Bisphosphonate zoledronic acid blocks secretory autophagy and inhibits bone resorptive functions in osteoclasts

Bisphosphonates (BPs) are the most widely used anti-resorptive agents and first-line drugs for managing bone-related diseases, such as osteoporosis, Paget disease of bone, and bone metastatic cancer. BPs are known to inhibit osteoclasts' functions, and recent studies have highlighted the importance of macroautophagy/autophagy in osteoclasts. However, the involvement of autophagy in BP-mediated inhibition of osteoclast functions remains unclear. In this study, we showed that BPs inhibit the bone resorptive functions of osteoclasts by blocking autophagy. At the non-apoptotic doses, zoledronic acid (ZOL) inhibited autophagy by blocking autophagic flux and delaying the degradation of autophagy-related proteins. ZOL also prevented the cleavage and secretion of secretory proteins such as CTSK, ACP5/TRAP, and MMP9 essential for bone resorption. Mechanistically, ZOL inhibits the prenylation of the RAB7 small GTPase, a key protein that is required for autolysosome formation. In vivo studies showed that osteoclast-specific rab7 conditional knockout mice exhibited osteopetrotic phenotypes. These findings provide insights into how BPs disrupt osteoclast function by blocking autophagy and suggest that targeting autophagy in osteoclasts could be a potential therapeutic approach for bone-related diseases.Abbreviations: ACP5/TRAP: acid phosphatase 5, tartrate resistant, ATG: autophagy related, 54 BafA1: bafilomycin A1, BECN1: beclin 1, BMM: bone marrow-derived macrophage, CQ: 55 chloroquine, CSF1/M-CSF: colony stimulating factor 1, CTSK: cathepsin K, CTX-I: C-terminal 56 telopeptide of type I collagen, FPPS: farnesyl pyrophosphate synthase, GFP: green fluorescent 57 protein, GGTI: geranylgeranyltransferase I inhibitor, LAMP1: lysosome associated membrane 58 protein 1, MAP1LC3/LC3: microtubule associated protein 1 light chain 3, MMP9: matrix 59 metallopeptidase 9, N-BPs: nitrogen-containing bisphosphonates, P1NP: procollagen type I N- 60 terminal propeptide, PGGT1B: protein geranylgeranyl transferase type I subunit beta, RABGGTB: 61 Rab geranylgeranyltransferase subunit beta, RFP: red fluorescent protein, SQSTM1/p62: 62 sequestosome 1, TNFSF11/RANKL: TNF superfamily member 11, ZOL: zoledronic acid.