Then the Caki-1 cells were transduced with the virus

s the protons; and the specialized chloride channel ClC7 provides the anions needed for electroneutrality. Lossof-function mutations in any of those genes cause severe autosomal recessive osteopetrosis , also known as malignant osteopetrosis. Bone degradation products are then taken up by endocytosis at the ruffled border and further degraded as they are transported by transcytosis to PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19667188 the facultative secretory domain at the top of the polarized osteoclast for secretion. The importance of intracellular vesicle trafficking was emphasized with the discovery of two proteins involved in that process which cause osteoclast bone disease when mutated. One is the sorting nexin, Snx10. Snx10 regulates endosome sorting and movement through the cell. Mutations in SNX10 account for roughly 4% of ARO in humans, often including an osteopetro-rickets phenotype, and Snx10 is required for osteoclast differentiation and function. The other is Plekhm1, a large, multi-domain protein that also causes osteopetrosis in humans and in the incisors absent rat when truncated, and which causes osteopenia with focal sclerosis when carrying a gain-of-function point mutation. Plekhm1 associates with late endosomes/early lysosomes, binding to the small GTPase Rab7 and interacting with Rubicon to regulate PI3 kinase-dependent vesicle movement. During our investigations described below, we used mass spectrometry to identify proteins that interact with Plekhm1 in screens of pull-downs of cell extracts. One protein thus identified was TRAFD1. TRAFD1 was first identified as an interferon- and lipopolysaccharide- inducible factor. TRAFD1 contains a TRAF-type zinc finger domain at its N-terminus and a TRAF6-binding motif in its middle region. The TRAF6 binding motif of TRAFD1 interacts 2 / 21 TRAFD1 in Osteoclast Activity with the C-terminal part of TRAF6 in monocytes, and TRAFD1 also physically interacts with other TRAF proteins . In monocytes/macrophages, TRAFD1 suppresses the inflammatory responses to innate immunity by inhibiting Toll-like receptor 4 dependent NF-B and MAPK activation. The interaction with TRAF6 led us to hypothesize that TRAFD1 may be important for osteoclast function. In the present study, we confirmed and mapped in detail the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19667299 interaction of TRAFD1 and Plekhm1 We also showed that TRAFD1 expression is up-regulated by RANKL, that it co-localizes with Plekhm1 to late endosomes, and that co-localization of TRAFD1 and Plekhm1 increases after RANKL stimulation. We also show here that TRAFD1 affects osteoclast acidification, mineral c-Met inhibitor 2 supplier solubilization, and secretion of tartrate-resistant acid phosphatase and cathepsin K. In ia/ia osteopetrotic rat osteoclasts which are unable to resorb bone, truncated Plekhm1 prevented TRAFD1 association with vesicles. Materials and Methods Animals All animals were obtained from our colonies of ia rats and C57BL/6J mice maintained at the University of Massachusetts Medical School under specific-pathogen-free conditions, and all procedures were in accordance with the NIH Guide for the Care and Use of Laboratory animals and were approved by the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School. Euthanasia was performed by inhalation anesthesia followed by decapitation. Antibodies The following antibodies were used in the study: rat anti-HA, mouse anti-FLAG M2, rabbit anti-lamin B1. Goat anti-Rab7, goat anti-ClC7, and mouse anti-CAII were purchased in Santa Cruz Biotechnology. Rabbit anti-R