Impaired Bone Formation in Pdia3 Deficient Mice

1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is crucial for normal skeletal development and bone homeostasis. Protein disulfide isomerase family A, member 3 (PDIA3) mediates 1α,25(OH)2D3 initiated-rapid membrane signaling in several cell types. To understand its role in regulating skeletal development, we generated Pdia3-deficient mice and examined the physiologic consequence of Pdia3-disruption in embryos and Pdia3+/− heterozygotes at different ages. No mice homozygous for the Pdia3-deletion were found at birth nor were there embryos after E12.5, indicating that targeted disruption of the Pdia3 gene resulted in early embryonic lethality. Pdia3-deficiency also resulted in skeletal manifestations as revealed by µCT analysis of the tibias. In comparison to wild type mice, Pdia3 heterozygous mice displayed expanded growth plates associated with decreased tether formation. Histomorphometry also showed that the hypertrophic zone in Pdia3+/− mice was more cellular than seen in wild type growth plates. Metaphyseal trabecular bone in Pdia3+/− mice exhibited an age-dependent phenotype with lower BV/TV and trabecular numbers, which was most pronounced at 15 weeks of age. Bone marrow cells from Pdia3+/− mice exhibited impaired osteoblastic differentiation, based on reduced expression of osteoblast markers and mineral deposition compared to cells from wild type animals. Collectively, our findings provide in vivo evidence that PDIA3 is essential for normal skeletal development. The fact that the Pdia3+/− heterozygous mice share a similar growth plate and bone phenotype to nVdr knockout mice, suggests that PDIA3-mediated rapid membrane signaling might be an alternative mechanism responsible for 1α,25(OH)2D3’s actions in regulating skeletal development.1α,25-二羟基维生素D3（1α,25(OH)2D3）对正常骨骼发育及骨稳态维持至关重要。蛋白二硫键异构酶家族A成员3（PDIA3）可介导多种细胞中由1α,25(OH)2D3启动的快速膜信号传导。为阐明其在调控骨骼发育中的作用，我们构建了Pdia3基因缺陷小鼠，并检测了Pdia3基因敲除对胚胎以及不同周龄Pdia3+/-杂合子小鼠的生理影响。未在出生个体中发现Pdia3纯合敲除小鼠，且胚胎发育第12.5天（E12.5）之后的胚胎中也未检测到此类个体，这表明Pdia3基因的靶向敲除会导致胚胎早期致死。通过对胫骨进行显微CT（µCT）分析可见，Pdia3基因缺陷还会引发骨骼表型异常。与野生型小鼠相比，Pdia3+/-杂合子小鼠的生长板出现扩张，且连接结构形成减少。组织形态计量学分析显示，Pdia3+/-杂合子小鼠的软骨肥大区细胞密度高于野生型小鼠的生长板。Pdia3+/-杂合子小鼠的干骺端小梁骨表现出年龄依赖性表型：其骨体积分数（BV/TV）与小梁数量均低于野生型小鼠，该表型在15周龄时最为显著。与野生型小鼠骨髓细胞相比，Pdia3+/-杂合子小鼠的骨髓细胞成骨分化能力受损，具体表现为成骨细胞标志物表达下调以及矿化沉积减少。综上，本研究结果为PDIA3对正常骨骼发育的必要性提供了体内实验证据。Pdia3+/-杂合子小鼠的生长板与骨表型与核维生素D受体（nVdr）敲除小鼠相似，这提示PDIA3介导的快速膜信号传导可能是1α,25(OH)2D3调控骨骼发育的另一替代机制。