Transcriptome Analysis of Differentially Expressed Genes and Molecular Pathways Involved During Osteoclast...

Bone remodeling relies on balanced osteoclast and osteoblast activity, with dysregulated osteoclast differentiation contributing to pathologies like osteoporosis. While RANKL/M-CSF signaling and master regulators (NFATc1, c-Fos) are established, comprehensive temporal dynamics of gene networks governing progressive osteoclastogenesis stages remain poorly characterized. We employed an integrated approach combining RANKL-induced osteoclast differentiation from murine bone marrow-derived macrophages (BMMs) with rigorous functional validation (TRAP staining, podosome visualization via rhodamine-phalloidin/DAPI, Western blotting for NFATc1/c-Fos/CTSK) and high-throughput RNA sequencing across critical time points (day 0, 1, 3, and 5). Subsequent bioinformatic analyses included differential expression profiling (DESeq2/edgeR), Gene Ontology (GO) and KEGG pathway enrichment, Gene Set Enrichment Analysis (GSEA), and temporal clustering using Mfuzz. Key transcriptional findings were confirmed by RT-qPCR. GO/KEGG/GSEA analyses revealed significant enrichment of DEGs in FA signaling, PI3K-Akt pathway, and cytoskeletal organization. Mfuzz clustering delineated distinct gene expression trajectories. We documented stage-specific expression kinetics: 23 FA-related genes showed dynamic shifts (e.g., upregulated Itgb3, Src, Pik3r3, Fn1; downregulated Itga6, Parvg); multiple MMPs (Mmp2, Mmp9, Mmp13-17) and TIMPs (Timp1-3) were progressively induced, while Mmp8, Mmp12, Mmp27 were suppressed. Cell fusion involved upregulated Tnfrsf11a, Nfatc1, Plcg1, Xkr8 alongside downregulated Trem2, Tyrobp, Ccr2, Rhoa, Casp3. This study provides the first comprehensive temporal transcriptome atlas of RANKL-induced osteoclastogenesis. It delineates stage-specific molecular reprogramming, revealing dynamic regulation of FA signaling components promoting adhesion/migration, complex MMP/TIMP induction balancing ECM degradation, and coordinated transcriptional networks enabling fusion. These findings significantly expand understanding beyond the core RANKL-NFATc1 axis, identifying novel stage-specific regulatory hubs and potential therapeutic targets within FA, PI3K-Akt, MMP/TIMP, and fusion pathways for osteoclast-driven bone diseases.