Transcriptional Activation of the Macrophage Migration-Inhibitory Factor Gene by the Corticotropin-Releasing Factor is Mediated by the Cyclic Adenosine 3',5'-Monophosphate Responsive Element-Binding Protein CREB in Pituitary Cells
Gérard Waeber,
Nancy Thompson,
Thierry Chautard,
Myriam Steinmann,
Pascal Nicod,
François P. Pralong,
Thierry Calandra and
Rolf C. Gaillard
Department of Internal Medicine B (G.W., N.T., M.S., P.N.)
Division of Infectious Disease (T.C.) Division of Endocrinology and
Metabolism (T.C., F.P.P., R.C.G.) Centre Hospitalier Universitaire
Vaudois 1011 CHUV Lausanne, Switzerland
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ABSTRACT
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Macrophage migration-inhibitory factor (MIF) has
recently been identified as a pituitary hormone that functions as a
counterregulatory modulator of glucocorticoid action within the immune
system. In the anterior pituitary gland, MIF is expressed in TSH- and
ACTH-producing cells, and its secretion is induced by CRF. To
investigate MIF function and regulation within pituitary cells, we
initiated the characterization of the MIF 5'-regulatory region of the
gene. The -1033 to +63 bp of the murine MIF promoter was cloned 5' to
a luciferase reporter gene and transiently transfected into freshly
isolated rat anterior pituitary cells. This construct drove high basal
transcriptional activity that was further enhanced after stimulation
with CRF or with an activator of adenylate cyclase. These
transcriptional effects were associated with a concomitant rise in ACTH
secretion in the transfected cells and by an increase in MIF gene
expression as assessed by Northern blot analysis. A cAMP-responsive
element (CRE) was identified within the MIF promoter region which, once
mutated, abolished the cAMP responsiveness of the gene. Using this
newly identified CRE, DNA-binding activity was detected by gel
retardation assay in nuclear extracts prepared from isolated anterior
pituitary cells and AtT-20 corticotrope tumor cells. Supershift
experiments using antibodies against the CRE-binding protein CREB,
together with competition assays and the use of recombinant CREB,
allowed the detection of CREB-binding activity with the identified MIF
CRE. These data demonstrate that CREB is the mediator of the
CRF-induced MIF gene transcription in pituitary cells through an
identified CRE in the proximal region of the MIF promoter.
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INTRODUCTION
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Macrophage migration-inhibitory factor (MIF), originally described
as a T cell product, has been identified recently in several endocrine
tissues including the pituitary gland, the Leydig cells of the testis,
the granulosa cells of the ovary, and the insulin-secreting ß-cells
of the endocrine pancreas (1, 2, 3, 4, 5). MIF is secreted by immune and
endocrine cells and acts on target tissues to modulate glucocorticoid
action and functions also locally as an auto- and paracrine regulator
of hormonal secretion (6, 7, 8). In Leydig cells, the secreted MIF acts as
a modulator of inhibin production (3). In the endocrine pancreas, MIF
is produced by the insulin-secreting cells and potentiates the
glucose-induced insulin secretion in an autocrine fashion (5). In
rodent and human pituitary gland, MIF expression is high (2, 9). The
overall MIF content within this gland has been estimated to be
approximatively 0.05% of total pituitary protein as compared with
0.2% and 0.08% for ACTH and PRL, respectively (2, 8). Cellular and
subcellular localization of MIF within the pituitary gland has shown
that it is colocalized in ACTH- and, to a lesser extent, in
TSH-producing cells (2). The release of MIF from these corticotropic
cells is stimulated by CRF and by various proinflammatory stimuli such
as lipopolysaccharide (LPS) in vitro and in vivo
(1). The function of MIF has been only partially elucidated, yet the
factor seems to be a crucial regulator of the host response to various
stimuli by counterregulating glucocorticoid action within the immune
system (6).
MIF gene expression has been studied in different cell systems. In
monocytes/macrophages, MIF mRNA and protein expression are stimulated
by LPS and tumor necrosis factor-
(10). In the endocrine pancreas,
glucose is a potent regulator of MIF gene expression in the
insulin-secreting cells. In the pituitary gland, LPS stimulates MIF
gene expression and the subsequent release of MIF protein (1, 5). It is
also well established that CRF plays a major physiological role in
regulating ACTH secretion as well as POMC gene expression (11). These
effects are mediated, in part, by the activation of the cAMP-dependent
protein kinase A pathway (11, 12, 13). We therefore investigated whether
similar regulators of POMC gene expression could also be involved in
the control of MIF gene transcription. Herein we report the
identification of a functional cAMP-responsive element (CRE) in the
proximal region of the MIF
promoter.1 Once mutated, the
cAMP responsiveness of the MIF gene was lost. Furthermore, the
CRE-binding protein CREB was identified as the trans-acting
factor that mediates this effect through the MIF CRE. Since the binding
of CRF to its receptor in ACTH-producing cells triggers the
cAMP-dependent pathway, it is likely that the subsequent
phosphorylation of CREB by the cAMP-dependent protein kinase A acts
thereafter as a transactivator of several cAMP-reponsive genes,
including the MIF gene.
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RESULTS
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Functional Activity of the -1033/+63 bp Region of the MIF Promoter
in Isolated Rat Pituitary Cells
We first examined whether the -1033/+63 bp region of the MIF
promoter was sufficient to drive reporter gene expression in pituitary
cells. To this end, this promoter region was fused to a luciferase
reporter gene (MIFluc construct), transiently transfected into isolated
primary rat anterior pituitary cells. A high basal transcriptional
activity for the MIFluc construct was detected in unstimulated primary
rat anterior pituitary cells (2.6 ± 0.3 x 103
relative light units (RLU), 14-fold over the promoterless parent
vector) (Fig. 1A
). The MIFluc transfected
cells were then incubated for 14 h with CRF (0.5 and 5 ng/ml) or
forskolin (FSK, 10-6 M). This transcriptional
activity was found to be responsive to CRF, and these effects were
mimicked by the use of an activator of adenylate cyclase, FSK (2.6
± 0.3 x 103 in unstimulated cells to 3.5 ± 0.7
x 103, 4.8 ± 0.6 x 103, 7.5 ±
0.98 x 103 RLU with 0.5, 5 ng of CRF and FSK,
respectively; Fig. 1A
). To assess the response of the cells to the
various stimuli, ACTH concentrations were measured in the culture media
of all transfected primary anterior pituitary cells. As depicted in
Fig. 1B
, the treatment with CRF or FSK increased ACTH release. The
fold-increase of ACTH release induced by these treatments was higher
compared with that of the luciferase activity since the basal ACTH
secretion of unstimulated cells is much lower than the basal
transcriptional activity measured in unstimulated cells 48 h after
transfection.

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Figure 1. Transcriptional Activity of the MIF Promoter in Rat
Isolated Anterior Pituitary Cells
A, The MIF promoter fused to the luciferase gene was transiently
transfected into freshly isolated primary anterior pituitary cells. The
construct drove high transcriptional activity (2.6 ± 0.3 x
103 RLU, 13-fold over the promoterless plasmid) that was
further enhanced with 0.5 ng/ml of CRF (3.5 ± 0.7 x
103 RLU) or 5 ng/ml of CRF (4.8 ± 0.6 x
103 RLU) or FSK (7.5 ± 0.9 x 103
RLU). The bars represent the mean of 11 separate
experiments done in duplicate or triplicate ± SEM.
***, P < 0.001 vs. unstimulated
transfected cells. B, ACTH secretion of transfected anterior pituitary
cells. Cells were stimulated for 14 h with various agonists, and
the ACTH content of cell-conditioned media was analyzed by RIA as
reported (28). Basal ACTH secretion of cells transfected with the
MIFluc construct was 8.5 ± 1.9 ng/ml. With 0.5 and 5 ng/ml of CRF
or with FSK, ACTH concentrations increased to 23.1 ± 1.6 ng/ml,
51 ± 13 ng/ml, 54 ± 12 ng/ml, respectively. ***,
P < 0.001 vs. unstimulated
transfected cells.
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In another set of experiments, primary anterior pituitary cells were
isolated and incubated for 14 h with CRF (5 ng/ml) or FSK
(10-6 M). Total RNA was extracted from these
cells and analyzed by Northern blotting for MIF and ß-actin gene
expression. As shown in Fig. 2
, MIF gene
expression was induced 1.8- and 2.8-fold by CRF and FSK, respectively
(basal MIF/bactin, 0.66 ± 0.09; CRF, 1.21 ± 0.21; FSK,
1.9 ± 0.31). Taken together, these data demonstrate that the
-1033 to +63 bp region of the murine MIF promoter drives high basal
transcriptional activity in freshly isolated primary anterior pituitary
cells that is further enhanced by the activation of the cAMP protein
kinase A pathway. These effects were paralled by a similar increase in
MIF mRNA expression.

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Figure 2. Expression and cAMP-Dependent Regulation of MIF
mRNA in Isolated Rat Pituitary Cells
Northern blotting analysis (inset) of MIF mRNA (0.6 kb)
and ß-actin mRNA (1.8 kb) expression in isolated rat anterior
pituitary cells incubated with CRF (5 ng/ml) or FSK (1 x
10-6 M). The quantitative assessment of
MIF/ß-actin RNAs expression was done by laser densitometric scanning.
The experiments were repeated three times in duplicate. *,
P < 0.05; **, P < 0.01
vs. unstimulated cells.
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Identification of a Functional CRE in the Proximal Region of the
MIF Promoter
A putative CRE is located -48 to -41 bp in the murine MIF
promoter (14). Interestingly, this CRE site is partially conserved in
the human MIF gene where it appears -12 nucleotides upstream from the
transcriptional start site (15). This identified murine
mifCRE is highly conserved when compared with several other
functional CREs identified in the 5'-flanking regions of other
hormones, such as the human gonadotropin, the rat somatostatin, and the
bovine PTH genes (16, 17, 18). A construct was then generated from the
parent MIFluc plasmid where the wild-type CRE was mutated. When
transiently transfected into isolated rat pituitary cells, the mutated
MIF promoter drove lower basal reporter gene expression (-26%; wild
type promoter, 1.5 ± 0.5 x 103 RLU; mutated
promoter, 1.1 ± 0.2 x 103 RLU) compared with
the wild type MIF promoter (Fig. 3
, A and
B). The cAMP responsiveness of the mutated construct was dramatically
reduced compared with the wild-type construct (3.7-fold induction to
1.5-fold with the mutated CRE). By contrast, the cells transfected
either with wild-type or with the mutated mifCRE
construct produced a similar amount of ACTH (Fig. 3
, A and B). These
data imply that the identified mifCRE is the major
cis-regulatory element responsible for the cAMP
responsiveness of the MIF gene.

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Figure 3. Functional CRE in the Proximal Region of the MIF
Promoter
A, The -1033/+63 bp MIF promoter fused to the luciferase reporter gene
construct was transfected into primary anterior pituitary cells and
incubated with or without FSK for 14 h. This treatment increased
by 3.7-fold the basal luciferase activity (basal 1556 ± 492 RLU
to stimulated 5879 ± 1261 RLU, mean ± SEM of
three separate experiments done in duplicate). The ACTH released in the
same transfected cells was induced by 3.6-fold by the activation of the
cAMP protein kinase A pathway. ***, P < 0.001
vs. unstimulated transfected cells. B, The CRE located
at -41 to -48 bp of the MIF promoter was mutated by deleting four
nucleotides (ACGT) of the core element in the wild-type MIF luciferase
construct. Once transfected into pituitary cells, the basal
transcriptional activity of this construct decreased by 26% compared
with the wild-type promoter (wild type, 1556 ± 492 RLU; mutated
CRE construct, 1149 ± 224 RLU). Furthermore, the cAMP
responsiveness of the gene was dramatically reduced to 1.5-fold with
the mutated construct (basal 1149 ± 492 RLU to stimulated
1683 ± 315 RLU, mean ± SEM of three separate
experiments done in duplicate) while the ACTH release was conserved in
the same transfected cells (4.3-fold stimulation with FSK compared with
basal).
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The CRE-Binding Protein CREB Is the Mediator of the cAMP
Responsiveness of the MIF Gene in Rat Pituitary Cells
To determine whether DNA-binding activity may interact with the
identified mifCRE, electrophoretic mobility shift assays
(EMSA) were performed using nuclear extracts of AtT-20 and freshly
isolated rat anterior pituitary cells. In the first set of experiments
(Fig. 4
, A and B),
32P-labeled probes corresponding to the mifCRE
or to the somatostatin CRE motif were incubated with nuclear
extracts of rat anterior pituitary cells. The somatostatin
CRE motif has been previously shown to be a preferential binding site
for the CRE-binding protein CREB (19, 20). As shown in Fig. 4A
, a
similar DNA-binding complex was present using either the
mifCRE or the somatostatin CRE motif.
Furthermore, this DNA-protein complex is competed with an excess of
unlabeled wild-type mifCRE or with the
somatostatin CRE but not with the mutated mifCRE
oligonucleotides (Fig. 4B
). CREB was therefore suspected to be part of
the complex bound to the mifCRE. We then conducted
experiments using as probe the mifCRE incubated with nuclear
extracts obtained from rat anterior pituitary or AtT-20 cells where the
DNA-binding complex was disrupted by the addition of CREB antiserum but
not with the preimmune serum (Figs. 4B
and 5A
). Bacterially expressed recombinant
CREB (rCREB, 2.5 µg) was similarly able to bind to the
mifCRE, and this DNA-binding complex was disrupted once CREB
antiserum was added to the mixture of probe and recombinant CREB (Fig. 5B
). These data thus demonstrate that CREB participates in the
DNA-binding complex of the mifCRE and CREB is present in
nuclear extracts obtained from AtT-20 and freshly isolated rat anterior
pituitary nuclear extracts.

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Figure 4. Similar DNA-Binding Activity to the
mifCRE and to the SomatostatinCRE Motif
Is Detected in Nuclear Extracts of Rat Anterior Pituitary Cells
A, EMSA performed with 32P-labeled mifCRE or
with a somatostatinCRE motif using nuclear extracts from
anterior pituitary cells. As the somatostatinCRE was
previously shown to be the preferential binding site to the CREB
nuclear protein, the similar pattern of DNA binding activity detected
with the smsCRE and with the mifCRE
suggests that CREB is able to bind to the identified
mifCRE and is present in rat anterior pituitary nuclear
extracts. B, Competition experiments using 100- and 500-fold excess of
cold wild-type mifCRE, somatostatinCRE
(smsCRE), or cold mutated mifCRE
oligonucleotides in EMSA. The binding activity with labeled
mifCRE is competed with an excess of wild-type
mifCRE or smsCRE but not with the mutated
CRE oligonucleotides. The DNA-protein binding complex is disrupted by
the addition of the anti-CREB serum but not with the preimmune serum.
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Figure 5. The CRE-Binding Protein CREB Is Part of the
DNA-Binding Complex of the mifCRE Detected in Rat
Anterior Pituitary and AtT-20 Nuclear Extracts
A, EMSA with mifCRE probe and nuclear extracts from
AtT-20 or rat anterior pituitary cells. The DNA-protein binding complex
is disrupted by the addition of the anti-CREB serum but not with the
preimmune serum. B, Bacterially expressed CREB binds to the
mifCRE probe by EMSA and the DNA-binding complex is
disrupted in the presence of the anti-CREB serum but not with the
preimmune serum.
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DISCUSSION
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In this study, we report the identification of a functional CRE in
the proximal region of the MIF promoter. Mutational analysis of this
CRE decreased basal transcriptional activity by 26% and dramatically
reduced the cAMP responsiveness of the gene. These functional data were
obtained by transfecting freshly isolated rat anterior pituitary cells
with a reporter gene construct and, to our knowledge, this has never
been previously reported. Furthermore, the cAMP responsiveness of the
MIF reporter construct was obtained not only with the activator of the
adenylate cyclase, FSK, but also with the hypothalamic factor CRF. This
recapitulates, in part, the effects of CRF upon POMC gene expression
and ACTH secretion (11). CRF regulates positively POMC promoter
activity, and this effect is mediated by cFOS and cJUN via a AP1 site
located in exon 1 of the gene (13). Regarding the MIF gene, we clearly
demonstrate herein that the cAMP responsiveness of the gene involves
the binding of CREB to the identified mifCRE. Interestingly,
MIF release from pituitary cells was previously described to occur at
lower CRF concentrations than those required to induce ACTH release,
suggesting that different pathways of regulated secretion by
corticotrope cells are present for MIF and ACTH and/or that the cAMP
responsiveness of these genes are mediated by different factors (2).
This last hypothesis may be correct since CREB is the activated factor
by the cAMP/protein kinase A pathway (19, 20) that confers cAMP
responsiveness to the MIF gene while the AP1 complex seems to be more
important to confer cAMP responsiveness to the POMC gene (13).
The identified mifCRE, compared with other CREs, is
located proximal to the start site of the gene, and this CRE is
partially conserved in the human mif gene (14, 15, 18). The
cAMP core element TGACGTCA in the MIF promoter is perfectly conserved
with several other CREs found in cAMP-responsive genes such as the
human gonadotropin, the rat somatostatin, and the bovine PTH (16, 17, 18).
However, the flanking nucleotides of the core CRE octamer are not
conserved within any of these genes, and the mifCRE is
flanked with nucleotides quite different from the above citated genes.
This could be of importance since these flanking nucleotides have been
shown to bind CREB-associated proteins that modulate CREB-binding
activity and therefore influence the level of cAMP responsiveness of
these genes (18, 21). Using EMSA, we first demonstrate that similar
DNA-binding complexes are detected in pituitary nuclear extracts when
using the mifCRE or the preferential binding site of CREB,
the somatostatin CRE. Second, we show that this binding
activity is specific and competed by an excess of unlabeled
mifCRE or the somatostatinCRE but not with the
mutated mifCRE oligonucleotides used in the functional
study. Third, we demonstrate that similar binding complexes are
detected using mifCRE as probe in the corticotrope AtT-20
cell line or in anterior pituitary cells and that CREB antibody
disrupts the DNA-binding complex, implying that this factor is part of
the complex. Fourth, we show that recombinant CREB is able to bind to
the newly identified mifCRE. Taken together, these data
strongly support the contention that CREB is the mediator of the
CRF-induced MIF gene transcription in anterior pituitary cells through
an identified CRE in the proximal region of the MIF promoter.
MIF was originally described as a T cell cytokine and recently
rediscovered as an humoral factor present in several endocrine tissues
(1, 3, 4, 5, 7, 8). Interestingly, the MIF gene is expressed and its
translated product secreted from several endocrine cells that are
tightly regulated by the cAMP/protein kinase A pathway. MIF is secreted
from Leydig cells under the control of LH to modulate, as a paracrine
hormone, inhibin production (3). MIF is also present in granulosa cells
of the ovary where FSH mediates several functions, including the
phosphorylation of CREB, thereby inducing cAMP responsiveness of the
inhibin subunit-
, the proenkephalin, or the aromatase genes
(22, 23, 24). In the endocrine pancreas, the MIF gene is expressed in the
ß-cells, and its regulated secretion potentiates glucose-induced
insulin secretion (5). Insulin release is also tightly controlled by
activators of the cAMP/protein kinase A pathway, which includes the
incretin hormones glucagon-like-peptide I and the gastric inhibitory
peptide (25). It remains to be tested whether the cAMP responsiveness
described herein for the MIF gene in the anterior pituitary cells is
also present in other endocrine cells where MIF is expressed. This
could be of importance because the dysregulation of the cAMP/protein
kinase A pathway has been shown to be involved in several endocrine
diseases. In some somatotrope tumors, Gs protein mutations have been
described that may be responsible for a constitutive activation of the
cAMP/protein kinase A pathway (26). On the other hand, a decreased
responsiveness of the insulin-secreting ß-cells to the incretin
hormones is suspected to participate in some forms of diabetes (25). It
remains to be elucidated whether situations occur in which altered MIF
gene expression participates in any clinical situations, such as the
host response to stress or endotoxemia during the activation of the
hypothalamic-pituitary-adrenal axis, in diabetes or in any reproductive
disorders. Some of the MIF properties have been elucidated. These
include the fact that MIF is now recognized as a circulating hormone,
secreted by various endocrine cells and by the immune cells to act as a
counterregulating factor of glucocorticoid action (1, 3, 5, 6, 8). Our
work helps to further understand how MIF may regulate cell functions by
identifying an important site within the MIF gene which is regulated by
cAMP in anterior pituitary cells. CRF mediates transcriptional
activation of the MIF gene through an identified CRE by the binding of
phosphorylated CREB, and the cAMP responsiveness of the MIF gene
differs from the one observed with the POMC gene.
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MATERIALS AND METHODS
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Plasmid Construction
The -1033 to +63 bp of the murine MIF promoter was
PCR-amplified from mouse genomic DNA (NMRI strain) and subsequently
cloned into the Nhe/BglII sites of the promoterless pGL3
basic (Promega, Madison, WI) luciferase reporter vector (MIFluc). The
sequences of the oligonucleotides used in the PCR are the following:
5'- TAGCTAGCCAGAACTGGTAGTCCCCA-3' and 5'-TGAGATCTGGACCGGAGGACGGTAC-3'.
The PCR conditions were 94 C for 10 min and then 30 cycles at 94 C, 50
C, and 72 C each for 1 min with Life Tech taq polymerase (Life
Technologies, Gaithersburg, MD). Two clones were completely sequenced
and found to be identical. However, there are 18 nucleic acids which
are different from the previously reported mouse 129SV promoter
sequence (14); none of the differences being in the identified
mifCRE. They may be due to the different strain origin of
the mouse DNA. The mutated MIFluc construct was generated by digesting
the wild-type construct with the restriction enzyme AatII,
T4 polymerase treated in the presence of dNTPs, and subsequently
ligated; 4 bp of the core of the CRE (ACGT) were therefore deleted.
This mutation was verified by nucleic acid sequencing.
Cell Culture
The AtT-20 cells were obtained from the ATCC and cultured in
DMEM supplemented with 10% FCS and antibiotics. Rat anterior pituitary
cells were isolated exactly as described previously (27). Ovine CRF and
FSK were obtained from Sigma (Buchs, Switzerland). The ACTH RIA was
performed as described previously (28).
Transfection Studies
All constructs were transfected into isolated rat anterior
pituitary cells using the cationic reagent DOTAP in solution as
recommended by the supplier (Boehringer Mannheim, Rotkreuz,
Switzerland). Six micrograms of plasmid DNAs were used for 1 to
1.3 x 106 cells and incubated for 48 h. Fourteen
hours before harversting the cells, the medium was changed and the
various treatments were included in the medium. The cells were
harvested with Promega lysis buffer, the cellular debris was removed,
and the supernatant was collected. Protein concentrations were
determined using the Bio-Rad protein assay (Bio-Rad Laboratories,
Richmond, CA). Luciferase activity was measured twice with 1030 µg
protein cell extracts from each transfected plate according to the
protocol of Brasier et al. (29). Each set of experiments was
repeated three to 11 times in duplicate or triplicate.
EMSA
Nuclear extracts were prepared according to the method of Dent
and Latchmann (30). The R1090 anti-CREB serum was produced by
immunization of rabbits with a synthetic peptide consisting of amino
acids 92124 of CREB-127 and described previously (31). The clone for
the recombinant CREB and the R1090 antibody were kindly provided by
J. F. Habener. The sequences of the oligonucleotides used in the
gel retardation assays are as follows. mifCRE sense 5'-
GGACGTAGTCTGACGTCAGCGGA-3' and antisense 5'-CGCCTCCGCTGACGTCAGACTAC-3';
the mutated mifCRE sense 5'-TGGGACGTAGTCTGCAGCGGAGG-3' and
antisense 5'-TCCGCCTCCGCTGCAGACTACGTC-3'; the somatostatin CRE sense
5'-AGAGATTGGCTGACGTCAGAGA-G-3' and antisense
5'-CTAGCTCTCTGACGTCAGCCAA-T-3' (Promega). Complementary sense and
antisense oligonucleotides were hybridized and gel purified and 2.5
pmol were filled in by the Klenow fragment of DNA polymerase I in the
presence of [
32P]dCTP, and the remaining free
nucleotides were removed on a G-50 column. Approximatively 0.1 ng of an
end-labeled double-stranded oligonucleotide probe was incubated with
1.5 µg nuclear extract and 1.5 µg polydeoxyinosinic-deoxycytidylic
acid (Sigma) for 20 min on ice in 20 µl of 15 mM HEPES,
pH 7.8, 75 mM NaCl, 1.5 mM MgCl2, 1
mM EDTA, 1 mM dithiothreitol, and 10%
glycerol. Samples were separated on a 6% nondenaturating
polyacrylamide gel using Tris-acetate as running buffer. The gels were
fixed in a solution of 10% acetic acid and 30% methanol, dried, and
exposed to Hyperfilm-MP (Amersham, Buckinghamshire, U.K.). When
antibodies were used in EMSA, the preimmune or the CREB antiserum R1090
was incubated with the nuclear extracts for 20 min on ice before the
probe was added.
RNA and Northern Blot Analysis
Total RNA was isolated by the method of Chomczynski and Sacchi
(32). The Northern blot and hybridization were performed with 15 µg
total RNA as described previously (5).
Statistics
Transfection studies were carried out three to 11 times in
duplicate or triplicate. RNA expression studies were conducted three
times in duplicate. Data are expressed as mean ± SEM
and compared by a nonparametric Mann-Whitney U or
Wilcoxon/Kruskal-Wallis tests.
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ACKNOWLEDGMENTS
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We are grateful to J. F. Habener for the kind gift of the
R1090 anti-CREB antiserum and the recombinant CREB protein (Howard
Hughes Institute, Laboratory of Molecular Endocrinology, Massachusetts
General Hospital, Boston, MA). We wish to thank Marco Giacomini for
expert technical assistance and Juan Ruiz for the help in statistical
analysis.
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FOOTNOTES
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Address requests for reprints to: Gérard Waeber, M.D., Department of Internal Medicine B, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland.
G.W. is supported by a career award from the Swiss National Science
Foundation (Grants 3231915.91/2 and 3249673.96) and the Placide
Nicod Foundation. T.C. is supported by the Swiss National Science
Foundation (32-48916.96 and 32-49129.96) and R.C.G. by Grants
31-039749.93/1 and 31-50748.97 from the same foundation.
1 The nucleic acid sequence of the cloned murine
MIF promoter region has been submitted to the GenBank database with
accession number AF 033192. 
Received for publication October 2, 1997.
Revision received January 8, 1998.
Accepted for publication January 27, 1998.
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REFERENCES
|
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-
Bernhagen J, Calandra T, Mitchell RA, Martin SB, Tracey
KJ, Voelter W, Manogue KR, Cerami A, Bucala R 1993 MIF is a
pituitary-derived cytokine that potentiates lethal endotoxaemia. Nature 365:756759[CrossRef][Medline]
-
Nishino T, Bernhagen J, Shiki H, Calandra T, Dohi K, Bucala R 1995 Localization of macrophage migration inhibitory factor (MIF) to
secretory granules within corticotrophic and thyrotrophic cells of
pituitary gland. Mol Med 1:781788[Medline]
-
Meinhardt A, Bacher M, McFarlene JR, Metz CN, Seitz J, Hedger
MP, De Kretser DM, Bucala R 1996 Macrophage migration inhibitory factor
production by Leydig cells: evidence for a role in the regulation of
testicular function. Endocrinology 137:50905095[Abstract]
-
Fujimoto WS, Mizue Y, Nishihira J 1997 Macrophage migration
inhibitory factor in the human ovary: presence in the follicular fluids
and production by granulosa cells. Biochem Mol Biol Int 41:805814[Medline]
-
Waeber G, Calandra T, Roduit R, Haefliger J-A, Bonny C,
Thompson N, Thorens B, Temler E, Meinhardt A, Bacher M, Metz CN, Nicod
P, Bucala R 1997 Insulin secretion is regulated by the
glucose-dependent production of islet ß cell macrophage migration
inhibitory factor. Proc Natl Acad Sci USA 94:47824787[Abstract/Free Full Text]
-
Calandra T, Bernhagen J, Metz CN, Spiegel LA, Bacher M,
Donnelly T, Cerami A, Bucala R 1995 MIF as a glucocorticoid-induced
modulator of cytokine production. Nature 377:6871[Medline]
-
Bernhagen J, Calandra T, Cerami A, Bucala R 1994 Macrophage
migration inhibitory factor is a neuroendocrine mediator of
endotoxaemia. Trends Microbiol 2:198201[Medline]
-
Bucala R 1996 MIF rediscovered: cytokine, pituitary
hormone, and glucocorticoid-induced regulator of the immune
response. FASEB J 10:16071613[Abstract/Free Full Text]
-
Tampanaru-Sarmesiu A, Stefaneanu L, Thapar K, Kovacs K,
Donnelly T, Metz CN, Bucala R 1997 Immunocytochemical localization of
macrophage migration inhibitory factor in human hypophysis and
pituitary adenomas. Arch Pathol Lab Med 121:404410[Medline]
-
Calandra T, Bernhagen J, Mitchell RA, Bucala R 1994 The
macrophage is an important and previously unrecognized source of
macrophage migration inhibitory factor. J Exp Med 179:18951902[Abstract]
-
Lundblad JR, Roberts JL 1988 Regulation of
proopio-melanocortin gene expression in pituitary. Endocr Rev 9:135158[Medline]
-
Chen R, Lewis KA, Perrin MH, Vale WW 1993 Expression cloning
of a human corticotropin-releasing-factor receptor. Proc Natl Acad Sci
USA 90:89678971[Abstract]
-
Boutillier AL, Monnier D, Lorang D, Landblad JR, Roberts JL,
Loeffler JP 1995 Corticotropin-releasing hormone stimulates
proopiomenalocortin transcription by cFOS-dependent and -independent
pathways:characterization on an AP1 site in Exon 1. Mol Endocrinol 9:745755[Abstract]
-
Bacher MR, Bernhagen J, Pushkarskaya T, Seldin MF, R Bucala R 1995 Cloning and characterization of the gene for mouse macrophage
migration inhibitory factor (MIF). J Immunol 154:38633870[Abstract/Free Full Text]
-
Paralkar V, Wistow G 1994 Cloning the human gene for
macrophage migration inhibitory factor MIF. Genomics 19:4851[CrossRef][Medline]
-
Jameson JL, Deutsch PJ, Gallagher GD, Jaffe RC, Habener
JF 1987 Trans-acting factors interact with a cyclic AMP response
element to modulate expression of the human gonadotropin alpha gene.
Mol Cell Biol 7:30323040[Medline]
-
Montminy MR, Sevarino KA, Wagner JA, Mandel G, Goodman RH 1986 Identification of a cyclic AMP reponsive element within the rat
somatostatin gene. Proc Natl Acad Sci USA 83:66826686[Abstract]
-
Deutsch PJ, Hoeffler JP, Jameson JL, Habener JF 1988 Cyclic
AMP and phorbol ester-stimulated transcription mediated by similar DNA
elements that bind distinct proteins. Proc Natl Acad Sci USA 85:79227926[Abstract]
-
Meyer TE, Habener JF 1993 Cyclic AMP response element binding
protein (CREB) and related transcription activating DNA binding
proteins. Endocr Rev 14:269290[Medline]
-
Montminy M, Gonzalez GA, Yamamoto KK 1990 Regulation of
cAMP-inducible genes by CREB. Trends Neurosci 13:184188[CrossRef][Medline]
-
Miller CP, Lin JC, Habener JF 1993 Transcription of the rat
glucagon gene by cyclic AMP response element-binding protein CREB is
modulated by adjacent CREB-associated proteins. Mol Cell Biol 13:70807090[Abstract]
-
Fitzpatrick SL, Richards JS 1994 Identification of a cyclic
adenosine 3',5'-monophosphate-response element in the rat aromatase
promoter that is required for transcriptional activation in rat
granulosa cells and R2C Leydig cells. Mol Endocrinol 8:13091319[Abstract]
-
Pei L, Dodson R, Schoderbek WE, Maurer RA, Mayo KE 1991 Regulation of the alpha inhibin gene by cyclic adenosine
3',5'-monophosphate after transfection into rat granulosa cells. Mol
Endocrinol 5:521534[Abstract]
-
Comb M, Birnberg NC, Seasholtz A, Herbert E, Goodman HM 1986 A
cyclic AMP- and phorbol ester-inducible DNA element. Nature 323:353356[Medline]
-
Thorens B, Waeber G 1993 Glucagon-like peptide-1 in the
control of insulin secretion in the normal state and in NIDDM. Diabetes 42:12191225[Medline]
-
Landis CA, Masters SB, Spada A, Pace AM, Bourne HR, Vallar L 1989 GTPase inhibiting mutations activate the alpha chain of Gs and
stimulate adenylyl cyclase in human pituitary tumours. Nature 340:692696[CrossRef][Medline]
-
Kehrer P, Turnill D, Dayer J-M, Muller AF, Gaillard RC 1988 Human recombinant interleukin-1 beta and -alpha, but not recombinant
tumor necrosis factor alpha stimulate ACTH release from rat anterior
pituitary cells in vitro in a prostanglandin E2 and cAMP
independent manner. Neuroendocrinology 48:160166[Medline]
-
Rees L, Cook DM, Kendall JW, Allen CF, Kramer RM 1971 A
radioimmunoassay for rat plasma ACTH. Endocrinology 89:254261[Medline]
-
Brasier AR, Tate JE, Habener JF 1989 Optimized use of the
firefly luciferase assay as a reporter gene in mammalian cell lines.
Biotechniques 7:11161121[Medline]
-
Dent CL, Latchman DS 1993 Transcription Factors: A Practical
Approach. Oxford University Press, New York
-
Waeber G, Meyer TE, Lesieur M, Hermann HL, Gérard N,
Habener JF 1991 Developmental stage-specific expression of the cyclic
AMP response element binding protein CREB during spermatogenesis
involves alternative exon splicing. Mol Endocrinol 10:14191430
-
Chomczynski P, Sacchi N 1993 Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem 162:156159[CrossRef]