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• W2097471645 abstract "CCAAT/enhancer-binding protein ϵ (C/EBPϵ) plays a critical role in terminal myeloid differentiation. Differentiation is an integrated process of cell cycle arrest, morphological change, functional maturation, and apoptosis. However, the molecular networks underlying these events in C/EBPϵ-induced differentiation remain poorly understood. To reveal these mechanisms, we performed a detailed molecular analysis of C/EBPϵ-induced differentiation using an inducible form of C/EBPϵ. The activation of C/EBPϵ induced growth arrest, morphological differentiation, the expression of CD11b and secondary granule proteins, and apoptosis in myeloid cell lines. Unlike C/EBPα, C/EBPϵ dramatically up-regulated p27 with a concomitant down-regulation of cdk4/6 and cyclin D2/A/E. Moreover, the anti-apoptotic proteins Bcl-2 and Bcl-x were down-regulated, whereas pro-apoptotic protein Bax remained unchanged. Using a variety of mutants, we revealed that these events were all regulated by the N-terminal activation domain of C/EBPϵ. Interestingly, some of the differentiation processes such as the induction of secondary granule protein genes were clearly inhibited by c-Myc; however, inhibition of apoptosis by Bcl-x did not affect the entire differentiation processes. These data indicate the N terminus of C/EBPϵ to be solely responsible for most aspects of myeloid differentiation, and these events were differentially affected by c-Myc. CCAAT/enhancer-binding protein ϵ (C/EBPϵ) plays a critical role in terminal myeloid differentiation. Differentiation is an integrated process of cell cycle arrest, morphological change, functional maturation, and apoptosis. However, the molecular networks underlying these events in C/EBPϵ-induced differentiation remain poorly understood. To reveal these mechanisms, we performed a detailed molecular analysis of C/EBPϵ-induced differentiation using an inducible form of C/EBPϵ. The activation of C/EBPϵ induced growth arrest, morphological differentiation, the expression of CD11b and secondary granule proteins, and apoptosis in myeloid cell lines. Unlike C/EBPα, C/EBPϵ dramatically up-regulated p27 with a concomitant down-regulation of cdk4/6 and cyclin D2/A/E. Moreover, the anti-apoptotic proteins Bcl-2 and Bcl-x were down-regulated, whereas pro-apoptotic protein Bax remained unchanged. Using a variety of mutants, we revealed that these events were all regulated by the N-terminal activation domain of C/EBPϵ. Interestingly, some of the differentiation processes such as the induction of secondary granule protein genes were clearly inhibited by c-Myc; however, inhibition of apoptosis by Bcl-x did not affect the entire differentiation processes. These data indicate the N terminus of C/EBPϵ to be solely responsible for most aspects of myeloid differentiation, and these events were differentially affected by c-Myc. CCAAT/enhancer-binding protein ϵ (C/EBPϵ) 3The abbreviations used are: C/EBPϵ, CCAAT/enhancer-binding protein ϵ; ER, ligand-binding domain of estrogen receptor; 4-HT, 4-hydroxytamoxifen; G-CSF, granulocyte colony-stimulating factor; IL, interleukin; FACS, fluorescence-activated cell sorter; GFP, green fluorescent protein; cdk, cyclin-dependent kinase; STAT, signal transducers and activators of transcription. 3The abbreviations used are: C/EBPϵ, CCAAT/enhancer-binding protein ϵ; ER, ligand-binding domain of estrogen receptor; 4-HT, 4-hydroxytamoxifen; G-CSF, granulocyte colony-stimulating factor; IL, interleukin; FACS, fluorescence-activated cell sorter; GFP, green fluorescent protein; cdk, cyclin-dependent kinase; STAT, signal transducers and activators of transcription. is a member of the C/EBP family of transcription factors that plays a critical role in late granulopoiesis (1Lekstrom-Himes J.A. Stem Cells. 2001; 19: 125-133Crossref PubMed Scopus (106) Google Scholar, 2Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-28548Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar). The loss of C/EBPϵ in mice leads to the terminal differentiation failure of granulocytes and eosinophils (3Yamanaka R. Barlow C. Lekstrom-Himes J. Castilla L.H. Liu P.P. Eckhaus M. Decker T. Wynshaw-Boris A. Xanthopoulos K.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13187-13192Crossref PubMed Scopus (302) Google Scholar). Neutrophils in C/EBPϵ-deficient mice display an abnormal morphology such as hyposegmentation of the nuclei. These mice tend to die from opportunistic infections caused by the defective neutrophil functions such as an impaired chemotaxis and superoxide productions and the lack of secondary and tertiary granule proteins (i.e. lactoferrin, gelatinase B) (3Yamanaka R. Barlow C. Lekstrom-Himes J. Castilla L.H. Liu P.P. Eckhaus M. Decker T. Wynshaw-Boris A. Xanthopoulos K.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13187-13192Crossref PubMed Scopus (302) Google Scholar, 4Lekstrom-Himes J. Xanthopoulos K.G. Blood. 1999; 93: 3096-3105Crossref PubMed Google Scholar, 5Verbeek W. Lekstrom-Himes J. Park D.J. Dang P.M. Vuong P.T. Kawano S. Babior B.M. Xanthopoulos K. Koeffler H.P. Blood. 1999; 94: 3141-3150Crossref PubMed Google Scholar). This phenotype can be explained by the fact that C/EBPϵ works downstream of granulocyte colony-stimulating factor (G-CSF), which plays a critical role in the development and function of neutrophils (6Nakajima H. Ihle J.N. Blood. 2001; 98: 897-905Crossref PubMed Scopus (67) Google Scholar). An aberrant C/EBPϵ function is actually linked to a variety of disease processes in humans. A mutation of the C/EBPϵ gene is the cause of human secondary granule deficiency (7Lekstrom-Himes J.A. Dorman S.E. Kopar P. Holland S.M. Gallin J.I. J. Exp. Med. 1999; 189: 1847-1852Crossref PubMed Scopus (157) Google Scholar, 8Dinauer M.C. Lekstrom-Himes J.A. Dale D.C. Hematology (Am. Soc. Hematol. Educ. Program). 2000; : 303-318Crossref PubMed Google Scholar). In human leukemia, C/EBPϵ is reported to be the critical target of PML-RARα, a leukemic fusion product of chromosomal translocation t(15;17) (9Park D.J. Chumakov A.M. Vuong P.T. Chih D.Y. Gombart A.F. Miller Jr., W.H. Koeffler H.P. J. Clin. Investig. 1999; 103: 1399-1408Crossref PubMed Scopus (164) Google Scholar, 10Truong B.T. Lee Y.J. Lodie T.A. Park D.J. Perrotti D. Watanabe N. Koeffler H.P. Nakajima H. Tenen D.G. Kogan S.C. Blood. 2003; 101: 1141-1148Crossref PubMed Scopus (93) Google Scholar). Taken together, C/EBPϵ is considered to play a critical role in normal granulocyte differentiation and leukemogenesis, and therefore, a full understanding of the molecular networks surrounding C/EBPϵ is essential to elucidate the mechanism of myeloid differentiation and leukemogenesis.The differentiation of hematopoietic cells is an integrated process of morphological change, functional maturation, growth arrest, and apoptosis. We have previously shown that C/EBPϵ retarded cellular growth by some unknown mechanisms when it was expressed in cell lines (6Nakajima H. Ihle J.N. Blood. 2001; 98: 897-905Crossref PubMed Scopus (67) Google Scholar). In addition, C/EBPϵ-/- myeloid progenitors have an increased rate of proliferation (11Verbeek W. Wachter M. Lekstrom-Himes J. Koeffler H.P. Leukemia. 2001; 15: 103-111Crossref PubMed Scopus (27) Google Scholar). C/EBPα, another C/EBP family member that is essential for early granulopoieis (12Zhang D.E. Zhang P. Wang N.D. Hetherington C.J. Darlington G.J. Tenen D.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 569-574Crossref PubMed Scopus (750) Google Scholar), induces growth arrest and differentiation in a manner similar to C/EBPϵ (13Yamanaka R. Lekstrom-Himes J. Barlow C. Wynshaw-Boris A. Xanthopoulos K.G. Int. J. Mol. Med. 1998; 1: 213-221PubMed Google Scholar). C/EBPα inhibits the kinase activity of cyclin-dependent kinase (cdk) 4 and cdk6 by direct protein-protein interactions to inhibit cell cycle progression (14Wang H. Goode T. Iakova P. Albrecht J.H. Timchenko N.A. EMBO J. 2002; 21: 930-941Crossref PubMed Scopus (80) Google Scholar, 15McKnight S.L. Cell. 2001; 107: 259-261Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 16Wang H. Iakova P. Wilde M. Welm A. Goode T. Roesler W.J. Timchenko N.A. Mol. Cell. 2001; 8: 817-828Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 17Harris T.E. Albrecht J.H. Nakanishi M. Darlington G.J. J. Biol. Chem. 2001; 276: 29200-29209Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 18Timchenko N.A. Wilde M. Nakanishi M. Smith J.R. Darlington G.J. Genes Dev. 1996; 10: 804-815Crossref PubMed Scopus (344) Google Scholar). The interaction occurs through the N terminus (amino acids 175-187) of C/EBPα, and this facilitates the proteasomal degradation of cdk4 and cdk6. The N-terminal region of C/EBPα also interacts with E2F to inhibit its function, and this region is critically required for granulocyte and adipocyte differentiation in vivo (19Porse B.T. Pedersen T.A. Xu X. Lindberg B. Wewer U.M. Friis-Hansen L. Nerlov C. Cell. 2001; 107: 247-258Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). As for C/EBPϵ, it has also been reported to repress the E2F activity by direct protein-protein interactions (20Gery S. Gombart A.F. Fung Y.K. Koeffler H.P. Blood. 2004; 103: 828-835Crossref PubMed Scopus (49) Google Scholar). However, the precise molecular mechanism regarding how C/EBPϵ regulates growth arrest remains largely unknown. Moreover, the mechanism regulating the induction of apoptosis and secondary granule protein genes by C/EBPϵ still remains unclear.In this study, we established an inducible system of C/EBPϵ to reveal the molecular targets of C/EBPϵ during differentiation. In addition, we performed a detailed structure-function analysis of C/EBPϵ to map the responsible regions for various aspects of differentiation. We herein demonstrate that C/EBPϵ up-regulates p27 and down-regulates cyclins/cdks to induce growth arrest during differentiation. In addition, C/EBPϵ induces apoptosis by down-regulating anti-apoptotic proteins, Bcl-2 and Bcl-x. These effects are mediated by the N-terminal activation domain of C/EBPϵ, and this domain is also responsible for the induction of secondary granule protein genes. An overexpression of c-Myc prevented the induction of secondary granule protein genes, whereas Bcl-x did not affect the differentiation processes. These results demonstrate that the molecular pathways of C/EBPϵ leading to growth arrest, apoptosis, and the functional maturation all emanate from the N-terminal activation domain, and these pathways were differentially affected by c-Myc.EXPERIMENTAL PROCEDURESCells—32D (6Nakajima H. Ihle J.N. Blood. 2001; 98: 897-905Crossref PubMed Scopus (67) Google Scholar), LG, and LGM3 (kindly provided by Dr. T. Honjo) cells and their transfectants are cultured in an RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, 100 μg/ml streptomycin, 2 mm l-glutamine, and 2.5 units/ml of recombinant murine IL-3. All of the cell lines were cultured at 37 °C in a humidified atmosphere with 5% CO2. 32D/DN-STAT3 cells and 32D/ϵ cells were described previously (6Nakajima H. Ihle J.N. Blood. 2001; 98: 897-905Crossref PubMed Scopus (67) Google Scholar). 32D, LG, and LGM3 cells expressing C/EBPϵ-ER were generated by infecting C/EBPϵ-ER retrovirus. Briefly, cells in a logarithmic growth phase were suspended in medium containing retrovirus with 10 μg/ml of polybrene (Sigma) and 10 units/ml of murine IL-3. An equal amount of medium (RPMI 1640, 10% fetal bovine serum, 2.5 units/ml of murine IL-3) was supplemented after 5 h of infection, and GFP-positive cells were sorted by FACS vantage (Becton Dickinson) after several days. To avoid clonal variation, pools of sorted cells were used for all experiments. The intensities of GFP in established cell lines were checked periodically by FACS, showing that GFP expressions remained constant during experiments.Retroviral Constructs—cDNAs for full-length C/EBPϵ and their mutants were fused in-frame to a mutated (G525R) ligand-binding domain of murine estrogen receptor and subcloned into MSCV-IRES-GFP retrovirus vector. cDNA for the ligand-binding domain (amino acids 281-599) of murine estrogen receptor was amplified by PCR using bone marrow cDNA as a template, and the G525R mutation that confers a selective response to 4-hydroxytamoxifen was introduced using QuikChange site-directed mutagenesis kit (Stratagene). A variety of C/EBPϵ mutants were generated by PCR using full-length C/EBPϵ cDNA as a template. cDNAs for Bcl-x and c-Myc were also amplified by PCR using bone marrow cDNA as a template and subcloned into MSCV-IRES-GFP retrovirus vector. All of the amplified sequences were verified by DNA sequencing.Transfection and Retrovirus Production—Retroviral constructs were transfected into PlatE producer cell line (21Morita S. Kojima T. Kitamura T. Gene. Ther. 2000; 7: 1063-1066Crossref PubMed Scopus (1337) Google Scholar) using FuGENE 6 (Roche Applied Science) according to the manufacturer's protocol. Retroviral supernatants were harvested after 48 h of transfection, filtered, and stored at -80 °C until use.4-Hydroxytamoxifen— 4-Hydroxytamoxifen (4-HT; Sigma) was dissolved in EtOH (stock concentration at 5 mm) and was used at the final concentration of 1 μm in all experiments.Gel Shift Assay—Nuclear extracts were made from LG/CEBPϵ-ER cells stimulated with 1 μm of 4-HT for various times indicated as described previously (22Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3907) Google Scholar). Cell extracts (10 μg of total protein) were incubated with 2 μg of poly(dI-dC) for 15 min and then with double-stranded C/EBP consensus oligonucleotide probes (5′-TGCAGATTGCGCAATCTGCA-3′) labeled with [γ-32P]ATP for 30 min on ice. For supershift, anti-C/EBPϵ (C-22; Santa Cruz) or anti-ER (MC-20; Santa Cruz) antibodies were added to the reactions. The resulting complexes were resolved on 4.5% polyacrylamide gel in 1× TBE (89 mm Tris, 89 mm boric acid, 2 mm EDTA) buffer. The gels were dried and visualized by autoradiography.Western Blot Analysis—Protein extracts were prepared as previously described (6Nakajima H. Ihle J.N. Blood. 2001; 98: 897-905Crossref PubMed Scopus (67) Google Scholar, 23Nakajima H. Brindle P.K. Handa M. Ihle J.N. EMBO J. 2001; 20: 6836-6844Crossref PubMed Scopus (92) Google Scholar). The extracts (30 μg/lane) were separated on 4-20% SDS-PAGE gels and transferred to PROTRAN BA85 membrane (Schleicher & Schuell). The membranes were probed sequentially with primary antibodies and horseradish peroxidase-conjugated anti-rabbit or anti-mouse Ig polyclonal antibody (Amersham Biosciences). Bound antibodies were visualized by ECL (Amersham Biosciences). The primary antibodies of anti-cdk4 (C-22), anti-cdk6 (C-21), anti-cyclin D2 (34B1-3), anti-cyclin A (H-432), anti-cyclin E (M-20), anti-p21 (C-19), and anti-Bax (N-20) were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-p27, Bcl-x, Bcl-2, Rb, and β-tubulin antibodies were from Pharmingen (San Diego, CA).Northern Blot—Total RNA was extracted from 1 × 107 cells by TRIzol (Invitrogen) according to the manufacturer's protocol. The RNA samples (20 μg/lane) were separated on formaldehyde-denaturing 1.0% agarose gel and transferred onto Hybond N+ membrane (Amersham Biosciences). Full-length cDNAs of lactoferrin, gelatinase-B, leukocyte elastase, and lysozyme (kindly provided by Dr. Dan Tenen, Harvard University) were used as probes. All of the probes were labeled using a Rediprime kit (Amersham Biosciences). Hybridizations with 32P-labeled probes were carried out in ExpressHyb buffer (Clontech) according to the manufacturer's protocol. The membranes were washed in 2× SSC, 0.1% SDS washing buffer for 30 min at room temperature with several buffer changes, followed by washing twice in 0.1× SSC, 0.1% SDS for 15 min at 42 °C. The membranes were then exposed on XAR film (Kodak) at -80 °C for 1-5 days.Flow Cytometry—The cells were stained as described previously (6Nakajima H. Ihle J.N. Blood. 2001; 98: 897-905Crossref PubMed Scopus (67) Google Scholar) with anti-Gr-1-fluorescein isothiocyanate or anti-Mac-1α (CD11b)-fluorescein isothiocyanate antibodies (Pharmingen). A FACS analysis was performed using FACSCalibur flow cytometer (Becton Dickinson), and the data were analyzed using the Cell Quest software package.Cell Cycle Analysis—1 × 106 cells were suspended and stained in hypotonic buffer containing propidium iodide (0.1% Triton X-100, 1 mm Tris-HCl, pH 8.0, 3.4 mmol/liter sodium citrate, and 0.1 mm EDTA, 50 μg/ml propidium iodide). The DNA content was analyzed by FAC-SCalibur (Becton Dickinson) using the Cell FIT software package (Becton Dickinson).DNA Fragmentation Analysis—The cells (1 × 106) were washed twice with ice-cold phosphate-buffered saline and lysed in a lysis buffer (10 mm Tris-HCl, pH 7.4, 10 mm EDTA, 0.5% Triton X-100) at 4 °C for 10 min. After centrifugation of the lysates at 12,000 × g for 5 min at 4 °C, the supernatants were collected and transferred to new tubes. The supernatants were treated with 50 μg/ml of RNase A (Sigma) for 1 h at 37 °C and then with 100 μg/ml of proteinase K (Sigma) for another hour. DNA was extracted with phenol-chloroform, precipitated in ethanol, and centrifuged for 20 min at 12,000 × g. DNA was dissolved in TE buffer (10 mm Tris-HCl, pH 7.4, 10 mm EDTA) and separated on a 2% agarose gel. The fragmentation of DNA was visualized by ethidium bromide staining of the gel.RESULTSExpression of C/EBPϵ Correlates with Up-regulation of p27— C/EBPα, another C/EBP family member, induces cell cycle arrest through modulation of p21, cdk2, and cdk4 (14Wang H. Goode T. Iakova P. Albrecht J.H. Timchenko N.A. EMBO J. 2002; 21: 930-941Crossref PubMed Scopus (80) Google Scholar, 15McKnight S.L. Cell. 2001; 107: 259-261Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 16Wang H. Iakova P. Wilde M. Welm A. Goode T. Roesler W.J. Timchenko N.A. Mol. Cell. 2001; 8: 817-828Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 17Harris T.E. Albrecht J.H. Nakanishi M. Darlington G.J. J. Biol. Chem. 2001; 276: 29200-29209Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 18Timchenko N.A. Wilde M. Nakanishi M. Smith J.R. Darlington G.J. Genes Dev. 1996; 10: 804-815Crossref PubMed Scopus (344) Google Scholar). Therefore, we hypothesized that C/EBPϵ induces withdrawal from the cell cycle similarly to C/EBPα by modulating a family of cdk or cdk inhibitors. To get some insight into the molecular mechanism of cell cycle arrest induced by C/EBPϵ, we first examined the role of two major cdk inhibitors, p21 and p27 in 32D cells treated by G-CSF. C/EBPϵ is up-regulated by G-CSF in these cells where it plays a critical role in inducing differentiation to the granulocytes (6Nakajima H. Ihle J.N. Blood. 2001; 98: 897-905Crossref PubMed Scopus (67) Google Scholar). Interestingly, p27 protein is markedly up-regulated in G-CSF treated 32D cells (Fig. 1), whereas the p21 protein level did not change substantially (data not shown). We speculated that C/EBPϵ could thus be involved in the up-regulation of p27 and therefore examined the p27 protein levels in 32D cells overexpressing C/EBPϵ (32D/ϵ). As expected, p27 protein was markedly up-regulated in these cells (Fig. 1). We also examined 32D cells expressing dominant negative signal transducers and activators of transcription (STAT) 3 (32D/DN-STAT3). These cells do not differentiate but rather proliferate in G-CSF at a slower rate compared with those in IL-3. Because G-CSF induces C/EBPϵ in these cells, we speculated that this lead to the induction of p27. As expected, p27 was clearly up-regulated in 32D/DN-STAT3 cells cultured in G-CSF, but not in IL-3. These data show a clear correlation between C/EBPϵ and p27 and suggest that p27 could be the critical target of C/EBPϵ during cell cycle arrest.C/EBPϵ Induces Morphological Differentiation, Growth Arrest, and Apoptosis in Early Myeloid Cell Line—To further explore the molecular mechanism of growth arrest induced by C/EBPϵ, we created an inducible form of C/EBPϵ that is a fusion of full-length C/EBPϵ and the mutated ligand-binding domain of estrogen receptor (C/EBPϵ-ER) and examined its effect in various hematopoietic cell lines. This fusion protein can be activated selectively by an estrogen antagonist, 4-HT. As shown in Fig. 2A, stimulation of cells expressing C/EBPϵ-ER with 4-HT induced specific binding to C/EBP-oligonucleotides within 30 min by gel shift analysis, thus showing that this protein is working properly.FIGURE 2Effects of C/EBPϵ on growth, differentiation, and apoptosis of myeloid cells. LG cells expressing C/EBPϵ-ER were generated and examined for the outcome of C/EBPϵ activation. A, DNA binding activity of C/EBPϵ-ER protein induced by 4-HT. The cells were treated with IL-3 (10 ng/ml) or 4-HT for the times indicated, and a gel shift analysis was performed as described under “Experimental Procedures. “ Supershifts were performed by adding anti-ER or anti-C/EBPϵ antibodies to the extract of 4-HT for 120 min. TheDNA-binding complex of C/EBPϵ-ER and its supershifted complexes (s.s.) are indicated by arrows. The asterisk indicates endogenous C/EBP proteins. The schematic structures of C/EBPϵ and C/EBPϵ-ER proteins are shown on the left. AD, activation domain; DBD, DNA-binding domain; LZ, leucine zipper motif; ER, ligand-binding domain of estrogen receptor. B, cell growth. LG cells transfected with vector alone are shown in red, and LG cells expressing C/EBPϵ-ER are shown in blue. The cells were counted every day and diluted to maintain cell concentrations between 1 × 105/ml and 8 × 105/ml. The cumulative cell numbers are shown. C, cell cycle analysis. The cells were treated with vehicle (EtOH) or 4-HT for the times indicated, and the percentages of cells in G0/G1 or sub-G0/G1 were analyzed by FACS. D, apoptosis. The cells were treated with vehicle (EtOH) or 4-HT, and apoptotic cells were enumerated under a microscopy at the times indicated. A fragmentation of DNA was examined as described under “Experimental Procedures. “ E, cell morphology. Cells treated with vehicle (EtOH) or 4-HT for 4 days were cytospun onto glass slides and stained by Wright-Giemsa staining. Original magnification was 1000× or 400×. The closed and open arrowheads indicate cells with granulocyte and macrophage morphology, respectively. F, expression of Mac-1α and Gr-1. The expressions of Mac-1α and Gr-1 were analyzed by FACS as described under “Experimental Procedures,” and their positivity was plotted in graphs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We introduced the C/EBPϵ-ER expression vector into several IL-3-dependent hematopoietic cell lines including 32D, LG, and LGM3 and then examined the effect on their growth and differentiation. As shown in Fig. 2B, the induction of the C/EBPϵ-ER activity by 4-HT clearly impaired the growth of these cell lines within 3 days of stimulation even in the presence of IL-3. Cell cycle analysis revealed that induction of C/EBPϵ activity led to a rapid accumulation of cells in G0/G1 (Fig. 2C). Notably, this was accompanied by enhanced cell death with apoptosis as revealed by the cell morphology, an increased percentage of cells in sub-G0/G1, and DNA ladder formation (Fig. 2, C and D). A significant fraction (up to 35% at day 6) of LG cells also arrested in sub-G0/G1 by 4-HT stimulation; however, this was not as dramatic as that of LG/CEBPϵ-ER cells and hence was regarded as a nonspecific, toxic effect of 4-HT. A morphological analysis revealed that treating LG/CEBPϵ-ER and LGM3/CEBPϵ-ER cells for 5 days with 4-HT induced a clear differentiation to the myeloid lineage, mainly mature granulocytes (Fig. 2E, closed arrowheads, and data not shown). We observed differentiation to the macrophage-like cells in a significant fraction of LG/CEBPϵ-ER and LGM3/CEBPϵ-ER cells (open arrowheads in Fig. 2E and data not shown), thus indicating that C/EBPϵ could also be directing cells to a monocytic lineage in these cells. Differentiation to a myeloid lineage was also confirmed by a FACS analysis showing the 4-HT-treated LG/CEBPϵ-ER cells to express increased levels of Mac-1α (CD11b) and Gr-1 (Fig. 2F). These data indicate that the induction of the C/EBPϵ activity is sufficient to induce myeloid differentiation, growth arrest, and apoptosis in myeloid cell lines.C/EBPϵ Induces the Up-regulation of p27 and the Down-regulation of Cyclins and Cyclin-dependent Kinases—To reveal the mechanism of growth arrest and apoptosis induced by C/EBPϵ, we analyzed the expression of various molecules involved in cell cycle regulation and apoptosis by Western blot (Fig. 3A). The most striking difference induced by C/EBPϵ-ER induction is the robust up-regulation of p27. In sharp contrast, cyclin D2, cyclin A, cyclin E, cdk4, and cdk6 were clearly down-regulated after 4 days of 4-HT treatment. In addition, hypophosphorylation of Rb protein was specifically observed in LG/CEBPϵ-ER cells treated with 4-HT. It should be mentioned that EtOH alone caused similar changes of some protein expressions seen in the 4-HT-treated LG/CEBPϵ-ER cells (cyclin D2, cdk4/6 in Fig. 3 and Bcl-2 in Fig. 4). However, the extent of the changes were more dramatic in 4-HT-treated cells, and moreover, it was not observed in other experiments (see Fig. 6C). Therefore, we speculate this could be due to the specific experimental condition or to the background activity of the C/EBPϵ-ER protein, which is commonly seen in inducible systems. Collectively, these data demonstrate that p27 is indeed the downstream target of C/EBPϵ, as suggested by the previous experiment. Moreover, C/EBPϵ not only up-regulates p27 but also down-regulates various cyclins and cdks. This suggests that the concomitant modulation of positive and negative regulators for cell cycle may thus be the major underlying mechanism for C/EBPϵ-induced growth arrest during myeloid differentiation.FIGURE 3C/EBPϵ regulates various cell cycle molecules during differentiation. A, LG cells transfected with vector alone or C/EBPϵ-ER were treated with vehicle (EtOH) or 4-HT (1 μm) for the times indicated, and a Western blot analysis was performed as described under “Experimental Procedures.” B, cells were treated with vehicle (EtOH) or 4-HT (1 μm) for 4 days, and then cycloheximide (10 μg/ml) was added. After treating cells with cycloheximide for 30 min, cellular amount of p27 protein was chased serially by Western blot analysis at the indicated time points.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4C/EBPϵ regulates anti-apoptotic but not pro-apoptotic proteins during differentiation. LG cells transfected with vector alone or C/EBPϵ-ER were treated with vehicle (EtOH) or 4-HT (1 μm) for the times indicated, and a Western blot analysis was performed as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Structure-function analysis of C/EBPϵ. A, schematic presentation of C/EBPϵ-ER mutants and the summary of their effects on differentiation. AD1, activation domain 1; AD2, activation domain 2; RD, repressor domain; BR, basic region; L-Z, leucine zipper motif; F, full-length C/EBPϵ. Expressions of each mutant protein in cell lines are shown in the lower panel (indicated by asterisks). B, growth curve. LG cells transfected with each mutants were examined for their growth in the presence or absence of 4-HT. The cells were counted every day and diluted to keep the cell concentrations between 1 × 105/ml and 8 × 105/ml. C, effects of N0, ΔA1, and ΔA2 mutants of C/EBPϵ-ER on cell cycle- or apoptosis-related molecules. LG cells expressing full-length (F) or mutant C/EBPϵ-ER were stimulated with vehicle control (EtOH) or 4-HT (1 μm), and the expressions of p27, cdk4, cyclin D2 and Bcl-2 were analyzed by Western blot. D, induction of secondary granule protein genes. 32D cells expressing full-length (F) or mutant C/EBPϵ-ER were stimulated with either vehicle control (lanes C; EtOH) or 4-HT for 3 days and analyzed for lactoferrin gene expression by Northern blot. Lanes 0, unstimulated cells. 28 S rRNA is shown as a loading control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Structure-function analysis of C/EBPϵ. A, schematic presentation of C/EBPϵ-ER mutants and the summary of their effects on differentiation. AD1, activation domain 1; AD2, activation domain 2; RD, repressor domain; BR, basic region; L-Z, leucine zipper motif; F, full-length C/EBPϵ. Expressions of each mutant protein in cell lines are shown in the lower panel (indicated by asterisks). B, growth curve. LG cells transfected with each mutants were examined for their growth in the presence or absence of 4-HT. The cells were counted every day and diluted to keep the cell concentrations between 1 × 105/ml and 8 × 105/ml. C, effects of N0, ΔA1, and ΔA2 mutants of C/EBPϵ-ER on cell cycle- or apoptosis-related molecules. LG cells expressing full-length (F) or mutant C/EBPϵ-ER were stimulated with vehicle control (EtOH) or 4-HT (1 μm), and the expressions of p27, cdk4, cyclin D2 and Bcl-2 were anal" @default.
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• W2097471645 title "N-terminal Region of CCAAT/Enhancer-binding Protein ϵ Is Critical for Cell Cycle Arrest, Apoptosis, and Functional Maturation during Myeloid Differentiation" @default.
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