The Kruppel-like factor KLF4 is a critical regulator of monocyte differentiation
2007; Springer Nature; Volume: 26; Issue: 18 Linguagem: Inglês
10.1038/sj.emboj.7601824
ISSN1460-2075
AutoresMark W. Feinberg, Akm Khyrul Wara, Zhuoxiao Cao, Maria A. Lebedeva, Frank Rosenbauer, Hiromi Iwasaki, Hideyo Hirai, Jonathan P. Katz, Richard L. Haspel, Susan Gray, Koichi Akashi, Julie Segre, Klaus H. Kaestner, Daniel G. Tenen, Mukesh K. Jain,
Tópico(s)Chronic Myeloid Leukemia Treatments
ResumoArticle30 August 2007free access The Kruppel-like factor KLF4 is a critical regulator of monocyte differentiation Mark W Feinberg Corresponding Author Mark W Feinberg Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Akm Khyrul Wara Akm Khyrul Wara Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Zhuoxiao Cao Zhuoxiao Cao Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Maria A Lebedeva Maria A Lebedeva Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Frank Rosenbauer Frank Rosenbauer Harvard Institutes of Medicine, Boston, MA, USA Search for more papers by this author Hiromi Iwasaki Hiromi Iwasaki The Department of Cancer and Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author Hideyo Hirai Hideyo Hirai Harvard Institutes of Medicine, Boston, MA, USA Search for more papers by this author Jonathan P Katz Jonathan P Katz Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Richard L Haspel Richard L Haspel Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA, USA Search for more papers by this author Susan Gray Susan Gray Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Koichi Akashi Koichi Akashi The Department of Cancer and Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author Julie Segre Julie Segre National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Klaus H Kaestner Klaus H Kaestner Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Daniel G Tenen Daniel G Tenen Harvard Institutes of Medicine, Boston, MA, USA Search for more papers by this author Mukesh K Jain Corresponding Author Mukesh K Jain Cardiovascular Division, Department of Medicine, Case Cardiovascular Research Institute, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Mark W Feinberg Corresponding Author Mark W Feinberg Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Akm Khyrul Wara Akm Khyrul Wara Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Zhuoxiao Cao Zhuoxiao Cao Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Maria A Lebedeva Maria A Lebedeva Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Frank Rosenbauer Frank Rosenbauer Harvard Institutes of Medicine, Boston, MA, USA Search for more papers by this author Hiromi Iwasaki Hiromi Iwasaki The Department of Cancer and Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author Hideyo Hirai Hideyo Hirai Harvard Institutes of Medicine, Boston, MA, USA Search for more papers by this author Jonathan P Katz Jonathan P Katz Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Richard L Haspel Richard L Haspel Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA, USA Search for more papers by this author Susan Gray Susan Gray Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Koichi Akashi Koichi Akashi The Department of Cancer and Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author Julie Segre Julie Segre National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Klaus H Kaestner Klaus H Kaestner Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Daniel G Tenen Daniel G Tenen Harvard Institutes of Medicine, Boston, MA, USA Search for more papers by this author Mukesh K Jain Corresponding Author Mukesh K Jain Cardiovascular Division, Department of Medicine, Case Cardiovascular Research Institute, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Author Information Mark W Feinberg 1, Akm Khyrul Wara1, Zhuoxiao Cao1, Maria A Lebedeva1, Frank Rosenbauer2, Hiromi Iwasaki3, Hideyo Hirai2, Jonathan P Katz4, Richard L Haspel5, Susan Gray1, Koichi Akashi3, Julie Segre6, Klaus H Kaestner4, Daniel G Tenen2 and Mukesh K Jain 7 1Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA 2Harvard Institutes of Medicine, Boston, MA, USA 3The Department of Cancer and Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA, USA 4Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA 5Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA, USA 6National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA 7Cardiovascular Division, Department of Medicine, Case Cardiovascular Research Institute, Case Western Reserve University, Cleveland, OH, USA *Corresponding authors: Cardiovascular Division, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA. Tel.: +1 617 525 4381; Fax: +1 617 525 4380; E-mail: [email protected] Cardiovascular Division, Department of Medicine, Case Cardiovascular Research Institute, Case Western Reserve University, Wolstein Research Building, 2103 Cornell Road, Room 4537, Cleveland, OH 44106, USA. Tel.: +1 216 368 3607; Fax: +1 216 368 0556; E-mail: [email protected] The EMBO Journal (2007)26:4138-4148https://doi.org/10.1038/sj.emboj.7601824 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Monocyte differentiation involves the participation of lineage-restricted transcription factors, although the mechanisms by which this process occurs are incompletely defined. Within the hematopoietic system, members of the Kruppel-like family of factors (KLFs) play essential roles in erythrocyte and T lymphocyte development. Here we show that KLF4/GKLF is expressed in a monocyte-restricted and stage-specific pattern during myelopoiesis and functions to promote monocyte differentiation. Overexpression of KLF4 in HL-60 cells confers the characteristics of mature monocytes. Conversely, KLF4 knockdown blocked phorbol ester-induced monocyte differentiation. Forced expression of KLF4 in primary common myeloid progenitors (CMPs) or hematopoietic stem cells (HSCs) induced exclusive monocyte differentiation in clonogenic assays, whereas KLF4 deficiency inhibited monocyte but increased granulocyte differentiation. Mechanistic studies demonstrate that KLF4 is a target gene of PU.1. Consistently, KLF4 can rescue PU.1–/– fetal liver cells along the monocytic lineage and can activate the monocytic-specific CD14 promoter. Thus, KLF4 is a critical regulator in the transcriptional network controlling monocyte differentiation. Introduction Hematopoietic stem cells (HSCs) may generate committed progenitor cells that lose the capacity to self-renew and ultimately differentiate along a specific lineage to form mature blood cells. Control of hematopoiesis is a complex process requiring the coordinated expression of stage-specific transcription factors that allow for subsequent induction of lineage-restricted genes and cell surface receptors (Tenen et al, 1997; Friedman, 2002). Cytokines and growth factors may then increase lineage-committed progenitor cell populations depending, in part, on the expression levels of lineage determination transcription factors. In the myeloid pathway, mature monocytes and granulocytes arise from bipotential granulocyte/macrophage progenitors (GMPs) that, in turn, arise from multipotential common myeloid progenitors (CMPs); CMPs may also give rise to bipotential megakaryocyte/erythrocyte progenitors (MEPs) (Akashi et al, 2000; Traver et al, 2001). Identification of transcription factors that participate in controlling progenitor cell fate decisions in the myeloid pathway has been of considerable interest with therapeutic implications for a variety of conditions including leukemia, anemia, and chronic inflammatory diseases, among others. Gene knockout experiments in mice have identified several transcription factors as critical regulators of different aspects of myeloid development (Tenen et al, 1997; Friedman, 2002). For example, disruption of the Ets transcription factor PU.1 in mice resulted in multiple hematopoietic defects, including a reduction of not only mature macrophages and granulocytes, but also B and T lymphocytes and NK cells (Scott et al, 1994; McKercher et al, 1996; Colucci et al, 2001). Indeed, the presence of PU.1 is required for the development of CMPs and common lymphoid progenitors (CLPs) from HSCs (Iwasaki et al, 2005). These findings place PU.1 upstream in the transcriptional hierarchy of specifying progenitor cell fate and raise the possibility that additional factors may participate at specific stages to restrict lineage commitment along single myeloid pathways. For example, GATA-1, a transcription factor essential in specifying progenitors along erythrocyte, megakaryocyte, mast cell, and eosinophil lineages (at the expense of monocyte/granulocyte lineages), interacts with PU.1 to inhibit its function and vice versa (Rekhtman et al, 1999; Zhang et al, 1999; Nerlov et al, 2000). Examples of this type of mutual antagonism exist throughout the hematopoietic system (Orkin, 2000). The balance between monocyte and granulocyte differentiation may be regulated, in part, by mutual antagonism between PU.1 and the CCAAT enhancer-binding protein-α (C/EBP-α). C/EBP-α-deficient mice exhibit a complete block in neutrophil differentiation with normal monocyte maturation (Zhang et al, 1997). Recent mechanistic studies have revealed that C/EBP-α physically interacts with PU.1 and may contribute to the specification of myeloid progenitors to the granulocyte lineage depending on the expression ratio of these two factors (Reddy et al, 2002; Dahl et al, 2003) (Radomska et al, 1998; Reddy et al, 2002). Interestingly, mice carrying a graded reduction of PU.1 expression to 20% of normal levels develop acute myeloid leukemia (Rosenbauer et al, 2004). Thus, stage-specific expression of various transcription factors helps dictate not only cell fate decisions in the myeloid lineage, but also the development of malignant transformation (Tenen, 2003; Rosenbauer et al, 2004). The Kruppel-like family of transcription factors (KLFs) are critical regulators of cellular development, growth, and differentiation. (Bieker, 1996; Feinberg et al, 2004). Two examples highlight the critical role of this family of genes in hematopoietic development. KLF1, or EKLF (erythroid Kruppel-like factor), is expressed primarily in red blood cells. Gene targeting experiments revealed that KLF1 is necessary for γ- to β-globin switching during erythrocyte development (Nuez et al, 1995; Perkins et al, 1995). KLF2, or LKLF (lung Kruppel-like factor), is highly expressed in T cells, and targeted disruption of KLF2 verified an essential role for this factor in programming and maintaining naïve T-cell quiescence (Kuo et al, 1997). Because of the importance of KLF1 and KLF2 in different hematopoietic lineages, we hypothesized that a related Kruppel-like zinc-finger (ZF) protein may regulate the differentiation of precursor cells along the monocyte/macrophage cell pathway. Using a low-stringency homology screening strategy, we identified KLF4 as being highly expressed in monocytes. KLF4 was initially identified in the epithelial lining of the gut and skin (Garrett-Sinha et al, 1996; Shields et al, 1996) and gene targeting experiments have verified a critical role for this factor in these tissues (Segre et al, 1999; Katz et al, 2002) as well as in embryonic cells (Takahashi and Yamanaka, 2006) and cornea (Swamynathan et al, 2006). Recently, we demonstrated that KLF4 can regulate iNOS expression and TGF-β signaling in activated macrophages (Feinberg et al, 2005) and Noti et al (2005) identified that KLF4 can repress the CD11d promoter in leukemic cell lines. However, the functional role of KLF4 in myeloid cell development has not been defined. Herein, we found that among myeloid cells, KLF4 is expressed principally in monocytes and is induced in a stage-specific manner during myelopoiesis. Overexpression of KLF4 in promyelocytic HL-60 cells or in primary CMPs or HSCs from bone marrow restricts these cells along the monocyte/macrophage pathway at the expense of other myeloid lineages and confers the morphologic, genetic, and functional characteristics of a mature monocyte. We further show that KLF4 is a downstream target gene of PU.1 and is capable of binding to the monocyte-specific CD14 promoter. Taken together, these data support an important role for KLF4 as a key transcriptional regulator of monocyte differentiation. Results Identification of KLF4 The full-length cDNA of KLF4 was identified through low-stringency homology screening of a rat monocyte/macrophage cDNA library using the ZF domain of KLF1/EKLF as a probe. Analysis of the 1422-base pair open reading frame revealed a 474-amino-acid protein with three Cys2/His2 zinc fingers at the C-terminus and a proline-rich N-terminus (data not shown). A GenBank search of three out of nine clones isolated revealed that our factor is identical to the rat homolog of GKLF/EZF, also known as KLF4 (Garrett-Sinha et al, 1996; Shields et al, 1996; Higaki et al, 2002). Expression of KLF4 in human hematopoietic cell lines To understand the pattern of KLF4 expression in human hematopoietic cells, we analyzed total RNA isolated from primary human peripheral blood monocytes and seven human hematopoietic cell lines by Northern blotting (Figure 1A). A single, intense 3.5-kb band corresponding to KLF4 was detected in RNA from human peripheral blood monocytes, the monocyte-like THP-1 cell line, and the histiocytic U-937 cell line. In contrast, KLF4 mRNA was undetectable in RNA derived from the HeL (erythrocyte), Jurkat (T lymphocyte), Raji (B lymphocyte, immature), and U-266 (B lymphocyte, mature) cells. To assess the expression of KLF4 protein in myeloid cells, we harvested total cell extracts from human peripheral blood monocytes and THP-1, U-937, and HL-60 cells and performed Western blot analysis with a polyclonal KLF4 antiserum. KLF4 protein was detected in human peripheral blood monocytes and THP-1 and U-937 cells but not in promyelocytic HL-60 cells (Figure 1B). Thus, among hematopoietic cells, KLF4 mRNA and protein are expressed in monocytes and monocyte-like cell lines. Figure 1.Expression of KLF4 in human monocytes, hematopoietic cell lines, and during monocyte and granulocyte differentiation. (A) Northern blot analysis of KLF4 demonstrates a monocyte-enriched expression pattern. The cell types tested were human peripheral blood monocytes (1° Monocytes), THP-1 (monocytic leukemia), U-937 (histiocytic leukemia), HeL (erythrocyte), Jurkat (T cell), Raji (immature B cell), and U-266 (mature B cell). (B) Western blot analysis of KLF4 protein expression in human monocytes and human myeloid cell lines. (C) Northern blot analysis of HL-60 cells shows expression of KLF4 in TPA-differentiated monocytes, but not in RA-differentiated granulocytes, whereas CD11b is expressed in both cell types. Download figure Download PowerPoint As shown in Figure 1A and B, KLF4 is expressed in several cell lines committed to the monocytic lineage (THP-1 and U-937 cells). In contrast, KLF4 is not expressed in the uncommitted bipotential cell line HL-60. These cells can differentiate along the monocytic or the granulocytic pathway when treated with 12-O-tetradecanoyl phorbol-13 acetate (TPA) or retinoic acid (RA), respectively. Therefore, we reasoned that HL-60 cells would provide a useful in vitro system for studying the role of KLF4 in monocytic differentiation. First, we determined whether TPA and RA treatment led to KLF4 expression in HL-60 cells. KLF4 mRNA was undetectable in HL-60 control cells (vehicle alone) and in cells treated with RA, but was markedly induced in cells treated with TPA (Figure 1C). In contrast, CD11b mRNA (a marker of differentiation that does not distinguish monocytes from granulocytes) was induced by TPA as well as RA. Taken together, these observations demonstrate that KLF4 expression is restricted to monocytes but not granulocytes (Figure 1C). KLF4 induces a monocytic phenotype in HL-60 cells To determine whether KLF4 participates directly in monocytic differentiation, we retrovirally infected HL-60 cells with either full-length KLF4 or an empty virus control (EV) and analyzed the cells for various myeloid markers 4 days later. Exogenous expression of KLF4 was verified by Northern and Western analyses (Figure 2A and B). In comparison with EV-infected cells, we noticed a marked induction of the myeloid markers CD11b and CD14 (Figure 2A). Consistent with a monocytic phenotype, KLF4 expressing cells also expressed higher levels of c-fms (Figure 2A). To quantitate the induction of various hematopoeitic cell surface markers, we performed fluorescence-activated cell sorter (FACS) analyses on both EV- and KLF4-infected HL-60 cells. In comparison with EV-infected cells, KLF4 induced monocytic markers for CD11b (81.7 vs 2.6%) and CD14 (75.8 vs 6.5%), while having no effect on the granulocytic marker CD66b or the lymphocytic markers CD3 and CD19 (Figure 2C). As expected, there were no significant differences in the expression of cell surface markers between the GFP-negative cell populations (Figure 2D). Thus, KLF4 induces monocytic but not granulocytic or lymphocytic markers in HL-60 cells. Figure 2.Retroviral overexpression of KLF4 in HL-60 cells promotes features of mature monocytes. HL-60 cells were retrovirally infected with either an empty virus (EV) control or KLF4 construct as described in Materials and methods. (A) Northern blot analysis shows that KLF4 overexpression was capable of inducing a number of myeloid differentiation markers such as CD11b, CD14, PU.1, and c-fms. KLF4 (exo) is exogenous KLF4 mRNA expression. (B) Western blot analysis of exogenous KLF4 protein. (C) FACS analysis was performed on EV or KLF4 transduced cells and revealed high induction for myeloid differentiation markers CD11b (81.7 vs 2.6%; P<0.000002) and CD14 (75.8 vs 6.5%; P<0.00003) in response to KFL4 overexpression. There were no differences using antibodies to CD66b (granulocytes), CD3 (T lymphocytes), or CD19 (B lymphocytes). (D) Percent positivity for each marker in EV or KLF4-overexpressing cells from three independent experiments. (E) Cytospin preparations from EV or KLF4-overexpressing HL-60 cells were stained by Wright–Giemsa staining and viewed at × 100. (F) KLF4 knockdown inhibits HL-60 TPA-induced monocyte differentiation. HL-60 cells were incubated with morpholino oligonucleotide specific to KLF4 or nonspecific (NS) control and then allowed to differentiate in the presence of TPA (100 ng/ml) for 48 h. (G) Marked reduction (∼5-fold, right) of adherent and differentiated HL-60 cells after KLF4 knockdown. Light microscopy (left, × 100) of HL-60 cells after NS or AS-KLF4 incubation as described in panel F. (H) The growth rate of EV or KLF4-infected cells counted over 6 days. (I) Cells overexpressing EV or KLF4 were analyzed for DNA contents. (J) Northern blot analysis shows that KLF4 induces p21WAF1 and inhibits cyclin D1. EtBr, ethidium bromide. Download figure Download PowerPoint Both monocytes and granulocytes bear characteristic morphologic features. To determine if KLF4 overexpression affects cellular morphology, we performed cytospin analyses. As shown in Figure 2E, HL-60 cells infected with KLF4 exhibited marked morphologic changes. In comparison with EV-infected cells, the KLF4-overexpressing cells are larger, with increased cytoplasmic size and smaller more condensed indented nuclei. In addition, KLF4-overexpressing cells bear ruffled edges, are less basophilic, and contain cytoplasmic vacuoles. These characteristics are consistent with a monocytic phenotype in these cells (Collins, 1987). To verify whether KLF4 directly participates in regulating PU.1 and monocytic gene expression, we used morpholino antisense oligonucleotides to knockdown KLF4 expression during HL-60 cell TPA-induced monocytic differentiation. As shown in Figure 2F and G, in comparison with the nonspecific control morpholino, KLF4 knockdown resulted in a significant reduction in PU.1 and monocytic differentiation markers, as well as impairment of cell adhesion and cytoplasmic spreading. Furthermore, KLF4 overexpression induced HL-60 cell G1-growth arrest, p21WAF1, and inhibited cyclin D1 (Figure 2H–J). Finally, we demonstrate that overexpression of KLF4 allows precursor HL-60 cells to become functionally mature monocytes capable of adhering to a stimulated endothelial monolayer and undergoing phagocytosis (Supplementary Figure 1). Taken together, these data indicate that KLF4 is a critical regulator of HL-60 monocytic differentiation. KLF4 transactivates the monocytic CD14 promoter To define the mechanism(s) underlying the ability of KLF4 to induce expression of monocytic markers, we performed transient transfection studies using the proximal −474-bp CD14 in HeLa cells. We observed an ∼30-fold induction of the CD14 promoter by KLF4 (Figure 3A). In contrast, KLF4 repressed the smooth muscle cell-specific promoter SM-α-actin (Figure 3A). To assess whether other KLF family members are capable of activating a myeloid promoter, we cotransfected KLF4, KLF2/LKLF, KLF5/IKLF, or KLF15 with the monocytic-specific CD14 promoter. As demonstrated in Figure 3B, KLF4 induced the CD14 promoter by 35-fold, whereas KLF2, KLF5, or KLF15 transactivated the CD14 promoter no more than the ZF domain of KLF4 alone. Finally, the induction by KLF4 required both the ZF DNA-binding domain of KLF4 as well as the non-ZF domain (aa 1–388), as each construct alone had little effect on the CD14 promoter (Figure 3B). Collectively, these data suggest that in comparison with several other KLF family members, KLF4 is able to promote the monocytic differentiation marker CD14 and this requires intact KLF4. Figure 3.KLF4 transactivates the monocytic CD14 promoter and binds to DNA through KLF sites. Transient transfection experiments were performed with either 0.5 μg of pcDNA3 or KLF4, along with the respective promoter-luciferase reporter constructs in HeLa cells. Relative luciferase values are reported after correcting for β-galactosidase. (A) KLF4 induces the CD14 promoter, whereas it represses the non-myeloid smooth muscle (SM) α-actin promoter. (B) Transient transfection studies were performed comparing KLF4, KLF4 DNA-binding domain only (ZnF-KLF4), and several other KLF family members (KLF2, KLF5, and KLF15). Only full-length KLF4 and not other KLFs can transactivate the monocytic CD14 promoter. (C) Loss of the proximal and distal KLF sites results in marked reduction of KLF4 induction of CD14 promoter. (D) Electrophoresis mobility shift assays (EMSAs) were performed using GST or GST-KLF4-Flag-purified protein on the proximal and distal KLF DNA-binding sites. A specific band (arrow) for KLF4 demonstrates binding only to a radiolabeled oligonucleotide probe containing either the wild-type KLF proximal (−92 to −82) or distal (−288 to −278) site, but not to a mutant site, and may be supershifted in the presence of an α-Flag antibody. Download figure Download PowerPoint KLF4 induces the CD14 promoter through DNA binding To better define how KLF4 may induce monocytic-specific markers, we analyzed the −474-bp CD14 promoter, as it is active almost exclusively in monocytes/macrophages. Analysis of this promoter region revealed two potential KLF4-binding sites (open boxes, Figure 3C). One of these sites (−288 to −278) contains three partially overlapping KLF-binding sites. A 5′ deletion downstream to this site (−277-bp CD14 promoter) revealed a ∼48% decrease in KLF4 transactivation (data not shown). Site-directed mutation of the proximal (−88 to −83) KLF site within the −474-bp CD14 promoter also resulted in a ∼61% reduction of activity by KLF4 (Figure 3C). However, when the two KLF sites were mutated within the −474-bp CD14 promoter, we found that there was an ∼84% reduction in activity by KLF4 (Figure 3C). These data suggest that KLF4 can transactivate the CD14 promoter by binding to KLF sites. Members of the Kruppel-like family bind to specific DNA elements (5′-CNCCC-3′) to exert their function. To assess the ability of KLF4 to bind DNA within the −474-bp CD14 promoter, we performed electrophoretic mobility shift assays using Flag-tagged GST-KLF4 and a radiolabeled oligonucleotide probe containing the KLF sites (−288 to −278) and (−92 to −82) of the −474-bp CD14 promoter. As shown in Figure 3D, in comparison to GST alone, incubation of GST-KLF4 resulted in a dominant DNA–protein complex (arrow) that bound to each of these sites. These complexes are specific as they cannot bind to mutated radiolabeled probes and can be supershifted with an α-Flag antibody. Thus, KLF4 is able to bind to KLF sites within the −474-bp CD14 promoter. Expression of KLF4 mRNA in primary bone marrow-derived myeloid progenitor cells To assess KLF4 expression in primary progenitor cells, we isolated several populations of myeloid progenitors or HSCs by multicolor FACS system, as described previously (Akashi et al, 2000; Miyamoto et al, 2002). As shown in Figure 4A (left), real-time PCR analyses revealed that KLF4 mRNA expression progressively increased from the HSC to the GMP stage, whereas it decreased in MEPs. Expression of the Ets transcription factor PU.1 also increased in a similar manner as KLF4 from the HSC to the GMP stage, with weaker expression in MEPs (Figure 4A, right). These findings raise the possibility that KLF4 may participate in regulating myeloid differentiation. Figure 4.Enforced KLF4 instructs CMPs to preferentially induce monocyte differentiation. (A) Stage-specific expression of endogenous KLF4 during myeloid differentiation. qPCR analysis for KLF4 (left) or PU.1 (right) was performed on mouse bone marrow-derived myeloid progenitors (HSC, hematopoietic stem cell; CMP, common myeloid progenitor; GMP, granulocyte macrophage progenitors; MEP, megakaryocyte erythrocyte progenitors). (B–F) Transduction of CMPs or HSCs. Bone marrow-derived CMPs or HSCs were isolated, transduced with control (EV), KLF4, or PU.1 retrovirus as indicated, sorted for GFP positivity, and assessed for differentiation in methylcellulose colony assays. Approximately 3–5% of cells were GFP positive (B). (C) Effect of KLF4 overexpression in CMPs on various hematopoietic lineages identified based on morphology. Control retrovirus-infected cells demonstrated a spectrum of all the various myeloid lineages, whereas KLF4-overexpressing cells exhibited predominant monocytic differentiation. (D) Morphology of KLF4-overexpressing CMPs. Light microscopy (top) and Wright–Giemsa staining (bottom) show that KLF4-transduced cells exhibit morphologic characteristics of monocytes. (E) PU.1 overexpression in CMPs promotes both monocytic and granulocytic differentiation. (F) KLF4 overexpression in HSCs promotes monocytic differentiation, whereas PU.1 promotes both monocytic and granulocytic differentiation. Data are representative of three independent experiments and the same results were obtained. Download figure Download PowerPoint KLF4 overexpression restricts CMPs or HSCs to the monocytic lineage Because KLF4 expression is induced in a stage-specific manner during primary myeloid differentiation, we hypothesized that overexpression of KLF4 in CMPs may promote monocytic differentiation at the expense of other lineages. To address this, we retrovirally infected CMPs with either EV (Ctrl) or KLF4, isolated the GFP positive cells (Figure 4B), and allowed them to grow in methylcellulose medium in a cocktail of cytokines capable of differentiating the CMPs along each of the lineages after 5 days of culture. Remarkably, overexpression of KLF4 in CMPs resulted in the preferential commitment and differentiation of nearly all cells to the monocytes/macrophage lineage in comparison with EV (Ctrl) cells (Figure 4C). Furthermore, this was accompanied by a reduction of cells from other lineages (granulocytes, erythrocytes, or megakaryocytes). Light microscopy of KLF4-overexpressing cells demonstrated many adherent cells, and cytospun preparations verified that these cells had enlarged vacuolated cytoplasms with condensed, often indented, nuclei—morphological characteristics typical of mature mo
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