Biology subjects

Bonewald, L. F.

Publications and source records attributed to Bonewald, L. F..

4 recordsLinked to original sources

bioRxiv2025

Osteocyte Differentiation Requires Glycolysis, But Mature Osteocytes Display Metabolic Flexibility

Recent research has identified metabolic pathways which play key roles in the differentiation and function of osteoblasts and osteoclasts. However, the mechanisms by which osteocytes, the most numerous cells in bone, meet their energetic demands are still unknown. To address this, we used the IDG-SW3 osteocyte cell line to examine changes in metabolism during differentiation from late osteoblasts to mature osteocytes. There was a significant increase in the expression of glycolysis genes (including Pkm and Ldha), glucose consumption and lactate production during late differentiation of these cells. This was concurrent with the onset of the expression of mature osteocyte markers. Inhibition of glycolysis using the glucose analogue 2-deoxy-d-glucose (2-DG) inhibited IDG-SW3 cell mineralization and differentiation into osteocytes. To examine the effect of glycolysis inhibition on mature osteocytes, we treated differentiated IDG-SW3 cells and long bone osteocytes with 2-DG. Glycolysis inhibition resulted in decreased expression of the bone formation inhibitor Sost and mineralization inhibitor Fgf23. Concurrently, there was an increase in genes associated with lipolysis (Lpl) fatty acid {beta}-oxidation (Ppar{delta} and Cpt1a). Treatment of differentiated IDG-SW3 cells with the unsaturated fatty acid oleic acid increased Cpt1a expression and downregulated Sost and Fgf23. Application of mechanical stress to IDG-SW3 cells resulted in upregulation of oxidative metabolism, Ppar{delta} and Cpt1a expression. Long and short chain acylcarnitines were increased in the cortical bone of axially loaded tibiae compared to non-loaded controls, indicative of increased {beta}-oxidation. Overall, our data suggests that while glycolysis is essential for osteocyte differentiation, mature osteocytes are metabolically flexible. Furthermore, {beta}-oxidation may play an important role in the osteocyte response to mechanical stress.

cell biology↗

bioRxiv2023

Deletion of FNDC5/Irisin modifies murine osteocyte function in a sex-specific manner

Irisin, released from exercised muscle, has been shown to have beneficial effects on numerous tissues but its effects on bone are unclear. We found significant sex and genotype differences in bone from wildtype (WT) mice compared to mice lacking Fndc5 (KO), with and without calcium deficiency. Despite their bone being indistinguishable from WT females, KO female mice were partially protected from osteocytic osteolysis and osteoclastic bone resorption when allowed to lactate or when placed on a low-calcium diet. Male KO mice have more but weaker bone compared to WT males, and when challenged with a low-calcium diet lost more bone than WT males. To begin to understand responsible molecular mechanisms, osteocyte transcriptomics was performed. Osteocytes from WT females had greater expression of genes associated with osteocytic osteolysis and osteoclastic bone resorption compared to WT males which had greater expression of genes associated with steroid and fatty acid metabolism. Few differences were observed between female KO and WT osteocytes, but with a low calcium diet, the KO females had lower expression of genes responsible for osteocytic osteolysis and osteoclastic resorption than the WT females. Male KO osteocytes had lower expression of genes associated with steroid and fatty acid metabolism, but higher expression of genes associated with bone resorption compared to male WT. In conclusion, irisin plays a critical role in the development of the male but not the female skeleton and protects male but not female bone from calcium deficiency. We propose irisin ensures the survival of offspring by targeting the osteocyte to provide calcium in lactating females, a novel function for this myokine.

physiology↗

bioRxiv2022

Single Cell Cortical Bone Transcriptomics Defines Novel Osteolineage Gene Sets Altered in Chronic Kidney Disease

Due to a lack of spatial-temporal resolution at the single cell level, the etiologies of the bone dysfunction caused by diseases such as normal aging, osteoporosis, and the metabolic bone disease associated with chronic kidney disease (CKD) remain largely unknown. To this end, flow cytometry and scRNAseq were performed on long bone cells from Sost-cre/Ai9+ mice, and pure osteolineage transcriptomes were identified, including novel osteocyte-specific gene sets. Clustering analysis isolated osteoblast precursors that expressed Tnc, Mmp13, and Spp1, and a mature osteoblast population defined by Smpd3, Col1a1, and Col11a1. Osteocytes were demarcated by Cd109, Ptprz1, Ramp1, Bambi, Adamts14, Spns2, Bmp2, WasI, and Phex. We validated our in vivo scRNAseq using integrative in vitro promoter occupancy via ATACseq coupled with transcriptomic analyses of a conditional, temporally differentiated MSC cell line. Further, trajectory analyses predicted osteoblast-to-osteocyte transitions via defined pathways associated with a distinct metabolic shift as determined by single-cell flux estimation analysis (scFEA). Using the adenine mouse model of CKD, at a time point prior to major skeletal alterations, we found that gene expression within all stages of the osteolineage was disturbed. In sum, distinct populations of osteoblasts/osteocytes were defined at the single cell level. Using this roadmap of gene assembly, we demonstrated unrealized molecular defects across multiple bone cell populations in a mouse model of CKD, and our collective results suggest a potentially earlier and more broad bone pathology in this disease than previously recognized.

cell biology↗

bioRxiv2020

Sclerostin directly stimulates osteocyte synthesis of fibroblast growth factor-23

Osteocyte produced fibroblast growth factor 23 (FGF23) is the key regulator of serum phosphate (Pi) homeostasis. The interplay between parathyroid hormone (PTH), FGF23 and other proteins that regulate FGF23 production and serum Pi levels is complex and incompletely characterised. Evidence suggests that the protein product of the SOST gene, sclerostin (SCL), also a PTH target and also produced by osteocytes, plays a role in FGF23 expression, however the mechanism for this effect is unclear. Part of the problem of understanding the interplay of these mediators is the complex multi-organ system that achieves Pi homeostasis in vivo. In the current study, we sought to address this using a unique cell line model of the osteocyte, IDG-SW3, known to express FGF23 at both the mRNA and protein levels. In cultures of differentiated IDG-SW3 cells, both PTH1-34 and recombinant human (rh) SCL remarkably induced Fgf23 mRNA expression dose-dependently within 3 hours. Both rhPTH1-34 and rhSCL also strongly induced C-terminal FGF23 protein secretion. Secreted intact FGF23 levels remained unchanged, consistent with constitutive post-translational cleavage of FGF23 in this cell model. Both rhPTH1-34 and rhSCL treatments significantly suppressed mRNA levels of Phex, Dmp1 and Enpp1 mRNA, encoding putative negative regulators of FGF23 levels, and induced Galnt3 mRNA expression, encoding N-acetylgalactosaminyl-transferase 3 (GalNAc-T3), which protects FGF23 from furin-like proprotein convertase-mediated cleavage. The effect of both rhPTH1-34 and rhSCL was antagonised by pre-treatment with the NF-{kappa}{beta} signalling inhibitors, BAY11 and TPCK. RhSCL also stimulated FGF23 mRNA expression in ex vivo cultures of human bone. These findings provide evidence for the direct regulation of FGF23 expression by sclerostin. Locally expressed sclerostin via the induction of FGF23 in osteocytes thus has the potential to contribute to the regulation of Pi homeostasis.

physiology↗