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Originally published In Press as doi:10.1074/jbc.M311991200 on January 30, 2004

J. Biol. Chem., Vol. 279, Issue 17, 17772-17784, April 23, 2004
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PE-1/METS, an Antiproliferative Ets Repressor Factor, Is Induced by CREB-1/CREM-1 during Macrophage Differentiation*

Dominique Sawka-Verhelle{ddagger}, Laure Escoubet-Lozach{ddagger}, Amy L. Fong{ddagger}, Kelly D. Hester{ddagger}, Stephan Herzig§, Patricia Lebrun§, and Christopher K. Glass{ddagger}||

From the Departments of {ddagger}Cellular and Molecular Medicine and Medicine, University of California at San Diego, La Jolla, California 92093 and the §Peptide Biology Laboratories, Salk Institute for Biological Studies, La Jolla, California 92037-1002

Received for publication, October 31, 2003 , and in revised form, January 26, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The molecular mechanisms involved in regulating the balance between cellular proliferation and differentiation remain poorly understood. Members of the Ets-domain family of transcription factors are candidates for proteins that might differentially regulate cell cycle control and cell type-specific genes during the differentiation of myeloid progenitor cells. The Ets repressor PE-1/METS has been suggested to contribute to growth arrest during terminal macrophage differentiation by repressing Ets target genes involved in Ras-dependent proliferation. An important feature of this regulatory model is that PE-1/METS is itself induced by the program of macrophage differentiation elicited by M-CSF. Here, we present evidence that the PE-1/METS gene is a transcriptional target of the cyclic AMP response element-binding protein-1 (CREB-1). CREB-1 expression is dramatically up-regulated during macrophage differentiation and phosphorylation of CREB-1 and the related factor CREM-1 are stimulated by M-CSF in a SAPK2/p38-dependent manner. Chromatin immunoprecipitation experiments demonstrate that CREB-1/CREM-1 are recruited to the PE-1/METS promoter as well as to the promoters of other genes that are up-regulated during terminal macrophage differentiation. Overexpression of CREB-1 stimulates the activities of the PE-1/METS, and macrosialin promoters, while expression of a dominant negative form of CREB-1 during macrophage differentiation inhibits expression of the PE-1/METS and macrosialin genes. Inhibition of CREB function also results in reduced expression of CD54 and impaired cell adhesion. Taken together, these findings reveal new roles of CREB-1/CREM-1 as regulators of macrophage differentiation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Understanding the molecular mechanisms that coordinately regulate cellular proliferation and differentiation remain central problems in biology. This problem is exemplified by the program of hematopoiesis, in which committed progenitor cells proliferate and differentiate in response to both lineage-specific and multilineage colony stimulating factors (1, 2). The program of macrophage differentiation can be recapitulated in vitro by culturing bone marrow progenitor cells in the presence of MCSF (3, 4). Progenitors that express the M-CSF receptor proliferate and differentiate, while the remaining cells die. Analysis of macrophage-specific markers such as scavenger receptor-A (SR-A)1 and macrosialin transcripts indicates that they become markedly up-regulated late in the differentiation program, with maximal expression occurring after 5-7 days of M-CSF treatment (5).

Several lines of evidence suggest that the Ras signaling pathway contributes to both the proliferation and differentiation responses of macrophages to M-CSF. Using CSF-1R (MCSF receptor)-transduced fibroblasts as a model system, dominant negative inhibitors of Ras signaling block M-CSF-dependent proliferation and inhibit M-CSF-dependent differentiation (6). Ets-domain transcription factors often act as crucial transcriptional effectors of Ras signaling pathways. In the hematopoietic system, positively acting Ets factors that include Ets-1, Ets-2, and PU-1, cooperating with other classes of transcription factors are thought to play critical roles in mediating both mitogenic and lineage-specific differentiation responses to colony stimulating factors (7-11). The involvement of Ets activators in both Ras-dependent differentiation and Ras-dependent proliferation events raises an apparent paradox; maintenance of Ras signaling is required for expression of cell type-specific genes during terminal macrophage differentiation, while the proliferation response of Ras signaling is inhibited.

Ets repressors are candidates for proteins that might differentially regulate programs of proliferation and differentiation. Ets repressor factor (ERF) and PE-1/METS constitute a related subfamily of Ets repressors that have been demonstrated to be capable of inhibiting cellular proliferation (12-14). We recently reported that the induction of the Ets repressor PE-1/METS (PU-Ets related-1/Mitogenic Ets transcriptional suppressor) during macrophage differentiation contributes to terminal cell cycle arrest by repressing transcription of cell cycle control genes that include c-Myc, c-Myb, and Cdc2 (14). While PE-1/METS exhibits an overlapping DNA binding specificity with Ets2 and other Ets activators, it does not appear to inhibit transcription of macrophage-specific genes that are activated by AP1-Ets ternary complexes such as SR-A (15, 16). These findings formed the basis for a model by which PE-1/METS contributes to terminal macrophage differentiation by selectively repressing Ets target genes involved in Ras-dependent proliferation while sparing genes that are targets of Ras-dependent differentiation.

An important feature of this regulatory model is that PE-1/METS is itself induced by the program of macrophage differentiation elicited by M-CSF. Indeed, murine bone marrow cells treated with serum and M-CSF for several days show an increase of the protein and mRNA levels of PE-1/METS. Here, we investigated the regulation of PE-1/METS expression during macrophage differentiation by studying the PE-1/METS promoter. We found that the sequence from -65 to +55 of the PE-1/METS transcriptional start site is sufficient to confer basal promoter activity and mediate a transcriptional response to serum and the phorbol ester TPA. The PE-1/METS proximal promoter contains at least two binding sites important for this activity. One of these sites is recognized by the cyclic AMP response element-binding proteins, CREB-1 and CREM-1, which are themselves induced during macrophage differentiation. CREB-1 and CREM-1 are phosphorylated in macrophages by SAPK2/p38 in response to serum and M-CSF and CREB-1 activates the PE-1/METS promoter. Inhibition of SAPK2/p38 activity or expression of a dominant negative form of CREB-1 (A-CREB) that interferes with the function of CREB-1, CREM-1, and ATF-1 blocks up-regulation of PE-1/METS during macrophage differentiation. Expression of A-CREB also inhibits expression of the macrosialin and CD54 genes and causes a defect in cell adhesion. Taken together, our data indicate that CREB-1/CREM-1 are involved in PE-1/METS expression and suggest new roles for CREB-1/CREM-1 as regulators of the macrophage differentiation process.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The Val12-H-Ras plasmid that encodes for a constitutively active form of Ras has been described previously (17). Guinea pig anti-PE-1/METS antiserum has been described previously (14). Anti-phospho-ERK1/2 was purchased from Cell Signaling Technology, Inc. (Beverly, MA). Anti-CREB-1 and {beta}-actin (sc186) (sc271) were supplied by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). PD98059, U0126, U0124, and SB203580 were purchased from Calbiochem (La Jolla, CA). The human CREB-1 cDNA, pZeo A-CREB, and the phospho-CREB-1 antibody were kind gifts from M. Montminy (Salk Institute, San Diego, CA). The pRc/RSV FLAG rat CREB-1 vector was a generous gift from R. Goodman (Vollum Institute, Portland, OR) (18). Oligonucleotides were purchased from Operon (Qiagen Inc., Valentia, CA) and Invitrogen, Inc. (Carlsbad, CA). The macrosialin-luciferase reporter gene that contains 7 kb of the 5'-flanking region of macrosialin linked to {Delta}5'-PSV2 luciferase vector (19) has been described previously (20). The SR-A S10A1 plasmid containing the SR-A promoter linked to {Delta}5'-PSV2 luciferase vector has been described previously (15, 16).

DNA Constructs—The PE-1/METS promoter was isolated from a BAC plasmid (Genome System Inc., St Louis, MO) containing a 200-kb genomic DNA fragment that encompasses the PE-1/METS gene. A 4-kb EcoRV fragment containing exon 1 was identified by Southern blotting and subcloned. The METS (-1662; +1683) construct was obtained by PCR using the 4-kb EcoRV fragment as a template, the sense primer 5'-ACGCGTCGACCACTGTGCCATCCTCCGTCC-3' and the antisense primer 5'-GCCAAGCTTGATGCTACAGCCTGCTTTCAT-3'. SalI and HindIII sites were incorporated into the primers as indicated by underline. The PCR product was cut with SalI and HindIII and subcloned into a lacZ reporter gene vector at the SalI and HindIII sites. METS luciferase constructs were obtained by subcloning PCR-amplified products into a BamHI- and HindIII-digested {Delta}5'-PSV2 luciferase vector. The primers used for the different METS promoter constructs are indicated in Table I. METS (+555; +1683) construct was obtained by ligation of {Delta}5' PSV2-cut BamHI-HindIII with a 1.1-kb fragment obtained after the double digestion with BamHI and HindIII of the 4-kb EcoRV fragment. METS (-2011; +55) {Delta}IRE1, {Delta}IRE2, {Delta}IRE1/{Delta}IRE2, mGAAA, mTGAC, mGTCA, and mIRE1/2 constructs were obtained by site-directed mutagenesis of double-stranded DNA using the QuickChange kit (Invitrogen, Inc.) and cloned into the {Delta}5'-PSV2 luciferase vector. The mutant METS (-2011; +55) {Delta}IRE1 and METS (-2011; +55) {Delta}IRE2 contain deletions of GGAAGTGA and GTCACTTCC, respectively. The mutant METS (-2011; +55) {Delta}IRE1/{Delta}IRE2 contains a double deletion of IRE1 and IRE2. The point mutations made to make the METS (-2011; +55) mGAAA, mTGAC, mGTCA, and mIRE1/2 are listed in Fig. 5A.


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  		TABLE I
Primers used to PCR-amplify METS luciferase constructs

 



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  		FIG. 5.
IRE1 and IRE2 are required for CREB-dependent activation of the PE-1/METS promoter. A, sequence of the PE-1/METS proximal promoter. IRE1 and IRE2 contain two inverted repeat elements separated by 15 nucleotides. Point mutations introduced to make the mGGAA, mTGAC, mGTCA, and mIRE1/2 are indicated in bold letters. B, point mutants of the PE-1/METS promoter (-2011; +55) driven luciferase reporter gene were cotransfected with empty vector, pRc/RSV FLAG CREB, or pZeo A-CREB as indicated into RAW 264.7 macrophages using Superfect Reagent as described under "Experimental Procedures." Cells were also transfected with a CMV-driven Renilla luciferase reporter to normalize for transfection efficiency. Twenty-four hours post-transfection, luciferase activity was measured and normalized to Renilla luciferase activity. Results are the mean ± S.E. of triplicates.

 
Cell Culture and Transfection—Bone marrow-derived macrophages were obtained from 6- to 10-week-old Swiss Webster mice (Charles River Laboratories, Wilmington, MA). Macrophages were cultured in RPMI 1640 (Invitrogen, Inc) supplemented with 20% FBS (Gemini, Woodland, CA) and 30% L cell-conditioned medium as a source of M-CSF. The adherent cells were harvested at the indicated day of incubation. CV-1 cells and RAW 267.4 macrophages were cultured in DMEM (Invitrogen Inc.) supplemented with 10% FBS. CV-1 cells were transfected with LipofectAMINE Reagent (Invitrogen, Inc.) for 6 h following the manufacturer's recommendations. RAW 267.4 macrophages were either transfected with LipofectAMINE Reagent for 6 h or with Superfect Reagent (Qiagen) for 3 h, as indicated, according to the manufacturer's instructions. Medium was changed to Dulbecco's modified Eagle's medium supplemented with 10% FBS, and cells were assayed for luciferase activity 16 h (LipofectAMINE) or 24 h (Superfect) later. THP-1 (monocytic leukemia) and U937 (histocytic leukemia) were incubated with RPMI 1640 supplemented with 10% FCS and transfected by electroporation as previously described (21) using 10-20 µg of total plasmid DNA. THP-1 and U937 cells were supplemented with 0.5 or 10% FBS and treated with TPA (10-7 M) for 16 h depending on the cells and on the experiments. Cells were harvested and luciferase activity was determined as described previously (19). {beta}-Galactosidase activity was assayed using Galacto-Light TM (Tropix, Bedford, MA). Renilla luciferase activity was assayed using Renilla luciferase assay system, (Promega, Madison, WI). {beta}-Galactosidase activity or Renilla luciferase activity was used to normalize PE-1/METS promoter-driven luciferase activity, as indicated, and is expressed as relative promoter activity in arbitrary units. {beta}-Galactosidase activity was normalized to luciferase activity and expressed as relative promoter activity in arbitrary units.

Preparation of Nuclear Extracts—Nuclear extracts from CV-1 cells and bone marrow-derived macrophages were obtained as described previously (22). After centrifugation, lysates were removed and pellets were solubilized in 1 mM phenylmethylsulfonyl fluoride and protease inhibitor mixture (Roche Applied Science). Pellets were vortexed at 4 °C for 15 min and then nuclear extracts were collected by centrifugation at 13,000 rpm for 15 min at 4 °C. Quantification of protein was performed using a modified Bradford assay (Bio-Rad), and extracts were stored at -80 °C.

Electrophoretic Mobility Shift Assay (EMSA)—Nuclear extracts (10 µg) were incubated for 15 min at 4 °C with EMSA buffer (150 mM KCl, 20 mM HEPES, 2 mM dithiothreitol, 0.1% Nonidet P-40, 1 µg of poly-(dIdC)) in a total volume of 18 µl. For supershift experiments, 2 µg of antibody were added to the mixture for 30 min at 4 °C. Probes (5 x 104 cpm) were added and the incubation continued at room temperature for 20 min. Polyacrylamide gels (5%) containing 2.5% (v/v) glycerol and 0.5% Tris borate/EDTA were prerun in 0.5% Tris borate/EDTA buffer at 4 °C for 30 min at 300 volts. After loading the samples, the gel was run at 4 °C for 2 h at 300 volts prior to autoradiography. Double-stranded oligonucleotide probes were labeled with T4 polynucleotide kinase in presence of [{gamma}-32P]ATP. The sequences of sense strands of the double-strand oligonucleotides were as follows: IRE1/IRE2, 5'-CCCAGAGTGGAAGTGACGAAAGCCGCCGCGGAGTCACTTCCTTCTGG-3'; IRE1, 5'-CTGGCCCAGAGTGGAAGTGACGAAAGCCGCC-3'; IRE1{Delta}GGAAGTGAC, 5'-CTGGCCCAGAGTGAAAGCCGCC-3'; IRE1mGGAA, 5'-CTGGCCCAGAGTTTAAGTGACGAAAGCCGCC-3'; IRE1{Delta}TGAC, 5'-CTGGCCCAGAGTGGAAGGAAAGCCGCC-3'; IRE2, 5'-GAAAGCCGCCGCGGAGTCACTTCCTTCTGGGTGGATGA-3'.

Primer Extension—Primer extension assays were performed as described (23) with the substitution of Superscript (Invitrogen, Inc.) for avian myeloblastosis virus reverse transcriptase. In brief, 10 µg of total RNA isolated from bone marrow-derived macrophages after 7 days of M-CSF incubation were hybridized at 65 °C for 90 min to end-labeled primer located in exon 1 5'-TCTCCGGCGCCGTCCGTCTACACTC-3'. Reverse transcription was performed for 1 h at 42 °C with Superscript in the appropriate buffer. RNA was degraded with RNase H (Invitrogen, Inc.), and cDNA was isolated by phenol-chloroform extraction. Parallel sequencing reactions of a PE-1/METS genomic clone containing exon 1 was performed using the Sequenase kit (Amersham Biosciences). Both the sequencing reaction and the cDNA obtained from the primer extension were separated on 8% acrylamide/7 M urea sequencing gel, and visualized by autoradiography.

Western Blot Analysis—Macrophages were washed with ice-cold phosphate-buffered saline, and the pellet of cells resuspended in radio-immune precipitation assay lysis buffer as described previously (24). 50 µg of total proteins was separated on a NuPage Novex Bis-Tris 4-12% gel (Invitrogen, Inc.) and transferred onto a nitrocellulose membrane. The membrane was probed with different antibodies: anti-METS (1/1000), anti-CREB-1 (1/1000), anti-phospho-CREB-1 (1/2000), anti-phospho-ERK1/2 (1/1000), and anti-{beta}-actin (1/1000). Proteins were revealed with a chemiluminescence detection system (ECL) (Pierce).

Real Time PCR Analysis—Total RNA samples from bone marrow-derived macrophages cultured for different lengths of time with M-CSF were prepared using the RNeasy Mini Kit (Qiagen). 1 µg of DNase-treated RNA was used to generate cDNA using an oligo(dT) oligodeoxynucleotide primer following the protocol for Superscript. Primers used for analysis of SR-A, macrosialin, and GAPDH expression have been described previously (25). The following additional primers were used for analysis of PE-1/METS expression: METS, 5'-GACAGCCTCCGGCAGTCA-3' (forward), 5'-GCTCCGAATGCTGTCCACTT-3' (reverse), and 5'-CCCAGGGAAAGGAGGAACAGACCCA-3' (probe). Equal amounts of cDNA were used in triplicate and amplified with the Taqman master Mix provided by PerkinElmer Applied Biosystems. Nanograms of products were calculated using the standard curve method and normalized against GAPDH.

Chromatin Immunoprecipitation (ChIP) Assay—Cells were cross-linked with 1% formaldehyde at room temperature for 10 min. Chromatin extracts were obtained as previously described (26). Sonication was performed 20 times for 10 s each at Power 7 (MICROSONTM, Ultrasonic Cell Disruptor, Model XL) resulting in DNA fragments between 150 and 600 bp in size. Immunoclearing was performed for 6 h at 4C using 2 µg of sheared salmon sperm DNA (Invitrogen Inc.), 15 µl of normal rabbit serum (The Jackson Laboratory, Bar Harbor, ME) and 45 µl of protein A-Sepharose (50% slurry in dilution buffer) (Sigma). Supernatants were collected and submitted to immunoprecipitation with 5 µl of polyclonal anti-CREB-1 antibody overnight at 4C. In parallel, supernatants were incubated with normal rabbit serum as controls. Then 45 µl of protein A-Sepharose were added and the incubation was continued for 4 h. Precipitates were washed and extracted with 1% SDS (v/v), 0.1 M NaHCO3, and heated at 65 °C overnight to reverse the cross-linking. DNA fragments were purified on QIAquick Spin columns (Qiagen). Finally, 3 µl from a 30-µl extraction were amplified by PCR for 30 cycles with the primers described in Table II.


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  		TABLE II
Specific primers used to amplify by PCR different promoter regions precipitated by CHIP experiments

 
Adenovirus Infection—Bone marrow-derived monocytes were separated by Ficoll gradient purification and cultured for 3 days with serum 20% FBS and 30% L-cell-conditioned medium as a source of M-CSF. Adherent cells were washed and infected for 3 h with 2.5 x 108 adenovirus particles/10 cm dish conferring expression of either GFP alone or GFP and A-CREB (27). Cells were washed and cultured for 3 more days with RPMI 1640 supplemented with serum and L-cell conditioned medium. Supernatant and adherent cells were harvested and subjected to flow cytometry analysis or fluorescence-activated cell sorting of GFP-positive cells.

Flow Cytometry Analysis—For flow cytometry analysis, cells were preincubated with anti-mouse CD16/CD32 antibody (Fc Block, BD Pharmingen, La Jolla, CA) for 15 min at room temperature then incubated with anti-mouse CD54 for 10 min at room temperature (Pharmingen). Cells were then submitted for analysis on a FACScan using the Cell Quest software (BD Biosciences).

GenBankTM Accession Number—The nucleotide sequence of the murine PE-1/METS gene reported in this article has been submitted to the GenBankTM/EBI Data Bank with the accession number AY274927 [GenBank] .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
PE-1/METS Is Up-regulated during Macrophage Differentiation—To better understand the regulation of PE-1/METS expression during macrophage differentiation, we analyzed PE-1/METS protein and RNA levels during bone marrow-derived macrophage differentiation (Fig. 1). Murine bone marrow progenitor cells were cultured with serum and M-CSF for 2-7 days to induce macrophage differentiation. Cells were subjected to Western blot and real-time PCR analysis in order to quantify protein and mRNA levels, respectively. Protein analysis indicated low levels of PE-1/METS up to 4 days of M-CSF treatment, then a progressive increase from day 5 to day 7 of culture (Fig. 1A). The real-time PCR assay indicated a marked up-regulation of PE-1/METS mRNA from day 1 to day 7 of culture (Fig. 1B). Taken together, these data confirm that PE-1/METS is up-regulated during macrophage differentiation and suggest that this regulation is transcriptional.



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  		FIG. 1.
PE-1/METS is up-regulated during macrophage differentiation; structure of PE-1/METS gene. Murine bone marrow progenitor cells were cultured in the presence of serum and M-CSF to induce macrophage differentiation as described under "Experimental Procedures." At different days of cell culture, whole cell extracts and total RNA were prepared from adherent cells and analyzed by Western blot and real-time PCR, respectively. A, Western blot using anti-PE-1/METS antibody. B, real-time PCR was performed with specific primers for PE-1/METS and GAPDH. Nanograms of PE-1/METS mRNA were calculated using the standard curve method and normalized against GAPDH. C, schematic diagram of the intron-exon structure of PE-1/METS gene starting at the transcription site. Black boxes correspond to the coding sequence of PE-1/METS. D, transcription start sites revealed by primer extension analysis using 10 µg of total RNA from murine bone marrow-derived macrophages after 7 days of M-CSF incubation. Lane 5 represents primer extension products resulting from priming with the antisense primer exon 1. Lanes 1-4 represent sequencing reactions of genomic DNA from murine bone marrow-derived macrophages after 7 days of M-CSF using the same primer. E, sequence of the transcription and translation start sites of PE-1/METS. +1 was assigned to the A nucleotide from the first transcription start site. The ATG translation start site is located 65 nucleotides downstream of the transcription start site on the mRNA.

 
Cloning and Characterization of PE-1/METS Gene—To investigate molecular mechanisms controlling PE-1/METS gene expression, the PE-1/METS gene was isolated from a BAC plasmid and the exon structure was defined (Fig. 1C). The PE-1/METS gene is composed of 5 exons and 4 introns, with the translation start located in exon 2. The transcription start sites were determined by primer extension analysis using total RNA from murine bone marrow-derived macrophages cultured for 7 days with M-CSF. An oligonucleotide complementary to PE-1/METS mRNA located in the exon 1 (primer exon 1) was used for primer extension experiments. Two extension products were obtained. They are separated by 3 nucleotides and start with AG (Fig. 1D). We have utilized the convention in which the +1 corresponds to the first nucleotide of the first transcription start (Fig. 1E). In this nomenclature, nucleotide A from the initiation codon (ATG) in the mature mRNA transcript is +65 and is located in exon 2.

Functional Analysis of the PE-1/METS Promoter—As primary macrophages cannot be effectively transfected with reporter plasmids, functional analysis of transcriptional regulatory regions of the METS gene was performed using U937 and THP-1 monocytic leukemia cells and the RAW264.7 macrophage cell line. To identify regions of the METS gene important for promoter activity, transient transfection assays were performed (Fig. 2). A 3.2-kb DNA fragment containing nucleotides -1662 to +1683 linked to a lac Z reporter gene, conferred a 7-fold increase in {beta}-galactosidase expression compared with the empty vector in Raw 264.7 cells (Fig. 2A). Regions upstream of exon 1 and exon 2 were also cloned upstream of a luciferase reporter and analyzed in Raw 264.7 cells (Fig. 2B). The METS (-2011; +55) construct that contains 2011 nucleotides upstream of the transcription start and 55 nucleotides of exon 1 directed expression of luciferase activity whereas the METS (+555; +1683) construct containing information upstream of exon 2 did not. The PE-1/METS promoter (-2011; +55) also directed reporter gene activity in human THP-1 monocytic leukemia cells cultured in 0.5% serum and was further activated by culture with 10% serum or the phorbol ester TPA (Fig. 2C). Additive effects were observed with the combination of 10% serum and 10-7 M TPA (5-fold increase).



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  		FIG. 2.
Localization of PE-1/METS promoter. The indicated 5'-flanking regions of PE-1/METS were linked to a {beta}-galactosidase reporter gene (A) or luciferase reporter gene (B and C) and transfected into Raw 264.7 macrophages using LipofectAMINE (A and B) or THP-1 monocytic leukemia cells (c) as described. A and B, 16 h after transfection, cells were harvested and subjected to luciferase and {beta}-galactosidase assays. To control for different transfection efficiencies, PE-1/METS promoter activities were normalized to the activity of the {beta}-actin promoter in both cell types. Results expressed as relative promoter activity correspond to {beta}-galactosidase activity (A) and luciferase activity (B and C) normalized to {beta}-actin luciferase activity and {beta}-actin {beta}-galactosidase activity, respectively. C, 6 h after transfection, THP-1 cells were cultured with 0.5 or 10% serum and treated with or without TPA (10-7 M) for 16 h. Cells were harvested and luciferase activity measured and normalized with {beta}-actin {beta}-galactosidase activity. Results are the mean ± S.E. of triplicates and are representative of three independent experiments.

 
In order to localize cis active elements within the PE-1/METS proximal promoter, the METS (-2011; + 55) construct was subjected to several 5' and 3' deletions. Luciferase activities of these mutants were measured in the human monocytic leukemia cell line U937 cultured with 10% serum (Fig. 3A). Deletion of the 3'-sequence from nucleotides +55 to -45 abolished promoter activity whereas deletion of 5'-sequences from -2011 to -162 had modest effects on luciferase activities. A further truncation to -65 reduced, but did not abolish PE-1/METS promoter activity. Interestingly, an increase of promoter activity was found with deletion of nucleotides from -2011 to -290, suggesting that this region could contain binding sites for transcription factors involved in the repression of PE-1/METS transcription. These deletion constructs were also analyzed in Raw 264.7 cells and identical results were observed (data not shown).



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  		FIG. 3.
The PE-1/METS proximal promoter is induced by TPA and serum. A, indicated 5'- and 3'-deletion mutants of the PE-1/METS promoter extending from -2011 bp to +55 bp were assessed for promoter activity in human U937 histocytic leukemia cells. Cells were cultured with 10% serum and lysed 16 h post-transfection. Luciferase activities were measured, normalized to {beta}-galactosidase activity and expressed as relative promoter activity. B, cells were transfected with PE-1/METS proximal promoter (-65; +55) and cultured in 10% serum. Six hours after transfection, cells were treated with or without TPA (10-7 M) for 16 h. Luciferase activities were measured, normalized to {beta}-galactosidase activity, and expressed as relative promoter activity. C, cells were transfected with the PE-1/METS proximal promoter (-65; +55) luciferase reporter gene and the plasmid encoding a constitutive active form of Ras (Val12H-Ras) as indicated. After 6 h of transfection, cells were resuspended into medium containing 0.5 or 10% serum for 16 h. Luciferase activity was measured, normalized to {beta}-galactosidase activity and expressed as relative promoter activity. Results are the mean ± S.E. of triplicates and are representative of three independent experiments.

 
Treatment of U937 cells transfected with METS (-65; +55) construct with 10-7 M TPA and 10% serum for 12 h resulted in a 2.5-fold and a 2-fold increase of luciferase activity, respectively (Fig. 3, B and C). Furthermore, cotransfection of a constitutively active form of Ras (Val12 H-Ras) with the METS (-65; +55) construct increased the luciferase activity (2-fold) in 0.5% serum and reached approximately the same activity obtained when cells were cultured with 10% serum (Fig. 3C). These data indicate that activation of Ras signaling stimulates PE-1/METS proximal promoter activity and suggest that the Ras pathways are fully activated in these cells with 10% serum.

Analysis of the nucleotide sequence of the METS -65; +55 region revealed two identical inverted repeat elements (IREs) separated by 15 nucleotides referred to as IRE1 and IRE2 (Figs. 4A and 5A) that are also conserved in the human PE-1/METS gene. Computer-assisted analysis of the PE-1/METS proximal promoter revealed several potential binding sites for sequence-specific transcription factors. The IRE sequences contain potential binding sites for Ets transcription factors such as Ets-1 and Ets-2, and ATF transcription factor family members such as ATF-1, ATF-2, CREB-1, and CREM-1. To determine whether these sequences are required for PE-1/METS promoter function, IRE1 and IRE2 were deleted individually or together in the context of the METS (-2011; + 55) construct. Mutants were assayed for promoter activity in THP-1 cells cultured with 10% serum. Deletion of IRE1 or IRE2 resulted in decreased promoter activity, with deletion of IRE1 resulting in a more substantial loss of activity (7-fold decrease) compared with IRE2 deletion (3-fold decrease). When both sites were deleted, the promoter activity was totally abolished, indicating that both IRE1 and IRE2 contain cis-active elements required for this activity (Fig. 4A).



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  		FIG. 4.
Both IRE1 and IRE2 mediate activation of the PE-1/METS gene and CREB-1/CREM-1 binds IRE1. A, PE-1/METS promoter extending from -2011 bp to +55 bp was subjected to deletion of IRE1 and IRE2 sites individually or together and assayed for promoter activity in THP-1 cells in addition to {beta}-actin-{beta}-galactosidase. After 6 h of transfection, cells were resuspended into medium containing 10% serum for 16 h. Luciferase activity was measured, normalized to {beta}-galactosidase activity and expressed as relative promoter activity. Results are the mean ± S.E. of triplicates and representative of three independent experiments. B-E, EMSA using 10 µg of nuclear extracts from CV-1 cells and 32P-labeled probes as indicated on the panels. The sequences of the probes are described under "Experimental Procedures." For supershift experiments, 2 µg of CREB-1/CREM-1 antibody were added to the mixture. DNA-protein complexes are indicated with arrows.

 
CREB-1/CREM-1 Bind to IRE1—In order to characterize proteins binding to these sites, EMSA were performed with nuclear extracts prepared from simian kidney fibroblast CV-1 cells cultured with 10% serum. CV-1 cells were chosen for this initial level of analysis because nuclear extracts derived from primary macrophages exhibit high proteolytic activity that cannot be completely blocked by protease inhibitors. Incubation of CV-1 nuclear extracts with a probe that contains both IRE1 and IRE2 resulted in formation of a cluster of bands (Fig. 4B). Four of them appeared to be specific because they could be competed with unlabelled IRE1/IRE2 probe in a concentration-dependent manner. The presence of CREB-1/CREM-1 in at least one of these complexes was confirmed by supershift with an antiCREB-1/CREM-1 antibody (Fig. 4C). In contrast, antibodies to ATF-1, ATF-2, Ets-1, Ets-2, METS, ERF, c-jun, junD, junB, c-fos, Elk-1, c-maf, and PU-1 did not alter the presence or migration of these (data not shown).

In order to define the CREB-1/CREM-1 binding site, EMSAs and supershift assays with CREB-1/CREM-1 antibody were performed with a series of probes containing wild type and mutant sequences of IRE1 and/or IRE2 (Fig. 4, D and E). CREB-1 binds specifically to IRE1 since CREB-1/CREM-1 antibody supershifted one of the DNA-protein complexes obtained with the IRE1 probe (Fig. 4D, lanes 3 and 4). DNA-protein complexes identified with IRE1/IRE2 probe were not found when EMSAs were performed with IRE2 probe. Instead a more slowly migrating DNA-protein complex was found (Fig. 4E, lane 1) that was not altered by CREB-1/CREM-1 antibody, suggesting that protein complexes bound to IRE2 do not contain CREB-1 or CREM-1 (Fig. 4E, lane 3). In order to precisely identify the CREB-1 site, several mutant probes containing sequences surrounding IRE1 were used for EMSA analysis. Deletion of the entire GGAAGTGAC sequence from the IRE1 probe eliminated the formation of specific protein-DNA complexes and exhibited no CREB-1/CREM-1 binding (Fig. 4D, lanes 5 and 6). Similarly, deletion of TGAC alone, which forms a half-site for CREB-1 family members, abolished DNA-protein complex formation and CREB-1/CREM-1 binding (Fig. 4D, lanes 9 and 10). From these experiments, we concluded that CREB-1/CREM-1 recognizes the TGAC sequence in IRE1. Nevertheless, the GGAA site in IRE1 seems to play a role in CREB-1/CREM-1 binding because we observed a decrease in both the specific protein-DNA complexes and CREB-1/CREM-1 binding when the GGAA site in the IRE1 probe is mutated (Fig. 4D, lanes 7 and 8).

To investigate the role of CREB-1/CREM-1 in regulation of the PE-1/METS promoter, the effects of overexpression of a dominant negative form of CREB, A-CREB, which inhibits DNA binding of CREB-1, CREM-1, and ATF-1, were evaluated in RAW cells. Overexpression of A-CREB drastically reduced the activity of the wild-type promoter (Fig. 5B). Point mutations were also introduced into the PE-1/METS promoter luciferase reporter in order to more precisely establish the roles of specific transcription factor binding sites in IRE1 and IRE2 for transcriptional activation of the PE-1/METS gene (Fig. 5A). Disruption of the Ets family binding site in IRE1 (mGGAA) or the CREB-1 family binding sites in IRE1 (mTGAC), IRE2 (mGTCA), or in both IRE1 and IRE2 (mIRE1/2) resulted in a significant loss of PE-1/METS reporter gene activation (Fig. 5B, black bars), indicating functional importance of each of these sites. Interestingly, the dominant negative form of CREB had little or no effect on the residual activities of any of these mutants (Fig. 5B, white bars). Thus, even though the electro-phoretic mobility shift assays only demonstrated binding of CREB-1/CREM-1 to IRE1, these results suggest that the METS promoter is regulated in a CREB-dependent manner through both IRE1 and IRE2 and not through other proximal sites in the promoter.

Overexpression of CREB-1 Increases the Activities of the PE-1/METS, Macrosialin, and SR-A Promoters in CV-1 Cells—Cotransfection of wild-type CREB-1 failed to significantly potentiate transcription of the wild-type or mutant reporter genes in RAW cells, perhaps due to high levels of endogenous CREB-1 expression (data not shown). To independently examine whether CREB-1 can function as an activator of PE-1/METS transcription, the METS (-2011; +55) promoter construct was transfected into CV-1 cells together with either an empty vector or a vector directing expression of the human CREB-1 cDNA. Cells were assayed for promoter activity 24 h after transfection. CREB-1 expression stimulated the activity of the PE-1/METS promoter ~3-fold in these cells (Fig. 6A).



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  		FIG. 6.
CREB-1/CREM-1 are induced and phosphorylated during macrophage differentiation, and activate PE-1/METS, SR-A, and macrosialin promoters. A, luciferase reporter genes under the control of the PE-1/METS promoter (-2011 bp to +55 bp), the macrosialin promoter (7-kb 5'-flanking region) or the SR-A promoter (-245; +72) were cotransfected with either a plasmid directing CREB-1 expression or an empty vector into CV-1 cells. In addition, cells were cotransfected with a renilla luciferase reporter under the control of the CMV promoter. Luciferase and Renilla luciferase activities were measured 24-h post-transfection. Luciferase activity was normalized to Renilla luciferase activity and expressed as relative promoter activity. Results are the mean ± S.E. of triplicates and are representative of three independent experiments. B and C, murine bone marrow-derived macrophages were cultured with serum and M-CSF for 2, 4, 6, and 8 days. Whole cell extracts were prepared and subjected to Western blot using CREB-1/CREM-1 antibody (B) and phospho-CREB-1/CREM-1 antibody (C). D, ChIP experiments were carried out in murine bone marrow progenitor cells and in murine bone marrow-derived macrophages cultured for 7 days. Following formaldehyde cross-linking, genomic sequences corresponding to PE-1/METS, SR-A, and macrosialin were subjected to PCR amplification following immunoprecipitation of protein-DNA products with CREB-1/CREM-1 antibody. Preimmune rabbit serum was used as a control. As a negative control, primers directed against 3'-end of the PGC-1 gene have been used for the PCR.

 
SR-A and macrosialin are macrophage-specific genes that are up-regulated during terminal differentiation. Previous studies have demonstrated that activation of these genes involves cis active elements that are recognized by AP-1 family members (20, 28, 29). Because CREB binding elements have been suggested in the macrosialin promoter (30) and computer analysis (31) revealed several potential CREB-1 binding sites in the murine and human SR-A promoters, we investigated whether these promoters could also be activated by CREB-1. We found that CREB-1 expression stimulated SR-A and macrosialin promoter activity 3-fold and 2-fold, respectively (Fig. 6A).

CREB-1/CREM-1 Are Induced and Phosphorylated during Macrophage Differentiation—To investigate whether the ability of CREB-1 to activate the PE-1/METS promoter is relevant to regulation of PE-1/METS expression in macrophages, levels of CREB-1 and phospho-CREB-1 protein were evaluated during the differentiation of murine bone marrow progenitor cells in response to M-CSF. Western blot analysis indicated low levels of CREB-1 up to 4 days of M-CSF-dependent differentiation, with a progressive increase observed from day 4 to day 8 of culture (Fig. 6B). CREM-1 protein levels are also increased, although less dramatically than CREB-1. In addition, Western blot analysis performed with an anti-phospho-CREB-1 antibody that also recognizes phospho-CREM-1 indicated that CREB-1 and CREM-1 became phosphorylated after 4 days of bone marrow differentiation, with the extent of phosphorylation progressively increasing through day 8 of culture (Fig. 6C).

CREB-1 Is Recruited to PE-1/METS, SR-A, and Macrosialin Promoters during Macrophage Differentiation—To determine whether CREB-1/CREM-1 binds to PE-1/METS promoter during macrophage differentiation, ChIP experiments were performed. Specific primers were designed to amplify the PE-1/METS promoter region (-161/-31) containing the CREB-1 binding site. ChIP experiments were carried out in bone marrow progenitor cells and in bone marrow-derived macrophages cultured for 7 days with M-CSF (Fig. 6D). Immunoprecipitations of chromatin from bone marrow-derived macrophages using anti CREB-1/CREM-1 IgG significantly enriched the PE-1/METS promoter sequence as compared with immunoprecipitations using normal rabbit serum. In contrast, relative enrichment of the PE-1/METS promoter was not observed using chromatin obtained from bone marrow progenitor cells, confirming that CREB-1/CREM-1 is recruited to the PE-1/METS promoter during macrophage differentiation. In addition, CREB-1/CREM-1 antibody precipitated the SR-A and macrosialin promoters (Fig. 6D). CREB-1/CREM-1 binding to these promoters increased during macrophage differentiation as was observed for the PE-1/METS promoter. Taken together, these findings indicate that CREB-1/CREM-1 are induced, phosphorylated and recruited to the PE-1/METS, macrosialin and SR-A promoters during macrophage differentiation.

Mechanism of CREB-1 Phosphorylation—We next investigated the signaling pathway(s) responsible for CREB-1 activation in macrophages. CREB-1 has been described to be phosphorylated at Ser-133 by mitogen and stress-activated protein kinase-1 (MSK1) in response to cell activation with growth factors and phorbol ester (32, 33), while CREM-1 is phosphorylated on conserved Ser-117 (34). MSK1 is activated in vitro and in vivo by two different classes of mitogen-activated protein kinase (MAPK), ERKs, and stress-activated protein kinases SAPK2/p38 (35). In order to elucidate which kinases are involved in CREB-1/CREM-1 phosphorylation in macrophages, we treated bone marrow-derived macrophages with specific MEK inhibitors that inhibit the ERK1/2 pathway (PD98059 and U0126) or a SAPK2/p38 inhibitor (SB203580). Cells were serum-deprived for 12 h, treated for 1 h with specific inhibitors, and then stimulated 15 min with 10% serum or 10 min with M-CSF (10 ng/ml). Whole cell extracts were prepared and subjected to Western blot analysis with either phospho-CREB-1 antibody or with phospho-ERK1/2 antibody to check the inhibitor effect of the drugs (Fig. 7A). Treatment of cells with two different ERK inhibitors U0126 and PD98059 prevented phosphorylation of ERK1/2, but CREB-1/CREM-1 phosphorylation in response to serum activation remained unchanged as compared with Me2SO and U0124 treatments (Fig. 7A). In contrast, M-CSF-dependent (Fig. 7B) and serum-dependent (data not shown) phosphorylation of CREB-1 and CREM-1 was decreased when cells were treated with SB203580. This effect was dose-dependent with the maximum effect observed when cells were treated with 5 µM SB203580, suggesting that the SAPK2/p38 pathway is responsible for CREB-1/CREM-1 phosphorylation during macrophage differentiation.



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  		FIG. 7.
An inhibitor of SAPK2/p38 blocks CREB-1 and CREM-1 phosphorylation and PE-1/METS expression during macrophage differentiation. A and B, murine bone marrow-derived macrophages cultured with serum and M-CSF for 7 days were serum-deprived for 12 h and treated for 1 h with MEK inhibitors; PD98059 (25 µM) or U0126 (10 µM) and negative controls; U0124 (10 µM) and Me2SO (A), or 0, 1, or 5 µM of SAPK2/p38 inhibitor (SB 203580) (B). Cells were stimulated for 15 min with 10% serum or 10 min with M-CSF (10 ng/ml) as indicated. Whole cell extracts were prepared and subjected to Western blot analysis using phospho-CREB-1 and phospho-ERK1/2 antibodies (A) and phospho-CREB-1 and CREB-1 antibodies (B). Bone marrow-derived macrophages separated by conventional Ficoll gradient purification were cultured with serum and M-CSF for 4 and 5 days. SB 203580 (5 µM) or Me2SO were added to the culture at day 0, day 3 and day 5. Whole cell extracts (C) and RNA (D) were prepared. C, whole cell extracts prepared after 4 and 5 days of culture were subjected to Western blot analysis using PE-1/METS, phospho-CREB-1, and CREB-1 antibodies. {beta}-Actin antibody is used to control the amount of loaded proteins. D, RNA prepared after 6 days of culture was subjected to real time PCR with specific primers for PE-1/METS and GAPDH. Nanograms of PE-1/METS mRNA were calculated using the standard curve method and normalized against GAPDH.

 
In order to investigate the role of SAPK2/p38 on PE-1/METS expression, we differentiated bone marrow progenitor cells in the presence of SB203580. As shown in Fig. 1A, PE-1/METS becomes markedly up-regulated between 4 and 5 days of macrophage differentiation in response to M-CSF. Treatment of bone marrow progenitor cells with SB203580 during the differentiation process did not significantly affect macrophage proliferation, but did result in a viable, mostly non-adherent population of cells at day 5 of culture that became reattached to the culture plates at day 6 (data not shown). SB203580 treatment at days 0, 3 and 5 completely inhibited phosphorylation of CREB-1 and CREM-1 in whole cell extracts derived from cells harvested at day 4 or 5 of M-CSF-dependent differentiation as detected by Western blot analysis with anti-phospho-CREB-1 antibody (Fig. 7C). CREB-1 and CREM-1 protein levels were also reduced in SB203580-treated cells, while expression of {beta}-actin was not affected (Fig. 7C). Treatment of cells with SB203580 decreased PE-1/METS expression at both days 4 and 5 of M-CSF-dependent differentiation as compared with Me2SO-treated cultures. Consistent with these results, real-time PCR analysis demonstrated a 2-fold decrease of PE-1/METS mRNA levels in cells treated with SB203580 as compared with Me2SO treatment (Fig. 7D). These results support a role of SAPK2/p38 in CREB-1/CREM-1 phosphorylation and PE-1/METS expression bone marrow-derived macrophage differentiation.

A Dominant Negative Form of CREB-1 Inhibits PE-1/METS and Macrosialin Expression—As an independent line of investigation into the potential role of CREB-1 as an inducer of PE-1/METS expression, bone marrow progenitor cells were infected with an adenovirus expressing either green fluorescent protein (GFP) alone or an adenovirus directing expression of GFP and a dominant negative form of CREB-1 (A-CREB) (27). A-CREB inhibits the activities of CREB-1, CREM-1 and ATF-1, but not other basic-leucine zipper transcription factors (36). Cells were infected on day 3 of differentiation and cultured in M-CSF for 3 additional days prior to analysis. GFP-positive cells were isolated using fluorescence-activated cell sorting and analyzed for transcription of specific mRNAs by quantitative real-time PCR. A-CREB had no effect on the expression of the macrophage marker F4/80 (data not shown) but significantly inhibited the expression of PE-1/METS and macrosialin (Fig. 8A). Despite the presence of CREB-1 on the SR-A promoter determined by ChIP (Fig. 6D) and the ability of CREB-1 to stimulate SR-A promoter activity in CV-1 cells (Fig. 6A), expression of A-CREB did not significantly alter SR-A expression. Intriguingly, macrophages expressing A-CREB became non-adherent at day five of culture (Fig. 8B), suggesting a role of CREB-1 in the regulation of cell adhesion. Evaluation of proteins involved in cell adhesion, including CD54/ICAM1 (37, 38) revealed a significant reduction in CD54 expression on cells expressing A-CREB (Fig. 8C). No significant change in CD11b expression was observed (data not shown).



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  		FIG. 8.
Expression of a dominant-negative form of CREB (A-CREB) decreases PE-1/METS, macrosialin, and CD54 expression and inhibits cell adhesion. A, bone marrow progenitor cells isolated by conventional Ficoll gradient purification were cultured with serum and M-CSF for 3 days before being infected for 3 h with adenovirus expressing GFP alone or adenovirus expressing GFP and A-CREB. Three days later, GFP-positive cells were analyzed for expression of PE-1/METS, SR-A and macrosialin transcripts by quantitative real-time PCR. Expression values are normalized to GAPDH mRNA levels. B, GFP-positive cells that express A-CREB are non-adherent, while cells expressing GFP alone exhibit normal adherence to bacterial culture dishes. Photographs illustrate representative fields of cells in which GFP fluorescence provides an indication of overall cell shape. C, FACS analysis using CD54 antibody of the cell population infected with adenovirus expressing GFP alone (green lane) or GFP and A-CREB (purple area). M1 represents the CD54+ cell population.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
While substantial progress has been made in identifying factors that are required for proliferation and differentiation of specific cell types, there remains a relatively poor understanding of how proliferation is coordinated with the differentiation of specific cells in vivo and the mechanisms that ultimately ensure permanent exit from the cell cycle in terminally differentiated cells. Although differentiation and proliferation are often viewed as antagonistic, Ras signaling pathways appear to play important roles in both of these processes in macrophages. Our previous studies presented evidence that PE-1/METS could dissociate Ras-dependent proliferation from Ras-dependent differentiation programs by selectively inhibiting expression of cell cycle control genes (14). Up-regulation of PE-1/METS during the program of macrophage differentiation would therefore be predicted to contribute to permanent growth arrest through its ability to functionally interact with general cell cycle control proteins, such as members of the Rb family (14). While PE-1/METS is not sufficient in itself to block macrophage proliferation, the present studies indicating that it is induced by the program of macrophage differentiation elicited by M-CSF supports the hypothesis that it is a component of the regulatory machinery that ultimately results in terminal growth arrest.

To identify the mechanisms responsible for the regulation of PE-1/METS expression, we characterized the PE-1/METS promoter and found that it is activated in response to TPA, serum and a constitutively active form of Ras (Fig. 2). Functional analysis of the promoter led to the characterization of a minimal region (-65 bp to +55 bp from the transcriptional start site) that was sufficient to confer activation by serum and TPA. Nucleotide sequence analysis revealed two inverted repeat elements separated by 15 nucleotides, IRE1 and IRE2, which are also conserved in the human PE-1/METS gene (Celera data base (hCG39826). Deletion or point mutation of these elements individually or together in the context of the METS (-2011; +55) promoter revealed that both sites are important for transcriptional activity (Figs. 4A and 5B). Several lines of evidence indicate that CREB-1/CREM-1 stimulate PE-1/METS transcription through these sites. First, computer searches only identified potential CREB response elements within IRE1 and IRE2. Antibody-perturbated electrophoretic mobility shift assays demonstrated that CREB-1/CREM-1 bind to IRE-1 and chromatin immunoprecipitation assays documented the presence of CREB-1/CREM-1 on the PE-1/METS promoter in primary macrophages. Finally, a dominant negative form of CREB significantly inhibited the activity of wild-type PE-1/METS promoter activity in RAW cells, but did not inhibit the residual activities of promoters containing mutations in IRE1 or IRE2.

Despite the fact that the IRE1 and IRE2 sites are identical, CREB-1 bound selectively to oligonucleotides containing IRE1, indicating the importance of additional sequence beyond the conserved IRE1 (Fig. 4D). The consensus CRE contains an 8-bp palindrome with the sequence 5'-TGACGTCA-3'. This sequence mediates cAMP induction of the somatostatin promoter and is also highly conserved in the promoters of other genes regulated by cAMP (39). While the consensus CRE is not present in the PE-1/METS promoter, the selective binding of CREB-1 to IRE1 can be explained by studies demonstrating that the minimum sequence required for a functional CRE is the downstream half-site, 5'-CGTCA-3' (40). This minimum half-site is present in IRE1 whereas the first C of the 5'-CGTCA-3' is absent in IRE2. IRE2 appears to also be important for PE-1/METS promoter activity, but the factors that bind to this element remain to be identified.

The current studies also indicate that the conserved GGAA motif in IRE1 and IRE2 are important for regulation of PE1/METS expression (Fig. 5B). It is likely that these sites bind additional factors that cooperate with CREB-1/CREM-1. The GGAA represents the core binding motif for members of the Ets-domain family of transcription factors, raising the intriguing possibility that PE-1/METS could negatively regulate its own expression. Studies thus far have failed to document negative regulation by PE-1/METS, or its structural homolog, ERF (data not shown).

Role of CREB-1 in Regulation of PE-1/METS—By analyzing PE-1/METS and CREB-1/CREM-1 expression during macrophage differentiation, we were able to intimately correlate the expression and activation of CREB-1/CREM-1 to the expression of PE-1/METS. Furthermore, expression of the dominant-negative A-CREB not only inhibited the activity of the transiently transfected PE-1/METS promoter, but reduced expression of endogenous PE-1/METS in primary macrophages. The activation of CREB-1 due to Ser-133 phosphorylation starts between 4 and 6 days of M-CSF culture (Fig. 6C) and precedes the increase in PE-1/METS expression between 5 and 7 days of culture (Fig. 1, A and B). CREM-1 also exhibits a similar pattern of phosphorylation using this antibody. ChIP experiments confirm that CREB-1/CREM-1 are recruited to the PE-1/METS promoter in vivo during murine bone marrow differentiation in macrophages (Fig. 6D). This recruitment may follow the increase of CREB-1/CREM-1 expression during macrophage differentiation or the increase of CREB-1/CREM-1 phosphorylation. Many studies largely performed in vitro suggest that CREB-1 is constitutively bound to the CRE and that transcriptional activation is due to specific phosphorylation on serine residues that promote recruitment of the transcriptional co-activator CBP (41, 42). However, chromatin immunoprecipitation experiments with CREB-1 and the hCRH promoter in NPLC cell line, indicated that the interaction between CREB-1 and the promoter increases after cAMP stimulation (43). Moreover, phosphorylation at serine 133 of CREB-1 may increase the strength of the binding of CREB-1 to certain promoters, notably those with an asymmetric CRE, like the CREB-1 site found in the PE-1/METS promoter (40). ChIP assays performed using bone marrow-derived macrophages differentiated after 4, 5, 6 and 7 days of M-CSF culture, indicated the same level of CREB-1/CREM-1 recruited to the PE-1/METS promoter (data not shown). It therefore appears that CREB-1/CREM-1 does not need to be activated by serine phosphorylation to bind to the PE-1/METS promoter and its recruitment to the CRE is dependent on its expression during macrophage differentiation.

CREB-1 Phosphorylation in Macrophages—Transactivation by CREB-1 requires a specific phosphorylation event that promotes recruitment of the transcriptional co-activator CBP. The CREB-1 phosphorylation site (Ser-133) is classically phosphorylated by PKA (44). Several lines of evidence indicate that CREB-1 can also be phosphorylated by several other kinases that are activated by a variety of signals. It has been shown that growth factors NGF (nerve growth factor) and EGF (epidermal growth factor), through their tyrosine kinase receptors, induce a kinase cascade that involves Ras signaling pathways (45, 46). MEKs, ERK1, ERK2, and Rsk are involved in CREB-1 phosphorylation (47). An alternative signaling route, involving SAPK2/p38 and MAPKAP-2 kinases that are targets of FGF (fibroblast growth factor) and TNF (tumor necrosis factor)-activated signaling cascades also lead to CREB-1 phosphorylation (48). The treatment of bone marrow-derived macrophages with several kinase inhibitors suggests that SAPK2/p38 is responsible for CREB-1 phosphorylation in response to M-CSF (Fig. 7B). Deak et al. (35) have identified a novel family of CREB kinases, the mitogen- and stress-activated kinases (MSKs) that are activated by both growth factors and stress stimuli. MSK1, the major substrate of SAPK2/p38 kinase, is the best candidate to mediate CREB-1/CREM-1 phosphorylation in macrophages (32, 35).

It has been shown that SAPK2/p38 plays an important role in cell differentiation for several different cell types. The differentiation of 3T3-L1 cells into adipocytes and the differentiation of PC12 cells into neurons both require SAPK2/p38 (49, 50). Moreover, the SAPK2/p38 pathway was found to be necessary and sufficient for SKT6 differentiation into hemoglobinized cells as well as C2C12 differentiation into myotubes (51). The transcription factors C/EBP, CREB-1, and MEF2C were suggested to be downstream of the SAPK2/p38 pathway and would participate in the process of differentiation mentioned above.

Potential Roles of CREB-1/CREM-1 in Regulation of Macrophage Differentiation—The present studies provide evidence for roles of CREB-1/CREM-1 in regulating aspects of macrophage differentiation. CREB-1/CREM-1 are induced and activated by M-CSF and stimulate the activities of the macrosialin and PE-1/METS promoters. The loss of adherence in macrophages treated with the SAPK2/p38 inhibitor or expression of A-CREB suggests additional roles of this pathway in specific programs of gene expression that are induced during macrophage differentiation (Fig. 8, B and C). The finding that ACREB inhibited expression of CD54 is consistent with a recent report that CREB regulates CD54 expression, adhesion and cell morphology in U937 leukemia cells (52). It will be of interest to determine the spectrum of genes that are regulated by CREB-1/CREM-1 to further define their roles in macrophage differentiation.

The ability of CREB-1 to stimulate PE-1/METS expression suggests it may play an indirect role in terminal growth arrest in some contexts. Our previous studies suggested that PE-1/METS displaces Ets activators from cell cycle control genes such as c-myc, c-myb, and cdc2, but not from genes that are activated by AP1/Ets ternary complexes (14). CREB-1 has been suggested to play anti-proliferative roles in other cell types. For example, Arnould et al. (53) demonstrated that phosphorylated CREB induced by mitochondrial dysfunction contributes to cell cycle arrest. Moreover, Houglum et al. (54) have shown that proliferation of hepatic stellate cells is inhibited by phosphorylation of CREB-1 on Ser-133. It will be of interest to explore the possibility that anti-proliferative roles of CREB-1 are mediated, at least in part, through activation of PE-1/METS.


   FOOTNOTES
 
The atomic coordinates and structure factors (code AY274927 [GenBank] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* These studies were supported in part by National Institutes of Health Grant HL59694-06, by postdoctoral fellowships (to D. S.-V.; ARC and Leukemia and Lymphoma Society (Grant 5388-02), and L. E.L; ARC), and by NCI, National Institutes of Health Cancer Cell Biology Training Grant T32-CA67754 (to A. L. F). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed. Tel.: 858-534-6011; Fax: 858-822-2127; E-mail: cglass{at}ucsd.edu.

1 The abbreviations used are: SR-A, scavenger receptor-A; FBS, fetal bovine serum; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation assay; GFP, green fluorescent protein; CREB, cAMP response element-binding protein; IRE, inverted repeat elements; TPA, 12-O-tetradecanoylphorbol-13-acetate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back


   ACKNOWLEDGMENTS
 
We thank M. Montminy for the CREB-1 cDNA, pZeo A-CREB, adenovirus expressing GFP, A-CREB and CREB-1 antibody, R. Goodman for the pRc/RSV FLAG CREB expression vector, Andrew C. Li for his help in performing the real-time PCR analysis, Jean Lozach for his assistance in bioinformatic analysis and Alexandra Zulueta for assistance in preparation of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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