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

Abstract

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 on Nmp4 chromatin immunoprecipitation sequencing (ChIP-seq) data. Mice (12 w) 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.

Nuclear Matrix Protein 4 Is a Novel Regulator of Ribosome Biogenesis and Controls the Unfolded Protein Response via Repression of Gadd34 Expression

“…20). The reported NMP4-binding site in the Gadd34 promoter in MC3T3-E1 cells is illustrated in Fig. 1A (20), and we posited that NMP4 alters transcriptional expression of Gadd34 mRNA.…”

“…mTORC1 and c-MYC are potent inducers of ribosome biogenesis (27,28), and prior ChIP-Seq analyses indicated that NMP4 can bind to the promoter of the c-Myc gene (Fig. 1A) (20). There were increased levels of c-Myc mRNA in the Nmp4 Ϫ/Ϫ cells as compared with WT (Fig.…”

“…Ϫ/Ϫ mice are as previously described (20). The local Institutional Animal Care and Use Committee approved all husbandry practices and experimental procedures.…”

“…Cell Culture-Mesenchymal stem progenitor cells (MSPCs) were isolated from bone marrow using a Ficoll gradient as described (20,22) and were cultured in Mesencult Media with Mesencult Stimulatory Supplement (StemCell Technologies).…”

“…Targeted deletion of Nmp4 in mice enhances bone response to PTH and BMP2 and protects these animals from osteopenia. Furthermore, ChIP-Seq analyses of NMP4-binding sites in preosteoblasts, embryonic stem cells, and two blood cell lines suggest that NMP4 binds to the promoters of genes encoding UPR regulators and modulates their gene expression (20).…”