Fig 1.
Surface of Orai1fl/fl and Orai1fl/fl-Runx2cre fourth lumbar vertebrae.
Bruker CTvox software-generated three-dimensional images of vertebrae reconstructed from microCT scans at 5 μm resolution. All animals were homozygous for floxed Orai1; the conditional knockouts (lower panels) are Runx2-cre positive. A. Representative vertebrae from control Orai1fl/fl animals without cre recombinase, the control group. Apart from sites of blood vessel entry, the surface of the bone is smooth (green arrows), typical for mice at 16 weeks of age. B. Representative vertebrae from Orai1fl/fl-Runx2cre animals, the conditional knock out (cKO). In contrast to the control vertebrae, the bone surface appears irregular (red arrows) with patchy darker areas representing regions of reduced bone.
Fig 2.
Cross sections of vertebral cortex from Orai1fl/fl controls versus Orai1fl/fl-Runx2-cre conditional KO (cKO) animals and ribs 5–6 of Orai1fl/fl versus cKO animals. A.
Wild type cortex with typical smooth bone showed relatively uniform thickness compared with Orai1fl/fl-Runx2cre (cKO) animals. Images of H&E stained histologic sections from upper lumbar vertebrae (top panels) had irregularly thinned regions (arrow) but no other distinguishing features). Images 1 mm wide; images of microCT scans of lower lumbar vertebrae (middle and lower panels) 1.4 mm wide, excluding trabecular bone for clarity; for trabecular structure see Fig 3. This is in keeping with the appearance of the bone surface in the three-dimensional reconstructions (see Fig 1). B. Cortical bone thickness, though variable, is reduced on average in Orai1fl/fl-Runx2cre (Orai1 cKO) animals (p = 0.009, N = 8). C. Cortical bone density by integrating mineral in 8 bit images. A difference comparing 6 measurements, at p = 0.08, was not significant, and the difference was only ~ 2% of the total. D. Cross sections of ribs 5 and 6 compared in Orai1fl/fl and cKO animals. Surfaces were uniformly smooth, in contrast to vertebral cortex (A). E. In contrast to vertebral bone, rib cortical bone thickness did not vary. F. In rib cortical bone a difference between Orai1fl/fl and cKO animals was barely significant, p = 0.04, and difference was < 5%.
Fig 3.
Key features of Orai1fl/fl and conditional KO animals on static and dynamic histomorphometry. A.
Bone volume/total volume (BV/TV) of vertebrae. Vertebral bone is significantly reduced (N = 7, p = 0.01) in the Orai1fl/fl-Runx2cre (Orai1 cKO) mice compared to Orai1fl/fl controls without cre recombinase. The greater variability in the conditional-knockout bone is thought to reflect variable cre excision; this variability is also apparent in the vertebral cortex thickness (Fig 2B; see text). B. Trabecular thickness is significantly decreased in the Orai1 conditional knockout mice (N = 7, p <0.01). C. Trabecular number also decreased significantly in the conditional knockout animals (N = 7, p<0.02). D. Trabecular spacing increased in keeping with reduced trabecular number in cKO animals (N = 7, p <0.03) E. Example of xylenol orange (red) and calcein (green) fluorescent labels in wild type and conditional KO bones. Interval between double labels represents the bone formation rate in active osteoblasts. The interlabel distance was not statistically different between the control and cKO (not illustrated). See also Fig 3 J below for Ob.S/BS. Fields shown are 100 μm wide. F. The proportion of calcein labeled bone surface in wild type and Orai1fl/fl-Runx2cre bone (Orai1 cKO) was not statistically different (N = 4). G. The bone formation rate at 4 months of age was significantly reduced in the Orai1fl/fl-Runx2cre mice (Orai1 cKO) compared to Orai1fl/fl controls (N = 4, p = 0.011). H. Resorbed surface, 10–15% if bone surface, did not vary significantly between Orai1fl/fl and cKO animals. I. Formation of osteoclasts in vitro from spleen macrophages with RANKL and CSF-1 did not vary between the Orai1fl/fl and cKO, in contrast to reduced osteoclast formation previously reported in cells from LysM-cre Orai1fl/fl animals [16]. J. Osteoblast surface area (bone formation surface) (Ob.S/BS) estimated as cuboidal bone lining cell area [16]; it did not vary mesurably.
Fig 4.
Variable efficacy of Runx2-cre in deleting flox/flox Orai1 in osteoblasts in bone sections.
It is often assumed that promoter-cre constructs uniformly delete the target in all cells of an organ. Recent findings suggest this is sometimes not the case (see Text). We tested this by antibody labeling of Orai1fl/fl and Orai1fl/fl-Runx2cre bone. A. Rabbit antibody detects Orai1 in osteoblasts (upper left panel). Absence of antibody eliminates labeling (lower left panel). All sections are from one wild type animal. Left and right panels are of the same section. Osteoblasts are shown independently with phalloidin rhodamine (right panels). Fields are 200 μm across. B. In the upper panels, in an Orai1fl/fl animal, osteoblasts are shown with the antibody at high power (fields 200 microns wide) and in phase of the same field (right). In the lower panels, the same field of a conditional KO animal (Orai1fl/fl-Runx2cre). Note that some conditional KO cells (Orai1fl/fl-Runx2cre) do not label (arrows, phase and antibody label, lower panels). C. Lower power fields, 350 μm, of Orai1 labeled osteoblasts (surrounding tissue fluorescence is an artifact) in Orai1fl/fl bone (top) and Orai1fl/fl-Runx2cre conditional KO bone (bottom). Osteoblasts in the wild type label strongly, including surface cells of the bone (arrows, top frame). Some, but not all, of the osteocytes in the conditional KO label (arrows, bottom panel). D. Western blot for Orai1 in cells expressing or not expressing the protein (See Fig 5). Thirty-five μg loads of cell protein from the isolates indicated were run on denaturing SDS-PAGE and blotted. This blot using the very specific Alomone antibody shows a trace of Orai1 even in the osteoblast cKO preparation (left lane) and reduced, but not absent, Orai1 in the MSC cKO (right lane) is shown in the upper panel, in each case relative to Orai1fl/fl controls. The beta actin re-blot (lower panel) confirms similar protein loads. Densitometry showed that labeling in the osteoblast cKO (left lane) relative to Orai1fl/fl was reduced by 93%.
Fig 5.
Calcium activated calcium release in cells from Oraifl/fl control and Runx2cre-Oraifl/fl mice and the effect of conditional KO of Orai1 on osteoblasts mRNAs in cells differentiated for one week from MSCs in osteoblast medium.
A. Intracellular calcium was measured by Fura2 [26] (top panel). Cells were treated with thapsigargin in the absence of extracellular calcium. Only the wild type cells show calcium entry upon extracellular calcium repletion. B. Examples of Fura2 signals from control and conditional KO cells, with false color reflecting calcium concentration in cells (scale is shown in upper right corner of each panel). A scale bar for size for the photomicrographs is shown below the bottom panel. C. GAPDH expression by PCR in Orai1fl/fl and cKO cells. Differences were not significant. D. Expression of RunX2 in in Orai1fl/fl and cKO cells. Differences were also not significant, indicating that recombination with floxed Orai1 was not due to changes in RunX2 transcription. E. Expression of Orai1 mRNA was greatly and significantly reduced in cKO cells selected for lack of Orai1 expression (see Fig 5D). F. Expression of Alkaline phosphatase was greatly reduced (but not completely absent), in keeping with cellular expression of alkaline phosphatase shown below in Fig 6B. G. Expression of Col I a1 was reduced but had high variability and did not reach significance. H. Expression of osteocalcin was significantly reduced in the cKO cells. I. Expression of osterix (SP7) was significantly reduced in the cKO cells. J. Expression of ATF4 (CREB2) was significantly reduced in the cKO cells.
Fig 6.
Elimination of Orai1 results in profoundly reduces OB differentiation and mineralization from OB precursors.
Osteoblasts isolated as described in the methods section from control Orai1fl/fl or Runx2-cre floxed (Orai1fl/fl-Runx2-cre) conditional knock-out animals, incubated for two weeks in osteoblast mineralization medium and analyzed by histologic staining. Each well illustrated is 2 cm across. Note that the cell culture data show a (pure) knockout phenotype, while the in vivo data are of a mosaic, so the larger changes in the Orai1fl/fl-Runx2-cre are expected relative to in vivo data. A. Von Kossa staining for mineral. Wild type cells made mineral nodules, but there were only rare and small nodules in Orai1 knockout cell cultures. Representative culture wells for control (Orai1fl/fl) and conditional knockout (cKO Orai1fl/f with RunX2-cre) cells are shown on the left. Staining was quantified for four samples of each genotype. Mineralized matrix production appeared significantly reduced in cultures of Orai1-deficient osteoblasts (p < 0.0001, N = 4).B. Alkaline phosphatase activity. Representative cultures are shown on the left. Results quantified from four samples of each genotype are shown on the right. Wild type cells produced much more alkaline phosphatase, with small nodules of high activity as in the silver stain for mineral (A), there was uniformly less alkaline phosphatase in Orai1 null cells (p = 0.0002, N = 4). C. Oil red O staining for adipocytes. Representative cultures are shown on the left. Staining was quantified for four samples of each genotype with results shown on the right. There was no evidence of increased adipogenic differentiation in Orai1 null cells, with minimal labeling in cultures of either genotype. This applies only to osteoblast medium, and it is possible that strong adipocyte induction might show effects on adipocytes (see Discussion). D. Von Kossa staining of Orai1fl/fl and cKO cells at 2.5 times the magnification shown in (A). The individual clusters of labeled cells in the Orai1fl/fl represent nodules of bone forming cells; these are greatly reduced in the cKO.