Upregulation of Runt-Related Transcription Factor-2 Through CCAAT Enhancer Binding Protein-β Signaling Pathway in Microglial BV-2 Cells Exposed to ATP

We have shown constitutive expression of the master regulator of osteoblastogenesis, runt-related transcription factor-2 (Runx2), by microglia cells outside bone. Here, we attempted to evaluate the pathological significance of Runx2 in microglial BV-2 cells exposed to ATP at a high concentration. Marked upregulation of Runx2 transcript and protein expression was seen in cells exposed to 1mM ATP for a period longer than 30min without inducing cytotoxicity. The Runx2 upregulation by ATP was prevented by extracellular and intracellular Ca²⁺ chelators, while thapsigargin upregulated Runx2 expression alone without affecting the upregulation by ATP. A calmodulin antagonist prevented the upregulation by ATP, with calcineurin inhibitors being ineffective. Although ATP markedly increased nuclear levels of nuclear factor of activated T cell-2 (NFAT2), Runx2 promoter activity was not simulated by the introduction of either NFAT1 or NFAT2, but facilitated by that of CCAAT enhancer binding protein-α (C/EBPα), C/EBPβ and nuclear factor (erythroid-derived 2)-like-2 (Nrf2). Exposure to ATP up-regulated C/EBPβ and Nrf2, but not C/EBPα, expression, in addition to increasing nuclear levels of respective corresponding proteins. Runx2 upregulation by ATP was deteriorated by knockdown of C/EBPβ but not by that of Nrf2, however, while exposure to ATP up-regulated matrix metalloproteinase-13 (Mmp13) expression in a Runx2-dependent manner. Overexpression of Runx2 up-regulated Mmp13 expression with promoted incorporation of fluorescent beads into BV-2 cells without ATP. These results suggest that extracellular ATP up-regulates Runx2 expression through activation of the C/EBPβ signaling in a calmodulin-dependent manner to play a pivotal role in phagocytosis in microglial BV-2 cells.