GSE112694
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. Overall design: 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 a-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.