Novel biochemical and functional insights into nuclear Ca(2+) transport through IP(3)Rs and RyRs in osteoblasts.
We report the first biochemical and functional characterization of inositol trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) in the nuclear membrane of bone-forming (MC3T3-E1) osteoblasts. Intact nuclei fluoresced intensely with anti-RyR (Ab(34)) and anti-IP(3)R (Ab(40)) 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 Ab(40), anti-RyR-1, anti-RyR-2 (Ab(129)), and anti-RyR-3 (Ab(180)). Only anti-RyR-1 and Ab(40) showed bands corresponding, respectively, to full-length RyR-1 ( approximately 500 kDa) and IP(3)R-1 (approximately 250 kDa). Band intensity was reduced by just approximately 20% after brief tryptic proteolysis of intact nuclei; this confirmed that isolated nuclear membranes were mostly free of endoplasmic reticular contaminants. Finally, the nucleoplasmic Ca(2+) concentration ([Ca(2+)](np)) was measured in single nuclei by using fura-dextran. The nuclear envelope was initially loaded with Ca(2+) via Ca(2+)-ATPase activation (1 mM ATP and approximately 100 nM Ca(2+)). Adequate Ca(2+) loading was next confirmed by imaging the nuclear envelope (and nucleoplasm). Exposure of Ca(2+)-loaded nuclei to IP(3) or cADP ribose resulted in a rapid and sustained [Ca(2+)](np) elevation. Taken together, the results provide complementary evidence for nucleoplasmic Ca(2+) influx in osteoblasts through nuclear membrane-resident IP(3)Rs and RyRs. Our findings may conceivably explain the direct regulation of osteoblastic gene expression by hormones that use the IP(3)-Ca(2+) pathway.
Adebanjo
O A
OA
Division of Endocrinology and Metabolism, Mount Sinai School of Medicine, and Bronx Veterans Affairs Geriatric Research Education and Clinical Center, New York 10029, USA.
Biswas
G
G
Moonga
B S
BS
Anandatheerthavarada
H K
HK
Sun
L
L
Bevis
P J
PJ
Sodam
B R
BR
Lai
F A
FA
Avadhani
N G
NG
Zaidi
M
M
D016475:3T3 Cells; D000818:Animals; D015220:Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling; D002467:Cell Nucleus / Q000378:metabolism*; D007150:Immunohistochemistry; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors; D051379:Mice; D009685:Nuclear Envelope / Q000378:metabolism; D010006:Osteoblasts / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D019837:Ryanodine Receptor Calcium Release Channel; D015544:Inositol 1,4,5-Trisphosphate