Expression of Jagged1 gene in macrophages and its regulation by hematopoietic growth factors

Experimental Hematology 29 (2001) 850–855

Expression of Jagged1 gene in macrophages and its regulation by hematopoietic growth factors Kouji Nomaguchia, Shinya Suzua, Muneo Yamadaa, Hirotoshi Hayasawaa, and Kazuo Motoyoshib a Biochemical Research Laboratory, Morinaga Milk Industry Co., Ltd., Kanagawa, Japan; Third Department of Internal Medicine, National Defense Medical College, Saitama, Japan

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(Received 12 October 2000; revised 23 February 2001; accepted 2 March 2001)

Objective. Serrate/Jagged and Delta are cell surface ligands for Notch receptors that may influence hematopoietic cell fate decisions and are known to be expressed in bone marrow stromal cells. In a series of screenings of cDNAs constructed by a cDNA library subtraction technique, we identified Jagged1, one of the Notch ligands, as a gene up-regulated by macrophage colony-stimulating factor (M-CSF) in bone marrow macrophages. Therefore, we compared stromal cells and macrophages for expression of Notch ligands including Jagged1 and analyzed the regulation of their expression by cytokines. Materials and Methods. Murine bone marrow macrophages were prepared by culturing femoral bone marrow cells with M-CSF. Primary bone marrow fibroblastic stromal cells were prepared by a culture system that we recently developed. The expression of Notch ligands was analyzed by either Northern blot analysis or reverse transcriptase polymerase chain reaction. Results. The bone marrow macrophages expressed Jagged1 but not Jagged2 and Delta1 at a level that was detectable by Northern blot analysis. Expression of the Jagged1 gene was markedly up-regulated by growth factors for the cells, i.e., M-CSF, granulocyte-macrophage colony-stimulating factor, and interleukin-3. Expression of Jagged2 and Delta1 seldom was affected by the stimuli. The primary bone marrow fibroblastic stromal cells, and murine stromal cell lines, such as PA6 and ST2, also expressed Jagged1 transcript, at levels comparable to the steady-state level in macrophages. However, expression of the Jagged1 gene was little affected when these cells were stimulated with fibroblastic growth factor and plateletderived growth factor. Conclusions. We demonstrated that bone marrow macrophages as well as stromal cells constitutively produced Jagged1 and that the expression was markedly up-regulated by hematopoietic growth factors, M-CSF, granulocyte-macrophage colony-stimulating factor, and interleukin-3. The results highlight the involvement of macrophages and these growth factors in hematopoietic cell fate decisions via the production of Jagged1. © 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc.

Ligands for Notch receptors have been identified, including Serrate and Delta in Drosophila, and Lag-2 and Apx-1 in Caenorhabditis elegans (reviewed in [1]). Two Notch ligand families have been identified in mammals: Jagged1 and Jagged2 for the Jagged/Serrate family [2–7], and Delta1 and Delta3 for the Delta family [8,9]. They encode membraneintegrated proteins having an extracellular domain containing a variable number of epidermal growth factor-like reOffprint requests to: Kazuo Motoyoshi, M.D., Third Department of Internal Medicine, National Defense Medical College, Namiki 3-2, Tokorozawa, Saitama 359-8513, Japan; E-mail: [email protected]

peats that modulate the affinity of the interaction with their receptors [4]. The extracellular domain also contains a conserved region unique to the families of ligands: a DSL (Delta/Serrate/Lag-2) domain essential for Notch binding and activation [10]. The Jagged/Serrate family ligands contain a conserved cysteine-rich region that is not present in Delta family ligands [11]. Like the ligands, multiple Notch receptor homologues have been described in higher vertebrates, including Notch1 through Notch4 in rodents and humans (reviewed in [12]). The physical interaction between Notch receptors and DSL ligands is not fully understood. However, a recent study demonstrated that Jagged1 binds to

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Notch1, Notch2, and Notch3, suggesting that Jagged1 is a ligand for multiple Notch receptors [4]. Notch and their ligands have been shown to play an important role in cell fate decisions during oogenesis, embryogenesis, and metamorphosis (reviewed in [1]). Hematopoiesis is a continuous developmental process in which pluripotent stem cells and their progeny make sequential cell fate decisions (reviewed in [12]). Therefore, Notch signaling could play a role in the hematopoietic system. Recent studies demonstrated the expression of Notch1 and Notch2 in CD34 cells, CD34Lin subset, CD14 monocytes, B cells, and T cells (reviewed in [12]). Several studies indicated that Notch1 is critically involved in T-cell lineage development (reviewed in [12]). However, the role of Notch signaling in regulating myelopoiesis remains ambiguous. Inconsistent results were obtained when primary hematopoietic progenitor cells were stimulated by Jagged1. Varnum-Finney et al. [13] and Jones et al. [14] observed a moderate increase in colony formation, whereas Walker et al. [15] observed no effect or a slight decrease in colony formation. Milner et al. [16] and Carlesso et al. [17] showed that ectopic expression of constitutively active forms of Notch1 inhibits differentiation of myeloid progenitor cell lines, whereas Shelly et al. [18] indicated that Notch is required for erythroid differentiation to proceed and a more recent study by Schroeder and Just [19] showed that activation of Notch1 promotes differentiation of a myeloid cell line. Therefore, more studies are needed to clarify the function of Notch signaling in myelopoiesis. Likewise, it will be necessary to analyze the expression and regulation of Notch ligands in specific cell types within the hematopoietic microenvironment because the ligands are transmembrane proteins and, therefore, direct cell-to-cell contact is required for the biologic effect of the ligands. We screened a cDNA library constructed by a cDNA subtraction technique [20] to isolate genes whose expression in murine bone marrow macrophages was up-regulated by macrophage colony-stimulating factor (M-CSF) [21]. One of the genes identified through this approach encoded murine Jagged1. Although the expression of Notch ligands by cultured primary murine fetal liver stroma, cultured primary murine bone marrow stroma, and stromal cell lines has been reported [13], expression of the ligands in bone marrow macrophages and regulation of the expression by cytokines remains to be investigated. In this study, we compared macrophages with stromal cells for the expression of several Notch ligands and determined whether the expression in these cells could be regulated by cytokines.

Materials and methods Library subtraction The murine bone marrow macrophages were prepared by culturing femoral bone marrow cells from C57BL/6 mice (Charles River, Kanagawa, Japan) with 100 ng/mL recombinant human (rh) M-CSF for 7 days [22]. The purity of the macrophage preparations usually

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was more than 97% as determined by enzymatic analysis (nonspecific esterase staining). Cells were factor depleted for 12 hours in RPMI 1640 medium (Life Technologies, Stafford, TX, USA) containing 15% fetal calf serum (FCS) and then treated with 100 ng/mL rhM-CSF for 3 hours. Poly (A) RNA from untreated cells or from those treated with rhM-CSF was prepared using a mRNA separator kit (Clontech, Palo Alto, CA, USA). cDNA library construction and library subtraction were performed according to the manufacturer’s instructions (PCR-Select cDNA subtraction kit; Clontech). The cDNA fragments of the subtracted cDNA library were cloned into pCR2.1 vector (Invitrogen, Carlsbad, CA, USA). Randomly isolated clones were further analyzed by Northern hybridization using total RNA from unstimulated- and rhM-CSF–stimulated bone marrow macrophages. Cell culture The murine bone marrow stromal cell lines PA6 [23] and ST2 [24] were obtained from the Riken Cell Bank (Tsukuba, Japan). They were routinely maintained in RPMI 1640 medium supplemented with 10% FCS. Northern blot analysis Bone marrow macrophages were prepared as described earlier. These cells were M-CSF depleted for 12 hours in RPMI 1640 medium containing 15% FCS. They then were treated with either rhM-CSF (100 ng/mL), recombinant murine (rm) granulocyte-macrophage colony-stimulating factor (GM-CSF, 10 ng/mL; Life Technologies), rm interleukin-3 (rmIL-3, 10 ng/mL; Life Technologies), rh platelet-derived growth factor-BB (PDGF-BB, 10 ng/mL; Genzyme, Cambridge, MA, USA), or rh basic fibroblast growth factor (bFGF) (10 ng/mL; Genzyme-Techne, Minneapolis, MN, USA) for various periods. PA6 and ST2 cells were cultured for 12 hours in RPMI 1640 medium containing 1% FCS and then treated with either rhPDGF-BB (10 ng/mL) or rh-bFGF (10 ng/mL) for various periods. The primary bone marrow fibroblastic stromal cells were prepared by a culture system developed by Deryugina et al. [25] and Yamada et al. [22]. Total RNA from these cells was isolated using RNAzol B reagent (TEL-TEST, Friendswood, TX, USA), electrophoresed on agarose gels, and transferred to a nylon membrane (Hybond N; Amersham, Uppsala, Sweden). The membrane was prehybridized with Rapid-Hyb Buffer (Amersham) for 2 hours and hybridized with radiolabeled cDNA probes. Murine Jagged1 cDNA containing part of the coding region was obtained by reverse transcriptase polymerase chain reaction (RT-PCR) using the primers 5-TGCAGCTGTCAATCACT TCG-3 and 5-CAGAATGACGCTTCCTGTCG3 (GenBank accession number NM013822) and radioisotopically labeled using a Megaprime DNA Labeling System (Amersham) and [-32P]-dCTP (Amersham). Membranes were washed twice for 10 minutes at room temperature with 2 SSC/0.1% sodium dodecyl sulfate (SDS) and once for 10 minutes at 65C in 0.1 SSC/0.1% SDS. The membranes were exposed to x-ray film (BioMax MS; Eastman Kodak, Rochester, NY, USA). A cDNA probe for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) used as a loading control was purchased from Clontech. Immunoblot analysis Bone marrow macrophages were factor depleted for 12 hours in RPMI 1640 medium containing 15% FCS. Cells then were treated with rhM-CSF (100 ng/mL) for various periods and solubilized with lysis buffer (1 PBS, 1% Nonidet P-40, 1 mM EDTA, 0.1% SDS, and 0.5% Na-deoxycholate) containing protease inhibitors (1

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mM phenylmethylsulfonyl fluoride, 1 g/mL aprotinin, 1 g/mL leupeptin, and 1 g/mL pepstatin). The cell lysates were centrifuged and the resulting supernatants subjected to immunoblot analysis. The protein concentrations of the cleared cell lysates were determined using BCA protein assay reagent (Pierce, Rockford, IL, USA). The cleared cell lysates containing equal amounts of proteins were resolved by SDS-PAGE under reducing conditions and the proteins transferred to a nylon membrane (Hybond-P; Amersham). The membrane was probed with antibodies to Jagged1 (C-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The antibodies were visualized with horseradish peroxidase-coupled anti-immunoglobulin (705-0350147; Jackson Immuno Research Laboratories, West Grove, PA, USA) using the Enhanced Chemiluminescence Western Blotting Detection System (Amersham) according to manufacturer’s instructions.

Results Expression of Notch ligands in bone marrow macrophages To characterize genes whose expression is affected by M-CSF, we used the cDNA library subtraction technique [20]. We

Figure 1. Northern blot analysis of total RNA from bone marrow macrophages for Jagged1 expression. (A) Bone marrow macrophages were M-CSF depleted for 12 hours () and then restimulated with M-CSF for the indicated periods. Cells at the exponential growth stage (EXPO.) also were collected. Total RNA (2 g/lane) was electrophoresed on agarose gels, blotted to membrane, and hybridized with probe to Jagged1. To verify the amount of RNA loaded, the same blots were hybridized with G3PDH probe. (B) Cells were pretreated with cycloheximide (10 g/mL) for 1 hour before M-CSF stimulation. Time of M-CSF stimulation was 3 hours.

prepared a cDNA library of murine bone marrow-derived macrophages cultured in M-CSF–free medium and subtracted this cDNA library from that of cells stimulated with the growth factor for 3 hours [21]. Clones isolated randomly from the subtracted library were screened by Northern blot analysis. The study identifies a number of novel or known genes (Suzu et al., unpublished data). One of the genes characterized by these procedures was that encoding murine Jagged1. The cDNA fragment obtained from the subtracted cDNA library and used for Northern analysis corresponded to the 3 untranslated region of the murine Jagged1 transcript. Thus, we prepared a cDNA probe corresponding to the coding region of Jagged1 and reexamined expression of the gene in bone marrow macrophages (Fig. 1A). The bone marrow macrophages harvested at the exponential growth stage expressed Jagged1 transcript at a detectable but relatively low level (Fig. 1A). However, when the cells were stimulated with M-CSF, a marked increase in Jagged1 expression was observed. The maximal induction was observed within 1.5 hours after M-CSF stimulation, and high-level expression was maintained thereafter (Fig. 1A). Because the protein synthesis inhibitor cycloheximide did not affect the expression level of this gene (Fig. 1B), de novo protein synthesis was not required for its induction. Induction of Jagged1 protein by M-CSF was confirmed by immunoblot analysis. The inducted expression in macrophages was detected within 2 hours after stimulation with M-CSF, and an elevated level of expression was maintained for at least 12 hours (Fig. 2). We also found that Jagged1 could be induced by other growth factors for macrophages, i.e., GM-CSF and IL-3 (Fig. 3). Although Jagged1 mRNA was induced more strongly by MCSF than by GM-CSF or IL-3, the kinetics of induction of the gene by GM-CSF or IL-3 appeared to be similar to that

Figure 2. Immunoblot analysis of total cell extracts from bone marrow macrophages for Jagged1 expression. Bone marrow macrophages were M-CSF depleted for 12 hours () and then restimulated with M-CSF for the indicated periods. Cells at the exponential growth stage (EXPO.) also were collected. The cleared cell lysates containing equal amounts of protein (2 g/lane) were electrophoresed on SDS-PAGE under reducing conditions, blotted to membrane, and probed with anti-Jagged1 antibody (upper panel). To verify the amount of protein loaded, the gel was stained with silver. Molecular weight markers (in Da) are shown on the left side.

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Figure 3. Northern blot analysis of total RNA from bone marrow macrophages showing induction of Jagged1 following exposure to M-CSF, IL-3, or GM-CSF. Bone marrow macrophages were factor-depleted for 12 hours () and then stimulated with either M-CSF, IL-3, or GM-CSF for the indicated periods. Total RNA (2 g/lane) was electrophoresed, blotted to membrane, and hybridized with probe to Jagged1 or G3PDH.

by M-CSF (Fig. 3). Addition of recombinant soluble M-CSF receptor/human IgG1 Fc chimera protein (Genzyme-Techne) inhibited Jagged1 induction by M-CSF (data not shown). The addition of neutralizing antibodies to GM-CSF or IL-3 (R & D Systems, Minneapolis, MN, USA) inhibited the induction by GM-CSF or IL-3, respectively (data not shown). We sought to determine whether other Notch ligands, Jagged2 and Delta1, are expressed in macrophages. However, we could not detect the transcripts of these genes by Northern blot analysis, even when the cells were stimulated with M-CSF (data not shown). Thus, expression of Jagged2 or Delta1 in bone marrow macrophages seemed to be extremely low. In a preliminary experiment in which we performed RT-PCR analysis followed by Southern blotting, we observed a slight induction of the Delta1 gene but not the Jagged2 gene by M-CSF stimulation (data not shown).

Expression of Notch ligands in stromal cells An earlier study in which the expression of Notch ligands was analyzed by RT-PCR showed that Jagged1 mRNA was detectable in bone marrow stromal cultures [14]. In the present study, we also found that the murine stromal cell line PA6 expressed Jagged1 mRNA at a level detectable by Northern blot analysis (Fig. 4). We next analyzed the level of Jagged1 expression after stimulation with mitogens (PDGF-BB and bFGF) for PA6 cells [22]. Jagged1 was upregulated after 1 to 2 hours of stimulation with these mitogens and then decreased after 4 hours of simulation (Fig. 4). This up-regulation was not as obvious as that in primary bone marrow macrophages upon stimulation with M-CSF. Similar results were obtained using another murine stromal cell line, ST2 (data not shown). These mitogens did not affect expression of Jagged1 in bone marrow macrophages (data not shown). Finally, we compared macrophages with stromal cells for expression of Jagged1 mRNA. The basal level of Jagged1 expression in PA6 cells was slightly higher than that in other cell preparations, ST2 cells, primary bone marrow stromal cells, and unstimulated macrophages (Fig. 5).

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Figure 4. Northern blot analysis of total RNA from stromal cell line PA6 for Jagged1 expression. PA6 cells were allowed to grow confluent and then to be quiescent by replacing them in medium with 1% FCS for 12 hours (). Cells then were stimulated with either PDGF-BB or bFGF for the indicated periods. Cells at the exponential growth stage (EXPO.) also were collected. Total RNA (2 g/lane) was electrophoresed, blotted to membrane, and hybridized with probe to Jagged1 or G3PDH. Exposure time is longer than that shown in Figure 5.

However, it was obvious that the level in M-CSF–stimulated bone marrow macrophages was much higher than that in PA6 cells (Fig. 5). The level of expression of Jagged2 and Delta1 mRNA in PA6, ST2, and primary stromal cells was extremely low when assessed by Northern blot analysis and RT-PCR analysis (data not shown). Discussion In this study, we demonstrated the constitutive production of Jagged1 by bone marrow macrophages, at a level comparable to that by bone marrow stromal cells (Fig. 4). Jagged1 was shown to be expressed in cultured primary bone marrow stroma, fetal liver stroma, and stromal cell lines [13,14]. A recent study also reported strong expression of Jagged1

Figure 5. Comparison of Jagged1 gene expression between stromal cells and macrophages. Total RNAs were obtained from two different preparations of primary stromal cells, PA6 cells and ST2 cells. These cells were collected at the exponential growth stage. Alternatively, total RNA was obtained from macrophages that were M-CSF depleted for 12 hours () or restimulated with M-CSF for 1.5 hours. Total RNA (2 g/lane) was electrophoresed, blotted to membrane, and hybridized with probe to Jagged1 or G3PDH.

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transcript in terminally maturing cells, including mast cells and megakaryocytes [26]. In contrast to Jagged1, Jagged2 and Delta1 were found to be expressed relatively weakly in bone marrow stromal cells [14]. In contrast, it was reported that endothelial cells expressed high levels of Jagged2 [27]. The present study in which we performed Northern blot analysis and RT-PCR suggested that bone marrow macrophages scarcely expressed Jagged2 and Delta1. Thus, it is likely that the expression pattern of Notch ligands in macrophages is similar to that in stromal cells. A noteworthy finding of our study is that production of Jagged1 protein in macrophages increased markedly on stimulation with M-CSF, GM-CSF, and IL-3 (Fig. 3). The study provides the first evidence that expression of Notch ligands can be regulated by extracellular stimuli, such as cytokine stimulation. The observation is in contrast with the finding that Jagged1 expression in bone marrow stromal cells was not affected by PDGF-BB or bFGF (Fig. 4), or that Delta1 expression in human umbilical vein endothelial cells was not affected by interferon- and interleukin-1 [28]. Transcription factors of the Rel/NF- B family are critical regulators of genes that function in inflammation, cell, proliferation and apoptosis (reviewed in [29]). Bash et al. [30] demonstrated that Jagged1 but not Delta1 is a Rel/NF B–responsive gene. Both c-Rel and RelA induced Jagged1 gene expression, whereas a mutant defective for transactivation did not [30]. Furthermore, Jagged1 transcript was upregulated by endogenous NF- B activation, and this effect was inhibited by a dominant mutant of I B, a physiologic inhibitor of NF- B [30]. In a preliminary experiment, we found that Jagged1 expression in macrophages also was induced by tumor necrosis factor-, a well-known physiologic inducer of NF- B activation (data not shown). Thus, induction of the Jagged1 gene by M-CSF, GM-CSF, or IL-3 might be mediated by activation of NF- B. Brach et al. [31] reported that M-CSF could induce expression of its own gene in monocytes, and this induction was associated with detection of NF- B protein in nuclear extracts. The finding that Jagged1 is expressed in bone marrow macrophages and up-regulated markedly by cytokines might suggest that macrophages in the hematopoietic microenvironment play a role in hematopoiesis through production of Jagged1. Several studies were done with coculture systems in which primary hematopoietic progenitor cells were cultured on control feeder layers or on feeder layers engineered to express Jagged1 [13–15]. The feeder layer cells used in these studies included a stromal cell line S17 [14], a fibroblastic cell line NIH3T3 [13], and a myoblast cell line C2C12 [15]. Importantly, ambivalent results were obtained from these studies. In some studies, a moderate increase in colony formation was observed [13,14], whereas in other studies no effect or a decrease in colony formation was seen [15]. The difference in the outcome of coculture experiments might be due to the differences among feeder layer cells in the types or amounts of cytokines made or ad-

hesion molecules expressed. Macrophages are a physiologic source of Jagged1 and produced larger amounts of protein in response to cytokine stimulation. Therefore, the use of unstimulated or cytokine-stimulated macrophages as feeder layer cells seems to be more appropriate to clarify the physiologic function of Jagged1. More recent studies indicate that Notch signaling also may regulate apoptosis. For example, Ohishi et al. [32] reported that incubation of human peripheral monocytes with plastic-immobilized Delta1 in the presence of M-CSF led to apoptotic cell death. We therefore tested the possibility that Jagged1 influences the growth of macrophages in an autocrine manner. A murine Jagged1 cDNA encoding the entire extracellular portion was fused with a cDNA for the Fc portion of human IgG (FE-J1-Fc) [4], and the resulting plasmid was transfected into COS7 cells. The media conditioned by COS7 cells, which contained soluble Jagged1 protein, were added to the cultures of bone marrow macrophages or peritoneal macrophages at various concentrations. However, we did not observe any difference in growth and morphology between control media-treated macrophages and the cells treated with the conditioned media containing soluble Jagged1 (data not shown). More studies are needed to elucidate the significance of Jagged1 expression in macrophages. Acknowledgments We thank H. Hirai for providing us with a plasmid containing fulllength extracellular region of mJagged1 cDNA, and N. Wakimoto and A. Yasuda for technical assistance.

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