The function of the calcium channel Orai1 in osteoclast development

Abstract

To determine the intrinsic role of Orai1 in osteoclast development, Orai1-floxed mice were bred with LysMcre mice to delete Orai1 from the myeloid lineage. PCR, in situ labelling and Western analysis showed Orai1 deletion in myeloid-lineage cells, including osteoclasts, as expected. Surprisingly, bone resorption was maintained in vivo, despite loss of multinucleated osteoclasts; instead, a large number of mononuclear cells bearing tartrate resistant acid phosphatase were observed on cell surfaces.An in vitro resorption assay confirmed that RANKL-treated Orai1 null cells, also TRAP-positive but mononuclear, degraded matrix, albeit at a reduced rate compared to wild type osteoclasts. This shows that mononuclear osteoclasts can degrade bone, albeit less efficiently. Further unexpected findings included that Orai1 fl/fl -LysMcre vertebrae showed slightly reduced bone density in 16-week-old mice, despite Orai1 deletion only in myeloid cells; however, this mild difference resolved with age. In summary, in vitro analysis showed a severe defect in osteoclast multinucleation in Orai1 negative mononuclear cells, consistent with prior studies using less targeted strategies, but with evidence of resorption in vivo and unexpected secondary effects on bone formation leaving bone mass largely unaffected. 2 of 15 | ROBINSON et al.

The calcium channel Orai1 is required for osteoblast development: studies in a chimeric mouse with variable in vivo Runx-cre deletion of Orai-1

“…Animals and Genotyping. Orai1 fl/fl mice were generated by flanking Orai1 exons 2/3 with loxP as previously described [16,19]. Mice were backcrossed to C57Bl/6 for a minimum of 10 generations and then crossed with Runx2-cre mice (Tg(Runx2-icre)1Jtuc) [20] for conditional deletion in the osteoblast lineage.…”

“…Histomorphometry and Histology. Static histomorphometry was performed as described [16]. Briefly, lumbar vertebrae were fixed overnight in 3.7% formalin, then transferred to 70% ethanol for micro-CT analysis using Bruker Skyscan 1272 with a bone density cutoff of 150 mg/cm 2 , at 5 µm resolution.…”

“…Osteoblasts on glass coverslips were loaded with 2 µM Fura-2 AM-ester (Invitrogen) for 30 minutes at 25 °C in 107 mM NaCl, 7.2 mM KCl, 1.2 mM MgCl 2 , 11.5 mM glucose, 20 mM HEPES, 1 mM CaCl 2 , at pH 7.2. Washed cells were allowed to de-esterify dye for 30 minutes at 25°C as described [16]. Ca 2+ measurements used a Leica DMI 6000B fluorescence microscope controlled by Slidebook Software (Intelligent Imaging; Denver, CO).…”

“…Effect of Orai1 deletion on store-operated calcium entry. MSCs were collected from Orai1fl/fl and Orai1 f/f -Runx2-cre mice before differentiating into osteoblasts in vitro and measuring store-operated calcium entry as described [16]. Cells were treated with thapsigargin in the absence of extracellular calcium to deplete the endoplasmic reticulum of calcium and initiate the store-operated calcium response.…”

“…Loss of Orai1 also causes a wide range of changes in cardiovascular function [6], neuronal function [7, 8], tooth formation [9, 10] and others [11]. We [12, 13, 14, 15, 16] and others [17, 18], demonstrated that Orai1 contributes to bone differentiation and maintenance in conventional knockout mice and in vitro cell lines.…”