Disc degeneration primarily contributes to chronic low back and neck pain. Consequently, there is an urgent need to understand the spectrum of disc degeneration phenotypes such as fibrosis, ectopic calcification, herniation, or mixed phenotypes. Amongst these phenotypes, disc calcification is the least studied. Ectopic calcification, by definition, is the pathological mineralization of soft tissues, widely studied in the context of conditions that afflict vasculature, skin, and cartilage. Clinically, disc calcification is associated with poor surgical outcomes and back pain refractory to conservative treatment. It is frequently seen as a consequence of disc aging and progressive degeneration but exhibits unique molecular and morphological characteristics: hypertrophic chondrocyte-like cell differentiation; TNAP, ENPP1, and ANK upregulation; cell death; altered Pi and PPi homeostasis; and local inflammation. Recent studies in mouse models have provided a better understanding of the mechanisms underlying this phenotype. It is essential to recognize that the presentation and nature of mineralization differ between AF, NP, and EP compartments. Moreover, the combination of anatomic location, genetics, and environmental stressors, such as aging or trauma, govern the predisposition to calcification. Lastly, the systemic regulation of calcium and Pi metabolism is less important than the local activity of PPi modulated by the ANK-ENPP1 axis, along with disc cell death and differentiation status. While there is limited understanding of this phenotype, understanding the molecular pathways governing local intervertebral disc calcification may lead to developing disease-modifying drugs and better clinical management of degeneration-related pathologies.

Skeletal stem/progenitor cell (SSPC) senescence is a major cause of decreased bone regenerative potential with aging, but the causes of SSPC senescence remain unclear. In this study, we revealed that macrophages in calluses secrete prosenescent factors, including grancalcin (GCA), during aging, which triggers SSPC senescence and impairs fracture healing. Local injection of human rGCA in young mice induced SSPC senescence and delayed fracture repair. Genetic deletion of Gca in monocytes/macrophages was sufficient to rejuvenate fracture repair in aged mice and alleviate SSPC senescence. Mechanistically, GCA binds to the plexin-B2 receptor and activates Arg2-mediated mitochondrial dysfunction, resulting in cellular senescence. Depletion of Plxnb2 in SSPCs impaired fracture healing. Administration of GCA-neutralizing antibody enhanced fracture healing in aged mice. Thus, our study revealed that senescent macrophages within calluses secrete GCA to trigger SSPC secondary senescence, and GCA neutralization represents a promising therapy for nonunion or delayed union in elderly individuals.

Fibrous dysplasia (FD) is a rare, disabling skeletal disease for which there are no established treatments. Growing evidence supports inhibiting the osteoclastogenic factor receptor activator of nuclear kappa-B ligand (RANKL) as a potential treatment strategy. In this study, we investigated the mechanisms underlying RANKL inhibition in FD tissue and its likely indirect effects on osteoprogenitors by evaluating human FD tissue pre- and post-treatment in a phase 2 clinical trial of denosumab (NCT03571191) and in murine in vivo and ex vivo preclinical models. Histological analysis of human and mouse tissue demonstrated increased osteogenic maturation, reduced cellularity, and reduced expression of the pathogenic Gα_s variant in FD lesions after RANKL inhibition. RNA sequencing of human and mouse tissue supported these findings. The interaction between osteoclasts and mutant osteoprogenitors was further assessed in an ex vivo lesion model, which indicated that the proliferation of abnormal FD osteoprogenitors was dependent on osteoclasts. The results from this study demonstrated that, in addition to its expected antiosteoclastic effect, denosumab reduces FD lesion activity by decreasing FD cell proliferation and increasing osteogenic maturation, leading to increased bone formation within lesions. These findings highlight the unappreciated role of cellular crosstalk between osteoclasts and preosteoblasts/osteoblasts as a driver of FD pathology and demonstrate a novel mechanism of action of denosumab in the treatment of bone disease.

While hypoxic signaling has been shown to play a role in many cellular processes, its role in metabolism-linked extracellular matrix (ECM) organization and downstream processes of cell fate after musculoskeletal injury remains to be determined. Heterotopic ossification (HO) is a debilitating condition where abnormal bone formation occurs within extra-skeletal tissues. Hypoxia and hypoxia-inducible factor 1α (HIF-1α) activation have been shown to promote HO. However, the underlying molecular mechanisms by which the HIF-1α pathway in mesenchymal progenitor cells (MPCs) contributes to pathologic bone formation remain to be elucidated. Here, we used a proven mouse injury-induced HO model to investigate the role of HIF-1α on aberrant cell fate. Using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics analyses of the HO site, we found that collagen ECM organization is the most highly up-regulated biological process in MPCs. Zeugopod mesenchymal cell-specific deletion of Hif1α (Hoxa11-CreER^T2; Hif1a^fl/fl) significantly mitigated HO in vivo. ScRNA-seq analysis of these Hoxa11-CreER^T2; Hif1a^fl/fl mice identified the PLOD2/LOX pathway for collagen cross-linking as downstream of the HIF-1α regulation of HO. Importantly, our scRNA-seq data and mechanistic studies further uncovered that glucose metabolism in MPCs is most highly impacted by HIF-1α deletion. From a translational aspect, a pan-LOX inhibitor significantly decreased HO. A newly screened compound revealed that the inhibition of PLOD2 activity in MPCs significantly decreased osteogenic differentiation and glycolytic metabolism. This suggests that the HIF-1α/PLOD2/LOX axis linked to metabolism regulates HO-forming MPC fate. These results suggest that the HIF-1α/PLOD2/LOX pathway represents a promising strategy to mitigate HO formation.

The autonomic nervous system plays a crucial role in regulating bone metabolism, with sympathetic activation stimulating bone resorption and inhibiting bone formation. We found that fractures lead to increased sympathetic tone, enhanced osteoclast resorption, decreased osteoblast formation, and thus hastened systemic bone loss in ovariectomized (OVX) mice. However, the combined administration of parathyroid hormone (PTH) and the β-receptor blocker propranolol dramatically promoted systemic bone formation and osteoporotic fracture healing in OVX mice. The effect of this treatment is superior to that of treatment with PTH or propranolol alone. In vitro, the sympathetic neurotransmitter norepinephrine (NE) suppressed PTH-induced osteoblast differentiation and mineralization, which was rescued by propranolol. Moreover, NE decreased the PTH-induced expression of Runx2 but enhanced the expression of Rankl and the effect of PTH-stimulated osteoblasts on osteoclastic differentiation, whereas these effects were reversed by propranolol. Furthermore, PTH increased the expression of the circadian clock gene Bmal1, which was inhibited by NE-βAR signaling. Bmal1 knockdown blocked the rescue effect of propranolol on the NE-induced decrease in PTH-stimulated osteoblast differentiation. Taken together, these results suggest that propranolol enhances the anabolic effect of PTH in preventing systemic bone loss following osteoporotic fracture by blocking the negative effects of sympathetic signaling on PTH anabolism.

Syndactyly type V (SDTY5) is an autosomal dominant extremity malformation characterized by fusion of the fourth and fifth metacarpals. In the previous publication, we first identified a heterozygous missense mutation Q50R in homeobox domain (HD) of HOXD13 in a large Chinese family with SDTY5. In order to substantiate the pathogenicity of the variant and elucidate the underlying pathogenic mechanism causing limb malformation, transcription-activator-like effector nucleases (TALEN) was employed to generate a Hoxd13Q50R mutant mouse. The mutant mice exhibited obvious limb malformations including slight brachydactyly and partial syndactyly between digits 2-4 in the heterozygotes, and severe syndactyly, brachydactyly and polydactyly in homozygotes. Focusing on BMP2 and SHH/GREM1/AER-FGF epithelial mesenchymal (e-m) feedback, a crucial signal pathway for limb development, we found the ectopically expressed Shh, Grem1 and Fgf8 and down-regulated Bmp2 in the embryonic limb bud at E10.5 to E12.5. A transcriptome sequencing analysis was conducted on limb buds (LBs) at E11.5, revealing 31 genes that exhibited notable disparities in mRNA level between the Hoxd13Q50R homozygotes and the wild-type. These genes are known to be involved in various processes such as limb development, cell proliferation, migration, and apoptosis. Our findings indicate that the ectopic expression of Shh and Fgf8, in conjunction with the down-regulation of Bmp2, results in a failure of patterning along both the anterior-posterior and proximal-distal axes, as well as a decrease in interdigital programmed cell death (PCD). This cascade ultimately leads to the development of syndactyly and brachydactyly in heterozygous mice, and severe limb malformations in homozygous mice. These findings suggest that abnormal expression of SHH, FGF8, and BMP2 induced by HOXD13Q50R may be responsible for the manifestation of human SDTY5.

Extracellular matrix (ECM) stiffening is a typical characteristic of cartilage aging, which is a quintessential feature of knee osteoarthritis (KOA). However, little is known about how ECM stiffening affects chondrocytes and other molecules downstream. This study mimicked the physiological and pathological stiffness of human cartilage using polydimethylsiloxane (PDMS) substrates. It demonstrated that epigenetic Parkin regulation by histone deacetylase 3 (HDAC3) represents a new mechanosensitive mechanism by which the stiffness matrix affected chondrocyte physiology. We found that ECM stiffening accelerated cultured chondrocyte senescence in vitro, while the stiffness ECM downregulated HDAC3, prompting Parkin acetylation to activate excessive mitophagy and accelerating chondrocyte senescence and osteoarthritis (OA) in mice. Contrarily, intra-articular injection with an HDAC3-expressing adeno-associated virus restored the young phenotype of the aged chondrocytes stimulated by ECM stiffening and alleviated OA in mice. The findings indicated that changes in the mechanical ECM properties initiated pathogenic mechanotransduction signals, promoted the Parkin acetylation and hyperactivated mitophagy, and damaged chondrocyte health. These results may provide new insights into chondrocyte regulation by the mechanical properties of ECM, suggesting that the modification of the physical ECM properties may be a potential OA treatment strategy.

Wnt/β-catenin signaling is critical for various cellular processes in multiple cell types, including osteoblast (OB) differentiation and function. Exactly how Wnt/β-catenin signaling is regulated in OBs remain elusive. ATP6AP2, an accessory subunit of V-ATPase, plays important roles in multiple cell types/organs and multiple signaling pathways. However, little is known whether and how ATP6AP2 in OBs regulates Wnt/β-catenin signaling and bone formation. Here we provide evidence for ATP6AP2 in the OB-lineage cells to promote OB-mediated bone formation and bone homeostasis selectively in the trabecular bone regions. Conditionally knocking out (CKO) ATP6AP2 in the OB-lineage cells (Atp6ap2^Ocn-Cre) reduced trabecular, but not cortical, bone formation and bone mass. Proteomic and cellular biochemical studies revealed that LRP6 and N-cadherin were reduced in ATP6AP2-KO BMSCs and OBs, but not osteocytes. Additional in vitro and in vivo studies revealed impaired β-catenin signaling in ATP6AP2-KO BMSCs and OBs, but not osteocytes, under both basal and Wnt stimulated conditions, although LRP5 was decreased in ATP6AP2-KO osteocytes, but not BMSCs. Further cell biological studies uncovered that osteoblastic ATP6AP2 is not required for Wnt3a suppression of β-catenin phosphorylation, but necessary for LRP6/β-catenin and N-cadherin/β-catenin protein complex distribution at the cell membrane, thus preventing their degradation. Expression of active β-catenin diminished the OB differentiation deficit in ATP6AP2-KO BMSCs. Taken together, these results support the view for ATP6AP2 as a critical regulator of both LRP6 and N-cadherin protein trafficking and stability, and thus regulating β-catenin levels, demonstrating an un-recognized function of osteoblastic ATP6AP2 in promoting Wnt/LRP6/β-catenin signaling and trabecular bone formation.