Figure 5.

PKCα induces ERK-dependent upregulation of TGFβR1 and Runx2 at the transcriptional level.A, IEC-18 cells were treated with vehicle, 100 nM PMA or 20 μg/ml DiC₈ for 2 h and incubated with ethynyl uridine (EU) for the final 1 h. Labeled RNA was then isolated using the Click-IT Nascent RNA Capture Kit and quantified by RT-qPCR. Data show levels of nascent TGFβR1 mRNA normalized to nascent 18S rRNA. B, FET colon cancer cells were transfected with a TGFβR1 promoter construct driving expression of Gaussia luciferase. Twenty-four hours after transfection, cells were treated with 100 nM PMA for 2 h. Data show relative luciferase activity from PMA-treated cells normalized to luciferase activity from vehicle-treated cells. C, IEC-18 cells were pretreated with vehicle, 5 μM BIM, or 4 μM Gö6976 prior to addition of PMA (100 nM) or DiC₈ (20 μg/ml) for 2 h. Levels of Runx2 mRNA (normalized to 18S RNA) were then assessed by RT-qPCR. D, as in A except that data show relative levels of nascent Runx2 mRNA. E, as in C except that cells were pretreated with vehicle or 1 μM SCH772984 prior to addition of PMA or DiC₈. F, IEC-18 cells were pretreated with Gö6976 as indicated prior to addition of vehicle (C), 100 nM PMA (P) or 20 μg/ml DiC₈ (D) for 2 h and levels of the indicated proteins were determined by Western blotting. Numbers below the blots represent quantification of Runx2 band intensity relative to loading control (average ± SD from three independent experiments). Data in B, C, and E are averages ± SEM of at least three independent experiments, and data in A and D are averages ± SEM of two independent experiments. ∗p < 0.05. ∗∗p < 0.01. BIM, bisindolylmaleimide I; DiC8, 1,2-dioctanoyl-sn-glycerol; IEC-18, intestinal crypt-like cells; PKCα, protein kinase C α; PMA, phorbol 12-myristate 13-acetate; TGFβR1, transforming growth factor-β receptor 1; ERK, extracellular signal-regulated kinase 1/2; Runx2, runt-related transcription factor 2.