MGP 与 BMP-4 和 BMP-2 相互作用并支持正常和恶性造血干细胞/祖细胞,Blood

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MGP 与 BMP-4 和 BMP-2 相互作用并支持正常和恶性造血干细胞/祖细胞
Blood ( IF 21.0 ) Pub Date : 2018-11-29 , DOI: 10.1182/blood-2018-99-110138
Aya Yokoi ₁ , Kana Kuronuma ₁ , Tomoya Fukuoka ₁ , Chie Goto ₁ , Miki Matsuo ₁ , Eri Adachi ₁ , Naoko Okada ₁ , Hikaru Tsutsumi ₁ , Mirano Kajitani ₁ , Natsumi Hasegawa ₁ , Shigetaka Asano ₂ , Mitsuhiro Ito _{1,

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Affiliation

摘要通用转录共调控复合物Mediator是RNA聚合酶II全酶的亚复合物，是多种细胞内信号的终点汇聚，通过募集通用起始因子和形成功能性预起始复合物来启动转录。在介质亚基中，MED1 对造血至关重要：已知它可用于参与造血生态位功能的基因的转录，以及造血前体细胞的分化（例如，RARα 和 VDR 介导的骨髓单细胞生成、GATA1 介导的红细胞巨核细胞生成和 iNKT 细胞发育）。Med1-/- 间充质基质细胞中的减毒基因是基质 Gla 蛋白 (MGP)，它是 BMP-SMAD 信号的调节剂。由于 MGP 在骨组织中大量表达并且据报道与生态位因子 BMP-4 和 BMP-2 相互作用，我们假设 MGP 可能通过与这些 BMP 的相互作用调节造血中的生态位功能。我们通过由骨髓 (BM) 间充质基质细胞和 BM 造血细胞共培养的体外生态位模型测试了这种可能性。当小鼠骨髓造血细胞与 OP-9 或 MS-5 小鼠骨髓基质细胞在 MGP 封闭抗体存在下共培养时，骨髓造血细胞的生长和 DNA 合成减弱，长期培养起始细胞的数量减少。与受支持的造血干细胞/祖细胞 (HSPCs) 相对应的细胞 (LTC-ICs) 减少。我们接下来询问 MGP 是否也可作为恶性 HSPCs 的利基因子，为此，我们利用 MB-1 细胞，急变危象 CML 衍生的成髓细胞白血病细胞。MB-1 细胞依赖于共培养的 BM 基质细胞并具有白血病干细胞 (LSC) 的特征。当 MB-1 细胞与 OP-9 或 MS-5 BM 基质细胞在抗 MGP 抗体存在下共培养时，MB-1 细胞的数量，以及鹅卵石形成的数量，即 LSC 性质， MB-1 细胞，显着减毒。因此，MGP 似乎对造血细胞具有营养作用，并在我们的生态位模型中支持正常和恶性 HSPC。MGP 在 mRNA 和蛋白质水平上都在 BM 基质细胞中大量表达，但在正常造血细胞中却很少表达。然而，MB-1 白血病细胞（可能是异位的）表达了有意义的高水平 MGP。在正常 BM 造血细胞与 OP-9 或 MS-5 BM 基质细胞共培养期间，一天后，MGP 被显着和短暂地诱导。在 MS-5 或 OP-9 BM 基质细胞中表达的 MGP 在用 BM 造血细胞培养 1 天后同样被瞬时诱导，BM 造血细胞通过 transwell 装置与这些 BM 基质细胞物理分离，表明分泌的体液因子诱导BM基质细胞中的MGP表达。对于恶性情况，MGP在MB-1白血病细胞和OP-9或MS-5 BM基质细胞中的表达均呈振荡天数波动。由于HSPCs的支持需要它们与BM基质细胞的结合，诱导的MGP表达显然不足以支持HSPCs，诱导的MGP的作用可能是间接的。因此，我们接下来询问据报道与 MGP 相关的 BMP-4 和 BMP-2，也被共培养诱导，并与 MGP 联合用于 HSPCs 支持。事实上，在共培养过程中，BMP-4 在几天内被迅速和短暂地诱导，同时与 MGP 一起，随后持续诱导 BMP-2，持续了一个多星期。然而，当使用 transwell 装置与解离的 BM 造血细胞一起培养时，BM 基质细胞既不产生 BMP-4 也不产生 BMP-2。因此，BMP2/BMP4 的诱导依赖于造血细胞和基质细胞的物理结合。GST-pulldown 试验和哺乳动物双杂交试验证实 MGP 与 BMP-4 和 BMP-2 特异性相互作用。为了阐明针对 MGP 的阻断抗体的作用机制，我们测试了该抗体是否抑制了 MGP 和 BMP-4 之间的相互作用。确实，一系列 GST pulldown 测定揭示了抗体的添加特异性地消除了 GST-MGP 和 BMP-4 之间的相互作用。这些结果表明，MGP 可能至少部分地通过与 BMP-4 和 BMP-2 的相互作用和调节 BMP-4 和 BMP-2 的作用作为正常和恶性 HSPC 的利基因子。披露没有需要声明的相关利益冲突。

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MGP Interacts with BMP-4 and BMP-2 and Supports Normal and Malignant Hematopoietic Stem/Progenitor Cells

Abstract The general transcriptional coregulatory complex Mediator, subcomplex of the RNA polymerase II holoenzyme, is the endpoint convergence of a variety of intracellular signals, and initiates transcription through recruitment of general initiation factors and formation of a functional preinitiation complex. Among the Mediator subunits, MED1 is crucial for hematopoiesis: it is known to be employed in transcription of genes involved in hematopoietic niche function, as well as in differentiation of hematopoietic precursor cells (e.g., RARα- and VDR-mediated myelomonopoiesis, GATA1-mediated erythromegakaryopoiesis, and iNKT cell development). Among the attenuated genes in Med1-/- mesenchymal stromal cells was matrix Gla protein (MGP), modulator of the BMP-SMAD signals. As MGP is abundantly expressed in bone tissues and reportedly interacts with niche factors BMP-4 and BMP-2, we hypothesized that MGP might modulate niche function in hematopoiesis through the interaction with these BMPs. We tested this possibility by an in vitro niche model composed of a coculture of bone marrow (BM) mesenchymal stromal cells and BM hematopoietic cells. When mouse BM hematopoietic cells were cocultured with OP-9 or MS-5 mouse BM stromal cells in the presence of the blocking antibody against MGP, the growth and DNA synthesis of BM hematopoietic cells were attenuated and the number of long-term culture-initiating cells (LTC-ICs), which corresponded to the supported hematopoietic stem/progenitor cells (HSPCs), decreased. We next asked if MGP also functioned as a niche factor for malignant HSPCs, To this end, we utilized MB-1 cells, blast crisis CML-derived myeloblastic leukemia cells. MB-1 cells are dependent on cocultured BM stromal cells and possess characteristics of leukemia stem cells (LSCs). When MB-1 cells were cocultured with OP-9 or MS-5 BM stromal cells in the presence of the anti-MGP antibody, the number of MB-1 cells, as well as the number of cobblestone formation, i.e., LSC nature, of MB-1 cells, was profoundly attenuated. Therefore, MGP appears to have a trophic effect on hematopoietic cells and to support normal and malignant HSPCs in our niche model. MGP was, both at mRNA and protein levels, expressed abundantly in BM stromal cells but scarcely in normal hematopoietic cells. However, MB-1 leukemia cells (presumably ectopically) expressed a meaningfully high level of MGP. During the coculture of normal BM hematopoietic cells with OP-9 or MS-5 BM stromal cells, MGP was induced prominently and transiently after one day. MGP, expressed in MS-5 or OP-9 BM stromal cells, was likewise transiently induced after 1 day of culture with BM hematopoietic cells that were physically dissociated from these BM stromal cells by the transwell apparatus, indicating that secreted humoral factor(s) induced the MGP expression in the BM stromal cells. As for a malignant situation, the expressions of MGP in both MB-1 leukemia cells and OP-9 or MS-5 BM stromal cells were fluctuated in an oscillated days period. Since the support of HSPCs required their association with BM stromal cells, the induced MGP expression was apparently insufficient for HSPCs support, and the action of induced MGP may be indirect. Therefore, we next asked if BMP-4 and BMP-2, which reportedly associate with MGP, were also induced by the coculture and were employed in HSPCs support jointly with MGP. Indeed, during the coculture, BMP-4 was rapidly and transiently induced within a few days, simultaneously with MGP, followed by a subsequent and sustained induction of BMP-2 that lasted for over a week. However, neither BMP-4 nor BMP-2 was produced by BM stromal cells when cultured with dissociated BM hematopoietic cells using the transwell apparatus. Therefore, the induction of BMP2/BMP4 was dependent on physical association of hematopoietic cells and stromal cells. GST-pulldown assays and mammalian two-hybrid assays confirmed that MGP specifically interacted with both BMP-4 and BMP-2. To elucidate the mechanism of action of the blocking antibody against MGP, we tested if the antibody inhibited the interaction between MGP and BMP-4. Indeed, serial GST pulldown assays disclosed that the addition of the antibody specifically abolished the interaction between GST-MGP and BMP-4. These results indicate that MGP might act as a niche factor for normal and malignant HSPCs, at least in part, through interacting with, and modulating the action of, BMP-4 and BMP-2. Disclosures No relevant conflicts of interest to declare.

更新日期：2018-11-29

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