目的:骨髓间充质基质细胞(BMSCs)是骨重建过程中成骨前体细胞的主要来源,直接参与骨质疏松症(OP)的进展。然而,参与OP的BMSCs的具体机制值得大量研究。最初,我们的生物信息学分析揭示了OP患者成骨细胞(OB)中Asporin(ASPN)和蛋白聚糖连接蛋白1(HAPLN1)的显著上调及其可能的蛋白相互作用.因此,本研究旨在探讨ASPN和HAPLN1对BMSCs成骨分化的影响,OBs的细胞外基质(ECM)矿化,和破骨细胞生成,希望能为OP的治疗提供研究依据。
方法:使用GSE156508数据集进行分析和筛选,以获得OP患者OB中差异表达的基因,然后通过STRING进行谓词分析。通过卵巢切除术(OVX)诱导OP小鼠模型,并测定ASPN和HAPLN1表达。从OVX小鼠中分离出BMSCs和骨髓巨噬细胞(BMMs),诱导成骨分化和破骨细胞生成,分别。在击倒实验之后,我们评估了BMSCs的成脂分化和成骨分化。成骨(OPN,OCN,和COL1A1)和破骨细胞(Nfatc1和c-Fos)标记蛋白的表达。分析ASPN与HAPLN1的结合。
结果:通过生物信息学方法在OP患者的OB中以及在OVX小鼠的骨组织中观察到ASPN和HAPLN1的高表达及其蛋白相互作用。ASPN与OVX小鼠BMSCs中的HAPLN1相互作用。ASPN/HAPLN1敲除增加ALP,OPN,OCN,BMSCs中的COL1A1蛋白表达和ECM矿化,同时降低BMMs中的Nfatc1和c-Fos表达。ASPN和HAPLN1的同时敲低加重了这些作用。
结论:我们的结果表明ASPN与HAPLN1协同抑制BMSCs的成骨分化和OB的ECM矿化,并促进OP的破骨细胞生成。
OBJECTIVE: Bone marrow mesenchymal stromal cells (BMSCs) are major sources of osteogenic precursor cells in bone remodeling, which directly participate in osteoporosis (OP) progression. However, the involved specific mechanisms of BMSCs in OP warrant mass investigations. Initially, our bioinformatics analysis uncovered the prominent up-regulation of Asporin (ASPN) and proteoglycan link protein 1 (HAPLN1) in osteoblasts (OBs) of OP patients and their possible protein interaction. Hence, this study aimed to explore the effects of ASPN and
HAPLN1 on osteogenic differentiation of BMSCs, extracellular matrix (ECM) mineralization of OBs, and osteoclastogenesis, hoping to offer research basis for OP treatment.
METHODS: GSE156508 dataset was used for analysis and screening to acquire the differentially expressed genes in OBs of OP patients, followed by the predicative analysis via STRING. OP mouse models were induced by ovariectomy (OVX), and ASPN and
HAPLN1 expression was determined. BMSCs and bone marrow macrophages (BMMs) were isolated from OVX mice and induced for osteogenic differentiation and osteoclastogenesis, respectively. After knockdown experiments, we assessed adipogenic differentiation and osteogenic differentiation in BMSCs. Osteogenic (OPN, OCN, and COL1A1) and osteoclast (Nfatc1 and c-Fos) marker protein expression was determined. The binding of ASPN to
HAPLN1 was analyzed.
RESULTS: High expression of ASPN and HAPLN1 and their protein interaction were observed in OBs of OP patients via bioinformatics and in bone tissues of OVX mice. ASPN interacted with
HAPLN1 in BMSCs of OVX mice. ASPN/HAPLN1 knockdown increased ALP, OPN, OCN, and COL1A1 protein expression and ECM mineralization in BMSCs while decreasing Nfatc1 and c-Fos expression in BMMs. These effects were aggravated by the simultaneous knockdown of ASPN and
HAPLN1.
CONCLUSIONS: Our results indicate that ASPN synergises with HAPLN1 to suppress the osteogenic differentiation of BMSCs and ECM mineralization of OBs and promote the osteoclastogenesis in OP.