Targeted Overexpression of Osteoactivin in Cells of Osteoclastic Lineage Promotes Osteoclastic Resorption and Bone Loss in Mice

This study sought to test whether targeted overexpression of osteoactivin (OA) in cells of osteoclastic lineage, using the tartrate-resistant acid phosphase (TRAP) exon 1B/C promoter to drive OA expression, would increase bone resorption and bone loss in vivo. OA transgenic osteoclasts showed ∼2-fold increases in OA mRNA and proteins compared wild-type (WT) osteoclasts. However, the OA expression in transgenic osteoblasts was not different. At 4, 8, and 15.3 week-old, transgenic mice showed significant bone loss determined by pQCT and confirmed by μ-CT. In vitro, transgenic osteoclasts were twice as large, had twice as much TRAP activity, resorbed twice as much bone matrix, and expressed twice as much osteoclastic genes (MMP9, calciton receptor, and ADAM12), as WT osteoclasts. The siRNA-mediated suppression of OA expression in RAW264.7-derived osteoclasts reduced cell size and osteoclastic gene expression. Bone histomorphometry revealed that transgenic mice had more osteoclasts and osteoclast surface. Plasma c-telopeptide (a resorption biomarker) measurements confirmed an increase in bone resorption in transgenic mice in vivo. In contrast, histomorphometric bone formation parameters and plasma levels of bone formation biomarkers (osteocalcin and pro-collagen type I N-terminal peptide) were not different between transgenic mice and WT littermates, indicating the lack of bone formation effects. In conclusion, this study provides compelling in vivo evidence that osteoclast-derived OA is a novel stimulator of osteoclast activity and bone resorption

Erythroid Promoter Confines FGF2 Expression to the Marrow after Hematopoietic Stem Cell Gene Therapy and Leads to Enhanced Endosteal Bone Formation

Fibroblast growth factor-2 (FGF2) has been demonstrated to be a promising osteogenic factor for treating osteoporosis. Our earlier study shows that transplantation of mouse Sca-1+ hematopoietic stem/progenitor cells that are engineered to express a modified FGF2 leads to considerable endosteal/trabecular bone formation, but it also induces adverse effects like hypocalemia and osteomalacia. Here we report that the use of an erythroid specific promoter, β-globin, leads to a 5-fold decrease in the ratio of serum FGF2 to the FGF2 expression in the marrow cavity when compared to the use of a ubiquitous promoter spleen focus-forming virus (SFFV). The confined FGF2 expression promotes considerable trabeculae bone formation in endosteum and does not yield anemia and osteomalacia. The avoidance of anemia in the mice that received Sca1+ cells transduced with FGF2 driven by the β-globin promoter is likely due to attenuation of high-level serum FGF2-mediated stem cell mobilization observed in the SFFV-FGF2 animals. The prevention of osteomalacia is associated with substantially reduced serum Fgf23/hypophosphatemia, and less pronounced secondary hyperparathyroidism. Our improved stem cell gene therapy strategy represents one step closer to FGF2-based clinical therapy for systemic skeletal augmentation

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Effects of one-week of dietary calcium repletion on plasma P1NP level in osteocyte <i>Igf1</i> cKO mutants and in corresponding WT mice.

<p>Four-week-old osteocyte <i>Igf1</i> cKO mice (n = 8) and age- and gender-matched WT control mice (n = 10) were fed either the calcium-deficient or the calcium-containing diet for two weeks followed by 1 week calcium-containing diet. Plasma samples were obtained at the beginning of the experiment (basal), and after three days and 7 days of calcium repletion. Results are shown as mean ± SEM. A shows the actual basal level of P1NP; and B shows the effect of the dietary calcium repletion in the two strains, which is reported as relative percentage changes of the basal level of WT control mice fed the calcium-containing diet for east indicated test time period. *P<0.05.</p

Effects of two-week dietary calcium restriction on plasma calcium (A), PTH (B), phosphorus (C), and FGF23 (D) of osteocyte <i>Igf1</i> cKO mice and those of WT mice.

<p>Four-week-old female osteocyte <i>Igf1</i> cKO mice and age- and gender-matched WT control mice were fed a mouse diet containing either <0.01% calcium (calcium-deficient diet) or 1.2% calcium (control diet) for two weeks. At the end of the calcium depletion, blood samples were collected from each mouse for measurements of calcium (Ca), PTH, phosphorus, and FGF23. Results are shown in relative percentage of the respective level of each metabolite in corresponding control mice that were fed the control calcium-containing diet (mean ± S.E.M). The number of animals in each test group is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115897#pone.0115897.t002" target="_blank">Table 2</a>. *P<0.05; **P<0.01; and ***P<0.001. P = N.S. (not significant), where P>0.05. </p

Proposed cellular mechanisms contribute to bone repletion-induced bone formation (A) or to loading-induced bone formation (B). Please refer to text for details.

<p>Proposed cellular mechanisms contribute to bone repletion-induced bone formation (A) or to loading-induced bone formation (B). Please refer to text for details.</p

Schematic representation of the experimental design of the dietary calcium depletion and repletion experiment.

<p>The indicated numbers of 4-week-old female <i>Igf1</i> osteocytic cKO mutants and age- and gender-matched WT littermates were each divided into four groups: 1) depletion control groups, which were fed the calcium sufficient diet (with 1.2% calcium) for 2 weeks; 2) depletion experimental groups, which were fed the calcium deficient diet (with <0.01% calcium) for the same 2 weeks; 3) repletion control groups, which were fed the calcium sufficient diet for 3 weeks; and 4) repletion experimental groups, which were fed the calcium deficient diet for two weeks followed by the calcium sufficient diet for one week. At the end of each regimen, all mice were sacrificed and blood drawn for measurements of plasma parameters. Femurs were isolated, cleaned, fixed with formalin and subjected pQCT and histomorphometry measurements. Tibias were isolated, and total RNA was isolated for real-time RT-PCR measurements.</p

Effects of two weeks of dietary calcium depletion followed by one week of dietary calcium repletion on dynamic bone formation histomorphometric parameters of at the endosteal surface of the secondary spongiosia of the distal femur.

<p>E = endosteal; L.Pm = tetracycline labeling surface (dL.Pm + ½ sL.Pm); B.Pm = bone surface; MAR = mineral apposition rate; BFR = bone formation rate.</p><p>P = N.S. (not significant), when P>0.05.</p><p>* P<0.05; ** P<0.01; *** P<0.001, when compared to respective control mice on the control diet.</p><p>Effects of two weeks of dietary calcium depletion followed by one week of dietary calcium repletion on dynamic bone formation histomorphometric parameters of at the endosteal surface of the secondary spongiosia of the distal femur.</p

Sequence of PCR primer sets for the test mouse genes.

<p>Sequence of PCR primer sets for the test mouse genes.</p

Comparison of cortical cross-sectional bone parameters at the mid-shaft of femurs between WT and <i>Igf1</i> cKO mice after two-week dietary calcium depletion and/or after one-week dietary calcium repletion.

<p>Results are shown as mean ± SEM.</p><p>^# The control group are mice receiving normal calcium-containing diet throughout.</p><p>^a P<0.001; ^b P<0.01; ^c P<0.05; when compared to corresponding WT littermates.</p><p>P = N.S., not significant, when P>0.05.</p><p>Comparison of cortical cross-sectional bone parameters at the mid-shaft of femurs between WT and <i>Igf1</i> cKO mice after two-week dietary calcium depletion and/or after one-week dietary calcium repletion.</p