武汉云克隆科技股份有限公司

Abstract：

Increased synthesis of endothelin-1 (ET-1) by human vascular endothelial cells (HVECs) in response to hypoxia underscores persistent vasoconstriction observed in patients with pulmonary hypertension. The molecular mechanism whereby hypoxia stimulates ET-1 gene transcription is not well understood. Here we report that megakaryocytic leukemia 1 (MKL1) potentiated hypoxia-induced ET-1 transactivation in HVECs. Disruption of MKL1 activity by either a dominant negative mutant or small interfering RNA mediated knockdown dampened ET-1 synthesis. MKL1 was recruited to the proximal ET-1 promoter region (−81/+150) in HVECs challenged with hypoxic stress by the sequence-specific transcription factor serum response factor (SRF). Depletion of SRF blocked MKL1 recruitment and blunted ET-1 transactivation by hypoxia. Chromatin immunoprecipitation analysis of the ET-1 promoter revealed that MKL1 loss-of-function erased histone modifications consistent with transcriptional activation. In addition, MKL1 was indispensable for the occupancy of Brg1 and Brm, key components of the chromatin remodeling complex, on the ET-1 promoter. Brg1 and Brm modulated ET-1 transactivation by impacting histone modifications. In conclusion, our data have delineated a MKL1-centered complex that links epigenetic maneuverings to ET-1 transactivation in HVECs under hypoxic conditions.

摘要：

在肺动脉高压患者中观察到人血管内皮细胞(HVECs)使得内皮素-1(ET-1)合成增加强调了缺氧加重血管收缩作用。但是缺氧刺激ET-1基因转录的分子机制尚不清楚。研究表明MKL1增强了人血管内皮细胞缺氧诱导内皮细胞的ET-1活化。通过显性失活突变体或小干扰RNA介导的敲低抑制ET-1合成破坏MKL1活性。在人血管内皮细胞中MKL1聚集于近端ET-1启动子区（−81/+150），受到序列特异性转录因子血清应答因子（SRF）对低氧应激的影响。SRF耗竭阻断MKL 1的再吸收，使ET-1在缺氧时减弱。ET-1启动子的染色质免疫共沉淀分析表明，MKL1失活清除了组蛋白修饰，与转录激活相一致。此外，ET-1启动子的研究表明，MKL1作为染色质重塑复合物的关键组分，同时对于Brg1和Brm在ET-1启动子上的占据是必不可少的。 Brg1和Brm 通过影响组蛋白修饰调节ET-1的活化。本研究数据描述了一个以MKL1为中心的复合物，该复合体将表观遗传操纵与缺氧条件下HVECs中内皮素-1(ET-1)的激活联系起来。

使用试剂原文信息：Enzyme-linked immune absorbance assay (ELISA) was performed using supernatant collected from cultured HVECs or rat pulmonary artery homogenates according to manufacturer’s instructions (USCN Life Sciences).