Department of Biochemistry, School of Life Sciences, University of Hyderabad, India.
FEBS J. 2020 Dec;287(24):5411-5438. doi: 10.1111/febs.15329. Epub 2020 May 1.
Thrombocytopenia is characterized by low platelet count and is typically observed among all preterm and low birthweight neonates admitted to the neonatal intensive care unit. Although the underlying cause for this predisposition is unclear, recent studies have proposed that the intrinsic inability of neonatal hematopoietic stem/progenitor cells to produce mature polyploid megakaryocytes (MKs) may result in delayed platelet engraftment. The developmental and molecular differences between neonatal and adult MKs are not yet fully understood. Previously, we had reported that the key MK transcription factor RUNX1, which is crucial for the regulation of MK specification and maturation, is down-regulated in neonatal MKs when compared with adult MKs. In humans, loss-of-function mutations in RUNX1 cause familial platelet disorder, which is characterized by thrombocytopenia, indicating its crucial role in MK development. However, information about its cross talk with developmentally regulated signaling pathways in MKs is lacking. In this study, we performed a differential gene expression analysis in MKs derived from human cord blood (CB) and adult peripheral blood (PB) CD34 cells. Further, validation and correlation studies between RUNX1 and transforming growth factor beta (TGF-β) were performed in a differentiating megakaryocytic cell line model. The analysis revealed that TGF-β pathway was the main pathway affected between CB- and PB-MKs. RUNX1 is reported to be a modulator of TGF-β signaling in several studies. The correlation between RUNX1 and TGF-β pathway was analyzed in the PMA-induced megakaryocytic differentiating K562 cells, which exhibit mature megakaryocytic features. The RT profiler PCR array analysis revealed that TGF-β pathway components were up-regulated in the PMA-induced megakaryocytic differentiating cells. Furthermore, our study indicated that human TGF-β1 promotes cytosolic calcium (Ca ) activity and MK maturation. We noticed that TGF-β1 increased intracellular free Ca ([Ca ]i) via reactive oxygen species-mediated activation of transient receptor potential (TRP) ion channels. Moreover, we observed that decreased cytosolic Ca activity in the siRUNX1-transfected cells was associated with down-regulation of TRP ion channel expression. Finally, we demonstrated that TGF-β/SMAD signaling augments the development of MKs derived from CB-CD34 . Present data suggest that RUNX1/TGF-β pathway cross talk is crucial for MK maturation.
血小板减少症的特征是血小板计数低，通常见于所有入住新生儿重症监护病房的早产儿和低出生体重儿。尽管这种易感性的潜在原因尚不清楚，但最近的研究表明，新生儿造血干/祖细胞产生成熟多倍体巨核细胞（MK）的内在能力可能导致血小板植入延迟。新生儿和成人 MK 之间的发育和分子差异尚未完全理解。之前，我们曾报道过关键的 MK 转录因子 RUNX1，它对于 MK 特化和成熟的调节至关重要，与成人 MK 相比，在新生儿 MK 中下调。在人类中，RUNX1 的功能丧失突变导致家族性血小板疾病，其特征是血小板减少，表明其在 MK 发育中起关键作用。然而，关于其与 MK 中发育调节信号通路的交叉对话的信息尚不清楚。在这项研究中，我们对来自人脐血（CB）和成人外周血（PB）CD34 细胞的 MK 进行了差异基因表达分析。此外，在分化的巨核细胞系模型中对 RUNX1 和转化生长因子β（TGF-β）之间的验证和相关性研究进行了研究。分析表明，CB-MK 和 PB-MK 之间受影响的主要途径是 TGF-β 途径。在几项研究中，RUNX1 被报道为 TGF-β 信号的调节剂。在 PMA 诱导的巨核细胞分化 K562 细胞中分析了 RUNX1 与 TGF-β 途径之间的相关性，该细胞表现出成熟巨核细胞的特征。RT 谱 PCR 阵列分析显示，PMA 诱导的巨核细胞分化细胞中 TGF-β 途径成分上调。此外，我们的研究表明，人类 TGF-β1 促进细胞质钙离子（Ca ）活性和 MK 成熟。我们注意到 TGF-β1 通过活性氧物质介导的瞬时受体电位（TRP）离子通道的激活增加细胞内游离钙（[Ca ]i）。此外，我们观察到在 siRUNX1 转染细胞中细胞浆 Ca 活性降低与 TRP 离子通道表达下调有关。最后，我们证明了 TGF-β/SMAD 信号增强了来自 CB-CD34 的 MK 的发育。目前的数据表明，RUNX1/TGF-β 途径的相互作用对于 MK 成熟至关重要。
