Abstract
Osteoporosis is a bone loss disorder that mainly affects the elderly population and reduces the quality of life for patients12,21. Current treatments slows down bone degradation instead of promoting bone formation, emphasizing the need for osteoanabolic therapeutics. Cellular communication network factor 3 (CCN3) has been identified as an osteoanabolic hormone that stimulates skeletal stem cell (SSC) activity and enhances bone remodeling2. However, the molecular target and downstream signaling pathways remains unclear. Structural modeling indicates that CCN3 binds to Discoidin Domain Receptor 2 (DDR2), a collagen-binding receptor tyrosine kinase that plays a role in bone remodeling26. This study aimed to provide insight on whether a CCN3-DDR2 signaling pathway promotes SSC osteogenesis and whether targeting this pathway may mitigate bone loss disorders. This study had three aims: (1) investigate whether lentiviral DDR2 knockdown affects SSC osteogenesis with or without CCN3 in vitro, (2) assess whether DDR2 expression varied in young and aged murine long bones, and (3) examine whether DDR2 knockdown prevents CCN3 treated SSC-derived ossicle formation in vivo. To establish DDR2 function in vitro, primary human SSCs were isolated, transduced with a lentiviral DDR2 knockdown (KD) or GFP control, and sorted via fluorescence-activated cell sorting. Osteogenic differentiation, with or without recombinant CCN3, was quantified by Alizarin Red staining. No statistically significant changes were observed, but trends suggested DDR2 knockdown might affect osteogenic potential in the presence of CCN3. Further study and validation are needed. To determine DDR2 expression in aging bone, wild-type young and aged mice femurs were immunohistochemically stained to detect DDR2 localization. Young mice displayed higher DDR2 signal in the growth plate, periosteum, and bone marrow than aged mice in all skeletal compartments. The reduced DDR2 expression in aged bone suggests an age-related decrease in DDR2. To explore DDR2’s role in bone formation in vivo, human SSCs with or without DDR2 knockdown were subcutaneously transplanted into young NSG mice. After an eight-week engraftment, ossicle development was analyzed by histology. Low sample recovery made statistical analysis unfeasible. DDR2 KD transplants contained minimal bone formation and DDR2 KD + CCN3-treated transplants had no mineralized tissue. Data suggest that DDR2 is needed for human SSC-mediated osteogenesis independent of CCN3. These findings suggest a relationship between CCN3 and DDR2 signaling. This research highlights DDR2’s potential function in SSC-mediated osteogenesis. However, due to high variability in this study, the functional effects of DDR2 knockdown remains inconclusive. Future investigations should enhance DDR2 knockdown efficiency, increase sample size, and investigate transcriptomic analyses. Further research into the CCN3-DDR2 signaling axis could contribute to the development of osteoanabolic therapeutics targeting osteoporosis and other bone loss disorders.