TARGET GENE ACTIVATION BY 1,25-DIHYDROXYVITAMIN D-3 IN OSTEOSARCOMA CELLS IS INDEPENDENT OF CALCIUM INFLUX

Analogs of the seco-steroid hormone, 1,25-dihydroxyvitamin D-3 [1,25-(OH)(2)D-3] can preferentially stimulate genomic or nongenomic signaling pathways in osteoblasts. In this study, we used 1,25-(OH)(2)D-3 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)(2)D-3-stimulated Ca2+ influx and genomic and nongenomic pathways leading to osteoblast activation. Northern blotting experiments demonstrated that an analog of 1,25-(OH)(2)D-3, 1,24-dihydroxy-22-ene-24-cyclopropyl D-3, 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 D-3 or 1,25-(OH)(2)D-3. 1-Deoxy analogs of 1,25-(OH)(2)D-3, 25-hydroxy-16-ene-23-yne-D-3, or 25-hydroxy-23-yne-D-3, 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)(2)D-3-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)(2)D-3 is not required for target gene activation.