ROS-Notch3通路在TGF-β1诱导的血管平滑肌细胞分化中的作用及其调控机制

TGF-β signaling has been studied consistently in vascular remodeling and pathogenesis. However, the interplay between TGF-β and other signaling in these events still remains under investigation, particularly in the differentiation of vascular smooth muscle cells (VSMC). We have previously disclosed a new pathway in VSMC, reactive oxygen species (ROS)-Notch3 pathway. Afterwards, we found that ROS-Notch3 pathway was also involved in TGF-β1 induced VSMC differentiation, and TGF-β1 stimulation elevated Notch3 activity. The current study is aimed to understand the function of ROS-Notch3 pathway in TGF-β induced VSMC differentiation and the underlying mechanisms. Experiments are carried out to further confirm role of ROS-Notch3 in TGF-β induced VSMC differentiation and the specific pathway how TGF-β1 activates Notch3, to study the interaction between Notch3 ICD and p-Smad, and whether ROS level affects the interaction, to understand the impact of ROS-Notch3 pathway on TGF-β signaling and p-Smad activity in regulating transcription of VSMC differentiation markers，and to demonstrate role of MAPK signaling in ROS-Notch3 pathway and VSMC differentiation. The involved methods include biochemical and molecular biology techniques. Our results will facilitate the understanding of signal transduction in vascular remodeling and pathogenesis, and provide theoretical and experimental evidences in developing new therapeutic strategy against vascular diseases.

TGF-β通路在血管重构及病变中已成为研究的热点，然而这过程中与其它信号间的交互调控机制仍尚未明确，尤其在血管平滑肌细胞（VSMC）分化方面有待于进一步深入。申请者前期研究已经揭示了VSMC中活性氧自由基（ROS）-Notch3新通路。随后初步发现ROS-Notch3通路参与TGF-β1对VSMC的分化调控，并且TGF-β1能激活Notch3。本课题拟采用生化和分子生物学方法，进一步确认上述调控作用和Notch3具体激活途径，研究Notch3活性区与p-Smad的结合作用以及ROS对两者结合活性的影响，明确ROS-Notch3对TGF-β信号活性和p-Smad调控分化基因转录的影响，确定MAPK信号与新通路的关系及在分化中的作用，从而阐明ROS-Notch3通路在VSMC分化调控过程中的功能和机制。我们的研究成果将有助于了解血管重构及病变的信号转导机制，为治疗策略的研究提供理论和实验依据。

前期研究发现ROS-Notch3通路可能参与TGF-β1诱导的细胞分化，但具体机制尚未明确。本研究利用具有平滑肌分化标志基因表达的肺成纤维细胞IMR-90发现了ROS-MAPKs-Notch3通路对成纤维细胞分化的调控作用及其机制。发现在低ROS水平时，ERK1/2信号占优势，对细胞分化表现为抑制作用；在TGF-β1刺激ROS水平升高时，p38、JNK1/2和Notch3信号被激活和增强，ERK1/2信号占劣势，对细胞分化表现为促进作用。此外，课题组发现抗氧化剂N-乙酰半胱氨酸（NAC）对Notch3蛋白具有显著下调作用。并在肿瘤细胞中阐明了相关机制和功能，即NAC通过溶酶体途径降低Notch3蛋白水平，这种调控不依赖于ROS水平，并且可显著抑制多种Notch3依赖性肿瘤细胞的恶性表型。总之，课题组基本完成了本项目的预定目标，并发现了Notch3新的调控机制及功能。本项目的完成对细胞分化异常的内在调控机制及相关疾病，以及对肿瘤的治疗和预防皆有较为重要的意义。