Microarray detection of gene expression changes induced by 1,25(OH)(2)D(3) and a Ca(2+) influx-activating analog in osteoblastic ROS 17/2.8 cells.

Abstract

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) treatment of osteoblastic ROS 17/2.8 cells initiates membrane-initiated rapid responses through activation of Ca(2+) influx and longer-term nuclear receptor-mediated changes in gene expression. Ca(2+) influx triggers a change in the phosphorylation state of the bone matrix protein, osteopontin (OPN), detectable at 3 h and prior to nuclear receptor-mediated events. This study aimed to determine if Ca(2+) influx induced by 1,25(OH)(2)D(3) would produce nuclear receptor-independent changes in gene expression. We employed a rat cDNA microarray strategy to screen the transcriptional changes at 3 h of treatment with 1,25(OH)(2)D(3) and with an analog of 1,25(OH)(2)D(3) (25(OH)-16ene-23yne-D(3) [AT]) that we previously showed to activate Ca(2+) influx without binding to the nuclear receptor. Arrays also were screened with cDNA from ROS 17/2.8 cells treated for 24 h, when nuclear receptor-mediated transcriptional events would occur. Rat gene filters (GeneFilter, Research Genetics) were hybridized with labeled cDNA probes from treatment groups. Among 5000 different clones on the array filters, we identified a family of genes which were altered 2-fold or greater following treatment with 1,25(OH)(2)D(3) or analog AT for 3 h. Cluster analysis also revealed genes whose expression was significantly up-regulated at 24 h, including OPN. Analysis of rapid changes in gene expression revealed changes affecting a diverse range of cellular pathways and functions, including protein kinases and phosphatases, Ca(2+) signaling, cell adhesion and secretion. These findings provide clear evidence of rapid changes in gene expression associated with Ca(2+) influx mediated by 1,25(OH)(2)D(3), and shed light on the nuclear-receptor independent signaling pathway affecting OPN phosphorylation.