A goal of osteoporosis therapy is to restore lost bone with structurally sound tissue. Mice lacking the transcription factor nuclear matrix protein 4 ( Nmp4, Zfp384, Ciz, ZNF384) respond to several classes of osteoporosis drugs with enhanced bone formation compared with wild-type (WT) animals. Nmp4−/− mesenchymal stem/progenitor cells (MSPCs) exhibit an accelerated and enhanced mineralization during osteoblast differentiation. To address the mechanisms underlying this hyperanabolic phenotype, we carried out RNA-sequencing and molecular and cellular analyses of WT and Nmp4−/− MSPCs during osteogenesis to define pathways and mechanisms associated with elevated matrix production. We determined that Nmp4 has a broad impact on the transcriptome during osteogenic differentiation, contributing to the expression of over 5,000 genes. Phenotypic anchoring of transcriptional data was performed for the hypothesis-testing arm through analysis of cell metabolism, protein synthesis and secretion, and bone material properties. Mechanistic studies confirmed that Nmp4−/− MSPCs exhibited an enhanced capacity for glycolytic conversion: a key step in bone anabolism. Nmp4−/− cells showed elevated collagen translation and secretion. The expression of matrix genes that contribute to bone material-level mechanical properties was elevated in Nmp4−/− cells, an observation that was supported by biomechanical testing of bone samples from Nmp4−/− and WT mice. We conclude that loss of Nmp4 increases the magnitude of glycolysis upon the metabolic switch, which fuels the conversion of the osteoblast into a super-secretor of matrix resulting in more bone with improvements in intrinsic quality.

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Activation of bone anabolic pathways is a fruitful approach for treating severe osteoporosis, yet FDA‐approved osteoanabolics, eg, parathyroid hormone (PTH), have limited efficacy. Improving their potency is a promising strategy for maximizing bone anabolic output. Nmp4 (Nuclear Matrix Protein 4) global knockout mice exhibit enhanced PTH‐induced increases in trabecular bone but display no overt baseline skeletal phenotype. Nmp4 is expressed in all tissues; therefore, to determine which cell type is responsible for driving the beneficial effects of Nmp4 inhibition, we conditionally removed this gene from cells at distinct stages of osteogenic differentiation. Nmp4‐floxed (Nmp4fl/fl) mice were crossed with mice bearing one of three Cre drivers including (i) Prx1Cre+ to remove Nmp4 from mesenchymal stem/progenitor cells (MSPCs) in long bones; (ii) BglapCre+ targeting mature osteoblasts, and (iii) Dmp1Cre+ to disable Nmp4 in osteocytes. Virgin female Cre+ and Cre− mice (10 weeks of age) were sorted into cohorts by weight and genotype. Mice were administered daily injections of either human PTH 1‐34 at 30 μg/kg or vehicle for 4 weeks or 7 weeks. Skeletal response was assessed using dual‐energy X‐ray absorptiometry, micro‐computed tomography, bone histomorphometry, and serum analysis for remodeling markers. Nmp4fl/fl;Prx1Cre+ mice virtually phenocopied the global Nmp4−/− skeleton in the femur, ie, a mild baseline phenotype but significantly enhanced PTH‐induced increase in femur trabecular bone volume/total volume (BV/TV) compared with their Nmp4fl/fl;Prx1Cre− controls. This was not observed in the spine, where Prrx1 is not expressed. Heightened response to PTH was coincident with enhanced bone formation. Conditional loss of Nmp4 from the mature osteoblasts (Nmp4fl/fl;BglapCre+) failed to increase BV/TV or enhance PTH response. However, conditional disabling of Nmp4 in osteocytes (Nmp4fl/fl;Dmp1Cre+) increased BV/TV without boosting response to hormone under our experimental regimen. We conclude that Nmp4−/− Prx1‐expressing MSPCs drive the improved response to PTH therapy and that this gene has stage‐specific effects on osteoanabolism. © 2022 American Society for Bone and Mineral Research (ASBMR).

Mesenchymal stem/progenitor cells (MSPCs) have immunomodulatory activity. We previously observed that osteoprogenitors and CD8+T cells are increased in the bone marrow (BM) of mice lacking the transcription factor Nmp4. Transcriptome analysis of MSPCs from Nmp4−/− and wild type (WT) mice revealed significant changes in genes related to innate immune responses, including reduced expression of Tlr3 and Mda5 in the null MSPCs. Loss of Nmp4 increased mRNA translation, ribosomal biogenesis, and Gadd34 expression in MSPCs; collectively, these processes likely promote the unfolded protein response (UPR), expanding the processing capacity of the endoplasmic reticulum. Therefore, we hypothesized that disabling Nmp4 would improve innate immune cell function. To test this, Nmp4−/− mice (C57BL6) were infected intranasally with influenza A/PR8 (H1N1; 150pfu). Despite having similar virus load in the lung, H1N1-infected Nmp4−/− mice lost less body weight than WT mice post infection and had improved survival. Infected Nmp4−/− mice had fewer infiltrating neutrophils (PMNs; CD11b+/Ly6G+) and monocytes (CD11b+/SiglecFNeg/Ly6GNeg/Ly6C+) in bronchoalveolar lavage (BAL) but more BAL macrophages (CD11c+/SiglecF+); CD4+ and CD8+ T cells were not different between genotypes. Ex vivo, Nmp4-deficiency did not alter the differentiation of BM marrow-derived macrophages; however, Chil3/Ym1 and Alox15 mRNA expression was reduced in Nmp4−/− macrophages polarized with IL-4/IL-13, suggesting cell-autonomous regulation of M2 macrophage activation by Nmp4. Our data point to Nmp4 as a novel regulator of innate immunity and influenza response in mice.