Figure 6.

PKCα activation increases the transcriptional activity of Runx2 through ERK signaling.A, IEC-18 cells were treated with vehicle or 100 nM PMA for 1 h and 2 h, and TGFβR1 mRNA expression was determined by RT-qPCR. B, IEC-18 cells were treated with vehicle (-) or 100 nM PMA for 0.5 h, 1 h, and 2 h, and Runx2 protein expression was determined by Western blotting. Numbers below the blots indicate relative band intensity (mean ± SD) for Runx2 normalized to corresponding loading control. C, protein extracts from IEC-18 cells were treated with λ-phosphatase as indicated and analyzed by Phos-tag gel electrophoresis (upper panel) or SDS-PAGE (lower panel) and immunoblotted for Runx2 and β-actin. Each panel is from the same blot, and dashed lines indicate where lanes have been rearranged for clarity. D, IEC-18 cells were treated with vehicle (C), 100 nM PMA (P), or 20 μg/ml DiC₈ (D) for 15 min and analyzed by Phos-tag gel electrophoresis, SDS-PAGE, and immunoblotting as in C. E, IEC-18 cells were treated with vehicle (C) or 100 nM PMA (P) for 15 min in the presence or absence of 1 μM SCH772984. Extracts were divided and one portion was treated with λ-phosphatase as indicated before analysis by Phos-tag gel electrophoresis (top panels) or SDS-PAGE (middle and lower panels) and immunoblotting for the indicated proteins. Lanes five and six show effects of λ-phosphatase treatment on the extracts shown in lanes 1 and 2, respectively. The dashed line is as in C. F, IEC-18 cells were treated with vehicle (C) or 100 nM PMA (P) for 2 min, 5 min, or 10 min. Phosphorylation/activation of ERK and Runx2 were determined by Western blotting and Phos-tag gel analysis, respectively. G, IEC-18 cells were transfected with p6OSE-luc firefly luciferase reporter along with TK-Renilla luciferase transfection efficiency control. After 24 h, cells were treated with 100 nM PMA or 20 μg/ml DiC₈ in the presence or absence of 1 μM SCH772984 for 2 h, and luciferase activity was determined. Data show p6OSE driven firefly luciferase relative to TK-Renilla activity. Data in B–F are representative of at least three independent experiments, and data in A and G are the average ± SEM of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Arrow in D–F indicates slower mobility species of Runx2 induced by PKC agonist treatment. DiC8, 1,2-dioctanoyl-sn-glycerol; ERK, extracellular signal-regulated kinase 1/2; IEC-18, intestinal crypt-like cells; PKCα, protein kinase C α; PMA, phorbol 12-myristate 13-acetate; Runx2, runt-related transcription factor 2; TGFβR1, transforming growth factor-β receptor 1.