Novel roles of C1q tumor necrosis factor-related proteins (CTRPs) in relaxin receptor biology

我研究了松弛蛋白受体和结合伙伴在细胞分化中的作用。我的实验室建立了松弛素(RLX)和INSL3作为细胞运动和细胞外基质(ECM)侵袭的新介质。我们还展示了另一种松弛素样多肽Insl5在小鼠血糖稳态中的新功能，并发现人和小鼠大肠的肠内分泌细胞是INSL5的特异性产生细胞。最近，我的实验室发现了一个新的松弛蛋白受体RXFP1的结合伙伴。分泌的C1qTNF相关肽CTRP8蛋白在结构上不同于RLX。CTRP8与RXFP1结合，激活信号通路，促进细胞迁移和基质侵袭。这一建议是我开创性CTRP8-RXFP1工作的继续，并使用分子建模和定向突变来确定对RXFP1结合重要的特定CTRP8残基。我使用体内标记来捕获触发CTRP8诱导的信号事件的RXFP1胞内域的新结合因子。我们首次展示了CTRP8-RXFP1通过增加特定DNA修复蛋白的表达和活性来保护DNA免受损伤的新作用。我将对这一发现进行扩展，并确定CTRP8-RXFP1对细胞中频繁发生的氧化应激反应的影响。我将测量线粒体产生的反应性氧化自由基(ROS)，评估线粒体基因，并使用3D核成像、线粒体基因表达谱和蛋白质分析来量化ROS诱导的氧化DNA碱基损伤。我发现CTRP8-RXFP1参与了STAT3信号转导，并增加了促进DNA修复和细胞存活的关键蛋白的表达。我将研究CTRP8-RXFP1促进细胞存活和突变DNA损伤修复的分子机制。我的实验室有令人信服的证据表明，CTRP1是CTRP8的近亲，是一种新的RXFP1激动剂。CTRP1和CTRP8共享RXFP1结合域，我们发现CTRP1需要RXFP1来增强细胞运动性和激活特定的信号通路。CTRP1在组织中的广泛表达可能对RXFP1信号在组织中的表达具有重要意义。与CTRP8-RXFP1类似，我们将演示CTRP1-RXFP1的物理相互作用，并确定CTRP1在细胞运动、基质侵袭和DNA保护中的作用。我们使用新的Ctrp1小鼠品系(过度表达和KO)来评估Ctrp1-Rxfp1在博莱霉素诱导的肺纤维化中的保护作用。我们使用我们的眼创伤愈合小鼠模型，并确定CTRP1和CTRP8修复眼表损伤的能力。我的建议将在RXFP这一令人兴奋的新兴研究领域取得关键进展，在这笔赠款的任期内，将为总共三名研究生和最多五名夏季/合作伙伴学生提供培训机会。

I study the role of relaxin receptor and binding partners on cell differentiation. My lab established relaxin (RLX) and INSL3 as novel mediators of cell motility and extracellular matrix (ECM) invasion. We also demonstrated a new function of Insl5, another relaxin-like peptide, in glucose homeostasis in mice and identified enteroendocrine cells of the human and mouse large intestine as specific producers of INSL5. Recently, my lab discovered a new binding partner to relaxin receptor RXFP1. The secreted C1qTNF-related peptide CTRP8 protein is structurally distinct from RLX. CTRP8 binds to RXFP1 and activates signaling pathways resulting in enhanced cell migration and matrix invasion. This proposal is a continuation of my pioneering CTRP8-RXFP1 work and uses molecular modelling and targeted mutagenesis to identify specific CTRP8 residues important for the binding of RXFP1. I use in-vivo labeling to capture novel binding factors to intracellular domains of RXFP1 that trigger CTRP8-induced signaling events. We were the first to show a novel role of CTRP8-RXFP1 in the protection against DNA damage by increased expression and activity of defined DNA repair proteins. I will expand on this finding and determine the impact of CTRP8-RXFP1 on oxidative stress responses which occur frequently in cells. I will measure mitochondrial production of reactive oxidative radicals (ROS), assess mitochondrial genes, and quantify ROS-induced oxidative DNA base lesions using 3D nuclear imaging, mitochondrial gene expression profiling and protein analysis. I showed that CTRP8-RXFP1 engages STAT3 signaling and increases the expression of key proteins promoting DNA repair and cell survival. I will study the molecular mechanisms by which CTRP8-RXFP1 promotes cell survival and mutagenic DNA damage repair. My lab has compelling evidence implicating CTRP1, a close relative of CTRP8, as a new RXFP1 agonist. CTRP1 and CTRP8 share the RXFP1 binding domain and we showed that CTRP1 requires RXFP1 to enhance cell motility and activate specific signaling pathways. The broad tissue expression of CTRP1 may have important implications for RXFP1 signaling in tissues. Like for CTRP8-RXFP1, we will demonstrate the physical CTRP1-RXFP1 interaction and identify the role of CTRP1 in cell motility, matrix invasion and DNA protection. We use new Ctrp1 mouse strains (overexpressing and KO) to assess the protective functions of Ctrp1-Rxfp1 in bleomycin-induced lung fibrosis. We employ our ocular wound healing mouse model and determine the ability of CTRP1 and CTRP8 to repair ocular surface lesions. My proposal will generate key advances in this exciting emerging field of RXFP research and, for the tenure of this grant, will provide training opportunities for a total of three graduate students and up to five summer/COOP students.