Involvement of aquaporin 9 in osteoclast differentiation.
Aquaporins (water channels) selectively enhance water permeability of membranes. Since osteoclast differentiation includes a dramatic increase in cell volume, we hypothesize that aquaporin(s) is/are critical for the formation of the multinucleated osteoclast from its mononuclear precursor. Our studies employ two cell models, bone marrow macrophages (BMMs) and the murine macrophage-like cell line, RAW264.7, as osteoclast precursors. Receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL) and macrophage-colony-stimulating factor or RANKL alone were used to induce osteoclast differentiation in BMMs or RAW264.7 cells, respectively. We first used qualitative reverse transcription (RT)-PCR to examine which of the aquaporins are expressed in osteoclasts and in their precursor cells. Out of the 10 aquaporins examined, only aquaporin 9 (AQP9) was expressed in osteoclast-lineage cells. AQP9 has unique aqueous pore properties mediating the passage of a wide variety of non-charged solutes in addition to water. Western analyses using specific antibodies revealed a higher AQP9 level in RANKL-treated than in untreated cells. Quantitative real-time RT-PCR analyses also demonstrated higher AQP9 mRNA levels in RANKL-treated cells. Finally, we examined the effect of phloretin, an AQP9 inhibitor, on RANKL-induced osteoclast differentiation. Cells were incubated with RANKL for 5 days, and phloretin was added for the last 2 days, when most fusion occurs. A dramatic reduction in osteoclast size and in the number of nuclei per osteoclast was observed in cultures containing phloretin. The inhibitor did not have a significant effect on the number and size of mononuclear phagocytes in cultures not treated with RANKL. Our results suggest a role for AQP9 in osteoclast differentiation, specifically in the fusion process.
Aharon
Refael
R
Humphrey Center for Experimental Medicine and Cancer Research, The Hebrew University Faculty of Medicine, Jerusalem 91120, Israel.
Bar-Shavit
Zvi
Z
D000818:Animals; D020346:Aquaporins / Q000378:metabolism / Q000502:physiology*; D001854:Bone Marrow Cells / Q000166:cytology; D002352:Carrier Proteins / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D008264:Macrophages / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D010010:Osteoclasts / Q000378:metabolism; D010586:Phagocytes / Q000378:metabolism; D010693:Phloretin / Q000494:pharmacology; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B
C491638:Aqp9 protein, mouse; D020346:Aquaporins; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D010693:Phloretin