Figure 6. Depletion of Cdh1 leads to inactivation of Smurf1 and augmentation of the MEKK2 signaling pathway to induce osteoblast differentiation.
A-C. Depletion of Cdh1 in primary mouse calvarial osteoblasts promotes osteoblast differentiation. Primary mouse calvarial osteoblast cells were derived from neonatal mice and infected with the indicated lentiviral shRNA constructs. The infected cells were selected with 1 μg/ml puromycin for 72 hours to eliminate non-infected cells and the efficiency of Cdh1 depletion was examined by anti-Cdh1 immunoblot analysis in (A). Afterwards, the generated cells were incubated in osteoblast differentiation medium for 21 days before the Fast Blue staining for ALP activity (shown as blue staining) and von Kossa staining for minerilization (shown as black staining) in (B). Additionally, the Cdh1 knockdown efficiency and the expression of various characteristic osteoblast markers were determined by real-time PCR analysis in (C). Three sets of independent experiments were performed to generate each data point in (C) and the error bars represent means ± SD.
D-E. The osteoblast differentiation is also increased in Cre-encoding lentivirus-induced Cdh1 depletion in primary Cdh1F/F calvarial osteoblast cells (empty vector (EV) lentivirus was used as a negative control). The infected cells were selected with 1 μg/ml puromycin for 72 hours to eliminate non-infected cells, the resulting cells were then incubated in osteoblast differentiation medium for 21 days before Fast Blue staining and von Kossa staining in (D). Cre-encoding lentivirus-induced Cdh1 depletion and the expression of various characteristic osteoblast markers were monitored by real-time PCR analysis in (E). Three sets of independent experiments were performed to generate each data point in (E) and the error bars represent means ± SD.
F. Ectopic expression of WT-Cdh1 and ΔC-box-Cdh1, but not N174-Cdh1 could suppress the increased osteoblast differentiation phenotype in Cdh1-depleted primary calvarial osteoblast cells. Primary calvarial osteoblast cells derived from Cdh1F/F mice were co-infected with Purolentivirus encoding Cre (or a lentiviral vector encoding EGFP as a negative control) and the indicated Hygro-lentiviral vector encoding WT-Cdh1 and various Cdh1 mutants. The infected cells were selected with 1 μg/ml puromycin and 200 μg/ml hygromycin for 96 hours to eliminate non-infected cells. Afterwards, the generated cells were incubated in osteoblast differentiation medium for 21 days before von Kossa staining.
(see also Figure S6)