RNA-seq analysis of osteogenic differentiating mouse wild type (WT) and Zfp384 (Nmp4) knockout mesenchymal stem/progenitor cells (MSPCs)

A goal of osteoporosis therapy is to restore lost bone with structurally sound tissue. Mice lacking the transcription factor Nmp4 respond to several classes of osteoporosis drugs with enhanced bone formation compared to wild type (WT) animals. To address how loss of Nmp4 maximizes anabolic output we used RNA-seq and network analyses, along with biochemical and bone mechanical measurements to define this anti-anabolic axis. Several independent preparations of expanded mesenchymal stem/progenitor cells (MSPCs) were derived from individual Nmp4-/- and WT mice. The Nmp4-/- cells exhibited accelerated and enhanced mineralization. Loss of Nmp4 significantly altered the expression of over 5,000 genes. While Nmp4 status did not alter the mRNA expression of Runx2 and Sp7, key regulators of osteoblast differentiation, Nmp4-depletion enhanced expression of genes that drive osteogenesis and conversion to aerobic glycolysis, a key step in bone anabolism. Functional studies confirmed that Nmp4-/- MSPCs exhibited an enhanced capacity for glycolytic conversion. Several canonical pathways sensitive to Nmp4 status controlled protein production and delivery and as predicted the Nmp4-/- cells showed elevated collagen translation and secretion. Expression of matrix genes that contribute to bone material-level mechanical properties were elevated in Nmp4-/- cells. Mechanical analysis of femurs from Nmp4-/- mice treated with osteoporosis therapies resulted in enhanced bone material properties compared to WT mice. In conclusion, disabling Nmp4 converts the osteoblast into a super-secretor by metabolically reprogramming the cell to support the concomitant elevated matrix production and expansion of the cell’s delivery capacity. Resolving this pathway will guide advances in osteoporosis therapy. To compare transcriptome profiles of non-differentiating and osteogenic-differentiating WT and Nmp4-/- MSPCs, cells were seeded into 12-well plates at either 10,000 cells/well (25 cells/mm2) or 25,000 cells/well (62 cells/mm2). The cells seeded at the lower density were maintained in Mesencult MediaTM + Mesencult Stimulatory SupplementTM (non-differentiating medium) for 3 days post-seeding and then harvested for total RNA. Cells plated at the higher density were maintained in α-MEM complete medium throughout the experiment. At 48 hours post-seeding the medium was refreshed with the ascorbic acid, dexamethasone, and BGP supplement. These cells were harvested at 7 days post-seeding as early osteogenic cells. For each condition there were four technical replicates and 1 biological replicate.

骨质疏松症治疗的目标在于利用结构完整的骨组织修复丢失的骨量。缺失转录因子Nmp4的小鼠，在接受多种骨质疏松症药物治疗后，骨形成能力较野生型（wild type, WT）动物显著增强。为阐明Nmp4缺失如何最大化合成代谢产出，本研究通过RNA测序（RNA-seq）与网络分析，结合生物化学及骨力学检测手段，明确了这一抗合成代谢调控轴。研究人员从单只Nmp4敲除（Nmp4-/-）与WT小鼠中分离得到多批次独立扩增的间充质干细胞/祖细胞（mesenchymal stem/progenitor cells, MSPCs）。Nmp4-/-细胞的矿化过程加速且增强。Nmp4缺失显著改变了5000余个基因的表达水平。尽管Nmp4的存在与否并未改变成骨细胞分化关键调控因子Runx2与Sp7的mRNA表达水平，但Nmp4敲除可增强驱动成骨作用及向有氧糖酵解表型转化的基因表达——而这正是骨合成代谢的关键环节。功能实验证实，Nmp4-/- MSPCs的糖酵解转化能力显著提升。多种受Nmp4状态调控的经典通路参与蛋白质合成与转运过程，正如预期，Nmp4-/-细胞的胶原翻译与分泌水平显著升高。参与调控骨材料级力学特性的基质基因在Nmp4-/-细胞中的表达水平显著上调。对接受骨质疏松症药物治疗的Nmp4-/-小鼠股骨进行力学分析，结果显示其骨材料特性较WT小鼠更为优异。综上，失活Nmp4可通过代谢重编程将成骨细胞转化为超级分泌细胞，以支持同步升高的基质合成与细胞转运能力扩增。解析这一调控通路将为骨质疏松症治疗的研发提供重要指导。为比较未分化与成骨分化状态下WT及Nmp4-/- MSPCs的转录组谱，研究人员将细胞以10000个/孔（25个细胞/mm²）或25000个/孔（62个细胞/mm²）的密度接种至12孔板。接种于低密度的细胞在接种后3天内均维持于Mesencult Media™ + Mesencult Stimulatory Supplement™（未分化培养基）中，随后收集总RNA。接种于高密度的细胞在整个实验过程中均维持于完全α-最低必需培养基（α-MEM）中。接种后48小时，更换添加了抗坏血酸、地塞米松与骨钙蛋白（BGP）补充剂的培养基，该组细胞于接种后7天收获，作为早期成骨细胞。每个实验条件设置4个技术重复与1个生物学重复。