Role of GI OSTERIX in Gut and Bone Biology

项目总结： 几十年来，人们已经知道肠道通过影响骨矿物质的平衡来深刻地调节骨矿物质的动态平衡。 成骨细胞和降解破骨细胞的发育和功能。在手机上 和分子水平，肠道如何影响骨骼，以及骨骼和胃肠道(GI)上皮 开发在功能上是否同步仍在很大程度上不得而知。本提案中概述的工作构建了 关于我们新的和令人兴奋的发现，锌指蛋白OSTERIX，到目前为止 被认为是骨骼特异的主要转录因子，在肠道隐窝中强势表达。OSTERIX 该基因在骨外组织中的表达尚未见报道。我们在胃肠道上皮细胞中发现了OSTERIX Osterix-Cre介导的磷酸酶SHP2在小鼠骨骼发育中的作用 PTPN11等位基因缺失。随后，我们证实OSTERIX启动子是活性的，并且 OSTERIX蛋白在胃肠道隐窝基础干细胞(CBCs)中表达。鉴于OSTERIX在骨骼中的核心作用 我们推测GI隐窝细胞中的OSTERIX可能参与了GI上皮细胞的发育 和骨骼矿物质的动态平衡。在这些新发现的基础上，我们将采用R21融资机制并 确定OSTERIX如何调节GI CBC的自我更新和多谱系分化，以及 研究胃肠道上皮细胞中的OSTERIX是否以及如何调节成骨细胞和破骨细胞的功能 骨矿物质动态平衡。在具体目标#1中，我们将测试OSX保持CBC干性的假设 并利用转基因小鼠结合多种方法调节多血统分化 包括标记共定位、活细胞谱系追踪、器官培养、组织学和 免疫组织化学染色。在具体目标#2中，我们将测试GI OSX调节OB和OB的假设 胃肠道上皮细胞生长因子表达调控破骨细胞功能与骨矿物质平衡 和荷尔蒙。方法包括新骨形成分析、RNAScope®、组织形态计量学、 骨代谢标记物的免疫染色和生化分析将在本研究中应用。

PROJECT SUMMARY: It has been known for decades that the gut profoundly regulates bone mineral homeostasis by influencing the development and function of bone-forming osteoblasts and bone-degrading osteoclasts. At the cellular and molecular levels, how the gut affects the bone and whether skeletal and gastrointestinal (GI) epithelium development is functionally synchronized remains largely unknown. The work outlined in this proposal builds on our novel and exciting discovery that the zinc finger protein OSTERIX, which to this point has been considered a bone-specific master transcription factor, is robustly expressed in the intestinal crypts. OSTERIX expression has not yet been reported in extraskeletal tissues. We discovered OSTERIX in GI epithelium while examining the role of the phosphatase SHP2 in mouse skeletal development using Osterix-Cre-mediated deletion of PTPN11 foxed alleles. Subsequently, we confirmed that the OSTERIX promoter is active and OSTERIX protein is expressed in the GI crypt base stem cells (CBCs). Given OSTERIX’s central role in bone development, we hypothesize that OSTERIX in the GI crypt cells may participate in GI epithelial development and bone mineral homeostasis. Building on these novel findings we will take the R21 funding mechanism and determine how OSTERIX modulates the self-renewal and multiple lineage differentiation of the GI CBCs, and examine whether and how OSTERIX in the GI epithelium modulates osteoblast and osteoclast function and bone mineral homeostasis. In Specific Aim #1 we will test the hypothesis that OSX maintains CBC stemness and regulates multilineage differentiation using genetically modified mice in combination of approaches including marker colocalization, live cell-lineage tracing, organoid culture, histology, and immunohistochemical staining. In Specific Aim #2 we will test the hypothesis that GI OSX regulates OB and osteoclast function and bone mineral homeostasis by modulating the expression of GI epithelial growth factors and hormones. Approaches including new bone formation assays, RNAScope®, histomorphometry, immunostaining, and biochemical assays of bone metabolic markers will be applied in this study.