Stimulation of the B cell surface receptor CD40 induces transcriptional activation and protein expression. To determine which proteins are required for the CD40-mediated B cell activation, we performed a two-dimensional gel electrophoresis of the WEHI 231 B cell lysates. We report in this study the identification of one protein in which the expression was remarkably induced following CD40 stimulation. It was the p190 Rho guanine nucleotide exchange factor (GEF), p190RhoGEF, a recently identified GEF that is specific for RhoA. Overexpression of either p190RhoGEF or RhoA (Q63L), a constitutively active form of RhoA, mimics the effects of CD40 stimulation, such as changes in cellular structure and NF-κB activation. These p190RhoGEF overexpression effects are abrogated when coexpressed with a dominant negative form of RhoA (T19N). We also provide evidence for the CD40-mediated cellular changes that are abrogated in cells that are overexpressed with the dominant negative form of either p190RhoGEF (Y1003A) or RhoA (T19N).
CD40, a B cell surface receptor, delivers key activation signals to the B cell. This leads to the proliferation, differentiation, isotype switching of Ig gene, secretion of cytokines, and up-regulation of many surface molecules (1, 2, 3). Considerable progress has been made in identifying the initial signaling events after CD40 stimulation. This includes the involvement of the TNFR-associated factor proteins, activation of several protein kinases, and consequent activation of nuclear transcription factors (4, 5). These signaling events are likely to further regulate the protein expression changes that are required to perform complex cellular responses to CD40 ligation.
To identify the CD40-induced changes in the protein expression that might regulate the B cell activation, we performed a two-dimensional (2D)3 gel electrophoresis (6, 7) of the lysates from the WEHI 231 B cells, either CD40-activated or rested. We analyzed one protein spot on the 2D gels; it was remarkably increased in its expression following a 48-h CD40 stimulation. We identified the protein as the p190 Rho guanine nucleotide exchange factor (GEF), p190RhoGEF. For this identification, we used the peptide mass fingerprinting method (8) that used a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry (MS).
Recently, p190RhoGEF was cloned and identified as specific for the RhoA small G protein (9, 10). In neuronal cells, the overexpression of this protein mimics activated RhoA in stimulating cytoskeletal contraction and preventing neurite outgrowth (9, 10). However, this GEF has never been reported to function in immune cells. We report in this study the p190RhoGEF role in B cell activation following CD40 stimulation. First, we demonstrate that the expression of this GEF is remarkably enhanced after the CD40 stimulation of the WEHI 231 B cells, which correlates with the cellular structural changes. We also show that the transient overexpression of p190RhoGEF mimics the function of activated RhoA. This modulates the filamentous actin (F-actin) activity that results in the size and shape changes of the cells, as well as activating the NF-κB. We also demonstrate that the transient overexpression of the dominant negative form of p190RhoGEF (Y1003A) or RhoA (T19N) blocks CD40-mediated activation effects. This indicates the crucial roles of these proteins in the CD40-mediated B cell activation.
Materials and Methods
Cell culture and treatment
Maintenance of the WEHI 231 mouse B lymphoma line and isolation of splenic B cells were described previously (11). When stimulated, the cells (WEHI 231: 5 × 105/ml, splenic B cells: 1 × 106/ml) were incubated with anti-CD40 mAb (R&D Systems, Minneapolis, MN) at 1 μg/ml for indicated times.
The expression plasmid for p190RhoGEF (pcDNA3-HA) was kindly provided by Dr. W. Moolenaar (The Netherlands Cancer Institute, Amsterdam, The Netherlands) (9). A single-point mutant, Y1003A, was prepared as described previously (9) using a QuickChange mutagenesis kit (Stratagene, La Jolla, CA). The expression plasmids for the wild-type (WT) RhoA (pRK5-myc), a constitutively active mutant (Q63L), and a dominant-negative mutant (T19N) were gifts from Dr. G. M. Bokoch (The Scripps Research Institute, La Jolla, CA). The reporter plasmids of NF-κB luciferase and CMV-β-gal were generous gifts from Dr. E. Clark (University of Washington, Seattle, WA) and Dr. G. MacGregor (Emory University, Atlanta, GA), respectively. The green fluorescence protein (GFP) plasmid was obtained from Clontech Laboratories (Palo Alto, CA).
Transient transfections and reporter gene assay
The cells (1–2 × 107) were resuspended in a 400 μl cytomix intracellular buffer (12) before electroporation at 200 V/65 ms with a BTX-T820 (Genetronics, San Diego, CA). The cells were transfected with 40 μg of the vector, or the indicated expression plasmid DNA, along with 5 μg of the GFP plasmid for immunocytochemistry. Reporter gene assays were performed as described previously (11) and luminescence was determined using Luminoskan TL Plus (Bio-Orbit, Turku, Finland).
RNA isolation and RT-PCR
Total RNA from the splenic B cells (5 × 106) that were either unstimulated or stimulated with anti-CD40 was isolated using the TRIzol reagent (Life Technologies, Rockville, MD). It was converted to cDNA with an oligo(dT) primer (Life Technologies). A fragment of either p190RhoGEF or β-actin was amplified and the PCR products were separated on a 1.2% agarose gel.
The cells were fixed with 3.7% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 for 5 min. After being blocked with 1% BSA for 1 h, the samples were incubated with anti-p190RhoGEF, 187 (13) (Dr. B. Margolis, University of Michigan, Ann Arbor, MI) or anti-RhoA (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h. A 30-min incubation with FITC- or Texas Red-conjugated secondary Ab (Kirkegaard & Perry Laboratories, Gaithersburg, MD or Vector Laboratories, Burlingame, CA) followed. Visualization of the actin filaments was conducted by incubating the cells with TRITC-phalloidin (Sigma-Aldrich, St. Louis, MO) for 1 h. The samples were analyzed using a Zeiss Axiovert 100 M inverted microscope that was equipped with a 100× Plan-Apochromat objective and a Zeiss LSM 510 confocal attachment (Zeiss, Oberkochen, Germany).
The cells were blocked with normal rabbit serum (The Jackson Laboratory, Bar Harbor, ME). This was followed by fixation/permeabilization with a Cytofix/Cytoperm solution (BD PharMingen, San Diego, CA). The fixed/permeabilized cells were stained for p190RhoGEF using Ab 187 and FITC-conjugated secondary Ab. These cells (1 × 106) were resuspended in a HEPES buffer and analyzed using a FACSCalibur and CellQuest software (BD Biosciences, Mountain View, CA). Ten thousand events were acquired from each sample. Light scatter and fluorescence signals were analyzed as dot plots of side scatter (SSC-H) vs fluorescence intensity channel 1 (FL1).
2D gel electrophoresis and MS
Immobiline DryStrips (pH 3–10 nonlinear, 7 cm; Amersham Pharmacia Biotech, Piscataway, NJ) were loaded with 1 mg of the whole-cell extracts prepared with a 4% Nonidet P-40 solution similarly as described previously (14). Isoelectric focusing was performed at room temperature using an IPGphor electrophoresis unit (Amersham Pharmacia Biotech). The equilibrated strips were inserted onto SDS-7% PAGE gels for the second dimension. The 2D protein gels were then stained with Coomassie brilliant blue R-250 (Sigma-Aldrich). An individual protein spot was excised from 2D gels. Then the destaining procedure and in-gel digestion with trypsin followed. A mass analysis of the resultant peptide mixtures was performed using MALDI-TOF-MS (Voyager-DE STR; PerkinElmer, Wellesley, MA) that operated in the positive-ion reflector mode. Spectra were collected over the mass range of 800-3500 Da and calibrated with standard peptides. The protein identification was by peptide mass fingerprinting using MS-Fit.
Results and Discussion
Induced expression of p190RhoGEF following CD40 stimulation
CD40 is an important surface receptor that is involved in the activation and maturation process of B cells during the humoral immune response, as well as in the development of memory cells (1, 2, 3, 4, 5). To identify specific proteins that are expressed differentially during long-term activation by CD40 on B cells, we used the proteome approach. This has been rapidly and successfully applied to the analyses of many biological phenomena (14, 15, 16) by resolving the expressed proteins of the cell. Peptide sequencing and identification then followed.
The protein expression profile, which was resolved on 2D gels of lysates from the WEHI 231 B cells that were either rested (control) or stimulated with anti-CD40 for 48 h, revealed a dramatic change following CD40 ligation, especially at high molecular mass ranges (Fig. 1,A). One protein spot (indicated by an arrowhead) showed remarkable size increase after the CD40 stimulation. A MALDI-TOF peptide mass analysis of the tryptic digest of this spot identified it as p190RhoGEF, a recently described GEF specific for the RhoA small G protein (9, 10). This RhoA-binding GEF of 190 kDa belongs to the Dbl family of GEFs that stimulate the exchange of GDP to GTP. The CD40-induced expression of p190RhoGEF was further confirmed by an immunoblot analysis with a specific Ab for p190RhoGEF, 187 (Fig. 1,B), as well as at the message level by RT-PCR: the induction appears at 12 h, is greatest at 24 h, and diminishes 48 h after CD40 stimulation (Fig. 1 C).
CD40-induced cellular changes and the colocalization of p190RhoGEF with RhoA
The enhanced p190RhoGEF expression after a 48-h CD40 stimulation was also shown by an indirect immunofluorescence for endogenous p190RhoGEF in WEHI 231 B cells (Fig. 2,A, 50–60% induction). When stained together with RhoA, the colocalization of these two proteins was seen in the cytoplasm toward the plasma membrane after a 48-h CD40 ligation (Fig. 2,A). In these cells, the CD40-induced cellular changes were also noted for their size and shape (60–80% response). Analyses of the CD40-stimulated cells using flow cytometry also quantitatively demonstrated cellular changes in size and shape (SSC-H) as well as an increase in the p190RhoGEF expression (FL1). As seen in Fig. 2 B, the CD40-induced increase in FL1 and SSC-H (R1) was 5- and 6-fold, respectively, over the unstimulated control. A previous study reported that p190RhoGEF mimics activated RhoA in stimulating cytoskeletal contraction and preventing neurite outgrowth when overexpressed in a neuronal cell (9). However, this GEF does not function in lymphocytes. A remarkable increase in the p190RhoGEF expression after CD40 ligation strongly implicates the significant role of this protein on B cell activation. Our results, combined with previous reports, imply the possibility that at the colocalization site, the CD40-induced p190RhoGEF regulates RhoA activation. This causes cellular changes since these factors have been implicated in modulating cytoskeletal structures (17, 18).
Overexpression of p190RhoGEF mimics CD40-induced cellular changes
Because both the p190RhoGEF expression and cellular structure are changed after the CD40 stimulation, we tested to see if the p190RhoGEF overexpression alone (in the absence of CD40 stimulation) mimics the cellular changes that are mediated by CD40 ligation. We transiently transfected WEHI 231 B cells with a GFP plasmid for the transfection control, together with either a vector or a full-length p190RhoGEF plasmid. Consequent size and shape changes of these cells, if any, were visualized by staining F-actin using rhodamine-conjugated phalloidin in conjunction with a confocal microscopy.
As seen in Fig. 3,A, in the cells that were transfected with a vector plasmid that was determined by the GFP expression, CD40 stimulation induced cellular shape and size changes (∼75%). The CD40-induced changes in the vector-transfected WEHI 231 B cells indicate that these cells are able to respond to CD40 ligation. Indeed, we confirmed the enhanced p190RhoGEF expression after the CD40 stimulation in these vector-transfected cells by staining them with anti-p190RhoGEF Ab, 187 (Fig. 3,A). As expected, the cells that were transfected transiently with only the p190RhoGEF plasmid showed cellular changes in the absence of CD40 stimulation (Fig. 3,B, ∼70%). However, in the cells that were transfected with a dominant-negative form of p190RhoGEF (Y1003A), the observed cellular changes could not be determined either in the absence or presence of CD40 stimulation (Fig. 3 C, >90%). These results strongly suggest that cellular changes after the CD40 stimulation are possibly mediated by the enhanced function of p190RhoGEF.
Because p190RhoGEF specifically activates the RhoA small G protein (9, 10), we then examined whether the changes that were shown in the cells that were transfected with the p190RhoGEF plasmid, or in cells that were stimulated with anti-CD40, may be a result of the increased RhoA activity. As shown in Fig. 3,D, the WEHI 231 B cells that were transfected with the dominant negative of RhoA (T19N) showed no cellular changes, with or without CD40 stimulation (>90%). Moreover, the simultaneous overexpression of RhoA (T19N) and p190RhoGEF abrogated the cellular changes that were seen in the cells that were transfected with p190RhoGEF alone (Fig. 3,E, ∼70%). Also, distinct cellular changes were similarly observed in the WEHI 231 B cells that were transfected transiently with a constitutively active form of RhoA (Q63L), as seen in Fig. 3 F (>90%). These results strongly imply that the enhanced activation of the RhoA small G protein mediates the p190RhoGEF function after the CD40 stimulation in WEHI 231 B cells.
Overexpression of p190RhoGEF enhances the NF-κB activation
Because the p190RhoGEF overexpression mimicked the same cellular changes that were induced by CD40 ligation, we examined whether the overexpression of this protein also induces the activation of NF-κB that is seen in the WEHI 231 B cells after the CD40 stimulation. Because our data demonstrated that p190RhoGEF functions through RhoA (the activity of which is known to induce changes in the cytoskeletal structure as well as gene transcription), we also examined whether the activity of RhoA plays a role in the activation of NF-κB by the p190RhoGEF overexpression. For these experiments, the WEHI 231 B cells were transiently transfected with the p190RhoGEF plasmid alone, or in combination with either a constitutively active or a dominant negative form of RhoA along with a reporter plasmid for NF-κB.
Compared with the reporter activity that is induced by the CD40 ligation in the cells that were transfected with the vector plasmid, the WEHI 231 B cells that were transiently transfected with only the WT RhoA showed similar activity. However, in the cells that were transfected with either p190RhoGEF or a constitutively active form of RhoA (Q63L), the NF-κB reporter activity was augmented ∼2-fold or >4-fold. In contrast, the cells that were transfected with the dominant negative form of RhoA (T19N) showed a significant reduction in the reporter activity to the basal level, shown in the vector-transfected resting cells (Fig. 4). Additionally, the p190RhoGEF-induced reporter activity was further enhanced in the cells that were cotransfected with the constitutively active form of RhoA (Q63L), but returned to the basal in the cells that were cotransfected with the dominant-negative form of RhoA (T19N) (Fig. 4). Furthermore, in the cells that were transfected with the dominant negative of either p190RhoGEF (Y1003A) or RhoA (T19N), the NF-κB reporter activity was not induced by CD40 stimulation (data not shown).
These results show that similar to the cellular changes, the CD40-mediated NF-κB activation in WEHI 231 B cells requires the activities of RhoA and p190RhoGEF. In this study, the activity of RhoA is in part regulated by the enhanced p190RhoGEF expression after CD40 ligation. However, a recent study on neuronal cells also showed that p190RhoGEF can directly bind to the c-Jun N-terminal kinase (JNK)-interacting protein, JIP-1 (13), which is reported to serve as a substrate for JNK (19) and as a scaffolding protein for JNK activation (20). Therefore, further study will be required to determine the possibility that p190RhoGEF may control a wider range of cellular processes than those that are predicted by its GDP/GTP exchange activity.
In summary, we identified p190RhoGEF as a protein that is enhanced in its expression after CD40 stimulation in WEHI 231 B cells that use the proteome approach. We further demonstrated that this protein plays a significant role in the CD40-mediated B cell activation through the regulation of RhoA activity. However, it is yet to be determined which upstream signals that follow the CD40 stimulation control the expression and activity of p190RhoGEF. Further studies on the specific contribution of the enhanced expression and activity of p190RhoGEF to the CD40-mediated functional outcome would help us to characterize the molecular mechanisms for the B cell maturation and differentiation.
We thank Drs. Wouter Moolenaar, Gary Bokoch, and Ben Margolis for providing valuable reagents, and Dr. Gary Koretzky for a critical review of the manuscript, as well as his valuable comments.
This work was supported in part by the Korea Science and Engineering Foundation through the Center for Cell Signaling Research at Ewha Womans University and the Basic Research Program Grant 1999-1-212-002-3 and by the Korea Research Foundation Grant KRF-2002-015-EP0083. Y.J.H. was supported in part by the Brain Korea 21 Program from the Korea Ministry of Education.
Abbreviations used in this paper: 2D, two-dimensional; F-actin, filamentous actin; GFP, green fluorescence protein; JNK, c-Jun N-terminal kinase; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; MS, mass spectrometry; WT, wild type; GEF, guanine nucleotide exchange factor; SSC-H, side scatter; FL1, fluorescence intensity channel 1.