In Vivo and In Vitro Analysis of Cathepsin D in Bone Homeostasis and Osteoporosis.

OBJECTIVES: To explore the role of cathepsin D (CTSD), a lysosomal aspartyl protease, as a potential biomarker and therapeutic target in the context of osteoporosis. METHODS: Utilizing single-cell sequencing data from the Gene Expression Omnibus (GEO) database, we identified differential expression patterns of CTSD in human femoral head tissues between osteoporotic and non-osteoporotic states. Human mesenchymal stem cells (MSCs) were treated with recombinant human CTSD and/or osteogenic induction medium and the role of CTSD on osteoblast differentiation were investigated by RT-qPCR, western blot, immunofluorescence staining and alizarin red staining. The role of CTSD on osteoporotic changes was further verified in ovariectomized mice. RESULTS: In vitro analyses of human mesenchymal stem cells (MSCs) treated with recombinant human CTSD (rhCTSD) under osteogenic induction conditions revealed modulation of osteogenic marker expression, including ALP, COL1A1, and RUNX2. Histological assessments using Alizarin Red staining and immunofluorescence corroborated these findings, demonstrating that CTSD influences osteoblast differentiation and matrix mineralization. In vivo studies in mice further supported the role of CTSD, showing that perturbations in this enzyme result in alterations in bone mineral density and volume characteristic of osteoporotic changes. CONCLUSION: Collectively, these findings implicate CTSD as a regulator of bone homeostasis and support its potential as a novel target for therapeutic intervention in osteoporosis.