Role of GI OSTERIX in Gut and Bone Biology

项目概要： 几十年来，人们已经知道肠道通过影响骨矿物质的稳态来深刻地调节骨矿物质的稳态。 骨形成成骨细胞和骨降解破骨细胞的发育和功能。在细胞 和分子水平，肠道如何影响骨骼以及骨骼和胃肠道（GI）上皮是否 发育在功能上是同步的，这在很大程度上仍然是未知的。本提案中概述的工作 我们的新的和令人兴奋的发现，锌指蛋白OSTERIX，这一点已经 被认为是骨特异性主转录因子，在肠隐窝中稳健表达。OSTERIX 在皮肤外组织中的表达尚未报道。我们在胃肠道上皮中发现了Osterix， 使用Osterix-Cre-介导的方法检测磷酸酶SHP 2在小鼠骨骼发育中的作用， PTPN 11缺失等位基因。随后，我们证实了OSTERIX启动子是有活性的， OSTERIX蛋白在GI隐窝基底干细胞（CBC）中表达。鉴于OSTERIX在骨骼中的核心作用， 因此，我们推测胃肠道腺细胞中的Osterix可能参与胃肠道上皮的发育 和骨矿物质平衡。在这些新发现的基础上，我们将采取R21资助机制， 确定OSTERIX如何调节GI CBC的自我更新和多谱系分化，以及 检查GI上皮中的OSTERIX是否以及如何调节成骨细胞和破骨细胞功能， 骨矿物质平衡在具体目标#1中，我们将测试OSX保持CBC干性的假设 并使用遗传修饰小鼠结合以下方法调节多谱系分化： 包括标记物共定位、活细胞谱系追踪、类器官培养、组织学， 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 通过调节GI上皮生长因子的表达调节破骨细胞功能和骨矿物质稳态 和荷尔蒙。方法包括新骨形成测定、RNAScope®、组织形态测定、 骨代谢标志物的免疫染色和生化测定将在本研究中应用。

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.