PINK1-driven mitophagy regulates RANKL-induced osteoclastogenesis in bone-marrow macrophages

Background : Excessive bone resorption by osteoclasts is a hallmark of osteoporosis, yet the molecular mechanisms that govern osteoclast differentiation remain incompletely defined. By integrating in-silico public transcriptomic dataset analysis with in-vitro validation, we sought to identify novel regulators of osteoclastogenesis. Methods: Differential‑expression and STRING network analyses of two independent RNA‑seq datasets of RANKL‑stimulated macrophages (GSE172007, GSE272401) were performed with limma , clusterProfiler and CytoHubba. For in-vitro analyses, RAW264.7 macrophages were transfected with a PINK1 over‑expression vector (OE‑PINK1) or PINK1‑specific siRNA (si‑PINK1) and exposed to RANKL + M‑CSF. Osteoclast viability (CCK‑8), apoptosis/mitophagy proteins (Bax, Bcl‑2, Beclin‑1, LC3‑II), mitochondrial membrane potential (JC‑1) and intracellular ROS were quantified. Expression of osteoclast markers (TRAP, Cathepsin K, NFATc1, c‑Fos) was assessed by qRT‑PCR and Western blot. Results: In-silico screening highlighted PTEN‑induced kinase 1 (PINK1) as a top‑degree hub within 220 high‑confidence, RANKL‑responsive genes enriched for osteoclast differentiation pathways. In vitro, PINK1 over‑expression (i) increased cell viability, (ii) raised Bax and lowered Bcl‑2, (iii) elevated Beclin‑1 and LC3‑II, (iv) preserved mitochondrial ΔΨm and suppressed ROS, and (v) up‑regulated TRAP, Cathepsin K, NFATc1 and c‑Fos. Conversely, PINK1 silencing produced the opposite effects, depolarizing ΔΨm and provoking ROS accumulation. Conclusion: Overall, our combined in‑silico and experimental approach identifies PINK1-mediated mitophagy as a pivotal driver of RANKL‑induced osteoclastogenesis. PINK1 couples mitochondrial quality control to osteoclast survival and resorptive gene expression, making it a promising therapeutic target for osteoporosis and other bone‑resorptive disorders.