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1 Division of Endocrinology and Metabolism, Mount Sinai School of Medicine, and Bronx Veterans Affairs Geriatric Research Education and Clinical Center, New York 10029; 2 School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; and 3 National Institute of Medical Research, London, NW7 1AA United Kingdom
We report the first biochemical and functional characterization of inositol trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) in the nuclear membrane of bone-forming (MC3T3-E1) osteoblasts. Intact nuclei fluoresced intensely with anti-RyR (Ab34) and anti-IP3R (Ab40) antisera in a typically peripheral nuclear membrane pattern. Isolated nuclear membranes were next subjected to SDS-PAGE and blotted with isoform-specific anti-receptor antisera, notably Ab40, anti-RyR-1, anti-RyR-2 (Ab129), and anti-RyR-3 (Ab180). Only anti-RyR-1 and Ab40 showed bands corresponding, respectively, to full-length RyR-1 (~500 kDa) and IP3R-1 (~250 kDa). Band intensity was reduced by just ~20% after brief tryptic proteolysis of intact nuclei; this confirmed that isolated nuclear membranes were mostly free of endoplasmic reticular contaminants. Finally, the nucleoplasmic Ca2+ concentration ([Ca2+]np) was measured in single nuclei by using fura-dextran. The nuclear envelope was initially loaded with Ca2+ via Ca2+-ATPase activation (1 mM ATP and ~100 nM Ca2+). Adequate Ca2+ loading was next confirmed by imaging the nuclear envelope (and nucleoplasm). Exposure of Ca2+-loaded nuclei to IP3 or cADP ribose resulted in a rapid and sustained [Ca2+]np elevation. Taken together, the results provide complementary evidence for nucleoplasmic Ca2+ influx in osteoblasts through nuclear membrane-resident IP3Rs and RyRs. Our findings may conceivably explain the direct regulation of osteoblastic gene expression by hormones that use the IP3-Ca2+ pathway.
nuclear calcium channels; bone formation; osteoblasts; osteoporosis; ryanodine receptors; inositol 1,4,5-trisphosphate receptors
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