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. 2007 Sep 19;26(18):4138-48.
doi: 10.1038/sj.emboj.7601824. Epub 2007 Aug 30.

The Kruppel-like factor KLF4 is a critical regulator of monocyte differentiation

Mark W Feinberg  1 , 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
Affiliations

The Kruppel-like factor KLF4 is a critical regulator of monocyte differentiation

Mark W Feinberg et al. EMBO J. .

Abstract

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.

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Figures

Figure 1
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.
Figure 2
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.
Figure 3
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.
Figure 4
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.
Figure 5
Figure 5
KLF4 rescues monocyte differentiation in PU.1-null cells and is a PU.1 target gene. (A) Northern and Western blot analyses demonstrate absence of KLF4 expression in PU.1−/− fetal liver cells (Anderson et al, 2001, 1999) in comparison to wild-type (WT) macrophages. (B) Retroviral overexpression of KLF4 in PU.1−/− fetal liver cells induces myeloid differentiation markers CD11b and CD45 to levels achieved by PU.1 itself. (C) Semi-quantitative RT–PCR analysis shows that KLF4-overexpressing cells express M-CSFR and not G-CSFR. In contrast, PU.1-overexpressing cells induce both M-CSFR and G-CSFR. HPRT is shown as a loading control. (D) Northern blot analysis of PU.1 overexpression in HL-60 cells induces KLF4 mRNA expression. EtBr, ethidium bromide. (E) PU.1 induces the −1.0 kb KLF4 promoter ∼15-fold, whereas mutation of a putative-PU.1 DNA-binding site markedly decreases PU.1 transactivation. (F–G) PU.1 can bind to the PU.1 site in the KLF4 promoter, as verified by (F) electrophoretic mobility shift assay (EMSA) and by ChIP studies (G).
Figure 6
Figure 6
KLF4 deficiency in CMPs or HSCs decreases monocyte and increases granulocyte differentiation. (A–D) Clonogenic analyses of CMPs or HSCs from Klf4loxP mice. CMPs were transduced with retrovirus carrying Cre recombinase or EV (Ctrl). GFP-positive CMPs or HSCs were isolated 36 h after the retroviral infection and subjected to methylcellulose assays for 7 days. Cre-mediated excision of KLF4 (B, D) corresponded with a reduction in monocytes and increase in granulocytes (A, C). Data are representative of three independent experiments, and the same results were obtained.
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