Figure 4. Impaired p38 MAPK pathway activation in TAK1-deficient osteoblasts.

(A) Tak1^fl/fl CalvOb were infected by vector or cre-expressing lentivirus and cultured under differentiation conditions in the absence or the presence of BMP2/7 (50 ng/ml). ALP activity and mineralization were analyzed by colorimetric assay (upper) and Von Kossa staining (lower), respectively. Values are mean + SD. (B) Tak1^fl/fl CalvOb infected by vector or cre lentivirus were serum starved for 12 hours before BMP2/7 (100 ng/ml) stimulation at different time points. Cell lysates were then immunoblotted with the indicated antibodies. (C) Immunohistochemistry showing phosphorylation levels of MKK3/6 and p38 in a coronal section of the calvarium of Tak1^fl/fl and Tak1^osx mice. (D) Primary WT CalvOb were cultured under differentiation conditions in the absence or the presence of the p38 inhibitor, and then ALP activity was analyzed by colorimetric assay (upper) and Fast Blue staining (lower). Values are mean + SD. (E and F) Human MSCs were infected with vector, MKK6-glu, or MKK6-K82A and cultured under osteoblast differentiation conditions; then ALP activity and mineralization were analyzed by colorimetric assay and Von Kossa staining, respectively (E). Alternatively, cells were cultured under osteoblast differentiation conditions in the presence of DMSO, MEK1/2, JNK, or p38 inhibitor (F). Values are mean + SD. Original magnification, ×100.