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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-{alpha} (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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo). 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. 1AGo). 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. 1BGo, 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.

 
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. 2Go, 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.

 
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. 3Go, 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. 3Go, 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).

 
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. 4Go, 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. 4AGo, 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. 4BGo). 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. 4BGo and 5AGo). 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. 5BGo). 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-{alpha}, 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 10–30 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 92–124 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 [{alpha}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.


    ACKNOWLEDGMENTS
 
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.


    FOOTNOTES
 
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 32–31915.91/2 and 32–49673.96) 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. Back

Received for publication October 2, 1997. Revision received January 8, 1998. Accepted for publication January 27, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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