In addition to -La mAb and -La rAb, in some experiments we also used rabbit anti-La Phospho-Ser366 antibody (Abcam, 61800, referred to as -p366 La rAb) that specifically recognizes phosphorylated human La (phosphoSer366). a therapeutic target. Subject terms: Cell biology, Biochemistry, Bone Bone maintenance in health and disease depends on bone-resorbing osteoclasts. Whitlock gene product). La, also referred to as LARP3 and La autoantigen, is generally recognized as an abundant and ubiquitous RNA-binding VAV1 protein26. La has a nuclear localization sequence (NLS) at its C-terminus in addition to other intracellular trafficking signals27 that result in La being observed almost exclusively in the nucleus of human cells28. The best-characterized function of nuclear La is to protect precursor tRNAs from exonuclease digestion through specific interactions between Las highly conserved, N-terminal La domain and the 3 ends of tRNA. In addition to its AZD9567 nuclear functions, La shuttles to the cytoplasm29 and assists in the correct folding of some mRNAs, acting as an RNA chaperone30. In a few specialized biological processes (e.g., apoptosis, viral infection, serum starvation), La protein is non-phosphorylated at phospo-Ser-366, loses its NLS via proteolytic cleavage, and this low molecular weight (LMW) species traffics to the surface of the cells27,31C34. However, the biological function of this cleaved, surface La, if any, is unknown. Here, we report that osteoclast formation is accompanied by and depends on drastic changes in the steady-state level, molecular species, and AZD9567 intracellular localization of La protein. We demonstrate that human and murine La functions as a regulator of osteoclast fusion and impacts osteoclasts ability to resorb bone. Surprisingly, La, present in primary human monocytes, nearly disappears in M-CSF-derived osteoclast precursors. RANKL-induced commitment to osteoclastogenesis drives the reappearance of La protein at the surface of committed, fusing osteoclasts. As osteoclast fusion plateaus, LMW La disappears and higher molecular weight, phosphorylated, full-length protein (FL-La) is observed within the nuclei of mature, multinucleated osteoclasts. Perturbing La expression, cleavage or surface function inhibits osteoclast fusion, while exogenous, surface La promotes fusion. Moreover, the mechanism by which La promotes osteoclast fusion is independent of AZD9567 Las ability to interact with RNA through its highly conserved La domain. Indeed, a C-terminal portion of La, lacking the La domain and RNA recognition motif 1 (RRM1) is sufficient to promote fusion between human osteoclasts. Our findings indicate that, while La protein plays an ancient, well-described and essential role in the RNA biology of all eukaryotes, La has been adapted in mammals to also serve as an osteoclast fusion manager. In this highly specific role on the surface of fusing osteoclasts, La may present a promising target for the treatment of bone diseases stemming from perturbed bone turnover. Results Formation of multinucleated osteoclasts involves La protein Human osteoclastogenesis was modeled by treating primary monocytes with M-CSF to derive mononucleated osteoclast precursors to which recombinant RANKL was subsequently added to obtain multinucleated osteoclasts that readily resorb bone18 (Fig.?1a, b, Fig.?S1aCc). Osteoclast precursors begin fusing at ~2 days following RANKL addition and after ~5 days reach sizes (~ 5C10 nuclei/cell) characteristic of mature multinucleated osteoclasts10,35,36 (Fig.?1b, Fig.?S1c). Open in a separate window Fig. 1 Osteoclastogenic differentiation is accompanied by drastic changes in the steady-state levels and localization of La molecular species.a Representative images of stages of osteoclastogenic derivation of human monocytes after M-CSF (6 days, referred to as M-CSF) and after M-CSF (6 days) followed by M-CSF?+?RANKL (5 days, RANKL), respectively. (Magenta = Phalloidin-Alexa488, Cyan = Hoechst). b Quantification of the number of fusion events normalized to the total number of nuclei observed over time following RANKL addition. (in human osteoclast precursors treated with siRNA at day 1 post-RANKL addition. (mouse (FD) and a wild-type littermate (WT). Explants were either cultured with M-CSF alone, or M-CSF and Doxy. c ELISA quantification of mRANKL produced in FD vs WT cultures.