Figure 3

Inhibition of the MEK1/2-ERK1/2 pathway stimulates tibia growth, while p38 MAPK is required for CNP-induced bone growth. Mouse E15.5 tibiae were harvested and cultured for six days in the presence of control or CNP (1 μM) and vehicle (DMSO) or MEK1/2-ERK1/2 pathway inhibitors PD98059 (10 μM) and U0126 (10 μM) (A). Though both PD98059 and U0126 stimulated basal bone growth, inhibition of the MEK1/2-ERK1/2 pathway did not further enhance CNP-induced bone growth (*: p < 0.05 when comparing control/inhibitors to control/vehicle; #: p < 0.05 when comparing CNP/vehicle to control/vehicle; p > 0.05 when comparing CNP/vehicle to CNP/inhibitors). Tibiae were incubated with control or CNP and pharmacological inhibitors of the p38 MAPK pathway (SB202190 or PD169316, 10 μM each) or an inactive analog (SB202474, 10 μM) (B). p38 inhibition did not effect basal bone growth significantly, but did suppress CNP-induced bone growth (*: p < 0.05 when comparing CNP/inhibitors to CNP/SB202474; #: p < 0.05 when comparing CNP/SB202474 to control/SB202474). Bone growth was measured over an extended time course of eight days, showing that CNP continued to significantly influence growth on day 8, while SB202190 reversed these effects (C). Bones from each treatment were weighed under different conditions, and it was found that p38 inhibition reversed the effects of CNP on weight (D). Protein extracts from primary chondrocytes cultured with control, CNP (10^-6M), or 8-(4-cpt) cGMP (0.1 mM) for 10 minutes were examined for phosphorylation of the p38 activators MKK3/6 by western blot analysis (E). Both treatments increased phosphorylation of MKK3/6, supporting the stimulation of p38 MAP kinase activity by CNP signaling. Immunohistochemistry with an antibody against phosphorylated p38 demonstrates markedly higher signal in CNP-treated tibiae when compared to control bones (F).