Leukotriene B4 (LTB4) is a lipid mediator with potent chemoattractant properties and rapidly generated from activated innate immune cells such as neutrophils, macrophages, and mast cells. Previously, our laboratory identified two types of LTB4 receptors, which are LTB4 type-1 receptor (BLT1) and type-2 receptor (BLT2). Several groups have certified the pivotal roles of BLT1 in peripheral leukocytes. BLT1-deficient mice have revealed that LTB4-BLT1 pathway appears to play important roles in various inflammatory diseases, such as allergen-induced airway hyperresponsiveness, autoimmune arthritis and atherosclerosis. Although BLT1 is exclusively expressed in peripheral leukocytes, suggesting the stringent regulation of its transcription, the precise mechanism of the BLT1 expression is not fully understood. In this thesis, I present two findings of the transcriptional regulation in human promyelocytic leukemia cell line (HL-60 cells), in which the BLT1 expression is upregulated by stimulation with retinoic acid (RA).

First, I analyzed a core-promoter contributing to the BLT1 expression. The core-promoter of BLT1 possesses a crucial site (GC box) for the recognition of specificity protein 1 (Sp1). Interestingly, western blot analyses and electrophoretic mobility shift assays revealed that Sp1 is not produced in HL-60 cells, and that other proteins which belong to Kruppel-like factor (KLF) family could bind to this site. It has been reported that KLF1, KLF2 and KLF3 are specifically expressed in hematopoietic cells. I confirmed the expressions of these factors in HL-60 cells by PCR and western blot analyses, and examined the abilities of these factors for the activation of the BLT1 promoter by reporter assays. Thus, some KLFs, e.g., KLF1, can work as regulators for the BLT1 expression in HL-60 cells.

The core-promoter activity is not enhanced directly by the stimulation with RA in HL-60 cells, indicating the possible existence of enhancer region(s) at other loci. Therefore, I searched for the region involving in the enhanced expression of BLT1 in my second theme. DNase I-hypersensitivity analyses revealed an activated region in the RA-stimulated cells, termed AE-BLex, at the intron-I:exon-II boundary of the BLT1 gene. By the reporter assays, I elucidated that AE-BLex acts as an enhancer for the BLT1 promoter. AE-BLex possesses two acute myeloid leukemia 1 (AML1/Runx1) recognition sites. These sites are pivotal for the enhancer function because the BLT1 expression was impaired by the knockdown of AML1 in HL-60 cells. The enhancement of the BLT1 expression during the RA-induced differentiation of HL-60 cells is due to a loosening of the chromatin structure around AE-BLex, which leads to the incremental binding of AML1. The AML1/AE-BLex complex was further confirmed in other BLT1-expressing leukemia cell lines and human peripheral leukocytes. Thus, AML1 enhances the BLT1 expression through binding to AE-BLex, which is accessible in leukocytes. This thesis shows a part of the regulation of the BLT1 expression in HL-60 cells, and may be helpful to understand the transcriptional mechanisms in leukocytes.

Schematic model of BLT1 expression in leukocytes

The gradation of each bar indicates the expression levels of KLFs, AML 1 and BL T1 during hematopoiesis. In HSCs (hematopoietic stem cells), the BLTI gene is silenced due to inactivation of the BLTI promoter and AE-BLex. The chromati n structures of the BLTI promoter and AE-BLex are altered by historic acetylation during the terminal differentiation. GTFs containing KLFs are recruited to the BLT1 promoter. Following recruitment to AE-BLex, AML 1 facilitates the activity of the BLT1 promoter. Upregulated BLTI expression is crucial for inflammation and the immune response in leukocytes (e.g., neutrophils, cosi nophi Is, activated T-cells, dendritic cells and macrophages). GTFs; general transcription factors, P; BLTI promoter.

本研究は、白血球走化因子であるロイコトリエンB4(LTB4)の第一受容体(BLT1)の白血球における発現調節機構を明らかにするため、ヒト骨髄性白血病細胞株HL-60をレチノイン酸にて分化誘導する系を用いて、プロモーターに作用する転写因子の探索とエンハンサー領域の特定を試みたものであり、下記の結果を得ている。

1. Electrophoretic mobility shift assayの結果より、HL-60細胞においてBLT1のプロモーター領域内のSp1結合サイト(転写活性に必要)へのSp1以外の転写因子の結合を見出した。またウエスタンブロット解析の結果より、HL-60細胞ではSp1が検出出来なかった。

2. Reporter gene assay、Electrophoretic mobility shift assayの結果から、この転写因子がSp1と同様にSp1結合サイトを認識するKLFファミリーのメンバーである可能性を示唆した。

3. レチノイン酸刺激したHL-60細胞におけるBLT1のmRNA増加は、レチノイン酸がBLT1のプロモーターへ直接作用するためではない事をreporter gene assay やchromatin immunoprecipitation (ChIP) assayより示した。さらにDNase I-hypersensitivity assayの結果から、レチノイン酸反応エレメント(AE-BLex)がBLT1のイントロン1とエクソン2の間に存在する事を明らかにした。

4. AE-BLexはエンハンサー活性を持つ事、さらにこの活性には血球の分化・成熟に寄与する転写因子であるAML1の結合サイトが重要である事をreporter gene assayの結果より明らかにした。siRNAを用いたAML1ノックダウン実験ではAML1の発現低下とともにBLT1のmRNA量の減少も観察した。またChIP assayでは、レチノイン酸刺激によりAE-BLex付近のヒストンのアセチル化が上昇し、AML1の結合が増加する事も示唆した。

5. 血球系細胞株において、BLT1が発現している細胞ではAE-BLex へのAML1の結合が見られたのに対し、未発現の細胞ではAE-BLex 付近のヒストンのアセチル化およびAML1の結合が検出出来なかった。またヒト末梢血由来の白血球においても、AE-BLex 付近においてAML1が結合している事を明らかにした。

以上、本論文は、ヒト骨髄性白血病細胞株HL-60において、KLFファミリーがBLT1のプロモーターに作用しうる可能性と、AML1がエンハンサーを介してBLT1の発現に寄与している事を明らかにした。本研究はこれまで未知に等しかった、白血球におけるBLT1の発現調節の解明に重要な貢献をなすと考えられ、学位の授与に値するものと考えられる。