Increased p190RhoGEF expression in activated B cells correlates with the induction of the plasma cell differentiation.

Abstract

Previously, we demonstrated that the p190 Rho guanine nucleotide exchange factor (p190RhoGEF) was induced following CD40 stimulation of B cells. In this study, we examined whether p190RhoGEF and a downstream effector molecule RhoA are required for B cell differentiation. Expression of p190RhoGEF positively correlated with the expression of surface markers and transcriptional regulators that are characteristic of mature B cells with plasma cell (PC) phenotypes. Moreover, either the overexpression of p190RhoGEF or the expression of a constitutively active RhoA drove cellular differentiation toward PC phenotypes. B cell maturation was abrogated in cells that overexpressed p190RhoGEF and a dominant-negative form of RhoA simultaneously. CD40-mediated maturation events were also abrogated in cells that overexpressed either dominant-negative p190RhoGEF or RhoA. Together, these data provide evidence that p190RhoGEF signaling through RhoA in CD40-activated B cells drives the induction of the PC differentiation.

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EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 44, No. 2, 138-148, February 2012
Copyright ⓒ 2012 by the The Korean Society for Biochemistry and Molecular Biology
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Increased p190RhoGEF expression in activated B cells
correlates with the induction of the plasma cell differentiation
Yun Jung Ha1, Ji Hye Jeong1, Yuna Park1
and Jong Ran Lee1,2,3,4
1Division of Life and Pharmaceutical Sciences
2Department of Life Science
College of Natural Sciences
3Center for Cell Signaling and Drug Discovery Research
Ewha Womans University
Seoul 120-750, Korea
4Corresponding author: Tel, 82-2-3277-3762;
Fax, 82-2-3277-3760; E-mail, jrlee@ewha.ac.kr
http://dx.doi.org/10.3858/emm.2012.44.2.009
Accepted 22 November 2011
Available Online 23 November 2011
Abbreviations: AID, activation-induced deaminase; Bcl-6, B-cell
lymphoma protein-6; Blimp-1, B lymphocyte-induced matura-
tion protein-1; CA, constitutively active; DN, dominant negative;
EV, empty vector; GC, germinal center; HA, hemagglutinin; IB,
immunoblot; IRF-4, interferon regulatory factor-4; MFI, mean flu-
orescence intensities; Pax5, paired-box-protein 5; PC, plasma
cell; PE, phycoerythrin; PerCP, peridinin chlorophyll protein;
p190RhoGEF, p190 Rho guanine nucleotide exchange factor;
XBP-1, X-box-binding protein-1
Abstract
Previously, we demonstrated that the p190 Rho gua-
nine nucleotide exchange factor (p190RhoGEF) was
induced following CD40 stimulation of B cells. In this
study, we examined whether p190RhoGEF and a down-
stream effector molecule RhoA are required for B cell
differentiation. Expression of p190RhoGEF positively
correlated with the expression of surface markers and
transcriptional regulators that are characteristic of ma-
ture B cells with plasma cell (PC) phenotypes.
Moreover, either the overexpression of p190RhoGEF
or the expression of a constitutively active RhoA drove
cellular differentiation toward PC phenotypes. B cell
maturation was abrogated in cells that overexpressed
p190RhoGEF and a dominant-negative form of RhoA
simultaneously. CD40-mediated maturation events
were also abrogated in cells that overexpressed either
dominant-negative p190RhoGEF or RhoA. Together,
these data provide evidence that p190RhoGEF signal-
ing through RhoA in CD40-activated B cells drives the
induction of the PC differentiation.
Keywords: antigens, CD40; ARHGAP35 protein, hu-
man; B-lymphocytes; CD40; cell differentiation;
p190RhoGEF; plasma cells; rhoA GTP-binding pro-
tein
Introduction
Many of the signaling molecules and transcription
factors that regulate B cell fate decisions have
been well characterized. Key transcriptional regu-
lators, including B lymphocyte-induced maturation
protein-1 (Blimp-1), X-box-binding protein-1 (XBP-1),
and interferon regulatory factor-4 (IRF-4), are re-
quired for plasma cell (PC) differentiation and func-
tion (Calame, 2001; Shapiro-Shelef and Calame,
2005). Blimp-1 is a major regulator of PC differ-
entiation and generation of Ab-secreting cells
(Turner et al., 1994; Shaffer et al., 2002, 2004;
Shapiro- Shelef et al., 2003). Blimp-1 suppresses
the transcriptional repressor paired- box-protein 5
(Pax5), which is essential for the commitment of
lymphoid progenitors to the B cell lineage, and also
functions in late B cell development and activation
by inducing activation-induced deaminase (AID)
expression (Delogu et al., 2006; Klein et al., 2006;
Nera et al., 2006). AID, a novel member of the
RNA editing cytidine deaminase family, is specifi-
cally expressed in germinal center (GC) B cells
and is essential for somatic hypermutation and iso-
type switching/class switch recombination (Gray et
al., 1994; Muramatsu et al., 2000; Xu et al., 2007).
Blimp-1 suppresses another transcriptional re-
pressor, B cell lymphoma protein-6 (Bcl-6), a major
regulator of GC B cells (Ye et al., 1997; Delogu et
al., 2006; Nera et al., 2006). Blimp-1 is thought to
act upstream of XBP-1, a transcriptional regulator
of the secretory pathway in PC (Reimold et al.,
2001). Recently, IRF-4 has also been identified as
a second major gene in PC differentiation (Klein et
al., 2006; Sciammas et al., 2006).

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B cell differentiation by p190RhoGEF expression 139
Figure 1. p190RhoGEF protein levels and changes after CD40 stimulation of B cells of various maturational stages. (A) Mouse splenic B cells (1 × 106/ml)
and the B cell lines WEHI 231, BAL17, CH12.LX (5 × 105/ml) were stimulated with isotype control Ig or anti-CD40 at a concentration of 1 μg/ml for 48 h.
(B) HEK 293T cells were transfected with plasmids carrying an empty vector (EV), WT, or DN p190RhoGEF (HA). (C) WEHI 231 B cells were stably trans-
fected with MIGR1 (EV), WT, or DN p190RhoGEF. Lysates were prepared from these cells, subjected to a SDS-PAGE (6% or 8% gels), transferred to ni-
trocellulose, and probed with an Ab specific for p190RhoGEF (A-C) or HA (B). Equal loading of the protein samples was demonstrated by anti-β actin IB
in (A). (D, E) Mouse splenic B cells (1 × 106/ml) were stimulated with control Ig or anti-CD40 at a concentration of 1 μg/ml. After 48 h of stimulation, cells
were fixed, permeabilized, and stained for endogenous p190RhoGEF, CD23, and IgD. The fluorescence intensities of cells stained with an isotype control
Ab or antiserum were all corrected (data not shown). The results from flow cytometry are presented as a dot plot, showing IgD versus CD23 positive cells.
For the cell populations in R1, R2, and R3 as indicated in (D), the population of cells (%) and the expression level of p190RhoGEF as mean fluorescence
intensity (MFI) in the absence (control) and presence of CD40 stimulation is presented in (E). The results shown are representative of three independent
experiments.
CD40 is an important cell surface receptor for
Ag-dependent and T helper cell-regulated terminal
differentiation of naïve B cells into Ab-secreting
plasma and memory cells. The molecular mecha-
nisms underlying the developmental coordination
of CD40 stimulation with Ig secretion has been well
characterized by many groups (Gray et al., 1994;
Kawabe et al., 1994; Benson et al., 2007). However,
the context in which CD40-CD154 signaling acts to
activate transcription factors that regulate B cell
fate decisions is not clearly understood.
Previously, we identified the p190 Rho guanine
nucleotide exchange factor (p190RhoGEF) as a
protein that is strongly induced in response to CD40
stimulation of B cells (Lee et al., 2003). We also
demonstrated that the expression of p190RhoGEF
is correlated with changes in cellular structure fol-
lowing CD40 stimulation in WEHI 231 B cells (Lee
et al., 2003). Furthermore, cells that transiently
overexpressed p190RhoGEF behaved as if they
expressed a constitutively active (CA) form of
RhoA (Q63L), whereas transient overexpression of
dominant negative (DN) forms of p190RhoGEF
(Y1003A) or RhoA (T19N) blocked the CD40-medi-
ated activation effects (Lee et al., 2003). These re-
sults suggest that there may exist a relationship
between p190RhoGEF, the morphological changes
in CD40-activated B cells, and additional B cell ma-
turation and differentiation events.
In the present study, we examined whether
p190RhoGEF expression is correlated with the ex-
pression of specific surface markers and transcrip-
tional regulators that are characteristic of both ma-
ture and differentiated B cells. We also examined
whether the activities of p190RhoGEF and RhoA
are required for CD40-mediated B cell maturation
and differentiation. Our results demonstrate that
the expression of p190RhoGEF correlates with the
expression of the cell surface markers CD138
(syndecan-1) and CXCR4, as well as the tran-
scription factors Blimp-1, XBP-1, and IRF-4. The
expression of these molecules was also induced
following CD40 stimulation, as well as by over-
expressing either wild type (WT) p190RhoGEF or a

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140 Exp. Mol. Med. Vol. 44(2), 138-148, 2012
Figure 2. Expression of p190RhoGEF, CD138, and B220 in B cells at various maturational stages. (A, B) Mouse splenic B cells (1 × 106/ml) were stimu-
lated with control Ig or anti-CD40 at a concentration of 1 μg/ml. After 72 h of stimulation, cells were fixed, permeabilized, and stained for endogenous
p190RhoGEF, B220, and CD138. The fluorescence intensities of cells stained with an isotype control Ab or antiserum were all corrected (data not shown).
The results from flow cytometry are presented as a dot plot, showing the B220 positive cells versus the CD138 positive cells. The data shown are repre-
sentative of three independent experiments. For the cell populations indicated in (A), expression of p190RhoGEF is presented as MFI in (B). Data are pre-
sented as the mean (n = 4) ± SD. (C, D) The B cell lines, BAL17, CH12.LX, and NS-1 were fixed, permeabilized, and stained for endogenous
p190RhoGEF, CD138, and B220. The results were obtained as described above. For the cell populations indicated in (C), expression of p190RhoGEF is
presented as MFI in (D). Data are presented as the mean (n = 3) ± SD.
CA form of RhoA (Q63L). Collectively, these data
suggest a specific contribution of p190RhoGEF to
CD40-mediated differentiation and that p190RhoGEF
may be a crucial component of T cell-dependent B
cell maturation and differentiation.
Results
Changes in expression of surface markers and
p190RhoGEF by CD40 stimulation
We previously reported that a 48 h stimulation of
CD40 leads to increases in p190RhoGEF expression
and causes characteristic structural changes in B
cells (Lee et al., 2003). Because the cellular struc-
ture of B cells changes during cell maturation, we
tested whether p190RhoGEF expression corre-
lates with stages in the maturation of B cells follow-
ing CD40 stimulation. Analysis of the protein levels
of p190RhoGEF in B cells of various maturational
stages also demonstrated that there was an in-
crease in p190RhoGEF protein levels following
CD40 stimulation of WEHI 231 and splenic B cells,
but not the mature B cell lines, BAL17 and
CH12.LX (Figure 1A). The specificity of the poly-
clonal Ab generated against p190RhoGEF was
characterized in HEK 293T cells transfected with
hemagglutinin (HA)-p190RhoGEF expression plas-
mids (Figure 1B) and in WEHI 231 B cells in which
p190RhoGEF was overexpressed (Figure 1C).
As B cell maturation progresses, expression of
CD23 is increased and expression of IgD is
decreased. We further analyzed expression of the
surface markers IgD and CD23. We used flow cy-

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B cell differentiation by p190RhoGEF expression 141
Figure 3. Expression changes of transcriptional regulators after CD40
stimulation and in response to p190RhoGEF and RhoA down-regulation
in B cells at various maturational stages. (A) cDNA was prepared from B
cells that were either untreated (control) or stimulated for 24 h with an-
ti-CD40 (1 μg/mL). (B) cDNA was prepared from BAL17 and NS-1 cells
that were stably transfected with MIGR1 (EV), p190RhoGEF (Y1003A) or
RhoA (T19N). The PCR products for p190RhoGEF, RhoA, Blimp-1,
XBP-1, IRF-4, and GAPDH were separated as described in “Methods.”
The data shown are representative of three independent experiments.
Values indicate normalization for GAPDH.
tometry to analyze whether CD40 stimulation
caused changes in the expression of IgD and
CD23. Changes in the expression level of IgD ver-
sus CD23 in mouse splenic B cells treated with an-
ti-CD40 are shown in Figure 1D. B cell population
was defined as three groups (R1: CD23low, R2:
CD23medium, R3: CD23low) based on the level of
CD23 expression. Results in Figure 1D and 1E
demonstrate that CD40 stimulation of these B cells
results in a change in maturation stage, indicated
as a percentage of cell population in R1-R3.
p190RhoGEF expression correlates to changes in
cellular maturation both in unstimulated control and
CD40 stimulation of splenic B cells (Figure 1E).
Expression of p190RhoGEF is correlated with the
expression of CD138 and transcriptional regulators
driving PC differentiation
To determine whether p190RhoGEF expression in-
duced by CD40-stimulated B cell activation is in-
volved in PC differentiation, we correlated the ex-
pression of p190RhoGEF with the expression of a
specific surface molecule, CD138, which has been
previously implicated as a PC marker (Calame,
2001; Shapiro-Shelef and Calame, 2005). B cell
population was separated by the expression level
of B220 and CD138 (R1: B220highCD138low, R2:
B220highCD138high, R3: B220lowCD138high). By flow
cytometry, we found that following 72 h of CD40
stimulation, the B cell population expressing CD138
on the surface increased from -7.4% to -20% (R2
and R3) in mouse splenic B cells (Figure 2A).
Accordingly, the expression of p190RhoGEF was
analyzed in the regions (R1, R2, and R3) sepa-
rated by the expression level of B220 and CD138.
As shown in Figure 2B, p190RhoGEF expression
increased in R2 and R3 in mouse splenic B cells
over the expression level in R1 both in unstimulated
controls and following CD40 stimulation. CD40
stimulation also increased p190RhoGEF expression
-2-fold in R1compared to the level of unstimulated
controls.
To directly correlate
p190RhoGEF with PC differentiation, we analyzed
the expression levels of B220 versus CD138 in the
mature B cell lines with PC phenotypes, BAL17
and CH12.LX, and in plasmacytoma NS-1 cells. As
shown in Figure 2C, populations demonstrating
distinct maturation stages differ slightly when
R1-R3 are compared. Further analysis of the ex-
pression of p190RhoGEF in these cell populations
demonstrated that p190RhoGEF expression is
much higher in cells that show more characteristics
of PC with higher population in R3 (CH12.LX and
NS-1 cells with higher population in R3 vs. BAL17
the expression of
cells with lower population in R3 (Figure 2D).
Because the transcriptional regulators, Blimp-1,
XBP-1, and IRF-4 are known to play key roles in
PC development (Turner et al., 1994; Reimold et
al., 2001; Shaffer et al., 2002; Shapiro-Shelef et al.,
2003; Shaffer et al., 2004; Klein et al., 2006;
Sciammas et al., 2006), we next examined whether
p190RhoGEF expression also correlated with the
expression of these transcriptional regulators. The
expression of Blimp-1, XBP-1, and IRF-4 was com-
pared with the CD40-induced changes in these
regulators compared to the unstimulated controls.
As shown in Figure 3A, CD40 stimulation greatly
enhanced the expression of these transcripts in
WEHI 231 and splenic B cells. In contrast, the ex-
pression of Blimp-1, XBP-1, and IRF-4 is high in
BAL17 and CH12.LX cells, independent of CD40
stimulation (Figure 3A).
Similarly, p190RhoGEF expression was con-
stitutively high in BAL17 and CH12.LX cells, but
was induced in WEHI 231 and splenic B cells after

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142 Exp. Mol. Med. Vol. 44(2), 138-148, 2012
Figure 4. Direct effects of manipulating the activity of p190RhoGEF and RhoA on B cell maturation and downstream transcriptional regulators. WEHI 231
B cells were stably transfected with MIGR1 (EV), WT p190RhoGEF, DN p190RhoGEF (Y1003A), CA RhoA (Q63L), DN RhoA (T19N), or a combination of
WT p190RhoGEF and DN RhoA (T19N). (A) cDNA was prepared from these stably transfected cells. The PCR products for p190RhoGEF, RhoA, Blimp-1,
XBP-1, IRF-4, Bcl-6, AID, and GAPDH were separated as described in “Methods.” The data shown are representative of three independent experiments.
Values indicate normalization for GAPDH. (B) These stably transfected cells were removed from the cultures and then washed, counted, and recultured in
fresh medium for 24 h at 1 × 106 cells/ml. Some EV-transfected cells were also incubated with a medium alone (black bars) or with a combination of
CD40 and IL4 as positive controls (white bars). Supernatants were harvested and serially diluted, and secreted Ig was measured by anti-IgM and anti-IgG
ELISA as described in “Methods.” Values of the optical densities at 490 nm in triplicate supernatant samples were collected in each independent
experiment. Data are presented as the mean of three independent experiments (n = 3) ± SD, *P ＜ 0.05, **P ＜ 0.01, compared with cells transfected
with EV. (C, D) These stably transfected cells were fixed, stained with anti-CD138 or anti-CXCR4, and analyzed by standard flow cytometry. The fluo-
rescence intensities of cells stained with an isotype control Ab or antiserum were all corrected (data not shown).The bar graphs show the expression of
CD138 (C) or CXCR4 (D), as indicated by MFI. Data are presented as the mean of three independent experiments (n = 3) ± SEM, **P ＜ 0.01, ***P ＜
0.001, compared with cells transfected with EV.
CD40 stimulation (Figures 1A and 3A). These re-
sults strongly suggest that the expression of
p190RhoGEF mediates the induction of key tran-
scriptional regulators, Blimp-1, XBP-1, and IRF-4,
which regulate B cell maturation and differentiation.
Because p190RhoGEF specifically activates
RhoA (Gebbink et al., 1997; van Horck et al.,
2001), we next examined whether the activities of
p190RhoGEF and its downstream effector RhoA
are required during PC differentiation. As shown in
Figure 3B, the expression levels of the major
transcriptional regulators of PC maturation and
differentiation, Blimp-1 and XBP-1 were greatly
reduced in BAL17 cells transfected with a DN form
of p190RhoGEF (Y1003A) or RhoA (T19N). These
results strongly suggest that one function of
p190RhoGEF is to maintain the expression of key
transcriptional regulators of B cell maturation and
differentiation. In contrast, the DN form of either
p190RhoGEF or RhoA had no effect on the
expression of these transcription factors in NS-1
cells (Figure 3B). These results also suggest that
p190RhoGEF functions in maturing B cells, but not
in completely differentiated plasmacytoma cells.