Homeostatic cell-cycle control by BLyS: Induction of cell-cycle entry but not G1/S transition in opposition to p18INK4c and p27Kip1.
ABSTRACT Cell-cycle entry is critical for homeostatic control in physiologic response of higher organisms but is not well understood. The antibody response begins with induction of naive mature B cells, which are naturally arrested in G(0)/G(1) phase of the cell cycle, to enter the cell cycle in response to antigen and cytokine. BLyS (BAFF), a cytokine essential for mature B cell development and survival, is thought to act mainly by attenuation of apoptosis. Here, we show that BLyS alone induces cell-cycle entry and early G(1) cell-cycle progression, but not S-phase entry, in opposition to the cyclin-dependent kinase inhibitors p18(INK4c). Independent of its survival function, BLyS enhances the synthesis of cyclin D2, in part through activation of NF-kappaB, as well as CDK4 and retinoblastoma protein phosphorylation. By convergent activation of the same cell-cycle regulators in opposition to p18(INK4c), B cell receptor signaling induces cell-cycle entry and G(1) progression in synergy with BLyS, but also DNA replication. The failure of BLyS to induce S-phase cell-cycle entry lies in its inability to increase cyclin E and reduce p27(Kip1) expression. Antagonistic cell-cycle regulation by BLyS and p18(INK4c) is functionally linked to apoptotic control and conserved from B cell activation in vitro to antibody response in vivo, further indicating a physiologic role in homeostasis.
- SourceAvailable from: Matthew H Cato[Show abstract] [Hide abstract]
ABSTRACT: BAFF is a soluble factor required for B cell maturation and survival. BAFF-R signals via the noncanonical NF-κB pathway regulated by the TRAF3/NIK/IKK1 axis. We show that deletion of Ikk1 during early B cell development causes a partial impairment in B cell maturation and BAFF-dependent survival, but inactivation of Ikk1 in mature B cells does not affect survival. We further show that BAFF-R employs CD19 to promote survival via phosphatidylinositol 3-kinase (PI3K), and that coinactivation of Cd19 and Ikk1 causes a profound block in B cell maturation at the transitional stage. Consistent with a role for PI3K in BAFF-R function, inactivation of PTEN mediates a partial rescue of B cell maturation and function in Baff(-/-) animals. Elevated PI3K signaling also circumvents BAFF-dependent survival in a spontaneous B cell lymphoma model. These findings indicate that the combined activities of PI3K and IKK1 drive peripheral B cell differentiation and survival in a context-dependent manner.Cell Reports 11/2013; · 7.21 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: B-lymphocytes are integral to host defense against microbial pathogens and are associated with many autoimmune diseases. The B-cell receptor implements B-cell self-tolerance based on the antigen specificity, and B-cell-activating factor receptor (BAFF-R) imposes homeostatic control. While shaping the repertoire, the immune tolerance process also culls mature B cells into distinct populations. The activation response of B cells is tailored to the type of pathogen attack and is facilitated by T-cell help via CD40/CD40L interaction and/or innate cell help via toll-like receptors in conjunction with BAFF receptors and ligands. Activated effector B cells not only produce antibodies, but also produce a variety of cytokines to enhance and suppress the immune response. Not surprisingly, B cells play multiple roles in both humoral and cellular immune responses during infection and autoimmune pathogenesis. Here, we discuss how gene expression and signaling networks regulate peripheral B-cell tolerance, B-cell effector functions and emerging therapies targeting B-cell signaling in autoimmune diseases.Immunologic Research 11/2013; · 3.53 Impact Factor
Homeostatic cell-cycle control by BLyS: Induction of
cell-cycle entry but not G1?S transition in opposition
to p18INK4cand p27Kip1
Xiangao Huang*†, Maurizio Di Liberto*†, Adam F. Cunningham‡, Lin Kang*, Shuhua Cheng§, Scott Ely*, Hsiou-chi Liou§¶,
Ian C. M. MacLennan‡, and Selina Chen-Kiang*¶?
Departments of *Pathology and§Medicine and¶Graduate Program in Immunology and Microbial Pathogenesis, Weill Medical College of Cornell University,
1300 York Avenue, New York, NY 10021; and‡University of Birmingham Medical School, Birmingham B15 2TT, United Kingdom
Edited by Arthur Weiss, University of California, San Francisco, CA, and approved November 4, 2004 (received for review August 18, 2004)
Cell-cycle entry is critical for homeostatic control in physiologic
response of higher organisms but is not well understood. The
antibody response begins with induction of naı ¨ve mature B cells,
which are naturally arrested in G0?G1 phase of the cell cycle, to
enter the cell cycle in response to antigen and cytokine. BLyS
(BAFF), a cytokine essential for mature B cell development and
we show that BLyS alone induces cell-cycle entry and early G1
cell-cycle progression, but not S-phase entry, in opposition to the
cyclin-dependent kinase inhibitors p18INK4c. Independent of its
survival function, BLyS enhances the synthesis of cyclin D2, in part
through activation of NF-?B, as well as CDK4 and retinoblastoma
protein phosphorylation. By convergent activation of the same
cell-cycle regulators in opposition to p18INK4c, B cell receptor
signaling induces cell-cycle entry and G1 progression in synergy
with BLyS, but also DNA replication. The failure of BLyS to induce
S-phase cell-cycle entry lies in its inability to increase cyclin E and
reduce p27Kip1expression. Antagonistic cell-cycle regulation by
BLyS and p18INK4cis functionally linked to apoptotic control and
conserved from B cell activation in vitro to antibody response in
vivo, further indicating a physiologic role in homeostasis.
BAFF ? cyclin D2 ? cyclin E ? cyclin-dependent kinase ? B cell receptor
Our understanding of this process has been limited by a lack of
experimental cellular systems that can be manipulated easily in
vivo and ex vivo. Naı ¨ve mature B cells are naturally arrested in
the G0?G1phase of the cell cycle. They can be induced to enter
the cell cycle in response to antigen and cytokine stimulation,
and after clonal expansion they differentiate terminally to
G1-arrested antibody-secreting plasma cells. Each successive
step is controlled by the cell cycle in concert with apoptosis, and
differentiation stage-specific B cells can be identified by cell
surface markers and isolated for ex vivo analysis. The antibody
response, therefore, is an exceptional mammalian system for
elucidating cell-cycle control of the timing and magnitude of
In mammalian cells, cytokines and growth factors regulate
cell-cycle entry and G1to S phase cell-cycle progression mainly
by modulating the balance between positive cell-cycle regulators
[(cyclins and cyclin-dependent kinases (CDKs)] on the one hand
and CDK inhibitors (CDKIs) on the other (1). One specific
CDKI, p18INK4c(p18) (2, 3), is regulated by IL-6 (4) and is
essential for the antibody response. p18 is required for G1
cell-cycle arrest and terminal differentiation of antibody-
secreting plasma cells (5). It also may control cell-cycle entry at
the beginning of an antibody response, because it attenuates B
cell proliferation before and after immunization and in mito-
genic stimulation in vitro (5, 7, 32). Moreover, p18-mediated
cell-cycle control is functionally linked to homeostasis, as indi-
egulation of cell-cycle entry and exit critically controls
homeostasis in physiologic responses of higher organisms.
cated by the acceleration of apoptosis of nonsecreting plasma-
cytoid cells in the absence of p18 (5).
BLyS (BAFF) is a cytokine of the tumor necrosis factor family
(8, 9), whose receptors (BR3, BCMA, and TACI) are expressed
nearly exclusively on B cells (10–12). It is required for mature B
cell development (12–15) and plasma cell survival (16), and it
promotes the antibody response (17, 18) and Ig class switch
recombination (19). A role for BLyS in the development of
autoimmunity (20, 21) and the fatal plasma cell cancer, multiple
by attenuating apoptosis (18, 24) regardless of the cell-cycle
status (18), presumably through activation of two NF-?B path-
ways (18, 25–27) and the downstream antiapoptotic Bcl-2 and
Bcl-xL genes (18, 26, 28). Although it is generally assumed that
attenuation of apoptosis underlies the diverse biological func-
tions of BLyS, other possibilities have not been ruled out. BLyS
alone does not induce S-phase cell-cycle entry (18). However,
cyclin D2, the major D-type cyclin expressed in B cells and
activated in B cell receptor (BCR) signaling (29, 30), is a target
of NF-?B activation (31). This knowledge raises the possibility
that BLyS may induce individual G1cell-cycle regulators such as
cyclin D2, although by a means that is insufficient to induce S
phase entry. In this way, BLyS would cooperate with p18 in
homeostatic control of B cell activation by regulating both the
cell cycle and apoptosis.
To understand cell-cycle control of the antibody response
better, we investigated the control of cell-cycle activation by p18
and BLyS in BCR signaling in vitro and in the T cell-independent
antibody response in vivo. We present direct evidence that in
cell-cycle entry and mid-G1progression. Cell-cycle activation by
BLyS is attenuated by CDKIs, p18 in early G1and p27 in late G1.
Both BLyS and p18 are required for optimal B cell survival
during antigen stimulation in vitro and in the antibody response
in vivo, further suggesting cooperative homeostatic cell-cycle
control by BLyS and p18.
Materials and Methods
Mice and Isolation of Resting B Cells. p18?/?and p18?/?mice (32)
(7–11 weeks, age-matched) were immunized i.p. with 10 ?g of
4-hydroxy-3-nitrophenylacetyl (NP) coupled to Ficoll (NP-
Ficoll) (24:1; Biosearch) in 250 ?l of PBS, alone or together with
This paper was submitted directly (Track II) to the PNAS office.
Freely available online through the PNAS open access option.
Abbreviations: BCR, B cell receptor; CDK, cyclin-dependent kinase; CDKI, CDK inhibitor;
CFSE, carboxyfluorescein diacetate succinimidyl ester; NP, 4-hydroxy-3-nitrophenylacetyl;
NP-Ficoll, NP coupled to Ficoll; Rb, retinoblastoma protein; pS-Rb, phosphorylation of
serine on Rb.
†X.H. and M.D. contributed equally to this work.
?To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2004 by The National Academy of Sciences of the USA
www.pnas.org?cgi?doi?10.1073?pnas.0406111101 PNAS ?
December 21, 2004 ?
vol. 101 ?
no. 51 ?
a daily i.p. injection of 10 ?g of BLyS or PBS. p27-deficient
(p27?51/?51) mice (33) were kindly provided by Andrew Koff
(Memorial Sloan–Kettering Cancer Center, New York). Bcl-2
transgenic mice (Em?-bcl-2–22) were purchased from The
Jackson Laboratory. High-density (resting B) and low-density
(activated B and plasma) cells were isolated from splenocytes
from the 60–70% and 50–60% interfaces of a discontinuous
Percoll gradient, respectively (18). The resting B cells were
?96% pure based on the presence of B220, CD19, and IgM, and
the absence of CD3.
B Cell Activation in Vitro. Resting B cells were cultured (4 ? 105
cells?ml) in RPMI medium 1640 containing 10% heat-
inactivated FCS (HyClone) in the absence or presence of goat
together with recombinant soluble human BLyS (50 ng?ml) as
described in ref. 18. HIV-1 transactivator of transcription-cyclin
E (34) was added to cultures together with a lipopolysaccharide
inhibitor polymyxin B sulfate (200 units?ml) (Sigma), in the
presence or absence of BLyS (30 ng?ml). Apoptotic cells were
characterized with the annexin V FITC Apoptosis Detection Kit
(Calbiochem). Flow cytometry was performed to determine the
expression of CD21, CD23, and CD86 using FITC-anti-mouse
CD21?CD35, phycoerythrin-anti-mouse CD23 (Fc? RII), and
phycoerythrin-anti-mouse CD86 (B7–2) (Pharmingen), and
changes in cell size by forward scattering analysis. Cell shape was
visualized by phase contrast microscopy using a Zeiss Axioplan
Analysis of BrdUrd Uptake and Carboxyfluorescein Diacetate Succin-
imidyl Ester (CFSE) Labeling. BrdUrd (5-bromo-2-deoxyuridine)
uptake was determined by flow cytometry as described in ref. 18,
in resting B cells after incubation with BrdUrd (5 ?g?ml; Sigma)
for 2 h or in low-density splenic B cells isolated from NP-Ficoll-
immunized mice 2 h after i.p. injection of BrdUrd (50 mg per kg
of body weight). Labeling resting B cells with CFSE (Molecular
Probes) and analysis of cell division by the CFSE dilution by flow
cytometry were performed as described in ref. 35. The CFSE-
labeled cells were cultured in RPMI medium 1640 (4 ? 105cells
per ml) in the absence or presence of anti-IgM (5 ?g?ml) or daily
addition of BLyS (50 ng?ml), or both.
Immunoblotting. Whole-cell lysates were prepared from viable
resting B cells before and after in vitro incubation in a buffer
containing 300 mM NaCl, 20 mM Hepes (pH 7.9), 0.2% Nonidet
1 ?g?ml pepstatin, 1 ?g?ml leupeptin, 2 mM sodium orthovana-
date, 10 mM ?-glycerol phosphate, and 1 mM PMSF. Proteins
were resolved on a 4–12% NuPAGE gel (Invitrogen) and
analyzed with one of the following antibodies: mouse monoclo-
nal antibody to human retinoblastoma (Rb) (Pharmingen) or
human CDK6 (Cell Signaling Technology, Beverly, MA); rabbit
polyclonal antibodies to pSer807?811 of human Rb (Cell Sig-
naling Technology), mouse CDK4, mouse cyclin D2, mouse p27,
human CDK2, or rat cyclin E; or goat anti-human actin (all
from Santa Cruz Biotechnology). Signals were developed with
the enhanced chemiluminescence system (ECL, Amersham
Real-Time RT-PCR. Total RNA was isolated from resting B cells
before and after in vitro incubation by using the TRIzol reagent
(Invitrogen). The first strand cDNA was synthesized by using
SuperScript III (Invitrogen) and subjected to real-time RT-PCR
by using the Assays-on-Demand gene expression mixes specific
for cyclin D2 and 18S ribosomal RNA and the TaqMan Uni-
versal PCR Master Mix (Applied Biosystems). Reactions were
carried out in the ABI PRISM 7900 HT Sequence Detection
System (Applied Biosystems). The relative amount of products
was determined by the comparative threshold cycle method
according to the manufacturer’s instructions.
p18INK4cand BLyS Antagonistically Regulate the Cell Cycle and Coop-
eratively Control Homeostasis. To understand cell-cycle control of
the antibody response, we investigated the control of cell-cycle
entry by p18 and BLyS in BCR signaling in mouse resting splenic
B cells. Stimulation of BCR by cross-linking with anti-IgM [3–10
?g?ml, or molar equivalents of the F(ab?)2fragment] induced
DNA replication in a dose-dependent manner by 45 h, as
determined by pulse-labeling (2 h) with BrdUrd (Fig. 1 A and B).
Removal of p18 greatly enhanced DNA replication (Fig. 1B),
demonstrating that p18 is required to attenuate cell-cycle entry
in BCR signaling.
However, p18-deficient (p18?/?) B cells were consistently
more apoptotic than p18?/?B cells, before and after stimulation
with anti-IgM, even at a limiting concentration (5 ?g?ml) (Fig.
1C). They also were less efficiently protected by BLyS (Fig. 1C).
Because there are no appreciable differences in the proportions
among resting B cell subsets between p18?/?and p18?/?mice
(X.H., M.D., and S.C.-K., unpublished data), p18 and BLyS may
protect mature B cells from apoptosis through nonredundant
mechanisms and cooperate in homeostatic control in BCR
To address this possibility, we confirmed that p18 attenuated
the cell cycle in BCR signaling by BrdUrd pulse-labeling; twice
as many p18?/?as p18?/?B cells were in the S phase by day 4
of anti-IgM stimulation (Fig. 1D). Because p18 inhibits CDK4
and CDK6 in early G1 (2, 3), S-phase entry apparently was
accelerated when the threshold for CDK4?6 activation was
lowered by the removal of p18. As expected (18), BLyS alone did
not induce DNA replication (Fig. 1D). However, it profoundly
increased the proportion of replicating p18?/?and p18?/?B cells
in anti-IgM costimulation. This increase is not likely due to
preferential protection of replicating B cells by BLyS, because
anti-IgM activated B cells were less well protected by BLyS (Fig.
1C). Rather, these results suggest that BLyS enhances G1?S
transition in BCR signaling in opposition to p18.
Cell division in BCR signaling was similarly accelerated by the
absence of p18 because substantially more p18?/?than p18?/?
cells had divided once or twice in 4 days of anti-IgM stimulation
as assessed by the dilution of CFSE (Fig. 1E). BLyS markedly
increased the proportion of B cells that had completed at least
two cell divisions in anti-IgM costimulation, and this increase
was amplified in p18?/?B cells (Fig. 1E). However, accelerated
division of p18?/?B cells seems to be counterbalanced by
enhanced apoptosis, because live p18?/?and p18?/?B cells
accumulated comparably in anti-IgM stimulation and in co-
stimulation with anti-IgM and BLyS, albeit to higher levels. (Fig.
1F). Considering the substantial increases in DNA replication
and cell division in BLyS and anti-IgM costimulation (Fig. 1 D
and E), these results cannot be explained solely by a survival
function of BLyS. BLyS therefore may have additional functions
acting in concert with p18 to control homeostasis of mature B
cells in BCR signaling.
BLyS Induces Cell-Cycle Entry in Opposition to p18INK4cThrough
Activation of Cyclin D2 and CDK4. Activation of the cell cycle by
BLyS without inducing G1?S transition is the most likely of these
functions. Closer inspection of resting B cells within 24 h of BLyS
stimulation revealed a change in cell shape, from spherical to
by a modest increase in cell size based on forward-scattering
analysis in flow cytometry (see Fig. 6, which is published as
supporting information on the PNAS web site). Moreover, BLyS
augmented CD23 expression as reported in ref. 36 (Fig. 6) and
induced an early B cell activation marker, CD86, although also
www.pnas.org?cgi?doi?10.1073?pnas.0406111101Huang et al.
less compared with that in anti-IgM stimulation (Fig. 2A).
Neither the change in cell shape and size nor the induction of
CD23 and CD86 by BLyS requires p18 (Fig. 2A and data not
shown). Thus, BLyS independently induces cell growth and
cellular differentiation characteristic of early B cell activation by
BCR stimulation. Together with the ability of BLyS to accelerate
S-phase entry and cell division in BCR signaling (Fig. 1 D and
E), these results suggest that in addition to attenuation of
and its catalytic partners, CDK4 and CDK6, by BLyS and p18 near
the onset of DNA replication on day 2. As a functional assay, we
determined CDK4?6-specific phosphorylation of Ser807/811on Rb
(pS-Rb) (37, 38), which is indicative of early G1cell-cycle progres-
sion. Cyclin D2 and CDK4 were modestly expressed in freshly
isolated resting B cells and reduced to an undetectable level during
incubation in the medium. They were increased coordinately by
anti-IgM stimulation to modestly activate pS-Rb (Fig. 2B, lanes
1–3). Importantly, BLyS similarly elevated the levels of cyclin D2
and CDK4, as well as Rb. This increase led to greater phosphor-
ylation of Rb than BCR signaling based on the ratios of pS-Rb to
Rb (Fig. 2B, lane 4). Costimulation with BLyS and anti-IgM
5). This enhancement was not due to preservation or amplification
of a specific subset of resting splenic B cell (T1, T2, marginal, and
follicular), because the proportions among them were largely
maintained in the presence of BLyS, while shifting in the absence
indicates that CDK4 activation is specific and may be rate-limiting
for serine phosphorylation of Rb. Collectively, these results dem-
onstrate that BLyS alone induces pS-Rb by coordinated activation
of cyclin D2 and CDK4 and that through convergent activation of
these positive early G1cell-cycle regulators BLyS and BCR signal-
ing synergistically induces G1cell-cycle progression.
The absence of p18 profoundly enhanced pS-Rb in BCR and
BLyS signaling, alone or together (Fig. 2B, lanes 6–10). This
enhancement was accompanied by a striking increase in the
levels of cyclin D2, CDK4, Rb, and even CDK6 in BLyS
signaling. Because DNA replication in p18?/?B cells on day 2
was modest in BCR signaling and barely detectable in BLyS
signaling, these increases were restricted to G1(Fig. 1B and data
not shown). Given that the p18 function is dependent on CDK4
and not CDK6 (39), enhanced pS-Rb in the absence of p18 is
mediated mainly by CDK4. Thus, p18 suppresses pS-Rb by
inhibiting both the CDK4 catalytic activity and the synthesis of
cyclin D2 and CDK4 proteins in either BCR or BLyS signaling.
BLyS and p18 therefore antagonistically control cell-cycle entry
through opposing regulation of cyclin D2, CDK4, and Rb
BLyS Activates Cyclin D2 Independent of Its Survival Function Through
NF-?B. To verify that BLyS induces cell-cycle entry independent
of its survival function, the BLyS response was further charac-
Bcl-2. Cyclin D2 protein and pS-Rb were enhanced by BLyS
stimulation in these cells, although to a lesser extent than their
wild-type littermate controls (Fig. 2C). The basis for the blunted
BLyS response in B cell-overexpressing Bcl-2 is unknown, but
BLyS clearly regulates cyclin D2 and pS-Rb independent of its
survival function. BLyS activates NF-?B (18), which regulates
both cyclin D2 and CD86 (31, 34). Real-time RT-PCR analysis
revealed that the cyclin D2 RNA was expressed in resting B cells,
serum levels (0.5–10%), and then increased by 12 h BLyS
with indicated anti-IgM concentration for 24 (Upper) and 45 (Lower) h. The horizontal bars represent the average of three independent analyses. (C) The
percentage of apoptotic (annexin V??PI?) resting p18?/?and p18?/?B cells stimulated with anti-IgM (5 ?g?ml) or BLyS (50 ng?ml), or both, for 48 h. The error
bars indicate three independent analyses. (D and E) BrdUrd uptake (D) and CFSE dilution (E) of cells incubated as in C for 4 days. The CFSE level in undivided cells
(triangle), the reduction of CFSE by successive cell division (dotted line, right to left), and the percentage of cells in each cell division (numbers in each panel,
top to bottom) are indicated. (F) The number of live cells was determined by trypan blue staining in triplicate cultures. The results in C–F are representative of
four independent experiments.
Antagonistic cell-cycle control by p18 and BLyS. (A) Cell-cycle activation by BCR stimulation. p18 inhibits CDK4?6 in early G1, p27 inhibits cyclin E?CDK2
Huang et al.
December 21, 2004 ?
vol. 101 ?
no. 51 ?
stimulation (2-fold in p18?/?cells and 3- to 4-fold in p18?/?cells)
to levels comparable with those before incubation (Fig. 2D).
These results confirmed that BLyS activates cyclin D2 synthesis.
The greater increase in the cyclin D2 protein by BLyS in the
absence of p18 (Fig. 2B) is therefore due in part to enhanced
RNA synthesis, and not solely to stabilization of the cyclin D2
protein in complex with CDK4. The time course parallels NF-?B
activation in resting B cells and its diminution in vitro (18),
consistent with NF-?B mediating the in vivo growth factor
signals including BLyS for transcriptional activation of cyclin D2
in resting B cells. Indeed, inhibition of NF-?B activation with
SN50, a cell-permeable peptide that blocks nuclear translocation
of p50 (NF-?B1)-containing complexes, ablated the induction of
cyclin D2 RNA by BLyS (Fig. 2D). BLyS therefore enhances
cyclin D2 synthesis, at least in part, through NF-?B-mediated
transcriptional activation independent of the BLyS survival
BLyS and p18INK4cAntagonistically Regulate Cyclin D2 and the Cell
Cycle in the Antibody Response. The BLyS action is concentration-
dependent (18). We therefore addressed the physiologic rele-
vance of cell-cycle and homeostatic controls by p18 and BLyS in
the antibody response to NP-Ficoll, a well characterized T
cell-independent antigen. BrdUrd uptake and splenic B cell
expansion after immunization was greater in p18-deficient mice
than in their wild-type counterparts (Fig. 3 A and B). However,
although administration of BLyS increased the number of
splenic B cells as expected, the proportional increase in the
p18-deficient mice was blunted, reaching a level similar to that
of the BLyS-treated wild-type mice despite a higher baseline
(Fig. 3B). These results suggest that p18 attenuates the cell cycle
and cooperates with BLyS in homeostatic control of splenic B
cell expansion in the NP antibody response, as in BCR signaling
in vitro (Fig. 1 B and F).
pS-Rb was detected along with a low level of cyclin D2 and
strong CDK4 expression in splenic B cells at the height of
replication on day 4 after immunization (Fig. 3C, lane 1). This
finding indicates that CDK4 is activated by multiple in vivo
signals, but induction of cyclin D2 is more specific and is
rate-limiting for Rb phosphorylation in the antibody response.
Consistent with this possibility, the cyclin D2 and Rb protein
3C, lane 2), more prominently in p18?/?splenic B cells than in
their wild-type counterparts (Fig. 3C, lanes 3 and 4). Again, in
keeping with regulation of CDK4 in vivo being more complex,
the CDK4 level remained unchanged (Fig. 3C, lanes 1–4). BLyS
and p18, therefore, antagonistically regulate cyclin D2 synthesis
and Rb phosphorylation in the antibody response. The remark-
able conservation of antagonistic cell-cycle regulation and co-
operative homeostatic control by BLyS and p18, from cell-cycle
entry in BCR signaling in vitro to asynchronously replicating
splenic B cells in antigen-specific antibody response, indicate
their physiologic role.
BLyS Fails to Induce G1?S Cell-Cycle Transition Due to Its Inability to
Increase Cyclin E or Reduce p27.Wethenaddressedthebasisforthe
failure of BLyS to induce G1?S cell-cycle transition. Activation
accompanied by both an increase in cyclin E, which is necessary
for S-phase entry, and a decrease in p27Kip1, which inhibits the
catalytic activity of cyclin E?CDK2 (1) (Fig. 4A). This finding is
consistent with observations in total splenic B cells (34, 40). In
B cells cultured in the absence (shaded area) or presence (red open area) of
D2, CDK4, and CDK6, in cells cultured in A for 0 and 48 h (B) and in resting B
cells isolated from Bcl-2 transgenic mice (Tg) and wild-type controls (wt) after
stimulation with BLyS for 48 h (C). The ratio of pS-Rb to total Rb was deter-
mined by phosphoimaging (B). The actin level was used for a loading control.
p18?/?and p18?/?B cells (?16), after 16-h incubation in medium containing
0.5% serum (0), plus an additional 12 h in the absence (?) or presence (?) of
BLyS (Left) or an additional 6 h in the absence (?) or presence (?) of BLyS plus
serum levels. The data are representative of four independent experiments.
BLyS induces cell-cycle entry in opposition to p18. (A) Phase contrast
response. (A) FACS analysis of BrdUrd uptake in activated splenic B cells of
p18?/?and p18?/?mice on days (d) after NP-Ficoll immunization. (B) The
number of total splenic B cells in p18?/?and p18?/?mice on day 5 after
immunization, with administration of PBS (circle) or BLyS (triangle). (C) Im-
munoblot of pS-Rb, Rb, CDK4, and cyclin D2 in activated p18?/?and p18?/?
splenic B cells isolated on day 4 after immunization, with or without BLyS
administration. The results are representative of three independent
BLyS and p18 antagonistically regulate the cell cycle in the antibody
www.pnas.org?cgi?doi?10.1073?pnas.0406111101 Huang et al.
striking contrast, BLyS signaling modestly reduced cyclin E and
sustained p27 expression, although maintaining CDK2 expres-
sion as BCR signaling (Fig. 4A). BLyS did not, however,
interfere with the regulation of cyclin E and p27 by anti-IgM in
This set of findings poses two related questions. The first is
whether p27 inhibits G1?S transition after cell-cycle entry is
induced by BLyS. The second is whether cyclin E is required for
the initial G1?S transition in response to BLyS, considering that
in mouse embryonic fibroblasts cyclin E is required for cell-cycle
reentry after serum starvation but not for cell-cycle progression
(41). Transduction of a HIV-1 transactivator of transcription-
cyclin E protein (34) into resting B cells led to a dose-dependent
increase of S-phase cells (3% at 33 nM and 6% at 66 nM). Cyclin
E therefore can bypass G1activation and induce S-phase entry
in resting B cells (Fig. 4B). Importantly, stimulation of HIV-1
transactivator of transcription-cyclin E transduced cells with
BLyS profoundly increased the proportion of S-phase cells
(25%) (Fig. 4B), demonstrating an extraordinary complemen-
tation between induction of cell-cycle entry by BLyS and ex-
pression of cyclin E. This complementation was amplified in
p27-deficient B cells, as evidenced by the heightened proportion
of S-phase cells (40%) (Fig. 4B). Thus, the failure of BLyS to
drive cells past G1?S transition lies in its inability to increase
cyclin E and reduce p27.
induces cell-cycle entry and early G1progression and that p18,
an early G1CDKI required for plasma cell differentiation, also
attenuates cell-cycle entry in BCR signaling. BLyS and p18
function antagonistically in G1cell-cycle control and coopera-
tively in homeostatic control of B cell activation and the antibody
response. On this basis, we propose a ‘‘homeostatic cell-cycle
control’’ model (Fig. 5).
In this model, BLyS and BCR signaling cooperatively control
diverge in the control of G1?S transition through differential
regulation of cyclin E and p27. In addition, cell-cycle control by
BLyS and p18 is linked functionally to apoptotic control. BLyS
induces resting B cells to enter the cell cycle by activating the
synthesis of cyclin D2, CDK4, and Rb, but not the S phase due
to insufficient cyclin E and sustained p27 expression. Conse-
quently, BLyS signaling does not lead to the release of a putative
apoptotic factor, thereby preserving its full survival function. By
contrast, BCR signaling induces sequential phosphorylation of
Rb by cyclin D2-CDK4?6 and cyclin E-CDK2, G1?S transition
and the release of the apoptotic factor. In BLyS and BCR
costimulation, synergistic phosphorylation of Rb by cyclin D2-
CDK4 accelerates early G1progression and facilitates cyclin E
CDK2 phosphorylation and the release of the putative apoptotic
factor. This G1 acceleration, in turn, compromises the BLyS
survival function, before and during G1?S transition. p18 effec-
tively attenuates G1 progression by dual mechanisms, direct
inhibition of the CDK4 activity and suppression of cyclin D2 and
CDK4 synthesis, and attenuates the release of the putative
antiapoptotic factor. In this way, the BLyS survival function is
modulated by the control of cell-cycle entry and G1?S transition
according to the strength of BCR and BLyS signaling and the
expression of p18 and p27.
This model is supported by the following findings: (i) the p18
function is CDK4-dependent (39); (ii) cyclin D2 is the major D
cyclin expressed in B cells (30) and is required for cell-cycle
activation early in BCR signaling in vitro (29); (iii) cyclin D2 is
transcriptionally activated by BLyS by means of NF-?B activa-
tion but independent of the BLyS survival function (Fig. 2); (iv)
attenuation of apoptosis by BLyS is dose-dependent but cell-
cycle-independent (18); and (v) cell-cycle and homeostatic con-
trols of B cell expansion by BLyS and p18 is conserved from B
cell activation in vitro (Figs. 1 and 2) to the antibody response
cyclin E in resting p18?/?and p18?/?B cells cultured for 0 or 48 h as indicated.
The level of actin was used for a loading control. (B) FACS analysis of BrdUrd
uptake in resting p27?/?and p27?51/?51B cells cultured in the absence or
presence of BLyS, 33 nM (1?) or 66 nM (2?) of HIV-1 transactivator of
transcription cyclin E (E), or both, for 48 h in the presence of the lipopolysac-
charide inhibitor. Similar results were obtained from five independent exper-
iments regardless of the presence of the lipopolysaccharide inhibitor.
CDK6 by the INK4 CDKIs, and CDK2 by the Cip?Kip CDKIs. (B Top) BLyS induces
of Rb and early G1 progression, but not S-phase entry. The BLyS survival
the CDK4 activity and the induction of cyclin D2 and CDK4, p18 attenuates
pS-Rb and G1progression as well as apoptosis. (Middle) BCR signaling induces
serine phosphorylation of Rb as in BLyS signaling, which facilitates threonine
phosphorylation of Rb by cyclin E?CDK2 and G1?S transition. Without inter-
vention, the outcome favors apoptosis because of the release of an apoptosis
factor. p18 attenuates the cell cycle and apoptosis as in BLyS signaling.
(Bottom) BLyS and BCR costimulation leads to synergistic induction of cyclin
D2 and CDK4, pS-Rb, accelerated G1progression, and G1?S transition and
increases the propensity of apoptosis. The balance between survival and
apoptosis is subject to composite control by the strength of BCR and BLyS
signaling and the presence of p18.
A model of homeostatic cell-cycle control. (A) Inactivation of CDK4?
Huang et al.
December 21, 2004 ?
vol. 101 ?
no. 51 ?
Incomplete cell-cycle activation by BLyS may in fact be a way
both to prevent resting mature B cells from premature replica-
tion, thereby maintaining the resting B cell repertoire, and to
facilitate their activation by antigen and cytokines. Precedent of
complementary cell-cycle induction exists in cell-cycle control of
T cell activation, where cell-cycle reentry and progression re-
quires two sequential signals: T cell receptor stimulation, which
induces cyclins and CDKs but not G1?S transition, and IL-2
signaling, which inactivates p27 (ref. 42 and A. Koff, personal
communication). Reconstitution of S-phase entry by BLyS and
cyclin E, particularly in p27-deficient B cells (Fig. 4), suggests
of integrating cytokine signals for cell-cycle control in B cell
activation. BLyS regulates nearly all functions of mature B cells
(12–21), most likely according to signaling strength, considering
of autoantigen-engaged B cells (6). Cooperative cell-cycle and
apoptotic control may underlie the diverse biological functions
of BLyS. Additional experimental proof certainly will be re-
quired to validate this model, but the stage is set to explore
further cell-cycle control of homeostasis in B cell immunity.
We thank David Hilbert and Human Genome Sciences for the recom-
binant human BLyS, Andrew Koff for the p27-deficient mice and
insightful comments, and Lee Kiang for a critical reading of the
manuscript. This work was supported by British Medical Research
Council Program research grants (to I.C.M.M.) and National Institutes
of Health Grants CA 80204 and AR 49436 and a Specialized Center of
Research grant from the Leukemia and Lymphoma Society (to S.C.-K.).
1. Sherr, C. J. & Roberts, J. M. (1999) Genes Dev. 13, 1501–1512.
2. Guan, K. L., Jenkins, C. W., Li, Y., Nichols, M. A., Wu, X., O’Keefe, C. L.,
Matera, A. G. & Xiong, Y. (1994) Genes Dev. 8, 2939–2952.
3. Hirai, H., Roussel, M. F., Kato, J. Y., Ashmun, R. A. & Sherr, C. J. (1995) Mol.
Cell. Biol. 15, 2672–2681.
5. Tourigny, M. R., Ursini-Siegel, J., Lee, H., Toellner, K. M., Cunningham, A. F.,
Franklin, D. S., Ely, S., Chen, M., Qin, X. F., Xiong, Y., et al. (2002) Immunity
J. G. (2004) Immunity 20, 441–453.
7. Latres, E., Malumbres, M., Sotillo, R., Martin, J., Ortega, S., Martin-Caballero,
J., Flores, J. M., Cordon-Cardo, C. & Barbacid, M. (2000) EMBO J. 19,
8. Moore, P. A., Belvedere, O., Orr, A., Pieri, K., LaFleur, D. W., Feng, P.,
Soppet, D., Charters, M., Gentz, R., Parmelee, D., et al. (1999) Science 285,
9. Schneider, P., MacKay, F., Steiner, V., Hofmann, K., Bodmer, J. L., Holler, N.,
Ambrose, C., Lawton, P., Bixler, S., Acha-Orbea, H., et al. (1999) J. Exp. Med.
10. Gross, J. A., Johnston, J., Mudri, S., Enselman, R., Dillon, S. R., Madden, K.,
Xu, W., Parrish-Novak, J., Foster, D., Lofton-Day, C., et al. (2000) Nature 404,
11. von Bulow, G. U. & Bram, R. J. (1997) Science 278, 138–141.
12. Yan, M., Brady, J. R., Chan, B., Lee, W. P., Hsu, B., Harless, S., Cancro, M.,
Grewal, I. S. & Dixit, V. M. (2001) Curr. Biol. 11, 1547–1552.
13. Gross, J. A., Dillon, S. R., Mudri, S., Johnston, J., Littau, A., Roque, R., Rixon,
M., Schou, O., Foley, K. P., Haugen, H., et al. (2001) Immunity 15, 289–302.
14. Harless, S. M., Lentz, V. M., Sah, A. P., Hsu, B. L., Clise-Dwyer, K., Hilbert,
D. M., Hayes, C. E. & Cancro, M. P. (2001) Curr. Biol. 11, 1986–1989.
S., Dobles, M., Frew, E. & Scott, M. L. (2001) Science 293, 2111–2114.
16. O’Connor, B. P., Raman, V. S., Erickson, L. D., Cook, W. J., Weaver, L. K.,
Ahonen, C., Lin, L. L., Mantchev, G. T., Bram, R. J. & Noelle, R. J. (2004) J.
Exp. Med. 199, 91–98.
17. Balazs, M., Martin, F., Zhou, T. & Kearney, J. (2002) Immunity 17, 341–352.
18. Do, R. K., Hatada, E., Lee, H., Tourigny, M. R., Hilbert, D. & Chen-Kiang,
S. (2000) J. Exp. Med. 192, 953–964.
19. Litinskiy, M. B., Nardelli, B., Hilbert, D. M., He, B., Schaffer, A., Casali, P. &
Cerutti, A. (2002) Nat. Immunol. 3, 822–829.
N., Kelley, M., Chang, D., Van, G., et al. (2000) Proc. Natl. Acad. Sci. USA 97,
21. Mackay, F., Woodcock, S. A., Lawton, P., Ambrose, C., Baetscher, M.,
22. Moreaux, J., Legouffe, E., Jourdan, E., Quittet, P., Reme, T., Lugagne, C.,
Moine, P., Rossi, J. F., Klein, B. & Tarte, K. (2004) Blood 103, 3148–
23. Novak, A. J., Darce, J. R., Arendt, B. K., Harder, B., Henderson, K.,
Kindsvogel, W., Gross, J. A., Greipp, P. R. & Jelinek, D. F. (2004) Blood 103,
24. Batten, M., Groom, J., Cachero, T. G., Qian, F., Schneider, P., Tschopp, J.,
Browning, J. L. & Mackay, F. (2000) J. Exp. Med. 192, 1453–1466.
25. Claudio, E., Brown, K., Park, S., Wang, H. & Siebenlist, U. (2002) Nat.
Immunol. 3, 958–965.
26. Hatada, E. N., Do, R. K., Orlofsky, A., Liou, H. C., Prystowsky, M., MacLen-
nan, I. C., Caamano, J. & Chen-Kiang, S. (2003) J. Immunol. 171, 761–768.
27. Kayagaki, N., Yan, M., Seshasayee, D., Wang, H., Lee, W., French, D. M.,
Grewal, I. S., Cochran, A. G., Gordon, N. C., Yin, J., et al. (2002) Immunity 17,
28. Hsu, B. L., Harless, S. M., Lindsley, R. C., Hilbert, D. M. & Cancro, M. P.
(2002) J. Immunol. 168, 5993–5996.
29. Solvason, N., Wu, W. W., Parry, D., Mahony, D., Lam, E. W., Glassford, J.,
Klaus, G. G., Sicinski, P., Weinberg, R., Liu, Y. J., et al. (2000) Int. Immunol.
30. Tanguay, D. A. & Chiles, T. C. (1996) J. Immunol. 156, 539–548.
31. Hinz, M., Krappmann, D., Eichten, A., Heder, A., Scheidereit, C. & Strauss,
M. (1999) Mol. Cell. Biol. 19, 2690–2698.
32. Franklin, D. S., Godfrey, V. L., Lee, H., Kovalev, G. I., Schoonhoven, R., Chen-
Kiang, S., Su, L. & Xiong, Y. (1998) Genes Dev. 12, 2899–2911.
33. Kiyokawa, H., Kineman, R. D., Manova-Todorova, K. O., Soares, V. C.,
Hoffman, E. S., Ono, M., Khanam, D., Hayday, A. C., Frohman, L. A. & Koff,
A. (1996) Cell 85, 721–732.
35. Lyons, A. B. & Parish, C. R. (1994) J. Immunol. Methods 171, 131–137.
36. Gorelik, L., Cutler, A. H., Thill, G., Miklasz, S. D., Shea, D. E., Ambrose, C.,
Bixler, S. A., Su, L., Scott, M. L. & Kalled, S. L. (2004) J. Immunol. 172,
37. Knudsen, E. S. & Wang, J. Y. (1996) J. Biol. Chem. 271, 8313–8320.
38. Zarkowska, T. & Mittnacht, S. (1997) J. Biol. Chem. 272, 12738–12746.
39. Pei, X. H., Bai, F., Tsutsui, T., Kiyokawa, H. & Xiong, Y. (2004) Mol. Cell. Biol.
40. Solvason, N., Wu, W. W., Kabra, N., Wu, X., Lees, E. & Howard, M. C. (1996)
J. Exp. Med. 184, 407–417.
41. Geng, Y., Yu, Q., Sicinska, E., Das, M., Schneider, J. E., Bhattacharya, S.,
Rideout, W. M., Bronson, R. T., Gardner, H. & Sicinski, P. (2003) Cell 114,
42. Firpo, E. J., Koff, A., Solomon, M. J. & Roberts, J. M. (1994) Mol. Cell. Biol.
www.pnas.org?cgi?doi?10.1073?pnas.0406111101Huang et al.