Inhibition of the MEK/ERK Signaling Pathway Blocks a Subset of B Responses to Antigen

This information is current as James D. Richards, Shaival H. Davé, Chih-Hao G. Chou, of September 25, 2021. Alusha A. Mamchak and Anthony L. DeFranco J Immunol 2001; 166:3855-3864; ; doi: 10.4049/jimmunol.166.6.3855 http://www.jimmunol.org/content/166/6/3855 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Inhibition of the MEK/ERK Signaling Pathway Blocks a Subset of B Cell Responses to Antigen1

James D. Richards,2 Shaival H. Dave´,3 Chih-Hao G. Chou, Alusha A. Mamchak, and Anthony L. DeFranco4

Signal transduction initiated by B cell Ag (BCR) cross-linking plays an important role in the development and activation of B cells. Therefore, considerable effort has gone into determining the biochemical signaling events initiated by the BCR and delineating which events participate in specific biological responses to Ag. We used two inhibitors of mitogen-activated kinase/extracellular signal-regulated kinase kinase (MEK) 1 and MEK2, PD98059, and U0126, to assess the role the Ras-mitogen- activated protein kinase pathway plays in several BCR-induced responses. PD98059 or U0126 treatment substantially inhibited the BCR-induced activation of the extracellular signal-regulated kinase (ERK) forms of mitogen-activated protein kinase in the immature B cell line WEHI-231, in immature splenic B cells, and in mature splenic B cells. However, MEK-ERK inhibition did Downloaded from not block BCR-induced growth arrest or of WEHI-231 cells or apoptosis of immature splenic B cells, indicating that the MEK-ERK pathway is not required for these events. In contrast, PD98059 and U0126 treatment did inhibit the up-regulation of specific BCR-induced , including the factor Egr-1 in WEHI-231 and mature splenic B cells, and the CD44 adhesion molecule and CD69 activation marker in mature splenic B cells. Moreover, both inhibitors suppressed BCR-induced proliferation of mature splenic B cells, in the absence and in the presence of IL-4. Therefore, activation of the MEK-ERK pathway is necessary for a subset of B cell responses to Ag. The Journal of Immunology, 2001, 166: 3855–3864. http://www.jimmunol.org/

cell Ag receptor (BCR)5 signaling plays important roles cally causes them to enter into an anergic state or undergo apo- in the development, survival, and activation of B lym- ptosis, responses that promote immunological tolerance to self- phocytes. The first result of BCR engagement is the ac- Ags. In contrast, mature B cells contacting Ag enter G phase of B 1 tivation of Src family and Syk protein tyrosine kinases. These ki- the cell cycle and up-regulate many proteins involved in adhesion nases trigger a complex network of signaling pathways and Ag presentation to Th cells. Strong BCR stimulation can also downstream of the receptor, including the activation of phospho- induce B cell proliferation (3). Th cells enhance this proliferation lipase C (PLC)-␥1 and -␥2, phosphatidylinositol (PI) 3 kinase, the and induce B cell differentiation into the Ab-secreting by guest on September 25, 2021 Ras-Raf-mitogen-activated protein kinase/extracellular signal-reg- state by providing cell-cell contact signals via CD40 and by re- ulated kinase kinase (MEK)-extracellular signal-regulated kinase leasing , such as IL-4 and IL-5 (3). (ERK) mitogen-activated protein (MAP) kinase pathway, and Both genetic and pharmacologic approaches have been used to phosphorylation of the Vav proto-oncogene product (1–3). The determine how individual signaling pathways participate in these resulting signals quickly reach the nucleus and alter expres- biological responses to Ag. For example, wortmannin, which in- sion. The ultimate effects on the B cell are profound and vary hibits PI 3 kinase, has been shown to block BCR-induced apopto- depending on the maturation state of the cell and on the additional sis in a human immature B cell line (4). In addition, gene disrup- signals the cell receives. Ag contact with immature B cells typi- tion experiments demonstrate that PI 3 kinase participates in B cell development and proliferation as well (5, 6). Experiments with pharmacologic agents that mimic the second messengers resulting ␥ Department of Microbiology and Immunology, G. W. Hooper Foundation, University from PLC- activation, and with mutant cell lines defective in this of California, San Francisco, CA 94143 pathway, have demonstrated its importance for BCR-induced Received for publication November 29, 1999. Accepted for publication January growth arrest and apoptosis of B cell lines (7–10) and proliferation 8, 2001. of mature splenic B cells (11, 12). Moreover, mice and cell lines The costs of publication of this article were defrayed in part by the payment of page with targeted mutations in the signaling proteins Vav (13, 14) and charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. HS1 (15, 16) both exhibit decreased B cell responses to Ag. 1 This work was supported by National Institute of Allergy and Infectious Diseases By comparison, relatively little is known about the role(s) of the Grant AI20038. Ras-Raf-MEK-ERK MAP kinase pathway in B cell responses to 2 Current address: Department of Biology, University of Utah, 257 South 1400 East, Ag. When in its active GTP-bound state, Ras activates the serine/ Salt Lake City, UT 84112. threonine protein kinase Raf, which phosphorylates and activates 3 Current address: University of Pittsburgh School of Medicine, Pittsburgh, PA the protein kinases MEK1 and MEK2. The MEK1/2 kinases in 15261. turn phosphorylate and activate the classical p44 and p42 MAP 4 Address correspondence and reprint requests to Dr. Anthony L. DeFranco, G. W. kinases, also called ERK 1 and 2, respectively (17). This pathway Hooper Foundation, Box 0552, University of California, San Francisco, San Fran- cisco, CA 94143-0552. E-mail address: [email protected] has been found to be important in many cell types for receptor- 5 Abbreviations used in this paper: BCR, B cell Ag receptor; PLC, phospholipase C; induced biological responses, such as proliferation, growth arrest, PI, phosphatidylinositol; MEK, mitogen-activated protein kinase/extracellular signal- and differentiation (17–22). In B cells, Ras-ERK MAP kinase ac- regulated kinase kinase; ERK, extracellular signal-regulated kinase; MAP, mitogen- activated protein; BrdUrd, 5-bromo-2Ј-deoxyuridine; JNK, c-Jun N-terminal kinase; tivation correlates with BCR-induced growth arrest and apoptosis SRE, serum response element. in immature B cells, and cell-cycle entry and activation in mature

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 3856 MEK-ERK CASCADE IN B CELL RESPONSES TO ANTIGEN

B cells (3, 23, 24). We used two structurally and mechanistically Isolation and stimulation of mature splenic B distinct MEK1/2 inhibitors, PD98059 and U0126 (25–27), to in- ϫ Female (C57BL/6 DBA/2) F1 (BDF1) mice from Simonsen Laboratories vestigate the role that this pathway plays in these BCR-induced (Gilroy, CA) or Charles River Breeding Laboratories (Hollister, CA) were events. Both inhibitors suppressed BCR-induced activation of the sacrificed at 2–4 mo of age. Small resting B cells were isolated from the ERK MAP kinases in all B cells examined. These inhibitors also spleen as described (30). Cells were collected from the 60%/70% Percoll suppressed up-regulation of the early response gene product Egr-1 interphase, washed, and rested in normal medium at 37°C for 30 min to 2 h before treatment with DMSO or inhibitors. The purity of the splenic B cell in mature splenic B cells and in the WEHI-231 immature B cell preparations was typically at least 96% B220ϩ, as verified by flow line and partially blocked the induction of the CD44 adhesion pro- cytometry. tein and CD69 activation marker in mature splenic B cells. In Splenic B cells (1 ϫ 107 cells/ml) were stimulated with goat anti-mouse addition, both inhibitors blocked BCR-induced proliferation of IgM and/or IL-4 (50 U/ml) for the indicated times and washed twice with ice-cold PBS containing 1 mM Na VO before lysis. mature splenic B cells. In contrast, MEK-ERK inhibition failed to 3 4 suppress BCR-induced growth arrest or apoptosis of WEHI-231 ERK2 and JNK immunocomplex protein kinase assays cells and did not inhibit BCR-induced apoptosis in primary imma- ture splenic B cells. Thus, the Ras-ERK MAP kinase pathway is Cells were stimulated for 4 min (ERK2 assays) or for 15 min (JNK assays), important for mediating some, but not all, specific B cell responses and subsequently lysed as previously described (31). Analysis of the ki- netics of activation of these MAP kinases indicated that strong inductions to Ag. were reproducibly seen at these times. Anti-JNK and anti-ERK2 immuno- precipitations were performed as described (28). Immunocomplex protein kinase assays were performed on the immunoprecipitates as described (30),

with GST-Elk-1 as the substrate for ERK2 and GST-Jun(1–79) as the sub- Downloaded from Materials and Methods strate for JNK. Reaction products were resolved by SDS-PAGE, trans- Abs and other reagents ferred to nitrocellulose, and detected by autoradiography. Quantitation of kinase activity was performed using a PhosphorImager (Molecular Dy- Affinity-purified goat-anti-mouse IgM was obtained from Jackson Immu- namics, Sunnyvale, CA). To calculate inhibition of kinase activity by noResearch (West Grove, PA). The anti-IgD mAb H␦a/1 was generously ϭ Ϫ PD98059, we used the formula: Percent inhibition (1 – ((stimpd un- provided by Fred Finkelman, University of Cincinnati, as a mouse ascites Ϫ ϫ stimpd)/(stim unstim))) 100. Blots were reprobed with anti-ERK2 Abs fluid. Mouse rIL-4 was from Genzyme (Cambridge, MA). Hybridomas (1:1000) to ensure that all reactions contained equal amounts of ERK2. HO13.4 (anti-Thy1), 53.6.172 (anti-CD8), and GK1.5 (anti-CD4) were ob- http://www.jimmunol.org/ tained from American Type Culture Collection (Manassas, VA). Anti- ERK2 (C-14), anti-c-Jun N-terminal kinase (JNK) 1 (C-17), and anti-Egr-1 Egr-1 and anti-phosphoMAP kinase immunoblotting (C-19) Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, For examining Egr-1 up-regulation, cells (5 ϫ 106 cells/lane) were stim- CA). Anti-ACTIVE MAP kinase polyclonal Ab (pTEpY) was purchased ulated for 1 h and lysed in SDS-PAGE sample buffer, resolved by SDS- from Promega (Madison, WI). PE- and FITC-conjugated RA3–6B2 (anti- PAGE, and transferred to nitrocellulose. The anti-Egr-1 Ab was used at B220), FITC-conjugated GL-1 (anti-CD86), FITC-conjugated anti-CD44, 1:500 for detection. General SDS-PAGE and immunoblotting procedures FITC-conjugated anti-CD69, and anti-CD16/CD32 were purchased from were as described (2). For anti-phosphoMAP kinase immunoblotting time PharMingen (San Diego, CA). 5-Bromo-2Ј-deoxyuridine (BrdUrd) was course experiments, 1 ϫ 107 splenic B cells/ml or 2.5 ϫ 106 WEHI-231 purchased from Sigma (St. Louis, MO). Anti-BrdUrd-FITC was purchased cells in 0.5 ml were stimulated with 10 ␮g/ml goat anti-mouse IgM for the from Becton Dickinson (San Jose, CA). -TriColor was obtained indicated times, washed, and lysed in SDS-PAGE sample buffer. Samples by guest on September 25, 2021 3 from Caltag (South San Francisco, CA). [ H]Thymidine was purchased were resolved by SDS-PAGE, transferred to nitrocellulose, and blotted from NEN (Boston, MA). PD98059 and the GST-Elk-1 fusion protein were with anti-ACTIVE MAP kinase Ab (pTEpY) (diluted 1:1000). For anti- purchased from New England Biolabs (Beverly, MA). U0126 was pur- IgM dose-response experiments, cells were stimulated with the indicated chased from Calbiochem (La Jolla, CA). The GST-Jun(1–79) fusion pro- concentrations of anti-IgM for 4 min before washing, lysis, SDS-PAGE tein was prepared as described (28). PD98059 and U0126 were dissolved and immunoblotting. in DMSO and stored at Ϫ20°C until used. Growth arrest and apoptosis of WEHI-231 cells Cell culture For growth arrest, WEHI-231 cells were plated in triplicate at 2 ϫ 104 Splenic B cells were cultured in RPMI 1640 medium supplemented with cells/well in 96-well plates and stimulated with goat-anti-mouse IgM for 3 ␮ 10% FCS (Life Technologies, Rockville, MD), 2 mM pyruvate, 20 mM 28 h. Cells were pulsed with [ H]dT (1 Ci/well) for the last 4 h, harvested ␮ onto glass-fiber filters (Wallac, Gaithersburg, MD), and the amount of in- glutamine, 50 M 2-ME, and penicillin/streptomycin. WEHI-231 cells 3 were cultured as previously described (2). In all experiments, PD98059, corporated [ H]dT was determined using a Betaplate reader (Wallac). For ϫ 5 U0126, or DMSO carrier was added to the splenic B cells or WEHI-231 apoptosis, WEHI-231 cells were plated at 1.5 10 cells/ml in 12-well plates, stimulated, and cultured for 48 h. Cell survival was measured by cells 1 h before stimulation, unless otherwise indicated. Cells were stim- ␮ ulated and cultured in medium that always contained PD98059, U0126, or ability to exclude 1 g/ml propidium iodide, as determined by flow cy- an equivalent volume of DMSO carrier. tometry, or by annexin V staining following protocols supplied by the manufacturer.

Isolation and apoptosis of immature splenic B cells Proliferation and survival of mature splenic B cells Three-week-old BALB/c mice were obtained from The Jackson Laboratory Proliferation of murine splenic B cells was measured either by incorpora- (Bar Harbor, ME). Mice were given 500-rad whole-body irradiation 13.5 tion of [3H]thymidine or incorporation of BrdUrd. For [3H]dT incorpora- days before sacrifice (29). A single cell suspension of the spleen cells in tion assays, splenic B cells were plated in triplicate in 96-well plates (2.5 ϫ either ice-cold RPMI 1640 or DMEM with 10% FCS was treated with an 106 cells/ml), in the presence of anti-IgM Ϯ 50 U/ml IL-4, and cultured for equal volume of 17 mM Tris-HCl and 1.44% ammonium chloride for 5 min 45 h. Cells were pulsed with [3H]dT for the last5hofculture, and incor- on ice to lyse erythrocytes. The T cells and IgDϩ B cells were lysed by poration was determined as described above. In vitro BrdUrd-labeling ex- incubating in a cocktail containing anti-CD4, anti-CD8, anti-Thy1.2, and periments were performed as described (30). Briefly, RBC-depleted H␦a/1 anti-IgD ascites on ice for 30 min, followed by complement lysis at splenocytes were plated at 5 ϫ 105 cells/ml, stimulated, and cultured for 37°C for 30 min. The isolated immature splenocytes were IgMhigh, IgDlow, 48 h. BrdUrd (10 ␮M) was added during the final 24 h. Cells were fixed, B220high, and negative for Gr1, CD3, and Mac-1 as shown by flow cy- permeabilized with 0.5% Tween 20 in PBS, the DNA denatured, and the tometry. The immature splenic B cells at a density of 2–4 ϫ 106/ml were cells subsequently were costained with anti-B220 and anti-BrdUrd and incubated for 30 min to 1 h with 20 ␮M PD98059 and then stimulated with analyzed by flow cytometry. The data are presented as the percentage of various concentrations of anti-IgM in the presence or absence of 20 ␮M B220ϩ cells that are BrdUrdϩ. For survival experiments, RBC-depleted PD98059 for 18 h. The apoptosis of immature splenic cells was quantified splenocytes or purified splenic B cells were cultured for 45 h. B220ϩ cells by annexin V/PI staining (Oncogene Research Products, Cambridge, MA) were then tested by flow cytometry for their ability to exclude 1 ␮g/ml following the manufacturer’s protocol. propidium iodide. The Journal of Immunology 3857

Up-regulation of cell surface molecules WEHI-231 cells (data not shown). As with the time course assays, RBC-depleted splenocytes were cultured at 5 ϫ 105 or 1 ϫ 106 cells/ml. U0126 inhibited ERK phosphorylation more completely than did Ј PD98059. Cells were stimulated with goat anti-mouse whole Abs or F(ab )2 frag- ments for 18 h. Cells were harvested, Fc receptors blocked with anti-CD16/ CD32, and subsequently costained with anti-B220 and anti-B7-2 (CD86), anti-CD44, or anti-CD69 and analyzed by flow cytometry. Only live (as PD98059 inhibits the BCR-induced up-regulation of Egr-1, determined by propidium iodide exclusion or by forward- and side-scatter CD44, and CD69 profiles) B220ϩ cells were analyzed. BCR-stimulated B cells up-regulate the expression of many tran- scription factors (38, 39). This up-regulation leads to changes in Results the expression of proteins important for cell-cycle regulation, cell- PD98059 and U0126 inhibit activation of the ERK MAP kinases , and activation of T cells. One BCR-induced tran- in WEHI-231 and purified splenic B cells scription factor is Egr-1, which is important for the BCR-induced expression of the adhesion proteins, CD44 and ICAM-1 (40, 41). In vitro protein kinase assays were performed to determine In addition, putative Egr-1 binding sites exist in the promoter of whether PD98059 inhibited the BCR-induced activation of the the gene encoding the human CD69 activation marker (42). BCR- ERK2 form of MAP kinase, as expected. As previously reported induced transcription of a reporter gene linked to the egr-1 pro- (23, 32, 33), stimulation of the WEHI-231 immature B cell line moter can be blocked by dominant negative forms of Ras and Raf with anti-IgM Abs substantially increased the activity of ERK2 (39). Therefore, we wished to determine whether BCR-induced

(Fig. 1A). This activation was inhibited in a dose-dependent man- Egr-1, CD44, and CD69 protein expression required MEK activity. Downloaded from ner by PD98059 at a 4-min time point (Fig. 1A). PD98059 also PD98059 treatment (20 ␮M) greatly suppressed anti-IgM-induced inhibited ERK2 activation over a 24-h time course (see below and up-regulation of Egr-1 in mature splenic B cells (Fig. 2A) and in data not shown). At low concentrations of stimulating Ab, ERK2 WEHI-231 B lymphoma cells (Fig. 2B). In addition, both ␮ activation was always greatly inhibited by 5–20 M PD98059 and PD98059 and U0126 partially inhibited the BCR-induced up-reg- ␮ in some experiments completely blocked. Inhibition by 10–20 M ulation of CD44 and CD69 in mature splenic B cells (Fig. 2, C–E). PD98059 was substantial but not absolute at higher doses of stim- In contrast, PD98059 and U0126 did not inhibit BCR-induced ex- http://www.jimmunol.org/ ulating Ab, consistent with previous results in other systems (26). pression of B7-2 (CD86) (Fig. 2F), a protein that provides an im- Likewise, 5–10 ␮M PD98059 suppressed ERK2 activation in pu- portant costimulatory signal for Th cells (43). rified murine splenic B cells stimulated with anti-IgM (Fig. 1B) and anti-IgM plus IL-4 (Fig. 1C). IL-4 did not reproducibly in- crease or decrease BCR-induced ERK2 activation at a 4-min time MEK inhibitors do not inhibit BCR-induced growth arrest or point (Fig. 1C) and it did not alter the magnitude and kinetics of apoptosis in WEHI-231 B cells or apoptosis in immature splenic BCR-induced ERK2 activation over a 2-h time course (data not B cells shown). Moreover, PD98059 was equally as effective at blocking BCR stimulation induces WEHI-231 B cells to arrest in the G1 BCR-induced ERK2 activity in the presence or absence of IL-4 phase of the cell cycle and then undergo apoptosis (7–9, 44, 45). by guest on September 25, 2021 (compare Fig. 1, H and I). In contrast, PD98059 in this dose range To determine whether the MEK-ERK pathway is important for did not decrease or increase the BCR-induced activation of JNK, these events, we pretreated WEHI-231 cells with varying amounts another MAP kinase protein that is downstream of the MEK fam- of PD98059 or U0126 before stimulation with anti-IgM Abs. No ily member MKK4 (34), in purified splenic B cells (Fig. 1D)orin inhibition of BCR-induced growth arrest was seen, as assessed by WEHI-231 B cells (data not shown). PD98059 also did not block incorporation of [3H]thymidine (Fig. 3, A and B). However, 20 ␮M the BCR-induced activation of the tyrosine kinase Syk or the ap- PD98059 and 10 ␮M U0126 did reproducibly decrease basal pro- pearance of tyrosine phosphoproteins in the lysates of BCR-stim- liferation of WEHI-231 cells, suggesting that the MEK-ERK path- ulated B cells (data not shown). Therefore, PD98059 seemed to way may be required for the proliferation of this cell line. inhibit MEK1/2, and, thus, ERK specifically in B cells, as it does PD98059 and U0126 also failed to inhibit the BCR-induced apo- in other cell types up to concentrations of at least 50 ␮M (25, 26, ptosis of these cells, as determined by propidium iodide exclusion 35, 36). (Fig. 3, C and D), and by annexin V staining (data not shown). The ERK kinases must be dually phosphorylated by MEK1/2 on These data suggest that the MEK-ERK pathway is not required for specific threonine and tyrosine residues in the activation loop to anti-IgM-mediated growth arrest or apoptosis in WEHI-231 cells. become activated (37). Thus, immunoblotting with Abs that rec- Immature B cells from spleen or bone marrow also undergo ognize the dually phosphorylated forms of ERK1 and ERK2 is a apoptosis in response to Ag receptor stimulation (46). To deter- useful assay for both MEK1/2 action and ERK activity. Inhibition mine whether inhibition of MEK would affect these events, we of ERK1/2 phosphorylation by 20 ␮M PD98059 and by a second, used autoreconstitution of sublethally irradiated mice (29) as a structurally and mechanistically distinct MEK1/2 inhibitor, 10 ␮M method for isolating sufficient numbers of immature B cells. The U0126, therefore, was examined by anti-phosphoMAP kinase im- isolated immature B cells were incubated in culture with or with- munoblotting. Both inhibitors blocked ERK phosphorylation over out 20 ␮M PD98059 and with various concentrations of polyclonal a 4-h time course in purified splenic B cells (Fig. 1E) and in anti-IgM. Although there was a substantial spontaneous apoptosis WEHI-231 B cells (Fig. 1F). Similar to the above protein kinase of these immature B cells after 18 h of incubation in culture, the assays, the inhibition of ERK phosphorylation provided by fraction of cells that had initiated apoptosis, as judged by staining PD98059 was significant but incomplete. (However, note that by with annexin V, increased appreciably in response to low concen- the end of the time course, ERK phosphorylation was inhibited trations of anti-IgM (Fig. 4A). Similar results were obtained when back down to levels approximating baseline.) In contrast to apoptosis was judged by failure to exclude propidium iodide, rep- PD98059, U0126 blocked BCR-induced phosphorylation of ERK resenting a late step in apoptosis (data not shown). PD98059 treat- completely or almost completely at all time points in both cell ment did not decrease the fraction of immature B cells that were types. Both inhibitors also blocked ERK phosphorylation over a apoptotic upon stimulation with anti-IgM, whereas it did partially wide range of stimulating Ab in purified B cells (Fig. 1G) and in inhibit ERK2 activation (by 50–72% in the experiment shown in 3858 MEK-ERK CASCADE IN B CELL RESPONSES TO ANTIGEN Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. PD98059 inhibits BCR-induced activation of ERK1/2. WEHI-231 B cells (A) or murine splenic B cells (B and C) were treated with PD98059 or DMSO carrier for 1 h, stimulated for 4 min with anti-IgM Abs Ϯ 50 U/ml IL-4, and lysed in a 1% Triton X-100-containing lysis buffer. ERK2 was immunoprecipitated, and its activity was assessed by in vitro kinase assay using GST-Elk-1 as the substrate. Reaction products were separated by SDS-PAGE and visualized by autoradiography. The position of GST-Elk-1 is indicated. D, Murine splenic B cells were treated with PD98059 or DMSO for 1 h, stimulated for 15 min, and lysed, as above. JNK1 was immunoprecipitated and its activity was measured by in vitro kinase assay, using GST-Jun(1–79) as the substrate. E and F, Murine splenic B cells (E) or WEHI-231 B cells (F) were treated with DMSO, 20 ␮M PD98059 or 10 ␮M U0126, stimulated for the indicated times with 10 ␮g/ml anti-IgM Abs, and lysed. Lysates were separated by SDS-PAGE and immunoblotted with anti-ACTIVE MAP kinase Ab, which recognizes dually phosphorylated forms of ERK1 and ERK2 (P-ERK1 and P-ERK2, respectively). G, Murine splenic B cells were treated with DMSO, 20 ␮M PD98059 or 10 ␮M U0126, stimulated with the indicated concentrations of anti-IgM Abs for 4 min, and lysed. Lysates were immunoblotted with anti-ACTIVE MAP kinase Ab. The membranes shown in A–C and E–G were reprobed with anti-ERK2 Abs to show equal loading between lanes. A representative example is shown in G. H and I, In three separate experiments, the percent inhibition of BCR-induced ERK2 activity in splenic B cells (as measured by in vitro kinase assays) was calculated for 5 ␮M and 10 ␮M PD98059 at the indicated concentrations of anti-IgM, for cells stimulated with anti-IgM (H) or anti-IgM plus 50 U/ml IL-4 (I). These individual values were averaged to generate the average percent inhibition of ERK2 activity, which is shown. Error bars indicate SEM.

Fig. 4B), as seen in the other B cell populations tested. It should PD98059 alone, anti-IgM alone, and the cultures containing be noted that PD98059 treatment by itself often increased the both agents. It may be that stimulation of the Ras-ERK MAP fraction of cells that had initiated apoptosis. In some experi- kinase pathway by some element of the culture conditions (se- ments, this effect was small, or, as in the example shown in Fig. rum, for example) provides a survival signal, such that inhibi- 4A, moderate. In other experiments, the fraction of immature B tion of this pathway leads to greater apoptosis even in the ab- cells initiating apoptosis was similar in the cultures treated with sence of BCR stimulation. The Journal of Immunology 3859 Downloaded from

FIGURE 2. PD98059 selectively inhibits BCR-induced up-regulation of Egr-1, CD44, and CD69. Murine splenic B cells (A) or WEHI-231 B cells (B) were treated with PD98059 or DMSO carrier, stimulated with anti-IgM Abs for 1 h, and lysed. Lysates were immunoblotted with anti-Egr-1 Abs (upper panels). The membrane was also probed with anti-ERK2 Abs to verify equal loading (lower panels). C–F, RBC-depleted splenocytes were incubated with http://www.jimmunol.org/ Ј the indicated concentrations of PD98059, U0126, or DMSO carrier for 1 h, then stimulated with anti-IgM whole Abs or F(ab) 2 fragments for 18 h. Expression of CD44 (C and D), CD69 (E), and B7-2/CD86 (F) was analyzed by flow cytometry, and the data are presented according to the nature of the induction. Anti-CD44 staining revealed a single population of cells whose intensity of CD44 expression increased with stimulation. C and D, Median fluorescence of anti-CD44 staining. In contrast, anti-CD69 and anti-B7-2 (CD86) staining revealed two distinct populations of cells, expressors and nonexpressors. E and F, The percentage of cells that express CD69 and B7-2, respectively.

PD98059 and U0126 inhibit proliferation of BCR-stimulated pharmacologic inhibitors of MEK1 and MEK2, PD98059 and by guest on September 25, 2021 splenic B cells U0126, to inhibit activation of this pathway in WEHI-231 B lym- In contrast to its effect on immature B cells, BCR engagement phoma cells, in immature splenic B cells, and in mature splenic B cells. PD98059 inhibits MEK1/2 specifically and does not block causes mature B cells to enter the G1 phase of the cell cycle and to proliferate. Addition of -derived cytokines such as IL-4 activation of the closely related MEK family members MKK3 or enhances this proliferative response (47–49). We wished to de- SEK/MKK4, which are upstream of JNK and p38 MAP kinases, termine whether the MEK-ERK pathway is important for BCR- and it does not inhibit numerous other serine/threonine, tyrosine, induced proliferation of murine splenic B cells. Anti-IgM treat- or lipid kinases (Fig. 1D and data not shown) (25, 26, 35, 36). ment induced B cell proliferation in a [3H]thymidine U0126 also specifically inhibits MEK1/2, without blocking the incorporation assay, and PD98059 blocked this proliferation in activation of JNK, p38 MAP kinase, or several other protein ki- a dose-dependent manner (Fig. 5A). In agreement with earlier nases (27, 50, 51). In vitro immunocomplex kinase assays dem- reports (47–49), the addition of IL-4 greatly enhanced the pro- onstrated that PD98059 was able to substantially inhibit BCR-in- liferation of the anti-IgM-stimulated B cells (Fig. 5B, note dif- duced ERK2 activity in a dose-dependent manner (Fig. 1, A–C). ferent scale on x-axis). PD98059 was also able to suppress B Likewise, anti-phosphoMAP kinase blots determined that U0126 cell proliferation in the presence of IL-4, but to a lesser degree and PD98059 both inhibited BCR-induced ERK1/2 activation over than when cells were stimulated with anti-IgM alone. IL-4 in- a wide range of times and doses of stimulation (Fig. 1, E–G). creases [3H]thymidine incorporation in this assay both by acting Treatment with PD98059 also inhibited the BCR-induced expres- as a B cell survival factor and by increasing the percentage of sion of the early response gene product Egr-1, demonstrating the B cells that enter the cell cycle (47–49). We investigated importance of the MEK-ERK kinase pathway for the expression of whether a block in MEK-ERK activity would inhibit one or this transcription factor. This result is consistent with previous both of these effects. As shown in Fig. 5C, the percentage of B studies demonstrating the requirement of promoter serum response cells that entered S phase in response to anti-IgM plus IL-4 was elements (SREs) for egr-1 expression (52), and the ability of dom- significantly diminished by treatment with either PD98059 or inant negative mutants of Ras and Raf to block egr-1 promoter- U0126. U0126 proved to be the more potent inhibitor, because driven reporter in response to BCR cross-linking 10 ␮M U0126 was sufficient to block proliferation essentially (39). Anti-IgM-induced proliferation of splenic B cells was also completely. In contrast, neither inhibitor substantially altered B substantially inhibited by PD98059 and U0126, in the absence and cell survival in the presence of IL-4 (Fig. 5D). presence of IL-4. Thus, MEK1/2 activity and presumably ERK activity play important roles in BCR-induced proliferation. In Discussion contrast, this pathway is not required for the BCR-induced expres- We wished to study the role(s) that the Ras-Raf-MEK-MAP kinase sion of B7-2 (CD86) in mature splenic B cells, for growth arrest pathway plays in B cell responses to Ag. To do this, we used two and apoptosis of the immature WEHI-231 B cell line, or for 3860 MEK-ERK CASCADE IN B CELL RESPONSES TO ANTIGEN Downloaded from http://www.jimmunol.org/

FIGURE 3. PD98059 and U0126 do not inhibit BCR-induced growth arrest or apoptosis of WEHI-231 immature B cells. WEHI-231 cells were incubated with PD98059, U0126, or DMSO carrier for 1 h. Cells were left unstimulated or were stimulated with 0.37, 1.1, or 10 ␮g/ml anti-IgM Abs. A 3 3 and B, Cells cultured in PD98059 (A) or U0126 (B) were stimulated for 28 h, pulsed with [ H]thymidine for the last 4 h, and [ H]thymidine incorporation by guest on September 25, 2021 was measured. C and D, Cells cultured in PD98059 (C) or U0126 (D) were stimulated for 48 h. Apoptosis was measured by the inability to exclude 1 ␮g/ml propidium iodide, as determined by flow cytometry. A–C, Single representative experiments. D, The average of three separate experiments. Error bars indicate SEM.

BCR-induced apoptosis of immature splenic B cells. In addition to liferation (11–15). Just as ERK kinases activate Elk-1 and Sap-1a, demonstrating that some BCR-induced effects are not dependent each of the above signaling pathways are likely to activate other on the MEK-ERK signaling pathway, these results demonstrated pre-existing transcription factors. For example, the PLC-␥ path- that PD98059 and U0126 did not exert significant toxicity or non- way activates the serine/threonine phosphatase calcineurin, which specific inhibition of B cell functions. Moreover, because these dephosphorylates and activates NFAT (56). Together, these pre- two structurally and mechanistically distinct inhibitors gave very existing but newly activated transcription factors induce expres- similar results, this strongly suggests that they are indeed working sion of early response , many of which, like egr-1, encode via the MEK-ERK MAP kinase pathway and not via some other transcription factors themselves. These secondary transcription unknown actions. factors likely control the expression of many other genes important Our results indicating that activation of the Ras-ERK MAP ki- for events such as proliferation and interaction with Th cells (3). nase pathway is important for proliferation of splenic B cells are Indeed, Egr-1 has been implicated in the BCR-induced expression consistent with results obtained in other cell types, such as fibro- of CD44 and ICAM-1 (see below). Different signaling pathways, blasts and PC12 cells, where this pathway also contributes to cel- then, could regulate the expression of different cell cycle genes by lular proliferation (17–22). The mechanism by which ERK pro- virtue of the transcription factors they activate. Some cell-cycle motes BCR-mediated proliferation is not yet defined, although it is genes may require the activation of transcription factors controlled likely to involve changes in gene expression; BCR stimulation by one signaling pathway and others by multiple pathways. rapidly induces changes in expression of several important cell The induction of proliferation by the BCR is known to be dra- cycle regulators, such as cyclins, cyclin-dependent kinases, and matically enhanced by IL-4. We have found that this enhancement cell-cycle inhibitors (53–55). The ERK kinases phosphorylate and is not paralleled by an increase in ERK activation (Fig. 1C and data activate several transcription factors, including Elk-1 and SAP-1a, not shown). Thus, IL-4 appears to promote B cell proliferation by which help enhance transcription of genes whose promoters con- its action on other signaling pathways, perhaps those involving tain SREs (34). Indeed, the egr-1 early response gene contains IRS-1, IRS-2, or Stat-6 (57–61). Interestingly, high concentrations several SREs and is rapidly induced upon BCR cross-linking, and of PD98059 only partially inhibited proliferation in response to ERK activity was required for Egr-1 expression (Fig. 2). anti-IgM and IL-4, whereas it completely or almost completely In addition to the Ras-ERK MAP kinase pathway, HS1, Vav, inhibited proliferation to similar or even higher doses of anti-IgM and the PLC-␥ pathway are also important for BCR-mediated pro- alone. This difference in proliferative inhibition occurred despite The Journal of Immunology 3861

U0126 (Fig. 2D), consistent with the hypothesis that Egr-1 plays a significant role in this induction. These data provide evidence that in addition to its role in BCR-induced proliferation, the Ras-ERK MAP kinase pathway is important for the up-regulation of specific proteins involved in cell-cell interactions and B cell trafficking. In addition, putative Egr-1 binding sites are found in the human CD69 promoter (42), and the Ras pathway is involved in its up- regulation in T cells (63, 64). Our data demonstrate that CD69 up-regulation is MEK1/2-dependent in murine B cells (Fig. 2E) and are consistent with a role for Egr-1 in its up-regulation. These data are also consistent with recently published results that PD98059 inhibits the BCR-induced up-regulation of Egr-1 mRNA (65). However, in contrast to Egr-1, this group found that BCR-induced induction of Egr-2 mRNA was not significantly blocked by PD98059. The persistence of Egr-2 expression in PD98059-treated B cells may explain why CD44 and CD69 up- regulation is only partially inhibited by PD98059 and U0126. Whereas in most situations the Ras-ERK MAP kinase pathway Downloaded from seems to be involved in promoting or differentiation, this pathway has been implicated in growth arrest in murine fi- broblasts (20–22), and ERK activity also has been reported to be important for apoptosis in some instances (24, 66, 67). However, we observed no inhibition in BCR-induced growth arrest or apo- ptosis in WEHI-231 B cells treated with PD98059 or with U0126, http://www.jimmunol.org/ FIGURE 4. PD98059 fails to inhibit anti-IgM-induced apoptosis of im- which blocks BCR-induced MEK-ERK activity almost completely mature splenic B cells. Mice were sublethally irradiated to deplete mature (Fig. 3). Moreover, 20 ␮M PD98059 also failed to inhibit BCR- B cells, and the immature B cells of the spleen were harvested at 13.5 days induced apoptosis in immature splenic B cells (Fig. 4A), although Ϫ after irradiation. A, Purified IgD B cells were cultured in the absence or this same concentration of PD98059 dramatically blocked BCR- ␮ presence of 20 M PD98059 and the presence of the indicated concentra- induced proliferation and Egr-1 and CD69 up-regulation in mature tion of polyclonal goat anti-mouse IgM Abs for 18 h. The percentage of apoptotic cells was determined by flow cytometry using annexin V and splenic B cells. propidium iodide staining. Shown is the percent of cells positively stained In contrast to our results, Lee and Koretzky (24) found that by annexin V. B, The ability of 20 ␮M PD98059 to inhibit activation of overexpression of MKP-1, a phosphatase that can dephosphorylate ERK2 in anti-IgM-stimulated immature splenic B cells (same preparation and inactivate ERK1/2, did block apoptosis of WEHI-231 cells. by guest on September 25, 2021 of cells as in A) was assessed as in Fig. 1. The percent inhibition of ERK This conclusion was based on experiments examining apoptosis at activity ranged between 50 and 72%. The membrane was also immuno- early times when Ͻ20% of cells had undergone apoptosis, so it is blotted with anti-ERK2 Abs to verify equal loading (lower panel). — in- possible that MKP-1 overexpression delayed but did not prevent dicates a blank lane. apoptosis. However, it should be noted that we did not observe inhibition of apoptosis by PD98059 even at relatively early times when apoptosis had occurred in a minority of cells (data not the fact that ERK activation was suppressed equally well in B cells shown). Alternatively, it is possible that MKP-1 overexpression stimulated in the absence or presence of IL-4 (Fig. 1, H and I). exerted its effects by dephosphorylating other MAP kinases, such Thus, it appears that BCR-induced B cell proliferation has a de- as JNK or p38 MAP kinase. MKP-1 has been demonstrated to creased requirement for MEK/ERK signaling events in the pres- dephosphorylate and inhibit the activity of JNK and p38 in many ence of IL-4. However, this proliferation of B cells was still de- cell types, including lymphocytes (68–71), and BCR cross-linking pendent on a low level of ERK activation, because it was has been reported to activate JNK and p38 (Fig. 1D) (23, 33, 72– completely blocked by the more effective MEK1/2 inhibitor, 75). Moreover, Graves and colleagues (74) have presented data U0126. MEK-ERK activity appears to be required for cell-cycle indicating that JNK and p38 (73, 74) MAP kinases are involved in entry (Fig. 5C) rather than for survival (Fig. 5D). BCR-induced apoptosis in human B cell lines, although contrast- BCR-stimulated mature B cells also up-regulate adhesion mol- ing data have been presented that p38 is not required for apoptosis ecules and a variety of other cell surface proteins, some of which are important for promoting interaction with Th cells. MEK-ERK in WEHI-231 cells (75). If MKP-1 overexpression inactivates JNK inhibition failed to block up-regulation of B7-2 (CD86) (Fig. 2F), and/or p38 in B cells, as it does in other cell types, this could a protein that provides a costimulatory signal to T cells through its explain the block in BCR-induced apoptosis reported by Lee and interaction with CD28 (43). In contrast, BCR-induced up-regula- Koretzky (24). Indeed, MKP-1 blocks UV-induced apoptosis in tion of the transcription factor Egr-1 was suppressed by PD98059, U937 human leukemia cells, apparently by inhibiting JNK activity as mentioned above. Egr-1 expression has been implicated in the (70). In contrast to MKP-1 overexpression, PD98059 and U0126 up-regulation of the adhesion proteins CD44 and ICAM-1 (40, 41). appear to inhibit ERK activity specifically without inhibiting JNK CD44 has been proposed to facilitate B to and re- or p38 (Fig. 1) (25, 26, 35, 36, 50, 51). Moreover, neither inhibitor tention in secondary lymphoid follicles, where B cells interact with blocked BCR-induced apoptosis in our experiments, even when T cells (40), and ICAM-1 expression helps mediate adhesion be- BCR-induced ERK activity was blocked almost completely (Figs. tween Ag-presenting B cells and Th cells during T cell activation, 3 and 4). Thus, whereas JNK and/or p38 activity may be required and subsequent stimulation of the B cells (3, 62). Up-regulation of for BCR-induced apoptosis of immature B cells, we conclude that CD44 expression was partially blocked by PD98059 (Fig. 2C) and ERK activity likely is not required. 3862 MEK-ERK CASCADE IN B CELL RESPONSES TO ANTIGEN Downloaded from http://www.jimmunol.org/

FIGURE 5. PD98059 and U0126 inhibit BCR-induced proliferation in the absence and presence of IL-4. A and B, Murine splenic B cells were treated with PD98059 or DMSO and stimulated with anti-IgM (A) or anti-IgM plus 50 U/ml IL-4 (B) for 45 h. Cells were pulsed with [3H]thymidine for the last 5h.[3H]thymidine incorporation from a representative experiment, performed on the same preparation of B cells, is shown (note different scales on x-axis). Error bars indicate SEM. C, RBC-depleted splenocytes (5 ϫ 105/ml) were treated with PD98059, U0126, or DMSO and stimulated with anti-IgM plus 50 U/ml IL-4 for 48 h. BrdUrd (10 ␮M) was added for the final 24 h. Shown is the percentage of B220ϩ B cells that incorporated BrdUrd, indicative of entry by guest on September 25, 2021 into S phase of the cell cycle. An average of three experiments is shown. Error bars indicate SEM. D, The ability of 20 ␮M PD98059 or 10 ␮M U0126 to inhibit IL-4-mediated survival was assessed. RBC-depleted splenocytes were treated with PD98059, U0126, or DMSO and stimulated with anti-IgM plus 50 U/ml IL-4 for 45 h. Survival was measured by the ability to exclude propidium iodide. A representative experiment is shown.

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