From the Department of Medical Biochemistry,
Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo,
Norway and ¶ Medical Genetics, Department of Medical Biochemistry,
Göteborg University, SE-405 30 Göteborg, Sweden
Received for publication, January 10, 2003, and in revised form, March 3, 2003
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ABSTRACT |
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Forkhead/winged helix (FOX) transcription factors
are essential for control of the cell cycle and metabolism. Here, we
show that spleens from Mf2 In T cells, activation of the cAMP-dependent protein
kinase type I (PKA type I)1
pathway inhibits the downstream activation events that follow TCR
triggering (1). PKA type I is recruited to the TCR-CD3 complex
following T cell activation (2) and elicits an inhibitory pathway in
lipid rafts that negatively modulates signaling through the TCR. This
pathway involves activation of C-terminal Src kinase by PKA type I. Active C-terminal Src kinase subsequently inhibits Lck by
phosphorylation of the C-terminal regulatory tyrosine 505 (3). This
inhibitory mechanism determines the threshold of T cell activation with
implications in several disease conditions. In human immunodeficiency
virus infection, high levels of cAMP and thereby hyperactivation of PKA
type I leads to T cell hyporesponsiveness and T cell anergy (4, 5).
Conversely, a deficiency in PKA type I regulatory subunit expression
and PKA type I activity has been reported in the autoimmune disorder
systemic lupus erythematosus (6).
PKA is activated by the intracellular second messenger cAMP, leading to
dissociation of two active catalytic (C) subunits from a regulatory
dimer (R2) (reviewed in Ref. 7). The R subunit isoforms
(RI A complex set of mechanisms appears to regulate expression of the RI Winged helix/forkhead transcription factors FOXD1 (Freac4) (17, 18) and
FOXD2 (Freac9/FKHL17) (19) were both cloned from human kidney and share
identical DNA-binding domains but have very low similarity in other
regions. We have recently reported that FOXD1 and the related factor
FOXC2 function as inducers of RI Here, we report the finding that T cells from FOXD2 Reverse Transcription (RT)-PCR--
Complementary DNA was
synthesized by reverse transcription using a commercial kit
(Invitrogen, ThermoScriptTM RT-PCR System, catalog number
11146-024). Total cellular RNA (5 µg of Jurkat, T cell, B cell, or
monocyte total RNA) was reverse transcribed at 42 °C using both
oligo(dT)20 and random hexamer primers. Subsequently, the
cDNA was subjected to 45 PCR amplification cycles (95 °C for
30 s, 99 °C for 10 s, 59 °C for 1 min, and 70 °C for
2 min) and a final incubation at 72 °C for 10 min. Amplification products were separated on a 1% agarose gel stained with
ethidium bromide. The human FOXD2 sense 5'-GGA GAA CAG GCA AAC TCG GAG CAG T-3' and antisense 5'-CGA GTG ATC ACC AGG AGC GAA CTT G-3' primers
amplified a 197-bp fragment of the FOXD2 cDNA.
Real Time Quantitative RT-PCR--
Sequence-specific PCR primers
were designed for RNA Extraction and Northern Blot Analysis--
Total RNA from
homogenized mouse spleens was extracted by the guanidium
isothiocyanate/CsCl method as previously described (23), and Northern
blot analysis was performed using 20 µg of total RNA. The RNA was
denatured in 50% (v/v) formamide and 6% (v/v) formaldehyde, subjected
to electrophoresis in a 1.5% (w/v) agarose gel containing 6.7%
formaldehyde, and blotted on to a nylon membrane. Complementary DNA
probes for mouse RI Immunoblotting--
Jurkat cells and C57/BL6 mouse spleen
lymphocytes (splenocytes) were collected by centrifugation (10 min,
600 × g), washed once in cold phosphate-buffered
saline, and resuspended in 500 µl of a buffer containing 10 mM potassium phosphate, pH 6.8, 1 mM EDTA, 10 mM CHAPS (Sigma) and CompleteTM protease
inhibitor mix (1 tablet/10 ml; Roche Applied Science). Cell suspensions
were sonicated three times (Heat Systems Ultrasonics) and centrifuged
for 5 min at 12,000 × g. The supernatants were stored
at Mouse Spleen Lymphocyte Proliferation Assays--
Spleens from
adult C57/BL6 FOXD2 Construction of Reporter and Expression Plasmids--
Deletion
constructs from the RI Preparation, Stimulation, and Transfection of Cell Cultures for
Promoter Analyses--
Jurkat TAg cells (a human leukemic T
cell line stably transfected with SV40 large T antigen) were grown in
RPMI 1640 medium (Invitrogen) containing streptomycin (100 g/liter),
and penicillin (70 mg/liter) supplemented with 10% heat-inactivated
fetal calf serum. For transfections, 2 × 107 cells
were resuspended in 0.4 ml of Opti-MEM (Invitrogen) and transiently
transfected with a total of 20 µg of DNA (2.5 µg of CAT reporter
plasmid, 2 µg of internal luciferase control vector, 7.5 µg of
expression vector(s) for FOX and/or PKB, and the respective empty
vectors to a total of 20 µg) by electroporation (250 V/cm, 950 microfarads) in cuvettes with a 0.4-cm electrode gap. The cells were
then expanded in 20 ml of complete medium in 75-cm2 culture
flasks and incubated for 20 h. For stimulation, the cAMP agonist
8-CPT-cAMP was added to a final concentration of 100 µM after 20 h, and the cells were cultured for another 10 h.
Luciferase and CAT Assays--
The Jurkat cells were harvested
in reporter lysis buffer (Promega) 30 h after transfection. CAT
activities were measured using an organic phase extraction method (26)
and normalized for expression from the Luciferase-encoding vector pGL3
Control (Promega). The luciferase activities were measured using a
luciferase assay reagent (Promega) and a Wallac 1251 luminometer
(Amersham Biosciences, Helsinki, Finland).
FOXD2 Is Expressed in T Cells--
The presence of FOXD2 in
leukocytes was initially seen in a tissue blot screen (not shown), and
we went on to map the expression in leukocyte subsets (B and T cells
and monocytes) purified from human peripheral blood. Fig.
1 shows that FOXD2 is expressed in T
cells and monocytes but not detected in B cells as assessed by RT-PCR.
Reduced Levels of RI FOXD2 Tunes the Sensitivity of T Lymphocytes to cAMP in
Vivo--
To explore the impact of FOXD2 regulation of RI FOXD2 Induces the RI Co-expression of PKB We have previously shown that the forkhead transcription factor
FOXC2 induces RI The inductive effect of PKB In conclusion, our data indicate that FOXD2 is a physiological
regulator of RI/
(FOXD2
/
) mice have reduced
mRNA (50%) and protein (35%) levels of the RI
subunit of the
cAMP-dependent protein kinase. In T cells from
Mf2
/
mice, reduced levels of RI
translates functionally
into ~2-fold less sensitivity to cAMP-mediated inhibition of
proliferation triggered through the T cell receptor-CD3 complex. In
Jurkat T cells, FOXD2 overexpression increased the endogenous levels of
RI
through induction of the RI
1b promoter. FOXD2 overexpression
also increased the sensitivity of the promoter to cAMP. Finally,
co-expression experiments demonstrated that protein kinase
B
/Akt1 work together with FOXD2 to induce the RI
1b promoter
(10-fold) and increase endogenous RI
protein levels further. Taken
together, our data indicate that FOXD2 is a physiological regulator of
the RI
1b promoter in vivo working synergistically with
protein kinase B to induce cAMP-dependent protein kinase RI
expression, which increases cAMP sensitivity and sets the threshold for cAMP-mediated negative modulation of T cell activation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, RI
, RII
, and RII
) can combine with the
catalytic subunit to form either PKA type I
(RI
2C2/RI
2C2) or
PKA type II
(RII
2C2/RII
2C2)
holoenzymes that have different affinities for cAMP and thus activate
at low or high local concentrations of cAMP in the cell
(Kact of 50-100 nM
versus 200-400 nM, respectively) (8). PKA types
I and II are also targeted differently in the cell through binding to A
kinase-anchoring proteins (reviewed in Ref. 9). When RI subunits are
up-regulated, cAMP sensitivity of PKA increases and thereby lowers the
threshold for activation of cAMP-mediated downstream effects (10).
Approximately 75% of PKA in T cells is of type I, and RI
is the
dominating R isoform.
subunit of PKA. First, several promoters differentially regulate the
expression of alternatively spliced RI
mRNAs. Second, these
mRNAs contain different 5'-untranslated regions encoded by the
first exons that may exhibit different translation efficiency. Five
RI
splice variants have been cloned (RI
1a-e), two of which are
ubiquitously expressed (RI
1a and RI
1b) (11-13). In addition, RI
protein levels are regulated by mechanisms such as rapid
degradation of excess protein not associated with catalytic subunit,
resulting in a regulatory:catalytic ratio of ~1:1 (14). RI
expression is regulated by cAMP in many cell types, and regulation at
both the transcriptional and post-transcriptional level has been
reported (12, 15, 16).
expression in testicular Sertoli
cells and adipocytes, respectively (21,
22).2 Having discovered the
presence of FOXD2 in T cells, we wanted to examine whether FOXD2
functions as an inducer of RI
expression in T cells by a similar
mechanism as reported in adipocytes and testicular cells.
/
mice have
reduced levels of RI
1b mRNA and RI
protein and a decreased sensitivity to cAMP-mediated inhibition of immune function.
Furthermore, FOXD2 regulates the RI
1b promoter and works together
with both cAMP and PKB to induce the RI
1b promoter and elevate
endogenous levels of RI
protein. In conclusion, our data indicate
that FOXD2 is important for expression of RI
mRNA and RI
protein in T lymphocytes in vivo and thereby tunes the
sensitivity to cAMP and sets the threshold for cAMP-mediated immunomodulation.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin (forward primer, 5'-AGG CAC CAG GGC GTG
AT-3', and reverse primer, 5'-TCG TCC CAG TTG GTG ACG AT-3';
GenBankTM accession number NM_001101) using the Primer
Express software version 1.5 (Applied Biosystems, Foster City, CA).
Quantification of mRNA was performed using the ABI Prism 7700 (Applied Biosystems) as suggested by the manufacturer. Briefly, 100 ng
of RNA was reverse transcribed using TaqMan reverse transcription
reagents (Applied Biosystems). SyBr Green assays were performed using
2× SyBr Green Universal Master Mix (Applied Biosystems) and 300 nM sense and antisense primers and water made up to 25 µl. The specificity of the SyBr Green assays was assessed by melting
point analysis and gel electrophoresis. All of the samples were
analyzed in triplicate.
1b 5'-untranslated region (11) and human RI
cDNA were labeled with [
-32P]dCTP using the
megaprime DNA labeling system (Amersham Biosciences). Hybridization was
performed with 50% formamide at 42 °C, and the filters were washed
four times in a solution containing 0.1-0.5× SSC (300 mM
NaCl and 30 mM sodium citrate, pH 7.0) with 0.1% SDS at
50 °C for 30 min. The filters were subjected to electronic autoradiography in a Packard Instant Imager
-scintillation counter.
70 °C until analysis. The protein samples were diluted in SDS
sample buffer and denaturated for 5 min at 100 °C before loading
onto a one-dimensional SDS-polyacrylamide gel (4.0% stacking gel, 10%
separating gel). 20 µg of total protein were loaded in each lane,
subjected to electrophoresis, and subsequently transferred onto
polyvinyldifluoride membranes (Millipore, Bedford, MA) by electroblotting. The membranes were blocked overnight at 4 °C in a
solution containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20, and 5% milk and then incubated
with a mouse monoclonal antibody against human or rat RI
and RII
(1:250) (Transduction Laboratories, Lexington, KY) or a rabbit
polyclonal antibody against catalytic subunit (1:250) (C-20, Santa Cruz
Biotechnology Inc., Santa Cruz, CA) in blocking solution for 2 h
at room temperature. The membranes were washed in a solution containing
25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05%
Tween 20. Immunoreactive proteins were visualized by Supersignal
(Pierce) using horseradish peroxidase-conjugated mouse or rabbit
secondary antibodies (1:5000) (Jackson ImmunoResearch Laboratories,
Inc., West Grove, PA) and subjected to autoradiography. The films were
scanned, and the densities of bands were quantified using the Scion
Image package (downloaded from www.scioncorp.com).
/
(24) or wt mice were extracted and converted
into single-cell suspensions by squeezing through a cell strainer in
serum-free medium (RPMI 1640 medium, 1% non-essential amino
acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin/streptomycin).
Erythrocytes were depleted in a buffer containing 10 mM
KHO3, 155 mM NH4Cl, and 8.9 µM EDTA. Spleen lymphocytes (2.5 × 106/ml) suspended in the medium defined above supplemented
with 10% heat-inactivated fetal calf serum were seeded into anti-mouse CD3 T cell activation plates (Becton Dickinson) in a total volume of
100 µl and placed in a CO2 incubator at 37 °C for
48 h. After 48 h of culturing, the cells were pulsed with 5 µCi of [3H]thymidine, and 4 h later cultures were
harvested onto filter plates and subsequently analyzed by
-scintillation counting. When used, cAMP agonist (8-CPT-cAMP; 0-100
µM; BioLog, Life Science, Bremen, Germany) was added
prior to incubation.
promoter regions RI
1a+1b (
882 to +77),
RI
1a (
882 to
310), and RI
1b (
406 to +77) were inserted into
the pCAT basic reporter vector (12). The numbers are relative to the
downstream transcription start of promoter 1b. The expression vector
for FOXD2 was constructed by insertion of the coding region into pCB6+,
and the FOXD1 coding region was inserted in pEVRFO. Expression vectors
for wt or mutant PKB
/Akt1 isoforms were created in pCMV6 fused to a
C-terminal hemagglutinin epitope tag (25). All of the expression
vectors contained CMV promoters. The internal luciferase control vector
was pGL3 control (Promega).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
FOXD2 is expressed in leukocyte subsets.
Total cellular RNA from Jurkat cells and from purified human peripheral
blood B cells, T cells, and monocytes was reversed transcribed using
both oligo(dT)20 and random hexamers. Subsequently,
the cDNA was subjected to 45 PCR amplification cycles using primers
within the coding region of human FOXD2 cDNA followed by separation
and visualization of amplification products on 1% agarose gels stained
with EtBr. Relative -actin levels in the different leukocyte
subset samples determined by real time RT-PCR are presented
below. Nd, not determined.
mRNA and Protein in Spleens from
FOXD2
/
Mice--
To examine whether FOXD2 regulates
RI
expression in T cells by a similar mechanism as FOXC2 in
adipocytes (22) and FOXD1 in testicular Sertoli cells (21), we first
examined spleens from homozygous (
/
) and heterozygous (+/
) FOXD2
null mutant mice compared with wt (+/+) for levels of expression of
total RI
mRNA (Fig. 2A)
and levels of the RI
1b mRNA with the 1b leader exon originating
from the RI
1b promoter (Fig. 2B). We found that levels of
both total RI
mRNA and of the 1b splice variant were reduced by
40-50% in the FOXD2-deficient mice relative to wt, whereas RI
1a
mRNA was not detected (not shown). Western blotting showed that
RI
protein levels were decreased by 35% in FOXD2
/
compared with
wt spleens, whereas RII
levels were increased moderately (Fig.
2C). These observations indicate that FOXD2 regulates RI
mRNA and protein levels in T lymphocytes in vivo.
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Fig. 2.
Reduced RI mRNA
and protein expression in FOXD2
/
mice. A and
B, Northern blot analysis was performed using total RNA from
homogenized spleens from either homozygous (
/
) (black
bars) or heterozygous (+/
) (light gray bars) FOXD2
mutant and wt (+/+) (gray bars) mice (n = 2 in each group). The filters were hybridized with
32P-labeled probes for RI
cDNA (A) and
mouse RI
1b 5'-untranslated region (1b) (B). The levels
of RI
and RI
1b mRNA were analyzed by densitometric scanning,
normalized for 28 S RNA levels, and presented relative to wt set to 1 (bar chart). The elevated levels of RI
mRNA in FOXD2
+/
mice are due to overloading in lane 3. C, 20 µg of total protein from FOXD2
/
(black bar)
(n = 3) and wt (light gray bar)
(n = 2) spleen lymphocyte whole cell lysates were
examined by immunoblotting using antibodies against RI
and RII
.
Relative levels of total protein were assessed by Coomassie staining of
the blots and densitometry. The levels of RI
and RII
protein are
normalized for total protein levels and depicted as intensity relative
to wild type in the bar chart above.
on
cAMP-mediated inhibition of T lymphocyte function in vivo,
FOXD2
/
and wt spleen lymphocytes were activated by anti-CD3 and
subjected to lymphocyte proliferation assays in the absence and
presence of 8-CPT-cAMP at increasing concentrations (0-100
µM). The data from proliferation assays showed that
spleen lymphocytes deficient in FOXD2 were less sensitive to cAMP than
wt spleen lymphocytes as illustrated by the right-shifted inhibition
curve in Fig. 3A. When examining four null mutant and three wt mice it was evident that T
cells from all the mutant mice were less sensitive to inhibition of T
cell function by cAMP than wt littermates as illustrated by the
increase in IC50 values for cAMP inhibition of T cell
proliferation in the FOXD2
/
mice (Fig. 3B).
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Fig. 3.
Reduced sensitivity of FOXD2 /
spleen lymphocytes to cAMP. Spleen lymphocytes from
FOXD2
/
and wt mice were activated by CD3 in the absence and
presence of increasing concentrations of 8-CPT-cAMP (0-100
µM). After 48 h of culture, the cells were pulsed
with 5 µCi of [3H]thymidine, and 4 h later
cultures were harvested onto filter plates and subsequently analyzed by
-scintillation counting. A, data are shown as
proliferation at various concentrations of cAMP relative to untreated
cells (mean ± S.E.). IC50 values for FOXD2
/
(empty circles) and wild type (filled circles)
are indicated. One representative experiment is shown. B,
IC50 values (mean ± S.E.) obtained from four
FOXD2
/
(black bar) and three wt (light gray
bar) mice subjected to proliferation assays in the presence of
increasing concentrations of cAMP as in A.
1b Promoter and Increases the Sensitivity of
the Promoter to cAMP--
We next transfected Jurkat T cells with pCAT
reporter constructs containing either the RI
1a, the RI
1b, or the
total RI
1a+1b promoter region (Fig.
4A) together with expression
construct for FOXD2 or the related factor FOXD1 (Fig. 4B).
Reporter activity directed by constructs containing the RI
1a+1b and
RI
1b promoter, but not the construct containing the upstream RI
1a
promoter alone, was induced 2-4-fold by both FOXD2 and FOXD1. A
construct containing the RI
1a promoter with the addition of 1.8 kb
of 5'-flanking sequences showed no further effect on reporter
expression or induction by FOXD2 (not shown). However, elements
immediately upstream of RI
exon 1a appeared to induce basal activity
of the RI
1b promoter. Elevating the amounts of FOXD2 expression
vector up to 12-fold relative to the RI
1b reporter construct
demonstrated that maximal induction of the RI
1b promoter by FOXD2
was at least 5.8-fold (Fig. 4C). However, we observed no
more than 2-fold induction by introducing the corresponding amount of
FOXD1 expression vector, indicating that FOXD1 and FOXD2 share the same
function but display different efficacy in Jurkat cells. We next
explored whether expression of FOXD2 affected the responsiveness of the
RI
1b promoter to cAMP and transfected the RI
1b reporter vector
together with the expression vector for FOXD2 or the corresponding
empty vector (Fig. 4D). The cells were treated with 100 µM 8-CPT-cAMP for 10 h or left untreated, which
produced a 2-fold induction of the RI
1b promoter in the presence of
endogenously expressed FOXD2, but this induction was increased to
5-fold in cells overexpressing FOXD2, suggesting that FOXD2 partly
mediates the effect of cAMP in activating the RI
1b promoter. Similar
results were obtained using the RI
1a+1b construct as a reporter,
whereas the RI
1a reporter was not affected (data not shown).
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Fig. 4.
FOXD2 induces expression from the
RI 1b promoter and is implicated in mediating
the sensitivity of the promoter to 8-CPT-cAMP. A,
schematic depiction of the RI
promoter constructs used in this
study; RI
1a +1b (
882 to +77), RI
1a (
882 to
310), and
RI
1b (
406 to +77), inserted upstream of the CAT reporter gene.
B, 2.5 µg of promoter constructs RI
1a +1b, RI
1a, and
RI
1b or empty vector were transfected into Jurkat TAg cells (20 × 106) by electroporation, together with 7.5 µg of
expression vectors for FOXD2 (gray bars), FOXD1 (light
gray bars), or the corresponding empty expression vectors
(black bars), as well as a luciferase expression vector
(pGL3Control, 2.0 µg) as an internal control. The cell cultures were
harvested after 20 h and assayed for CAT and Luc activities.
C, the RI
1b construct (1.0 µg) was transfected together
with luciferase control vector (2 µg), 4-12 µg of FOXD2 expression
vector or 12 µg of FOXD1 expression vector, and empty vector to a
total of 20 µg. D, cells transfected with the RI
1b
reporter construct (2.5 µg) and 7.5 µg of FOXD2 expression vector
(right 2 bars) were treated with 8-CPT-cAMP (100 µM) for 10 h (light gray bars) or left
untreated (black bars). The data show reporter activities
normalized for expression of the internal luciferase reporter and
represent the means ± S.E. of three separate transfections
(B and D) or half-range (C). Activity
of the RI
1b reporter construct in the absence of FOXD2 expression
and cAMP was assigned the value of 1.
Enhances the Effect of FOXD2 on RI
Expression--
The inhibitory effect of PKB
on the FOXO subfamily
of forkhead transcription factors (FKHR, FKHR-L1, and AFX) is well
documented. Furthermore, we have recently reported that PKB
enhances
the effect of a FOXD family member, FOXD1, on the RI
1b promoter in testicular Sertoli cells but not in 3T3-L1 adipocytes (21). To
investigate whether PKB
is involved in FOXD-mediated activation of
RI
1b in T cells, we transfected Jurkat T cells with 2.5 µg of pCAT
reporter constructs containing the RI
1a+1b, 1a, or 1b promoters
together with expression vector for FOXD2 and/or PKB
(Fig.
5A). Although FOXD2
overexpression did not induce the RI
1b promoter more than 1.4-fold
in these experiments, we found that the combination of FOXD2 and PKB
induced the RI
1b promoter 10-fold, whereas PKB
alone induced
expression from the RI
1b promoter 5-fold, indicating that PKB
may
influence RI
expression through endogenous FOXD2 or by alternative
mechanisms in these cells. When FOXD1 and PKB
were co-transfected in
Jurkat cells, a 6.3-fold induction of the RI
1b promoter was observed
(Fig. 5B). To assess the importance of the localization of
PKB
for induction of RI
expression, we examined the effect of a
myristylated PKB
mutant compared with a wt kinase (Fig.
5C). Myristylated PKB is reported to be constitutively
membrane-bound and constitutively active (21, 25). We observed that
membrane-restricted PKB
alone had a modest inductive effect on the
basal expression from the RI
1b promoter and produced a 3.8-fold
induction in FOXD2-expressing cells, indicating that the activating
mechanism of PKB is enhanced by detachment of PKB from the membrane.
Expressed as a control, the kinase-dead PKB mutant had no effect on the
RI
1b promoter. We next examined the effects of FOXD2 and/or PKB
on endogenous RI
protein levels at 16 and 24 h
post-transfection by immunoblotting (Fig. 5D). We observed
that FOXD2 up-regulated RI
protein levels up to 2-fold, whereas the
combined expression of FOXD2 and PKB
elevated RI
-levels
2.7-fold.
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Fig. 5.
PKB works together
with FOXD2 and FOXD1 to induce expression from the
RI
1b promoter. Reporter constructs
RI
1a +1b, RI
1a, and RI
1b (depicted in Fig. 4) or
the corresponding empty pCATbasic reporter vector (2.5 µg) were
co-transfected with 7.5 µg of expression vector for FOXD2
(A) or FOXD1 (B) (light gray bars),
7.5 µg of expression vector for wt PKB
(very light gray
bar), a combination of FOXD2/wt PKB
or FOXD1/wt PKB
(gray bars), or the corresponding empty expression vectors
(black bars) in Jurkat TAg cells. C, Jurkat TAg
cells were transfected with RI
1b expression vector (2.5 µg)
together with 7.5 µg of expression vector for FOXD2 (right four
bars) or the corresponding empty vector (left four
bars). In addition, 7.5 µg of empty pCMV vector
(basal, black bars), expression vector for wt
PKB
(light gray bars), a myristylated (myr)
constitutively active PKB
mutant (gray bars), or a kinase
dead (dead) PKB
mutant (very light gray bars)
were co-transfected. The data from all of the reporter assays
(A-C) represent reporter activities normalized for
expression of the internal luciferase reporter (2.0 µg of
transfected) and represent the means ± S.E. of three separate
transfections. Activity of the RI
1b reporter construct in the
absence of FOXD2 and PKB
expression was assigned the value of 1. D, whole cell extract from Jurkat TAg cells transfected with
FOXD2 (light gray bars) expression vector (10 µg) and/or
wt PKB
(gray/very light gray bars) expression
vector (10 µg) or the corresponding empty vectors (black
bars) were collected at 16 and 24 h following transfection as
shown below and examined by immunoblotting using human RI
or C
antibodies. Levels of RI
are depicted as relative intensity
in the bar chart above.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
expression in adipocytes (22) and that FOXD1 share
the same function in testicular Sertoli cells (21). We report here the
finding of FOXD2 mRNA expression in leukocytes and the regulation
of RI
expression by FOXD2 in T cells. Spleen lymphocytes from FOXD2
null mutant mice had reduced levels of RI
mRNA and protein,
whereas expression of both FOXD2 and FOXD1 induced the RI
1b promoter
as well as RI
protein in a Jurkat cell line. Furthermore, in the
FOXD2-deficient mice, anti-CD3 activated spleen lymphocytes were less
sensitive to inhibition of proliferation by cAMP than cells from wt
mice as indicated by a right-shifted inhibition curve and elevated
IC50 values. The observation that sensitivity to cAMP is
reduced in FOXD2
/
may be due to the facts that (i) the levels of
RI
are reduced and PKAI
(RI
2C2) has a
3-4-fold higher affinity and lower Kact for
cAMP than the PKAII
(RII
2C2) enzyme also
present in T cells and (ii) PKAI
is the PKA isoform specifically
recruited to the TCR complex (2). In support of this, we observed an
up-regulation of RI
promoter activity and RI
protein in
FOXD2-overexpressing cells. The RI
1b promoter was induced 2-3-fold
by cAMP in Jurkat T cells and about 5-fold in normal T cells (not
shown). We also show here that overexpression of FOXD2 markedly
increases the sensitivity of the RI
1b promoter to cAMP in Jurkat
cells, which may also involve endogenous FOXD2. A similar connection
between FOXD1 and cAMP-mediated regulation of the RI
1b promoter has
been reported in Sertoli cells (21). We have previously also reported that translation of the RI
1b mRNA may be stimulated by cAMP in testicular Sertoli cells depending on specific sequences in the RI
1b
5'-untranslated region (15). Furthermore, our findings showed that both
cAMP and PKB
activate the RI
1b promoter to a similar extent in
the presence of FOXD2 in Jurkat T cells. However, in our assays the
effects of PKB
and cAMP did not appear to be additive (not shown). A
possible explanation for this may be that FOXD2 is a cross-road for
PKA/PKB
cross-talk or that a mechanism of cAMP-mediated activation
of PKB
is involved. Cyclic AMP has been demonstrated to
stimulate PKB activity independently of PKA (27, 28), and
phosphoinositide 3-kinase-independent activation of PKB by PKA has also
been reported (29). Notably, Jurkat cells lack PTEN (phosphatase and
tensin homolog detected at chromosome 10) and have a disturbed
phosphoinositide signaling (30).
on FOXD2 in T cells and on FOXD1 in
Sertoli cells is in contrast to the inhibitory effects reported on a
different family of forkheads, the FOXO family. PKB/Akt is a key player
in transduction of anti-apoptotic and proliferative signals in T cells,
and overexpression of active PKB
/Akt1 promotes T cell survival (31).
PKB is activated following proliferative signals through the
interleukin-2 receptor and the T cell receptor complex in a
phosphoinositide 3-kinase-dependent fashion (31, 32) and is
negatively regulated by the tumor suppressor PTEN (30). PKB has
the ability to antagonize apoptotic signals by inhibiting the function
of winged helix transcription factors of the FOXO family members (AFX,
FKHR, and FKHR-L1) via a direct phosphorylation and nuclear exclusion
(reviewed in Ref. 20). FOXD2 and FOXD1 share little sequence similarity
with FOXO proteins in regions outside the DNA-binding region, and we
did not find consensus PKB phosphorylation sites. However, the
possibility exists that FOXD2 competes for DNA-binding sites with FOXO
proteins and that nuclear exclusion of the FOXO family members by PKB
takes part in the synergistic effect of PKB and FOXD2. Based on the above, it is possible that PKB, in addition to its strong role in T
cell activation, in parallel participates in activating
immunomodulatory effects via the PKA pathway. In testicular Sertoli
cells, we observed that ectopically expressed FOXD2 did not regulate
the RI
1b promoter regardless of the presence of PKB
(not shown),
whereas FOXD1 and PKB
induced RI
1b 15-fold in these cells
(21).3 This led us to believe
that additional cell-specific factors are essential for the different
effects of the two FOXD proteins.
levels in T lymphocytes in vivo by
inducing the RI
1b promoter, working with PKB to further induce PKA
RI
expression. Through this mechanism FOXD2 may regulate PKA RI
levels that regulate sensitivity to cAMP and set the threshold for
negative modulation of T cell activation (Fig.
6).
View larger version (30K):
[in a new window]
Fig. 6.
Regulation of cAMP responsiveness by FOXD2
and PKB sets threshold for modulation of T
cell immune function. Binding of a ligand to G-protein-coupled
receptors (e.g. PGE2/EP-R) leads to the
activation of adenylyl cyclase, elevation of intracellular cAMP and
thereby activation of PKA type I. PKA type I has been demonstrated to
down-regulate mitogenic signaling through the TCR by activating
Cbp/PAG-associated C-terminal Src kinase by phosphorylation on S364.
C-terminal Src kinase inhibits Lck by phosphorylation of the C-terminal
inhibitory site (Tyr505). Activated Lck phosphorylates
tyrosine residues within the immunoreceptor tyrosine-based
activation motifs leading to the recruitment of ZAP-70, which next is
phosphorylated and activated by Lck. Activated ZAP-70 phosphorylates
the adaptor protein called "linker for activation of T cells"
(LAT). When phosphorylated, the linker for activation of T
cells serves as a scaffold that couples downstream signaling pathways
including phosphoinositide 3-kinase through interaction with its SH2
domain. PKB is recruited to the membrane via the phosphoinositide
3-kinase pathway and activated following stimulation of the TCR-CD3
complex and may travel to the nucleus. PKB
activity, released from
the membrane, may increase the inductive effect of FOXD2 on the RI
promoter. This would thereby elevate the levels of the RI
subunit of
PKA and tune PKA sensitivity and enhance the inhibitory effect of PKA
on T cell signaling. The second messenger cAMP may also induce RI
expression in a FOXD2-dependent fashion and may thereby
increase the sensitivity of its own effector system. Taken together,
FOXD2 may together with PKB
and cAMP, induce PKA RI
expression
and set the threshold for activation of PKA and negative modulation of
T cell activation by cAMP.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Tsutomu Kume and Dr. Brigid
L. M. Hogan for supplying the FOXD2/
mice, Dr. Naheed N. Ahmed
for providing the PKB
expression vectors, and Dr. Mary Weiss for
furnishing the mouse RI
1b sequence for design of an RI
1b-specific
mouse cDNA probe. We greatly appreciate the technical assistance of
Gladys Josefsen and Guri Opsahl.
![]() |
FOOTNOTES |
---|
* This work was supported by the Norwegian Cancer Society, the Norwegian Research Council, Anders Jahre's Foundation, Novo Nordic Research Foundation Committee, the Swedish Medical Research Foundation, the Arne and IngaBritt Lundberg Foundation, the Juvenile Diabetes Foundation, the Wallenberg Foundation, and the Swedish Foundation for Strategic Research (Nucleic Acid Program).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
4722851454; Fax: 4722851497; E-mail:
kjetil.tasken@basalmed.uio.no.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M300311200
2 K. A. Taskén, S. Ernstsson, H. K. Knutsen, L. M. Grønning, K. Taskén, and S. Enerbäck, manuscript in preparation.
3 M. K. Dahle, L. M. Grønning, and K. Taskén, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PKA, cAMP-dependent protein kinase; TCR, T cell receptor; wt, wild type; PKB, protein kinase B; CMV, cytomegalovirus; CHAPS, 3-[(3-cholamidopropyl)dimethyl-ammonio]1-propane sulfonate; RT, reverse transcription; CAT, chloramphenicol acetyltransferase.
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