OP0041 Essential role for TRPC1 channels in osteoclast fusion and in osteoporotic bone loss
Authors: D. Umlauf, H. Hidding, L.O. Lindemann, S. Frank, B. Dankbar, C. Cromme, M. Rupp, R. Stange, R.P. Marshall, M. Amling, A. Dietrich, M. Steiner, U. Kornak, A. Schwab, T. Pap, J. Bertrand
Journal: Annals of the Rheumatic Diseases (2013)

<h3>Background</h3> Directed monocyte migration, cell-cell contacts and fusion to multinucleated cells are necessary events in the process of osteoclast differentiation. Since transient receptor potential canonical channel 1 (TRPC1) has a pivotal role in these specific processes in other cell types, we investigated the role of TRPC1 in osteoclastogenesis and bone remodeling. <h3>Objectives</h3> To analyse the effect of TRPC1 knockout on in-vitro osteoclast differentiation and to study the skeletal phenotype of TRPC1-/- mice under physiological conditions as well as using an animal model of postmenopausal osteoporosis. <h3>Methods</h3> For all in-vitro experiments, bone marrow macrophages were isolated from TRPC1-/- mice and corresponding wild type (WT) controls and were cultured in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor kappa-B ligand. Osteoclast-like cells were characterized by staining for tartrate-resistant acid phosphatase (TRAP). Using quantitative real-time PCR mRNA levels of TRPC1 and nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATC1) were analysed. Time-lapse videomicroscopy was used to study osteoclast migration and fusion. The intracellular Ca<sup>2+</sup> concentration of osteoclasts was measured ratiometrically with the fluorescent Ca<sup>2+</sup> dye Fura-2. The skeletal phenotype of 16-week old mice was investigated by μCT-analyses of trabecular bone in the lumbar spine. Ovariectomy was performed on 12-week old sex- and aged-matched littermates as a model for human postmenopausal bone loss. <h3>Results</h3> PCR analyses revealed that TRPC1 is hardly expressed in monocytes and pre-osteoclasts, but gets upregulated during osteoclast-differentiation. In osteoclast formation assays, the loss of TRPC1 is associated with a significantly impaired osteoclast differentiation. As seen in μCT analysis, TRPC1-/- mice showed no differences in bone phenotype compared to WT mice under physiological conditions. However, in the ovariectomy model of estrogen-deficiency mediated bone loss, TRPC1-/- mice exhibited a reduced loss of trabecular bone volume (-28,9% in WT compared to -13,1% in TRPC1-/-) and bone tissue density (-3,2% in WT compared to +0,9% in TRPC1-/-) in LW5. Analysing the underlying signaling pathways, we found no differences in Ca<sup>2+</sup>-oscillations and store-operated calcium entries and no differences in NFATC1 expression. In time-lapse microscopy, however, we observed a reduced capacity of TRPC1-/- osteoclast precursors to migrate and to fuse, due to a reduced velocity, translocation and a reduced increase of cell area over time. <h3>Conclusions</h3> These results provide the first evidence that TRPC1 is necessary for migration and fusion of osteoclasts and that the loss of TRPC1 while not affecting physiological bone turnover has a clear effect on accelerated bone loss as seen in estrogen deficiency. Thus, our data implicate that TRPC1 may represent a potential target for treating rapid osteoporotic bone loss. <h3>Disclosure of Interest</h3> None Declared