Figure 5.
p38α Positively Regulated OPG Expression via CREB
(A) p38α−/− BM-MSCs showed a decreased expression of OPG and a modest increase in RANKL and M-CSF compared with WT BM-MSCs. Total RNA was isolated from WT and p38α−/− BM-MSCs and was used to determine the mRNA levels of Opg, Rankl, and M-csf. Data represent means ± SEM of three independent experiments, ∗∗p < 0.01.
(B) Prx1-Cre; p38αf/f mouse bones also showed a decrease in Opg mRNA levels and a modest increase in Rankl and M-csf. The femur bones were frozen and total RNA was isolated. Data represent means ± SEM of six independent experiments, ∗∗p < 0.01.
(C) Addition of OPG to the p38α−/− BM-MSCs co-cultured with normal monocytes impeded the enhanced osteoclast differentiation. The experiments were carried out as described in Figure 4D, except that OPG (20 ng/ml) was added to the cultures. Scale bar, 100 μm.
(D) p38α−/− BM-MSCs showed a decrease in CREB phosphorylation. WT and p38α−/− BM-MSCs were collected and lysed. The levels of CREB and p-CREB were determined by western blot.
(E) Knockdown of Creb led to a down-regulation of Opg in normal BM-MSCs. BM-MSCs were transfected with control or siRNA against Creb. qPCR was carried out to determine the mRNA levels of Opg. Right panel: western blot showing the knockdown of CREB. Data represent means ± SEM of three independent experiments, ∗∗p < 0.01.
(F) ChIP experiments showed that Opg promoter has two CREB binding sites at −501 to −601 and −1201 to −1301. Right panel: a diagram showing the binding sites of CREB in the promoter of Opg gene. Data represent means ± SEM of three independent experiments, ∗∗p < 0.01.