Genome-Wide Mapping and Interrogation of the Nmp4 Antianabolic Bone Axis

PTH is an osteoanabolic for treating osteoporosis but its potency wanes. Disabling the transcription factor nuclear matrix protein 4 (Nmp4) in healthy, ovary-intact mice enhances bone response to PTH and bone morphogenetic protein 2 and protects from unloading-induced osteopenia. These Nmp4-/- mice exhibit expanded bone marrow populations of osteoprogenitors and supporting CD8+ T cells. To determine whether the Nmp4-/- phenotype persists in an osteoporosis model we compared PTH response in ovariectomized (ovx) wild-type (WT) and Nmp4-/- mice. To identify potential Nmp4 target genes, we performed bioinformatic/pathway profiling onNmp4chromatin immunoprecipitation sequencing (ChIP-seq) data. Mice (12 weeks old) were ovx or sham operated 4 weeks before the initiation of PTH therapy. Skeletal phenotype analysis included microcomputed tomography, histomorphometry, serum profiles, fluorescence-activated cell sorting and the growth/mineralization of cultured WT and Nmp4-/- bone marrow mesenchymal stem progenitor cells (MSPCs). ChIP-seq data were derived using MC3T3-E1 preosteoblasts, murine embryonic stem cells, and 2 blood cell lines. Ovx Nmp4-/- mice exhibited an improved response to PTH coupled with elevated numbers of osteoprogenitors and CD8+ T cells, but were not protected from ovx-induced bone loss. Cultured Nmp4-/- MSPCs displayed enhanced proliferation and accelerated mineralization. ChIP-seq/gene ontology analyses identified target genes likely under Nmp4 control as enriched for negative regulators of biosynthetic processes. Interrogation of mRNA transcripts in nondifferentiating and osteogenic differentiating WT and Nmp4-/- MSPCs was performed on 90 Nmp4 target genes and differentiation markers. These data suggest that Nmp4 suppresses bone anabolism, in part, by regulating IGF-binding protein expression. Changes in Nmp4 status may lead to improvements in osteoprogenitor response to therapeutic cues. MC3T3-E1 subclone 4, PTH therapy, Nmp4 chromatin immunoprecipitation sequencing (ChIP-seq) Cells from ATCC (MC3T3-E1 subclone 4) were maintained in a-MEM supplemented with 100-IU/mL penicillin, 100-µg/mL streptomycin, 25-µg/mL amphotericin, 2mM L-glutamine (Gibco BRL), ascorbic acid (50 µg/mL; Sigma-Aldrich), and 10% fetal bovine serum (Sigma-Aldrich). Cells were seeded into twenty-one 150-mm plates at an initial density of 50,000 cells/plate (320 cells/cm2) and maintained in a-MEM complete medium plus ascorbic acid. On day 14 post-seeding, cells were treated with 25nM hPTH (1–34) or vehicle control for 1 hour before harvest.