Target gene activation by 1,25-dihydroxyvitamin D3 in osteosarcoma cells is independent of calcium influx.
Analogs of the seco-steroid hormone, 1,25-dihydroxyvitamin D3 [1,25-(OH)-2D3] can preferentially stimulate genomic or nongenomic signaling pathways in osteoblasts. In this study, we used 1,25-(OH)2D3 analogs and voltage-sensitive Ca2+ channel (VSCC) ligands, including dihydropyridines (Bay K 8644 and nitrendipine), in an osteosarcoma cell model to examine the relationship between 1,25-(OH)2D3-stimulated Ca2+ influx and genomic and nongenomic pathways leading to osteoblast activation. Northern blotting experiments demonstrated that an analog of 1,25-(OH)2D3, 1,24-dihydroxy-22-ene-24-cyclopropyl D3, increased messenger RNA (mRNA) levels of both osteopontin (OPN) and osteocalcin (OCN) without triggering Ca2+ influx through VSCCs. Nitrendipine (an inhibitor of L-type VSCCs) did not block the mRNA increase induced by either analog 1,24-dihydroxy-22-ene-24-cyclopropyl D3 or 1,25-(OH)2D3. 1-Deoxy analogs of 1,25-(OH)2D3, 25-hydroxy-16-ene-23-yne-D3, or 25-hydroxy-23-yne-D3, which stimulate Ca2+ influx, did not produce mRNA accumulation for OPN and OCN, consistent with their poor binding to nuclear receptors. Likewise, Bay K 8644, an agonist of VSCCs that produces Ca2+ influx, did not increase mRNA levels for OPN or OCN. Experiments using a construct derived from the sequence of the genomic OPN promoter region and a luciferase reporter confirmed the analog specificity in stimulating transcription. Together these results indicate that 1,25-(OH)2D3-mediated up-regulation of genes encoding OPN and OCN is independent of Ca2+ influx and suggest that the stimulation of Ca2+ influx by 1,25-(OH)2D3 is not required for target gene activation.
Khoury
R
R
Department of Basic Sciences, University of Texas, Houston 77030.
Ridall
A L
AL
Norman
A W
AW
Farach-Carson
M C
MC
D002117:Calcitriol / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D004095:Dihydropyridines / Q000494:pharmacology; D005786:Gene Expression Regulation; D016678:Genome; D015675:Osteocalcin / Q000235:genetics; D053495:Osteopontin; D012516:Osteosarcoma / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism; D012795:Sialoglycoproteins / Q000235:genetics; D013997:Time Factors; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured / Q000187:drug effects
D004095:Dihydropyridines; D012333:RNA, Messenger; D012795:Sialoglycoproteins; D015675:Osteocalcin; D053495:Osteopontin; D002117:Calcitriol; D002118:Calcium