Figure 5. Reciprocal relationship between ERK1/2 and p38 MAPK activities: Effect of ERK1/2 and p38 MAPK inhibitors on RUNX2 transcriptional activity and phosphorylation.
A. COS7 cells. Cells were transfected with a wild type RUNX2 expression vector and 6OSE2-Luc reporter and grown for 48h. Sixteen hours before harvest, cells were treated with 20 μM U0126 (ERK/MAPK inhibitor), 20 μM SB203580 (SB, a p38/MAPK inhibitor) or both compounds. Cells were assayed for luciferase activity (upper panel) and phosphorylation of RUNX2, ERK1/2 and p38 (lower panel). B. MC3T3-E1 clone 42 preosteoblasts. These cells contain stably transfected copies of a 1.3 kb murine osteocalcin gene 2 (mOG2) promoter driving a firefly luciferase reporter. Cells were cultured using differentiation conditions described in Methods and treated with U0126 or SB203580 for 16 hour before harvest. Upper panel: mOG2 promoter activity. Lower panel: RUNX2, ERK and p38 phosphorylation. C,D. Calvarial organ cultures. Calvaria were isolated from E18.5 day mouse embryos and cultured for 5 d as described in Methods +/− U0126 or/and SB203580. Cultures were stained for mineral using Alizarin red (upper panel). Lower panel: phosphorylation of RUNX2, ERK and p38. Statistically significant differences are indicated: (*), P<0.05; (**), P<0.01.