Figure 6

The MEKK1/MKK7/JNK signal transduction pathway contributes to EpCAM-dependent AP-1 transcription factor activity. (a) Pharmacologic inhibition of c-Jun N-terminal kinase (JNK) abrogates recombinant soluble extracellular epithelial cell adhesion molecule (rEpCAM)-mediated rescue of activator protein 1 (AP-1) transcription factor activity. Stably transduced CA1a (scrambled control shRNA construct (SCR) and sh2) cells were transiently transfected with AP-1 and control luciferase reporters. Six hours after transfection, cells were treated with SP600125 (JNK inhibitor) at 10 μM and 20 μM for one hour followed by addition of rEpCAM. Luciferase reporter activity was measured 16 hours later. (b) To directly evaluate JNK phosphorylation, SCR and sh2 CA1a and MCF-7 cells were serum starved for 12 hours, stimulated with 20 μM anisomycin for 10 minutes, and then immunoprecipitated overnight with a JNK antibody. JNK phosphorylation was evaluated by immunoblot using antibodies specific for p-JNK1/2 and total JNK1. Relative band density was quantified using ImageJ software, with the results indicated. (c) Constitutively active genetic constructs corresponding to mitogen activated protein (MAP) kinases in the JNK signal transduction pathway rescued c-Jun phosphorylation and AP-1 transcription factor activity following specific ablation of EpCAM in MCF-7 breast cancer cells. Stably transduced SCR or sh2 MCF-7 cells were transiently transfected with plasmids encoding HA-MEKK1, FLAG-MKK7-JNK1, FLAG-MKK7, and FLAG-JNK1. After 16 hours, cells were analyzed by immunoblot for c-Jun phosphorylation. Relative band density was quantified using ImageJ software, with the results indicated. (d) Duplicate samples were transiently transfected with control and AP-1 luciferase reporters, and AP-1 transcription factor activity was measured. (e) Expression of HA-MEKK1, FLAG-MKK7-JNK1, FLAG-MKK7, and FLAG-JNK1 was confirmed by immunoblot with antibodies specific for the indicated protein tag (HA or FLAG). The results are representative of two independent experiments. For AP-1 reporter assay in Figure a and d, P < 0.05 were considered to be statistically significant when compared between SCR, sh2 (*) and sh2, sh2 treated/transfected cells (**).