Bone Abstracts

Mutations in PLEKHM1 cause bone disease in humans and animals. Truncations causedeficient bone resorption by osteoclasts leading to osteopetrosis. A gain-of-function point mutation causes increased resorption leading to osteopenia. We and others have shown that PLEKHM1, a multi-modular protein, interacts with the small GTPase rab7 and is involved in vesicle trafficking, secretion, and membrane biogenesis. To investigate other interactions of PLEKHM1 we performed tandem affinity purification and mass spectrophotometry analysis, and we found that TRAFD1 (FLN29) also interacts with PLEKHM1. Reciprocal pull-down assays confirmed the interaction and localized the regions of interaction. Trafd1 is implicated in the inhibition of innate immune responses in monocytes/macrophages via Toll-like receptor signaling, but a role for Trafd1 in osteoclast biology has not been described. We found Trafd1 expression and its co-localization with PLEKHM1 increased when osteoclast differentiation was induced by receptor activator for nuclear factor kappa B ligand (RANKL). Decreasing Trafd1 expression by shRNA in RAW264.7 cells dramatically attenuated osteoclast formation, acidification, and hydroxyapatite resorption. Decreased osteoclast differentiation and resorptive activity in cells with Trafd1 knockdown did not result from suppression of essential osteoclast transcription factors or from decreased cellular levels of acidification factors (ClC7 chloride channel, cathepsin K, carbonic anhydrase II, and a3- vATPase). This suggests a defect in targeting these factors to their correct locations at the ruffled border. To confirm that, for our study we used osteoclasts isolated from incisors absent (ia) osteopetrotic rats. ia/ia rats have a naturally occurring mutation in PLEKHM1 which leads to production of truncated protein. Subcellular fractionations of vesicles isolated from osteoclasts from ia/ia rats show that, in contrast to WT osteoclasts, Trafd1 is unable to associate with early or late endosomes. In conclusion, our data demonstrate a novel role for Trafd1 in osteoclast vesicle transport and acidification. PLEKHM1 and Trafd1 interaction is critical for their function in osteoclasts.