In vitro synthesis of 1 alpha,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol by isolated calvarial cells.

The question of whether the skeleton metabolizes 25-hydroxycholecalciferol [25(OH)D3] to more-polar products was studied. Calvarial cells were dispersed from 16-day old chicken embryos by using collagenase and then grown in culture in serum-free medium. Confluent cell cultures were incubated with 7 nM 25(OH)[3H]D3 for 2 hr, and the vitamin D metabolites were then extracted. At least four polar metabolites were produced. Based on separation by Sephadex LH-20 chromatography followed by high-pressure liquid chromatography, two of these metabolites were identified as 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3]. These metabolites were also produced by cultured kidney cells but not by liver, heart muscle, or skin cells isolated from the same embryos. The specific activities of the calvarial 1- and 24-hydroxylases were similar in magnitude to those in isolated kidney cells. The specific activity of the calvarial 25(OH)D3:1-hydroxylase was inhibited by an 8-hr preincubation with 1,25(OH)2D3, whereas the 24-hydroxylase was enhanced. It is concluded that (i) vitamin D metabolism by isolated cells is organ-specific, (ii) calvarial cells produce active metabolites of vitamin D in significant amounts, (iii) vitamin D metabolism by calvarial cells is regulated by 1,25(OH)2D3, and (iv) locally produced, active metabolites could act locally, thereby adding a new dimension to the regulation of mineral metabolism by vitamin D metabolites

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

LOSS OF PROTEINPOLYSACCHARIDES AT SITES WHERE BONE MINERALIZATION IS INITIATED

In both ground sections and demineralized frozen sections of the rat tibial cortex, osteoid but not mature bone matrix stained for proteinpolysaccharides with the Alcian Blue and toluidine blue techniques. The loss of proteinpolysaccharide staining occurred precisely at the mineralizing front, which was identified by in vivo lead or procion markers, not only in normal animals but also in animals in which osteoid width was either increasing or decreasing. In vitro, both proteases and saccharidases abolished proteinpolysaccharide staining of osteoid. Critical electrolyte concentration and other procedures indicated that the major acid polysaccharide component in osteoid is chondroitin sulfate. Consistent with these findings, electron microprobe analyses revealed that sulfur concentration was high in osteoid but dropped abruptly as calcium concentration increased at the mineralizing front. The precise synchronization between loss of proteinpolysaccharides and onset of mineralization under various experimental conditions provides strong indirect evidence that the loss of these macromolecules is somehow involved in initiation of mineralization in bone. </jats:p