Figure 6. mTORC2/SGK1 activation is important for MEK1/2 deficiency-induced osteoblast differentiation.

(A) Immunoblot analysis showing phosphorylation levels of mechanistic target of rapamycin (mTOR) signaling components in Ctrl and dKO Obs. GAPDH was used as a loading control. (B–D) Ctrl and dKO Obs expressing sh-Scr or sh-Sgk1 shRNAs were cultured under osteogenic conditions. Alkaline phosphatase (ALP) activity (B), osteogenic markers expression (C), and mineralization (D) were assessed after 6 or 18 days of culture, respectively. (E) Conditioned medium (CM) collected from Ctrl and dKO Obs expressing sh-Scr or sh-Sgk1 shRNAs 6 days after osteogenic culture was added to transwell migration of mouse endothelial cells (EPOCs) and migrated cells were assessed 12 hr after incubation. (F) Mouse bone marrow-derived mesenchymal stromal cells (BMSCs) were cultured under osteogenic condition in the presence of CM, and ALP activity and Cola1 mRNA level were analyzed at day 6 of the culture. (G) Vehicle- or trametinib-treated Obs expressing sh-Scr or sh-Sgk1 shRNAs were treated with Mitotracker or TMRE 4 days after osteogenic culture and mitochondrial mass and membrane potential were assessed using flow cytometry, respectively. (H) Oxygen consumption rate (OCR) in Ctrl and dKO Obs expressing sh-Scr or sh-Sgk1 shRNAs after 6 days of culture. (I) Diagram showing the molecular actions of extracellular signal-regulated kinase (ERK) on bone formation. Data are representative of two or three independent experiments (A–H). A two-tailed unpaired Student’s t-test for comparing two groups (B–D) or ordinary one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test (E, F, H; B–F, H; error bars, standard deviation [SD] of biological replicates).