Bcl-xL Expression Correlates with Primary Macrophage Differentiation, Activation of Functional Competence, and Survival and Results from Synergistic Transcriptional Activation by Ets2 and PU.1

Depriving primary bone marrow-derived macrophages of colony-stimulating factor-1 (CSF-1) induces programmed cell death by apoptosis. We show that cell death is accompanied by decreases in the expression of anti-apoptotic Bcl-xL protein and the Ets2 and PU.1 proteins of the Ets transcription factor family. Macrophages require both priming and triggering signals independent of CSF-1 to kill neoplastic cells or microorganisms, and this activation of macrophage competence is accompanied by increased expression ofbcl-xL, ets2, andPU.1. Furthermore, we show that only Ets2 and PU.1, but not Ets1, function in a synergistic manner to transactivate thebcl-x promoter. The synergy observed between PU.1 and Ets2 is dependent on the transactivation domains of both proteins. Although other transcription factors like Fos, c-Jun, Myc, STAT3, and STAT5a are implicated in the activation of macrophage competence or in CSF-1 signaling, no synergy was observed between Ets2 and these transcription factors on the bcl-x promoter. We demonstrate that the exogenous expression of both Ets2 and PU.1 in macrophages increases the number of viable cells upon CSF-1 depletion and that Ets2 and PU.1 can functionally replace Bcl-xL in inhibiting Bax-induced apoptosis. Together, these results demonstrate that PU.1 and Ets2 dramatically increase bcl-x activation, which is necessary for the cytocidal function and survival of macrophages. Depriving primary bone marrow-derived macrophages of colony-stimulating factor-1 (CSF-1) induces programmed cell death by apoptosis. We show that cell death is accompanied by decreases in the expression of anti-apoptotic Bcl-xL protein and the Ets2 and PU.1 proteins of the Ets transcription factor family. Macrophages require both priming and triggering signals independent of CSF-1 to kill neoplastic cells or microorganisms, and this activation of macrophage competence is accompanied by increased expression ofbcl-xL, ets2, andPU.1. Furthermore, we show that only Ets2 and PU.1, but not Ets1, function in a synergistic manner to transactivate thebcl-x promoter. The synergy observed between PU.1 and Ets2 is dependent on the transactivation domains of both proteins. Although other transcription factors like Fos, c-Jun, Myc, STAT3, and STAT5a are implicated in the activation of macrophage competence or in CSF-1 signaling, no synergy was observed between Ets2 and these transcription factors on the bcl-x promoter. We demonstrate that the exogenous expression of both Ets2 and PU.1 in macrophages increases the number of viable cells upon CSF-1 depletion and that Ets2 and PU.1 can functionally replace Bcl-xL in inhibiting Bax-induced apoptosis. Together, these results demonstrate that PU.1 and Ets2 dramatically increase bcl-x activation, which is necessary for the cytocidal function and survival of macrophages. colony-stimulating factor-1 colony-stimulating factor-1 receptor phosphate-buffered saline interferon-γ lipopolysaccharide hemagglutinin signal transducer and activator of transcription mitogen-activated protein kinase fluorescence-activated cell sorting bone-derived macrophages upstream stimulatory factor-1 Fos-interacting protein interferon-α activation sequence Ets-binding sites The Ets family of transcription factors consists of ∼30 members conserved from sea urchins to man. Ets members contain a conserved DNA-binding domain of ∼85 amino acids known as the Ets domain (reviewed in Ref. 1Ghysdael J. Boureux A. Yaniv M. Ghysdael J. Oncogenes as Transcriptional Regulators: Retroviral Oncogenes. 1. Birkhaeuser Verlag, Basel, Switzerland1997: 29-88Crossref Google Scholar). Ets1, the progenitor of v-Ets found in the E26 retrovirus, and Ets2 are 97% conserved in the Ets domain, whereas PU.1/Spi.1 is highly divergent in this domain (37% identity) (2Wasylyk B. Hahn S.L. Giovane A. Eur. J. Biochem. 1993; 211: 7-18Crossref PubMed Scopus (811) Google Scholar). These differences in sequence identity of the Ets domain allow Ets1, Ets2, and PU.1 to bind to common as well as distinct optimal DNA target sequences. Ets proteins bind to DNA as monomers to activate transcription alone or in conjunction with other transcription factors binding to their respective sites (reviewed in Ref. 1Ghysdael J. Boureux A. Yaniv M. Ghysdael J. Oncogenes as Transcriptional Regulators: Retroviral Oncogenes. 1. Birkhaeuser Verlag, Basel, Switzerland1997: 29-88Crossref Google Scholar). PU.1 is expressed in early progenitor cells as well as in fully differentiated B cells, neutrophils, and macrophages. The importance of PU.1 in hematopoietic development has been confirmed by PU.1gene disruption studies showing that PU.1-deficient mice lack mature B cells, neutrophils, and macrophages (3McKercher S. Torbett B. Anderson K. Henkel G. Vestal D. Baribault H. Klemsz M. Feeney A. Wu G. Paige C. Maki R. EMBO J. 1996; 15: 5647-5658Crossref PubMed Scopus (929) Google Scholar, 4Scott E.W. Simon M.C. Anastasi J. Singh H. Science. 1994; 265: 1573-1577Crossref PubMed Scopus (1279) Google Scholar).ets2 null mice die early during embryonic development (5Yamamoto H. Flannery M.L. Kupriyanov S. Pearce J. McKercher S.R. Henkel G.W. Maki R.A. Werb Z. Oshima R.G. Genes Dev. 1998; 12: 1315-1326Crossref PubMed Scopus (255) Google Scholar). However, transgenic studies using a dominant-negative form of Ets2 under the control of a monocyte/macrophage-specific promoter have provided insight into the role of Ets2 in macrophages (6Jin D.I. Jameson S.B. Reddy M.A. Schenkman D. Ostrowski M.C. Mol. Cell. Biol. 1995; 15: 693-703Crossref PubMed Google Scholar). Abnormal macrophage development occurs in these transgenic mice during the first 40 days following birth, and peritoneal macrophages obtained from these transgenic animals do not have the characteristic macrophage morphology when cultivated in vitro with CSF-1.1 These results imply the importance of Ets2 in the development of macrophages, yet the molecular mechanisms by which Ets2 functions had not been elucidated in these studies. The level of PU.1 is highly abundant in immature myeloid progenitor cells, and this level of expression remains high throughout macrophage differentiation (7Aperlo C. Pognonec P. Stanley E.R. Boulukos K.E. Mol. Cell. Biol. 1996; 16: 6851-6858Crossref PubMed Scopus (37) Google Scholar, 8Stacey K. Fowles L. Colman M. Ostrowski M. Hume D. Mol. Cell. Biol. 1995; 15: 3430-3441Crossref PubMed Scopus (129) Google Scholar). In contrast, Ets2 is not expressed in early myeloid progenitors, but later in more mature myeloid cells (9Boulukos K.E. Pognonec P. Begue A. Gesquière J.C. Stéhelin D. Ghysdael J. EMBO J. 1988; 7: 697-705Crossref PubMed Scopus (77) Google Scholar, 10Boulukos K.E. Pognonec P. Sariban E. Bailly M. Lagrou C. Ghysdael J. Genes Dev. 1990; 4: 401-409Crossref PubMed Scopus (40) Google Scholar, 11Ghysdael J. Gegonne A. Pognonec P. Boulukos K. Leprince D. Dernis D. Lagrou C. Stéhelin D. EMBO J. 1986; 5: 2251-2256Crossref PubMed Scopus (9) Google Scholar), and Ets2 become rapidly up-regulated upon induction of macrophage differentiation or activation of primary macrophages (10Boulukos K.E. Pognonec P. Sariban E. Bailly M. Lagrou C. Ghysdael J. Genes Dev. 1990; 4: 401-409Crossref PubMed Scopus (40) Google Scholar). Bcl-xL is believed to be the key anti-apoptotic protein expressed in myeloid precursors and macrophages (12Chatterjee D. Han Z. Mendoza J. Goodglick L. Hendrickson E.A. Pantazis P. Wyche J.H. Cell Growth Differ. 1997; 8: 1083-1089PubMed Google Scholar, 13Lotem J. Sachs L. Cell Growth Differ. 1995; 6: 647-653PubMed Google Scholar, 14Okada S. Zhang H. Hatano M. Tokuhisa T. J. Immunol. 1998; 160: 2590-2596PubMed Google Scholar, 15Packham G. White E.L. Eischen C.M. Yang H. Parganas E. Ihle J.N. Grillot D.A.M. Zambetti G.P. Nunez G. Cleveland J.L. Genes Dev. 1998; 12: 2475-2487Crossref PubMed Scopus (100) Google Scholar, 16Sanz C. Benito A. Silva M. Albella B. Richard C. Segovia J.C. Insunza A. Bueren J.A. Fernandez-Luna J.L. Blood. 1997; 89: 3199-3204Crossref PubMed Google Scholar, 17Sevilla L. Aperlo C. Dulic V. Chambard J.C. Boutonnet C. Pasquier O. Pognonec P. Boulukos K.E. Mol. Cell. Biol. 1999; 19: 2624-2634Crossref PubMed Scopus (91) Google Scholar). In contrast, Bcl-2 is down-regulated in these systems. Recently, we showed that the induction of bcl-xL results from an increase inbcl-x promoter activity and that de novo protein synthesis is required for this activation of bcl-xtranscription (17Sevilla L. Aperlo C. Dulic V. Chambard J.C. Boutonnet C. Pasquier O. Pognonec P. Boulukos K.E. Mol. Cell. Biol. 1999; 19: 2624-2634Crossref PubMed Scopus (91) Google Scholar). The human bcl-x promoter contains nine potential EBS. The capacities of PU.1 and Ets2 to individually transactivate the bcl-x promoter led us to ask whether PU.1 and Ets2 could compete or act in synergy to transactivate this promoter. In parallel, we wanted to determine the biological relevance of the coexpression of PU.1, ets2 andbcl-xL in primary macrophages upon induction of proliferation and differentiation and activation of macrophage competence and during programmed cell death by apoptosis. 293 cells, NIH3T3 cells, and NIH3T3 cells exogenously expressing the CSF-1R (NIH3T3-cfms) (18Roussel M.F. Sherr C.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7924-7927Crossref PubMed Scopus (45) Google Scholar) were maintained in Dulbecco's modified Eagle's medium and 10% fetal calf serum. Primary bone marrow-derived cells were isolated from femurs of 2–3-month-old male C57BL/6 mice. Femurs were flushed with PBS to recover cells. After several washes in PBS, cells were cultivated either in Dulbecco's modified Eagle's medium, 20% fetal calf serum, and 30% conditioned medium from L cells as a source of CSF-1 (19Stanley E.R. Methods Enzymol. 1985; 116: 565-587Google Scholar) or in Dulbecco's modified Eagle's medium and different concentrations of human recombinant CSF-1. After 4–5 days, fully differentiated macrophages were obtained. When recombinant CSF-1 was used, the concentrations are indicated below. IFN-γ (100 units/ml) or LPS (10 μg/ml) was added to macrophages for 4 h. The BAC1.2F5 and BACets2.1D macrophage cell lines have been previously described (17Sevilla L. Aperlo C. Dulic V. Chambard J.C. Boutonnet C. Pasquier O. Pognonec P. Boulukos K.E. Mol. Cell. Biol. 1999; 19: 2624-2634Crossref PubMed Scopus (91) Google Scholar). For transfection studies, BAC1.2F5 macrophages were electroporated either with 5 μg of pRK-ets2 and 5 μg of pRK-PU.1 or with 5 μg of pRK-ΔPU.1 and 5 μg of pRK-Δets2 (for description of plasmids, see below) as previously described (7Aperlo C. Pognonec P. Stanley E.R. Boulukos K.E. Mol. Cell. Biol. 1996; 16: 6851-6858Crossref PubMed Scopus (37) Google Scholar). BAC1.2F5 cells constitutively expressing Ets2 (BACets2.1D) were electroporated with 10 μg of pRK5 or pRK-ΔPU.1. After electroporation, the cells were plated with CSF-1 in duplicate dishes until they became adherent. Dishes were washed two times in PBS, and then the cells were cultivated in Dulbecco's modified Eagle's medium and 10% fetal calf serum with or without 20% conditioned medium as a source of CSF-1. The number of viable cells was determined by trypan blue exclusion. The cloning of the 5′-regulatory sequences of the bcl-x gene upstream of the luciferase gene has been described previously (17Sevilla L. Aperlo C. Dulic V. Chambard J.C. Boutonnet C. Pasquier O. Pognonec P. Boulukos K.E. Mol. Cell. Biol. 1999; 19: 2624-2634Crossref PubMed Scopus (91) Google Scholar). Ets2, a dominant-negative mutant of Ets2 (Δ1–238Ets2), Ets1, and PU.1/Spi.1 were cloned into pRK5 (20Schall T.J. Lewis M. Koller K.J. Lee A. Rice G.C. Wong G.H.W. Gatanaga T. Granger G.A. Lentz R. Raab H. Kohr W.J. Goeddel D.V. Cell. 1990; 61: 361-370Abstract Full Text PDF PubMed Scopus (846) Google Scholar) to generate pRK-ets2, pRKΔ1–238ets2, pRK-ets1, and pRK-PU.1, respectively, as previously described (7Aperlo C. Pognonec P. Stanley E.R. Boulukos K.E. Mol. Cell. Biol. 1996; 16: 6851-6858Crossref PubMed Scopus (37) Google Scholar, 17Sevilla L. Aperlo C. Dulic V. Chambard J.C. Boutonnet C. Pasquier O. Pognonec P. Boulukos K.E. Mol. Cell. Biol. 1999; 19: 2624-2634Crossref PubMed Scopus (91) Google Scholar). A hemagglutinin (HA) epitope tag was inserted upstream of the first ATG codon of Ets2, ΔEts2, Ets1, or PU.1. ΔPU.1 was constructed by deleting sequences corresponding to the transactivation domains found in the first 144 amino acids. An HA tag was inserted upstream of the newly created ATG codon. 293 cells were transfected by the calcium phosphate coprecipitation method in 96-well dishes by adding cells in suspension to pXP-Bcl-xPr(45 ng) and to different concentrations of pRK-ets1, pRK-PU.1, or pRK-ΔPU.1 (4–256 ng) or of pRK5-ets2 or pRK5Δ1–238ets2 (0.25–256 ng). In these experiments, 5 ng of pCMV-βgal was used as an internal control for transfection efficiency. For experiments performed in 12-well dishes, 293 cells or NIH3T3 cells were transfected by the calcium phosphate coprecipitation method or with LipofectAMINE Plus (Life Technologies, Inc.), respectively, using 200 ng of the reporter construct in the presence of varying amounts of pRK5, pRK-ets2, or pRK-PU.1 as indicated in the figure legends with 20 ng of pCMV-βgal as an internal control for transfection efficiency as described above. One-half of the lysate was used to quantify transfected protein levels by Western analysis, and the other half was used to measure luciferase and β-galactosidase activities. For transfections in 24-well dishes, 293 cells or NIH3T3 cells exogenously expressing the CSF-1R were transfected by the calcium phosphate coprecipitation method or with LipofectAMINE Plus, respectively, as described above for 12-well dishes. AP-1 activity was also measured using the full-length pXP-Bcl-xPr reporter construct. pXP-Bcl-xPr was cotransfected in 24-well dishes with 200 ng of pRK5, pRK-fos, or pRK-jun (21Pognonec P. Boulukos K.E. Aperlo C. Fujimoto M. Ariga H. Nomoto A. Kato H. Oncogene. 1997; 14: 2091-2098Crossref PubMed Scopus (49) Google Scholar) and 20 ng of pCMV-βgal as described (17Sevilla L. Aperlo C. Dulic V. Chambard J.C. Boutonnet C. Pasquier O. Pognonec P. Boulukos K.E. Mol. Cell. Biol. 1999; 19: 2624-2634Crossref PubMed Scopus (91) Google Scholar). STAT activity was measured using the full-length pXP-Bcl-xPr reporter construct in the presence of STAT3 or STAT5a cloned into pRK5. For dimerization and activation of STAT proteins, NIH3T3-cfms cells were cotransfected with pXP-Bcl-xPr in 24-well dishes with varying amounts of STAT3, STAT5a, and Ets2. 12 h after transfection, the cells were placed in low serum conditions (0.5% fetal calf serum) for 24 h ad then stimulated with CSF-1 for 24 h. Several independent experiments using the different cell types and different DNA preparations were performed in triplicate or quadruplicate. Cell lysates were prepared as previously described (22Aperlo C. Boulukos K.E. Sage J. Cuzin F. Pognonec P. Genomics. 1996; 37: 337-344Crossref PubMed Scopus (12) Google Scholar). Briefly, 48–72 h after transfections, cells lysates were prepared in 25 mm Tris (pH 7.5), 10% glycerol, 1% Triton X-100, and 2 mm dithiothreitol and analyzed for luciferase (Promega) and β-galactosidase (Tropix Inc., Galactolight) activities as described by the manufacturers. For 96-well dishes, the luciferase and β-galactosidase activities were read on a MicrobetaTrilux 1450 luminescence counter (Wallac). All luciferase activities were corrected according to pCMV-βgal used as an internal control for transfection efficiency. Cells were washed twice in 1× PBS. Cells were then lysed in RNA Insta-Pure (Eurogentec) as described by the manufacturer. 5 μg of total RNA was loaded and electrophoresed on a 2.2 m formaldehyde-containing 1% agarose gel and then transferred to a nylon membrane (Amersham Pharmacia Biotech) as described by the manufacturer. Purifiedets2, PU.1/spi.1,bcl-xL, lysozyme M, IP10 (gift of T. A. Hamilton), and S26 cDNA fragments were used as probes. Probes with equal high specific activities were generated using the Stratagene Prime-It kit as described by the manufacturer. Prehybridization and hybridization were carried out at 42 °C in a solution of 6× SSC, 5× Denhardt's solution, 0.5% SDS, and 50% formamide containing 20 μg/ml denatured salmon sperm DNA. Normal stringency washes were performed at 50 °C using 0.1× SSC and 0.1% SDS. All mRNA transcripts were visualized after exposure to Biomax film (Eastman Kodak Co.) at −80 °C with Dupont Quanta Fast intensifying screens. Cells were lysed in Laemmli buffer, and equal amounts of total protein from each lysate were electrophoresed on 10–15% polyacrylamide/bisacrylamide gels. Migrated proteins were transferred to a Polyscreen polyvinylidene difluoride transfer membrane (PerkinElmer Life Sciences) as described by the manufacturer; immunoblotted using anti-HA tag (12CA5), anti-Bcl-xL/S (S-18, Santa Cruz Biotechnology), anti-PU.1 (T-21, Santa Cruz Biotechnology), anti-Ets2 (C-20, Santa Cruz Biotechnology), or anti-p42 MAPK (gift of Jacques Pouyssegur) antibodies; and revealed by ECL (Amersham Pharmacia Biotech) as described by the manufacturer. Apoptotic macrophage cells were detected with fluorescein isothiocyanate-conjugated annexin V (Roche Molecular Biochemicals). Interaction of annexin V with phosphatidylserines on the outer surface of cells was performed as described by the manufacturer with the following modifications. After incubating cells with annexin V and washing in binding buffer, they were fixed in PBS containing 3% paraformaldehyde for 15 min at 20 °C. Cells were washed in 1× PBS and incubated with a 1:5000 dilution of 4,6-diamidino-2-phenylindole for 5 min at 37 °C. Cells were then washed three times in 1× PBS and twice in water, and Moviol was added to the slide and mounted. At least 350 cells (in the absence of CSF-1) or 700 cells (in the presence of CSF-1) visualized by 4,6-diamidino-2-phenylindole staining were counted to determine the number of viable cells. The number of annexin V-positive cells was calculated with respect to the number of viable cells. 293T cells were seeded in 100-mm culture dishes and transfected the following day using the calcium phosphate procedure with 10 μg of various combinations of plasmid DNA as indicated in the figure legend. 3 μg of a green fluorescent protein reporter plasmid was included in all transfections. 20 h post-transfection, cells were scraped and labeled with Alexa-conjugated annexin V (Roche Molecular Biochemicals) following the manufacturer's instructions. The cells were then analyzed on a Beckman FACSCalibur using the FL1-H window to detect transfected cells (>50% of total cells), and the annexin V-positive cells found among these were quantitated using the FL3-H window. CSF-1 induces primary bone marrow-derived precursor cells to proliferate and then to differentiate into macrophages (BMM). We (23Aperlo C. Sevilla L. Guerin S. Pognonec P. Boulukos K.E. Cell Growth Differ. 1998; 9: 929-937PubMed Google Scholar) and others (24Keller J.R. Gooya J.M. Ruscetti F.W. Blood. 1996; 88: 863-869Crossref PubMed Google Scholar) have shown that cotreatment of hematopoietic progenitors or myeloblastic cells with leukemia inhibitory factor enhances the hematopoiesis process. We obtained murine BMM using different concentrations of CSF-1 (12 and 120 ng/ml) in the presence of 0.1 ng/ml leukemia inhibitory factor. Based on morphology (data not shown) and the expression of a macrophage-specific marker with bacteriolytic functions (lysozyme M (lysM)), BMM were obtained with both concentrations of CSF-1 used (Fig. 1 B). However, fewer macrophages were obtained with low doses of CSF-1 (Fig. 1 A). Northern analysis revealed that PU.1 was abundantly expressed under both culture conditions, paralleling the expression of lysozyme M. However, ets2 expression was detected when a high concentration of CSF-1 was used. It is worth noting that even small increases in Ets2 expression are sufficient to induce profound biological changes since a <2-fold induction of ets2mRNA expression has been shown to greatly affect bone and cartilage development in ets2 transgenic mice (25Sumarsono S.H. Wilson T. Tymms M.J. Venter D.J. Corrick C.M. Kola R. Lahoud M.H. Papas T.S. Seth A. Kola I. Nature. 1996; 379: 534-537Crossref PubMed Scopus (181) Google Scholar). Interestingly,bcl-xL expression was clearly activated whenets2 was expressed (Fig. 1 B). These results show that there is a correlation of ets2 andbcl-xL expression upon treatment of primary precursor cells with concentrations of CSF-1 necessary to induce maximal proliferation and subsequent differentiation. In addition to inducing proliferation and differentiation, CSF-1 is required for the survival of BMM. To determine the expression patterns of PU.1, ets2, and bcl-xL upon death or survival signals, we first treated bone marrow-derived cells with conditioned medium containing CSF-1 for 5 days to obtain BMM (control BMM, 0 h). BMM were then either starved of CSF-1 (0 ng) or maintained with decreasing amounts of CSF-1 (120, 12, or 6 ng/ml) for 36 h. Macrophages were photographed at 24 and 36 h. Visualized in Fig.2 A is the morphology of primary macrophages upon the different treatments. At 24 and 36 h, a higher number of floating, round, refractile cells corresponding to dying cells was observed at 0, 6, or 12 ng of CSF-1. The number of adherent macrophages also decreased in the absence or presence of low doses of CSF-1 compared with the number of cells maintained at 120 ng/ml CSF-1. Total numbers of viable cells were confirmed by trypan blue exclusion for each test condition at 36 h (Fig.2 B). To demonstrate that the cell death observed in the absence of CSF-1 was indeed due to programmed cell death by apoptosis, macrophages maintained with or starved of CSF-1 were incubated with annexin V, an early marker of apoptosis. As shown in Fig.3, in the absence of CSF-1, 15% of the remaining cells were labeled with annexin V. The lower number of 4,6-diamidino-2-phenylindole-stained CSF-1-depleted cells (52% fewer compared with CSF-1-treated cells) and the typical compacted aspect of these nuclei reflect that the apoptotic process was underway.Figure 3CSF-1 starvation of primary bone marrow-derived macrophages induces programmed cell death by apoptosis. Fully differentiated primary macrophages were maintained with or starved of CSF-1 for 24 h. Cells were labeled with annexin V and 4,6-diamidino-2-phenylindole (DAPI) as a control for nuclear staining of intact cells and were photographed (magnification × 100).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To confirm that the cell death observed in CSF-1-starved BMM is accompanied by decreases in Bcl-xL protein expression, Western blot analysis was performed using lysates obtained from BMM starved of CSF-1 for 24 h and then restimulated with decreasing amounts of CSF-1 (120, 60, 12, or 0 ng/ml). After migration and transfer, the blot was incubated with an anti-Bcl-xL/Santibody recognizing both anti-apoptotic Bcl-xL and pro-apoptotic Bcl-xS proteins. As shown in Fig.4 A, Bcl-xL levels decreased in the absence of CSF-1, correlating with increases in cell death. The pro-apoptotic Bcl-xS product migrating at ∼25 kDa was not detected in these experiments. The amount of Ets2 and PU.1 proteins also decreased with decreasing concentrations of CSF-1. The conclusion from these results is that there is a tight correlation between the levels of Ets2, PU.1, and Bcl-xL protein expression and CSF-1. p42 MAPK was used as a control for the amount of protein loaded on the gel. Macrophage cytocidal activity for killing neoplastic cells or microorganisms requires both priming and triggering signals. Priming and triggering by IFN-γ and LPS, respectively, rapidly down-regulate CSF-1R expression even in the presence of CSF-1 (26Baccarini M. Dello Sbarba P. Buscher D. Bartocci A. Stanley E.R. J. Immunol. 1992; 149: 2656-2661PubMed Google Scholar, 27Vairo G. Royston A.K. Hamilton J.A. J. Cell Physiol. 1992; 151: 630-641Crossref PubMed Scopus (55) Google Scholar). In addition, IFN-γ and LPS up-regulate bcl-xL, whose expression depends on de novo protein synthesis (14Okada S. Zhang H. Hatano M. Tokuhisa T. J. Immunol. 1998; 160: 2590-2596PubMed Google Scholar). To determine whether the up-regulation of bcl-xL correlates withPU.1 and ets2 expression independent of CSF-1, we treated primary macrophages with IFN-γ and LPS. As shown in Fig.4 B, a 4-h treatment with IFN-γ or LPS up-regulated the expression of bcl-xL, ets2, andPU.1. IP10 was used as a positive control of an mRNA abundantly induced after IFN-γ or LPS treatment (28Omori S.A. Wall R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11723-11727Crossref PubMed Scopus (63) Google Scholar). These results demonstrate that bcl-xL, ets2, and PU.1 are coexpressed in macrophages not only after a CSF-1 growth, differentiation, and survival stimulus, but also upon activation of macrophage functions independent of CSF-1. Our Northern and Western results show that PU.1, Ets2, and Bcl-xL are expressed upon CSF-1 survival, proliferation, or differentiation signals in primary macrophages. In addition, treatment of macrophages with IFN-γ and LPS, which down-regulates CSF-1 signaling (26Baccarini M. Dello Sbarba P. Buscher D. Bartocci A. Stanley E.R. J. Immunol. 1992; 149: 2656-2661PubMed Google Scholar, 27Vairo G. Royston A.K. Hamilton J.A. J. Cell Physiol. 1992; 151: 630-641Crossref PubMed Scopus (55) Google Scholar), also up-regulatesets2, PU.1, and bcl-xLexpression. Because PU.1 (17Sevilla L. Aperlo C. Dulic V. Chambard J.C. Boutonnet C. Pasquier O. Pognonec P. Boulukos K.E. Mol. Cell. Biol. 1999; 19: 2624-2634Crossref PubMed Scopus (91) Google Scholar) and Ets2 (17Sevilla L. Aperlo C. Dulic V. Chambard J.C. Boutonnet C. Pasquier O. Pognonec P. Boulukos K.E. Mol. Cell. Biol. 1999; 19: 2624-2634Crossref PubMed Scopus (91) Google Scholar, 29Smith J. Schaffner A. Hofmeister J.K. Hartman M. Wei G. Forsthoefel D. Hume D. Ostrowski M. Mol. Cell. Biol. 2000; 20: 8026-8034Crossref PubMed Scopus (65) Google Scholar) individually transactivate the bcl-x promoter, and PU.1 and Ets2 were present when we detected the bcl-xL transcript, we asked whether these transcription factors could compete or work together in activating bcl-x transcription. Human 293 cells were used in these studies for two reasons. First, it is not possible to transiently transfect primary macrophages due to their rapid cell death following addition of DNA to these cells (30Stacey K.J. Ross I.L. Hume D.A. Immunol. Cell Biol. 1993; 71: 75-85Crossref PubMed Google Scholar). Second, no endogenous Ets2 or PU.1 was detected by Western analysis (data not shown) in 293 cells, thereby eliminating potential contributions from endogenously expressed proteins. By Western analysis, we verified that exogenously added HA-tagged Ets1, ΔEts2, Ets2, ΔPU.1, and PU.1 were expressed at comparable levels in transiently transfected 293 cells (Fig.5 A). Visualized in Fig.5 B (left panels) are the levels of the tagged Ets2 and PU.1 proteins following transfection of human 293 cells. In these experiments, the levels of transfected HA-PU.1 decreased as the levels of transfected HA-Ets2 were increased to keep the exogenously added amounts of total Ets proteins constant (transfected Ets DNAs at 400 ng). Corresponding transactivation studies from the same transfected samples are also shown using the 5′-regulatory sequences of the bcl-x gene upstream of the luciferase gene as the reporter (Fig. 5 B). The -fold inductions were higher when both Ets2 and PU.1 were equally expressed (200 ng of each DNA) than when either protein was expressed alone, but at twice the amount (400 ng of DNA) (see Fig. 5 B). To verify that this observation is valid in other cell systems, similar experiments were performed using murine NIH3T3 cells (Fig. 5 B, right panels). Similar results were obtained using NIH3T3 cells, in which Ets2 and PU.1 transactivated the bcl-x promoter better (8-fold) than Ets2 (3-fold) or PU.1 (2-fold) alone. These experiments indicate that, under constant levels of EBS activity, transcriptional activation is more efficient when both PU.1 and Ets2 are present. In other words, keeping the exogenous levels of Ets proteins constant, PU.1 and Ets2 transactivate the bcl-x promoter better than PU.1 or Ets2 alone in two different cell types from two different species. To investigate in greater detail this apparent cooperative effect,bcl-x promoter activity was monitored in 96-well plates to allow for the analysis of a wide range of Ets2 versus PU.1 concentrations. As the concentrations of either PU.1 or Ets2 alone increased, an increase in transcriptional activation was observed (Fig.6). As the concentrations of both PU.1 and Ets2 increased, so did their capacities to transactivate thebcl-x promoter. The inductions observed with Ets2 and PU.1 together result from a synergistic (and not an additive) effect between these factors. To address whether other Ets proteins could synergize with PU.1 or Ets2, the following experiments were performed. 293 cells were transiently transfected with Ets2 and Ets1 (Fig.7 A) or Ets1 and PU.1 (Fig.7 B). In the 96-well assay at the concentrations of DNA used, Ets1 alone transactivated the bcl-x promoter approximately two times better than Ets2 (Fig. 7 A). As the concentrations of Ets1 or Ets2 increased, so did the luciferase activities. The relative bcl-x promoter activity was not at all affected by cotransfections with increasing concentrations of both Ets1 and Ets2. Similar results were obtained using Ets1 and PU.1 (Fig.7 B). Cotransfection with Ets1 and PU.1 did not re