Transcription factors that regulate monocyte/macrophage ... - CiteSeerX

Transcription factors that regulate monocyte/macrophage differentiation Annabel F. Valledor, Francesc E. Borra`s, Martin Cullell-Young, and Antonio Celada Departament de Fisiologia (Immunologia), Facultat de Biologia and Fundacio´ August Pi i Sunyer, Campus de Bellvitge, Universitat de Barcelona, Spain

Abstract: Although all the cells in an organism contain the same genetic information, differences in the cell phenotype arise from the expression of lineage-specific genes. During myelopoiesis, external differentiating signals regulate the expression of a set of transcription factors. The combined action of these transcription factors subsequently determines the expression of myeloid-specific genes and the generation of monocytes and macrophages. In particular, the transcription factor PU.1 has a critical role in this process. We review the contribution of several transcription factors to the control of macrophage development. J. Leukoc. Biol. 63: 405–417; 1998. Key Words: proliferation · development · knockout · gene expression

INTRODUCTION Hematopoiesis consists of a cascade of finely regulated events by which totipotent stem cells differentiate to all the cells present in blood. These include erythrocytes, lymphocytes, megakaryocytes, granulocytes, and monocytes/macrophages. Stem cells are present in bone marrow and have self-renewing capability. In response to specific growth factors, interleukins, and hormones, these cells undergo two sequential differentiating processes. The first is commitment, by which stem cells lose their self-renewing capability and differentiate to other cells with a more limited differentiating potential (which give rise to only one or sometimes two cell lineages). The second is maturation, which allows the terminal differentiation of those cells committed to a specific lineage [1]. Although all the cells in an organism contain the same genetic information, both the commitment and the maturation of hematopoietic cells arise from the gradual expression of lineage-specific genes. The action of critical transcription factors determines the selective expression of cell type-specific genes. These transcription factors can be either expressed constitutively or induced at a certain stage of cell differentiation. In this review we analyze those transcription factors involved in the regulation of monocyte/ macrophage differentiation.

MONOCYTE/MACROPHAGE DIFFERENTIATION TAKES PLACE IN SEQUENTIAL STAGES In bone marrow, interleukins IL-1, IL-3, and/or IL-6 induce heteromitosis in the stem cell. This division gives rise to a new stem cell and a pluripotent myeloid cell, also referred to as granulocyte-erythrocyte-megakaryocyte-macrophage colonyforming unit (GEMM-CFU; Fig. 1). In the presence of IL-1 and/or IL-3, this precursor is committed to becoming a progenitor of both macrophages and granulocytes known as granulocyte-macrophage colony-forming unit (GM-CFU). For this reason, both lineages are closely bound together throughout hematopoiesis and commonly referred to as the myelomonocytic lineage [2, 3]. At this point, IL-3 and granulocytemacrophage colony-stimulating factor (GM-CSF) induce the proliferation of these myeloid precursors, whereas macrophage colony-stimulating factor (M-CSF) induces not only their proliferation but also their differentiation to monocytic precursors. The first cell from a terminal monocytic stage that can be detected easily is the promonocyte, which shows a limited phagocytic capability (Fig. 1). Maturation of promonocytes and the subsequent generation of monocytes also require the presence of M-CSF. Monocytes are generally smaller than their immediate precursors, but they have a well-developed lyso-

Abbreviations: AML, acute myelogenous leukemia; AP-1, activating protein-1; APC, antigen-presenting cell; bHLH, basic region-helix-loop-helix; CBF, core binding factor; C/EBP, CAAT enhancer-binding protein; DIF, differentiation-induced factor; ES cell, embryonic stem cell; GAS, gammainterferon activation site; GEMM-CFU, granulocyte-erythrocyte-megakaryocytemacrophage colony-forming unit; GM-CFU, granulocyte-macrophage colonyforming unit; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN-a, interferon-a; IFN-b, interferon-b; IFN-g, interferon-g; IgG, immunoglobulin G; IL-1, interleukin-1; IL-3, interleukin-3; IL-6, interleukin-6; IRF-1, interferon regulatory factor-1; ISRE, interferon-stimulated response element; LIF, leukemia inhibitory factor; LPS, lipopolysaccharide; M-CSF, macrophage colony-stimulating factor; M-CSFR, macrophage colony-stimulating factor receptor; MHC, major histocompatibility complex; MRE, Myb response element; OSM, oncostatin M; PMA, phorbol 12-myristate 13-acetate; RB, retinoblastoma; SH2, src homology 2. Correspondence: Dr. Antonio Celada, Departament de Fisiologia (Immunologia), Facultat de Biologia (Universitat de Barcelona), Av. Diagonal, 645, 3a planta, 08028 Barcelona, Spain. E-mail: [email protected] Received September 29, 1997; revised December 8, 1997; accepted January 20, 1998.

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Fig. 1. Differentiation of stem cells to monocyte/macrophages. Growth factors involved in each stage are indicated with arrows. Curved arrows indicate the points where derivations to other lineages are generated. The name of each derived lineage is also indicated.

somal system and enhanced phagocytic capability. They usually lose their proliferative capability, although in certain conditions they can still undergo further cell division [1, 2]. Monocytes leave bone marrow and travel through peripheral blood vessels. Once they reach a tissue, they differentiate to macrophages by growing and increasing their lysosomal content, the amount of hydrolytic enzymes and the number and size of mitochondria, and the extent of their energy metabolism. Although they have the same origin, the function of macrophages depends on the tissue in which they reside: in the erythroblast centers in bone marrow they transfer iron to erythroblasts; in spleen they phagocytose erythrocytes; in other tissues they phagocytose microorganisms, cells, and residual products. Dendritic cells, microglia, osteoclasts, Kupffer cells, class A cells in joints, and Langerhans cells are all of the macrophage line. Although they derive from circulating monocytes, tissue macrophages have complete proliferative capability and do not depend on bone marrow monocytes to maintain their population [1, 2].

SEVERAL TRANSCRIPTION FACTORS CONTROL MACROPHAGE DEVELOPMENT The genetic manipulation of a number of cell models and the analysis of knockout mice have led to the identification of several transcription factors that are involved in one or more

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stages of macrophage differentiation. Two important groups can be established on the basis of these analyses. First, those transcription factors that are necessary for macrophage development either because they directly control the differentiation of myeloid cells, as do PU.1 and AML1, or because they are essential for the survival of stem cells and/or pluripotent myeloid precursors, like GATA-2, SCL, and c-Myb. Second, those transcription factors that activate or repress the expression of crucial genes, although there is no evidence that they are essential for macrophage development. These include NF-M/C/ EBPa, HOXB7, and c-Myc, which regulate intermediate stages of myeloid differentiation, and C/EBPb, EGR-1, IRF-1, NF-Y, and some Jun/Fos and Stat proteins, which control macrophage maturation. The down-regulation of some transcription factors contributes to the establishment of the macrophage lineage. The transcription factors involved in the maintenance of the stem cell population are repressed at early stages of myeloid differentiation, and some of those that participate in intermediate stages of myelopoiesis, e.g., c-Myc and c-Myb, are downregulated during macrophage maturation.

PU.1 IS ESSENTIAL FOR MACROPHAGE DEVELOPMENT The transcription factor PU.1 (Table 1) is the product of the Spi-1/Sfp-1 gene, a member of the ets proto-oncogene

TABLE 1. Transcription factor

PU.1 GATA-1 and GATA-2

SCL

c-Myb

Transcription Factors that Control Monocyte Development

Macrophages B lymphocytes Mast cells, neutrophils Pluripotent precursors IL-3-dependent myeloid cell lines Erythroid cells Megakaryocytes Mast cells, neutrophils Pluripotent precursors IL-3-dependent myeloid cell lines Erythroid precursors

c-Myc

Immature erythroid, myeloid, and lymphoid cells Smooth muscle Myeloid cells Hepatocytes Adipocytes Hematopoietic cells Nervous system Skeletal muscle Reproductive tissues Ubiquitous

HOXB7

Myeloid cells

EGR-1 Jun/Fos family Stat family IRF-1

Myeloid cells Ubiquitous Several cell types Myeloid cells

NF-Y

Ubiquitous

C/EBP family AML1

Putative expression pattern during monocyte development

Tissue expression

Specific function

During commitment of GM-CFU to M-CFU During maturation In pluripotent precursors, before myeloid commitment Repressed during the rest of macrophage differentiation

Transactivation of M-CSFR, GM-CSFR, FcgRI/IIIA, CD11b, CD18, CD14, Scavenger receptor I/II

During commitment of pluripotent precursors to GM-CFU Repressed during the rest of macrophage differentiation During commitment of GEMM-CFU to the monocyte lineage Repressed during maturation During commitment of GEMM-CFU to the monocyte lineage During maturation During commitment of GEMM-CFU to the monocyte lineage During maturation

Repression is necessary for induction of CD11b, lysozyme, morphological changes, inhibition of proliferation

During commitment of stem cells to the monocyte lineage During the first stages of maturation Repressed before the end of maturation During commitment of GM-CFU to the monocyte lineage During maturation During maturation During maturation During maturation During maturation of promonocytes

Repression of C/EBPa/b

During monocyte-to-macrophage differentiation

family [4, 5]. It binds to the DNA sequence GAGGAA (PU box) [4]. The central core of this sequence (GGAA) is typically recognized by members of the ets proto-oncogene family [6]. Nucleotides flanking this central core are involved in specificity- and affinity-related events, thus preventing distinct ets proteins from competing for the same promoter region [7]. PU.1 contains the following three functional domains: (1) the transactivating domain [8], a proline-rich region in this domain facilitates the formation of intramolecular bounds and the interaction with other proteins [9]; (2) the PEST domain (proline-, glutamic acid-, serine-, and threonine-rich region), which is a target for endopeptidases [10]. This is believed to account for the rapid turnover of the protein. In this domain there are also several serine-phosphorylation sites that determine conformational changes of PU.1 and mediate its interaction with other proteins. Indeed, phosphorylation of serines 41 and 45 is necessary for the role of PU.1 in macrophage proliferation (see below), whereas phosphorylation of serine 148 is required for the interaction of PU.1 with other proteins in B cells [11, 12]; (3) the DNA-binding domain, also known as the ets domain, which is a consensus motif present in all the ets

Transactivation of mim-1 and lysozyme Repression of M-CSFR Transactivation of M-CSFR, GM-CSFR, mim-1, and lysozyme Transactivation of M-CSFR

Repression of c-myc and c-myb Transactivation of IFN-b Inhibition of proliferation Transactivation of Ferritin H-chain and MHC class II

family members [13]. The crystal structure of the PU.1 ets domain in complex with DNA has recently been determined. This domain is similar to an a 1 b (winged) helix-turn-helix motif, but it has a novel loop-helix-loop architecture [14, 15]. Among hematopoietic cells, only a few mature cell types express PU.1. These include monocytes/macrophages, B lymphocytes, mast cells, and neutrophils [16, 17]. Mice carrying a mutation in the PU.1 locus were obtained by two independent groups. In one of the reports, individuals homozygotic for the mutation (knockout mice for PU.1) died at a late gestational stage. They had a defect in multiple hematopoietic lineages: macrophages, granulocytes, B cells, and T cells [18]. In the second report, PU.1 knockout mice were born alive and died of severe septicemia within 48 h. The analysis of these neonates revealed a lack of mature macrophages, neutrophils, B cells, and T cells. However, mice maintained in antibiotics survived up to 2 weeks and developed normal T cells and neutrophils. In contrast, these mice were deficient in macrophages and mature B cells [19]. Embryonic stem (ES) cells with the PU.1 gene disrupted were also analyzed. ES cells are a totipotent cell line that, in appropriate conditions in vitro, can differentiate to the

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myeloid and erythroid lineages. The loss of function of PU.1 in ES cells resulted in an intrinsic defect that inhibited normal macrophage development. In liquid cultures, though, a small number of cells had a macrophage-like morphology, suggesting that commitment to the myeloid lineage was occurring, but survival and/or proliferation of these cells was blocked. The expression of early myeloid genes, such as the GM-CSF receptor, but not that of genes associated with terminal differentiation, such as the M-CSF receptor, CD11b and FcgRI, was detected in PU.1-defective ES cells [20, 21]. All these results suggest that, although PU.1 is not essential for commitment to the myeloid lineage, it is required for normal differentiation of myeloid cells to macrophages. During myelopoiesis, differentiation to monocyte/macrophages can only take place efficiently if the cells respond to the macrophage-specific growth factor M-CSF. The expression of the receptor for this growth factor (M-CSFR) is thus essential for macrophage differentiation. This receptor, which is the product of the proto-oncogene c-fms, is a transmembrane glycoprotein with ligand-induced tyrosine kinase activity [22, 23]. PU.1 binds to a purine-rich sequence in the promotor of the c-fms gene, although this sequence is not identical to a typical PU box [24]. In murine bone marrow-derived macrophages, inhibition of PU.1 with an antisense construct impaired both the expression of the receptor for M-CSF and the consequent M-CSFdependent proliferation. In addition, overexpression of PU.1 increased the M-CSF-dependent proliferation of these cells [12]. Furthermore, treatment of human bone marrow-derived cells (enriched for the immaturity marker CD34) with PU.1 antisense oligodeoxynucleotides blocked their myeloid differentiation [25]. Taken together, these results suggest that PU.1 is a critical transcription factor for macrophage differentiation, survival, and proliferation because it controls the expression of the M-CSF receptor. PU.1 also transactivates other genes involved in the acquisition of a mature monocyte phenotype. This allows monocytes/ macrophages to carry out their immunological functions when an activation signal is generated. For instance, PU.1 controls the expression of genes that code for FcgRI [26] and FcgRIIIA [27], the high- and low-affinity receptors, respectively, for the constant region of immunoglobulin G (IgG). Once expressed on the cell surface, these molecules allow the macrophage to recognize and phagocytose IgG-opsonized bacteria. PU.1 also induces the expression of the adhesion molecules CD11b [28], CD18 [29], and CD14 [30] during myeloid differentiation. CD18 is the b chain of integrins and, when associated with CD11b, they both constitute the membrane glycoprotein Mac-1, which mediates the adhesion of monocytes to endothelium and subsequent diapedesis, and the phagocytosis of complementopsonized particles. CD14 is a membrane glycoprotein that specifically binds to lipopolysaccharide (LPS), a structural component of the bacterial wall. This recognition is a crucial step in triggering the microbactericidal function of the macrophage. Moreover, PU.1, along with other members of the ets family, mediates the expression of Scavenger receptors type I and II [31]. Expression of these molecules is restricted to cells of a monocytic origin and is maximal during the terminal

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differentiation of monocytes to macrophages. Scavenger receptors are involved in the capture and subsequent degradation of proteins that have been chemically modified at inflammation sites. A common feature of all these genes is the absence of TATA boxes in their promoters. It has been postulated that in most of these cases PU.1 can recruit factors, such as TFIID, that normally bind to TATA boxes and form part of the transcriptional initiation complexes [32]. In conclusion, the expression of PU.1 is essential for macrophage differentiation. This requirement may be determined by the capability of PU.1 to induce the expression of the M-CSF receptor. Without expression of this receptor on the cell surface, monocytic precursor cells cannot recognize their specific growth factor, and the survival, proliferation, and maturation of these cells are drastically impaired. This also explains the absence of macrophages in the PU.1 knockout mice. Moreover, PU.1 also controls the expression of a number of molecules that mediate some of the immunological actions of macrophages.

GATA-1, GATA-2, AND PLZF REGULATE THE MAINTENANCE OF THE STEM CELL POPULATION GATA-1, GATA-2, and PLZF are members of the zinc finger family of transcription factors. High expression of these proteins is detected in totipotent precursor cells before they undergo commitment, and also in purified CD341 progenitor cells. Expression of PLZF declines as stem cells are committed to any hematopoietic lineage, whereas expression of GATA-1 and GATA-2 remains high during the development of erythrocytes, megakaryocytes, and mast cells and is specifically repressed during the differentiation to the monocyte lineage [33, 34]. In mice lacking GATA-2 there was an absence of macrophages and other hematopoietic lineages, indicating that it is required for the survival of stem cells or pluripotent precursors [35]. Myb-Ets-transformed chicken hematopoietic progenitors differentiate to myeloblasts, erythrocytes, thromboblasts, and eosinophils. Constitutive expression of GATA-1 in these cells suppressed the expression of monocyte markers and induced reprogramming of myeloblasts to cells resembling eosinophils or thromboblasts [36]. Indeed, GATA-1 is known to transactivate the expression of several genes specific for the erythroid, megakaryocytic, and mast cell lineages [36, 37]. In conclusion, distinct members of the zinc finger family of transcription factors control the survival of stem cells or pluripotent precursors. However, there are marked differences between their actions. PLZF contributes to the survival of stem cells and blocks the general commitment of these cells. GATA-1 and GATA-2, apart from controlling the survival of stem cells, constitute lineage-determining transcription factors, which not only switch on genetic programs that lead to the generation of erythrocytes, megakaryocytes, and mast cells but also block macrophage differentiation.

SCL AND E2A CONTROL THE EARLY COMMITMENT OF STEM CELLS

c-Myb DETERMINES THE EXPRESSION OF MYELOID-SPECIFIC GENES AND REGULATES EARLY MYELOID DIFFERENTIATION

The SCL gene (Table 1), also known as Tal-1 or TCL-5, codes for a basic helix-loop-helix (bHLH) transcription factor. In immature hematopoietic cells, SCL is expressed in totipotent non-committed progenitor cells, in CD341 cells, and in early erythroid precursors [33, 38]. SCL knockout mice died in utero as a result of the absence of blood cells. A marked reduction in the erythroid and myeloid cell populations was observed when embryonic sac-derived cells from SCL knockout mice were cultured in vitro [39]. In addition, ES cells lacking SCL did not produce any hematopoietic lineage [40]. These observations suggest that SCL is required for the survival of stem cells or myelo-erythroid progenitor cells (GEMM-CFU). However, several lines of evidence indicate that, like GATA proteins, repression of SCL expression is required for further monocyte differentiation. First, TF-1 is a bipotent cell line that can differentiate to either erythrocytes or monocytes. Expression of SCL remains high during the erythroid differentiation of TF-1 cells, whereas it is severely down-regulated during monocyte differentiation. Stable transfection with SCL induced erythroid differentiation of TF-1 cells and interfered with monocyte development [38]. Second, stable hematopoietic hybrid cells can be obtained by cell fusion experiments. However, several attempts to fuse SCL-expressing erythroid cells with a myeloid WEHI3B/D1 cell line were unsuccessful [33], perhaps due to the inhibitory effect of SCL expression on myeloid cells. Third, Elwood et al. [41] were unable to express the full-length SCL protein in the WEHI3B/D1 cell line. Finally, constitutive expression of SCL in M1 myeloblastic leukemia cells impaired some, but not all, of the features associated with monocyte differentiation induced by leukemia inhibitory factor (LIF) or oncostatin M (OSM): the expression of CD11b, morphological changes, and the inhibition of proliferation [42]. Expression of SCL can thus selectively interfere with certain events during the differentiation to macrophages. Furthermore, E2A proteins are ubiquitous transcription factors that also belong to the bHLH family. They form heterodimers with SCL in undifferentiated myeloid cells. These heterodimers bind to the DNA sequence CANNTG (also called E box), where N represents any nucleotide, and are believed to block the transcriptional activation of any promoter containing this sequence [43]. When M1 cells are stimulated with IL-6 they undergo terminal macrophage differentiation. This is associated with a marked decrease in SCL expression and early suppression of the binding of these heterodimers to DNA [43]. It has been suggested that this blockage is caused by the high expression of Id1 and Id2, two dominant negative members of the HLH family that are induced by IL-6 in these cells. These proteins lack the basic DNA-binding region before the HLH domain and dimerize with E2A proteins [44], thus inhibiting the DNA binding of E2A-SCL heterodimers [43]. This mechanism may allow the expression, during terminal macrophage differentiation, of myeloid-specific genes initially repressed by E2A-SCL heterodimers.

c-myb was initially identified as the cellular homologue of two viral v-myb genes [45, 46]. It was later demonstrated that c-myb could also be a proto-oncogene associated with murine myeloid neoplasias [47, 48]. Myb proteins contain a unique DNAbinding domain, characterized by two or three imperfect 52-amino acid-long repeats (R1, R2, and R3). All these repeats form alpha-helix-like structures similar to those observed in HLH motifs. In addition, R2 and R3 repeats resemble basic domains [45] and are essential to recognize the consensus DNA sequence PyAAC(G/T)G [47], also known as MRE (Myb response element) [49]. Very close to the transactivation domain, there is a negative regulatory domain [50]. Changes in the phosphorylation state of c-Myb may regulate its activity on distinct promoters [51, 52]. In addition, c-Myb-specific transactivation may be mediated by the direct interaction of c-Myb with the nuclear co-activator CBP [53]. Several lines of evidence indicate that c-Myb regulates the initial stages of myelopoiesis. First, in subpopulations of normal human bone marrow-derived cells, c-Myb was mainly detected in immature myeloid precursors (Table 1) [54]. Second, c-Myb anti-sense oligodeoxynucleotides inhibited both erythroid and myeloid colony-forming units [55]. Third, mice lacking c-Myb died in utero after day 14 due to severe disruption of erythroid and myeloid development [56]. Expression of c-Myb is thus required for survival or proliferation of myeloid pluripotent precursors (GEMM-CFU). However, as with GATA proteins and SCL, expression of c-Myb is severely inhibited during terminal macrophage differentiation [57]. Enforced expression of an active form of c-Myb in hematopoietic cells derived from murine fetal liver resulted in the abnormal persistence of myeloid colony-forming units (GM-CFU) [58]. Furthermore, when HL60 myeloblastic leukemia cells were induced to differentiate to macrophages through the use of phorbol ester or vitamin D3, c-Myb expression disappeared well before cell proliferation was blocked. This suggests that inhibition of c-Myb does not take place only as a consequence of a decreased proliferation rate associated with monocytic maturation [59]. Three independent studies are consistent with this hypothesis. First, the expression of c-Myb was quickly inhibited in M1 myeloblastic leukemia cells induced to terminal differentiation with either IL-6 or LIF [60]. Second, the constitutive expression of c-Myb in murine bone marrow-derived cells blocked their ability to differentiate to mature macrophages [61]. And third, mature macrophages underwent dedifferentiation after transfection with v-myb oncogene [62]. On the basis of all these results, c-Myb appears to have two marked effects in macrophage development. First, a positive regulation of genes involved in the induction of proliferation of immature myeloid cells at the same time incompatible with terminal differentiation. Second, a negative regulation of genes involved in the induction of terminal differentiation. Indeed, opposing actions have been described for c-Myb and PU.1 in the control of transcription from the c-fms promoter in macro-

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phages. Although PU.1 acts as a transactivator, as described above, c-Myb acts as a repressor of the same promoter [63]. This is consistent with the inhibition of c-Myb expression during terminal monocyte differentiation [57, 58], as surface expression of the M-CSF receptor is essential for this process. Finally, the expression of mim-1, a myeloid-specific gene that has recently been cloned [62], is specifically regulated by c-Myb in cooperation with other transcription factors, such as NF-M [64] and ets-2 [65]. The exact function of mim-1 in myeloid cells remains undetermined.

C/EBP PROTEINS: DIFFERENT MEMBERS HAVE SPECIFIC FUNCTIONS DURING MACROPHAGE DEVELOPMENT CCAAT enhancer-binding proteins (C/EBP) constitute a family of transcription factors with a basic region-leucine zipper structure. The basic region interacts directly with DNA, whereas the leucine zipper domain forms an a-helical coil and is directly involved in homo/heterodimerization [66, 67]. In the hematopoietic system, expression of C/EBP proteins is limited to the myeloid lineage [68, 69]. However, not all the members of this family are expressed at the same time. C/EBPa is mainly expressed in undifferentiated pluripotent myeloid cells and gradually decreases with macrophage maturation, whereas expression of C/EBPb (also known as NF-IL-6, LAP, IL-6DBP, or AGP-EBP) and C/EBPd is up-regulated during macrophage maturation [68, 70]. NF-M, the chicken homologue of mammalian C/EBPb, is also rapidly induced and activated by phosphorylation during the monocyte differentiation of avian pluripotent myeloid precursors [71, 72]. C/EBPa regulates transcription from the c-fms promoter [73]. This transcription factor specifically binds to the region IIA, which is located upstream from the PU box (Fig. 2) and is critical for the c-fms promoter activity. The region II functions as a monocyte-specific enhancer [74]. In addition, C/EBPa also cooperates with PU.1 in controlling the expression of the GM-CSF receptor [75]. Thus, C/EBPa seems to regulate monocyte development by enhancing the transcriptional activation of the receptors for M-CSF and GM-CSF. Considering that C/EBPa is primarily expressed in undifferentiated pluripotent myeloid cells, the enhancement of the expression of the

Fig. 3. Model for the synergism between transcription factors c-Myb and NF-M. The model suggests that both c-Myb and NF-M recruit the coactivator CBP. Once recruited to the promoter, CBP may affect the transcriptional machinery by interacting with TFIIB. NF-M BS, NF-M binding site; MRE, Myb response element.

GM-CSF and M-CSF receptors may contribute to the commitment of these cells to the monocyte lineage. On the other hand, production and differentiation of macrophages is not blocked in C/EBPb knockout mice. However, activation of these cells is severely impaired [76]. This raises the possibility that C/EBPb controls certain maturation stages that provide macrophages with a functional activation potential. In accordance with this hypothesis, C/EBPb/NF-M cooperates synergistically with c-Myb in transactivating the expression of lysozyme and mim-1 (see above). Ectopic expression of c-Myb and C/EBPb in erythroid cells or fibroblasts was sufficient for the ectopic induction of mim-1 and lysozyme [69, 77]. Because the sequences recognized by these transcription factors are very close to each other in the mim-1 promoter, recruitment of the co-activator CBP by both transcription factors can be the cause of the synergistic activation of gene transcription [53, 78] (Fig. 3). Members of the C/EBP family other than C/EBPa can also bind to region IIA in the c-fms promoter [74], thus suggesting that C/EBPb, and perhaps C/EBPd, may contribute to the expression of the M-CSF receptor at late stages of monocyte maturation. Moreover, in U937 cells induced with PMA to differentiate to mature macrophages, the nuclear protein retinoblastoma (Rb) interacted directly with C/EBPb, thus enhancing the DNA-binding capability and transcriptional activity of C/EBPb [79]. In conclusion, different members of the C/EBP family have distinct functions in macrophage development. Although C/ EBPa regulates commitment to the monocyte lineage, C/EBPb/ NF-M controls macrophage maturation, most probably by endowing these cells with a functional activation potential.

AML1: ITS COMBINED ACTION WITH C/EBPa AND PU.1 IN THE CONTROL OF MACROPHAGE DIFFERENTIATION

Fig. 2. Schematic representation of the c-fms promoter. Region II functions as a monocyte-specific enhancer. It can be divided in the overlapping subregions A and B. C/EBP BS, C/EBP binding site, which is located in region IIA; AML1 BS, AML1 binding site, which is located in region IIB; PU box, PU.1 binding site.

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AML1 belongs to the core binding factor (CBF) family. All members of this family are heterodimers of a DNA-binding a-subunit (CBFa) and a b-subunit (CBFb). The AML1 gene codes for a CBFa subunit. CBFa molecules contain a region homologous to the Drosophila runt protein, a pair-rule gene involved in segmentation. The runt homology domain (rhd) confers AML1 the ability to bind to its DNA consensus site,

TGT/CGGT. CBFb does not bind to DNA directly, but enhances the DNA-binding rate of the a-subunit [80]. The expression of AML1 is not restricted to myeloid cells [81] and AML1 knockout mice show impaired development of all hematopoietic lineages [82]. In monocytic cells, AML1 recognizes a region in the c-fms promoter, region IIB, which is located between the region IIA and the PU box. The AML1/CBFb heterodimer cooperates synergistically with C/EBPa to activate this promoter and the distance between regions IIA and IIB is critical for this cooperation, thus suggesting that physical interaction between the AML1 and C/EBPa proteins is necessary [74] (Fig. 2). AML1 is believed to facilitate the action of other adjacent transcription factors, such as PU.1. The fact that the AML1binding site is located between those of C/EBPa and PU.1 in the c-fms promoter is consistent with this hypothesis [74]. In conclusion, the simultaneous expression of PU.1, C/EBP proteins, and AML1 during myelopoiesis leads to the myeloidspecific expression of the M-CSF receptor. The abnormal expression of any of these factors or the alteration of their functions may lead to aberrant expression of the M-CSF receptor. Indeed, the AML1 gene is involved in an (8/21) translocation frequently associated with certain types of acute myelogenous leukemia (AML). The (8/21) translocation product synergized with the normal transactivational activity of AML1. Expression of the M-CSF receptor was markedly increased at all stages of myeloid differentiation in many AML patients [83]. The overexpression of the M-CSF receptor, and especially its premature expression in early progenitors, may thus contribute to leukemogenesis in some types of AML.

normal conditions. The M1 cells that constitutively expressed c-Myc presented some features from an intermediate myeloid differentiation state and also maintained their proliferative capability in the absence of c-Myb [60]. All these results suggest that, like c-Myb, inhibition of c-myc expression does not take place as a consequence of the blockage of myeloblastic proliferation. Indeed, a novel function has been recently described for v-Myc and c-Myc in myelomonocytic cells; they down-regulate the expression of C/EBPa and b as well as inhibit the C/EBPa- and b-mediated transactivation of certain genes (e.g., mim-1 and lysozyme) [89]. It seems probable that suppression of c-Myc is necessary for the induction of C/EBPb, and perhaps that of d, during monocyte maturation [74]. In addition, c-Myc may also be responsible for the rapid downregulation of C/EBPa shortly after the induction of monocyte maturation [68]. Because c-Myc and C/EBPa are both expressed during the commitment of undifferentiated pluripotent cells to the monocyte lineage, we wonder whether c-Myc may negatively regulate the enhancing activity of C/EBPa on the expression of the M-CSF and GM-CSF receptors. After a certain stage in monocytic maturation, the terminal differentiation program is no longer dependent on the downregulation of c-Myc. From that point on, the induction of c-myc expression does not have any effect on the phenotype of mature macrophages [60]. Although both c-Myb and c-Myc negatively control terminal differentiation, their windows of action are restricted to a specific period of time during monocyte differentiation.

THE HOMEOBOX FAMILY: HOXB7 IS NECESSARY FOR MYELOMONOCYTE DIFFERENTIATION IN RESPONSE TO GM-CSF

C-MYC IS INVOLVED IN THE NEGATIVE CONTROL OF C/EBP PROTEINS IN MYELOID CELLS c-myc was one of the earliest proto-oncogenes studied. It was initially discovered as the cellular homologue of the viral gene v-myc [84]. The DNA-binding domain of c-Myc contains both a basic region-helix-loop-helix and a leucine zipper motif (b-HLHLeu zip), and is essential to bind to DNA and to interact with other proteins. Before it can recognize its binding site, CACGTG, and activate gene transcription, c-Myc must form a heterodimer with another transcription factor, Max [85, 86]. c-Myc is also able to inhibit gene expression by binding to a weak consensus sequence in the initiator element, TCA (11) YYYNY [87]. Like c-Myb, c-Myc is highly expressed in proliferating myeloblastic cells and is strongly repressed after induction of their terminal differentiation (Table 1). However, the repression of c-myc takes place later than that of c-myb [88]. It should be emphasized, though, that expression of c-myc can be detected in a large number of undifferentiated proliferating cell types and not only in myeloid cells. In M1 cells, c-myc expression is undetectable after 18 h post induction of terminal monocyte differentiation. Constitutive expression of c-myc in M1 cells blocked terminal differentiation at an intermediate stage in the progression of immature myeloblasts to mature macrophages. This is the point after which c-Myc expression is repressed in

Homeobox transcription factors contain a characteristic DNAbinding domain, the homeodomain, which is closely related to HLH domains. The expression of some members of this family, such as HOXB3 and HOXB4, is restricted to the stem cell population. Overexpression of HOXB4 in transplanted murine bone marrow cells resulted in a drastic expansion of stem cells without affecting normal peripheral blood cells [90]. Other homeobox transcription factors, such as HOXB7, participate in intermediate stages of monocyte differentiation. Murine and human bone marrow-derived cells express HOXB7 when stimulated with GM-CSF. HOXB7 antisense oligodeoxynucleotides specifically blocked the formation of granulocyte and macrophage colony-forming units (G-CFU and M-CFU) in response to GM-CSF [91, 92]. In HL60 myeloblasts, HOXB7 was only expressed during monocyte differentiation induced by vitamin D3 but not during macrophage differentiation induced by phorbol ester [92, 93]. Differentiation to a monocyte or to a macrophage phenotype can thus be established according to the pattern of expression of homeobox genes. On the basis of these results, HOXB7 seems to be required for the GM-CSFdependent proliferation and differentiation of early semicommitted cells. HOXB7 may also have a second function in the restriction to the monocyte phenotype during terminal differentiation.

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EGR-1: A ROLE IN THE RESTRICTION OF THE DIFFERENTIATION OF MYELOID PRECURSORS TO THE MACROPHAGE LINEAGE? EGR-1 belongs to the zinc finger family of transcription factors. EGR-1 was initially described in 3T3 fibroblast-derived cells as a growth factor-inducible gene [94] (Table 1). Later, EGR-1 was shown to be specifically induced as an immediate early gene during the differentiation of HL60 cells to macrophages rather than granulocytes [95]. The expression of EGR-1 is also induced in murine peritoneal macrophages and peripheral blood monocytes stimulated with GM-CSF or M-CSF and during the macrophage differentiation of U937 and M1 cells. In all cases, the maximal expression is found in mature cells [60]. Although EGR-1 is specifically expressed in macrophages, there are controversial results about its function. On one hand, the analysis of EGR-1 knockout mice demonstrated that it was not essential for macrophage differentiation. Macrophages were detected in peripheral blood and several tissues and they could be activated normally. Besides, no differences in myeloid cell differentiation were observed in the presence of M-CSF, GM-CSF, or G-CSF [96]. On the other hand, the genetic manipulation of myeloid cell lines gave EGR-1 a deterministic role in the development of macrophages. In the first place, EGR-1 antisense oligodeoxynucleotides blocked the ability of HL60 cells, U937 cells, and myeloblast-enriched bone marrowderived cells to differentiate to monocyte/macrophages. In addition, the constitutive expression of EGR-1 in HL60 cells blocked their ability to differentiate to granulocytes but did not affect monocyte differentiation [60]. In response to G-CSF, IL-3-dependent 32Dcl3 hematopoietic precursor cells undergo granulocyte differentiation. Ectopic expression of EGR-1 blocked terminal granulocytic differentiation of 32Dcl3 cells induced with G-CSF and endowed these cells with the ability to differentiate exclusively to the macrophagic lineage in response to GM-CSF [97]. Taken together, these results raise the possibility that EGR-1 promotes and restricts the differentiation of myeloid precursor cells to the monocytic lineage. If this is true, the results obtained from the analysis of knockout mice may indicate that EGR-1 is a dispensable factor and other redundant proteins can replace it in vivo.

MEMBERS OF THE JUN/FOS SUPERFAMILY PARTICIPATE IN MACROPHAGE MATURATION All members of the proto-oncogene fos/jun superfamily code for transcription factors with a leucine zipper domain, which is necessary for dimerization [98]. Dimers composed of Jun/Fos proteins are collectively named AP-1 (activating protein-1) and bind to a common DNA site [99]. Jun proteins can form dimers either with other Jun proteins or with c-Fos, whereas Fos proteins only dimerize with Jun proteins. The expression of three members of this superfamily, c-Jun, JunB, and JunD, is immediately induced during the monocytic differentiation of M1 cells [60]. The constitutive expression of c-Fos and, to a lesser extent, that of JunB increased the ability of M1 cells to differentiate to monocytes and reduced the 412



aggressiveness of the leukemic phenotype of these cells when transplanted to nude mice. Besides, c-Fos antisense oligodeoxynucleotides inhibited the differentiation of both normal myeloblasts and c-Fos-transfected M1 cells to mature macrophages [100]. All these observations indicate that some members of the Jun/Fos superfamily can contribute to the induction of terminal monocyte differentiation. The mechanisms for such positive control remain to be determined. The fact that mice lacking functional c-Fos do not show impaired monocyte development [101] is not out of keeping with this hypothesis, since different members of the Jun/Fos superfamily may replace c-Fos in the formation of functional AP-1 complexes during monocyte maturation.

THE STAT FAMILY: STAT3 AND DIF TAKE PART IN MACROPHAGE MATURATION The first Stat family members were identified as DNA-binding proteins in interferon (IFN)-regulated gene expression. Later, new Stat-like activities were found to be activated by distinct factors, including differentiating signals. Stat proteins contain a phosphotyrosine-binding SRC homology 2 (SH2) domain. The SH2 domain mediates intracellular signal transduction and is critical for the homo/heterodimerization of the protein and subsequent binding to DNA. The DNA-binding domain recognizes a consensus site, TTCC(G/C)GGAA, present in gammainterferon activation site (GAS) elements [102]. Some members of the Stat family control monocyte differentiation. Dominant negative mutants of Stat 3 blocked the growth arrest and subsequent macrophage maturation of M1 cells stimulated with IL-6. These dominant negative mutants inhibited the IL-6-induced repression of c-Myb and c-Myc expression [103]. This suggests that repression of c-Myb and c-Myc during monocyte maturation may be mediated by the activation of Stat 3. Monocyte differentiation of myeloid cells after stimulation with GM-CSF and M-CSF accompanied the activation of DIF (differentiation-induced factor) [104], a protein related to known isoforms of Stat 5. DIF was also activated after the maturation of U937 promonocytic cells stimulated with phorbol ester [105]. These results suggest that certain members of the Stat family, such as Stat 3 and DIF, may be part of a developmental program that leads to terminal macrophage differentiation. The development of knockout mice for these proteins may be a valuable tool to assess their exact contribution to myelopoiesis.

IRF-1 CONTRIBUTES TO THE LOSS OF PROLIFERATION ASSOCIATED WITH MACROPHAGE MATURATION IRF-1 (interferon regulatory factor-1) belongs to a family of transcription factors that are induced by interferons and have tumor suppressor activity [106, 107]. The members of the IRF family have a conserved DNA binding domain that recognizes the interferon-stimulated response element (ISRE) present in the promoter of many genes regulated by Interferon (IFN) type I (IFN-a and IFN-b) [108, 109].

The expression of IRF-1 can be also induced by cytokines other than IFN-a/b. IRF-1 was rapidly expressed in M1 cells induced with IL-6 or LIF to differentiate to monocytes. It is interesting to note that IRF-1 antisense oligodeoxynucleotides impaired the inhibition of proliferation associated with the terminal differentiation of these cells [60]. Indeed, IRF-1 activates the expression of the genes that code for IFN-a/b [110, 111] (Table 1). These cytokines play an autocrine role in the inhibition of macrophage proliferation. Expression of IFN-b was dependent on protein synthesis and took place after the expression of IRF-1 in M1 cells stimulated with IL-6 or LIF [60]. These results suggest that loss of the proliferative capability associated to terminal monocyte differentiation is determined by events such as the early induction of transcription factor IRF-1 and subsequent expression of the autocrine cytokines IFN-a/b.

NF-Y CONTROLS THE EXPRESSION OF GENES INVOLVED IN THE ACQUISITION OF A FUNCTIONAL MACROPHAGE PHENOTYPE NF-Y, also known as CP1 or CBF, is a ubiquitous heterotrimeric transcription factor formed by subunits NF-YA, NF-YB, and NF-YC, which are necessary for DNA binding. Like C/EBP proteins, NF-Y binds to the sequence CCAAT [112, 113]. Freshly isolated peripheral blood monocytes expressed NF-YB but not NF-YA and did not show detectable NF-Y activity. In contrast, there was an induction of NF-YA synthesis and subsequent NF-Y transcriptional activity during the terminal differentiation of monocytes to macrophages. Binding of NF-Y to the CCAAT box in the ferritin heavy-chain gene promoter and its subsequent transcriptional activation significantly increased during monocyte-to-macrophage differentiation [114]. Thus, the transcription factor NF-Y contributes to the acquisition of a functional macrophage phenotype by inducing the synthesis of ferritin. Mature macrophages are one of the few cell types specialized in iron storage and in the subsequent transfer of iron molecules to the erythroblast centers in bone marrow [1, 2]. Furthermore, NF-Y also controls the expression of MHC class II molecules [115], which are required for presentation of antigens to T cells. The induction of these molecules by IFN-g is one of the key features of mature macrophages.

CONCLUSIONS AND FUTURE PERSPECTIVES Macrophage development is a complex process that depends on specific external signals that modulate the expression or activation of several transcription factors. The combined action of inducible and ubiquitous transcription factors regulates the lineage-specific expression of a number of genes and the subsequent establishment of the macrophage phenotype. Known genes that determine a macrophage phenotype are those that code for the M-CSF receptor, lysozyme, the adhesion molecules CD11b and CD18, the LPS receptor (CD14), the Scavenger receptors, and the receptors for the constant region of IgG. On

the basis of the analysis of knockout mice and the genetic manipulation of cell lines and normal hematopoietic cells, a putative order of the contribution of transcription factors to monocyte differentiation is proposed in this review (Fig. 4). The survival of stem cells is regulated by a combination of transcription factors that include GATA-2, PLZF, SCL, and the homeobox proteins HOXB3 and HOXB4. Although expression of PLZF, and perhaps that of HOXB3 and HOXB4, is downregulated during the commitment of stem cells to pluripotent myeloid precursors (GEMM-CFU), GATA-1, GATA-2, SCL, and c-Myc still take part in this process. However, the expression of these factors must later be repressed to allow monocyte differentiation. SCL also seems to have a marked effect during the commitment to granulocyte/monocyte precursor cells (GMCFU). In this case, SCL may be expressed simultaneously to c-Myb, C/EBP proteins, AML1 and, again, c-Myc. The expression of SCL beyond this stage of differentiation is incompatible with the establishment of a monocyte phenotype, perhaps because the expression of myeloid-specific genes is repressed by SCL-E2A heterodimers. Indeed, constitutive expression of SCL blocks key features associated with monocyte differentiation: the expression of CD11b and lysozyme, the inhibition of proliferation and specific morphological changes. Further studies should be addressed to the analysis of genes repressed by SCL-E2A dimers and their contribution to monocyte differentiation. c-Myb controls the survival and proliferation of granulocyte/ macrophage precursor cells (GM-CFU) and the commitment of these cells to macrophagic precursor cells (M-CFU). In cooperation with C/EBPb, c-Myb activates the expression of myeloidspecific genes such as mim-1 and lysozyme. Efficient survival and proliferation of M-CFU cells, as well as subsequent monocyte maturation, takes place thanks to the expression of the receptor for the macrophage-specific growth factor M-CSF. PU.1 is essential for macrophage development because it activates the expression of the M-CSF receptor. In addition, C/EBPa and the AML1/CBFb heterodimer synergistically enhance the expression of this receptor. C/EBPa also cooperates with PU.1 in the regulation of the receptor for GM-CSF, another relevant growth factor in myelopoiesis. Indeed, the genetic program that leads to monocyte differentiation must be determined by combinations of transcription factors rather than by the action of single proteins. The joint expression of PU.1, C/EBP proteins, and AML1/CFBb during myelopoiesis may thus account for the myeloid-specific expression of the M-CSF and GM-CSF receptors. Besides, c-Myc, which is expressed simultaneously to C/EBPa in myeloblasts, inhibits C/EBPa-mediated transactivating activity. It would be interesting to assess whether this could negatively regulate the enhancing activity of C/EBPa on the expression of the M-CSF and GM-CSF receptors. Indeed, the overexpression of the M-CSF receptor in immature myeloid cells may cause cell transformation. During the generation of monocytic precursor cells (M-CFU) and the subsequent maturation of these cells, PU.1 also transactivates other genes involved in the acquisition of a functional macrophage phenotype. Some of these genes code for the adhesion molecules CD11b and CD18, the LPS receptor (CD14), the high- and low-affinity receptors for the constant

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Fig. 4. Putative transcription factors involved in the control of the different stages of monocyte differentiation. Transcription factors are depicted in boxes next to the stage referred to. The narrow arrows indicate the points where derivations to other lineages are generated. The name of each derived lineage is also indicated.

region of IgG (FcgRI and III, respectively), and the Scavenger receptors I and II. C/EBPb may also control some of the events that provide macrophages with a functional activation potential, but further studies are required to determine which macrophagespecific genes are controlled by this transcription factor. The expression of other transcription factors is also induced during monocyte maturation. These include EGR-1, HOXB7, NF-Y, and some members of the proto-oncogene Jun/Fos superfamily. The function of EGR-1 in monocyte differentiation is not clear. While studies in vitro give EGR-1 a deterministic role that restricts differentiation to the monocyte lineage, analysis of knockout mice indicates that EGR-1 expression is not necessary for macrophage development. The exact contribution of EGR-1 to monocyte differentiation is likely to remain an important subject of discussion. Some members of the Jun/Fos superfamily participate in the proliferation and subsequent differentiation of monocytic precursor cells (M-CFU), although the specific mechanisms for this regulation need to be determined. HOXB7 has a similar effect under the stimulus of GM-CSF. During the monocyte-to-macrophage differentiation, NF-Y induces the synthesis of ferritin and controls the expression of MHC class II molecules, thus endowing the 414



mature macrophage with the potential to act as an iron storage compartment and as an antigen-presenting cell. Finally, the loss of proliferation associated with monocytic maturation seems to be caused by two parallel mechanisms. First, the expression of c-Myb and c-Myc is repressed early during terminal monocyte differentiation. If expression of these proteins is maintained, especially that of c-Myc, the maturation of monoblasts is blocked. Indeed, the activation of certain Stat proteins during terminal monocyte differentiation may mediate the repression of c-Myb and c-Myc. Second, the expression of the transcription factor IRF-1 is induced in promonocytes. This factor activates the transcription of the genes that code for IFN-a/b, which act as autocrine signals that promote the inhibition of monocyte proliferation. The expression of key transcription factors is thus the basis for the regulation of multiple aspects of myelopoiesis. Most probably, novel transcription factors that take part in macrophage development will soon be discovered thanks to their binding to critical sequences in the promoters of lineagespecific genes. However, other parameters must also be taken into account when analyzing the transcriptional regulation of this process. Posttranslational modifications, such as phosphor-

ylation, and complex interactions between inducible and constitutive transcription factors are two mechanisms that control the transactivation/repression of different genes. Further work needs to be focused, then, on the study of the specific interactions of those transcription factors that are expressed simultaneously at a certain stage of macrophage differentiation. In addition, new approaches need to be followed to identify novel genes that might be targets of these transcription factors and to determine how their transcriptional regulation affects macrophage development.

ACKNOWLEDGMENTS This work was supported by grants from the CICYT (PB941549) and FISS (96/2050) to A. Celada.

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