AUTHOR=Li Hongyu , Liu Sirui , Miao Congcong , Lv Yan , Hu Ying 

TITLE=Integration of metabolomics and transcriptomics provides insights into enhanced osteogenesis in Ano5Cys360Tyr knock-in mouse model

JOURNAL=Frontiers in Endocrinology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2023.1117111

DOI=10.3389/fendo.2023.1117111

ISSN=1664-2392

ABSTRACT=<sec><title>Introduction</title><p>Gnathodiaphyseal dysplasia (GDD; OMIM#166260) is a rare autosomal dominant disorder characterized by diaphyseal sclerosis of tubular bones and cemento-osseous lesions in mandibles. GDD is caused by point mutations in the <italic>ANO5</italic> gene. However, the mechanisms underlying GDD have not been disclosed. We previously generated the first knock-in mouse model for GDD expressing a human mutation (p.Cys360Tyr) in <italic>ANO5</italic> and homozygous <italic>Ano5</italic> knock-in (<italic>Ano5<sup>KI/KI</sup></italic>) mice exhibited representative traits of human GDD especially including enhanced osteogenesis.</p></sec><sec><title>Methods</title><p>Metabolomics and transcriptomics analyses were conducted for wildtype (<italic>Ano5<sup>+/+</sup></italic>) and <italic>Ano5<sup>KI/KI</sup></italic> mature mouse calvarial osteoblasts (mCOBs) grown in osteogenic cultures for 14 days to identify differential intracellular metabolites and genes involved in GDD. Subsequently, related differential genes were validated by qRT-PCR. Cell proliferation was confirmed by CCK8 assay and calcium content in mineral nodules was detected using SEM-EDS.</p></sec><sec><title>Results</title><p>Metabolomics identified 42 differential metabolites that are primarily involved in amino acid and pyrimidine metabolism, and endocrine and other factor-regulated calcium reabsorption. Concomitantly, transcriptomic analysis revealed 407 differentially expressed genes in <italic>Ano5<sup>KI/KI</sup></italic> osteoblasts compared with wildtype. Gene ontology and pathway analysis indicated that <italic>Ano5<sup>Cys360Tyr</sup></italic> mutation considerably promoted cell cycle progression and perturbed calcium signaling pathway, which were confirmed by validated experiments. qRT-PCR and CCK-8 assays manifested that proliferation of <italic>Ano5<sup>KI/KI</sup></italic> mCOBs was enhanced and the expression of cell cycle regulating genes (<italic>Mki67</italic>, <italic>Ccnb1</italic>, and <italic>Ccna2</italic>) was increased. In addition, SEM-EDS demonstrated that <italic>Ano5<sup>KI/KI</sup></italic> mCOBs developed higher calcium contents in mineral nodules than <italic>Ano5<sup>+/+</sup></italic> mCOBs, while some calcium-related genes (<italic>Cacna1</italic>, <italic>Slc8a1</italic>, and <italic>Cyp27b1</italic>) were significantly up-regulated. Furthermore, osteocalcin which has been proved to be an osteoblast-derived metabolic hormone was upregulated in <italic>Ano5<sup>KI/KI</sup></italic> osteoblast cultures.</p></sec><sec><title>Discussion</title><p>Our data demonstrated that the <italic>Ano5<sup>Cys360Tyr</sup></italic> mutation could affect the metabolism of osteoblasts, leading to unwonted calcium homeostasis and cellular proliferation that can contribute to the underlying pathogenesis of GDD disorders.</p></sec>