Angiotensin II up-regulates osteopontin mRNA expression of RAW264.7 macrophages via p38MAPK signaling

Abstract

To investigate the changes of osteopontin (OPN) mRNA expression induced by angiotensin II (AngII) in RAW264.7 macrophages, and to determine the role of p38 mitogen-activated protein kinase (p38MAPK) signaling in up-regulation of OPN mRNA expression. RT-PCR was used for examining the OPN mRNA expression, and the phosphorylation of p38MAPK and activating transcription factor2 (ATF2) was detected by Western blot. (1) AngII (1 micromol/L) enhanced the expression of OPN mRNA in RAW264.7 macrophages with time-dependent, including 0.9 fold, 2.3 folds and 2.4 folds up-regulation at 6, 12, 24 h, respectively (all P < 0.01), whereas there was no significant change in negative control at 0 h and 24 h, suggesting that the mRNA expression of OPN is inducible. (2) Following excitation by Ang II in vitro, there was substantial up-regulation of OPN mRNA expression in RAW264.7 macrophages with dose-dependent; with a dose of 1 micromol/L incubated for 12 h, Ang II increased the OPN mRNA expression by 2.6 folds (P < 0.01). (3) Pre-treatment with SB202190 (5 micromol/L) for 30 min in RAW264.7 macrophages before AngII (1 micromol/L) used, the expression of OPN mRNA was inhibited by 61.7% (P < 0.01); but no marked inhibition had happened at the time of treatment with both SB202190 (5 micromol/L) and Ang II (1 micromol/L) added at the same time and with SB202190 (5 micromol/L) for 120 min after Ang II (1 micromol/L) used. (4) Pre-treatment with SB202190 (1, 5, 10 micromol/L) for 30 min in RAW264.7 macrophages before Ang II (1 micromol/L) used, the expression of OPN mRNA was decreased by 43.4%, 63.0% and 65.5%, respectively (all P < 0.01). (5) There was a maximal expression of p38MAPK phosphorylation at 15 approximately 30 min induced by Ang II (1 micromol/L) in RAW264.7 macrophages, and the maximal expression of phosphorylated ATF2 was at 30 approximately 45 min. (6) Pre-treatment with SB202190 (1, 5 micromol/L) for 30 min in RAW264.7 macrophages before Ang II (1 micromol/L) used, the expression of p38MAPK phosphorylation was inhibited markedly with dose-dependent, and the phosphorylation of p38MAPK was decreased by 61.7% (P < 0.01) after the SB202190 (5 micromol/L) was used. Angiotensin II may up-regulate osteopontin mRNA expression of RAW264.7 macrophages via p38MAPK signaling.