Modulation of Ets-1 Expression in B Lymphocytes is Dependent on the Antigen Receptor–mediated Activation Signals and Cell Cycle Status

Authors

  • R. Raghunandan,

    1. Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, OH, USA
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  • F. W. Frissora,

    1. Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, OH, USA
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  • N. Muthusamy

    Corresponding author
    1. Molecular Virology, Immunology and Medical Genetics and Veterinary BioSciences, The OSU Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA
    • Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, OH, USA
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Correspondence to: N. Muthusamy, Associate Professor of Medicine, Division of Hematology, Department of Internal Medicine, The Ohio State University, 455E, OSUCCC, 410, West 12th Avenue, Columbus, OH 43210, USA. E-mail: raj.muthusamy@osumc.edu

Abstract

In this report, we tested the hypothesis that Ets-1 transcription factor is modulated at the mRNA level during B cell antigen receptor (BCR)-induced cell-signalling events. Quiescent B cells express high levels of Ets-1 mRNA. Stimulation through the BCR results in time-dependent inhibition of Ets-1 mRNA expression in primary splenic B cells with maximal inhibition observed by 16-h post-stimulation. Inhibition of Ets-1 expression is specific to antigen receptor but not CD40-mediated activation. Antigen receptor–induced inhibition of Ets-1 mRNA can be mimicked by phorbol myristate acetate (PMA) and/or ionomycin. PMA but not ionomycin-induced inhibition of Ets-1 expression is rescued by the inhibitors of protein kinase C and MEK. Extended time-course analysis revealed a time-dependent cyclical pattern in the re-expression of Ets-1 mRNA. While resting cells revealed maximal Ets-1 mRNA expression, activation events that induced exit from G0/G1 or cells blocked in early S phase exhibited decreased Ets-1 mRNA levels. Interestingly, cells arrested at late G2 or M phase of the cell cycle failed to down modulate Ets-1 mRNA expression. Overexpression of Ets-1 in 70Z/3 B cell line caused abnormal accumulation of cells in S phase associated with increased cyclin A expression. Consistent with a requirement for Ets-1 in BCR-induced cell cycle entry, splenic B cells from mice deficient in Ets-1 showed defective antigen receptor–induced DNA synthesis and S phase entry. These results suggest a critical role for Ets-1 regulation during B cell activation and cell cycle entry.

Introduction

Ets-1 belongs to the Ets family of transcription factors that were originally identified in V-ets oncogene of the E26 avian retrovirus that causes erythroblastosis and myeloblastosis in chickens. The Ets proteins recognize a consensus GGA(A/T) motif on multiple target genes and regulate several cellular functions such as cell proliferation, differentiation, apoptosis and migration [1-4]. More than 30 members of the Ets family of proteins have been identified in mammalian cells. Multiple Ets family members such as Ets-1, Ets-2, Elf-1, Spi-B and PU.1 are expressed in lymphoid lineages [5-10]. Ets-1, the founding member of the Ets family of proteins is highly expressed in B and T cells. In B cells, Ets-1 is expressed in the immature and mature stages of development [11, 12]. Ets binding sites have been identified in several target genes such as mb-1, IgH, Igμ and btk In B cells [13-16]. Ets-1 has been shown to be a key regulator of immunoglobulin heavy chain gene enhancer In vitro [15, 17].

The function of the Ets family of transcription factors is highly regulated by signal-mediated events [3, 4]. The Ras-mediated activation of p44 extracellular signal regulated kinase-1 (ERK-1) mitogen activated protein (MAP) kinase leads to phosphorylation of Threonine 38 and Threonine 72 in Ets-1 and Ets-2 proteins, respectively [3]. The Ras-dependent phosphorylation of Ets-1 has been shown to transactivate promoters of early genes of cell proliferation such as JunB in vitro [18]. Ets-1 protein undergoes hyper-phosphorylation during the mitotic phase of cell cycle in T cell lines [19]. Consistent with a role in cell proliferation, expression of mutants of Ets-1 and Ets-2 in NIH 3T3 cells reverted Ras-induced transformation [20].

Early growth response genes such as Egr-1 and JunB that are expressed in response to antigen receptor cross-linking in B lymphocytes are regulated by Ets-1 [21]. Stimulation of B cells, through the surface IgM or IgD molecules, induced phosphorylation of Ets-1 protein in calcium-dependent manner [22]. The calcium-dependent phosphorylation of Ets-1 has been identified in four serine residues, which are clustered in a region adjacent to the DNA-binding Ets domain. Phosphorylation of Ets-1 results in the formation of an auto-inhibitory domain, which hinders the DNA-binding activity of the protein by 50-fold [23]. Calcium-dependent phosphorylation of Ets-1 at multiple serine residues is associated with decreased DNA-binding activity in T cells [24]. Thus, activation-induced phosphorylation of Ets-1 is targeted towards the inhibition of pre-existing protein as well as the inactivation of Ets-1 DNA-binding activity.

We report here additional level of regulation of Ets-1 at mRNA in primary splenic B cells and B cell lines. Evidence is presented for antigen receptor, but not CD40 mediated, inhibition of Ets-1 mRNA in B lymphocytes. A role for protein kinase C (PKC)- and MAP kinase-dependent signalling events is shown to be required for the inhibition of Ets-1 mRNA. Further, cell cycle-dependent regulation of Ets-1 mRNA, as well as an essential requirement for Ets-1 in antigen receptor–induced B cell S phase entry, is established using B cell lines and B cells from mice deficient in Ets-1 protein.

Materials and methods

Animals

Female ICR mice were obtained from the Harlan Sprague Dawley (Indianapolis, IN, USA). Ets-1−/− mice were bred at the Columbus Children's Research Institute Transgenic and Embryonic Stem Cell Core facility. The Ets-1+/+ and Ets-1−/− mice used in these studies are in 129Sv/J xC57Bl/6 background. All animals were housed in micro isolators in the barrier animal facility located at Columbus Children's Research Institute Vivarium at Columbus Children's Hospital. The experiments were carried out using mice that were 6–10 weeks of age.

Reagents

F(ab)2 goat anti-mouse IgM and purified goat anti-mouse IgM antibodies were purchased from Jackson Immuno-Research Laboratories Inc. (West Grove, PA, USA). Ficoll-Paque was obtained from Sigma (St. Louis, MO, USA). 32P-labelled dCTP was purchased from Amersham Life Science Inc. (Arlington Heights, IL, USA). An antibody cocktail used to deplete T cells was made from culture supernatants of RL-172.4 (L3T4) and 3.155 (Lyt2) hybridoma cell lines. Batch-tested rabbit complement, from Pel-Freeze Biological (Fayetteville, AR, USA), was used to perform antibody-dependent lysis of T cells. Anti-mouse CD40 (HM40-3) was obtained from BD Pharmingen (San Diego, CA, USA). Phorbol myristate acetate, ionomycin, bisindolylmaleimide, PD98059 and Autocamtide-2-related inhibitory peptide were obtained from Calbiochem (La Jolla, CA, USA).

B Cell preparation

Purified splenic B cells were prepared as described previously [25]. Briefly, single-cell suspensions of splenocytes were made in RPMI 1640 containing 10% foetal bovine serum, 2 mm l-Glutamine, non-essential amino acids and 5 × 10−5m β-Mercaptoethanol. The cells were incubated in a cocktail containing monoclonal anti-L3T4 (clone RL-172/4) and anti-Lyt2 (clone 3.1555) antibodies for 15 min on ice, followed by treatment with rabbit complement for 30 min at 37 °C. The resulting B cells were washed, resuspended in 2 ml of RPMI 1640 and layered over Ficoll-Hypaque, and then spun at 4 °C for 20 min at 750 g. Live B cells from the interface were used for the cultures. Staining with fluorescein-conjugated anti-IgM revealed >90% sIgM-positive B cells. Concanavalin A-induced proliferative responses of B cells obtained in this manner were decreased to 5% of the untreated spleen cells, indicating the depletion of most of the T cells.

Culture conditions and assay for B cell DNA synthesis

Purified resting B cells (2.5 × 105) were cultured in Costar (Cambridge, MA, USA) 96-well tissue culture plates in a final volume of 200 μl RPMI 1640 supplemented with 10% foetal bovine serum, 2 mm l-Glutamine (M), PenStrep, non-essential amino acids and 5 × 10−5m β-Mercaptoethanol. Goat anti-mouse IgM (10 μg/ml) or F (ab)2 goat anti-mouse IgM (10 μg/ml), α-CD40 (5 μg/ml) and/or lipopolysaccharide (LPS) (25 μg/ml) were used as indicated. Cells were cultured for 48 h in 5% CO2 at 37 °C and then pulsed with 1 μCi of 3[H] thymidine [(2 Ci/mmol) Amersham] for 18 h as indicated. The cultures were harvested onto glass fibre filters using a Skatron (Sterling, VA, USA) automatic cell harvester, and the filters were counted in a Beckman LS 6500 liquid scintillation counter (Beckman, Fullerton, CA, USA). Results are presented as the geometric mean of responses from triplicate cultures.

Stimulation of B cells

Purified splenic B cells or 70Z/3 B cells were prewarmed at 37 °C for 15 min, prior to stimulation. Cells (10 × 106/ml) were stimulated with anti-IgM (10 μg/ml), anti-CD40 (5 μg/ml), PMA (0.1 μg/ml), ionomycin (0.5 μg/ml) or LPS (25 μg/ml) as indicated in a 37 °C incubator at 5% CO2.

Northern blot analysis

Total RNA (15 μg) made from B cells (20 × 106) using Trizol reagent (Gibco BRL, Gland Island, NY) was separated on 1% RNA agarose gel (1XMOPS, 2% formaldehyde) as described previously [26]. The gel was rinsed with 10X SSC (1X SSC is 0.15 m NaCl, and 15 mm sodium citrate) and blotted overnight onto a nylon Hybond-N+ membrane (Amersham Pharmacia Biotech, England, UK) in 10X SSC. The transferred RNA was UV cross-linked, and the membrane was baked in a vacuum oven at 80 °C for 30 min. The membrane was then prehybridized for 2 h at 42 °C in a hybridization solution containing 0.5% SSPE (20XSSPE: 3 m NaCl, 0.2 m NaH2PO4, 0.02M EDTA, pH7.4), 5% denhardt's solution, 0.5% SDS, 50% formamide, 10% dextran sulphate and 3 mg sperm DNA. The membrane was then hybridized with fresh hybridization solution containing the indicated denatured 32P radiolabeled cDNA probes. The hybridization was carried out overnight at 42 °C, and the blot was washed twice with 2XSSPE and 0.5% SDS at 42 °C for 20 min each wash. This was followed by two washes using 0.5X SSPE and 0.5% SDS at 65 °C for 30 min each. The membrane was then exposed to X-ray film (Biomax MR Kodak, NY, USA). The relative level of RNA samples in each lane was determined by stripping the probe and re-probing the membrane with radiolabeled mouse β-actin cDNA probe. The densities of the bands were quantitated using a phosphorimager by Molecular Dynamics Fluorescence Scanning System (Sunnyvale, CA, USA). The murine Ets-1, β-actin or cyclin A cDNA probes were 32P radiolabeled using Life Technologies Random Priming Labeling Kit. The indicated cDNA probes (50 ng) were denatured 5 min at 95 °C and cooled on ice. Reaction mix containing dATP, dGTP, dTTP and Klenow fragment (five Units), random oligonucleotide primer, and 5 μl of dCTP (3000 Ci/mmol) was added to a total volume of 50 μl. After 1-h incubation at 37 °C, the reaction was stopped using 5 mm EDTA. The free probe was removed by centrifugation using a G-50 (Pharmacia) gel filtration column. Incorporated radioactivity was determined using a Beckman Scintillation counter (Brea, CA, USA).

Transient transfection assays

70Z/3 cells were cultured in RPMI 1640 medium containing 10% foetal bovine serum, 2 mm L-Glutamine, non-essential amino acids and 5 × 10−5 m β-Mercaptoethanol. Cells (10 × 106) were suspended in 250 µl of RPMI 1640 media and were electroporated with 15 µg of expression plasmids pSV-cEts1, pCGN-Ets2, Elf-1 (kind gift from Dr. Lee Johnson, The Ohio State University) using a Bio-Rad gene pulser (Hercules, CA, USA). Cells were rested on ice for 10 min and cultured for 16 h before cell cycle analysis.

Cell cycle analysis

Cells from the cultures were washed with phosphate buffered saline (PBS) containing 0.1% glucose and fixed in 100% ice cold ethanol, and then stained with propidium iodide (0.5 mg/10 ml) in sample buffer (PBS with 0.1% glucose) containing RNAse A (1000 U) for 30 min at room temperature. Flow cytometric analysis was performed on a Coulter Epics Elite ESP (Hialeah, FL) flow cytometer. Fifty thousand events were acquired from each sample, and the cell cycle analysis was performed using the Coulter DNA analysis software.

Results

Activation-induced inhibition of Ets-1 mRNA in B cells

Activation-induced phosphorylation and subsequent negative modulation of Ets-1 DNA-binding activity suggested possible regulation of Ets-1 at multiple levels, including mRNA expression. Purified resting splenic B cells expressed abundant levels of Ets-1 mRNA (Fig. 1). To determine the effect of antigen receptor–induced regulation of Ets-1 mRNA, purified splenic B cells were stimulated with goat anti-mouse IgM, a surrogate ligand for the antigen receptor. Activation of B cells through antigen receptor for 24 h resulted in >90% reduction in the expression of Ets-1 mRNA, compared with unstimulated cells maintained in culture for the same period of time (Fig. 1a). Occupancy of Fc receptor in B cells by intact antibody has been shown to deliver negative signalling in B cells. To test whether or not the anti-IgM-induced downregulation of Ets-1 mRNA is due to the direct activation of the antigen receptor or is dependent on Fc receptor–mediated negative signalling events, F (ab)2 fragment of goat anti-IgM which lacks the Fc receptor binding region was tested in parallel cultures. F (ab)2 anti-mouse IgM inhibited Ets-1 mRNA expression to the same extent as the intact anti-IgM antibody (Fig. 1a). The activation-induced downregulation of RNA expression is unique to Ets-1 as the levels of PU.1 and Elf-1 are not altered, in response to stimulation with anti-IgM (data not shown). B cell antigen receptor (BCR)-induced down regulation of Ets-1 appears as early as 2 h. This downregulation is progressively increased, and maximal inhibition was observed as late as 16 h tested in a time-dependent manner (Fig. 1b).

Figure 1.

B cell Antigen receptor–induced inhibition of Ets-1 mRNA expression in splenic B lymphocytes. (A) Antigen receptor–induced downregulation of Ets-1 expression in B cells. Purified splenic B cells (10 × 106/ml) were cultured in the presence of medium (none) (lane 1), F(ab)2 (lane 2) or intact anti-IgM (10 μg/ml) (lane 3) for 16 h. Expression of Ets-1 mRNA was analysed by Northern blot analysis using total RNA and Ets-1 cDNA probe as described in the section entitled 'Materials and methods'. The blots were subsequently re-probed with β-actin cDNA probe for loading control in each of the lanes. The results are representative of five independent experiments with similar outcomes. (B) Time-course analysis of antigen receptor–induced downregulation of Ets-1 expression in B cells. Purified splenic B cells (10 × 106/ml) were cultured in the presence of anti-IgM (10 μg/ml) [lane 1–5] for 0, 2, 4, 8 and 16 h. Expression of Ets-1 mRNA was analysed by Northern blot analysis using total RNA and Ets-1 cDNA probe as described in the section 'Materials and methods'. The blots were subsequently re-probed with β-actin cDNA probe for loading control in each of the lanes. The results are representative of three independent experiments with similar outcomes.

Protein kinase C- and Ca++-dependent signalling events in the regulation of Ets-1 mRNA

Stimulation of B cells through the antigen receptor activates PKC and intracellular calcium (Ca++) release. Pharmacological agents such as phorbol myristate acetate (PMA) and ionomycin activate PKC and elevate intracellular Ca++ respectively [27]. To directly determine the relative role of PKC and Ca++ signalling events in downregulation of Ets-1 mRNA, resting B cells were cultured independently with PMA and/or ionomycin. Northern blot analysis of total RNA from splenic B cells treated with PMA and ionomycin showed inhibition of Ets-1 mRNA expression comparable with cells treated with anti-IgM (Fig 2a). Interestingly, B cells treated with PMA or ionomycin independently inhibited Ets-1 mRNA expression to the levels comparable with PMA and ionomycin together, indicating an independent role for PKC and calcium signalling pathways in downregulation of Ets-1 (Fig. 2b). The activation-induced downregulation of Ets-1 is selective to PKC and calcium signalling events in B cells. Thus, activation with anti-IgM or PMA ±ionomycin, which trigger PKC and Ca++ pathways (Fig. 2), but not with anti-CD40 antibody induced downregulation of Ets-1 expression in B cells (Fig. 2).

Figure 2.

B cell Antigen Receptor–induced inhibition of Ets-1 mRNA expression is mimicked by phorbol myristate acetate (PMA) and/or ionomycin but not with anti-CD40 antibody. (A) Purified splenic B cells (10 × 106/ml) were cultured in the presence of medium (none) (lane 1), PMA (P) + Ionomycin (I) (lane 2) or anti-IgM (10 μg/ml) (lane 3) for 16 h. Expression of Ets-1 mRNA was analysed by Northern blot analysis using total RNA and Ets-1 cDNA probe as described in the section entitled 'Materials and methods'. The blots were subsequently re-probed with β-actin cDNA probe for loading control in each of the lanes. The results are representative three independent experiments with similar outcomes. (B) Purified splenic B cells (10 × 106/ml) were cultured in the presence of medium (lane 1), PMA (100 ng/ml) (lane 2), Ionomycin (500 ng/ml) (lane 3), anti-CD40 (5 μg/ml) (lane 4), anti-CD40 + P+I (lane 5) or P+I (lane 6) for 16 h. Expression of Ets-1 mRNA was analysed by Northern blot analysis using total RNA and Ets-1 cDNA probe as described in the section entitled 'Materials and methods'. The blots were subsequently re-probed with β-actin cDNA probe for loading control in each of the lanes. The results are representative three independent experiments with similar outcomes.

In an attempt to delineate the PMA and ionomycin-induced downstream signalling events that are required for the inhibition of Ets-1 expression, we tested the effect of the inhibitors of PKC, MEK and CaM kinase, the major kinases modulated by PMA and ionomycin signalling events. Bisindolylmaleimide is a potent inhibitor of PKC. Treatment of B cells with PMA resulted in >90% reduction in Ets-1 expression compared with the untreated control cells (Fig. 3a). The inhibition of Ets-1 mRNA expression by PMA was partially rescued by pretreatment of the B cells with bisindolylmaleimide indicating a role for PKC. Indeed the rescue effect of bisindolylmaleimide is specific for the PKC but not calcium-mediated signalling events because PMA, but not ionomycin-induced inhibition of Ets-1 mRNA, was rescued by this inhibitor (Fig. 3b). The failure of bisindolylmaleimide to rescue the ionomycin-induced Ets-1 downregulation indicated PKC-independent mechanism in ionomycin-induced Ets-1 mRNA modulation. As ionomycin is known to upregulate increased intracellular calcium, these studies indicate a calcium-dependent mechanism in ionomycin-induced downregulation of Ets-1 mRNA.

Figure 3.

Inhibition of Ets-1 expression in B cells is protein kinase C and MAP kinase dependent. (A) Purified splenic B cells (10 × 106/ml) were pretreated with kinase inhibitors Bis-indolylmalemide (Bis) (1 μm) or PD98059 (25 μm) for 2 h. The cells were then cultured in the presence of medium (Lane 1), phorbol myristate acetate (PMA) (100 ng/ml) (lane 2), PMA + Bis (lane 3) or PMA + PD98059 (lane 4) for 16 h. Expression of Ets-1 mRNA was analysed by Northern blot analysis using total RNA and Ets-1 cDNA probe as described in the section entitled 'Materials and methods'. The blots were subsequently re-probed with β-actin cDNA probe for loading control in each of the lanes. The results are representative three independent experiments with similar outcomes. (B) Purified splenic B cells (10 × 106/ml) were pretreated with kinase inhibitors Bis-indolylmalemide (Bis 1 μm) or PD98059 (25 μm) for 2 h. The cells were then cultured in the presence of medium (lane 1), ionomycin (Iono) (500 ng/ml) (lane 2), Iono + Bis (lane 3) or Iono + PD98059 (lane 4) for 16 h. Expression of Ets-1 mRNA was analysed by Northern blot analysis using total RNA and Ets-1 cDNA probe as described in the section entitled 'Materials and methods'. The blots were subsequently re-probed with β-actin cDNA probe for loading control in each of the lanes. The results are representative three independent experiments with similar outcomes.

Stimulation through the antigen receptor in B cells has been shown to activate p21ras [28]. Ras signalling events lead to subsequent activation of Raf/MEK/MAPK pathway. PD98059 inhibits MEK and in turn suppresses the activation of its substrate MAP kinase [29]. To determine the role of MAP kinase signalling in activation-induced inhibition of Ets-1 mRNA expression, B cells were stimulated with PMA or ionomycin, in the presence or absence of PD98059. PD98059 rescued Ets-1 mRNA expression, which was inhibited by PMA (Fig. 3a). However, PD98059 failed to rescue the ionomycin-mediated inhibition of Ets-1 expression (Fig. 3b) indicating a role for MEK/MAP kinase in PMA, but not ionomycin-induced inhibition of Ets-1 mRNA. Similar effects of bisindolylmaleimide or PD98059 on PMA or ionomycin-induced Ets-1 expression was further independently confirmed in the 70Z/3 B cell line (data not shown).

Cell cycle-dependent regulation of Ets-1 expression

Ets-1 is expressed in resting primary splenic B cells as well as in B cell lines such as 70Z/3, NFS and A20 (Fig 4 and data not shown). Similar to the observation made in primary B cells, stimulation with PMA ±ionomycin resulted in inhibition of Ets-1 expression in 70Z/3 B cells (Fig. 4a). Inhibitors of MAP kinase and PKC-rescued PMA, but not ionomycin, induced Ets-1 mRNA downmodulation similar to the observation made in primary B cells (Fig 3 and data not shown). PMA plus ionomycin-induced inhibition of Ets-1 expression is time dependent. Time-course analysis of PMA plus ionomycin-treated 70Z/3 cells revealed a cyclical pattern in Ets-1 expression. While downmodulation of Ets-1 expression was seen as early as 8 h, re-expression of Ets-1 at 16, 32 and 48 h was observed (Fig. 4b). The re-expression of Ets-1 in cycling 70Z/3 cells at 16-h intervals suggested a potential regulation of Ets-1 in cycling B cells. To determine whether Ets-1 downmodulation is correlated with cell cycle stage, we used pharmacological agents such as hydroxyurea, quercetin and nocodozole to block the cycling cells at specific stages in the cell cycle. Hydroxyurea inhibits nucleotide synthesis and thus arrests cells at the early S phase. Quercetin inhibits PI3Kinase and blocks the cells at the G2 phase of the cell cycle [30]. Nocodozole is a potent inhibitor of tubulin polymerization and cause cell cycle arrest at the M phase [31]. 70Z/3 cell cultures treated with hydroxyureashowed 51% cells in the S phase compared with 28% in untreated cells. Consistent with the S phase arrest, there was a 50% reduction in the cells in G0/G1 as well as G2/M phases of the cell cycle. Interestingly, Northern blot analysis of RNA isolated from cells treated with hydroxyurea exhibited a significant reduction (>90%) in the expression of Ets-1 mRNA (Fig. 4c). Quercetin and Nocodozole treated cultures that accumulated cells in G2 phase and G2-M phase, respectively, did not affect the expression of Ets-1 (Fig. 4c).

Figure 4.

Cell cycle-dependent regulation of Ets-1 expression in 70Z/3 B cells. (A) Activation-induced downregulation of Ets-1 in 70Z/3 pre-B cells. 70Z/3 pre-B cells (10 × 106/ml) were stimulated with media (lane 1), phorbol myristate acetate (PMA) 100 ng/ml (lane 2), Ionomycin (500 ng/ml) (lane 3) or PMA + ionomycin (lane 4) for 24 h. Expression of Ets-1 mRNA was analysed by Northern blot analysis using total RNA and Ets-1 cDNA probe as described in the section entitled 'Materials and methods'. The blots were subsequently re-probed with β-actin cDNA probe for loading control in each of the lanes. The results are representative three independent experiments with similar outcomes. (B) Time-course analysis of Ets-1 mRNA expression in 70Z/3 B cells. 70Z/3 B cells (10 × 106/ml) were stimulated with media (lane 1), or PMA and Ionomycin (lanes 2–6) for 0, 8, 16, 24, 32 and 48 h. Expression of Ets-1 mRNA was analysed by Northern blot analysis using total RNA and Ets-1 cDNA probe as described in the section entitled 'Materials and methods'. 18s RNA is shown as loading control in each of the lanes. The results are representative three independent experiments with similar outcomes. (C) Differential Effects of Hydroxy urea, Quercetin or Nocodozone on Ets-1 mRNA expression. 70Z/3 B cells (10 × 106/ml) were grown in the presence of media (none), hydroxy urea (1 mm) (HU), Quercetin (70 μm) (QU) or Nocodozole (40 ng/ml) (NO) for 24 h. Expression of Ets-1 mRNA was analysed by Northern blot analysis using total RNA and Ets-1 cDNA probe as described in the section entitled 'Materials and methods'. The blots were subsequently re-probed with β-actin cDNA probe for loading control in each of the lanes. The Ets-1 mRNA levels in each of the lanes are normalized with corresponding β-actin controls. Normalized Ets-1 expression in the presence of the inhibitors is compared with percentage of expression in the absence of the blockers, which is represented as 100%. The results are representative two independent experiments with similar outcomes.

Overexpression of Ets-1 induces cyclin A expression associated with enhanced cell cycle entry

The downregulation of Ets-1 mRNA expression in cells exiting from G0/G1 suggested a role for Ets-1 in cell cycle. To test the effect of constitutive expression of Ets-1 in the B cell cycle, 70Z/3 B cells were transfected with either PSV-empty vector control or PSV-cEts-1 expression vector. Overexpression of cEts-1 in these cells was confirmed by Northern blot analysis of RNA isolated from PSV-cEts-1 and control PSV-empty vector–transfected cells (Fig. 5a). When compared to the empty vector–transfected control lane, the RNA from cEts-1 transfected lanes contained increased levels of cyclin A expression, suggesting Ets-1 to be one of the potential regulators of cyclin A (Fig. 6a). Further, PSV-cEts-1 transfected cells showed an accumulation of cells in the S phase (36%) compared with the PSV-empty control vector transfected cells (16%).

Figure 5.

Overexpression of Ets-1 in B cells results in increased Cyclin A expression associated with cell cycle deregulation. 70Z/3 B cells were transiently transfected with either control psv-vector or psv-cEts-1 expression plasmids. (A) Expression of cEts-1 mRNA (A) was analysed by Northern blot analysis using total RNA and Ets-1 cDNA probe as described in the section 'Materials and methods'. The blots were subsequently re-probed with cyclin A cDNA probe. 23S RNA is shown as loading control for each of the lanes. (B) Cell cycle analysis of the psv-control or psv-cEts-1 vector transfected cells was performed using propidium iodide as described in the 'Materials and methods' section. The results are representative two independent experiments with similar outcomes. The percentage of cells in S phase (S) is shown.

Figure 6.

Defective S phase entry of B cells lacking Ets-1 in response to antigen receptor stimulation. (A) Purified resting splenic B cells (2.5 × 105/ml) from Ets-1+/+ or Ets-1−/− mice were stimulated in the presence of indicated concentrations of anti-IgM antibody for 48 h. The cells were then pulsed with 1 μCi of 3[H] thymidine for 18 h. The DNA synthesis was measured as counts per minute (CPM) of 3[H] Thymidine incorporated using a liquid scintillation counter. Results are presented as the geometric mean of responses from triplicate cultures. The results are representative of at least five independent experiments with similar outcomes. (B) Purified resting splenic B cells (2.5 × 105/ml) from Ets-1+/+ or Ets-1−/− mice were stimulated in the presence of 20 µg/ml of anti-IgM antibody for 36 h. Cell cycle analysis was performed using propidium iodide staining as described in the 'Materials and methods' section. The percentage of cells in S phase (S) in activated Ets-1+/+ and Ets-1−/− B cells is shown.

Ets-1 is required for BCR-induced B cell proliferation and S phase entry

Antigen receptor–induced modulation of Ets-1 in B cells suggested a potential role for Ets-1 in BCR-mediated B cell proliferation. To directly test the role of Ets-1 in antigen receptor–induced cell cycle entry and DNA synthesis, purified splenic B cells from Ets-1−/− mice and control Ets-1+/+ were stimulated through the antigen receptor. Triggering of B cells with 0, 10 and 20 µg/ml of goat anti-mouse IgM resulted in dose-dependent increase in DNA synthesis as evidenced by increase in 3H-thymidine incorporation in Ets-1+/+ but not Ets-1−/− B cells (Fig. 6a). Consistent with the defective DNA synthesis, cell cycle analysis of Ets-1−/− splenic B cells activated with anti-IgM exhibited >60% reduction in cells entering the S phase compared with Ets-1+/+ B cells from control littermate mice (Fig. 6b).

Discussion

Studies described in this report demonstrate, for the first time, antigen receptor–induced inhibition of Ets-1 mRNA in B lymphocytes. Two lines of evidences indicated that the effect of anti-IgM on Ets-1 mRNA expression is due to direct stimulation through the antigen receptor and not due to Fc receptor mediated inhibitory effect. First, the F(ab)2 fragment of anti-IgM, which lacks the Fc portion of the antibody molecule–induced Ets-1 inhibition comparable with that of intact anti-IgM antibody (Fig. 1a). Second, pharmacological agents such as PMA and ionomycin that mimic antigen receptor–mediated signalling events also induced inhibition of Ets-1 mRNA expression. Further, activation-induced inhibition of Ets-1 mRNA expression is restricted to antigen receptor but not CD40-mediated signalling events.

The BCR or PMA and ionomycin-induced inhibition of Ets-1 mRNA expression suggested a potential role for PKC and calcium-dependent signalling events. The ability of PMA or ionomycin to independently inhibit Ets-1 expression suggested independent roles for PKC and calcium-mediated signalling events in the inhibition of Ets-1 expression. The ability of PKC inhibitor, bisindolylmaleimide, to rescue PMA-induced downregulation of Ets-1 mRNA effect, establishes the involvement of the PKC pathway. The effect of bisindolylmaleimide is specific to PKC in these studies, as Ets-1 inhibition mediated by ionomycin is not affected by the same concentration of bisindolylmaleimide in parallel expression. The ability of bisindolylmaleimide to partially rescue the PMA-inhibited Ets-1 mRNA expression is likely due to the incomplete inhibition of PKC under the conditions tested or involvement of PKC-independent pathways.

PD98059 has been shown to inhibit MEK resulting in the inhibition of the MAP kinase pathway and arrest entry of cells into mitotic phase. Reversal of PMA-induced Ets-1 mRNA expression by PD98059 strongly suggests a possible role for the MAP kinase pathway in BCR-induced negative regulation of Ets-1 expression. It is likely that the inhibition of Ets-1 expression by BCR could be attributed to possible activation of the MAP kinase pathway. This is consistent with the ability of the BCR to utilize MAP kinase signalling pathway [32]. The inhibitor studies described here do not distinguish whether PD98059-mediated rescue of Ets-1 expression is directly due to the inhibition of MEK signalling or due to accumulation of cells in G2 phase, thus resulting in increased Ets-1 expression as observed in fibroblasts [33]. Consistent with the latter possibility, cells arrested with quercetin in G2 phase of cell cycle also revealed a lack of inhibition of Ets-1 in 70Z/3 B cells. The ability of bisindolylmaleimide and PD98059 to rescue PMA, but not ionomycin-induced inhibition of Ets-1 mRNA, strongly suggests a role for PKC and MEK signalling events in PMA, but not ionomycin-mediated regulation of Ets-1 expression. This further reiterates calcium-dependent signalling events in the regulation of Ets-1 protein function and mRNA expression. Thus, elevation of intracellular calcium induces phosphorylation of serine residues in the C-terminal inhibitory domain of the Ets-1 protein resulting in decreased DNA-binding activity [34]. Calcium-dependent downregulation of Ets-1 mRNA is in agreement with calcium-dependent phosphorylation and subsequent inhibition of DNA-binding activity [34]. Although the studies described here have identified the activation-induced downregulation of Ets-1 in B cells, it has not been established whether the decreased mRNA expression is indeed due to transcriptional regulation or due to altered mRNA stability in response to the stimuli. Nevertheless, it is interesting to note that Ets-1 is expressed in a resting state and activation induces phosphorylation and inhibition of DNA-binding activity, and decreased mRNA expression. The Ets-1 in resting cells could presumably regulate the expression of genes required for maintenance in a quiescence state. Consistent with this hypothesis, we have previously reported inability of Ets-1-deficient T cells to remain in quiescent state, thus undergoing apoptosis [35]. Alternatively, it is also possible that Ets-1 may be involved in the suppression of genes required for G0/G1 exit.

It is interesting to note that inhibition of Ets-1 is selective for BCR but not CD40-mediated B cell activation (Fig 2b). These differences could be attributed to selective BCR but not CD40-induced signalling events. In addition, stimulation of B cells with PMA and ionomycin in the presence of CD40 failed to downregulate Ets-1 expression, suggesting differential regulation of Ets-1 during BCR, PMA, ionomycin and CD40-induced stimuli. The studies using cell cycle blockers suggest a potential cell cycle-dependent regulation of Ets-1 expression. Thus, inhibition of Ets-1 mRNA expression in 70Z/3 B cell line blocked in S phase using hydroxyurea suggested S phase-dependent downregulation of Ets-1 expression. The inhibition of Ets-1 is restricted to S phase as blocking the cells with G2 blocker quercetin or M phase blocker nocodozole failed to inhibit Ets-1 expression, suggesting selective downregulation of Ets-1 in the S phase of the cell cycle. Increased expression of cyclin A in cells overexpressing Ets-1 indicates a potential role for Ets-1 in cyclin A-dependent cell cycle regulation. Cyclin A is required for the initiation and maintenance of S phase of cell cycle [36, 37]. Cyclin A-dependent phosphorylation of Ets-related protein MEF has been recently shown to restrict the activity of the Ets protein to the G1 phase of the cell cycle [38].

The defective S phase entry of Ets-1-deficient cells in response to BCR suggests an essential requirement for Ets-1 in antigen receptor-induced cell cycle entry. It is possible that the Ets-1 regulates target gene expression that allows lymphocytes to exit from G0/G1 and enter into S phase. Ongoing analysis of downstream targets of Ets-1 in lymphocytes will identify the Ets-1 regulated genes involved in B cell growth and differentiation.

Acknowledgment

We would like to thank Ms. Cindy McAllistor for technical help with the flow cytometry, Mr. GirishRajgolikar for the generation of Ets-1 mutant mice and the OSUCCC Transgenic Animal Shared Resource and the Embryonic stem cell core facility at the Nationwide Columbus Children's Hospital. This work was supported by grants from Ohio Cancer Research Associates to NM.

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