Skeletal development is a complex process which requires the tight regulation of gene activation and suppression in response to local signaling pathways. Among these pathways, Notch signaling is implicated in governing cell fate determination, proliferation, differentiation and apoptosis of skeletal cells-osteoblasts, osteoclasts, osteocytes and chondrocytes. Moreover, human genetic mutations in Notch components emphasize the critical roles of Notch signaling in skeletal development and homeostasis. In this review, we focus on the physiological roles of Notch signaling in skeletogenesis, postnatal bone and cartilage homeostasis and fracture repair. We also discuss the pathological gain- and loss-of-function of Notch signaling in bone and cartilage, resulting in osteosarcoma and age-related degenerative diseases, such as osteoporosis and osteoarthritis. Understanding the physiological and pathological function of Notch signaling in skeletal tissues using animal models and human genetics will provide new insights into disease pathogenesis and offer novel approaches for the treatment of bone/cartilage diseases.

Shohat-type spondyloepimetaphyseal dysplasia (SEMD) is a skeletal dysplasia that affects cartilage development. Similar skeletal disorders, such as spondyloepiphyseal dysplasias, are linked to mutations in type II collagen (COL2A1), but the causative gene in SEMD is not known. Here, we have performed whole-exome sequencing to identify a recurrent homozygous c.408+1G>A donor splice site loss-of-function mutation in DDRGK domain containing 1 (DDRGK1) in 4 families affected by SEMD. In zebrafish, ddrgk1 deficiency disrupted craniofacial cartilage development and led to decreased levels of the chondrogenic master transcription factor sox9 and its downstream target, col2a1. Overexpression of sox9 rescued the zebrafish chondrogenic and craniofacial phenotype generated by ddrgk1 knockdown, thus identifying DDRGK1 as a regulator of SOX9. Consistent with these results, Ddrgk1^–/– mice displayed delayed limb bud chondrogenic condensation, decreased SOX9 protein expression and Col2a1 transcript levels, and increased apoptosis. Furthermore, we determined that DDRGK1 can directly bind to SOX9 to inhibit its ubiquitination and proteasomal degradation. Taken together, these data indicate that loss of DDRGK1 decreases SOX9 expression and causes a human skeletal dysplasia, identifying a mechanism that regulates chondrogenesis via modulation of SOX9 ubiquitination.

ATRAID is essential for responses to the commonly prescribed osteoporosis drugs nitrogen-containing bisphosphonates.

Loss-of-function mutations in DDRGK1 have been shown to cause Shohat type spondyloepimetaphyseal dysplasia (SEMD). In zebrafish, loss of function of ddrgk1 leads to defects in early cartilage development. Ddrgk1^−/− mice show delayed mesenchymal condensation in the limb buds and early embryonic lethality. Mechanistically, Ddrgk1 interacts with Sox9 and reduces ubiquitin-mediated proteasomal degradation of Sox9 protein. To investigate the cartilage-specific role of DDRGK1, conditional knockout mice were generated by intercrossing Prx1-Cre transgenic mice with Ddrgk^fl/fl mice to delete its expression in limb mesenchymal cells. Mutant mice showed progressive severe shortening of the limbs and joint abnormalities. The growth plate showed disorganization with shortened proliferative zone and enlarged hypertrophic zone. In correlation with these findings, Sox9 and Col2a1 protein levels were decreased, while Col10a1 expression was expanded. These data demonstrate the importance of Ddrgk1 during growth plate development. In contrast, deletion of Ddrgk1 with the osteoblast-specific Osteocalcin-Cre and Leptin receptor-Cre lines did not show bone phenotypes, suggesting that the effect on limb development is cartilage-specific. To evaluate the role of DDRGK1 in cartilage postnatal homeostasis, inducible Agc1-Cre^ERT2; Ddrgkl^fl/fl mice were generated. Mice in which Ddrgk1 was deleted at 3 months of age showed disorganized growth plate, with significant reduction in proteoglycan deposition. These data demonstrate a postnatal requirement for Ddrgk1 in maintaining normal growth plate morphology. Together, these findings highlight the physiological role of Ddrgk1 in the development and maintenance of the growth plate cartilage. Furthermore, these genetic mouse models recapitulate the clinical phenotype of short stature and joint abnormalities observed in patients with Shohat type SEMD.

The expression of microRNAs (miRNAs) is dysregulated in many types of cancers including osteosarcoma (OS) due to genetic and epigenetic alterations. Among these, miR‐34c, an effector of tumor suppressor P53 and an upstream negative regulator of Notch signaling in osteoblast differentiation, is dysregulated in OS. Here, we demonstrated a tumor suppressive role of miR‐34c in OS progression using in vitro assays and in vivo genetic mouse models. We found that miR‐34c inhibits the proliferation and the invasion of metastatic OS cells, which resulted in reduction of the tumor burden and increased overall survival in an orthotopic xenograft model. Moreover, the osteoblast‐specific overexpression of miR‐34c increased survival in the osteoblast specific p53 mutant OS mouse model. We found that miR‐34c regulates the transcription of several genes in Notch signaling (NOTCH1, JAG1, and HEY2) and in p53‐mediated cell cycle and apoptosis (CCNE2, E2F5, E2F2, and HDAC1). More interestingly, we found that the metastatic‐free survival probability was increased among a patient cohort from Therapeutically Applicable Research to Generate Effective Treatments (TARGET) OS, which has lower expression of direct targets of miR‐34c that was identified in our transcriptome analysis, such as E2F5 and NOTCH1. In conclusion, we demonstrate that miR‐34c is a tumor suppressive miRNA in OS progression in vivo. In addition, we highlight the therapeutic potential of targeting miR‐34c in OS. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

ATP-dependent chromatin remodeling protein ATRX is an essential regulator involved in maintenance of DNA structure and chromatin state and regulation of gene expression during development. ATRX was originally identified as the monogenic cause of X-linked α-thalassemia mental retardation (ATR-X) syndrome. Affected individuals display a variety of developmental abnormalities and skeletal deformities. Studies from others investigated the role of ATRX in skeletal development by tissue-specific Atrx knockout. However, the impact of ATRX during early skeletal development has not been examined. Using preosteoblast-specific Atrx conditional knockout mice, we observed increased trabecular bone mass and decreased osteoclast number in bone. In vitro coculture of Atrx conditional knockout bone marrow stromal cells (BMSCs) with WT splenocytes showed impaired osteoclast differentiation. Additionally, Atrx deletion was associated with decreased receptor activator of nuclear factor κ-B ligand ( Rankl )/ osteoprotegerin ( Opg ) expression ratio in BMSCs. Notably, Atrx -deficient osteolineage cells expressed high levels of the neuropeptide cocaine- and amphetamine-regulated transcript prepropeptide ( Cartpt ). Mechanistically, ATRX suppresses Cartpt transcription by binding to the promoter, which is otherwise poised for Cartpt expression by RUNX2 binding to the distal enhancer. Finally, Cartpt silencing in Atrx conditional knockout BMSCs rescued the molecular phenotype by increasing the Rankl / Opg expression ratio. Together, our data show a potent repressor function of ATRX in restricting Cartpt expression during skeletal development.