Gene disruption of the calcium channel Orai1 results in inhibition of osteoclast and osteoblast differentiation and impairs skeletal development

Abstract

Calcium signaling plays a central role in the regulation of bone cells, although uncertainty remains with regard to the channels involved. In previous studies, we determined that the calcium channel Orai1 was required for the formation of multinucleated osteoclasts in vitro. To define the skeletal functions of calcium release-activated calcium currents, we compared the mice with targeted deletion of the calcium channel Orai1 to wild-type littermate controls, and examined differentiation and function of osteoblast and osteoclast precursors in vitro with and without Orai1 inhibition. Consistent with in vitro findings, Orai1(-/-) mice lacked multinucleated osteoclasts. Yet, they did not develop osteopetrosis. Mononuclear cells expressing osteoclast products were found in Orai1(-/-) mice, and in vitro studies showed significantly reduced, but not absent, mineral resorption by the mononuclear osteoclast-like cells that form in culture from peripheral blood monocytic cells when Orai1 is inhibited. More prominent in Orai1(-/-) mice was a decrease in bone with retention of fetal cartilage. Micro-computed tomography showed reduced cortical ossification and thinned trabeculae in Orai1(-/-) animals compared with controls; bone deposition was markedly decreased in the knockout mice. This suggested a previously unrecognized role for Orai1 within osteoblasts. Analysis of osteoblasts and precursors in Orai1(-/-) and control mice showed a significant decrease in alkaline phosphatase-expressing osteoblasts. In vitro studies confirmed that inhibiting Orai1 activity impaired differentiation and function of human osteoblasts, supporting a critical function for Orai1 in osteoblasts, in addition to its role as a regulator of osteoclast formation.

Figure 1. Skeletal phenotype and dentition of Orai1^−/− mice

A. Genotyping and gross appearance The KO mice were smaller than WT littermates. B. Radiographs of Orai1^−/− and WT mice showed no gross defects. Bones in the KO were decreased proportionately relative to overall mouse size. C. Radiographs of thoracic vertebrae and ribs. There is reduced bone thickness in the Orai1^−/− animal. D. Teeth in the Orai1^−/− mouse were slightly smaller than in WT mice, but sections showed thinning and irregularity of the enamel in Orai1^−/−. Similar sections through the gingival sulcus are shown to minimize effect of plane of section. Left two frames, 450 μm, right frames 110 μm across.

Figure 2. Analysis of Orai1^−/− skeletons by μCT and alcian blue/alizarin red staining

A. Thoracic vertebrae from WT and Orai1^−/− mice by μCT. Images (left and middle) are three-dimensional projections of the vertebrae (lowest is T12). Intact vertebrae (left), frontal sections with the virtual cut surfaces in blue (middle), and the cut surfaces separately (right). The vertebrae of Orai1^−/− mice are smaller, show cortical discontinuities and reduced trabecular bone. B. Skeletons stained with Alcian blue to identify cartilage and alizarin red to stain mineral. Comparison of skulls (upper frames) revealed retained cartilage in the KO (right) in areas mineralized in the WT (left). More cartilage remains in the skull around the nares in the Orai1^−/−, and cranial sutures are wider in the KO (arrows). The spines (middle frames) showed large regions of vertebrae remained cartilage in the KO (arrow) in contrast to WT, although there is regional variation. Replacement of cartilage by bone in the ulna and radius (lower frames) was incomplete in the KO. In controls, the shafts of the long bones were mineralized, with cartilage only at the joints.

Figure 3. Bone, cartilage, and osteoclasts in sections of WT and KO bone, and osteoclast activity in vitro when Orai1 activity is suppressed

A. Increased cartilage and decreased bone in KO mice. Decalcified sections from KO (right) and WT (left) third lumbar vertebrae stained with hematoxylin and eosin. In the Orai1^−/−, the vertebral body retains cartilage on all sides, and has sparse, thin trabeculae. The control retains cartilage only at growth plates (gray, laterally) and has robust trabeculae. The growth plates are at the left and right sides of each frame. Images are 1.8 mm across. B. Retention of woven bone in Orai1^−/− mice. Collagen bundles are visualized by birefringence in polarized light. The cortex of a WT mouse vertebra (left) is mainly mature lamellar bone (parallel layers of collagen, bright). The KO retains woven bone (criss-cross pattern). Normally, the first bone synthesized when cartilage is replaced is woven bone, but woven bone is resorbed and replaced by lamellar bone. Images are 300 μm across. C. Decreased tartrate resistant acid phosphatase (TRAP, magenta) in cartilage growth plates of Orai1^−/− mice. Histochemical staining was performed on frozen sections of vertebrae from WT (left) and KO (right). The sections are centered on inter-vertebral discs and show the cartilaginous growth plates, which normally have high osteoclastic activity. Prominent TRAP is seen in WT but much less in the KO. Images are 750 μm across. D. TRAP expressing cells in Orai1^−/− mice. Higher power showed that the TRAP positive cells (arrows) in Orai1^−/− are single small cells, generally not multinucleated. Image width is 750 μm. E. Reduced mineral resorption by osteoclasts differentiated in vitro when Orai1 is inhibited. Human osteoclast precursors were treated with RANKL to induce osteoclast differentiation, in the presence (right) or absence (middle) of the Orai1 inhibitor DCPA, on slides coated with calcified matrix. Substrate resorption was evaluated by silver nitrate staining. Areas where substrate was resorbed are clear. Resorption was inhibited by addition of 50 μM DCPA (right), compared to control (center). A no-cell control (left) has no resorption tracks. Note also that the resorption tracks in the Orai1 inhibited cultures are thin and threadlike, consistent with resorption by small, mainly mononuclear, cells. This reflects the paucity of multinucleated osteoclasts when Orai1 is inhibited. F. The area of degraded mineral substrate. Cleared matrix area in cultures without cells (left), RANKL treated control cells (center), and cells treated with RANKL and the Orai1 inhibitor (right), are shown. DCPA resulted in a significant decrease (p=0.01), in resorption. Activity in DCPA was clearly greater than the no cell control (p<0.01). In each case, resorption activity was measured in three cell cultures for each condition, and a second experiment showed equivalent results (not shown).

Figure 4. Osteoblast activity in Orai1^−/− mice

A. Reduced mineralization in Orai1^−/− mice. Fluorescent labeling of newly formed mineral in sections from WT (left) and KO (right) mice 2 days after calcein injection. Sections are of vertebrae with growth plate at the bottom; each image is 250 μm across. In the WT mice (left) there was calcein labeling along the bone surface (appositional growth), on trabeculae, and in the primary spongiosa, where bone replacing cartilage is remodeled. Labeled bone was markedly decreased in the Orai1^−/− mice (right). Calcifying cartilage also labels with calcein, though these labels are diffuse. Some calcein labeling of mineralized cartilage is seen in the KO. Images are 250 μm across. B. Alkaline phosphatase in vertebral bone of WT and Orai1 deficient mice. Decreased bone formation suggested that osteoblast numbers might be decreased. This was evaluated using an antibody for alkaline phosphatase that is highly expressed by active osteoblasts in bone. The total area labeling for alkaline phosphatase, measured in three sections of vertebrae, was decreased in Orai1^−/− compared to the labeling measured in three sections from WT mice (p=0.002). C. Osteoblast expression of Orai1 protein. Lysates from osteoblasts in basal (growth) or mineralizing (diff) medium were evaluated by Western blotting with anti-Orai1, and blots re-probed with anti-Actin to demonstrate equivalent sample loading. The results confirm expression of Orai1 by osteoblasts. Orai1 protein appeared increased in mineralizing osteoblasts in keeping with PCR results. D. Osteoblast expression of Orai1 mRNA. Human osteoblasts in basal medium (Growth) or in differentiation medium containing ascorbate, glycerol-2-phosphate and hydrocortisone (Diff) were evaluated for Orai1 mRNA by quantitative PCR, relative to GAPDH, at 2 weeks. Orai1 message was present in the osteoblasts, with increased expression found in mineralizing cells (n=5, p=0.002). Results at three weeks and in a repeat experiment were similar (not shown).

Figure 5. Effect of Orai1 inhibition on osteoblast differentiation and activity

A. Effect of the Orai1 inhibitor DCPA on alkaline phosphatase activity. Human osteoblasts were grown in basal medium (growth) or in medium with ascorbate, glycerol-2-phosphate and hydrocortisone (diff) to promote mineralization, with or without DCPA (or equivalent vehicle). Alkaline phosphatase activity (blue staining) was evaluated by light microscopy (upper row); a phase image of the same field is shown below. Little alkaline phosphatase activity was present in cells in growth medium. Alkaline phosphatase was substantially increased in cultures treated with ascorbate, 2-glycerol phosphate and hydrocortisone (diff) but this effect was blunted by DCPA. The phase images, showing cell monolayers with and without DCPA, indicate that the reduction in alkaline phosphatase did not simply reflect a difference in cell numbers in the presence of the inhibitor. Results were similar in repeat experiments (not shown). B. Alkaline phosphatase in cultures of human osteoblasts. In situ enzyme activity is shown for cultures (left to right) in growth medium, growth medium plus DCPA, differentiation medium, or differentiation medium plus DCPA. In differentiation medium, addition of DCPA reduced alkaline phosphatase activity. Wells are 3.5 cm across. C. Alkaline phosphatase activity. For each condition in (A), alkaline phosphatase is measured as signal at 450-490 nm in assays of replicate cultures. DCPA significantly reduced alkaline phosphatase in mineralizing osteoblasts (n=4, p<0.001). D. Mineral deposition by human osteoblasts is blunted by DCPA. Cells were cultured 3 weeks in basal medium (growth), or in mineralizing conditions (differentiation), each without or with DCPA. Alizarin red was used to stain calcium (bright red); representative cultures for each condition are shown (lower panel). As expected, little mineral was produced by osteoblasts in growth medium, with or without DCPA, but the photomicrographs show strong matrix labeling by alizarin red in cultures grown in differentiation medium without DCPA (control, upper left). However, when DCPA was added, alizarin red staining was mainly nonspecific (light red, upper right), without the focal strongly mineralized nodules that occur in differentiation medium alone. Wells are 3.5 cm in diameter; micrographs are 220 μm across.

Figure 6. Effect of Orai1 inhibition osteoblast differentiation

Each assay shows expression relative to GAPDH mRNA in the same sample, mean ±SD, n=3. Differences were examined by analysis of variance. Repeat experiments gave similar results (not shown). A. Expression of a transcriptional regulator of osteoblast differentiation, RUNX2. Human osteoblasts in growth medium (Gr), or in medium supplemented with ascorbate, glycerol-2-phosphate and hydrocortisone (Dif) are compared, without or with the Orai1 inhibitor DCPA (50 μM). Cells in mineralizing medium for 2 weeks showed significant up-regulation of RUNX2 compared to cultures in growth medium (p<0.001 without DCPA, p<0.01 with DCPA). The increase in RUNX2 with mineralizing medium was reduced by DCPA (p<0.001). While RUNX2 expression appeared lower in mineralizing medium at 21 days compared to 14 days, osteoblast RUNX2 expression was still significantly elevated (p<0.01) in the absence of DCPA. RUNX2 expression by osteoblasts in mineralizing medium plus DCPA for 21 days was not significantly different from pre-treatment or growth medium control cells. B. Alkaline phosphatase mRNA. Alkaline phosphatase was significantly increased after 14 (p<0.001) or 21 days (p<0.001) in mineralizing medium compared to growth controls. Alkaline phosphatase expression by osteoblasts in mineralizing medium plus DCPA was reduced relative to mineralizing medium alone (p<0.01). C. Expression of type 1 collagen (Col1A1). Expression was increased for osteoblasts in mineralizing medium (p<0.001) at 14 days. This increase was strongly attenuated by DCPA (p<0.001), though DCPA-treated cells in mineralizing medium expressed 30% more Col1A1 than cells in growth medium with DCPA (p<0.05). At 21 days, a reduced but still significant increase in Col1A1 expression was apparent in mineralizing medium without DCPA (p<0.01) but collagen expression in cultures with DCPA was no longer significantly elevated compared to cells in growth medium. D. Expression of osteoclast regulatory proteins. RANKL and osteoprotegerin mRNA was assayed in human osteoblasts in growth (Gr) or mineralizing medium (Dif), with or without DCPA, for 14 days. Unlike markers of osteoblastic differentiation, expression of RANKL and osteoprotegerin did not vary significantly with DCPA.