Microarray detection of gene expression changes induced by 1,25(OH)2D3 and a Ca2+ influx-activating analog in osteoblastic ROS 17/2.8 cells

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 Ca2+ influx and longer-term nuclear receptor-mediated changes in gene expression. Ca2+ 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 Ca2+ 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 Ca2+ 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(TM), 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, Ca2+ signaling, cell adhesion and secretion. These findings provide clear evidence of rapid changes in gene expression associated with Ca2+ influx mediated by 1,25(OH)(2)D-3, and shed light on the nuclear-receptor independent signaling pathway affecting OPN phosphorylation. (C) 2002 Elsevier Science Inc. All rights reserved.