Lansoprazole Upregulates Polyubiquitination of the TNF Receptor-Associated Factor 6 and Facilitates Runx2-mediated Osteoblastogenesis

•Lansoprazole facilitates osteoblastogenesis by upregulating Runx2, and accelerates fracture healing in rats.•Lansoprazole activates the noncanonical BMP-TAK1-p38 MAPK pathway in mesenchymal stem cells.•Lansoprazole enhances autopolyubiquitination of TRAF6 by tightly fitting in a pocket of a deubiquitination enzyme, CYLD.Lansoprazole is a widely used proton pump inhibitor (PPI) for acid-related diseases such as gastroduodenal ulcers and reflux esophagitis. We found that lansoprazole facilitates differentiation and maturation of bone-forming cells, osteoblasts, and accelerates fracture healing in a rat model of femoral fracture. Lansoprazole fits in a pocket of an enzyme that breaks multi-ubiquitin chains, and inhibits its enzymatic activity. This inhibition activates a noncanonical bone morphogenetic protein (BMP)-mediated signaling and enhances osteoblastic differentiation in cultured cells and in a rat model of femoral facture. Lansoprazole is a potential therapeutic agent for fracture healing via upregulation of osteoblastogenesis.

The transcription factor, runt-related transcription factor 2 (Runx2), plays a pivotal role in the differentiation of the mesenchymal stem cells to the osteochondroblast lineages. We found by the drug repositioning strategy that a proton pump inhibitor, lansoprazole, enhances nuclear accumulation of Runx2 and induces osteoblastogenesis of human mesenchymal stromal cells. Systemic administration of lansoprazole to a rat femoral fracture model increased osteoblastogenesis. Dissection of signaling pathways revealed that lansoprazole activates a noncanonical bone morphogenic protein (BMP)-transforming growth factor-beta (TGF-β) activated kinase-1 (TAK1)–p38 mitogen-activated protein kinase (MAPK) pathway. We found by in cellulo ubiquitination studies that lansoprazole enhances polyubiquitination of the TNF receptor-associated factor 6 (TRAF6) and by in vitro ubiquitination studies that the enhanced polyubiquitination of TRAF6 is attributed to the blocking of a deubiquitination enzyme, cylindromatosis (CYLD). Structural modeling and site-directed mutagenesis of CYLD demonstrated that lansoprazole tightly fits in a pocket of CYLD where the C-terminal tail of ubiquitin lies. Lansoprazole is a potential therapeutic agent for enhancing osteoblastic differentiation.