Integrating bulk transcriptomics and single-cell RNA sequencing reveals protein secretion pathway genes and regulatory mechanisms in osteoporosis

Abstract Osteoporosis (OP) is a systemic skeletal disorder in which altered protein-secretory homeostasis may contribute to impaired bone remodeling. We integrated bulk transcriptomic datasets GSE56815 and GSE2208 with the single-cell RNA-sequencing dataset GSE147287 to identify protein secretion pathway-associated genes and their cellular context in OP. Differential expression, weighted gene co-expression network analysis, three machine-learning algorithms, immune infiltration analysis, gene set enrichment analysis, regulatory network construction, and single-cell analyses were performed, followed by RT-qPCR validation. PPWD1 and TMEM50B were prioritized as key genes and were consistently upregulated in OP. Both genes were associated with proteasome, ribosome, and spliceosome pathways. Single-cell analysis highlighted bone marrow mesenchymal stem cells (BM-MSCs) as a relevant cell population and suggested differences in PPWD1 and TMEM50B expression along pseudotime, together with candidate CXCL12-CXCR4-mediated communication. RT-qPCR confirmed increased expression of both genes in OP samples. These findings identify PPWD1 and TMEM50B as protein secretion pathway-associated genes in OP and provide a transcriptomic framework linking proteostasis-related signatures, BM-MSC states, and osteoimmune alterations while requiring further functional validation.