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

Regulation of DNA Synthesis in Chick Calvaria Cells by Factors from Bone Organ Culture

Abstract Embryonic chick bones growing in organ culture release a substance into the culture medium which stimulates bone formation in previously untreated bones. This “conditioned” medium also enhances proliferation of monolayer cultures of chick calvaria cells in serum-free medium. The active principle is nondialyzable, indicating a molecular weight greater than 12,000 daltons. Dialysis also separates the mitogenic activity from a low-molecular-weight inhibitor. The amount of the mitogen found in conditioned medium increases as the rate of bone resorption increases in response to treatment with parathyroid hormone or 1,25-dihydroxyvitamin D3. Maximal stimulation of DNA synthesis in calvaria cells is evident with conditioned medium obtained 3 to 5 days after treatment of bone cultures with parathyroid hormone. The cells must be treated with the conditioned medium continuously for 20 hr in order to obtain peak enhancement of DNA synthesis; there is no detectable effect in the first 8 hr. In contrast, the inhibitor acts within 4 hr. The data suggest that the stimulatory factor acts to increase cell proliferation by promoting entry of cells into the S phase of mitosis. We conclude that this stimulator is a locally produced regulator of bone formation, probably acting via an increased proliferation in osteoblast precursor cells

Endogenous Inhibitor of Bone Cell Proliferation

Abstract A substance recovered from the medium of embryonic chick bones growing in organ culture inhibits the proliferation of chick calvarial bone cells in vitro. This inhibition of proliferation occurs within 3 hr after exposure of the cells to the inhibitory substance (IS) in a manner which does not appear to affect the protein synthesis or the gross morphology of the cells. The medium from which IS is obtained can be diluted 500-fold and still retain significant inhibitory activity. Cells in culture spontaneously escape the inhibition of a single dose of IS within 24 hr of exposure, however, upon further addition of IS to these cells inhibition of proliferation is again observed. Cells derived from chick bone as well as rat and human bone are more sensitive to the inhibition than are chick dermal fibroblasts and chick liver and muscle cells. IS is not destroyed after treatment of the conditioned medium with trypsin, phospholipase A2, RNase, neuraminidase, or heat. IS is also not a commonly produced prostaglandin. Estimates of the molecular weight of IS range from 6000 to 14,000 daltons. While these features do not disclose the exact nature of this substance, they are consistent with this substance being a small polypeptide without the exposed amino acids, lysine or arginine. We conclude that the presence of such a substance supports the concept of local regulation of bone metabolism

In vitro metabolism of 25-hydroxyvitamin D(3) by isolated rat kidney cells

Cells were dispersed from rat kidney after enzymatic digestion of the extracellular matrix. When the cells were suspended in a serum-free medium and incubated with (3)H-labeled 25-hydroxyvitamin D(3) (25-OH-D(3)) several polar metabolites, including 1,25-(OH)(2)[(3)H]D(3) and 24,25-(OH)(2)[(3)H]D(3) were produced. The specific activities of the 25-OH-D(3):1- and 24-hydroxylases in isolated rat kidney cells were 10-100 times greater than in avian kidney homogenates. The rates of production of 1,25-(OH)(2)D(3) and 24,25-(OH)(2)D(3) were linear over a wide range in cell densities (0.65-5.0 × 10(6) cells per ml) and substrate concentrations (3.5-70 nM). The rate of production of 24,25-(OH)(2)[(3)H]D(3) from 25-OH-[(3)H]D(3) by cells isolated from rats fed control diet was linear with time for up to 30 min, while the synthesis of 1,25-(OH)(2)[(3)H]D(3) was linear for over 90 min. The specific activity of the 25-OH-D(3):1-hydroxylase was increased in kidney cells from vitamin D-deficient rats (11.5 fmol/min per 10(6) cells) as well as calcium-deficient rats (8.1 fmol/min per 10(6) cells) when compared to cells from rats fed the control diet (2.0 fmol/min per 10(6) cells). Also, the specific activity of the 25-OH-D(3):24-hydroxylase was reduced in cells from the vitamin D-deficient rats (<0.2 fmol/min per 10(6) cells) and calcium-deficient rats (5.1 fmol/min per 10(6) cells) compared to the controls (15.2 fmol/min per 10(6) cells). On the basis of these results, as well as previous in vivo studies, we conclude that the metabolism of 25-OH-D(3) by freshly isolated rat kidney cells reflects the in vivo activities of the renal vitamin D-metabolizing enzymes and may prove useful as an assay

Effects of short-term calcium depletion and repletion on biochemical markers of bone turnover in young adult women

The skeletal responses to calcium depletion and repletion in rodents have been well characterized, but those in humans are poorly understood. The present study sought to evaluate the effects of short term dietary calcium depletion and repletion on biochemical markers of bone turnover in 15 young Caucasian women (age, 21-30 yr). The study contained 3 phases: 1) 5 days of a regular diet containing more than 800 mg/day calcium to establish baseline values (baseline phase), 2) 22 days of a restricted diet containing less than 300 mg/day calcium (depletion phase), and 3) 7 days of a normal diet containing more than 800 mg/day calcium (repletion phase). Serum and urine samples were obtained from each subject during the baseline phase; on the first, second, and last days of the depletion phase; and on the third and last days of the repletion phase. Serum levels of calcium, PTH, 1,25- dihydroxyvitamin D3, osteocalcin, and C-terminal type I procollagen peptide (PICP) and urinary levels of calcium and deoxypyridinoline were determined. Serum and urinary calcium levels were significantly reduced, and serum PTH and 1,25-dihydroxyvitamin D3 levels were markedly increased during depletion. These changes were completely reversed after 1 week of repletion. Depletion also rapidly and significantly increased the urinary deoxypyridinoline level, indicating increased bone resorption. The increase also returned rapidly to baseline upon repletion. Calcium depletion had contrasting effects on bone formation markers; whereas depletion significantly reduced the serum PICP level, it significantly increased serum osteocalcin level. Past histomorphometric studies in rodents indicated that the number of mature but inactive osteoblasts was increased during depletion despite an inhibition of bone formation. Thus, it is speculated that although the reduction in serum PICP reflected the depletion-associated inhibition of bone formation, the increase in serum osteocalcin could represent this depletion-related increase in osteoblast number. During repletion, serum osteocalcin remained elevated above baseline. PICP recovered from its depressed level and increased above baseline, a finding consistent with past histomorphometric findings of increased bone formation during repletion. In summary, this study confirms that 1) a short calcium depletion period produces calcium stress in young women, which leads to rapid stimulation of bone resorption and inhibition of bone formation; and 2) a subsequent calcium repletion period could lead to a compensatory increase in bone formation. In conclusion, the skeletal responses to calcium depletion/repletion in young women may be similar to those in rodents

Bone Cells on Microcarrier Spheres

Many of the experiments being done in our laboratory to gain a clearer understanding of bone metabolism require the use of cell culture techniques to examine the effects of ions or hormones on a specific cell population. Unfortunately, however, because the isolated osteoblasts and their precursors are anchorage-dependent cells, it is often difficult to obtain large numbers of these cells by standard tissue culture techniques. In an attempt to overcome these difficulties and thus obtain large numbers of these cells, we have adapted a technique of using microspheres (Superbeads, Flow Laboratories, or Cytodex 1, Pharmacia) to which cells will attach and proliferate.These microspheres are composed of diethylaminoethyl anion exchange groups bound to a dextran matrix. First discovered by van Wezel,1 and worked into research scale applications by Levine et al,2 this method offers all of the advantages of suspension cultures for large-scale cultivation of cells, while providin