Discoidin Domain Receptor 2, β1 Integrins and ECM Control of Bone Formation

盘状蛋白结构域受体2(DDR2)是一种非整合素胶原受体，具有重要但知之甚少的功能 骨骼功能。DDR2基因失活突变导致脊柱-骨骺发育不良(Smed-Short 四肢类型)，这是一种人类疾病，具有严重的头面部和骨骼异常。DDR2缺陷小鼠有一种 相似的表型包括头骨形状异常、缝合延迟融合和软骨缺陷 颅底同步软骨的生长/定向，长骨生长板缩短和减少 骨小梁和皮质骨量。骨骼中DDR2选择性失活的初步研究 祖细胞在骨谱系的细胞中具有不同的功能。研究将检验以下假设： DDR2是一种重要的骨形成调节因子，它在骨系细胞中发挥作用，感知胶原ECM壁龛 从而刺激SPC谱系的承诺和分化；在纤维胶原蛋白激活时，DDR2 与β-1整合素协同作用，刺激成骨细胞分化。目标是： 1.建立DDR2阳性细胞在颅面神经系统发育过程中的分布、命运和基因表达谱 骨骼发育。假设：在发育过程中，DDR2在SPC和更成熟的骨细胞中表达。 研究将：a)确定DDR2在骨骼发育过程中的表达时间进程和模式，b)确定 表达DDR2的细胞的谱系，c)定义了表达DDR2的细胞的分子特征及其 使用单细胞RNAseq的后代。 2.确定DDR2在颅面和附件骨骼中的细胞作用部位。假设：DDR2 在两种SPC中发挥作用，并致力于软骨和成骨细胞以刺激骨形成。目前尚不清楚 哪些骨骼细胞需要DDR2才能正常发挥功能；骨骼干细胞、分化细胞或两者兼而有之。A)定义 DDR2的细胞功能，将使用定向删除的方法来灭活Gli中的DDR2 颅面/骨骼祖细胞、软骨细胞和成骨细胞。B)确定DDR2是否控制 分化的SPC，我们将检测DDR2缺乏对Gli1 SPC谱系的影响。 3.研究DDR2与胶原结合的β-1整合素相互作用的机制。 假设：DDR2和β1整合素协同作用刺激细胞活性。胶原结合β-1整合素 和DDR2协同作用。这种协同作用的机制基础将在体外和 体内实验方法如下：a.检测成骨细胞中DDR2和整合素信号的相互作用 差异化。用遗传学方法检测DDR2和β1整合素在体内的相互作用。 拟议的研究将阐明DDR2的骨骼功能及其与β1整合素的相互作用。这是 一个非常重要但研究不足的领域，对理解细胞的作用具有重要意义- 骨质疏松症和糖尿病等骨病中的胶原相互作用。此外，骨靶向 DDR2激动剂如果被开发出来，可能成为一类新的骨合成代谢药物。

Discoidin domain receptor 2 (DDR2) is a non-integrin collagen receptor having important but poorly understood skeletal functions. Inactivating mutations in DDR2 cause spondylo-meta-epiphyseal dysplasia (SMED-short limb type), a human disorder with severe craniofacial and skeletal abnormalities. Ddr2-deficient mice have a similar phenotype including abnormal skull shape, delayed suture fusion and defective cartilage growth/orientation in synchondroses at the base of the skull, shortened long bone growth plates and reduced trabecular and cortical bone mass. Preliminary studies where Ddr2 was selectively inactivated in skeletal progenitors indicate distinct functions in cells of the bone lineage. Studies will test the following hypothesis: DDR2 is an important regulator of bone formation that functions in bone lineage cells to sense the collagenous ECM niche and thereby stimulate SPC lineage commitment and differentiation; on activation by fibrillar collagens, DDR2 synergistically interacts with β1 integrins to stimulate osteoblast differentiation. Aims are: 1. Establish the distribution, fates and gene expression profile of Ddr2-positive cells during craniofacial and skeletal development. Hypothesis: During development, Ddr2 is expressed in SPCs as well as more mature bone cells. Studies will: a) establish the time course and pattern of Ddr2 expression during skeletal development, b) define the lineage of Ddr2-expressing cells, c) Define the molecular signature of Ddr2-expressing cells and their progeny using single-cell RNAseq. 2. Define the cellular sites of action of DDR2 in the craniofacial and appendicular skeleton. Hypothesis: Ddr2 functions in both SPCs and committed chondrogenic and osteogenic cells to stimulate bone formation. It is not known which skeletal cells require DDR2 for normal function; skeletal SCs, differentiated cells or both. a) To define cellular functions of DDR2, a targeted deletion approach will be used to inactivate Ddr2 in Gli+ craniofacial/skeletal progenitors, chondrocytes and osteoblasts. b) To determine if Ddr2 controls the differentiation of SPCs, the effect of Ddr2 deficiency on the lineage of Gli1+ SPCs will be examined. 3. Examine the mechanistic basis for interactions between DDR2 and collagen-binding β1 integrins. Hypothesis: DDR2 and β1 integrins synergistically interact to stimulate cellular activity. Collagen-binding β1 integrins and DDR2 synergistically interact. The mechanistic basis for this synergy will be established using in vitro and in vivo approaches as follows: a. Examine interactions between DDR2 and integrin signaling during osteoblast differentiation. b. Use a genetic approach to detect Ddr2 and β1 integrin interactions in vivo. Proposed studies will elucidate the skeletal functions of DDR2 and its interactions with β1 integrins. This is a highly significant but understudied area with important implications for understanding the role of cell- collagen interactions in bone diseases including SMED, osteoporosis and diabetes. In addition, bone-targeted DDR2 agonists if developed could be a new class of bone anabolic agents.