immunosuppressive compounds known to man. Among its many effects on the immune system, TCDD produces a profound suppression of primary immunoglobulin-M (IgM) antibody responses against T-cell–dependent as well as T-cell–independent antigens under both in vivo and in vitro conditions. Cell separation/reconstitution experiments and direct addition studies employing immortalized B-cell lines demonstrate that suppression of antibody responses can be mediated in the absence of effects on accessory cells by directly targeting the B cell (Dooley and Holsapple, 1988Go; Sulentic et al., 1998Go). The molecular mechanism responsible for most of the biological effects produced by TCDD involves ligand-mediated activation of the aryl hydrocarbon receptor (AhR), which leads to nuclear translocation, the dissociation of HSP90 from the AhR cytosolic complex, and the dimerization of AhR with the aryl hydrocarbon receptor nuclear translocator (ARNT). The AhR/ARNT complex functions as a transcription factor regulating gene expression. Results from studies employing AhR null mice and cell lines that differ in their expression of AhR suggest that AhR is essential for the inhibition of humoral immune responses by TCDD (Sulentic et al., 1998Go; Vorderstrasse et al., 2001Go). Although the specific molecular mechanism responsible for the suppression of B-cell function has remained elusive, it is clear from numerous published studies that TCDD modestly inhibits proliferation at concentrations that strongly inhibit B cell differentiation into plasma cells and immunoglobulin secretion (Dooley and Holsapple, 1988Go; Luster et al., 1988Go; Morris and Holsapple, 1991Go; Morris et al., 1993Go; Tucker et al., 1986Go).

The gene product of Pax5, also termed B-cell-lineage–specific 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., 1992Go). Pax5 is present in pro-B, pre-B, and mature B cells but is down-regulated in plasma cells (Horcher et al., 2001Go; Nutt et al., 1997Go, 2001Go). In mice lacking Pax5, B-cell development is arrested at the pro-B–cell stage identifying Pax5 as an essential B-cell–lineage commitment factor (Urbanek et al., 1997Go). In mature B cells, Pax5 positively regulates the expression of CD19 (Kozmik et al., 1992Go) while strongly repressing genes associated with the plasma cell phenotype including immunoglobulin heavy chain (IgH), kappa light chain (Ig{kappa}), J chain, and XBP-1 (Neurath et al., 1994Go; Reimold et al., 1996Go; Rinkenberger et al., 1996Go; Roque et al., 1996Go). Pax5 DNA binding motifs are present within the IgH 3'{alpha} enhancer, Ig{kappa} 3' enhancer, J chain promoter, and XBP-1 promoter to which Pax5 is recruited to exert transcriptional repression (Maitra and Atchison 2000Go; Neurath et al., 1994Go, 1995Go; Rinkenberger et al., 1996Go; Roque et al., 1996Go). 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, 2000Go). 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{kappa}, 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{kappa}, and J chain mRNA levels and cellular XBP-1.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
TCDD was purchased from AccuStandard, Inc. (New Haven, CT) in 100% DMSO. The certificate of product analysis stated that the purity of TCDD was 99.1%, as determined by AccuStandard using gas chromatography/mass spectography (GC/MS). Dimethylsulfoxide (DMSO) and LPS were purchased from Sigma-Aldrich (St. Louis, MO).

Cell line.
The CH12.LX B-cell line derived from the murine CH12 B-cell lymphoma (Arnold et al., 1983Go) has been previously characterized by Bishop and coworkers (Bishop and Haughton, 1986Go) 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)Go. Briefly, 100 µl of supernatant or standard (mouse IgM {kappa} 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 peroxidase–linked 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 {kappa} 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 SDS–PAGE (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 1–2 h at room temperature. Rabbit anti-mouse Pax5 and XBP-1 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Donkey HRP–conjugated anti-rabbit IgG was purchased from Amersham Pharmacia Biotech. Immunochemical staining was performed as described in Williams et al.(1996)Go. 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 manufacturer’s 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 1Go. 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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TABLE 1 Forward and Reverse Primers for mRNA Analyses of Pax5, IgH, Ig{kappa}, J Chain, and ß-Actin
 
Electrophoretic mobility shift assay.
Nuclear protein was isolated as described in Herring and Kaminski (1999)Go from naive or LPS (5 µg/ml)-activated CH12.LX cells that were treated with 10 nM TCDD or the vehicle (0.01% DMSO). In brief, the cells were lysed with a buffer containing 10 mM HEPES (pH 7.6) and 1.5 mM MgCl2, and the nuclei were recovered by centrifugation (7000 x g for 10 min). The nuclei were lysed and the protein isolated in a hypertonic buffer (25 mM HEPES—pH 7.6, 1.5 mM MgCl2, 450 mM NaCl, 0.2 mM EDTA, and 10% glycerol). DNA oligomers containing the consensus sequence for Pax5 were purchased from Santa Cruz Biotechnology, Inc., and were end-labeled with [{gamma}-32P]dATP. Nuclear proteins (10 µg) were incubated in binding buffer (100 mM NaCl, 25 mM HEPES, 1.5 mM MgCl2, 0.2 mM EDTA, and 10% glycerol) with 0.5 µg of poly dI-dC and the labeled DNA probe (80,000 d.p.m.) for 30 min at room temperature. The binding of protein to the DNA was resolved in a 4.0% nondenaturing gel using PAGE, dried on 3-mm filter paper (Whattman, Hillsboro, OR) and quantified by autoradiography and densitometry.

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 Dunnett’s two-tailed t test.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Kinetics of LPS-Induced IgM Secretion in the Presence and Absence of TCDD
LPS treatment induces the AhR-expressing CH12.LX B cell line to differentiate into cells that morphologically and functionally resemble primary antibody secreting cells. We have previously reported that TCDD treatment of CH12.LX cells markedly inhibited IgH mRNA levels and IgM secretion as measured 72 h post-LPS activation (Sulentic et al., 1998Go, 2000Go). Our previous studies also demonstrated that, as with primary B cells, TCDD must be added within the first 24 h post-LPS activation of CH12.LX cells for IgM secretion to be inhibited (Crawford et al., 2003Go). Here, studies were performed to characterize the kinetics of IgM secretion in the presence and absence of TCDD within the first 72 h post-LPS activation. In the absence of an activation stimulus, the CH12.LX cells exhibited a modest degree of background IgM secretion, which gradually accumulated with time in the supernatant (Fig. 1Go). The activation of CH12.LX cells with LPS strongly induced IgM secretion that was first evident at 48 h and which continued to increase over the 72-h culture period. In the presence of 10 nM TCDD, IgM secretion was suppressed by >50% at 48 and 72 h, when compared to the LPS-activated control CH12.LX cells.



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FIG. 1. TCDD suppresses IgM secretion in LPS-activated CH12.LX cells. Supernatants were harvested at the indicated times from naive (NA) or LPS (5 µg/ml)–activated CH12.LX cells that were treated with 10 nM TCDD and/or the vehicle (VH, 0.01% DMSO). The supernatants were analyzed for IgM by sandwich ELISA. The supernatant IgM concentration is represented on the y-axis as µg/ml. Results represent the mean ± S.E. and are representative of at least four separate experiments, each with an n = 4. Statistical significance was determined using Dunnett’s two-tailed t test. *Denotes values that are significantly different from the corresponding time-matched vehicle control at p < 0.05.

 
Effects of TCDD on Pax5 in LPS-Activated B Cells
Based on the marked suppression produced by TCDD on IgM secretion, experiments were performed to assess the effects of TCDD on Pax5, a well-characterized repressor of B-cell differentiation. Western immunoblotting demonstrated that LPS activation of CH12.LX produced a time-dependent decrease in the levels of cellular Pax5 that began at 48 h and continued to decrease during the 72-h culture period (Fig. 2Go). At 72 h post-LPS activation, Pax5 was down-regulated by >80%, when compared to the time 0 control. In comparison, LPS-activated CH12.LX cells treated with TCDD demonstrated no change in cellular Pax5 at 48 h and only a 40% decrease at 72 h, when compared to the background level of cellular Pax5 at time 0 in nonactivated cells (i.e., no LPS treatment). Due to the fact that cellular Pax5 is regulated in part at the transcriptional level, Pax5 mRNA levels were monitored over the same time course under identical cell culture conditions. In LPS-activated control cells, Pax5 mRNA levels were modestly decreased at 24 h and continued to decrease during the 72-h culture period to about 50% of the background time 0 level observed in nonactivated cells (Fig. 3Go). In contrast, LPS-activated CH12.LX cells treated with TCDD exhibited no decrease in Pax5 mRNA levels.



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FIG. 2. TCDD alters cellular Pax5 in LPS-activated CH12.LX cells. CH12.LX cells were activated with LPS (5 µg/ml) and treated with TCDD (10 nM) and/or the vehicle (VH, 0.01% DMSO). Cell lysates were isolated at 0, 24, 48, or 72 h. Proteins (20 µg/lane) were resolved on a 10% SDS–PAGE gel and probed with an anti-Pax5 antibody. To control for protein loading, blots were stripped and reprobed for ß-actin. Immunoblots were quantified for Pax5 by densitometry. The adjusted volume (OD x area) for all samples were normalized to the loading control (ß-actin) and expressed as fold change from time 0. Results are representative of four separate experiments.

 


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FIG. 3. Effect of TCDD on Pax5 mRNA levels in LPS-activated CH12.LX cells. CH12.LX cells activated with LPS (5 µg/ml) were treated with TCDD (10 nM) and/or the vehicle (VH, 0.01% DMSO). Quantitative RT-PCR analysis for Pax5 and the loading control (ß-actin) was performed on RNA isolated at 0, 24, 48, or 72 h post-LPS activation. All samples were normalized to the loading control. The fold change in Pax5 mRNA levels relative to time 0, which was arbitrarily given the value of 1.0, is identified on the y-axis. Results represent the mean ± S.E. of four separate RNA isolations in a single experiment. Results are representative of three separate experiments. *Denotes values that are significantly different from the corresponding time-matched vehicle control at p < 0.05 using Dunnett’s two-tailed t test.

 
Since Pax5 is a transcription factor, electrophoretic mobility shift assays were performed to assess Pax5 DNA binding activity over the same 72-h culture period. At 24 h post-LPS activation, the magnitude of Pax5 DNA binding activity was variable as compared to the naïve control cells from one experiment to the next; however, in all of the experiments performed, no significant differences were observed in the magnitude of binding activity in the vehicle versus the TCDD-treated cells (Fig. 4Go). Conversely, at 48 and 72 h, Pax5 DNA binding activity was consistently decreased by 40–50% in LPS-activated control cells, when compared to the nonactivated controls and to TCDD-treated cells at each of the respective time points. Collectively, a strong concordance was observed between the changes in cellular Pax5 protein, mRNA levels, and DNA binding activity in both the vehicle and TCDD-treated cells, respectively. In addition, in contrast to the LPS-activated control cells, where a decrease in Pax5 mRNA levels, protein, and DNA binding activity occurred in a time-related manner over the 72-h culture period, the magnitude of decrease was noticeably less pronounced in cells treated with TCDD.



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FIG 4. Pax5 binding to an oligonucleotide containing a Pax5 binding element is altered by TCDD in LPS-activated CH12.LX cells. CH12.LX cells were activated with LPS (5 µg/ml) and treated with TCDD (10 nM) and/or the vehicle (VH, 0.01% DMSO). Nuclear protein was isolated at 0, 24, 48, or 72 h post-LPS activation. Nuclear protein (10 µg) and the radiolabeled Pax5 consensus oligonucleotide were loaded in each lane, resolved on a 4.0% nondenaturing PAGE gel, dried on 3-mm filter paper, and analyzed by autoradiography. Binding of Pax5 to the oligonucleotide was quantified by densitometry. The adjusted volumes (OD x area) for all samples are expressed as fold change from time 0. Results are representative of three separate experiments.

 
Kinetics of LPS-Induced IgH, Ig{kappa}, and J Chain mRNA Expression and Cellular XBP-1 in the Absence and Presence of TCDD
Pax5 has been shown to repress a number of genes that are highly expressed when the B cell terminally differentiates into a plasma cell, including IgH, Ig{kappa}, J chain, and XBP-1. All four Pax5-regulated targets were assayed during the first 72 h post-LPS activation in the absence and presence of TCDD. LPS activation of CH12.LX cells simultaneously induced mRNA levels of IgH, Ig{kappa}, and J chain in a time-dependent manner with significant increases in expression by 48 h, which further increased at 72 h (Figs. 5Go–7Go). In striking contrast, LPS-activated cells treated with TCDD exhibited almost no increase in IgH, Ig{kappa}, and J chain mRNA levels throughout the entire 72-h culture period, paralleling the TCDD-mediated suppression on IgM secretion.



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FIG. 5. TCDD suppressed IgM heavy chain mRNA levels in LPS-activated CH12.LX cells. CH12.LX cells, activated with LPS (5 µg/ml), were treated with TCDD (10 nM) and/or the vehicle (VH, 0.01% DMSO). Quantitative RT-PCR analysis for IgM heavy chain and the loading control, ß-actin, were performed on RNA isolated at 0, 24, 48, or 72 h post-LPS activation. All samples were normalized to the loading control. Fold change of IgM heavy chain mRNA levels is relative to the time 0, which was arbitrarily given the value of 1.0, control is identified on the y-axis. Results represent the mean ± S.E. of four separate RNA isolations per experiment. Results are representative of three separate experiments. *Denotes values that are significantly different from the corresponding time-matched vehicle control at p < 0.05 using Dunnett’s two-tailed t test.

 


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FIG. 7. TCDD suppressed the J chain mRNA levels in LPS-activated CH12.LX cells. CH12.LX cells, activated with LPS (5 µg/ml), were treated with TCDD (10 nM) and/or the vehicle (VH, 0.01% DMSO). Quantitative RT-PCR analysis for J chain and the loading control, ß-actin, was performed on RNA isolated at 0, 24, 48, or 72 h post-LPS activation. All samples were normalized to the loading control. The fold change of J chain mRNA levels relative to time 0, which was arbitrarily given the value of 1.0, is identified on the y-axis. Results represent the mean ± S.E. of four separate RNA isolations per experiment. Results are representative of three separate experiments. *Denotes values that are significantly different from the corresponding time-matched vehicle control at p < 0.05 using Dunnett’s two-tailed t test.

 
XBP-1 mRNA undergoes site-specific cleavage of a 26-nucleotide intron by the endoplasmic reticulum–associated endoribonuclease, IRE1{alpha}, as part of the unfolded protein response, followed by religation of the 5' and 3' fragments to yield a spliced XBP-1 mRNA with an altered reading frame (Calfon et al., 2002Go; Shen et al., 2001Go; Yoshida et al., 2001Go). Consequently, the two sizes of XBP-1 transcripts yield two distinct protein forms of XBP-1, a 54-kDa and a 30-kDa peptide. The 54-kDa splice variant of XBP-1 has been found to be more biologically active, possess greater stability as a transcriptional activator, and functionally is the more important of the two forms of XBP-1 (Calfon et al., 2002Go; Shen et al., 2001Go). Due to the presence of multiple forms of XBP-1 protein and their differential activity, its modulation during B-cell activation was assayed by Western immunoblotting. In control nonactivated cells, XBP-1 protein was not detected (Fig. 8Go). In LPS-activated control cells, p54XBP-1 and p30XBP-1 proteins were induced at 24 h and continued to increase throughout the 72-h culture period. Conversely, in the LPS-activated cells treated with TCDD, the magnitude of both p54XBP-1 and p30XBP-1 induction was markedly diminished at all of the time points assayed.



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FIG. 8. TCDD addition alters XBP-1 protein expression in LPS-activated CH12.LX cells. CH12.LX cells, activated with LPS (5 µg/ml), and treated with TCDD (10 nM) and/or the vehicle (VH, 0.01% DMSO). Cell lysates were isolated at 0, 24, 48, or 72 h post-LPS activation. Proteins (25 µg/lane) were resolved on a 10% SDS–PAGE gel and probed with an anti-XBP-1 antibody. To control for protein loading, blots were stripped and reprobed for ß-actin. Results are representative of three separate experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The focus of the present investigation was to characterize the effects of TCDD on Pax5, a crucial regulatory factor that controls B-cell differentiation. We demonstrated that in non-LPS-activated CH12.LX B cells, Pax5 was readily expressed at the mRNA and protein levels. In addition, nuclear extracts from nonactivated CH12.LX cells showed strong Pax5 DNA binding activity as assessed by electrophoretic mobility shift assays. Concordant with the functional role of Pax5 as a repressor of B-cell differentiation, Pax5 mRNA levels, protein, and DNA binding activity markedly declined during the 72 h after LPS activation (Neurath et al., 1994Go; Reimold et al., 1996Go; Rinkenberger et al., 1996Go; Roque et al., 1996Go; Usui et al., 1997Go). In contrast, B cells activated in the presence of TCDD failed to decrease Pax5 mRNA levels, protein, or DNA binding activity to the same extent as observed in the time-matched LPS-activated controls. The inability of TCDD-treated cells to down-regulate Pax5 is consistent with strong impairment of B-cell differentiation and decreased immunoglobulin secretion. Interestingly, similar to the effects produced by TCDD treatment here, Usui and coworkers showed a marked suppression in plasma cell formation and immunoglobulin secretion by transiently overexpressing Pax5 in a clonal variant of the CH12.LX line, CH12.LX.A2, directly demonstrating the importance of appropriate Pax5 regulation to B-cell function (Usui et al., 1997Go).

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{kappa}, J chain, and XBP-1. Based on this fact, the effect of TCDD on IgH, Ig{kappa}, 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{kappa}, J chain transcript levels, and XBP-1 protein were observed. The effect of TCDD in this respect was striking on IgH, Ig{kappa}, 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{kappa}, 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., 1996Go). Repression of IgH expression by Pax5 is mediated through its DNA binding within at least two distinct regulatory domains of the IgH 3'{alpha} enhancer, the hs1,2 and the hs4, thus decreasing IgH 3'{alpha} enhancer activity (Neurath et al., 1994Go, 1995Go). Interestingly, we have previously demonstrated strong inhibition of IgH mRNA levels (Sulentic et al., 1998Go, 2000Go) by TCDD treatment in LPS-activated CH12.LX cells that was closely correlated with a decrease in IgH 3'{alpha} enhancer activity (manuscript submitted for publication). In addition to the previously identified Pax5 binding sites, analysis of IgH 3'{alpha} enhancer led to the identification of several TCDD-inducible AhR binding motifs (i.e., DRE-like sites) within the IgH 3'{alpha} enhancer, one in the hs4 and one in the hs1,2 domain (Sulentic et al., 2000Go). Analysis of the hs4 domain alone (i.e., removed from the context of other regulatory IgH 3'{alpha} 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'{alpha} 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{kappa} has also been recently investigated but remains poorly understood. Similar to the IgH 3'{alpha} enhancer, Ig{kappa} also possesses a 3' enhancer that is crucial to the regulation of Ig{kappa} expression. Within the Ig{kappa} 3' enhancer, several Pax5 DNA binding sites have been identified that have been shown to repress enhancer activity (Maitra and Atchison 2000Go). Interestingly, the mechanism for Pax5 repression of Ig{kappa} 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., 1996Go; Scott et al., 1994Go). 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)Go, our observation that TCDD treatment interferes with Pax5 down-regulation is consistent with the suppression of Ig{kappa} mRNA levels and previously reported Ig{kappa} protein (Crawford et al., 2003Go) in LPS-activated B cells treated with TCDD.

As observed with IgH, Ig{kappa}, 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., 1996Go). 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., 2001Go). 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., 2001Go). 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., 2002Go). 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{kappa}, 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{kappa}, J chain mRNA, cellular XBP-1, and, ultimately, secreted IgM.



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FIG. 6. TCDD-suppressed Ig{kappa} mRNA levels in LPS-activated CH12.LX cells. CH12.LX cells, activated with LPS (5 µg/ml), were treated with TCDD (10 nM) and/or the vehicle (VH, 0.01% DMSO). Quantitative RT-PCR analysis for Ig{kappa} and the loading control, ß-actin, was performed on RNA isolated at 0, 24, 48, or 72 h post-LPS activation. All samples were normalized to the loading control. The fold change of Ig{kappa} light chain mRNA levels relative to time 0, which was arbitrarily given the value of 1.0, is identified on the y-axis. Results represent the mean ± S.E. of four separate RNA isolations per experiment. Results are representative of three separate experiments. *Denotes values that are significantly different from the corresponding time-matched vehicle control at p < 0.05 using Dunnett’s two-tailed t test.

 

    ACKNOWLEDGMENTS
 
This work was supported in part by funds from NIH Grants ES02520 to NEK and ES012245 to TRZ. D.R.B. is supported in part by a Michigan Agriculture Experimental Station Fellowship. We thank Dr. Geoffrey Haughton for the CH12.LX cells, Dr. Kathryn Brooks for the BCL-1 cells, Elaine Tam for technical assistance, and Kimberly Townsend for her assistance in preparing this manuscript for publication.


    NOTES
 
1 To whom correspondence should be addressed at Department of Pharmacology and Toxicology, Michigan State University, 315 National Food Safety and Toxicology Center, East Lansing, MI 48824. Fax: (517) 432-3218. E-mail: kamins11{at}msu.edu. Back


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