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
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.
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572 ROLE OF GSK3b IN MAST CELL FUNCTION