Download Transduction Cascade In Myeloid Cells: A Novel Cytokine Signal

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βc Cytokine Receptor-Induced Stimulation of
cAMP Response Element Binding Protein
Phosphorylation Requires Protein Kinase C
In Myeloid Cells: A Novel Cytokine Signal
Transduction Cascade
Elaina Gubina, Xu Luo, E. Kwon, Kathleen Sakamoto, Yu
Fang Shi and R. Allan Mufson
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2001 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2001; 167:4303-4310; ;
doi: 10.4049/jimmunol.167.8.4303
http://www.jimmunol.org/content/167/8/4303
␤c Cytokine Receptor-Induced Stimulation of cAMP Response
Element Binding Protein Phosphorylation Requires Protein
Kinase C In Myeloid Cells: A Novel Cytokine Signal
Transduction Cascade1
Elaina Gubina,2* Xu Luo,2* E. Kwon,† Kathleen Sakamoto,† Yu Fang Shi,* and
R. Allan Mufson3*
T
he myeloid-specific colony-stimulating factors IL-3 and
GM-CSF are T cell-derived cytokines that allow the immune system to modulate the hemopoietic system during
stress. Both these cytokines and IL-5 signal their target cells
through a heterodimeric receptor consisting of a ligand-specific
binding subunit (␣) and a common signaling subunit (␤c) (1). The
␤c receptor transduces both the signals that enhance cell survival
by suppression of apoptosis, and those that signal cell proliferation. The ␤c subunit signal transduction cascade regulating DNA
synthesis has received extensive analysis; however, the transduction pathways responsible for regulating cell survival genes are far
less well understood.
Recent evidence from human myeloid cell lines dependent on
IL-3 or GM-CSF suggests that a newly described ␤c receptor-
*Department of Immunology, Holland Laboratory/American Red Cross, Rockville,
MD 20855; †Division of Hematology/Oncology, UCLA School of Medicine, Los
Angeles, CA 90095
Received for publication November 9, 2000. Accepted for publication August
1, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
activated protein kinase C (PKC)4 signaling cascade may be involved in regulating cell growth and survival. The human
megakaryoblastic leukemia cell line MO7E requires either IL-3 or
GM-CSF for survival and long-term growth (2). Stimulation of
MO7E cells with IL-3 results in enhanced tyrosine kinase activity,
activation of a phosphatidylcholine-specific phospholipase C (PCPLC), sustained accumulation of diacylglycerol, and activation of
PKC (3). Incubation of these cells with either IL-3 or GM-CSF in
the presence of structurally unrelated PKC inhibitors reduces cytokine-dependent apoptotic suppression (2). In the IL-3/GM-CSFdependent human erythroleukemia cell line TF-1, inhibitors of
PKC, but not inhibitors of the cAMP-dependent protein kinase A
(PKA), block both the suppression of apoptosis as well as bcl-2
mRNA and protein expression (4). Inhibition of a PC-PLC upstream of PKC also specifically blocks expression of bcl-2 but not
c-myc in TF-1 cells stimulated with IL-3 (5). Furthermore, overexpression of the specific novel isoform PKC⑀ in TF-1 cells maintains bcl-2 expression and suppresses apoptosis in the absence of
cytokine (6).
A link between PKC and activation of specific downstream transcription factors that might regulate survival or proliferation genes
after ␤c receptor engagement remains unclear. However, studies of
the immediate early growth gene egr-1 have provided a unique
candidate transcriptional component. A CREB element has also
1
This work was supported by National Institutes of Health Research Grant R01
CA53609-06.
2
E.G. and X.L. made equally significant contributions to this paper.
3
Address correspondence and reprint requests to Dr. R. Allan Mufson at the current
address: Division of Immunology/Hematology Branch, National Cancer Institute,
6130 Executive Boulevard, Executive Plaza North, Room 513, Rockville, MD 20852.
E-mail address: [email protected].
Copyright © 2001 by The American Association of Immunologists
4
Abbreviations used in this paper: PKC, protein kinase C; CRE, cAMP response
element; MAP, mitogen-activated protein; CAT, chloramphenicol acetyltransferase;
PC-PLC, phosphatidylcholine-specific phospholipaseC; PKA, protein kinase A; CBP,
CREB binding protein; CaMK, calmodulin kinase; SRE, serum response element; ki,
association rate constant; TPA, 12-O-tetradecanoyl phorbol-13-acetate; JAK, Janus
kinase; ERK, extracellular signal-regulated kinase; MAPK, MAP kinase; rsk, ●●●.
0022-1767/01/$02.00
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We have recently shown that IL-3R occupancy activates a phosphatidylcholine-specific phospholipase C, and the sustained diacylglycerol accumulation subsequently activates protein kinase C (PKC). In human IL-3-dependent myeloid cells (TF-1), the novel
PKC⑀ isoform regulates bcl-2 expression and cell survival. The report of a PKC activatable cAMP response element (CRE) in the
bcl-2 promoter and a role for PKC in bcl-2 expression in B cells led us to the hypothesis that PKC phosphorylation activates
transcription factor CREB after IL-3R engagement. We found that IL-3 and GM-CSF induced phosphorylation of CREB on
Ser133 in TF-1 cells, and this phosphorylation was blocked by two structurally unrelated classes of PKC inhibitors. An inhibitor
of cyclic nucleotide-dependent kinases did not block this phosphorylation. IL-4, which is biologically active in these cells but does
not use the ␤ common subunit, did not phosphorylate CREB on Ser133. Inhibition of mitogen-activated protein kinase kinase
activity also inhibited IL3-induced CREB phosphorylation. The PKC inhibitors, but not a cyclic nucleotide-dependent kinase
inhibitor, blocked IL-3 activation of CRE-dependent transcription from an egr-1 promoter/chloramphenicol acetyltransferase
(CAT) reporter construction transiently transfected into TF-1 cells. Finally, TF-1 cells stably overexpressing PKC⑀, but not the
␦ isoform of PKC, enhanced CRE-dependent CAT expression from the promoter/reporter construction. Therefore, it is likely that
a PKC⑀ kinase cascade resulting in CREB phosphorylation represents a novel signal transduction cascade for regulating cellular
gene expression through the ␤ common cytokine receptor. The Journal of Immunology, 2001, 167: 4303– 4310.
4304
Materials and Methods
Reagents and DNA vectors
Purified recombinant human cytokines were purchased from BioSource
International (Camarillo, CA). Protein kinase inhibitors H-7 and HA1004
were purchased from Biomol (Plymouth Meeting, PA). The PKC inhibitor
bisindolylmaleimide I was purchased from two sources. The bisindolylmaleimide purchased from L.C. Laboratories (Woburn, MA) is designated
GF109023X, and from Calbiochem (San Diego, CA) it is designated
GO6908. The inhibitor from both sources gave exactly the same results.
The PKC-specific hexanamide inhibitor NPC15432 and the mitogen-activated protein (MAP) kinase (MAPK) kinase inhibitor PD89059 were purchased from L. C. Laboratories. Specific Abs recognizing CREB or CREB
phosphorylated on Ser133 were purchased from Upstate Biotechnology
(Lake Placid, NY). The anti-phosphoCREB Ab was a rabbit anti-peptide
Ab that was made against residues 123–126 of rat CREB and is specific
only for phosphorylated CREB. This anti-phosphosphoCREB Ab recognizes both rat and human phosphoCREB. The anti CREB Ab is a rabbit Ab
made against amino acids 5–24 of human CREB. The Ab does not recognize phosphoCREB. The egr-1 promoter/chloramphenicol acetyltransferase (CAT) reporter constructions used in the transient transfection studies have been described previously (7). Briefly, the 116 enhancer
construction is composed of a CRE, serum response element (SRE), and
TATTA box promoter linked to a CAT reporter gene, 116 M, which was
the 116 construction with the CRE deleted, and 56, which had both enhancer elements deleted and contained only the promoter (7). Previous
work using EMSAs and transient transfection assays has shown that cytokine activation of this enhancer/promoter construction is dependent on
activation of the CRE and the presence of CREB in transcription
complexes.
Cell culture
Wild-type TF-1 cells or stably transfected TF-1 cell lines were routinely
passaged in RPMI 1640 medium supplemented with 10% FCS and 10%
A5637 bladder carcinoma-conditioned medium as a source of cytokines.
Derivation of cell lines stably overexpressing PKC isoforms was previ-
ously described (6). PKC-transfected cell lines were periodically assayed
for appropriate isoform overexpression by immunoblotting. Protein kinase
inhibitors were used at 5- to 10-fold of the in vitro association rate constant
(ki) values for enzyme inhibition as recommended by the manufacturers
and the open literature.
Immunoblotting
Immunoblotting was performed as described previously (4, 6). Briefly, cell
lysates were prepared and proteins separated by SDS-PAGE. Proteins were
transferred to nitrocellulose filters and the filters were probed with appropriate Abs. The anti-P Ser133 CREB Ab was used first, and then the blots
were stripped and reprobed with the anti-CREB Ab. Probed blots were
developed with ECL reagents (Amersham, Little Chalfont, England), and
the developed blots were exposed to Kodak (Rochester, NY) X-AR film.
Transient transfection
TF-1 cells were deprived of cytokine, but maintained in serum for 12 h
before transfection. Aliquots of 107 cells were electroporated with 10 ␮g of
egr-1 promoter/CAT reporter plasmid DNA and 10 ␮g of control plasmid
containing ␤-galactosidase driven by a CMV promoter in RPMI 1640 medium without serum or antibiotics as previously described (6). After electroporation, cells were incubated for 10 min on ice. The cells were then
transferred into 10 ml of fresh serum-supplemented RPMI 1640 medium in
the absence or presence of different combinations of cytokines and inhibitors. The cells were continually maintained in the presence of serum to
obviate any serum stimulation in the experiments. Cells were incubated at
37°C for 6 h in a humidified CO2 incubator before lysis for enzyme assay.
All transfections were performed in triplicate, and triplicate CAT and ␤-galactosidase assays were conducted for each sample to correct for variation
in transfection efficiency.
CAT activity assay
CAT activity was measured using an enzyme assay system obtained from
Promega (Madison, WI). Briefly, cells were washed with PBS and resuspended in 300 ␮l of lysis buffer. After a 10-min incubation at 60°C, lysates
were centrifuged. Aliquots of supernatant (25 ␮l) were collected and mixed
with 5 ␮l of n-butrylCoA containing 0.5 ␮Ci of [3H]chloramphenicol. The
assay volume was adjusted to 125 ␮l with 0.25 M Tris, pH 8.0. The assay
mixture was incubated at 37°C for 24 h. The organic fraction was extracted
with xylene, and radioactivity was determined by liquid scintillation counting. All CAT activities were corrected for transfection efficiency as determined from results with the control ␤-galactosidase plasmid.
Results
Inhibitors of the PKC catalytic domain block IL-3 induction of
CREB phosphorylation
We began by examining the effect of IL-3 stimulation on CREB
phosphorylation in factor-deprived TF-1 cells. Cells were deprived
of cytokine for 12 h and restimulated with 10 ng/ml recombinant
human IL-3. Lysates from stimulated and unstimulated cells were
separated by PAGE and transferred to nitrocellulose. Blots were
probed with a rabbit Ab specifically recognizing human CREB
phosphorylated on Ser133. A very low level of CREB phosphorylation was observable in the factor-deprived cells, and the addition
of IL-3 induced a strong increase in phosphorylation at 15 min
(Fig. 1). The induced level of phosphorylation had declined significantly by 30 min. It has already been demonstrated that activation of the ␤cR does not induce changes in cAMP levels in TF-1
cells, HL-60 cells, or primary eosinophils (7, 12, 13). In addition,
PKA inhibitors do not inhibit GM-CSF-induced CREB phosphorylation (12, 14). Thus, a cyclic nucleotide-independent protein kinase must be responsible for CREB phosphorylation in ␤c cytokine-dependent cells. When TF-1 cells were preincubated for 5
min with 30 ␮M preferential PKC inhibitor H-7 and stimulated
with IL-3 in the presence of this inhibitor, the Ser133 phosphorylation of CREB was completely abrogated. A similar incubation
with an equimolar concentration of the structurally related preferential PKA inhibitor HA1004 had no effect on IL-3 stimulation of
Ser133 phosphorylation. H-7 has a ki for PKC that is almost 1 log
lower than HA1004, but they inhibit PKA equivalently. Therefore,
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been identified in the egr-1 gene promoter, and GM-CSF activation of this promoter required CREB phosphorylation on Ser133
(7). Recently, a positive regulatory region has been found in the
bcl-2 promoter, and this region contains a cAMP response element
(CRE) consensus sequence (GTGACGTCA) (8). Expression of
bcl-2 in B cells is dependent on this promoter element and phosphorylation of the associated trans-activating factor CREB, although participation of other upstream regulatory elements was
also required. GM-CSF-induced CREB phosphorylation is independent of activation of the cAMP/GMP-dependent kinases (7).
CREB is resident in the nucleus, but its binding to CREB binding
protein (CBP) is dependent on the phosphorylation of Ser133 in the
transactivation domain of CREB. The interaction of CREB/CBP
with transcriptional complexes presumably initiates transcription
from CRE-containing genes (9). Initially this phosphorylation was
determined to be PKA-dependent, and later calmodulin kinase
(CaMK) was also implicated in CREB phosphorylation (10). In B
lymphocytes, cross-linking of surface Ig with anti-Ig Ab triggers
CREB Ser133 phosphorylation (11). Interestingly, however, this
Ig-induced phosphorylation was determined to be independent of
PKA or CaMK, but appeared to depend on PKC-mediated
phosphorylation.
It thus seemed possible that CREB could be a downstream transcription factor responsive to ␤cR-mediated PKC activation, and
an important effector in a novel ␤cR cytokine signal transduction
cascade regulating growth and survival genes. We have found in
TF-1 cells that active PKC is required for CREB Ser133 phosphorylation in response to ␤cR cytokines. In addition, we have determined that PKC is required for ␤cR cytokine stimulation of
CREB-dependent transcription in these cells. Finally, using TF-1
cell lines stably overexpressing PKC isoform ⑀, we have shown
that a specific novel isoform of PKC is responsible for stimulating
CREB-dependent transcription.
IL-3/GM-CSF CREB PHOSPHORYLATION REQUIRES PKC
The Journal of Immunology
4305
FIGURE 1. Effect of PKC catalytic domain inhibitors on IL-3-induced phosphorylation of CREB. TF-1
cells were cytokine-deprived for 18 h and restimulated
with human rIL-3 for the indicated times. Restimulation
was performed in the absence or presence of a 30 ␮M
concentration of H-7 or HA1004 and 5 ␮M
GF109023X. TPA was used at 10 nM. Cell lysates were
prepared and immunoblotted with equal numbers of
cells loaded per lane. Immunoblots were probed with an
Ab specific for CREB phosphorylated on Ser133 (A).
Blots were subsequently stripped and reprobed with an
Ab specifically recognizing CREB (B). Probed blots
were developed by chemiluminescence and exposed to
x-ray film.
NPC-15342, an inhibitor of the PKC regulatory domain, also
blocks IL-3-induced CREB phosphorylation
Both H-7 and GF1090123X inhibit PKC by competing for ATP
binding at the catalytic domain of the enzyme. To further ensure
the specificity of our inhibition of CREB phosphorylation through
PKC, we also tested the effect of a specific hexanamide derivative
PKC inhibitor, NPC-15342, that binds to the regulatory domain
of PKC (15). Very few protein kinases besides PKC have diacylglycerol binding domains, and NPC-15342 binding to this site
lends great PKC specificity to this inhibitor. The ki for inhibition
of PKC by NPC-15342 in intact cells is ⬃30 ␮M (15). Fig. 2
shows that between 5 and 100 ␮M NPC-15342 there was a concentration-dependent decrease in IL-3-induced phosphorylation of
Ser133 CREB in factor-deprived TF-1 cells. CREB phosphorylation induced by phorbol ester (TPA) in TF-1 cells was also blocked
by NPC-15342. Thus, two classes of structurally unrelated PKC
inhibitors acting through two different sites on the enzyme block
IL-3-induced CREB phosphorylation at the expected concentrations. These data strongly verify a role for PKC-mediated phosphorylation in the activation of this transcription factor after ␤cR
engagement.
GM-CSF but not IL-4 induces PKC-dependent CREB
phosphorylation in TF-1 cells
To determine whether other cytokines that use the ␤c subunit also
stimulate a PKC-dependent phosphorylation of CREB, we analyzed the response of TF-1 cells to GM-CSF. The CREB Ser133
phosphorylation response to GM-CSF was maximal at 30 min after
stimulation (Fig. 3). The relatively specific PKC inhibitor
GF109023X also blocked GM-CSF-induced CREB Ser133 phos-
phorylation as did H-7. The PKA inhibitor HA1004 actually
seemed to enhance GM-CSF-induced Ser133 phosphorylation. To
determine whether cytokines that do not use the ␤c subunit also
induce PKC-dependent CREB phosphorylation, we examined the
level of CREB Ser133 phosphorylation in factor-derived cells after
the addition of IL-4. TF-1 cells express IL-4 receptors, and IL-4
initiates intracellular signaling in these cells (16). The IL-4R does
not use the ␤c subunit but shares subunits with the IL-2R (16 –18).
Fig. 4 shows that the rapid phosphorylation of CREB Ser133 observed with IL-3 and GM-CSF was not observed with IL-4. Examination of later time points also did not show any increased
levels of phosphorylation in response to IL-4 (data not shown).
Therefore, it appears that PKC activation of CREB in myeloid
cells may be limited to cytokines using the ␤cR subunit. Furthermore, work is necessary to extend these findings to other non-␤c
cytokines.
Does MAPK participate in a ␤c PKC-dependent CREB
phosphorylation?
Previous work has shown that PKC is an upstream regulator of
Raf-1 kinase, which is the kinase that activates MAP/extracellular
signal-related kinase (ERK) kinase-1 (MAPKK), the upstream activator of p42 MAPK. If ␤c activation of PKC also activates
MAPK, then we would expect that the cell-permeable specific
FIGURE 2. NPC-15342, an inhibitor of the PKC regulatory domain,
blocks IL-3-induced CREB phosphorylation. TF-1 cells were cytokinedeprived for 18 h and restimulated with recombinant human IL-3 for 15
min in the presence or absence of NPC-15342 between 5 and 100 ␮M. Cell
lysates were prepared and immunoblotted with equal numbers of cells
loaded per lane. Immunoblots were probed with an Ab recognizing CREB
phosphorylated on Ser133 and developed with chemiluminescence. Blots
were subsequently stripped and reprobed with an Ab recognizing CREB.
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when used as a contrasting pair at the appropriate concentrations,
they can discriminate PKA from PKC (3). GF109023X (bisindolylmaleimide) specifically inhibits PKC in the micromolar range
in cell culture without inhibiting PKA. Incubation of TF-1 cells
with 5 ␮M GF109023X also completely abrogated IL-3-induced
serine phosphorylation of CREB. The classic PKC activator 12O-tetradecanoyl phorbol-13-acetate (TPA) also induced a marked
increase in CREB phosphorylation in TF-1 cells. Both PKC inhibitors blocked TPA-induced CREB phosphorylation in TF-1 cells
by phorbol ester at the appropriate concentrations (Fig. 1). Neither
PKC inhibitor altered cell viability during the short time courses
examined. We have shown previously that the time course for the
induction of apoptosis with either cytokine withdrawal or PKC
inhibition is 48 h (4, 6). Thus, we would not expect to see any
apoptosis during the short time courses presented here.
4306
MAPK kinase inhibitor 2⬘-amino-3⬘ methoxyflavone, also known
as PD 98059, would inhibit CREB phosphorylation in our system.
This inhibitor blocks MAPK activation without inhibiting tyrosine
kinases, PKC, or phosphatidylinositol 3-kinase. It has been used
successfully to determine the role of MAPK in epidermal growth
factor signal transduction and nerve growth factor-induced apoptosis (19, 20). We have found that 10 ␮M PD 98059 blocks
GM-CSF-induced phosphorylation of CREB in TF-1 cells (Fig. 5).
The inhibition was concentration-dependent between 1.0 and 10
␮M (data not shown). The bisindolylmaleimide PKC inhibitor
GO6908 completely inhibited IL-3-induced CREB phosphorylation, as observed in other experiments. Fig. 6 demonstrates that
PKC was still activatable in TF-1 cells after inhibition of MAPK
with PD 98059. Addition of phorbol ester to PD 98059-treated
cells produced a strong activation of CREB phosphorylation as
determined by immunoblotting, whereas the GM-CSF-induced
CREB phosphorylation was blocked. All lanes were equally as
determined by stripping the blot and reblotting with an Ab specific
for CREB. The manufacturer’s specifications showed that the inhibitors of PKC used in these studies have no effect on MAPK
phosphorylation activity. (data not shown). The activation of
MAPK with these cytokines is independent of PKC.
FIGURE 5. Effect of MAPK kinase inhibitor PD89059 on the GMCSF-induced phosphorylation of CREB in TF-1 cells. TF-1 cells were
deprived of cytokine overnight and treated with 10 ng/ml GM-CSF or
without either 10 ␮M PD89059 or 5 ␮M PKC-specific bisindolylmaleimide I (GO6976) for 15 min. Cell lysates were prepared and immunoblotted
for phosphoCREB. Blots were stripped and reprobed with anti-tubulin Abs
to verify equal loading in each lane.
egr-1 early response gene (14, 21). The transcriptional activation
of this gene in response to the latter cytokines in TF-1 cells requires the presence of the CRE contained within the ⫺116 nucleotide region of the egr-1 promoter. This region also contains an
SRE, but no protection of the SRE in DNase I footprinting assays
was observed. SRE/CAT constructs were not IL-3- or GM-CSFresponsive in TF-1 cells in transient transfection assays (21). The
binding of phosphorylated CREB to the CRE was shown to be
essential for induction of a transcription response to GM-CSF or
IL-3 (14, 21); however, the protein kinases responsible for the
phosphorylation of the trans-activating CREB after ␤cR engagement have not been determined. We used a set of previously described egr-1 promoter/CAT reporter constructions to confirm a
role for PKC in CREB-dependent transcription in TF-1 cells.
Three constructions were examined using transient transfection
into TF-1 cells: 56, which only contains the TATA box promoter
of the egr-1 gene; 116, which contains the promoter linked to a
SRE and CRE; and 116 mutant in which the CRE has been deleted.
TF-1 cells were deprived of cytokine for 12 h and then cotransfected by electroporation.
PKC inhibitors block CRE-dependent transcription from the
egr-1 promoter in myeloid cells
Previous work has shown that GM-CSF and IL-3 activate signaling pathways that result in rapid and transient activation of the
FIGURE 4. The effect of IL-4 on CREB phosphorylation in TF-1 cells.
TF-1 cells were deprived of cytokine for 18 h and stimulated with 20 ng/ml
IL-4. Lysates were prepared and CREB phosphorylation determined as in
Fig. 3.
FIGURE 6. Effect of MAPK inhibitor PD 89059 on PKC activation in
TF-1 cells. TF-1 cells were deprived of cytokine overnight and treated with
or without either PD89059 or the PKC-specific inhibitor bisindolylmaleimide I (GO6976) at the indicated concentrations. Cells were then stimulated with phorbol ester (3 nM TPA) or GM-CSF (5 ng/ml) for 15 min. Cell
lysates were prepared and immunoblotted for phosphoCREB. Blots were
stripped and reprobed with anti-CREB Abs to verify equal loading in each
lane. CREB immunoblot is not shown, but it demonstrated equal CREB
protein loading in each lane.
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FIGURE 3. The effect of GM-CSF on CREB phosphorylation and the
requirement for PKC. TF-1 cells were cytokine-deprived for 18 h and
restimulated with IL-5 at optimal concentrations. Some stimulations were
performed in the presence of 30 ␮M PKC inhibitor H-7, 30 ␮M HA1004,
or 5 ␮M GF109023X (GF). Immunoblots were prepared with aliquots of
equal numbers of cells loaded in each lane and probed for both CREB
phosphorylated on Ser133 and CREB itself as previously described. Autoradiograms were digitized and scanned with the 1-D Gel Scan program.
PhosphoCREB intensities were corrected for loading differences by comparison to the appropriate CREB intensity value. Quantitative values are
compared with the corrected phosphoCREB value in the deprived lane.
IL-3/GM-CSF CREB PHOSPHORYLATION REQUIRES PKC
The Journal of Immunology
4307
Overexpression of PKC isoform ⑀ but not ␦ enhances CREBdependent transcription from the egr-1 promoter in myeloid
cells
We have recently shown that TF-1 cells stably overexpressing the
PKC⑀ isoform are resistant to apoptosis after cytokine withdrawal
and show enhanced expression of the bcl-2 survival gene (6).
These findings coupled with the recent demonstration of a role for
PKC-dependent phosphorylation of CREB in regulating bcl-2 expression in B cells led us to investigate the role of this novel PKC
isoform in regulating transcription from the CRE containing 116
promoter/reporter construction. Vector, PKC⑀, and PKC ␦ stable
TF-1 transfectants that we have previously described were used as
hosts for transient transfection experiments with the egr-1 promoter/CAT reporter constructions. Cells were deprived of cytokine
FIGURE 7. PKC inhibitor blocks transcription from the CRE in the
egr-1 promoter in TF-1 cells. TF-1 cells were deprived of cytokine and
transiently cotransfected by electroporation with egr-1 promoter/CAT reporter constructions. Transfected cells were subsequently stimulated with
human rIL-3 alone or in the presence of 5 ␮M PKC inhibitor bisindolylmaleimide GF109023X (GF) or 30 ␮M PKA inhibitor HA1004 for 4 h, and
cell lysates were prepared for CAT assays. All transfections were performed in triplicate, and the CAT activities are corrected for transfection
efficiency. The mean value is presented and the SE was ⬍10% of the mean.
ⴱ, p ⬍ .05 compared with IL-3-deprived cells using Student’s t test. The
experiment presented is representative of three that gave similar results
overnight, and equal numbers of viable cells of each type were
electroporated with the 116 construct and a control plasmid to
monitor transfection efficiency. Six hours after transfection, cells
were lysed, and lysates assayed for CAT activity. The 116 mutant
construction (missing the CRE) did not signal in any of the vector
or PKC-transfected cell lines described (data not shown). This result is similar to the lack of expression of this CRE-deleted construction observed in cytokine-stimulated wild-type cells (Fig. 7).
Fig. 8 shows that after transfection of the 116 construction (containing the CRE) into the ⑀1 and ⑀10 cell lines, the basal level of
CAT expression was reproducibly 2- to 2.5-fold greater than the
basal activity in either the vector or PKC␦-transfected cell lines. In
fact, it was observed that CAT expression was significantly inhibited in the PKC␦ 25 transfectant. Although the responses of the
transfected cell lines to IL-3 was somewhat reduced in these experiments compared with wild-type cells, the IL-3-stimulated levels in the PKC⑀-overexpressing cell lines were, nevertheless,
higher than in any of the other transfected cell lines stimulated
with cytokine and assayed for CAT expression.
PKC inhibitor GF109023X reduces CRE-dependent
transcription in PKC⑀-overexpressing cell lines
It has been previously demonstrated that increased levels of PKC
protein yield levels of enzyme activity comparable to or greater
Table II. PKC inhibitor GF109023X inhibits basal transcription from a
CRE-dependent promotera
Table I. Effects of cytokines on CREB-dependent transcription in wildtype TF-1 cells a
Cytokine
Deprived
⫹IL-3
⫹GM-CSF
⫹IL-4
CAT Activity (cpm ⫻ 10
⫺3
Inhibitor Concentration
(␮M)
CAT
Activity
0.00
0.10
1.00
2.00
5.0
37 ⫾ 3.3
31 ⫾ 3.5
31 ⫾ 6.3
22 ⫾ 3.4*
16 ⫾ 3.7*
)
30.0 ⫾ 2.8
57.8 ⫾ 0.56*
71.0 ⫾ 7.0*
25.5 ⫾ 2.4
a
TF-1 cells were transfected as described in Materials and Methods and then
stimulated with the indicated cytokines at 10 ng/ml. Six hours after stimulation, cells
were harvested and lysed for CAT assays. Transfections were performed in triplicate
and CAT assays corrected for transfection efficiency. The results presented are the
mean ⫾ SEM from one experiment. ⴱ, p ⬍ 0.05 compared to deprived cells using
Student’s t test. The experiment was repeated three times with similar results.
a
TF-1 cells were deprived of cytokine and transfected with the egr-1 116 construction. They were then incubated in medium containing the indicated concentration
of GF109023X for 6 h. Cells were then harvested and assayed for CAT activity. The
results presented are the means ⫾ SD from triplicate transfections, and the experiment
was repeated three times with similar results. ⴱ, p ⬍ .05 compared to noninhibitortreated control using Student’s t test.
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After stimulation for 4 h with cytokine, CAT activity was assayed. Transfection efficiency was corrected for in each transfection as previously described (6), and three individual transfections
were performed for each condition in each independent experiment. Preliminary experiments using a ␤-galactosidase reporter
driven by the CMV promoter indicated no difference in gene expression driven by the CMV promoter between control transfected
cells and those incubated with PKA and PKC inhibitors for the 6 h
required of the assays (data not shown). Table I shows that IL-3
and GM-CSF both stimulate the egr-1 enhancer promoter construction with increases in CAT expression of ⬃2-fold above unstimulated cells. This level of stimulation is similar to what has
been reported previously with these cytokines using this construction (14, 21). IL-4, which neither uses the ␤cR subunit nor stimulates CREB phosphorylation, does not drive this promoter construction. Fig. 7 shows that the 116 mutant construction lacking the
CRE has neither basal expression in TF-1 cells nor activation of
transcription by IL-3 (the 56 activity is background). The figure
further shows that PKC inhibition by GF109023X blocks the ability of IL-3 to stimulate CAT expression from the116 construction
containing the CRE. Incubation of transiently transfected cells
with the PKA inhibitor HA1004 does not inhibit IL-3 stimulation
of CREB-dependent transcription from 116 in these cells. H-7 had
the same effect as GF109023X (data not shown). Interestingly,
GF109023X also inhibits the basal level of transcription from this
construction; however, IL-3 is unable to restore the level of CAT
expression to the basal unstimulated level in the presence of the
inhibitor (Fig. 7, V6 116 ⫹ GF lane). We confirmed that the inhibition of CREB-dependent basal transcription is concentrationdependent for GF109023X between 1 and 5 ␮M (Table II). The
inhibitors did not alter the viability of the transfectants after incubation. This demonstrates that PKC is required for ␤c cytokine
stimulation as well as basal transcription from this CREB-dependent promoter.
4308
than what is obtained after enzyme activation with endogenous
levels of protein. To confirm that the enhanced transcription from
the 116 construct in the PKC⑀ cells was in fact due to the overexpression of the ⑀ isoform enzyme activity, we performed a transient transfection experiment in the presence of the bisindolylmaleimide-specific PKC inhibitor GF109023X (5 ␮M). Fig. 9 shows
that in the ⑀1 cell line both the basal and IL-3-stimulated levels of
CAT expression are inhibitable by the specific PKC inhibitor
GF109023X. A similar effect is observed in the vector 6 transfectants; however, as observed in the experiment presented in Fig. 8,
the ⑀1 cells expressed ⬃3-fold the level of CAT in the control
vector-transfected cells. Furthermore, in the presence of the inhibitor IL-3 was unable to restore basal CAT level to those measured
in control ⑀1 transfectants. Thus, the 3-fold enhancement in basal
CAT expression observed in ⑀1 after transient transfection compared with the expression from the vector 6 cell line requires active
PKC⑀.
Discussion
Using two different classes of relatively specific PKC inhibitors
and an Ab specific for the Ser133 phosphorylated form of CREB,
we have shown that CREB Ser133 phosphorylation is dependent on
active PKC in myeloid cells. In addition to H-7 and GF109023X,
which inhibit at the PKC catalytic domain, we have also shown
that NPC-15342, an inhibitor that binds to the unique PKC regulatory domain, also inhibits CREB phosphorylation. HA-1004,
which is highly structurally related to H-7, but has a 10-fold lower
ki for inhibiting PKA or CaMK compared with PKC, did not inhibit IL-3-induced CREB Ser133 phosphorylation at a concentration equimolar to H-7. In addition, direct activation of PKC by
phorbol ester in these cells also induced CREB Ser133 phosphorylation. In TF-1 cells, all the cytokine receptors using the ␤c subunit (i.e., IL-3 and GM-CSF) and inducing Janus kinase (JAK)-2
phosphorylation also induce phosphorylation of CREB on Ser133
through PKC. These cells also express biologically active IL-4R;
however, the IL-4R uses neither the ␤c subunit nor JAK-2 for
FIGURE 9. The effect of PKC inhibitor GF109023X on CRE-dependent egr-1 promoter activity in the PKC ⑀1-overexpressing cell line. PKC⑀
were cytokine-deprived and transfected with the egr-1 promoter/reporter
constructions. Transfected cells were then unstimulated, stimulated with 10
ng/ml IL-3, or 10 ng/ml IL-3 in the presence of protein kinase inhibitors,
and incubated at 37°C. At the end of the incubation, cells were lysed for
CAT assay. Transfections were performed in triplicate, and CAT activities
were corrected for transfection efficiency. The mean activity is presented
and the SEM was ⬍10% of the mean. ⴱ, p ⬍ .05 compared with the
corresponding noninhibitor-treated control using Student’s t test. The results presented are representative of three experiments with similar results.
signaling. Instead, it associates with the common ␥ chain of the
IL-2R and induces JAK-1 and JAK-3 phosphorylation in target
cells (16, 18). However, IL-4 did not induce CREB Ser133 phosphorylation in TF-1 cells. Thus, the activation of CREB phosphorylation through PKC may be part of a signal transduction cascade
shared among ␤cR-using cytokines, but this needs to be more extensively studied. Previous work on the GM-CSF induction of the
egr-1 gene also demonstrated phosphorylation of CREB on Ser133,
but the kinases involved were not identified (7).
These results are consistent with results on CREB Ser133 phosphorylation in B lymphocytes stimulated by cross-linking the surface IgGR (22). In B cells, down-regulation of PKC by extended
incubation with phorbol ester blocks IgG stimulation of CREB
phosphorylation. Furthermore, CD40 ligand and LPS, which do
not activate PKC in B cells, do not induce CREB phosphorylation.
Using relatively specific PKC inhibitors, it was also shown that
PKC but not PKA or CaMK are required for surface IgG-stimulated CREB phosphorylation in B cells (23). In T cells, TCRmediated CREB phosphorylation also requires PKC and is independent of PKA and CaMK activity (24). Thus, both myeloid and
lymphoid cells with receptors capable of inducing CREB phosphorylation use a PKC cascade.
To determine whether PKC activation was directly required for
␤cR cytokine induction of CREB phosphorylation we used an egr1-based promoter construction containing an SRE and a CRE
linked to a CAT reporter gene. We have shown that the induction
of CAT transcription from this construction was dependent on the
CRE and inhibited by the structurally unrelated PKC inhibitors
H-7 and GF109023X. The PKA and CaMK inhibitor HA 1004 did
not inhibit cytokine-induced transcription from the construct. Previous DNase I footprinting assays of the SRE in this construction
has shown no protection, and SRE/CAT constructs did not confer
GM-CSF or IL-3 responsiveness in transient transfection assays
(14, 21). The binding of phosphorylated CREB to the CRE in the
116 construction was shown to be the essential activation signal
for the induction of a transcription response to GM-CSF in experiments transfecting the promoter/reporter construction with a vector overexpressing CREB (14). Interestingly, we also observed a
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FIGURE 8. The activity of the CRE in the egr-1 promoter in TF-1 cells
overexpressing novel PKC isoform. TF-1 cell lines stably overexpressing
the PKC ␦ (␦8 and ␦25), PKC⑀ isoform (⑀1 and ⑀10), or stably transfected
with the expression vector (vector 6) were cytokine deprived and transiently transfected with the ⫺116 egr-1 promoter/reporter construction.
The transfected cells were either not stimulated or stimulated with 10 ng/ml
human rIL-3, and 6 h after incubation at 37°C were lysed and assayed for
CAT activity. Transfections were performed in triplicate, and CAT activities were corrected for transfection efficiency. The mean value is presented
and the SEM was ⬍10% of the mean. The (ⴱ) and (⫹) indicate that p ⬍
.05 compared with IL-3-deprived or IL-3-stimulated vector 6-transfected
cells, respectively, using Student’s t test. The experiment presented is representative of three that gave similar results.
IL-3/GM-CSF CREB PHOSPHORYLATION REQUIRES PKC
The Journal of Immunology
basal level of CAT expression in TF-1 cells, and this background
level was also reduced by preincubation with the PKC inhibitors.
CREB is resident in the nucleus, and our data would indicate that
even under unstimulated conditions PKC provides a low basal
level of phosphorylation. The CREB Ser133 immunoblots did not
reveal significant levels of basal CREB phosphorylation. Thus, the
CAT expression assays appear to be more sensitive readout for the
levels of endogenous CREB phosphorylation. The fact that IL-3
stimulation could raise CAT expression levels to a small degree in
the presence of a PKC inhibitor, but did not restore them to the
levels in noninhibitor-treated cells, suggests that PKC is required
for both the basal level of expression and the cytokine-stimulated
increase in expression.
It should be noted that it was previously reported that IL-3 did
not use CREB to stimulate transcription from this promoter (14).
However, the previous studies were conducted by cotransfecting
plasmids overexpressing CREB or a mutated CREB with the reporter promoter construct. The lack of stimulation in these experiments may have been due to the high levels of background expression from the control-mutated construction that obscured the
IL-3 stimulation. The experiments reported here using the endogenous CREB levels do not have this complication, and we were
consistently able to observe an IL-3 stimulation of transcription.
Our transient transfections of CRE reporter promoter constructions into stable PKC⑀-overexpressing cell lines confirmed a role
for this PKC isoform in cytokine-induced CREB phosphorylation
in myeloid cells, and leads us to suggest an important role for this
novel isoform in CREB phosphorylation. The PKC⑀ cells had far
higher levels of basal CAT gene expression than either the vectortransfected cells or the PKC␦-overexpressing cells. The fact that
one novel isoform (⑀) but not another (␦) can specifically enhance
CREB phosphorylation suggests an important role for this specific
isoform in regulating the activity of this transcription factor. Little
information is available on PKC isoform specificity for transcription factor phosphorylation, and it would be quite interesting to
know whether this isoform were also responsible for CREB phosphorylation in response to surface IgG cross-linking in B lymphocytes (22, 23). Although MSK-1 (24) and pp90 rsk-2 (ribosomal
S6 kinase) are possible CREB kinases (25, 26) during signal transduction, direct evidence that GM-CSF activates rsk-2 phosphorylation of CREB has been published (26). This kinase acts downstream of stimulation by the ERK/p38 MAPK cascade (27–29),
and PKC⑀ has been shown to activate the ERK-1/p38 MAPK cascade through phosphorylation of Raf-1 (30). Our experiments inhibiting MAPK kinase activity resulting in inhibition of IL-3-induced CREB phosphorylation implicates the MAPK cascade in the
␤cR-induced phosphorylation of CREB. Bitorff et al. (31) have
shown that in TF-1 cells both IL-3 and GM-CSF activate MAPK
activity. They showed that IL-3 and GM-CSF showed the same
degree of activation and kinetics of activation in TF-1 cells. Therefore, it is likely that a PKC activation of the MAPK cascade followed by rsk-2 phosphorylation of CREB is involved in ␤cR signal transduction. The incomplete inhibition of CREB
phosphorylation observed in our studies suggests that PKC itself
may also directly contribute to the phosphorylation of CREB. Several pieces of evidence suggest that PKC can phosphorylate CREB
directly. The Ser117 in the CREB analog CRE modulator (CRE)
that corresponds to 133 in CREB itself is phosphorylated directly
by PKC or RSK (32–33). It is also established experimentally in
vitro that the PKC⑀ isoform can directly phosphorylate CREB
(30). Investigation of the contribution of other PKC isoforms to the
␤c cytokine receptor-induced phosphorylation of CREB should be
undertaken. Taken together, our data would suggest that IL-3/GMCSF receptor engagement initiates a novel signal transduction
pathway in which receptor-associated tyrosine kinases activate a
PC-PLC, generating a sustained increase in diacylglycerol levels.
This is followed by stimulation of PKC⑀, and PKC⑀ induces phosphorylation of CREB through a combination of MAPK cascade
activation of RSK-2 kinase and possible direct phosphorylation by
PKC. The diagram in Fig. 10 presents a schematic of such a
pathway.
Such a novel pathway has not received significant attention in
studies of ␤cR cytokine signal transduction; however, recent data
on the regulation of bcl-2 expression and apoptotic suppression
emphasize the important functional role of such a signal transduction cascade. The activation of a PC-PLC and PKC⑀ probably
plays an important role in the regulation of genes containing an
egr-1-like promoter containing a CRE. Expression of bcl-2 and
perhaps other survival genes in response to IL-3 and GM-CSF is
likely regulated by this cascade (6 –10). In PKC⑀-overexpressing
TF-1 cells, the high levels of bcl-2 expression can be reduced by
PKC inhibitors. Overexpression of PKC⑀ in TF-1 cells enhances
the survival of these cells in the absence of cytokine and induces
high level sustained bcl-2 gene expression (10). Studies of the
bcl-2 promoter in B cells have revealed that this promoter contains
a positive regulatory region containing a CRE element (11). Crosslinking of surface Ig receptor on mature B cells leads to increased
bcl-2 promoter activity through a PKC-dependent pathway involving phosphorylation of CREB. Reagents that increased cAMP levels had no effect on the transcription from the CRE-containing
promoter element in these cells. Furthermore, a similar PKC-dependent phosphorylation of CREB was involved in the enhanced
bcl-2 expression observed in immature B cells treated with survival signals like phorbol ester (11). It would thus appear likely
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FIGURE 10. Schematic diagram of the role of PKC in ␤c cytokine receptor signal transduction. Activation of the ␤c subunit by IL-3, GM-CSF,
or IL-5 activates enhanced tyrosine kinase activity (LYN and JAK), resulting in activation of PC-PLC. PC-PLC activation results in sustained
increases in diacylglycerol that activate the novel PKC⑀ isoform. PKC⑀
either directly or through activation of MAPK kinase pathway phosphorylates Ser133 in CREB. This transcription factor phosphorylation induces
the transcription of survival genes like bcl-2, whose promoters contain
CRE elements.
4309
4310
that in ␤c-dependent myeloid cell lines regulation of bcl-2 gene
expression occurs through the activation of PKC⑀ and subsequent
phosphorylation of CREB Ser133 leading to CREB/CBP binding to
the CRE in the bcl-2 downstream regulatory region. A definite role
for CREB in regulating bcl-2 expression will require appropriate
experiments with a bcl-2 reporter promoter construction.
A role for CREB phosphorylation in the suppression of apoptosis has also been shown in human melanoma cells (34). A dominant negative form of CREB transfected into human melanoma
cells rendered them susceptible to apoptosis induced by increasing
intracellular calcium ion concentrations. In addition, this dominant
negative form also inhibited CAT expression from a CRE-dependent promoter reporter construction transfected into these cells.
Thus, CREB phosphorylation may be important in the regulation
of expression of genes contributing to cell survival and suppression of apoptosis in a number of different cellular systems.
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