Objective: To explore the role of lipocalin 2 (Lcn2) in bone development and senile osteoporosis.
Methods: Chromatin immunoprecipitation with H3K27AC and H3K9Me3 antibodies coupled with massively parallel sequencing (ChIP-Seq) was performed to analyze the changes in binding of modified histones at the Lcn2 gene locus in C3H10T1/2 mesenchymal stem cells (MSCs) induced with osteogenic induction medium for 3 days. ITRAQ-MS/MS and ELISA were used to compare Lcn2 protein expressions in the cancellous bone and the serum between 16- and 3-month-old male mice. The effect of treatment with recombinant Lcn2 protein on osteogenic differentiation of C3H10T1/2 cells was evaluated by detecting changes in ALP expression, and Western blotting was performed to examine the changes in OSX and OCN expression.
Results: The expression of Lcn2 protein in the cancellous bone and its serum levels were significantly higher in 16-month-old than in 3-month-old male mice (P < 0.01). In C3H10T1/2 cells, ALP expression level decreased significantly after treatment with recombinant Lcn2 protein (P < 0.05) accompanied by lowered protein expressions of OSX and OCN (P < 0.05). Analysis with ROSE software revealed a super enhancer in the Lcn2 gene region of C3H10T1/2 cells after osteogenesis induction. RNA-seq results confirmed that Lcn2 was among the top 4 up-regulated genes in C3H10T1/2 cells following osteogenic induction, and a super enhancer was also detected in Zbtb16 gene, which showed the highest up- regulation after osteogenic induction. Real- time quantitative PCR further confirmed that the mRNA level of Lcn2 increased sharply in C3H10T1/2 cells after osteogenic induction (P < 0.001).
Conclusion: Lcn2 is essential for normal osteogenic differentiation of MSCs, but its overexpression and excessive secretion in aging induce self-limited inhibition of osteogenic differentiation of MSCs.
目的: 探究脂质运载蛋白2（Lcn2）在骨发育和老年性骨质疏松的作用。
方法: C3H10T1/2 MSC细胞用成骨诱导液诱导3 d或不作处理（control），收集细胞核提取染色质，以H3K27A和H3K9Me3抗体进行免疫沉淀；ChIP-seq分析了诱导成骨分化前后C3H10T1/2间充质干细胞Lcn2基因座位的修饰组蛋白结合情况；ROSE软件（利用H3K27AC信号计算）显示成骨诱导后出现超级增强子；RNA-seq结果证实Lcn2在C3H10T1/2成骨诱导后上调的倍数在所有基因中位列第4；逆转录-实时定量PCR结果用于证实Lcn2的mRNA水平在C3H10T1/2成骨诱导后上调；蛋白质组学分析了16月龄C57BL/6J雄鼠相对于3月龄雄鼠股骨远端总蛋白表达谱的差异。iTRAQ-MS/MS用于分析，16月龄的老年雄鼠的松质骨中Lcn2蛋白表达相对于3月龄雄鼠的差异；ELISA检测3月龄和16月龄老年雄鼠血清中Lcn2含量；用50、100、200、500 ng/mL浓度的Lcn2重组蛋白处理C3H10T1/2后以ALP检测成骨分化能力，以Westem blot检测早期成骨转录因子OSX和晚期成骨指标OCN。
结果: iTRAQ-MS/MS结果显示，16月龄的老年雄鼠的松质骨中Lcn2蛋白表达相对于3月龄雄鼠明显上调（P < 0.01）。与此对应，ELISA结果示16月龄老年雄鼠血清中Lcn2含量比3月龄对照（control）雄鼠明显升高（P < 0.01）。ALP染色结果和定量结果显示加入100、200、500 ng/mL浓度Lcn2 RP后ALP表达减弱（P < 0.05，P < 0.01，P < 0.01）。Westem blot定量结果显示OSX在100、200、500 ng/mL浓度的Lcn2 RP组中的表达减低，呈剂量依赖性（P < 0.05）。Westem blot定量结果显示OCN在100、200、500 ng/mL浓度的Lcn2 RP组中的表达减低，呈剂量依赖性（P < 0.05，P < 0.01，P < 0.001）。ROSE软件（利用H3K27AC信号计算）显示成骨诱导后Lcn2基因区域出现超级增强子。RNA-seq结果证实Lcn2在C3H10T1/2成骨诱导后上调的倍数在所有基因中位列第4。C3H10T1/2成骨诱导后上调倍数最高的Zbtb16基因区域也在成骨诱导后出现超级增强子。逆转录-实时定量PCR进一步证实了Lcn2的mRNA水平在成骨诱导后急剧升高（P < 0.001）。
结论: Lcn2是间充质干细胞（MSCs）正常成骨分化所必须的，但在衰老时过度表达和分泌以自限性抑制MSCs的成骨分化。
Keywords: lipocalin 2; mesenchymal stem cells; osteogenic differentiation; senile osteoporosis.