Integrating rapid responses to 1,25-dihydroxyvitamin D 3 with transcriptional changes in osteoblasts: Ca 2+ regulated pathways to the nucleus

Abstract

It is well established that 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) treatment of target cells including osteoblasts activates both membrane-initiated rapid Ca 2+ responses linked to influx through voltage sensitive Ca 2+ channels (VSCCs) and longer term nuclear receptor-mediated changes in gene expression. We recently reported use of a cDNA microarray strategy to identify transcriptional changes after 3 and 24 h of treatment with 1,25(OH) 2D 3 and with an analog of 1,25(OH) 2D 3 (25(OH)-16ene-23yne-D 3 [AT]) that activates Ca 2+ influx without binding to the nuclear receptor. Among 5000 different clones on the array filters, we identified families of genes in osteoblasts that were altered two-fold or greater following treatment with 1,25(OH) 2D 3 or analog AT for 3 h. Cluster analysis further revealed complex patterns of changes in gene expression, indicative of multiple pathways to the nucleus. Evidenced by changes in target gene expression, activation of a Ca 2+/CaMK/CREB/CRE pathway clearly occurs and modulates expression of a variety of genes associated with changes in protein secretion including those involved in paracrine regulation of bone resorption, RANKL and osteoprotegerin (OPG). The changes in gene expression can be inhibited by L-type VSCC channel blockers, confirming the role of Ca 2+ entry in pathway activation. These findings provide clear evidence of rapid changes in gene expression associated with Ca 2+ influx after treatment with 1,25(OH) 2D 3, and open the door to novel nuclear receptor-independent signaling pathways that affect gene transcription.