水解磷酸脂受器調控血球幹細胞的骨髓分化之研究

Activation of lysophosphatidic acid receptors regulates differentiation of hematopoietic stem cells in myeloid lineage

林貫浤(Kuan-Hung Lin)

指導教授 ： 李心予

國立臺灣大學/生命科學院/生命科學系/博士(2019年)

https://doi.org/10.6342/NTU201902498

摘要

水解磷酸脂 (Lysophosphatidic acid, LPA) 是由細胞膜上面分離出來的一種小分 子水解磷脂質。經由活化細胞膜上面不同種類的水解磷酸脂受器，會在體內會產 生各式各樣的生理作用。在本研究當中，我們利用四種不同的實驗模式來對水解 磷酸脂受器進行分化的研究。結果發現水解磷酸脂受器二型會抑制紅血球之分化。 相反地，受器三型會促進紅血球之分化。另外一方面，因為紅血球和血小板來自 同一個前趨細胞 Megakaryocyte/erythroid progenitor (MEP)，因此我們想探討水解 磷酸脂對於血小板分化路徑之調控。研究結果發現，受器三型在血小板分化的過 程中反而扮演一個抑制分化的角色。我們更發現在不同時期的血球幹細胞，它們 的受器二型和三型的表現量有相當大的差異，而此表現之形式跟 GATA 因子的 表現量具有一定程度的重疊性。研究結果也顯示水解磷酸脂受器二型和三型的表 現量確實是經由 GATA 因子所調控，而在受器二型和三型的啟動子區域也確實 有 GATA 因子的接合位置。經由染色質免疫沈澱的結果也指出 GATA 因子確實 會接合到這些位置上，進而調控受器二型和三型的表現。這些研究結果應可對紅 血球或血小板相關疾病治療提供重要背景知識。

關鍵字

水解磷酸脂 ； 造血生成 ； 骨髓分化 ； 貧血

並列摘要

Lysophosphatidic acid (LPA) is a membrane-derived lysophospholipid that exists in plasma and platelet. It exerts its functions through activating various LPA receptors (LPARs), which belong to the family of G-protein coupled receptors (GPCRs). Activation of LPARs has been implicated for important roles during stem cell differentiation. However, how LPA affects human hematopoietic stem cell (HSC) differentiation and underlying lineages remains elusive. In our previous studies, we have demonstrated that activation of LPA receptor 3 (LPA3) promotes erythropoiesis in human HSC and zebrafish. In the present study, pharmacological approach was adopted to further elucidate the functions of LPA receptors during red blood cell (RBC) differentiation. Treatment of LPA2 agonist DMP and GRI977143 suppressed erythropoiesis, whereas activation of LPA3 by 2S-OMPT promoted RBC differentiation both in vitro (K562 and HSC) and in vivo (zebrafish and mice). Furthermore, our results demonstrated a negative role of LPA3 during platelet differentiation both in vitro and in vivo. Moreover, the expression pattern of LPA receptors correlated to the hematopoietic transcriptional factors GATA1 and GATA2 in different stages of myeloid progenitors. In addition, manipulation of these GATA factors affected the expression level of LPA2 and LPA3. By luciferase assays, we demonstrated that the promoter region of LPAR2 and LPAR3 were regulated by these GATA factors. Mutation of identified GATA-Binding-Sites (GBS) in the region abrogate the luciferase activities, suggested that LPA2 and LPA3 are transcriptionally regulated by GATA factors. In addition, chromatin immunoprecipitation assays confirmed the directly interaction between GATA factors and promoter region of LPARs. Taken together, our results suggested that the expression level of LPA2 and LPA3, which may be determined by GATA factors, is a regulatory switch for the lineage commitment in myeloid progenitors. The pharmacological activation of LPA receptor subtypes represents a novel strategy for regulating erythropoiesis.

並列關鍵字

Lysophosphatidic acid ； Hematopoiesis ； Myeloid differentiation ； Anemia

參考文獻

1. Orkin SH, Zon LI: Hematopoiesis: an evolving paradigm for stem cell biology. Cell 2008, 132(4):631-644.

2. Palis J, Robertson S, Kennedy M, Wall C, Keller G: Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse. Development (Cambridge, England) 1999, 126(22):5073-5084.

3. Palis J: Ontogeny of erythropoiesis. Current opinion in hematology 2008, 15(3):155-161.

4. Lacaud G, Kouskoff V: Hemangioblast, hemogenic endothelium, and primitive versus definitive hematopoiesis. Experimental hematology 2016.

5. Murray PDF: The Development in vitro of the Blood of the Early Chick Embryo. Proceedings of the Royal Society of London Series B, Containing Papers of a Biological Character 1932, 111(773):497-521.