Effects of nuclear PTHrP on bone formation and the skeletal microenvironment

描述（由申请人提供）：甲状旁腺激素（PTH）相关蛋白（PTHrP）通过其氨基端（n端）与PTH/PTHrP受体（PTH1R）的相互作用，首次被发现是恶性肿瘤（HHM）的体液性高钙血症（HHM）的病原体。从那时起，PTHrP一直参与调节骨形成和骨髓基质细胞（BMSC）向成骨细胞谱系的承诺，这导致PTH的n端结构域在骨质疏松症的管理中使用。在造血干细胞（HSC）生态位中，激活成骨细胞上的PTH1R可促进HSC活性和n端PTH在骨髓移植中的应用。虽然骨形成和HSC生态位调节都归因于n端，但没有关于PTHrP其他结构域作用的信息。由于PTHrP的一些功能归因于核定位序列（NLS），我们创造了缺乏PTHrP NLS的小鼠。本提案的目的是研究核PTHrP在骨形成和骨骼微环境（即HSC生态位）中的作用，其中提出了三个具体目标。首先，通过测量BMSC成骨、脂肪、肌和软骨分化指标，确定核PTHrP在体外成骨中的作用，并通过测量PTHrP类似物对骨形成的影响，确定体内PTHrP类似物对骨形成的影响。其次，通过评估软骨内和膜内骨化部位的微结构指标、与骨调节蛋白的相互作用以及骨调节基因的表达，研究核PTHrP对骨骼微结构、蛋白质合成和基因表达的作用。第三，通过测量BMSC和HSC中相关基因的表达，枚举BM HSC，确定核PTHrP在HSC生态位调控中的作用。我们假设NLS对n端PTHrP在骨形成和骨骼微环境调节中的作用是必要的和附加的。研究结果将保证研究含有nls的PTHrP类似物的使用，这可能对骨科、肿瘤学和血液学产生重大影响。与公共卫生相关：PTHrP是一种类似于PTH的蛋白质，是唯一被证实的骨质疏松症治疗方法。与PTH不同，PTHrP有一个称为NLS的区域，该区域将蛋白质带到细胞核，并可能有助于PTHrP更广泛的作用。我们将使用缺乏PTHrP的NLS的小鼠来研究NLS在骨形成和骨骼微环境调节中的作用，这可能导致使用含有NLS的PTHrP类似物作为骨骼合成代谢剂和在BM移植和相关疾病中用于HSC扩增。

DESCRIPTION (provided by applicant): Parathyroid hormone (PTH)-related protein (PTHrP) was first discovered as the causative agent of humoral hypercalcemia of malignancy (HHM) through the interaction of its amino-terminus (N-terminus) with the PTH/PTHrP receptor (PTH1R). Since then, PTHrP has been implicating in regulating bone formation and the commitment of BM stromal cells (BMSC) to the osteoblastic lineage, which has led to the use of the N-terminal domain of PTH in the management of osteoporosis. In the hematopoietic stem cell (HSC) niche, activation of the PTH1R on osteoblasts promotes HSC activity and the application of N-terminal PTH in BM transplantation. While both bone formation and HSC niche regulation are attributed to the N-terminus, no information exists regarding the role of other domains of PTHrP. Since several functions of PTHrP are attributed to the nuclear localization sequence (NLS), we have created a mouse lacking the NLS of PTHrP. The objectives of this proposal are to investigate the role of nuclear PTHrP on bone formation and the skeletal microenvironment, namely the HSC niche, of which three specific aims are proposed. First, determine the role of nuclear PTHrP on osteogenesis in vitro, by measuring indices of osteogenic, adipogenic, myogenic, and chondrogenic differentiation of BMSC, and in vivo by measuring the effects of PTHrP analogs on bone formation. Second, examine the role of nuclear PTHrP on skeletal microarchitecture, protein synthesis, and gene expression by assessing microstructural indices at sites of endochondral and intramembranous ossification, its interaction with bone regulatory proteins, and the expression of bone regulatory genes. Third, determine the role of nuclear PTHrP in the regulation of the HSC niche by measuring the expression of associated genes in BMSC and HSC and enumerating BM HSC. We hypothesize that the NLS is necessary and additive to the effects of N-terminal PTHrP in bone formation and skeletal microenvironment regulation. Findings will warrant investigating the use of NLS-containing PTHrP analogs, which could have significant implications in orthopedics, oncology and hematology. Relevance to public health: PTHrP is a protein similar to PTH, which is the only proven therapy for osteoporosis. Unlike PTH, PTHrP has a region called the NLS, which takes the protein to the cell nucleus and may contribute to PTHrP's more widespread effects. We will investigate the role of the NLS in bone formation and skeletal microenvironment regulation using a mouse lacking the NLS of PTHrP, which may lead to the use of NLS-containing PTHrP analogs as both a skeletal anabolic agent and for HSC expansion in BM transplantation and related disorders.