The Journal of Immunology
Glycogen Synthase Kinase 3b Activation Is a Prerequisite
Signal for Cytokine Production and Chemotaxis in Human
Madeleine Ra ˚dinger, Hye Sun Kuehn, Mi-Sun Kim, Dean D. Metcalfe, and Alasdair M. Gilfillan
In addition to regulating mast cell homeostasis, the activation of KIT following ligation by stem cell factor promotes a diversity of
mast cell responses, including cytokine production and chemotaxis. Although we have previously defined a role for the mammalian
production and chemotaxis. In this study, we provide evidence to support a role for glycogen synthase kinase 3b (GSK3b) in such
regulation in human mast cells (HuMCs). GSK3b was observed to be constitutively activated in HuMCs. This activity was
inhibited by knockdown of GSK3b protein following transduction of these cells with GSK3b-targeted shRNA. This resulted in
a marked attenuation in the ability of KIT to promote chemotaxis and, in synergy with Fc«RI-mediated signaling, cytokine
production. GSK3b regulated KIT-dependent mast cell responses independently of mammalian target of rapamycin. However,
evidence from the knockdown studies suggested that GSK3b was required for activation of the MAPKs, p38, and JNK and
downstream phosphorylation of the transcription factors, Jun and activating transcription factor 2, in addition to activation of the
transcription factor NF-kB. These studies provide evidence for a novel prerequisite priming mechanism for KIT-dependent
responses regulated by GSK3b in HuMCs. The Journal of Immunology, 2010, 184: 564–572.
KIT (2). In addition to its role in mast cell homeostasis, however,
mediated activation of KIT potently induces mast cell chemotaxis
(3, 4) and adhesion to extracellular matrix (5), supporting a role for
SCF in mast cell homing to their tissues of residence in vivo. Fur-
with aggregation of the FcεRI, also promotes the generation of
multiple cytokines and chemokines (5–7).
KIT is member of the growth factor receptors with inherent
tyrosine kinase activity family (8, 9). Dimerization of KIT, fol-
lowing SCF binding, activates its inherent tyrosine kinase activity
resulting in phosphorylation of specific tyrosine residues in the
cytoplasmic tail of KIT allowing recruitment of critical adaptor
and signaling molecules (10). These receptor–proximal events
lead to the initiation of multiple downstream signaling process,
eventually culminating in transcriptional regulation (8, 9, 11).
Despite a comprehensive understanding of these immediate sig-
(1). Mast cell growth, development, and survival are
naling events elicited by activated KIT, it is unclear how these
events subsequently differentially control the diverse group of
responses mediated by KIT. In exploring this differential regula-
tion, we recently described, however, that the mammalian target of
rapamycin complex (mTORC)1 cascade, which is activated
downstream of PI3K following challenge of either mouse or hu-
man mast cells with SCF (12), contributes to the regulation of
SCF-mediated chemotaxis and SCF/Ag-mediated cytokine pro-
duction (12). Nevertheless, because a substantial portion of these
responses remained following rapamycin-induced inhibition of
mTORC1 signaling, it was concluded that other signaling path-
ways apart from those regulated by mTORC1 participate in the
regulation of SCF-mediated chemotaxis and transcriptional acti-
vation, leading to cytokine and chemokine generation (12).
In this study, we present evidence to support a role for glycogen
synthase kinase 3b (GSK3b) in such regulation. GSK3b is
a ubiquitously expressed serine/threonine kinase, which has been
reported to play a role in the regulation of diverse cellular re-
sponses including cell growth, tumorigenesis, cell migration, and
cytokine generation (13–15). However, it is not fully understood
how GSK3b regulates these responses. In studies that use
knockdown of GSK3b expression, we now demonstrate that
GSK3b activation is a prerequisite signal for SCF-mediated che-
motaxis and SCF/Ag-mediated cytokine generation. Thus, this
may constitute a novel priming mechanism for specific mast cell
responses. The regulation of the chemotactic response by GSK3b
appears dependent on its modulation of JNK and p38-dependent
pathways, whereas the regulation of cytokine generation by
GSK3b may be explained by the differential regulation of tran-
scriptional control downstream of JNK and p38.
Materials and Methods
Mast cell culture
Primary human mast cells (HuMCs) were derived from CD34+peripheral
blood progenitor cells (16) obtained from normal volunteers following
informed consent under a protocol approved by the National Institutes of
Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Dis-
eases, National Institutes of Health, Bethesda, MD 20892
Received for publication September 3, 2009. Accepted for publication November 5,
This work was supported by the Division of Intramural Research, National Institute
of Allergy and Infectious Diseases, National Institutes of Health. M.R. is grateful for
support from the Swedish Heart-Lung Foundation.
Address correspondence and reprint requests to Dr. Alasdair M. Gilfillan, Laboratory
of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Building 10, Room 11C206, 10 Center Drive, MSC 1881, Be-
thesda, MD 20892-1881. E-mail address: firstname.lastname@example.org
The online version of this article contains supplemental material.
Abbreviations used in this paper: ATF2, activating transcription factor 2; GS, glycogen
synthase; GSK3b, glycogen synthase kinase 3b; HuMC, human mast cell; mTORC,
mammalian target of rapamycin complex; MKK, map kinase kinase; p, phospho; rHu,
recombinant human; SA, streptavidin; SCF, stem cell factor; sh, short hairpin.
Health internal review board. The cells were developed in StemPro-34
culture medium containing StemPro-34 supplement (Invitrogen, Carlsbad,
CA), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 mg/
ml), recombinant human (rHu) IL-3 (30 ng/ml, first week only), rHuIL-6
(100 ng/ml), and rHuSCF (100 ng/ml) (PeproTech, Rocky Hill, NJ). Ex-
periments were conducted 7–9 wk after the initiation of HuMC cultures.
Lentivirus short hairpin RNA transfection of 293T cells and
transduction of HuMCs
The following GSK3b-targeted short hairpin (sh)RNAs were purchased
from Sigma-Aldrich (St. Louis, MO): CCGGGTGTGGATCAGTTGGTA-
10552) (construct A); CCGGGACACTAAAGTGATTGGAAATCTCGA-
GATTTCCAATCACTTTAGTGTCTTTTTG (TRCN0000040002) (cons-
truct B); CCGGCCACTGATTATACCTCTAGTACTCGAGTACTAGAGG-
TATAATCAGTGGTTTTTG (TRCN0000039998) (construct C); CCGGC-
GGTTTTTG (TRCN0000039999) (construct D); CCGGGCAGGACAAGA-
CN000040000) (construct E);andCCGGCAACAAGATGAAGAGC-
(control nontarget control vector).The packaging vector (MissionLentiviral
packaging mix [Sigma-Aldrich]), the pLKO1 transfer vectors with GSK3b
shRNA, orcontrol shRNA(3.4 mg)were cotransfectedinto 293Tpackaging
cells (4 3 106cells) with FuGENE6 transfection reagent (Roche, Indian-
DMEM containing FBS (10%), L-glutamine (4 mM), penicillin (100 U/ml),
and streptomycin (100 mg/ml). Following 16–19 h of transfection, medium
resulting pellet resuspended in 3 ml prewarmed completeStemPromedium.
Transductionof HuMCswas conductedby transferingthe 3 ml resuspended
virus to a T75 cultureflask containing3–4 3 106HuMCs in 15 ml complete
StemPro medium. Two days postinfection, the medium was changed to vi-
rus-free complete StemPro medium, and antibiotic selection was initiated
7 posttransduction. Cytospins of HuMCs transduced with shControl or
cells treated with GSK3b-targeted shRNA are hereafter termed GSK3b
Flow cytometric analysis for Fc«RI and KIT surface expression
Cells were incubated in cytokine-free media for 4 h and washed twice in
PBS containing 0.1% BSA. Cells were then stained with CD117-PE (BD
Biosciences, San Jose, CA) or PE-conjugated isotype control (BD Bio-
sciences) and FcεRI-allophycocyanin (eBioscience, San Diego, CA) or
allophycocyanin-conjugated isotype control (eBioscience) for 1 h on ice.
Cell fluorescence was analyzed (10,000 events) on a gated forward light
scatter and side light scatter area previously determined as mast cell-
specific using a FACSCalibur flow cytometer (BD Biosciences) and as-
sociated CellQuest software.
human myeloma IgE (100 ng/ml; Calbiochem, La Jolla, CA), biotinylated
within the National Institute of Allergy and Infectious Diseases Core
Facility. The cells were then starved in cytokine-free StemPro medium for
4 h (for chemotaxis assay and corresponding cell lysate preparations), then
the cells were activated by the addition of SCF (30 ng/ml). For cytokine
release studies, HuMCs (1 3 106/ml) were sensitized in complete StemPro
culture medium overnight and the next day washed in culture medium and
triggered concurrently via KIT with SCF (30 ng/ml) and via the FcεRI
with streptavidin (SA) (100 ng/ml) for 6 h. In some experiments, cells
were pretreated with the mTOR inhibitor, rapamycin (100 nM) or the
PI3K inhibitor, wortmaninn (100 nM), (Calbiochem) for 20 min prior to
Real-time PCR analysis
HuMCs (2–3 3 106/sample) were sensitized overnight with biotinylated
human IgE (100 ng/ml) in complete StemPro medium. The following day,
cells were washed with the same medium three times to remove excess
IgE, then the cells were stimulated with SA (100 ng/ml) and SCF (30 ng/
ml) for 4 h. Total RNA was isolated from each preparation using the
RNeasy Mini Kit (Qiagen, Valencia, CA). One microgram of total cellular
RNAwas treated for genomic DNA contamination and reverse transcribed
using SA Biosciences Reverse Transcription reagents and oligo(dT) (SA
Biosciences, Fredrick, MD). Gene expression was analyzed using real-time
PCR on an ABI7500 SDS system (Applied Biosystems, Foster City, CA).
A common cytokine PCR array was purchased from SA Biosciences, and
real-time PCR was performed according to manufacturer’s instructions.
All reactions (two different HuMCs donors) were performed in triplicate
for 40 cycles. The relative fold expression levels of cytokines was calcu-
lated as follows: for each sample the threshold cycle (Ct) was determined
and normalized to the average of five different housekeeping genes in the
KIT (DCt). The DCt of treated or untreated cells was then subtracted from
untreated control shRNA-transduced cells (DDCt) and the relative fold
expression was calculated using the equation 2DDCt.
Cell-free supernatants from activated cells were harvested and cytokine
content was measured by using DuoSet ELISA System (R&D Systems,
Chemotaxis assays were performed using Transwell polycarbonate
membranes (8-mm pore size) (Corning, Corning, NY). HuMCs (1 3 105/
well) were incubated in cytokine-free StemPro medium for 4 h and then
resuspended in cytokine-free StemPro medium containing 0.5% BSA.
The cell suspension (100 ml) was placed in the upper chamber and
preincubated in the bottom chamber containing 600 ml cytokine-free
StemPro medium for 30 min at 37˚C. After 30 min, the inserts were
replaced into the bottom chambers with or without SCF (30 ng/ml). After
4 h, the migrated cells were collected in the bottom chamber and counted
Cell lysates were prepared as described previously (18). Aliquots of lysates
were loaded onto a 4–12% NuPage BisTris gel (Invitrogen) and following
electrophoresis, proteins were transferred onto nitrocellulose membranes.
The proteins were probed with following phospho (p)-specific Abs from
Cell Signaling Technology (Beverley, MA): p-AKT (S473), p-GSK3b
(S9), p-JNK (T183 and Y185), p-map kinase kinase (MKK) 3/6 (S189 and
S207), p-p38 (T180), p-GS (S641), p-mTOR (S2448), p-p70 S6K (T389),
p-4E-BP1 (T37 and T46), phospho-activating transcription factor 2 (ATF2)
(T71), p-NFkB (S536), p-NFkB (S276), total JNK, total p38, and total NF-
kBp65. p-cJun (S73) was from Upstate Biotechnology (Lake Placid, NY),
and p-GSK3a/b (Y279/Y216) was from Invitrogen. Total Syk, total KIT,
total Lyn, and total cJun Abs were from Santa Cruz Biotechnology (Santa
Cruz, CA). Immunoreactive proteins were visualized by probing with
HRP-conjugated secondary Abs and then by ECL (PerkinElmer, Wellesley,
MA). Quantitation of changes in protein phosphorylation were performed
using a Quantity One scanner (Bio-Rad, Hercules, CA).
Data are represented as the mean 6 SE. The statistical analyses were
performed by unpaired Student’s t test. Differences were considered sig-
nificant when p , 0.05. The n values represent experiments from multiple
Expression and knockdown of GSK3b in human mast cells
Disruption of the GSK-3b gene in mice leads to an embryonically
lethal phenotype (19), therefore, to explore the role of GSK3b in
human mast cell function, we elected to use a gene knockdown
approach. To achieve this, HuMCs were stably transduced with
GSK3b-targeted shRNA, using a lentivirus system. Five (A–E)
different constructs were examined for their ability to selectively
knockdown GSK3b expression. As a control, we used a scrambled
shRNA construct purchased from Sigma-Aldrich. The level of ex-
pression of GSK3b in the cells treated with the control-scrambled
shRNA was not substantially different from that observed in un-
treated cells (data not shown). Of the shRNAs targeting GSK3b,
four decreased GSK3b levels to varying degrees within the cells
following transduction. Of these, two (A and B) were selected for
(Fig. 1A). These constructs had little effect on the expression of
The Journal of Immunology 565
other molecules examined,includingphospholipase Cg1,Lyn, Syk,
KIT, and NF-kΒ (Fig. 1B). In addition, no marked differences were
seen in surface expression of KIT and FcεRI (Fig. 1C) in cells
transduced with GSK3b-targeted shRNA (86.7 6 1.7% double-
positive cells, n = 3]). There was also little change in the gross
morphology of the cells treated with the GSK3b-targeted shRNA
compared with control cells (Fig. 1D) or nontransduced cells (data
not shown). In subsequent studies, responses observed in GSK3b
knockdown HuMCs are compared with those observed in HuMCs
incubated with scrambled control shRNA.
Knockdown of GSK3b phosphorylation in quiescent and
activated mast cells
Optimal cytokine generation in HuMCs requires that the cells be
concurrently activated through KIT and the FcεRI (6, 20). We
therefore examined GSK3b phosphorylation under these con-
ditions following GSK3b knockdown. GSK3b activity is regu-
lated by the phosphorylation status of Y216 and S9. Y216 is
reported to be constitutively phosphorylated in resting cells thus
maintaining GSK3b in an active state (14, 21, 22). However, the
ability of GSK3b to phosphorylate its specific substrates, for ex-
ample, glycogen synthase (GS), requires priming of the substrate
by means of prior phosphorylation by an additional kinase (23,
24). In resting HuMCs, we similarly observed constitutive phos-
phorylation of Y216 (Fig. 2A, 2B). When the cells were activated
through KIT and FcεRI, we observed no consistent increase in the
phosphorylation of this residue. Similarly, the GSK3b substrate,
GS, was constitutively phosphorylated at S641 (Fig. 2A, 2C).
However, in contrast to the phosphorylation of GSK3b, this
phosphorylation was slightly elevated in cells activated through
KIT and FcεRI. Surprisingly, in light of this observation, we also
observed that the phosphorylation of the inhibitory S9 residue of
GSK3b was enhanced in the activated HuMCs (Fig. 2A, 2D).
Similar responses to the above were observed in HuMCs activated
via either FcεRI or KIT alone (data not shown).
As expected, because of the effective reduction in total GSK3b
protein in the GSK3b knockdown HuMCs, the phosphorylation of
GSK3b-Y216 and GSK3b-S9 was substantially reduced in both
means of a negative control, we observed no decrease in the SCF/
SA-dependent phosphorylation of AKT, a surrogate marker for
PI3K activation, in these cells (Fig. 2A, 2E). Taken together, these
data suggest that, in HuMCs, GSK3b is active under resting con-
ditions and that, upon cell activation through FcεRI and KIT, this
permits an increase in phosphorylation of the GSK3b substrate GS.
shRNA-induced GSK3b knockdown.
Effect of shRNA-induced knockdown of GSK3b on mast cell
Having successfully established knockdown of GSK3b activity in
HuMCs, we next investigated the role of GSK3b in mast cell
cytokine generation. For these studies, the cells were again cos-
timulated through KIT, via SCF, and the FcεRI, through sensi-
tizing with biotinylated IgE, and challenging with SA as the
response to either stimulus alone is not marked (20). To initially
screen the effect of GSK3b knockdown on multiple cytokines,
cells were stimulated for an optimal period of 4 h, based on
previous kinetic studies (25), then cytokine gene expression was
determined by a commercially available array. In these studies, we
observed a marked increase in mRNA’s for multiple cytokines,
including those for GM-CSF, IL-8, and IL-13 (Fig. 3A–C and
Supplemental Material). These responses were markedly attenu-
ated in GSK3b knockdown cells compared with the control cells
(Fig. 3A–C). On the basis of this initial screen, we examined the
amounts of GM-CSF and IL-8 protein present in the supernatants
of control and GSK3b knockdown HuMCs challenged with SCF
in the presence of SA. As demonstrated in Fig. 3D and 3E, there
was a marked reduction in SCF/SA-induced generation of GM-
CSF and IL-8 in the GSK3b knockdown HuMCs compared with
controls. The relative inhibition produced by the two constructs
(Fig. 3D, 3E) correlated with their relative abilities to knockdown
GSK3b protein levels (Fig. 1A). This close correlation was further
illustrated by plotting the relative expression of GSK3b protein
levels to that of IL-8 production (Fig. 3F).
Effect of shRNA-induced knockdown of GSK3b on mast cell
In addition to stimulating cytokine generation in mast cells, SCF is
a potent chemotactic agent for HuMCs (4). To thus explore
whether SCF-mediated chemotaxis was similarly dependent on
GSK3b, we next examined the relative ability of HuMCs to mi-
grate in response to SCF following GSK3b knockdown. From Fig.
3G, it can be seen that GSK3b knockdown HuMCs displayed
a reduced capacity to migrate toward SCF compared with control
cells. The extent of inhibition of migration again correlated to the
extent of knockdown of GSK3b observed in these studies, with
GSK3b shRNA B producing a greater knockdown of GSK3b and
migration than that produced by GSK3b shRNA A (Fig. 3G).
HuMCs. HuMCs transduced with scrambled shRNA (shContr) or shRNA
for GSK3b (two different constructs; shGSK3b-A and shGSK3b-B).
Whole-cell extracts were prepared and immunoblotted with anti-GSK3b,
anti-phospholipase Cg1(anti-PLCg1), anti-Lyn, anti-Syk, anti-KIT, and
anti–NF-kB Abs (A and B). FcεRI and KIT surface expression on HuMCs
transduced with scrambled shRNA (shContr) or shRNA for GSK3b
(shGSK3b) analyzed by flow cytometry (C). HuMCs transduced with
scrambled shRNA (shContr) or shRNA for GSK3b (shGSK3b) stained
with toluidine blue (D; original magnifications 340 [upper panels] and
3100 [lower panels]) (n = 6 in A and n = 2–3 in B–D).
shRNA-mediated knockdown of GSK3b in quiescent
566ROLE OF GSK3b IN MAST CELL FUNCTION
GSK3b regulates mast cell responses independently of mTOR
We next examined how GSK3b may regulate mast cell cytokine
production and chemotaxis. GSK3b has been reported to phos-
phorylate the mTOR regulator, tuberin (TSC2), in HEK293T cells
(26), thereby influencing the activation of the mTORC1 and
mTORC2 cascades. Our previous studies in HuMCs demonstrated
that the mTORC1 cascade, downstream of PI3K, contributes to
SCF/SA-mediated cytokine production and SCF-mediated chemo-
the PI3K-dependent activation of AKT, which phosphorylates and
activation. This results in the sequential phosphorylation and acti-
vation of mTOR and its downstream substrates: the transcriptional
regulators p70 ribosomal S6 kinase (p70S6K) and eukaryotic ini-
mTORC2 cascade leads to the phosphorylation (S473) and activa-
cytokine production in the GSK3b knockdown HuMCs may be
As before, GSK3b knockdown significantly reduced GSK3b
expression in HuMCs (Fig. 4A, 4B). However, there were minimal
differences in the basal and SCF/SA-induced phosphorylation of
mTOR, p70S6K, and 4E-BP1 in the GSK3b knockdown HuMCs
earlier, there was also no difference in the resting or SCF/SA in-
duced, mTORC2-dependent phosphorylation of AKT(S473) in the
2A, 2E). Similarly, the mTORC1 inhibitor, rapamycin, failed to
block the phosphorylation of GSK3b (S9, Y216) (Fig. 4F) despite
a marked inhibition of GSK3b (S9 only) by the PI3K inhibitor
wortmannin. Taken together, these data support the conclusion that
GSK3b regulates SCF/SA-induced mast cell responses in-
dependently of both the mTORC1 and mTORC2 cascades. We
cytokine production and chemotaxis by an alternative mechanism.
Regulation of the MAPK activation by GSK3b in human mast
The MAPKs, p38, and JNK, regulate transcriptional activation
pathways that contribute to mast cell function, including cytokine
generation (32). Thus, we next investigated whether the MAPK
cascade represented a key intermediary step in the regulation of
GSK3b-regulated mast cell cytokine production. As can be seen
in Fig. 5A, in contrast to the lack of effect on the mTOR cascades,
the increase in both JNK (Fig. 5A, 5B) and p-38 (Fig. 5A, 5C)
phosphorylation in response to SCF/SA was reduced in the
GSK3b knockdown HuMCs compared with the control treated
in quiescent and activated HuMCs. HuMCs, transduced
with scrambled shRNA (shContr) or shRNA for GSK3b
(shGSK3b-B), were sensitized overnight and then
stimulated with SA (100 ng/ml) and SCF (30 ng/ml) for
2 min as described in Materials and Methods. Whole-
cell extracts were prepared and immunoblotted with
anti–p-GSK3b(Y216), anti–p-GS (p-GS(S641)), anti–
p-GSK3b(S9), or anti–p-AKT(S473) Abs (A). Protein
loading of the samples was normalized by probing for
Syk. Data were generated by scanning the blots from
three to five independent experiments, and normalized
to the response obtained at 2 min with SCF/SA stimu-
lation in shContr-transduced HuMCs (B–E) (n = 3–5,
ppp , 0.001 and pppp , 0.0001, by Student’s t test).
Knockdown of GSK3b phosphorylation
The Journal of Immunology 567
cells. There was no effect on total p38 or JNK protein levels under
these conditions (Fig. 5A). The SCF/SA-induced phosphorylation
of MKK3/6, upstream of p38, was however also markedly reduced
in the GSK3b knockdown-HuMCs (Fig 5A, 5D).
Because the MAPKs JNK and p38 have also been shown to
regulate SCF/KIT-induced mast cell migration (33–35), we next
determined the phosphorylation status of these proteins under the
conditions used for the chemotaxis studies, i.e., stimulation with
SCF after a 4-h period of starvation in SCF-depleted media. As
shown in Fig. 5E–I, SCF-mediated MKK3/6, JNK, and p38 acti-
vation, but not total p38 or JNK protein, were significantly reduced
under these conditions. In contrast, SCF-induced AKT activation
was unaffected compared with control treated cells (Fig. 5E, 5I).
the MAPKs p38 and JNK, thus providing an explanation for the
reduced SCF-induced chemotaxis observed in the GSK3b knock-
down HuMCs. Furthermore, GSK3b-dependent, p38- and JNK-
for the reduced SCF/SA-induced cytokine production of served in
the GSK3b knockdown HuMCs. We thus next examined the SCF/
SA-induced phosphorylation of specific transcription factors in the
GSK3b knockdown HuMCs.
Transcription factors regulation by GSK3b
The MAPK pathway leads to the phosphorylation and, thus, regu-
lation of multiple transcription factors including Jun and ATF2.
production and other transcriptional-dependent processes. We
therefore examined whether GSK3b regulated the phosphorylation
of Jun, ATF2, and NF-kB. As can be seen in Fig. 6, the phosphor-
ylation of the AP-1 transcription factors c-Jun (Fig. 6A, 6B) and
ATF2 (Fig. 6A, 6C) in response to SCF/SA stimulation was greatly
reduced in GSK3b knockdown HuMCs compared with control-
treated cells. Furthermore, the phosphorylation of the p65 NF-kB
subunit was also significantly reduced in the GSK3b knockdown
cells compared with control-treated cells (Fig. 6A, 6D). Taken to-
gether, these data support the conclusion that GSK3b activation is
a prerequisite signal for the MAPK-dependent activation of c-Jun
SA-induced cytokine production.
In this study, we present evidence that supports a prerequisite role
for GSK3b in KIT-mediated mast cell chemotaxis and KIT/FcεRI-
mediated enhanced gene expression leading to cytokine pro-
duction in HuMCs. As reported in other cell types (14, 21, 22), in
quiescent HuMCs, GSK3b was determined to be constitutively
activated. This conclusion was supported by the observed basal
phosphorylation of the activating tyrosine residue (Y216) in
GSK3b, the phosphorylation of its substrate GS at S641, and the
reduction of these phosphorylation states in the GSK3b knock-
down cells (Fig 2). Although we did not observe a consistent in-
crease in phosphorylation of GSK3b at the Y216 position in
GM-CSF, IL-8 production, and SCF-mediated
chemotaxis in HuMCs. HuMCs transduced with
scrambled shRNA (shControl) or shRNA for
GSK3b (shGSK3b-B) were sensitized overnight
and then stimulated with SA (100 ng/ml) and SCF
indicated samples, reverse transcribed, and quan-
tified by real-time PCR. Relative expression of
described in Materials and Methods (A–C). Data
are shown from two independent experiments.
HuMCs, transduced with scrambled shRNA
(shContr) or shRNA for GSK3b (two different
constructs; construct A or B), were sensitized
were collected, and ELISAwas performed for hu-
man GM-SCF and IL-8 (D and E) (n = 6–9; pp ,
0.05, ppp , 0.001, and pppp , 0.0001, by Stu-
dent’s t test). The degree of GSK3b knockdown
inversely correlates with SCF/SA-mediated IL-8
release (F) (n = 6, Spearman correlation test). The
diamond symbol represents the means, and shown
SEs, of the values obtained from control cells (n =
6). These data were not included in the statistical
analysis of the correlation. HuMCs, transduced
with scrambled shRNA (shContr) or shRNA for
GSK3b (two different constructs; construct A or
B), were starved for 4 h and then stimulated with
SCF (30 ng/ml) for 4 h in a Transwell chemotaxis
assay (G) (n = 3–5; ppp , 0.001, by Student’s t
568ROLE OF GSK3b IN MAST CELL FUNCTION
response to SCF and/or SA, under the conditions used to examine
chemotaxis and cytokine production, there was an apparent in-
crease in the phosphorylation of GS at S641 under these con-
ditions, which was reduced in the GSK3b knockdown. The
constitutive Y216 phosphorylation of GSK3b may be due to the
cells being maintained in SCF. Indeed, when the cells were starved
of SCF for a prolonged period of time (overnight) prior to stim-
ulation, we were able to observe an SCF-dependent increase in the
phosphorylation of this residue. Thus, the phosphorylation of this
residue may be directly dependent on KIT. Regardless, our results
suggest that triggering of mast cells through KIT and/or FcεRI
facilitates the ability of GSK3b to phosphorylate its substrate(s)
without necessarily increasing its constitutive activity, a potential
mechanism of action that is elaborated upon below.
In addition to the phosphorylation of (Y216) in GSK3b, how-
ever, we observed that the inhibitory S9 residue GSK3b was also
phosphorylated in a PI3K-dependent manner following SCF/ SA
challenge (Figs. 2D, 4F). This phenomenon has also been reported
in monocytes, dendritic cells, and T cells, following exposure to
TLR2, TLR4, TLR5, and TLR9 agonists, Escherichia coli, and
viral peptide respectively (36–38). In our study, however, the
observed increased phosphorylation of GS, at least at early time
points, would suggest that downregulation of GSK3b activation
may occur latently to the constitutive activation. We have pre-
viously demonstrated that PI3K, and signals dependent on PI3K
activity, are delayed responses compared with other signals initi-
ated upon FcεRI or KIT activation (12). Thus, it is likely that any
response due to downregulating GSK3b activity would be chro-
nologically secondary to those regulated by GSK3b activation.
Nevertheless, these data do suggest that the ability of GSK3b to
phosphorylate its substrates may depend on the net balance be-
tween positive and negative regulation of GSK3b activity.
The marked reduction in the ability of SCF/SA to enhance IL-8,
IL-13, and GM-CSF mRNA levels and IL-8 and GM-CSF secre-
tion, associated with the diminution of GSK3b activity in the
GSK3b knockdown HuMCs (Fig 3F), strongly supports a re-
quirement for GSK3b activity in the regulation of KIT/FcεRI-
mediated cytokine production. This conclusion is further sup-
ported by the close statistical correlation between the degree of
GSK3b knockdown and IL-8 secretion. Similarly, the close cor-
relation between GSK3b knockdown and reduction in SCF-
induced chemotaxis in the GSK3b knockdown HuMCs also pro-
vides evidence for a prerequisite role for GSK3b in the SCF-in-
duced chemotactic response.
There are conflicting reports regarding the role of GSK3b in
cytokine production in other cells of hematopoietic lineage.
mast cell responses independently of mTOR. HuMCs,
transduced with scrambled shRNA (shContr) or
shRNA for GSK3b (shGSK3b-B), were sensitized
overnight and then stimulated with SA (100 ng/ml)
and SCF (30 ng/ml) for 10 min as described in Ma-
terials and Methods. Whole-cell extracts were pre-
pared and immunoblotted with anti-GSK3b, anti–p-
mTOR(S2448), anti–p-p70S6K(T389), and anti–p-
4E-BP1(T37, T46) Abs (A). Data in B–E were gen-
erated by scanning the blots from three to five in-
response obtained at 10 min with SCF/SA stimulation
(n = 3–5, ppp , 0.001 for comparison with SCF/SA
response in shContr-transduced HuMCs by Student’s t
test). In F, HuMCs were pretreated with rapamycin
(100 nM) or wortmaninn (100 nM) 20 min prior
stimulation with SCF or SA at the time indicated.
Whole-cell extracts were prepared and immuno-
(Y216), anti–p-GSK3b(S9), or anti–p-GS(S641) Abs.
Protein loading of the samples was normalized by
probing for Syk.
GSK3b regulates SCF/SA-mediated
The Journal of Immunology569
Treatment of monocytes with GSK3b inhibitors such as LiCl and/
or SB216763, or with GSK3b-targeted small interfering RNA, has
been reported to inhibit TLR2-, 4-, 5-, and 9-dependent release of
IL-1b, IL-6, TNF-a, IL-12, and IFN-g but to enhance TLR-
dependent production of IL-10 (36). GSK3b inhibitors were also
reported to inhibit E. coli-induced IL-12, IL-6, and TNF-a, but not
IL-10, release from dendritic cells (37). In contrast, in T cells,
GSK3b inhibitors were observed to enhance viral peptide-induced
IL-2 production, whereas overexpression of GSK3b in T cells
downregulated the response (38). This apparent dichotomy in the
GSK3b-dependent regulation of cytokine generation in the vari-
ous cell types may reflect the potential for GSK3b to both nega-
tively and positively regulate transcriptional signaling pathways
for cytokine production. Indeed, it is possible that, in addition to
HuMCs. HuMCs, transduced with scrambled
shRNA (shContr) or shRNA for GSK3b
(shGSK3b-B), were sensitized overnight and
then stimulated with SA (100 ng/ml) and
SCF (30 ng/ml) (A) or starved for 4 h then
stimulated with SCF (30 ng/ml) (E) for 2 min
as described in Materials and Methods.
Whole-cell extracts were prepared and im-
munoblotted (A and E) with anti–p-JNK
(T183, Y185), total JNK, anti–p-MKK3/6
(S189, S207), anti–p-p38 (T180), total p-38,
or p-AKT (S473) Abs. Protein loading of the
samples was normalized by probing for Syk
(A and E). Data in B–D and F–I were gen-
erated by scanning the blots in three to five
independent experiments and normalized to
the response obtained with SCF/SA stimu-
lation (B–D), or SCF response (F–I), in
shContr-transduced HuMCs (n = 3–5; pp ,
0.05, ppp , 0.001 and pppp , 0.0001, by
Student’s t test).
GSK3b regulates SCF/SA-
AP1 transcription factors and NF-kB activity in
HuMCs. HuMCs, transduced with scrambled shRNA
(shContr) or shRNA for GSK3b (shGSK3b-B), were
ng/ml) and SCF (30 ng/ml) for 30 min as described in
materials and methods. Whole-cell extracts were pre-
cJun, anti–p-ATF2(T71),oranti–p-NF-kB(S536) Abs
probing for Syk (A). Data in B–D were generated by
normalizing to the responses obtained with SA/SCF
ppp , 0.001, and pppp , 0.0001, byStudent’s t test).
GSK3b regulates SCF/SA-mediated
570ROLE OF GSK3b IN MAST CELL FUNCTION
regulating positive signals, negative signaling pathways may also
be regulated by GSK3b in mast cells. In this respect, it has been
suggested that the ability of AKT to enhance cytokine generation
through NF-AT activation in mouse mast cells may be due to
downregulation of GSK3b activity (39). Whether this may also be
true for HuMCs is unclear from the current study; however, the
induced phosphorylation of the inhibitory GSK3b S9 residue in
response to SCF/SA in HuMCs was reduced by the PI3K inhibitor
wortmannin (Fig. 4F).
There has emerged no common mechanistic explanation as to
how GSK3b may be exerting its regulatory influence on cytokine
generation and other processes in hematopoietic cells. As we have
previously demonstrated that the mTORC1 cascade contributes to
KIT/FcεRI-mediated cytokine production and KIT-mediated mast
cell chemotaxis (12), and as GSK3b has been proposed to regulate
the mTOR pathway through phosphorylation of tuberin (26), the
scenario existed that, in the HuMCs, GSK3b may be acting via
regulation of mTOR pathways. However, the observations that the
KIT/FcεRI-mediated phosphorylation of components of the
mTORC1 and mTORC2 cascades was not reduced in the GSK3b
knockdown HuMCs (Fig. 4), argues against this possibility. It has
been proposed, that the contrasting roles for GSK3b in TLR cy-
tokine production in monocytes may be explained by opposing
regulation of the transcription factors CREB and NF-kB through
competition for binding to a common coactivator protein CREB
binding protein (36). According to this model, GSK3b inhibition
would increase CREB activation allowing CREB to compete with
the p65 subunit of NF-kB for binding to CREB binding protein. In
our present study, however, although SCF/SA-induced phosphor-
ylation of the p65 subunit of NF-ĸB was observed to be signifi-
cantly reduced in the GSK3b knockdown HuMCs (Fig. 6 A, 6D),
we did not consistently observe an increase in CREB activity in
these cells (data not shown). Regardless, these data are in agree-
ment with other studies showing that GSK3b is required for NF-
kB activation (19).
The most remarkable defects that we observed, however, in the
GSK3b knockdown HuMCs were in the p38 and JNK MAPK
pathways and, particularly, in the respective downstream tran-
scription factors ATF2 and c-Jun. JNK activity has previously
been shown to regulate cytokine production mediated by AP1
transcription factors in both mouse bone marrow-derived mast
cells (32) and HuMCs (6) Similarly, both JNK and p38 have been
previously described to regulate mast cell chemotaxis (33–35).
Therefore, the reduced SCF/SA-induced cytokine production and
SCF-induced chemotaxis observed in the GSK3b knockdown
HuMCs may be explained by defective JNK and p38 signaling in
these cells (Fig. 5A–D).
these pathways may be explained by the unique manner in which
GSKb phosphorylates its substrates. As discussed, GSK3b sub-
acids 4–5 COOH-termini to the GSK3b phosphorylation sites for
is active in resting conditions, it cannot optimally phosphorylate its
substrates, until upon FcεRI or KIT kinase activation, the GSK3b
substrates become phosphorylated as a consequence of the activa-
then allow GSK3b to optimally phosphorylate its target signaling
proteins and hence transduce the signals required for gene expres-
sion leading to cytokine production and the processes required for
chemotaxis (Fig. 7). Of potential relevance may be the presence of
two highly conserved SxxxS/T sequences in MKK3 and MKK6,
and in MKK4 and MKK7, which are responsible for the phos-
found in MEK kinase 1 and MEK kinase 4, upstream kinases of
MKK4 and MKK7.Thus, phosphorylation of thesesites by GSK3b
following initial phosphorylation by a “priming” serine/threonine
GSK3b may regulatetheactivationofp38 andJNK andsubsequent
downstream transcription factors. In support of this conclusion, we
was markedly reduced in the GSK3b knockdown HuMCs (Fig. 5).
In summary, in this study, we have presented evidence to support
the conclusion that GSK3b is a prerequisite signal for KIT-
mediated chemotaxis and KIT/FcεRI-mediated cytokine pro-
duction in human mast cells. The regulation of cytokine genera-
tion by GSK3b could be explained by the differential regulation of
transcriptional control downstream of JNK and p38, as well as
transcriptional control of NF-kB p65 subunit, whereas the regu-
lation of the chemotactic response by GSK3b may be explained
by its modulation of JNK- and p38-dependent pathways. As with
other cells types, it is, however, possible that as-yet undefined
inhibitory pathways both regulating GSK3b activation and
activated GSK3b may regulate HuMC cytokine pro-
duction and chemotaxis. Under resting conditions,
GSK3b is constitutively active, because of phosphor-
ylation of the Y216 residue, but is unable to optimally
phosphorylate its substrates as they require initial
phosphorylation by a “priming” kinase to allow these
reactions to take place. Upon SCF/SA challenge,
GSK3b substrates are phosphorylated by priming
kinases, thus allowing GSK3b to phosphorylate and
activate these substrates. This leads to activation of the
JNK and p38 MAPK pathways, thereby initiation of to
chemotaxis, and downstream transcription factors and
NF-kB leading to cytokine production. GSK3b activity
is terminated upon phosphorylation at the S9 position
as a consequence of activation of PI3K. On the basis of
other reports, it is possible that GSK3b may also reg-
ulate an inhibitory pathway for NF-AT activation (39),
leading to cytokine production.
Potential model by which constitutively
The Journal of Immunology 571
regulated by GSK3b activity may play a role in human mast cell
biology. Thus, GSK3b may act as a central regulator for the
precise control of the signaling processes required for mast cell
chemotaxis and cytokine production.
The authors have no financial conflicts of interest.
1. Mekori, Y. A., and D. D. Metcalfe. 2000. Mast cells in innate immunity. Im-
munol. Rev. 173: 131–140.
2. Metcalfe, D. D. 2008. Mast cells and mastocytosis. Blood 112: 946–956.
3. Okayama, Y., and T. Kawakami. 2006. Development, migration, and survival of
mast cells. Immunol. Res. 34: 97–115.
4. Nilsson, G., J. H. Butterfield, K. Nilsson, and A. Siegbahn. 1994. Stem cell factor
is a chemotactic factor for human mast cells. J. Immunol. 153: 3717–3723.
A. Gray, J. Giddings, E. Peskett, et al. 2004. Essential role for the p110d phos-
phoinositide 3-kinase in the allergic response. Nature 431: 1007–1011.
6. Hundley, T. R., A. M. Gilfillan, C. Tkaczyk, M. V. Andrade, D. D. Metcalfe, and
M. A. Beaven. 2004. Kit and FcεRI mediate unique and convergent signals for
7. Vosseller, K., G. Stella, N. S. Yee, and P. Besmer. 1997. c-kit receptor signaling
through its phosphatidylinositide-39-kinase-binding site and protein kinase C:
role in mast cell enhancement of degranulation, adhesion, and membrane ruf-
fling. Mol. Biol. Cell 8: 909–922.
8. Roskoski, R., Jr. 2005. Structure and regulation of Kit protein-tyrosine kinase—
the stem cell factor receptor. Biochem. Biophys. Res. Commun. 338: 1307–1315.
9. Roskoski, R., Jr. 2005. Signaling by Kit protein-tyrosine kinase—the stem cell
factor receptor. Biochem. Biophys. Res. Commun. 337: 1–13.
10. Jensen, B. M., C. Akin, and A. M. Gilfillan. 2008. Pharmacological targeting of
the KIT growth factor receptor: a therapeutic consideration for mast cell dis-
orders. Br. J. Pharmacol. 154: 1572–1582.
11. Linnekin, D. 1999. Early signaling pathways activated by c-Kit in hematopoietic
cells. Int. J. Biochem. Cell Biol. 31: 1053–1074.
function of the mTORC1 pathway in mast cells. J. Immunol. 180: 4586–4595.
13. Cohen, P., and M. Goedert. 2004. GSK3 inhibitors: development and therapeutic
potential. Nat. Rev. Drug Discov. 3: 479–487.
14. Doble, B. W., and J. R. Woodgett. 2003. GSK-3: tricks of the trade for a multi-
tasking kinase. J. Cell Sci. 116: 1175–1186.
15. Hu, X., P. K. Paik, J. Chen, A. Yarilina, L. Kockeritz, T. T. Lu, J. R. Woodgett, and
L. B. Ivashkiv. 2006. IFN-g suppresses IL-10 production and synergizes with
TLR2 by regulating GSK3 and CREB/AP-1 proteins. Immunity 24: 563–574.
16. Kirshenbaum, A. S., J. P. Goff, T. Semere, B. Foster, L. M. Scott, and
D. D. Metcalfe. 1999. Demonstration that human mast cells arise from a pro-
genitor cell population that is CD34(+), c-kit(+), and expresses aminopeptidase
N (CD13). Blood 94: 2333–2342.
17. Kirshenbaum, A. S., and D. D. Metcalfe. 2006. Growth of human mast cells from
bone marrow and peripheral blood-derived CD34+pluripotent progenitor cells.
Methods Mol. Biol. 315: 105–112.
18. Tkaczyk, C., D. D. Metcalfe, and A. M. Gilfillan. 2002. Determination of protein
phosphorylation in Fc ε RI-activated human mast cells by immunoblot analysis
requires protein extraction under denaturing conditions. J. Immunol. Methods
19. Hoeflich, K. P., J. Luo, E. A. Rubie, M. S. Tsao, O. Jin, and J. R. Woodgett. 2000.
Requirement for glycogen synthase kinase-3b in cell survival and NF-kB acti-
vation. Nature 406: 86–90.
20. Jensen, B. M., M. A. Beaven, S. Iwaki, D. D. Metcalfe, and A. M. Gilfillan.
2008. Concurrent inhibition of kit- and FcεRI-mediated signaling: coordinated
suppression of mast cell activation. J. Pharmacol. Exp. Ther. 324: 128–138.
21. Hughes, K., E. Nikolakaki, S. E. Plyte, N. F. Totty, and J. R. Woodgett. 1993.
Modulation of the glycogen synthase kinase-3 family by tyrosine phosphoryla-
tion. EMBO J. 12: 803–808.
22. Harwood, A. J. 2001. Regulation of GSK-3: a cellular multiprocessor. Cell 105:
23. Fiol, C. J., A. M. Mahrenholz, Y. Wang, R. W. Roeske, and P. J. Roach. 1987.
Formation of protein kinase recognition sites by covalent modification of the
substrate: molecular mechanism for the synergistic action of casein kinase II and
glycogen synthase kinase 3. J. Biol. Chem. 262: 14042–14048.
24. Frame, S., P. Cohen, and R. M. Biondi. 2001. A common phosphate binding site
explains the unique substrate specificity of GSK3 and its inactivation by phos-
phorylation. Mol. Cell 7: 1321–1327.
25. Okayama, Y., D. D. Hagaman, and D. D. Metcalfe. 2001. A comparison of
mediators released or generated by IFN-g-treated human mast cells following
aggregation of Fc g RI or Fc ε RI. J. Immunol. 166: 4705–4712.
26. Inoki, K., H. Ouyang, T. Zhu, C. Lindvall, Y. Wang, X. Zhang, Q. Yang,
C. Bennett, Y. Harada, K. Stankunas, et al. 2006. TSC2 integrates Wnt and
energy signals via a coordinated phosphorylation by AMPK and GSK3 to reg-
ulate cell growth. Cell 126: 955–968.
27. Dann, S. G., A. Selvaraj, and G. Thomas. 2007. mTOR Complex1-S6K1 sig-
naling: at the crossroads of obesity, diabetes and cancer. Trends Mol. Med. 13:
28. Shah, O. J., J. C. Anthony, S. R. Kimball, and L. S. Jefferson. 2000. 4E-BP1 and
S6K1: translational integration sites for nutritional and hormonal information in
muscle. Am. J. Physiol. Endocrinol. Metab. 279: E715–E729.
29. Sarbassov, D. D., D. A. Guertin, S. M. Ali, and D. M. Sabatini. 2005. Phos-
phorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science
30. Jacinto, E., V. Facchinetti, D. Liu, N. Soto, S. Wei, S. Y. Jung, Q. Huang, J. Qin,
and B. Su. 2006. SIN1/MIP1 maintains rictor-mTOR complex integrity and
regulates Akt phosphorylation and substrate specificity. Cell 127: 125–137.
31. Wullschleger, S., R. Loewith, and M. N. Hall. 2006. TOR signaling in growth
and metabolism. Cell 124: 471–484.
32. Hata, D., J. Kitaura, S. E. Hartman, Y. Kawakami, T. Yokota, and T. Kawakami.
1998. Bruton’s tyrosine kinase-mediated interleukin-2 gene activation in mast
cells: dependence on the c-Jun N-terminal kinase activation pathway. J. Biol.
Chem. 273: 10979–10987.
33. Samayawardhena, L. A., and C. J. Pallen. 2008. Protein-tyrosine phosphatase a
regulates stem cell factor-dependent c-Kit activation and migration of mast cells.
J. Biol. Chem. 283: 29175–29185.
34. Samayawardhena, L. A., J. Hu, P. L. Stein, and A. W. Craig. 2006. Fyn kinase
acts upstream of Shp2 and p38 mitogen-activated protein kinase to promote
chemotaxis of mast cells towards stem cell factor. Cell. Signal. 18: 1447–
35. Sundstro ¨m, M., J. Alfredsson, N. Olsson, and G. Nilsson. 2001. Stem cell factor-
induced migration of mast cells requires p38 mitogen-activated protein kinase
activity. Exp. Cell Res. 267: 144–151.
36. Martin, M., K. Rehani, R. S. Jope, and S. M. Michalek. 2005. Toll-like receptor-
mediated cytokine production is differentially regulated by glycogen synthase
kinase 3. Nat. Immunol. 6: 777–784.
37. Rodionova, E., M. Conzelmann, E. Maraskovsky, M. Hess, M. Kirsch, T. Giese,
A. D. Ho, M. Zo ¨ller, P. Dreger, and T. Luft. 2007. GSK-3 mediates differen-
tiation and activation of proinflammatory dendritic cells. Blood 109: 1584–
38. Ohteki, T., M. Parsons, A. Zakarian, R. G. Jones, L. T. Nguyen, J. R. Woodgett,
and P. S. Ohashi. 2000. Negative regulation of T cell proliferation and interleukin
2 production by the serine threonine kinase GSK-3. J. Exp. Med. 192: 99–104.
39. Kitaura, J., K. Asai, M. Maeda-Yamamoto, Y. Kawakami, U. Kikkawa, and
T. Kawakami. 2000. Akt-dependent cytokine production in mast cells. J. Exp.
Med. 192: 729–740.
572 ROLE OF GSK3b IN MAST CELL FUNCTION