Plasma membrane-mediated non-genomic effects of T4, T3 and thyroid hormone metabolite tetrac on different aspects of mesenchymal stem cell biology and their signaling pathways

近年来，我们和其他人已经表明，间充质干细胞（MSCs）选择性地回到损伤部位，并通过分化和释放促血管生成因子在组织修复、伤口愈合和癌症中发挥重要作用。在上一个资助期内，我们首次证明了T4和T3是成纤维细胞/周细胞样细胞中MSC分化的重要刺激因子，并对MSC迁移、侵袭和募集具有直接刺激作用。这些作用被证明是通过非基因组机制介导的，通过avb3的四氯乙烯抑制。这一观察结果的可能分子机制之一是T4/ t3介导的MSCs缺氧反应网络的刺激，这在我们过去的资助期也得到了证实。作为我们过去研究的逻辑结果，在下一个资助期，我们将更详细地分析非基因组avb3介导的T4、T3和tetrac对MSCs迁移和MSCs介导的血管生成效应的机制基础，特别强调所涉及的信号通路的表征。为此，我们将分析T4， T3和tetrac对1)与MSC迁移相关的MSC细胞表面标记物，整合素和受体的表达，2)MSCs分泌血管生成因子及其对内皮细胞管形成的影响。根据其他人和我们自己小组的初步数据，PI3K/Akt， MAPK和典型Wnt通路是进一步分析其参与avb3介导的T4， T3和tetrac在MSC生物学中的非基因组效应的逻辑候选者。因此，在目前的提案中，我们将通过分析T4、T3在存在或不存在tetrac的情况下处理MSCs后的下游靶点，以及应用这些途径的特异性抑制剂后对MSC迁移和侵袭的影响来研究这些途径。此外，我们将利用我们长期以来在应用NIS作为治疗基因方面的专业知识，在体外和体内使用通路应答性报告基因试验来评估典型Wnt通路以及两个中央血管生成相关通路，即Tie-2和VEGF信号通路的激活状态。基于伤口愈合过程与肿瘤间质形成过程之间的强烈相似性，我们之前研究建立的肝癌异种移植小鼠模型将被用作慢性伤口/组织修复的稳健模型。这些研究将显著增强我们对甲状腺激素T4和T3以及四羟丙基在慢性伤口/组织修复中调控MSC生物学的关键作用的理解。同时，我们的研究将扩大我们对整合素avb3作为甲状腺激素作用介质的演变作用及其治疗意义的理解。

In the recent years we and others have shown that mesenchymal stem cells (MSCs) selectively home to sites of injury and play an important role in tissue repair, wound healing and cancer through differentiation and the release of proangiogenic factors. In the last funding period we demonstrated, for the first time, that T4 and T3 are important stimulators of MSC differentiation in fibroblast-/pericyte-like cells, and have direct stimulatory effects on MSC migration, invasion and recruitment. These effects were shown to be mediated through non-genomic mechanisms via avb3 by their tetrac inhibition. One of the possible molecular mechanisms for this observation is T4/T3-mediated stimulation of the hypoxia response network in MSCs which was also demonstrated in our past funding period. As a logical consequence of our past studies, in the next funding period we will analyze in more detail the mechanistic basis for the non-genomic avb3-mediated effects of T4, T3 and tetrac on the migration of MSCs and MSC-mediated angiogenic effects with a special emphasis on characterization of the signaling pathways involved. For this purpose we will analyze the effects of T4, T3 and tetrac on 1) the expression of MSC cell surface markers, integrins and receptors that are associated with MSC migration, 2) the secretion of angiogenic factors by MSCs and their effect on endothelial cell tube formation. Based on preliminary data by others and our own group, the PI3K/Akt, MAPK and canonical Wnt pathways are logical candidates for further analysis as to their involvement in avb3-mediated non-genomic effects of T4, T3 and tetrac in MSC biology. Therefore, in the current proposal these pathways will be investigated by analysis of downstream targets after treatment of MSCs with T4, T3 in the presence or absence of tetrac, and the effect on MSC migration and invasion after application of specific inhibitors of these pathways. In addition, the activation status of the canonical Wnt pathway as well as two central angiogenesis-related pathways, Tie-2 and VEGF signaling pathways, will be assessed by using pathway-responsive reporter assays in vitro and in vivo using NIS as a reporter gene making use of our longstanding expertise in the application of NIS as theranostic gene. Based on the strong analogies between the process of wound healing and tumor stroma formation, the liver cancer xenograft mouse model established in our previous studies will be used as a robust model of chronic wound/tissue repair. These studies will significantly enhance our understanding of the critical role of thyroid hormones T4 and T3 as well as tetrac in regulation of MSC biology in the context of chronic wound/tissue repair. At the same time, our studies will expand our understanding of the evolving role of integrin avb3 as mediator of thyroid hormone action and its therapeutic implications.