(Received for publication, August 8, 1996, and in revised form, October 4, 1996)
From the Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371, Japan
CD20 functions as a calcium-permeable cation
channel. When expressed in Balb/c 3T3 cells, CD20 accelerates the
G1 progression induced by insulin-like growth factor-I
(IGF-I). To further characterize how CD20 modulates the action of
IGF-I, we investigated whether the activity of CD20 channel was
affected by IGF-I. In quiescent cells expressing CD20, IGF-I increased
cytoplasmic free calcium concentration, [Ca2+]c,
which was reversed by the removal of extracellular calcium. In
contrast, IGF-I did not increase [Ca2+]c in cells
that did not express CD20. In perforated patch clamp recordings,
addition of IGF-I to the bath solution augmented the Ca2+
permeability, which was reversed by anti-CD20 antibody. In
cell-attached patch, calcium-permeable channel activity with unitary
conductance of 7 picosiemens was detected, which was abolished by
anti-CD20 antibody. The single channel activities were markedly
enhanced when IGF-I was included in the pipette solution, whereas IGF-I added to the bath solution was ineffective. When cells were first exposed to pertussis toxin, activation of the channel by IGF-I was
blocked. Transfection of cDNA for Gip2, a constitutive active form
of i2, activated the CD20 channel. These results
indicate that the CD20 channel is regulated by the IGF-I receptor by a mechanism involving pertussis toxin-sensitive G protein.
CD20 is a cell surface protein with a molecular mass of 35 kDa expressed in mature B lymphocytes (1-4). Monoclonal antibodies raised against CD20 affect the growth of B lymphocytes. Thus, most of the antibodies inhibit cell proliferation, whereas some are stimulatory. These observations led to the consideration that CD20 is involved in the regulation of cell growth in lymphocytes. The primary structure of CD20 has been determined by molecular cloning (5-7), and the predicted amino acid sequence indicated that CD20 is a transmembrane protein with four transmembrane domains with both C- and N-terminals located in the cytoplasm. Hence, the structure of CD20 resembles those of ion channels and ion transporters. Indeed, when expressed in fibroblasts, CD20 functions as a calcium-permeable cation channel (8). In lymphocytes, CD20 is phosphorylated by protein kinases including calmodulin-dependent protein kinase. Furthermore, CD20 associates with src family tyrosine kinases including p53/56lyn, p56lck, and p59fyn (9). Since an addition of monoclonal antibody against CD20 induces tyrosine phosphorylation of several proteins (10), CD20 may also participate in the protein tyrosine kinase cascade. Nevertheless, the regulatory mechanism modulating the activity of CD20 is largely unknown and the ligand that activates CD20 has not been identified.
To investigate the function of CD20 as a calcium-permeable channel, we stably expressed CD20 in Balb/c 3T3 fibroblasts (11). CD20 expressed in these cells functioned as a calcium-permeable channel and modulated the growth characteristics of these cells. Thus, CD20 expression accelerated cell cycle progression through the G1 phase and enabled the cells to progress to the S phase in medium containing low extracellular calcium (11). Insulin-like growth factor-I (IGF-I)1 is a progression factor that induces G1 progression (12). As described by Stiles et al. (12), IGF-I exerts its action in a cell cycle-dependent manner. When IGF-I is added to quiescent Balb/c 3T3 cells, it cannot induce cell cycle progression (12, 13). In contrast, cells progress toward the S phase in response to IGF-I when they are first exposed to platelet-derived growth factor followed by epidermal growth factor (14). These are referred to as primed competent cells (14, 15). Therefore, IGF-I exerts its progression activity specifically in primed competent, but not quiescent, cells. However, when CD20 is expressed in quiescent Balb/c 3T3 cells expressing CD20, at least some of the cells progress toward S phase in response to IGF-I (11). This result suggests that IGF-I can elicit progression, even in quiescent cells with the aid of CD20, and implies that a CD20-like protein is critical for the progression activity of IGF-I. If so, it is possible that the function of CD20 expressed in Balb/c 3T3 cells is modulated by IGF-I. In the present study, we investigated this notion. The results indicate that IGF-I, by acting on the IGF-I receptor, activates the channel activity of CD20.
Recombinant human IGF-I was supplied by Fujisawa Pharmaceutical Co. Ltd. (Osaka, Japan). Na[125I] was obtained from ICN Biomedicals (Costa Mesa, CA). [3H]Thymidine and [32P]dCTP were obtained from Dupont NEN. mAb against CD20 (CBL456) was purchased from Cymbus Bioscience Ltd. (Southampton, UK).
Cell CultureBalb/c 3T3 cells (clone A31) and Raji cells (B lymphoblastoid cell line) were provided by the RIKEN cell bank (Tsukuba, Japan). Balb/c 3T3 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (Life Technologies, Inc.). Raji cells were cultured in RPMI 1640 medium containing 10% fetal calf serum. These cells were cultured under humidified conditions of 95% air and 5% CO2 at 37 °C.
Transfection of cDNAThe inducible CD20 expression vector (CD20-pMEP4) was stably transfected into Balb/c 3T3 cells by electroporation as described previously (11). CD20 expressing quiescent Balb/c 3T3 cells were obtained by incubating confluent cells in Dulbecco's modified Eagle's medium containing 0.5% platelet-poor plasma and 80 µM ZnCl2 for 24 h. After the treatment with ZnCl2, all of the cells expressed CD20 (11).
Measurement of DNA SynthesisDNA synthesis was assessed by measuring [3H]thymidine incorporation into trichloroacetic acid-precipitable materials. Cells were cultured in 24-well plate (Falcon, Lincoln Park, NJ). Queiscent cells were incubated with 0.5 µCi/ml [3H]thymidine for 24 h in the presence of 1 nM IGF-I. The level of [3H]thymidine incorporation was measured as described by McNiel et al. (16).
Co-transfection of Gip2-pcDNAI and CD20-pMEP4Constitutively active Gi2 mutant (Gip2) expression vector, Gip2-pcDNA I was generously provided by Dr. H. Bourne of the University of California, San Francisco. Balb/c 3T3 cells were co-transfected with CD20-pMEP4 and Gip2-pcDNA I using a transfection reagent DOTAP (Boehringer Manhein, GmbH, Germany). Twenty-four hours after exposure to DNA, cells were selected for 3 weeks of culture in the presence of 100 µg/ml hygromycin B. Hygromycin-resistant colonies were independently picked up and screened by Northern blotting for high expression of Gip2 and CD20.
Measurement of Cytoplasmic Free CalciumThe cytoplasmic free Ca2+ concentration ([Ca2+]c) was monitored using fura-2, as described previously (11). Briefly, cells cultured on glass coverslips were incubated with 2 µM fura-2 acetoxymethyl ester (Dojin Laboratories, Kumamoto, Japan) for 20 min at room temperature (20-26 °C), then placed on a flow-through chamber mounted on the stage of TMD microscope (Nikon, Tokyo, Japan). The perifusion medium comprised 137 mM NaCl, 5 mM KCl, 1.25 mM CaCl2, 1 mM MgCl2, 5 mM glucose, and 10 mM Hepes/NaOH (pH 7.4). Dual wavelength microfluorometry of the fura-2 fluorescence was performed using CAM-230 (Nihon Bunko, Tokyo, Japan). The emission signals excited at both 340 and 380 nm and the ratio of these signals (340/380 ratio) was recorded. In some experiments, the cytoplasmic free Ca2+ concentration was calibrated as described elsewhere (17). Statistical significance was evaluated by analysis of variance.
Electrophysiological RecordingsThe perforated-patch (18)
and the cell-attached patch clamp techniques were applied for the
voltage-clamp studies. Micropipettes were pulled from borosilicate
glass capillaries and heat-polished at the tip. They had resistance
values between 4 and 8 megohms after filling with a pipette solution.
High resolution membrane currents were recorded using an EPC-9
patch-clamp amplifer (HEKA, Lambrecht, Germany) controlled by
"E9SCREEN" software on an Atari computer. All voltages were
corrected for a liquid junction potential between the bath and pipette
solutions. Voltage ramps were of 300-ms duration, covering a range of
100 to +100 mV. Capacitance and series resistance were canceled
before each voltage ramp using the automatic neutralization routine of
the EPC-9. For studies using the perforated whole-cell configuration,
the micropipettes for electrical recording were filled with a solution
containing 143 mM cesium-aspartate, 4 mM
MgSO4, 1 mM EGTA, 200 µg/ml nystatin (Sigma;
200 mg/ml; dissolved in dimethyl sulfoxide) and 10 mM HEPES
(pH 7.2, adjusted by adding CsOH). The bath contained 140 mM
N-methyl-D-glucamine-methanesulfonic acid, 10 mM Ca(OH)2, and 10 mM HEPES (pH
7.4). Statistical significance was evaluated by Student's t
test.
Single-channel currents were recorded as described by Hamill et
al. (19). The signal was stored on video tape after
analogue/digital conversion (Sony PCM 501 ES, modified by Shoshin EM
Corp., Okazaki, Japan). For studies using the cell-attached
configuration, the micropipettes were filled with a solution containing
110 mM BaCl2 or CaCl2, 200 nM tetrodotoxin, and 10 mM HEPES (pH 7.4, adjusted by adding Ba(OH)2 or Ca(OH)2). In some
experiments, Cl was replaced with aspartate. The bath
solution contained 137 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.25 mM
CaCl2, 5 mM glucose, and 10 mM
HEPES (pH 7.4, adjusted with NaOH). Single channel recordings were
analyzes by using the TAC program (HEKA). The total number of
functional channels (N) in the patch were estimated by
observing the number of peaks detected on the amplitude histogram. As
an index of channel activity, NPo (number of
channels multiplied by the open probability) was calculated as
![]() |
(Eq. 1) |
Cells were harvested, and the total RNA was isolated using Isogene and quantified spectrophotometrically. Total RNA (20 µg) was resolved by electrophoresis on 1.2% agarose gels containing 2.2 M formaldehyde, 20 mM MOPS (pH 7.0), 8 mM sodium acetate, and 1 mM EDTA, and transferred to a nylon membrane (Hybond-N++, Amersham Corp.). by means of capillary blotting in 10 × sodium citrate buffer. Hybridization was performed with a probe labeled with [32P]dCTP by random priming according to the manufacture's instructions (Pharmacia Biotech Inc.). The hybridized membrane was exposed to Kodak XAR film (Eastman Kodak Co.).
We showed that IGF-I stimulates DNA synthesis when added to
quiescent cells expressing CD20, whereas it has no stimulatory effect
on DNA synthesis in untransfected quiescent cells (12). As shown in
Fig. 1, IGF-I stimulated [3H]thymidine
incorporation in CD20-expressing quiescent cells and the effect of
IGF-I was inhibited by a monoclonal antibody (mAb) against CD20 in a
dose-dependent manner. This monoclonal antibody also
inhibited serum-induced DNA synthesis in Raji cells that express native
CD20 (data not shown). It should be noted that mAb against CD20 did not
affect DNA synthesis induced by IGF-I (Table I).
Furthermore, mAb against CD20 did not cross-react with IGF-I assessed
by Western blotting (data not shown).
|
IGF-I induces oscillatory changes in
[Ca2+]c in primed competent cells (17) by
activating IGF-operated calcium-permeable channels (15, 20). In
quiescent cells, however, IGF-I does not affect
[Ca2+]c (16). To test whether or not CD20 is
modulated by IGF-I, we first monitored the changes in
[Ca2+]c in response to IGF-I using quiescent
cells expressing CD20. Fig. 2A shows the
typical changes in [Ca2+]c in CD20-expressing
cells monitored by measuring the fluorescence of a Ca2+
indicator, fura-2. As we reported (11), increasing the extracellular calcium concentration from 10 µM to 2 mM
slightly increased [Ca2+]c. When 1 nM
IGF-I was added to the cells, [Ca2+]c further
increased (43 out of 43 cells). The increase in
[Ca2+]c induced by IGF-I was monophasic in 39 out
of 43 cells and the net increase in [Ca2+]c was
168 ± 46 nM (means ± S.E., n = 39), which was statistically significant (p < 0.01).
The [Ca2+]c of mock-transfected control and
untransfected cells did not change significantly under these
conditions. In some cells (4 out of 43 cells), IGF-I induced
oscillatory changes in [Ca2+]c (Fig.
2B). The effect of IGF-I was reproduced by 100 nM insulin (30 of 30 cells) (Fig. 2C). The
IGF-I-induced elevation of [Ca2+]c was dependent
on extracellular calcium and its removal (Fig. 2A) or the
addition of lanthanum (data not shown) abolished the elevated
[Ca2+]c. Elevation of
[Ca2+]c induced by IGF-I were completely
abolished by the mAb against CD20 and [Ca2+]c
returned to the level of that in cells incubated low-calcium containing
medium (6 out of 6 cells) (Fig. 2D). These results suggest
that IGF-I stimulated Ca2+ influx through the CD20 channel
in CD20-expressing cells.
Effect of IGF-I on the Activity of CD20 Channel in CD20-expressing Cells
Patch-clamp experiments were performed to record the changes in calcium permeability of the membrane induced by IGF-I. We previously reported that IGF-I increases the open probability of a calcium permeable cation channel in primed competent, but not quiescent cells (15, 20). To distinguish CD20 from the native IGF-operated Ca2+-permeable cation channel, we used quiescent cells.
First, we examined changes in Ca2+ conductance induced by
IGF-I using a nystatin-perforated whole-cell patch clamp. The
current-voltage (I-V) relationship was obtained by applying voltage
ramps from 100 to 100 mV. Fig. 3A shows a
comparison of the I-V curves obtained before and 3 min after the
addition of IGF-I. These inward currents were generated by
Ca2+, since Ca2+ is the only cation permeant in
this condition. At a holding potential of
100 mV, inward current in
cells treated with IGF-I was 446 ± 51% (means ± S.E.,
n = 42) of that in control cells, which was statistically significant (p < 0.01). The I-V curve
was not changed as a function of time in the absence of IGF-I (Fig.
3B). The Ca2+ current was completely abolished
by the mAb against CD20 (Fig. 3B) and, in addition, both
La3+ and Co2+ inhibited the Ca2+
currents (data not shown). Similar results were obtained when Ca2+ was replaced with Ba2+ as a charge
carrier. When 100 nM insulin was added, the calcium current
was similarly enhanced (Fig. 3C). It is notable that we could not identify IGF-I-stimulated Ca2+ permeability using
the conventional whole-cell patch clamp procedure. This may be due to
the fact that, in whole cell configuration, soluble components of the
cytosol are quickly dialyzed by the solution filling the pipettes.
Next, we recorded inward currents in cell-attached patches to further
characterize the action of IGF-I on Ca2+ influx in cells
expressing CD20. Fig. 4 shows a typical record obtained
from a cell-attached patch on a CD20-expressing cell with a high
concentration of barium (110 mM) in the pipette and a
holding potential of 40 mV. An inward current was observed when IGF-I
was present in the pipette solution (200 out of 218 patches) (Fig.
4B), whereas little channel activity was evident in a patch
without IGF-I (Fig. 4A). The IGF-I-activated inward current
was not detected when mAb against CD20 (2 µg/ml) was present in the
pipette solution (none of 32 patches) (Fig. 4C). IGF-I may
activate CD20 channels either directly or indirectly via a soluble
second messenger in the cytoplasm. To elucidate whether the CD20
channel is regulated by a diffusible second messenger, IGF-I was added
to the bath solution while recording from cell-attached patches with
110 mM Ba2+ in the pipette. IGF-I therefore had
access to its receptors located only outside the patch. In this
condition, CD20 channels were not activated (none of 28 patches) (Fig.
4D). This finding suggested that IGF-I augments channel
activity by a direct mechanism that does not involve a diffusible
second messenger. The I-V curve displayed a slope conductance of
7.0 ± 0.6 pS (means ± S.E., n = 8) and
extraporation of the data indicates a reversal potential of +20 mV
(Fig. 5). Again, a high concentration of insulin (100 nM) reproduced the effect of IGF-I (data not shown). Barium
was used as a charge carrier since calcium channels are generally more
permeable to this ion, which often permits a better resolution of
differences in single-channel amplitudes. However, when barium was
substituted for calcium ions, there was no significant difference in
the IGF-I-induced current. Moreover, current amplitudes were the same
whether chloride or aspartate was the anion in the pipette solution,
which confirmed that the currents were carried by an influx of cations
(Ca2+ or Ba2+), rather than by an outward anion
flux, which would display negative reversal potentials. Potassium
cannot be taken into account since this ion was not in the pipette
solution and a high concentration of barium inhibits K+
permeability. No voltage dependence was detected and inhibitors of
voltage-dependent calcium channel such as nifedipine and
verapamil had no effect on the IGF-I-activated currents.
Effect of Pertussis Toxin and Mastoparan on the Activity of CD20
To examine the involvement of pertussis toxin
(PTX)-sensitive G protein in IGF-I-induced activation, we studied the
effect of IGF-I in PTX-treated cells. When cells were pretreated with PTX (21), the addition of IGF-I in the pipette did not affect the
activity of CD20 channel (none of 30 patches) (Fig.
6A). Likewise, IGF-I did not increase calcium
current in PTX-treated cells (none of 18 cells) (Fig. 6B).
Additionally, IGF-I did not elevate [Ca2+]c in
PTX-treated cells (none of 40 cells) (Fig. 6C). Conversely,
50 pM mastoparan, an activator of
Gi/Go class of G proteins (22), markedly
stimulated the activity of the CD20 channels (43 out of 43 patches)
(Fig. 7A) whereas Mas 17, an analogue which
does not activate the G proteins, was ineffective (none of 14 patches)
(Fig. 7B).
Effect of Transfection of Gip2 on the Activity of the CD20 Channel
To further assess the role of a G protein in the
regulation of CD20 channel, we transfected Balb/c 3T3 cells expressing
CD20 with the cDNA for Gip2, a constitutive active form of
Gi2 protein (23). When Gip2 cDNA was expressed, the
activity of CD20 channel measured in the cell-attached patch was
markedly augmented without the addition of any ligand (32 of 32 patches) (Fig. 8A). When transmembrane
calcium current was measured in the whole-cell-perforated patch,
calcium permeability was greatly elevated (20 out of 20 cells)
(Fig. 8B).
In the present study, we examined whether the calcium-permeable
cation channel activity of CD20 is affected by IGF-I in CD20-expressing Balb/c 3T3 cells. The notion that CD20 channel is activated by IGF-I in
Balb/c 3T3 cells was supported by results obtained by three independent
methods: monitoring changes in [Ca2+]c,
measurement of whole-cell calcium current and the single channel
analysis. It was most explicitly demonstrated by using the perforated
patch clamp. As shown in Fig. 4, addition of IGF-I to the bath solution
increased the inward calcium current, which was reversed by an
anti-CD20 monoclonal antibody. Therefore, it is clear that IGF-I
activates the channel activity of CD20 expressed in Balb/c 3T3 cells.
The following observations supported the notion that IGF-I exerts its
effect via the IGF-I receptor. First, we showed previously that, at a
concentration of 109 M, IGF-I predominantly
acts on the IGF-I receptor but not on either the IGF-II receptor or the
insulin receptor (15). In CD20-expressing cells, 1 nM IGF-I
activated the channel activity of CD20. Second, a high concentration of
insulin, which does not bind to the IGF-II receptor (24), activated the
CD20 channel. Given that Balb/c 3T3 cells do not express significant
number of the insulin receptor, a high concentration of insulin bound to the IGF-I receptor and modulated the activity of CD20. At present, the precise mechanism by which the IGF-I receptor activates
calcium-permeable CD20 channels is not certain. The IGF-I receptor
structurally resembles the insulin receptor (24-26), and it consists
of
- and
-subunits. Ligand binding to the
-subunit results in
the activation of the intrinsic tyrosine kinase located in the
-subunit. It is generally accepted that receptor-associated tyrosine
kinase is needed for the signal transduction. Yet, the downstream
signal leading to the channel activation is not clear at present. Since a single channel current of CD20 was not activated by IGF-I added outside the patch, the regulatory mechanism by which IGF-I receptor activated the channel may be direct, not involving a soluble second messenger. In this regard, previous studies done in our laboratory showed that IGF-I-mediated calcium influx via the calcium-permeable cation channel is blocked by pertussis toxin (27). In addition, IGF-I-mediated calcium entry is blocked by GDP
S and conversely, augmented by GTP
S (28). These results indicated that IGF-I, by
acting on the IGF-I receptor, modulates the calcium-permeable channel
by a mechanism involving pertussis toxin-sensitive G protein. Similarly, IGF-I stimulates calcium influx in Chinese hamster ovary
cells by Gi-dependent mechanism (29), although
the precise mechanism by which IGF-I activates the channel via G
protein is not yet identified. As shown in Fig. 6A,
pertussis toxin also abolished the activation of CD20 by IGF-I.
Mastoparan, which directly activates pertussis toxin-sensitive G
proteins (22), stimulated the CD20 channels. Additionally, the CD20
channel was markedly activated by the co-expression of cDNA for
Gip2, a constitutive active form of Gi2. Hence, CD20 can be
directly activated by
i2 subunit. Taken together, the
mechanisms by which IGF-I activates CD20 and the IGF-operated channel
may be similar. Recently, Luttrell et al. (30) demonstrated
that activation of MAP kinase by IGF-I is attenuated by pertussis
toxin. Their results support the notion that a pertussis
toxin-sensitive G protein is involved in the signaling system activated
by IGF-I. Despite the fact that CD20 is not a natural effector molecule
of the IGF-I signaling system, it may provide a good model system with
which to study the regulation of the calcium-permeable channel by the
IGF-I receptor. Further study is needed to elucidate the mechanism by
which CD20 is regulated by the IGF-I receptor. The present results also
provide some insight into the nature of the IGF-operated
calcium-permeable channel. Despite of the fact that CD20 channel is
ectopically expressed in Balb/c 3T3 fibroblasts, the channel is
activated by the IGF-I receptor. This raises an interesting possibility
that the putative IGF-operated channel and CD20 may share some
structural homology.
CD20 is a cell-surface protein expressed in B lymphocytes. Although it has several functions in the signal transduction system in B cells (8-10), information regarding the ligand that activates CD20 is not available. The present results may provide some insight into the regulation of CD20 functions. An obvious candidate ligand that may activate CD20 is interleukin 4, since this cytokine and insulin share the signaling molecules, insulin receptor substrate-1 and -2 (31, 32). However, interleukin 4 does not activate CD20 channels in Raji cells.2 It is possible that a ligand that acts on a receptor system functionally resembling the IGF-I receptor activates the channel activity of CD20. Alternately, the ligand activating PTX-sensitive G proteins may activate the CD20 channel.
We thank Dr. M. Kato of the Nihon Medical College for suggestions and Kiyomi Ohgi for secretarial assistance.