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1 Division of Hematology/Oncology, Department of Medicine, Kobe University School of Medicine,Kobe 650-0017, Japan
2 Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan
3 Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, NY 10021-6399, USA
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Abstract |
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Introduction |
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Nuclear receptors are involved in differentiation processes of a variety of cells. A precedent of the proof that TRAP220 has a vital role in physiological nuclear receptor-mediated cellular differentiation program is the peroxisome proliferator-activated receptor (PPAR)
2-mediated adipogenesis (Ge et al. 2002). However, available information from TRAP220 knockout mice is limited because of early embryonic lethality of the null mice and the role of TRAP220 in other differentiation processes remains unrevealed. In the case of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-induced keratinocyte differentiation, TRAP220 appears to be unnecessary (Oda et al. 2003). Thus, the activity of TRAP220 in the said processes still remains controversial.
1,25-(OH)2D3 and all-trans retinoic acid (ATRA) are the key ligands of VDR and RAR, correspondingly, for myelomonocytic differentiation. Bone marrow hematopoietic progenitor cells differentiate into monocytes and granulocytes in the presence of 1,25-(OH)2D3 and ATRA, respectively (Koeffler et al. 1984; Gratas et al. 1993). These processes can be most efficiently simulated in a cell culture system using HL-60 cells, human promyelocytic leukemia cell line having a myeloid progenitor phenotype. HL-60 cells differentiate into monocytes with 1,25-(OH)2D3 stimulation while into granulocytes with ATRA (Breitman et al. 1980; Matsui et al. 1984).
In this study, focusing on hematopoiesis, the role of TRAP220 in 1,25-(OH)2D3-induced monopoiesis via VDR and ATRA-induced granulopoiesis through RAR is investigated. It is revealed that TRAP220 has a fundamental action in these physiological differentiation processes.
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Results |
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Since TRAP220 knockout (Trap220–/–) mice were mortal during the early embryonic period before definitive hematopoiesis within the hepatic primordia becomes dominant, the function of TRAP220 in adult hematopoiesis was mostly unknown. However, since these embryos appeared to have normal composition of nucleated erythroid cells, primary hematopoiesis was preserved (Ito et al. 2000; Zhu et al. 2000). The yolk sac hematopoietic progenitors were known to differentiate efficiently into definitive blood cells in vitro in the presence of appropriate amounts of cytokines (Matsuoka et al. 2001). Therefore, in order to elucidate the role of TRAP220 in nuclear receptor-mediated definitive hematopoietic cell differentiation, we first analyzed the differentiation potency of Trap220–/– yolk sac hematopoietic progenitor cells.
Yolk sac cells from E9.0 embryos of each genotype were collected and plated into methylcellulose media containing human recombinant (hr) erythropoietin (EPO), hr interleukin (IL)-6, hr granulocyte-colony stimulating factor (G-CSF), murine recombinant (mr) IL-3, and mr stem cell factor (SCF). Seven days after plating the cultures, growth of the following hematopoietic progenitor colonies was analyzed: colony-forming unit-granulocyte (CFU-G), colony-forming unit-monocyte/macrophage (CFU-M), colony-forming unit-granulocyte, monocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming unit-granulocyte, erythroid, monocyte/macrophage, megakaryocyte (CFU-GEMM) (Nakahata & Ogawa 1982). Each Trap220–/– colony had the characteristic morphological features of the definitive mature blood cells that were similar to those of the corresponding Trap220+/+ colony (Fig. 1B). Importantly, the number of CFU-M obtained from Trap220–/– embryos was significantly lower (twofold) than the Trap220+/+ controls (P < 0.001), but not extinct. The numbers of CFU-G, CFU-GM, BFU-E, and CFU-GEMM, however, were comparable (Fig. 1A). The total number of Trap220–/– colonies was also lesser compared to the control (P = 0.04) due to the decreased CFU-M counts (Fig. 1A).
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Impaired nuclear receptor-driven transcription in Trap220–/– MEFs
VDR could be the candidate activator for TRAP220 in monopoiesis, in view of the fact that TRAP220 is a coactivator for a variety of nuclear receptors (Yuan et al. 1998) and 1,25-(OH)2D3 induces monocytic differentiation (Koeffler et al. 1984; Matsui et al. 1984). The transient transfection and luciferase reporter assays in mouse primary embryonic fibroblasts (MEFs) revealing a defective thyroid hormone receptor (TR)-driven transcription but not affecting other activator functions in Trap220–/– cells were previously reported (Ito et al. 2000). Since enough backcrossed mice were available, the function of TRAP220 was determined conveniently in genetically uniform MEFs. In order to normalize the basal level of transcription, reporter construct containing the same promoter and Gal4-binding sites were used in all assays.
A strong Gal4-VDR-driven transcription was observed in control cells, but the response to 1,25-(OH)2D3 was attenuated in mutant MEFs, being reduced twofold in the Trap220–/– MEFs (Fig. 2A). Experiments for Gal4-TR
(Fig. 2B) and Gal4-PPAR (Fig. 2C) were performed similarly and results were alike. Notably, the basal activity in Gal4-PPAR-transfected MEFs (due to putative endogenous ligands) was also significantly diminished in null cells. In contrast, Gal4-RAR- and Gal4-RXR-driven transcriptions in Trap220–/– MEFs were apparently similar to wild-type controls (Fig. 2D,E), although both RAR and RXR were shown to strongly interact with TRAP220 in a ligand-dependent manner (Yuan et al. 1998). Transfection assays of intact exogenous TR and PPAR with luciferase reporters containing thyroid hormone response element (TRE) and peroxisome proliferator response element (PPRE), respectively, were done and results were similar (data not shown). These results were consistent with the outcome in colony formation assays of yolk sac cells, where monocytic differentiation was selectively inhibited in Trap220–/– colonies. However, interpretation should be cautiously done because the assays utilized overly expressed artificial receptors and transiently introduced artificial promoters. Thus conclusion could be reached only after vigorous studies on natural endogenous genes (see below for RAR and VDR), as was demonstrated for PPAR function (Ge et al. 2002).
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Since the yolk sac colony formation assays indicated the importance of TRAP220 in monocytic differentiation, it was determined whether TRAP220 or TRAP/Mediator complex could be induced during the differentiation processes.
Time courses of mRNA expression of several TRAP subunits (TRAP220, TRAP170 and TRAP240) and steroid receptor coactivator (SRC)-1 as representative of other types of coactivators, in response to treatment with two differentiation inducers, 1,25-(OH)2D3 and ATRA, were analyzed. HL-60 cells differentiated into monocytes or granulocytes with 1,25-(OH)2D3 or ATRA treatment, respectively (Breitman et al. 1980; Matsui et al. 1984). During treatment with 1,25-(OH)2D3, mRNAs of TRAP components, including TRAP220, were induced in 24 h, although SRC-1 mRNA was not. Importantly, the addition of ATRA also induced the expression of TRAP components, but not of SRC-1 (Fig. 3). On the other hand, although HL-60 cells also differentiate into monocytes/macrophages or granulocytes with 12-O-tetradecanoylphorbol-13-acetate (TPA) or dimethylsulfoxide (DMSO) treatment, respectively, through pathways independent of nuclear receptors (reviewed in Collins 1987), during treatment with TPA or DMSO, mRNA expressions of TRAP220 were not induced in 24 h (Fig. 4A). Taking these results into consideration, it is indicated that TRAP220 was induced not as a result of myelomonocytic differentiation but was ligand-dependently induced. Hence, these results indicated that TRAP220 (or TRAP/Mediator complex) could be employed in nuclear receptor-mediated differentiation processes, not only in monopoiesis but also granulopoiesis.
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The yolk sac colony formation assays demonstrated the crucial role of TRAP220 selectively in monopoiesis. However, since assay condition included pharmacological (excessive) amounts of cytokines, defects in weak contributions of inducers in other types of differentiation pathways could have been concealed. In this sense, it was important to analyze each receptor-mediated process, including the well-known retinoic acid-induced granulocytic differentiation, in a more refined system. HL-60 was an ideal cell line in which selective nuclear receptor-mediated differentiation processes could be finely monitored.
We first assessed the expression profiles of genes that are induced directly downstream of VDR and RAR, and of differentiation marker genes. As a control, HL-60 cells were treated with 1 x 10-8M TPA or 1.25% DMSO, wherein cells differentiated into monocytes or granulocytes, respectively. 1,25-dihydroxyvitamin D3 24-hydroxylase (VDH) and CD38 are the corresponding direct targets of VDR and RAR (Drach et al. 1994; Chen & DeLuca 1995), while gp91-phox (gp91) is the differentiation marker for both monocytes and granulocytes (Friedman 2002). Neither the target genes nor the differentiation marker gene was expressed without ligands. On one hand, HL-60 cells differentiated into monocytes and expressed gp91 with both 1,25-(OH)2D3 and TPA treatment, but the VDR target gene, VDH, was expressed when treated only with 1,25-(OH)2D3. On the other hand, differentiation into granulocytes and expression of gp91 occurred with both ATRA and DMSO treatment, but the RAR target gene, CD38, was expressed only when treated with ATRA. The expression of gp91 in HL-60 cells exposed to DMSO was so weak that it was detected only after 7 days treatment (Fig. 4A).
Inefficient differentiation of TRAP220 knockdown HL-60 cells under 1,25-(OH)2D3 or ATRA treatment
We then investigated the expression of these marker genes in HL-60 cells wherein TRAP220 expression was depleted. For this purpose, the small interfering RNA (siRNA) expression vector, which down-regulated TRAP220, was generated (Fig. 4B) and inoculated directly into HL-60 cell nuclei using the Nucleofector device (amaxa). The efficiency of the knockdown of TRAP220 at a mRNA level (Fig. 4C,D) and at a protein level (data not shown) were both approximately 50% after treatment with 1,25-(OH)2D3 or ATRA. As control, a backbone vector was introduced.
After the addition of 1,25-(OH)2D3, expression of both VDH and gp91 were lower in knockdown cells than in control cells (Fig. 4C, left panel). In contrast, TPA treatment did not affect VDH expression but generated equal expression of gp91 in both knockdown and control cells (Fig. 4C, right panel). These results denoted that 1,25-(OH)2D3-induced monocytic differentiation was suppressed in knockdown cells while TPA-induced monocytic differentiation was not.
Gene expressions of knockdown HL-60 cells treated with ATRA or DMSO were evaluated next. With ATRA treatment, expressions of both CD38 and gp91 were lesser in TRAP220 knockdown cells compared to the control (Fig. 4D, left panel). Meanwhile, no expression of CD38 and gp91 was observed during 24-h treatment with DMSO in both knockdown and control cells (Fig. 4D, right panel). After 168-h treatment, gp91 was weakly and equally induced in both cells (data not shown). However, since a 24-h treatment with DMSO was too short to differentiate HL-60 cells sufficiently and nucleofection in HL-60 cells efficiently continued only up to 24 h (data not shown), these data needed meticulous interpretation. Nevertheless, these results evidently implied that ATRA-induced granulocytic differentiation was inhibited in knockdown cells.
We also down-regulated CBP using the siRNA expression vector in HL-60 cells treated with 1,25-(OH)2D3 or ATRA. The results were completely the same as those when a backbone vector was introduced (data not shown). Therefore, the effects of the siRNA plasmid that down-regulated TRAP220 unlikely reflected a nonspecific repression of a siRNA, and TRAP220 might have a more dominant role than CBP in myelomonocytic differentiation processes in HL-60 cells.
Flow cytometric analyses of HL-60 cells, in which knockdown or control vector was introduced and further treated with 1,25-(OH)2D3 or ATRA, were also performed. In HL-60 cells, CD11b and CD14 were known cell surface markers induced by monocytic differentiation, while CD11b and CD38 were associated with granulocytic differentiation (Drach et al. 1994; Brackman et al. 1995). These marker expressions were selectively delayed in knockdown cells compared with the control when treated with 1,25-(OH)2D3 and ATRA (Fig. 5A,B). These data further confirmed the above conclusion that TRAP220 is essential not only in VDR-mediated monopoiesis, but in RAR-mediated granulopoiesis as well, in HL-60 cells.
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Discussion |
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2 and PPAR2-mediated adipogenesis (Ge et al. 2002). The present study provides novel evidence of its critical role in the differentiation of hematopoietic progenitor cells into monocytes and granulocytes as mediated by VDR and RAR, respectively. This is the second in vitro example of TRAP220's involvement in nuclear-receptor mediated differentiation. Yet, it is the first mechanistic analysis of TRAP220 in hematopoiesis proven of physiologic relevance. Normal reserves of Trap220–/– yolk sac hematopoietic progenitor cells
The current model proposes that the most primitive origin of hematopoietic cells is a common progenitor of hematopoietic and vascular cells called the hemangioblast (Kennedy et al. 1997). This model is recently proved in vivo in gastrulating mouse embryos. The hemangioblast appears first in the primitive streak and migrates on to the yolk sac where it is thought to differentiate into hematopoietic, endothelial, and vascular smooth muscles (Huber et al. 2004). The first embryonic hematopoietic stem cells (HSCs) and blood cells are observed in E7.5-9.5 murine yolk sacs (Godin et al. 1995). Although the HSCs for definitive hematopoiesis might be derived from the hemangioblasts that are distinct from the primitive streak hemangioblasts, these HSCs share a common feature with yolk sac HSCs (or hematopoietic progenitor cells). Namely, the yolk sac HSCs differentiate into myelomonocytic lineage cells and megakaryocytes, as well as erythroid cells, in standard in vitro colony formation assays. Furthermore, they can provide long-term multilineage reconstitution in conditioned newborn mice (Yoder et al. 1997). Thus, although yolk sac HSCs are distinct from definitive HSCs in that the former differentiate into nucleated red blood cells in vivo and that they do not differentiate into lymphocytes in vitro, they appear to be near equivalent to the latter and the distinctions could be attributed to the yolk sac environment. Therefore, yolk sac HSCs may be the feasible substitute in research on HSCs destined for definitive hematopoiesis.
In the study, Trap220–/– mice are deceased before definitive hematopoiesis becomes dominant. Their hepatic HSCs or aorta-gonads-mesonephros (AGM) cells are technically difficult to obtain due to the runty phenotype of the Trap220–/– embryos. However, since nucleated red blood cells appear to be normally synthesized in these embryos, yolk sac HSCs are assumed normal. Therefore, yolk sac cells are utilized to analyze hematopoietic differentiation in this study.
The number of Trap220–/– CFU-GEMM (as well as other types of colonies) is comparable with the wild-type control, as expected. This signifies normal number and quality of Trap220–/– yolk sac HSCs. In view of the fact that blood vessels are formed in the embryo, Trap220–/– hemangioblasts are normal as well. Hence, TRAP220 is dispensable in the formation of primitive streak hemangioblasts and further differentiation into yolk sac HSCs during embryonic development.
Attenuated monopoiesis and VDR function of Trap220–/– cells
Both the attenuated results on monopoiesis (twofold) of Trap220–/– yolk sac cells and VDR function (twofold) in Trap220–/– MEFs indicate that TRAP220 is necessary for an optimal VDR-mediated monocytic differentiation. These results contrast the observation in the study of VDR-mediated keratinocyte differentiation (Oda et al. 2003). According to the report, 1,25-(OH)2D3 controls keratinocyte proliferation and differentiation by directly regulating transcription. TRAP220 and SRC-3 potentiate 1,25-(OH)2D3-induced transcription during proliferation, but TRAP220 is ineffective in differentiation. However, since keratinocytes utilize other nuclear receptors, including PPAR, PPARß/
, and PPAR, for differentiation (Mao-Qiang et al. 2004) and VDR knockout mice show normal keratinocytes (Sakai & Demay 2000), the above conclusion requires cautious interpretation. Indeed, the heterodimeric partner, RXR, is a requisite for normal terminal differentiation of keratinocytes (Li et al. 2001). In analogy that TRAP220 is necessary for PPAR2-mediated adipocyte differentiation (Ge et al. 2002), it may be safe to predict that it also has a dominant role for the same group of nuclear receptor (PPAR)-mediated keratinocyte differentiation. A study of primary keratinocyte culture using a TRAP220-knockdown strategy is recommended to prove this hypothesis.
In the above assays, other colony formations are comparable and other nuclear receptor functions are unaffected. However, the conclusion of this piece of study is tentative for the following reasons: firstly, the colony formation assay was influenced by cytokines present in pharmacological concentrations, which directed the differentiation of hematopoietic progenitor cells into mature blood cells (i.e. granulocytes through G-CSF and erythroid cells through EPO), and probably masked the weak contributions of other activators which used TRAP220, and secondly, the luciferase assays employed an artificial and over-expressed system. Indeed, RAR, as well as VDR, functions are proven to be significantly attenuated when HL-60 cells were used to analyze endogenous target genes of endogenous RAR (and VDR) (see below). This finding implies the existence of redundant mechanisms in in vivo system. Indeed, although the number and morphology of these colonies, including CFU-G and CFU-GM, are comparable, the function of differentiated blood cells, including granulocytes and monocytes/macrophages, in the Trap220–/– colonies might be affected and should be thoroughly investigated in future.
GATA-1, 2 and 3 transcriptional activators under the GATA family, among which GATA-1 is the most pertinent (Yamaguchi et al. 1998), operate in the development and differentiation of hematopoietic stem cells toward T-lymphocytes, erythroid cells, and megakaryocytes (reviewed in Weiss & Orkin 1995). Previous reports demonstrate significant interaction of TRAP220 with GATA-1, 2, 3, 4 and 6 (Zhu et al. 2000; Crawford et al. 2002). It is possible that the interactions contribute to erythropoiesis and megakaryopoiesis at a more physiological situation. In one study, Trap220–/– embryos show potential abnormalities in erythrocytes by histological examination and accompanied elevation of embryonic EPO (Crawford et al. 2002). Yet, since the development of hepatic primordia appears to be retarded in the runty Trap220–/– embryos (our unpublished observation), further vigorous analyses are suggested.
Finally, the existence of residual number of CFU-M denotes the existence of redundant mechanism(s), as well. The mechanism(s) may involve a c-fms-mediated intracellular signal pathway (Sariban et al. 1985).
Significance of TRAP220 for myelomonocytic differentiation in HL-60 cells
HL-60 cells were used in more detailed and mechanistic analyses, although they derive from leukemia cells and are not strictly equivalent to normal myeloid progenitor cells. They are ideal for evaluations on monocytic and granulocytic differentiation of hematopoietic progenitor cells, with the associated nuclear receptors VDR and RAR. TRAP220 and other subunits of the TRAP/Mediator complex are induced during differentiation of 1,25-(OH)2D3- and ATRA-treated HL-60 cells. TRAP220, indeed, enhanced both VDR- and RAR-mediated differentiation under physiological concentrations of cytokines. The present study is the first to establish the biological significance of TRAP220 in RAR function and in myelomonopoiesis.
The TRAP220 knockdown cells still exhibit residual amounts of direct target genes, as well as differentiation marker genes, of VDR and RAR. This can be partially attributed to the residual expression of TRAP220 in knockdown cells, but other alternative pathways (e.g. p160 coactivators and other histone acetyltransferases or methyltransferases) which do not utilize TRAP220 very problably exist. The latter possibility may well be the case when the phenotype of VDR knockout mice is taken into account, where monocytes/macrophages appear to be functional in these mice (O’Kelly et al. 2002). The results of normal colony formation in CFU-G and CFU-GM also support this hypothesis.
Transforming growth factor-ß (TGF-ß) is a recognized negative regulator of growth at all stages of hematopoiesis (reviewed in Fortunel et al. 2000). It acts synergistically with 1,25-(OH)2D3 or tumor necrosis factor (TNF) to induce monocytic differentiation (De Benedetti et al. 1990; Testa et al. 1993). Previous studies illustrate the induction of terminal differentiation by 1,25-(OH)2D3 and ATRA requiring TGF-ß1 as an autocrine mediator. It is also suggested that it participates in the differentiation of leukemia cells (Testa et al. 1993). The signaling is mediated in part by Smad proteins. The level of phosphorylated Smad2/3 is the sensor of the interplay between TGF-ß or 1,25-(OH)2D3 and ATRA on HL-60 cells and it contributes to the balance between monocytic and granulocytic differentiation pathways (Cao et al. 2003). As Smad interacts directly with ARC105/TIG-1/PAQ, a subunit of TRAP/Mediator complex (Kato et al. 2002), a synergistic mechanism of two distinct activators (nuclear receptors and Smads) may exist through simultaneous binding of these activators with the TRAP/Mediator complex (via TRAP220 and ARC105, respectively) (Ptashne & Gann 1997). Crosstalk of these distinct pathways with regards to the functions of TRAP/Mediator subunits needs to be further investigated.
One might ask why the number of Trap220–/– CFU-G in the yolk sac analysis and Gal4-RAR function in Trap220–/– MEFs are normal. A likely explanation is that the redundancy in usage of signaling molecules and cofactors described above might be different in different cells and systems. Alternatively, TRAP220 might be more active a coactivator for VDR and TR than for RAR. Further studies are necessary to elucidate the precise mechanisms.
In conclusion, this research first exemplifies the crucial role of TRAP220 in optimal VDR- and RAR-mediated myelomonocytic differentiation. Further studies modifying the interaction of TRAP220 with RAR and VDR could provide techniques to control hematopoietic cell differentiation.
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Experimental procedures |
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Trap220–/– mice were described (Ito et al. 2000). The heterozygous mutant mice, backcrossed 10 times with C57BL6J mice, were used in this study. All animal experimentation was performed according to the applied guidelines in Kobe University School of Medicine Institute for Experimental Animals.
Colony-forming cell assay of yolk sac hematopoietic cells
Yolk sacs from E9.0 embryos from the same heterozygous crossing were dissected. They were then incubated in 0.1% collagenase (Yakult, Tokyo, Japan)/phosphate-buffered saline (PBS) without calcium and magnesium/20% fetal bovine serum (FBS) (JRH Biosciences, Lenexa, Kansas, USA) for 3 h at 37 °C to make a single cell suspension (Matsuoka et al. 2001). Each E9.0 yolk sac yielded approximately 5–10 x 104 cells.
Yolk sac cells (2 x 104) were plated in media containing 0.9% methylcellulose in Iscove's Modified Dulbecco's Medium (IMDM), 15% FBS, 1% bovine serum albumin, 200 µg/mL human transferrin (iron-saturated), 10 µg/mL insulin, 1 x 10–4M 2-mercaptoethanol, and 2 mM glutamine (MethoCult M3234; StemCell Technologies, Vancouver, Canada), with supplements of 3 U/mL hrEPO (kindly provided by Chugai Pharmaceutical Co. Ltd, Tokyo, Japan), 10 ng/mL hrIL-6, 10 ng/mL hrG-CSF, 10 ng/mL mrIL-3, and 50 ng/mL mrSCF (Pepro Tech EC Ltd, London, UK) in 35-mm suspension culture dishes. Colony types were determined on day 7 of incubation at 37 °C by in situ observation using an inverted microscope and according to the criteria (Nakahata & Ogawa 1982). Then CFU-G; CFU-M; CFU-GM; BFU-E and CFU-GEMM colonies were counted.
Transient transfection assay
MEFs were isolated from E10.5 embryos of the same heterozygous mating. MEFs were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco/Invitrogen) with 10% FBS.
For luciferase assay, MEFs were transiently transfected by lipofectamine (Gibco/Invitrogen) with activator and receptor constructs with or without ligands, as described (Ito et al. 2002). Briefly, activator expression vectors driven by the CMV promoter (pCDM8, Invitrogen) were used at a level of 25 ng. Reporter plasmids (100 ng) consisted of an SV40 promoter-luciferase reporter (pGL3-Promoter, Promega) with 5 Gal4 binding sites. The Renilla luciferase control reporter pRL-SV40 (Promega) was used for controls, and the dual luciferase activities were measured using Dual-Luciferase Reporter Assay System (Promega) (Ito et al. 2002).
Cell culture
HL-60 cells, generously provided by Dr Robert C. Gallo, were maintained in RPMI1640 medium (Gibco/Invitrogen) supplemented with 10% FBS in a humidified atmosphere flushed with 5% CO2 in air. Cells were induced to differentiate by treatment with 1 x 10–7M 1,25-(OH)2D3 (The Teijin Institute for Bio-Medical Research, Tokyo, Japan), 1 x 10–6 M ATRA (SIGMA), 1 x 10–8 M TPA (SIGMA), or 1.25% DMSO (SIGMA) in RPMI1640 medium at 37 °C for the durations described in the text.
Northern blot analysis
Total RNA was extracted from HL-60 cells by a guanidine thiocyanate method and subjected to an RNA blot analysis (Ito et al. 2000). The probes used were human fragments of the following: 0.8 kb TRAP220 cDNA, 1.6 kb TRAP170 cDNA, 0.9 kb TRAP240 cDNA, 1.0 kb glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA, 0.4 kb VDH cDNA, 0.5 kb CD38 cDNA, 0.4 kb gp91 cDNA, and lastly, 0.7 kb fragment of mouse SRC-1 cDNA.
Transient expression of siRNA in HL-60 cells (nucleofection)
A human U6 construct expressing siRNA vector (pAVU6 +27) was kindheartedly provided by Dr David R. Engelke (The University of Michigan, Ann Arbor, MI, USA) (Paul et al. 2002). A pair of oligodeoxynucleotides containing the 21-mer sense and anti-sense sequences of TRAP220, with a 4-mer insert of loop and 4 Ts, were synthesized, annealed, and cloned into the SalI-XbaI sites of pAVU6 +27. A pAVU6 +27 vector was used as a non-silencing control.
Cells (5 x 106) were collected by centrifugation, washed once with PBS equilibrated to room temperature, and gently resuspended in 10 µL of PBS and 90 µL of Cell Line Nucleofector Solution V (amaxa, Cologne, FRG). A DNA plasmid (1 µg) was added and introduced directly into the nuclei by the Nucleofector device (amaxa) with electrical setting T-04 according to the manufacturer's instruction. Immediately after nucleofection, 500 µL of RPMI1640 complete medium was added. Cell suspension was transferred to 35-mm suspension culture dishes filled with 4.5 mL of RPMI1640 complete medium, followed by incubation at 37 °C for 6 h. Then 1,25-(OH)2D3, ATRA, TPA, or DMSO was added to the medium and cells were incubated for an additional 24 h.
Flow cytometric analysis
Cells stained with fluorescein isothyocyanate (FITC)- or phycoerythrin (PE)-conjugated monoclonal antibodies were analyzed on a dual-laser cell sorter (FACScan; Becton Dickinson). Monoclonal antibody CD14 (Becton Dickinson) was conjugated with FITC, while CD11b (IMMUNOTECH) and CD38 (Becton Dickinson) were conjugated with PE.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: itomi{at}med.kobe-u.ac.jp
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Received: 10 July 2005
Accepted: 8 September 2005
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