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Originally published online as doi:10.1189/jlb.0205083 on October 21, 2005

Published online before print October 21, 2005
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(Journal of Leukocyte Biology. 2006;79:173-183.)
© 2006 by Society for Leukocyte Biology

The interferon-inducible gene, Ifi204, is transcriptionally activated in response to M-CSF, and its expression favors macrophage differentiation in myeloid progenitor cells

Jérémy Dauffy, Guy Mouchiroud and Roland P. Bourette1

Centre de Génétique Moléculaire et Cellulaire, UMR CNRS 5534, Villeurbanne Cedex, France

1 Correspondence: Centre de Génétique Moléculaire et Cellulaire, UMR CNRS 5534, 16 Rue Dubois, 69622 Villeurbanne Cedex, France. E-mail: bourette{at}biomserv.univ-lyon1.fr


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ABSTRACT
 
The interferon-inducible (Ifi)204 gene was isolated as a macrophage-colony stimulating factor (M-CSF)-responsive gene using a gene trap approach in the myeloid interleukin-3 (IL-3)-dependent FD-Fms cell line, which differentiates in macrophages in response to M-CSF. Here, we show that Ifi204 was transcriptionally activated in response to M-CSF, and FD-Fms cells decreased their growth and committed toward a macrophage morphology; this induction was abrogated when the differentiation signal of the M-CSF receptor was blocked; the Ifi204 gene was also induced during macrophage differentiation controlled by leukemia inhibitory factor; and the Ifi204 gene is expressed in different mature monocyte/macrophage cells. Finally, we showed that enforced expression of Ifi204 strongly decreased IL-3- and M-CSF-dependent proliferation and conversely, favored macrophage differentiation of FD-Fms cells in response to M-CSF. Altogether, these results demonstrate that the Ifi204 gene is activated during macrophage development and suggest that the Ifi204 protein may act as a regulator of the balance between proliferation and differentiation. Moreover, this study suggests that other members of the Ifi family might act as regulators of hematopoiesis under the control of hemopoietic cytokines.

Key Words: gene trap • myeloid differentiation • CSF-1 • HIN 200 family


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INTRODUCTION
 
During hematopoiesis, macrophage-colony stimulating factor (M-CSF or CSF-1) stimulates survival, proliferation, and differentiation of bone marrow (BM) myelomonocytic progenitors, leading to the production of blood monocytes and tissue macrophages [1 ]. The essential role of M-CSF in monocytic cell development in vivo was demonstrated by studying mice homozygous for the mutation osteopetrosis, which results in the absence of M-CSF [2 ], or with targeted disruption of the mouse M-CSF receptor (M-CSF-R) gene [3 ]. Indeed, both mutant mice exhibited similar depletion of circulating monocytes, peritoneal cavity membrane-activated complex 1 (Mac1)+ cells, and tissue macrophages.

All the biological effects of M-CSF are mediated by a single tyrosine kinase receptor, encoded by the c-fms proto-oncogene [4 , 5 ]. M-CSF binding to the M-CSF-R (or Fms) induces receptor dimerization and transautophosphorylation on specific tyrosines of the cytoplasmic domain, creating binding sites for Src homology 2-containing proteins, including Src family kinases (SFK), Mona/Gads and Grb2 adapters, p85 subunit of phosphatidylinositol-3 kinase (PI-3K), phospholipase C{gamma}2, and the ubiquitin-protein ligase c-Cbl. These molecules initiate multiple intracellular signaling pathways that cooperate to regulate gene expression, resulting in cell survival, proliferation, and differentiation [6 7 8 9 ]. Although M-CSF signaling pathways have been examined extensively, only few genes were shown to be activated in response to these pathways. Moreover, most of these are expressed at the late stage of differentiation, i.e., monocytes and macrophages [10 ], but few studies have looked for genes specifically activated in response to M-CSF during the early steps of commitment.

In an attempt to isolate M-CSF-responsive genes, we used an in vitro gene trap strategy [11 ], taking advantage of the retroviral vector ROSAßEGFP, which places the enhanced green fluorescent protein (EGFP) reporter gene under the control of the regulatory elements of genes disrupted by retroviral insertion. As a model, we used FDC-P1 cells expressing the murine M-CSF-R (FD-Fms cells). These cells correspond to myeloid progenitors that self-renew in the presence of interleukin-3 (IL-3), whereas in response to M-CSF, their growth rate decreases, and cells acquire macrophage morphology and markers [12 ].

Here, we show the transcriptional modulation by M-CSF of a trapped gene, interferon (IFN)-inducible 204 (Ifi204), which belongs to the hematopoietic Ifi nuclear antigens with 200 amino acid repeats (HIN200) family, implicated in regulation of proliferation and differentiation [13 ]. The Ifi family comprises five murine genes (Ifi202a, -202b, -203, -204, -205) and three human genes: IFI-16, myeloid cell nuclear differentiation antigen, and absent in melanoma [14 ]. The products of all these genes are thought to be involved in regulation of growth and differentiation, primarily by binding and modulating the activity of transcription factors or their regulators [14 ]. Ifi204 encodes a 72-kDa phosphoprotein (p204), which contains two LXCXE motifs that enable its binding to the retinoblastoma tumor suppressor protein (pRb) and mediate its antiproliferative activity [15 16 17 ]. p204 can also inhibit rRNA transcription by binding to upstream binding factor 1 transcription factor [18 ]. Ifi204 gene expression has been reported in monocytes and macrophages [19 ], but neither its function nor its regulation was yet described in progenitors of the myelomonocytic lineage.

In this study, we showed that M-CSF induces Ifi204 gene transcription during macrophage differentiation of FD-Fms cells and that this induction is linked to the M-CSF-R differentiation signal. Constitutive expression of Ifi204 in FD-Fms cells inhibited IL-3- and M-CSF-dependent proliferation, whereas M-CSF-induced macrophage differentiation was reinforced, suggesting that Ifi204 may act as a regulator of the balance between proliferation and differentiation during macrophage differentiation.


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MATERIALS AND METHODS
 
Growth factors, antibodies, and reagents
The source of M-CSF was a conditioned medium (CM) of Sf9 insect cells expressing recombinant murine M-CSF from a baculovirus vector [20 ]. As a source of IL-3, we used X63 cell-CM (IL-3-CM) [21 ]. Leukemia inhibitory factor (LIF) was a CM of COS cells transfected with the human LIF cDNA. Fms-like tyrosine kinase 3 ligand (FL) was from R&D Systems (Minneapolis, MN). IFN-{gamma} was from Peprotech (Rocky Hill, NJ). PP2, U0126, and LY294002 were from Calbiochem (San Diego, CA). Neutralizing rat monoclonal antibody (mAb) against mouse IFN-{alpha} (RMMA-1) and rabbit polyclonal antibody against mouse IFN-ß, from PBL Biomedical Laboratories (Piscataway, NJ), were used at 10 µg/ml and 1000 U/ml, respectively. Neutralizing anti-mouse IFN-{gamma} mAb, from R&D Systems, was used at 10 µg/ml. Rabbit antiserum against p204 was a gift of Dr. Peter Lengyel (Yale University, New Haven, CT) and was used at a 1:5000 dilution; rabbit antiserum against p85 was from Upstate Biotechnology (Lake Placid, NY; #06-195) and was used at a 1:1000 dilution.

Cell cultures
FDC-P1 cells expressing murine wild-type Fms (FD-Fms) or mutated Y807F Fms (FD-Fms Y807F) have been described previously [12 ]; they were maintained in Iscove’s modified Dulbecco’s medium (IMDM; Gibco, Grand Island, NY), supplemented with 5% fetal bovine serum (FBS; Dutscher) and 5% of IL-3-CM. M1-Fms [22 ], RAW 264.7 and P388D1 cells were cultivated in IMDM-10% FBS. BAC1.2F5 and NFS60/MAC cells were maintained in IMDM-10% FBS supplemented with 1000 U/ml M-CSF. BM-derived macrophages (BMM{Phi}) were obtained by cultivating BM cells (BMC) in IMDM-10% FBS supplemented with 1000 U/ml M-CSF; after 7 days of culture, adherent macrophages were recovered. To obtain BMM{Phi} precursors (BMMP), murine BMC were cultivated in IMDM-15% FBS supplemented with 5 ng/ml FL, as described previously [23 ]; after 6 days of culture, nonadherent cells were recovered and shifted for 1–4 days to IMDM-15% FBS medium supplemented with 1000 U/ml M-CSF to allow terminal differentiation into macrophages. Fibroblast {Psi}–2 packaging cells transfected with the ROSAßEGFP retroviral vector (see below) were selected by G418 (1 mg/ml) and maintained in IMDM-5% FBS. PlatE packaging cells [24 ] were maintained in IMDM-5% FBS supplemented with puromycin (1 µg/ml) and blasticidin (10 µg/ml). For macrophage differentiation, FD-Fms cells were cultivated in the presence of 5% IL-3-CM and 2500 U/ml M-CSF. For growth curves, cells were washed free of IL-3 and seeded at 2 x 104 cells/ml in IMDM-5% FBS supplemented with IL-3 (5% IL-3-CM) or IL3 + M-CSF (2500 U/mL) into 24-well culture plates. Viable cell number was determined daily, and cultures were split and fed. For clonal assay, washed cells were plated in IMDM supplemented with 1% methylcellulose, 10% FBS, and 5% IL-3-CM or 5% IL3-CM + 2500 U/ml M-CSF. Cultures (500 µL) were performed in 24-well culture plates, and colonies were scored after 7 days of culture.

Retroviral vectors and infections
The retroviral vector ROSAßEGFP, a gift of Dr. S. Gomez (INSERM U119, Marseille, France), was derived from the ROSAßgeo vector [11 , 25 ], and its construction will be described elsewhere (Sophie Gomez and Patrice Dubreuil, unpublished data). Briefly, this vector has a deletion of viral enhancers and a splice acceptor (SA) sequence in front of a promoterless EGFP gene, allowing the production of a chimeric transcript containing at least the ATG of EGFP, from which the EGFP protein expression can be obtained and monitored by fluorescence analysis. Moreover, the neo gene under the control of a Pgk promoter allows selection of infected cells by G418. FD-Fms cells were infected by a supernatant of {Psi}–2 producer cells. Individual clones of infected cells were selected by G418 (1 mg/ml) in 96-well plates for 10–15 days in the presence of IL-3. Plates were then duplicated in the presence of IL-3 or IL-3 + M-CSF, and EGFP expression was examined by fluorescein-activated cell sorter (FACS) 48 h later. For Ifi204 gene transfer, a BamHI-EcoRI polymerase chain reaction (PCR)-amplified, full-length Ifi204 murine cDNA (a gift of Dr. Peter Lengyel) was inserted in the corresponding restriction sites of pMX-IG retroviral vector [24 ]. FD-Fms cells were infected by coculture with PlatE packaging cells, transfected with the pMX-IG or pM(Ifi204)-IG retroviral vector. Infected cells positive for EGFP expression were selected twice by FACS and then plated in semi-solid or liquid cultures supplemented with IL-3 or IL-3 + M-CSF.

Isolation of 5' flanking sequences
cDNA, corresponding to gene-trapped EGFP fusion transcripts, were obtained using a 5'-rapid amplification of cDNA ends (RACE) kit (SMART RACE, BD Biosciences, San Jose, CA), according to the manufacturer’s instructions. Primers specific to EGFP were: EGFP-1 (primary PCR primer), 5'-AGATGGTGCGCTCCTGGACGTAGCCTT-3', and EGFP-2 (nested PCR primer), 5'-GTCGTGCTGCTTCATGTGGTCGGGGTA-3'. PCR products were gel-purified and sequenced using the EGFP-2 primer. Sequences obtained were compared with GenBank database using BLASTN algorithm [26 ].

Reverse transcriptase (RT)-PCR and Northern blot analysis
Total RNA was extracted from cells maintained in different culture conditions, using the RNeasy kit (Qiagen, Valencia, CA). For RT-PCR experiments, 2 µg RNA was reverse-transcribed using random hexamers and the Omniscript RT kit (Qiagen). One-tenth of the first-strand cDNA synthesis product was used as template for the PCR reactions using specific primers. PCR conditions were: 95°C 30 s, 55°C 30 s, and 72°C 60 s, repeated 34 times. Products were size-fractionated on a 1.5% ethidium bromide-containing agarose gel. Primers used were: Ifi204 sense 5'-CAGGGAAAATGGAAGTGGTG-3'; Ifi204 antisense 5'-CAGAGAGGTTCTCCCGACTG-3'; Ifi202a sense 5'-GGTCATCTACCAACTCAGAAT-3'; Ifi202b sense 5'-CATCTACCAACTCAGATCTTG-3'; Ifi202a and b antisense 5'-CTCTAGGATGCCACTGCTGTTG-3'; Ifi203 sense 5'-GATTGCCTCCAGAATCCTCA-3'; Ifi203 antisense 5'-GTTCACATCGGACACACAGG-3'; enolase sense 5'-TCACAGGCTGTTGAGCACAT-3'; enolase antisense 5'-TCACGTTCTTCAGGTTGTGG-3'; IFN-{alpha} total sense 5'-ATGGCTAGRCTCTGTGCTTTCCT-3'; IFN-{alpha} total antisense 5'-AGGGCTCTCCAGAYTTCTGCTCTG-3'; IFN-ß sense 5'-CATCAACTATAAGCAGCTCCA-3'; IFN-ß antisense 5'-TTCAAGTGGAGAGCAGTTGAG-3'; IFN-{gamma} sense 5'-TACTGCCACGGCACAGTCATTGAA-3'; IFN-{gamma} antisense 5'-GCAGCGACTCCTTTTCCGCTTCCT-3'; early growth response gene-1 (Egr-1) sense 5'-AATCCTCAAGGGGAGCCGAGCGAACA-3'; Egr-1 antisense 5'-GAGTAGATGGGACTGCTGCTGTCGTTGGA-3'; primers for Ifi202a, Ifi202b, IFN-{alpha}, and IFN-ß have been described previously [27 28 ]. For Northern blot analysis, 8 µg total RNA was resolved by formaldehyde-1% agarose gel electrophoresis, transferred onto Hybond-N+ membrane (Amersham Pharmacia Biotech, Little Chalfont, UK), and fixed by ultraviolet cross-linking and heating at 80°C for 2 h. Membranes were prehybridized in ExpressHyb hybridization solution (Clontech, Palo Alto, CA) for 30 min at 68°C and then hybridized with 1.5 ng/ml random-labeled probe (MegaprimeTM DNA labeling systems, Amersham Pharmacia Biotech) for 1 h at 68°C. After four washes with 2x saline sodium citrate and 0.05% sodium dodecyl sulfate (SDS) for 10 min at room temperature and then, two washes with 0.1x saline sodium citrate and 0.1% SDS for 20 min at 50°C, membranes were exposed to Kodak Biomax MS film plus amplifying screen at –80°C for 48 h. A 500-base pair (bp) probe was PCR-amplified from Ifi204 cDNA using specific primers. Membranes were stripped and reprobed with a 1-kb glyceraldehyde 3- phosphate dehydrogenase (GAPDH) probe as a loading control.

Western blotting
Cells were washed with phosphate-buffered saline and lysed in cold radio immunoprecipitation assay buffer (1% deoxycholic acid, 1% Triton X-100, 0.1% SDS, 50 mM Tris-base, 150 mM NaCl, 20 mM EDTA, pH 7.4) with the addition of a protease inhibitor cocktail (Roche Diagnostics, Meylan, France). Insoluble material was removed by centrifugation, and protein concentration was determined by the protein assay kit (Bio-Rad, Hercules, CA). Proteins from equalized cell lysates were separated on a SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and blotted with various antibodies as described previously [29 ]. Antibody binding was visualized using horseradish peroxidase-conjugated secondary antibodies (Sigma Chemical Co., St. Louis, MO) and enhanced chemiluminescence reagent (ECL+, Amersham Pharmacia Biotech).


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RESULTS
 
Isolation of Ifi204 as a gene induced by M-CSF
Gene expression induced by shifting FD-Fms cells from IL-3- to M-CSF-supplemented medium might result from M-CSF stimulation or IL-3 withdrawal. As the M-CSF differentiation signal is dominant over the IL-3 self-renewal signal in FD-Fms cells [12 , 30 ], we then looked for genes induced in the presence of IL-3 and M-CSF, thus avoiding genes that would be induced by IL-3 withdrawal. FD-Fms cells were infected using the ROSAßEGFP retroviral vector (Fig. 1A ), and cells with integration into a transcriptionally active gene would therefore produce EGFP protein. Clonal selection of infected cells was performed in 96-well plates in the presence of IL-3 and G418, and after 10–15 days, culture plates were duplicated into IL-3 or IL-3 + M-CSF-containing cultures (Fig. 1B) . After 2 days, cells cultivated in the presence of IL-3 self-renewed and exhibited a blastic morphology (Fig. 1C , left), whereas in the presence of IL-3 + M-CSF, cells slowed down their growth and differentiated, leading to a heterogeneous population with approximately 50% of the cells exhibiting a macrophage morphology (Fig. 1C , right). Induction of EGFP expression was examined by FACS analysis, and clones showing fluorescence in the presence of M-CSF + IL-3 but not in the sole presence of IL-3 were analyzed further.



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  		Figure 1. Experimental approach. (A) The ROSAßEGFP retroviral gene trap vector contains a neo gene under the control of the pgk promoter and a SA 5' to the ATG of a promotorless EGFP reporter gene; 3' long terminal repeat (LTR) is mutated ({Delta}). (B) FD-Fms cells were infected by supernatant of producing cells, and individual clones of infected cells were selected in the presence of IL-3 and G418. NeoR clones were duplicated and cultivated for 2 days in the presence of IL-3 or IL-3 + M-CSF; expression of EGFP was analyzed by FACS, and clones of interest, which expressed EGFP in the presence of IL-3 + M-CSF but not in the presence of IL-3 alone, were then studied. (C) May-Grünwal Giemsa (MGG) staining of FD-Fms cells maintained in the presence of IL-3 (left) or IL-3 + M-CSF for 3 days (right); arrowheads indicate two differentiated cells, and arrow indicates an undifferentiated cell.

Among selected clones, Clone 14 showed rapid induction of EGFP expression in the presence of IL-3 + M-CSF with a maximum of approximatively 40% of positive cells obtained after 2 days (Fig. 2A ). It is interesting that cells positive for EGFP expression corresponded to cells that differentiated (not shown). A coding sequence of 26 bp, flanking the proviral integration site, was obtained by 5'-RACE PCR using total RNA from Clone 14 cells cultivated in the presence of IL-3 + M-CSF for 2 days. Homology search using the GenBank database sequences indicated that this sequence corresponded to the 5' untranslated region of the Ifi204 gene.



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  		Figure 2. Ifi204 gene expression is induced by M-CSF in FD-Fms cells. (A) EGFP expression of FD-Fms Clone 14 cells was analyzed by FACS after shifting the cells from IL-3- to IL-3 + M-CSF-containing medium; profiles of cells maintained in IL-3-containing medium (thin lines) were overlaid with those of cells shifted to M-CSF-containing medium (thick lines) for 24, 48, or 72 h; numbers in the upper corner represent the percentage of EGFP-positive cells in the presence of IL-3 + M-CSF as compared with cells maintained in the presence of IL-3. (B) Ifi204 transcript expression was analyzed by RT-PCR (left) or Northern blot (right) using total RNA isolated from FD-Fms cells maintained in the presence of IL-3 or shifted to IL-3 + M-CSF-containing medium for different times; enolase or GAPDH transcript expression was used as loading controls for RT-PCR and Northern blot experiments, respectively. (C) p204 protein expression was analyzed in FD-Fms cells maintained in the presence of IL-3 or shifted to IL-3 + M-CSF-containing medium for 24, 48, or 72 h; cell lysates were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting using anti-p204 antibody. The blot was then stripped and reprobed with anti-p85 antibodies as a loading control. These data are representative of two experiments.

To confirm transcriptional activation of Ifi204 by M-CSF in a bulk population of FD-Fms cells, total RNA was extracted from cells maintained in the presence of IL-3 or shifted to a IL-3 + M-CSF-containing medium for different times and used as templates for RT-PCR using oligonucleotide primers specific for Ifi204. As shown in Figure 2B (left panel), Ifi204 was rapidly induced by M-CSF with a maximal expression after 15 h. Northern blot analysis confirmed this result, as the 2.4-kb Ifi204 transcript was only detected in total RNA from differentiating FD-Fms cells (Fig. 2B , right panel). Finally, the expression of p204, the product of the Ifi204 gene, was examined by Western blotting. As shown in Figure 2C , p204 was not expressed in FD-Fms cells maintained in the presence of IL-3 and became detectable 2 days after shifting the cells to IL-3 + M-CSF-containing medium. Altogether, these results demonstrated that Ifi204 gene expression was induced in FD-Fms cells in response to M-CSF.

Ifi204 gene induction is linked to the M-CSF differentiation signal
Proliferation and differentiation signals of the M-CSF-R could be uncoupled by mutation of tyrosine 807 of the receptor or by drug inhibition of SFK activity; in both cases, M-CSF-dependent differentiation was impaired, whereas proliferation increased [12 , 31 ]. We then examined if M-CSF could induce Ifi204 expression in FD-Fms cells treated with PP2, an inhibitor of SFK, and in FD C-91 cells expressing the Y807F Fms mutant (FD-Fms Y807F cells). As shown in Figure 3A , in both cases, M-CSF-dependent Ifi204 induction was abrogated. Concerning the FD-Fms Y807F cells, the lack of induction of Ifi204 by M-CSF was not a result of this peculiar cell population, as stimulation of these cells by IFN-{gamma} induced Ifi204 gene expression. These results demonstrated that differentiation signal induced by M-CSF controls the Ifi204 gene expression, depending on the SFK pathway.



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  		Figure 3. Ifi204 gene expression is associated with macrophage differentiation. Ifi204 transcript expression was analyzed by RT-PCR using total RNA isolated from (A, left) FD-Fms wild-type (WT) cells maintained in the presence of IL-3 or shifted to IL-3 + M-CSF-contaning medium for 24, 48, or 72 h, in the absence or the presence of the SFK inhibitor PP2 (10 µM); (A, right) FD-Fms Y807F cells maintained in the presence of IL-3 or shifted to IL-3 + M-CSF-contaning medium for 24, 48, or 72 h or stimulated by IFN-{gamma} for 30 h as a positive control. (B) Factor-independent M1-Fms cells cultivated in the absence of growth factor (no GF) or shifted to a culture medium supplemented with M-CSF or with LIF for 24, 48, or 72 h. (C) Different macrophage cell lines M-CSF-dependent (BAC1.2F5 and NFS60/Mac cells) or factor-independent (RAW264.7 and P388D1 cells) for their growth. (D) BMMP cultivated in the presence of FL or shifted to M-CSF-containing medium for 24, 48, 72, or 96 h and BMM{Phi} and peritoneal macrophages. (A–D) Enolase transcript expression was used as a loading control, and one out of two experiments is shown here.

As Ifi204 was shown to be associated with differentiation, we asked if its induction could be obtained in response to another cytokine that induces macrophage differentiation. For this purpose, we used the factor-independent myeloid progenitor cell line M1-Fms, which is able to differentiate into macrophages in response to M-CSF or LIF [32 ]. As shown in Figure 3B , Ifi204 was not expressed in undifferentiated M1-Fms cells, whereas it was strongly induced by LIF as well as by M-CSF.

Ifi204 expression was also examined in four different macrophage cell lines, which are factor-independent (RAW264.7 and P388D1 cells) or M-CSF-dependent (BAC1.2F5 and NFS60/MAC cells). As shown in Figure 3C , Ifi204 was detected in the four cell lines. We next investigated Ifi204 expression in a more physiological context than immortalized cell lines. For this, we took advantage of a primary culture system that permits massive in vitro expansion of BMMP. Indeed, cultivation of mouse BM cells in the presence of FL for 6 days results in a cell population mainly composed of morphologically immature precursors expressing the M-CSF-R; shifting these cells to M-CSF-containing medium allows their progressive differentiation toward macrophages [23 ]. As shown in Figure 3D , Ifi204 was expressed in BMMP, and expression slighly increased after shifting the cells to M-CSF-containing medium. Ifi204 expression was also detected in normal macrophages obtained from peritoneum or from BM cells cultivated 7 days in the presence of M-CSF (BMM{Phi}).

Altogether, results showed that Ifi204 is not expressed in different myeloid progenitor cell lines: FDC-P1 (Fig. 2B) , M1 (Fig. 3B) , and 32D (data not shown), whereas it is expressed significantly in four different macrophage cell lines (Fig. 3C) and in normal myelomonocytic cells (Fig. 3D) , cultivated or not in the presence of M-CSF. Moreover, Ifi204 is also a LIF-responsive gene during macrophage differentiation. This indicates that Ifi204 expression is linked to macrophage differentiation rather than to a specific cytokine.

Ifi204 and Ifi203 gene inductions by M-CSF in FD-Fms cells are IFN-independent
As IFN-{alpha}, -ß, and -{gamma} are produced by macrophages [10 ], we next investigated if Ifi204 gene induction by M-CSF in FD-Fms cells resulted from IFN production. As the Ifi204 gene is located in a cluster of genes, defined by their property as being inducible by IFNs, we first looked for activation of other members of the Ifi family in response to M-CSF. Ifi203 was induced significantly and rapidly in FD-Fms cells in response to M-CSF (Fig. 4A ). On the contrary, no induction of Ifi202a and Ifi202b was observed in FD-Fms cells, even after 2 days in the presence of M-CSF, yet their expression was clearly detected in Raw 264.7 macrophage cells (Fig. 4A) ; similarly, we have not been able to detect Ifi205 gene expression in FD-Fms cells stimulated by M-CSF (data not shown). We then analyzed IFN-{alpha}, -ß, and -{gamma} gene expression by RT-PCR in FD-Fms cells. As shown in Figure 4B , no IFN-{gamma} transcript could be detected in FD-Fms cells; in contrast, a significant induction of IFN-{alpha} and -ß was detected in FD-Fms cells but only after 15 h in the presence of M-CSF. At that time, Ifi203 and Ifi204 transcripts are already expressed, which strongly suggests that IFN production is not responsible for the early induction of Ifi203 and Ifi204 genes. To confirm this result, FD-Fms cells maintained in the presence of IL-3 or shifted to IL-3 + M-CSF-containing medium for 15 h or 24 h in the presence or the absence of a mix of neutralizing antibodies against IFN-{alpha}, -ß, or -{gamma}. Analysis of Ifi203 and Ifi204 gene induction showed no significant difference whether cells were treated or not by neutralizing antibodies (Fig. 4C) . These results demonstrated that induction of two Ifi genes, Ifi203 and Ifi204, is a direct consequence of the M-CSF signal. However, we cannot exclude that IFNs participate in the activation of these genes during the later stages of macrophage differentiation.



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  		Figure 4. Ifi204 and Ifi203 gene activation by M-CSF is IFN-independent. (A) Transcript expression of different Ifi family members was analyzed by RT-PCR using total RNA isolated from FD-Fms cells maintained in the presence of IL-3 or shifted to IL-3 + M-CSF-containing medium for different times; RNA from the RAW264.7 macrophage cell line was used as a positive control (Ctr+); shown is one out of three experiments. (B) Transcript expression of different IFN genes was analyzed by RT-PCR using total RNA isolated from cells maintained in the presence of IL-3 or shifted to IL-3 + M-CSF-containing medium for different times; RNA from peritoneal macrophage was used as a positive control (Ctr+); one out of two experiments is shown here. (C) Expression of Ifi204 and Ifi203 transcripts was analyzed by RT-PCR from FD-Fms cells maintained in the presence of IL-3 or shifted to IL-3 + M-CSF-containing medium for 15 h or 24 h in the presence (+) or absence (–) of a mix of neutralizing antibodies against IFN-{alpha} (10 µg/ml), IFN-ß (1000 U/ml), and IFN-{gamma} (10 µg/ml). (D) Expression of the Ifi204 transcript was analyzed by RT-PCR from FD-Fms cells maintained in the presence of IL-3 or shifted to IL-3 + M-CSF-containing medium (upper panel) or IL-3 + IFN-{gamma}-containing medium (lower panel) for 24 h or 48 h in the presence of 0.1% dimethyl sulfoxide (DMSO) as a control or different inhibitors disolved in DMSO: PP2 (10 µM), U0126 (10 µM), LY294002 (10 µM). (A–D) Enolase transcript expression was used as a loading control.

To determine if M-CSF and IFN-{gamma} used distinct signaling pathways to induce Ifi204, we used specific inhibitors of Src kinases (PP2), mitogen-activated protein kinases (MAPK; U0126), and PI-3K (LY294002). Ifi204 induction in response to M-CSF was abolished when MAPK or Src kinases were inhibited, whereas inhibition of PI-3K had no effect (Fig. 4D , upper panel). Conversely, Ifi204 induction in response to IFN-{gamma} was not altered when Src and MAPK were inhibited but was totally abolished when PI-3K was inhibited (Fig. 4D , lower panel). This result confirmed that Ifi204 induction by M-CSF was not mediated by the production of IFN and demonstrated that IFN-{gamma} and M-CSF used different signaling pathways to induce Ifi204 expression.

Ifi204 expression inhibits M-CSF-dependent proliferation and favors macrophage differentiation
To investigate a possible Ifi204 role during macrophage differentiation, Ifi204 cDNA was constitutively expressed in FD-Fms cells using the pMX-IG retroviral vector, which allows simultaneous expression of the gene of interest and EGFP from a single bicistronic mRNA [24 ]. Cells were infected in the presence of IL-3 by pM(Ifi204)-IG vector or by empty pMX-IG vector as a control, and infected cells were sorted twice by FACS for EGFP expression. Western blot analysis confirmed p204 expression in sorted FD-Fms/Ifi204 cells similar to that observed in transfected PlatE packaging cells (Fig. 5A ). Cells were then plated in liquid cultures for 3 days (Fig. 5B , left) or in semi-solid cultures for 1 week (Fig. 5B , right) in the presence of IL-3 or IL-3 + M-CSF. As compared with control cells infected with empty vector (FD-Fms/Mx cells), FD-Fms/Ifi204 proliferated much more slowly in liquid culture supplemented by IL-3 or by IL-3 + M-CSF (Fig. 5B , left), yet they showed no decreased viability (not shown). Similarly, in methylcellulose cultures, Ifi204 expression resulted in between a 80% and 90% decrease in colony numbers obtained in the presence of IL-3 or IL-3 + M-CSF as compared with FD-Fms/Mx cells (Fig. 5B , right). These results clearly showed that Ifi204 inhibits IL-3- and M-CSF-dependent proliferation. As expected, when EGFP expression was analyzed over the 3 days of culture, there was no significant decrease in control FD-Fms/Mx cells cultivated in the presence of IL-3 or IL-3 + M-CSF (data not shown). On the contrary, EGFP expression decreased in FD-Fms/Ifi204 cells maintained in the presence of IL-3, and a minority of cells expressed EGFP (and presumably also, Ifi204) after 3 days (Fig. 5C) . This suggested that sorted EGFP+ FD-Fms/Ifi204 cells with low or no expression of the transgenes had a selective advantage. It is remarkable that in the presence of IL-3 + M-CSF, EGFP expression in FD-Fms/Ifi204 cells remained stable, with only a slight decrease at day 3 (Fig. 5C) . As the only difference between these two culture conditions was that cells maintained in IL-3 + M-CSF had the ability to differentiate toward macrophages, we analyzed the extent of differentiation in each population. In flow cytometry, increased cellular granularity is a valuable measure for macrophage differentiation of FD-Fms cells, reflecting the increased number of vacuoles and granules observed in the cytoplasm [12 ]. After 2 days in the presence of M-CSF, approximatively 40% of FD-Fms/Mx cells were differentiated (Fig. 6A ) with a mean value of granularity of 200 (vs. 100 in the presence of IL-3). This result is consistent with previous studies showing that differentiation of FD-Fms cells is heterogeneous [12 , 33 ] leading to a mix of morphology from blastic to macrophage-like cells (Fig. 1C , right). When FD-Fms/Ifi204 cells were analyzed, a tremendous effect of p204 on differentiation was observed, as after 2 days in the presence of M-CSF, 90% of the cells were differentiated (Fig. 6A) with a mean value of granularity of 500. Morphology analysis confirmed the positive effect of p204 on macrophage differentiation, as the FD-Fms/Ifi204 cell population was almost exclusively composed of fully differentiated macrophage-like cells in contrast to the FD-Fms/Mx cell population (Fig. 6B) . Although FD-Fms/Ifi204 cells maintained in the presence of IL-3 conserved their blastic morphology (not shown), they exhibited a slight increase in granularity that disappeared after 2 days (Fig. 6A) . As this result suggested that Ifi204-enforced expression could activate the differentiation process, we investigated the expression of different myeloid-specific transcription factors 24 h after cell infection. Among those, Egr-1 [34 ] was not expressed in undifferentiated FD-Fms cells, maintained in the presence of IL-3, but was rapidly induced in FD-Fms cells differentiating in response to M-CSF (Fig. 6C , left panel). We observed that enforced expression of Ifi204 in FD-Fms cells induced a significant expression of Egr-1 in the sole presence of IL-3 (Fig. 6C , right panel), demonstrating that Ifi204 could actually induce molecular changes involved in macrophage differentiation.



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  		Figure 5. Enforced expression of Ifi204 in FD-Fms cells decreases IL-3- and M-CSF-dependent proliferation. FD-Fms cells were infected by empty MX-IG or M(Ifi204)-IG retroviral vectors in the presence of IL-3 and sorted for EGFP expression by FACS. (A) p204 protein expression was checked by Western blot analysis in FD-Fms/Ifi204 cells as compared with control FD-Fms/Mx cells; p204 expression was studied in corresponding transfected PlatE cells as a positive control; the blot was then stripped and reprobed with anti-p85 antibodies as a loading control. One out of two experiments is shown. (B, left) Cells were washed twice with IMDM and seeded at 2 x 104 cells/ml in liquid cultures supplemented with IL-3 or IL-3 + M-CSF; viable cell numbers were determined daily before cultures were split and fed. (B, right) Cells were washed twice with IMDM and seeded in methylcellulose cultures supplemented with IL-3 or IL-3 + M-CSF; after 7 days, colonies were scored. Data are mean of two independent experiments. (C) Cells seeded in liquid culture as described above were analyzed daily by FACS for EGFP expression; histograms represent the percentage of FD-Fms/Ifi204, EGFP-positive cells as compared with control FD-Fms/Mx cells cultivated in the same conditions, i.e., IL-3 or IL-3 + M-CSF. Data represent mean of three independent experiments.



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  		Figure 6. Enforced expression of Ifi204 in FD-Fms cells favors M-CSF-dependent macrophage differentiation. (A) Cells seeded in liquid culture as described in Figure 5B were analyzed daily by FACS for cell granularity [side scatter chanel (SSC)]; profiles of undifferentiated cells maintained in IL-3-containing medium (thin lines) were overlaid with differentiating cells shifted to M-CSF-containing medium (thick line) for each day and each cell population (control FD-Fms/Mx or FD-Fms/Ifi204 cells); the number in the upper corner of each panel indicates the percentage of differentiated M-CSF-stimulated cells as compared with control undifferentiated cells maintained in the presence of IL-3. These data are representative of the finding of two experiments. (B) Morphology of FD-Fms/Mx cells (left) and FD-Fms/Ifi204 cells (right) cultivated in the presence of IL-3 + M-CSF for 3 days; cells were cytocentrifuged onto glass slides, air-dried, and visualized after MGG staining. (C) Egr-1 transcript expression was analyzed by RT-PCR using total RNA isolated from FD-Fms cells maintained in the presence of IL-3 or shifted to IL-3 + M-CSF-containing medium for different times (left panel) or from FD-Fms cells infected by empty MX-IG or M(Ifi204)-IG retroviral vectors in the presence of IL-3 (right panel); enolase transcript expression was used as loading controls.


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DISCUSSION
 
In this study, we have isolated the Ifi204 gene as a M-CSF-responsive gene and demonstrated its rapid induction during macrophage differentiation, independently of the presence of IFN. Previous studies have suggested the involvement of the Ifi204 gene during myelomonocytic development. The Ifi204 gene is expressed in cells of the myelomonocytic lineage [19 ]. A set of genes characterized as IFN-stimulated genes, including Ifi204, distinguishes monocyte progenitors from granulocyte progenitors, independently of IFN signal [35 ]. Based on their structures, the human homologue of murine Ifi204 is presumably IFI-16 [36 ], although IFI-16 may function differently from its mouse homologue, as it negatively regulates the p53 pathway [37 ]. IFI-16 is expressed in uncommitted CD34+ precursors, and its expression is maintained during their differentiation along monocytic lineage but is down-regulated after commitment toward the granulocytic pathways [38 ].

The Ifi204 gene is among the numerous genes induced by IFNs, which are important regulators of cell growth, immunomodulation, and host resistance to tumors and viral infections. Different IFN-responsive genes, including IFI16, were up-regulated by the chimeric tyrosine kinase BCR-ABL1 in a myelomonocytic cell line, independently of autocrine action of IFN [39 ]. Our results indicate that some IFN-responsive genes could also be up-regulated by hematopoietic cytokines, such as M-CSF or LIF. Moreover, as shown by inhibitor studies, M-CSF uses a specific signal to up-regulate Ifi204 gene expression as compared with IFN. In FD-Fms cells, Ifi204 gene induction by M-CSF required activation of the SFK and MAPK signaling pathways, whereas it is strickly dependent on PI-3K activity in response to IFN. It is interesting that inhibition of SFK or MAPK activation leads to impaired M-CSF-induced differentiation, accompanied by a significant increase in proliferation [31 , 40 ], suggesting that Ifi204 induction may be linked to the M-CSF-R differentiation signal, which simultaneously decreases the cell growth, modifies the cell morphology, and functions toward a differentiated macrophage phenotype [12 ]. An Ets-related PU.1-binding site is present in the 5' flanking of the Ifi204 gene [19 ]; PU.1 plays a key role in gene regulation during macrophage differentiation [41 ] and is induced in myelomonocytic cells in response to different cytokines, including M-CSF. It is interesting that PU.1 is induced by M-CSF in FD-Fms cells (our unpublished result) and may provide a link between the M-CSF differentiation signal and Ifi204 gene induction.

We and others [12 , 30 ] have shown that the M-CSF differentiation signal negatively regulates M-CSF- and IL-3-dependent proliferation and that this dominant inhibitory influence of M-CSF was alleviated when tyrosine 807 of M-CSF-R was mutated. Our data suggest that p204 is a part of this negative signaling pathway, as Ifi204 induction was lost in FD-Fms Y807F cells, and enforced expression of Ifi204 negatively regulated M-CSF- and IL-3-dependent proliferation. Altogether, our results reveal that p204 acts as a global negative regulator of proliferation during macrophage differentiation, probably by acting on the Rb-E2F pathway [16 , 17 , 42 ], which is essential for cell-cycle progression of myelomonocytic cells [43 ]. This is consistent with the role of p204 as a negative regulator of growth in different nonhematopoietic cells [15 , 18 , 42 ] and with a recent report showing that enforced expression of Ifi204, as well as that of Ifi202 and Ifi205, decreases IL-3-dependent proliferation of murine hematopoietic cell lines [44 ].

Ifi204-enforced expression in FD-Fms cells enhanced macrophage differentiation in response to M-CSF but also induced the expression of Egr-1 in the absence of M-CSF. Egr-1 is a positive modulator of macrophage differentiation, which dictates development of myeloid progenitors along the macrophage lineage [34 , 45 , 46 ]; Egr-1 was not expressed in FD-Fms cells maintained in the presence of IL-3 but was induced rapidly in response to M-CSF, indicating that it is involved in the macrophage differentiation program. The ability of p204 to induce this myeloid-differentiation primary response gene in the absence of M-CSF strengthens the idea that p204 may drive molecular components specific to macrophage differentiation. This is in agreement with previous results showing that p204 plays an active role in cell differentiation. Thus, p204 was found to be an important regulator of myogenesis [47 , 48 ]. During myoblast differentiation into myotubes, Ifi204 is transcriptionaly activated under the control of MyoD, and its enforced expression leads to the fusion of C2C12 myoblasts into myotubes [48 ]. Mechanisms implicate p204 binding to inhibitor of differentiation (Id) proteins, which overcome the inhibition of the MyoD protein [47 ]. More recently, the role of Ifi204 in osteogenesis was unveiled in a study showing that Ifi204 expression increases during osteoblast differentiation and that overexpression of p204 enhances bone morphogenetic protein-induced osteoblast differentiation. In these cells, p204 binds to core-binding factor {alpha}-1, an essential transcriptional regulator of osteoblast differentiation and bone formation [49 ]. The primary molecular targets of p204 during macrophage differentiation remain to be determined, but Id proteins may represent good candidates. Although little is known about expression and function of the different Id proteins during myelomonocytic development, Id2 expression increases during induced myeloid differentiation of leukemic cells and has been reported in human monocytes and macrophages [50 ].

To our knowledge, this study is the first report demonstrating the regulation of members of the Ifi family by a non-IFN cytokine in hematopoietic cells. In FD-Fms cells, Ifi203 and Ifi204, but not Ifi202a, 202b, and 205, were transcriptionaly activated in response to M-CSF, suggesting that a specific combination of Ifi genes may correspond to a given signal at a peculiar stage of differentiation. It would then be interesting to investigate involvement of the different Ifi family members during lineage commitments and differentiation as controlled by hematopoietic cytokines and also a possible implication of Ifi family members in anarchic proliferation and loss of differentiation of leukemic cells.


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ACKNOWLEDGEMENTS
 
This work was supported by grants from the Ligue Nationale contre le Cancer (Labelisation 2004) and from the Centre National de la Recherche Scientifique. We are grateful to Dr. Gomez for the gift of the ROSAßEGFP vector and helpful discussions, and we appreciate the generosity of Dr. Lengyel for Ifi204 reagents. We thank Dr. Kitamura for the gift of the pMX-IG vector and PlatE cells. We give a special thanks to Dr. Paul A. Algate.

Received February 9, 2005; revised August 24, 2005; accepted August 31, 2005.


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Y. Luan, X.-P. Yu, K. Xu, B. Ding, J. Yu, Y. Huang, N. Yang, P. Lengyel, P. E. Di Cesare, and C.-j. Liu
The Retinoblastoma Protein Is an Essential Mediator of Osteogenesis That Links the p204 Protein to the Cbfa1 Transcription Factor Thereby Increasing Its Activity
J. Biol. Chem., June 8, 2007; 282(23): 16860 - 16870.
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