Fig. 6.

Identification of the dominant cis-element in RTA promoter activated by TPA. (A) Deletion analysis of the RTA promoter to identify the region responsive to TPA induction. Bjab cells transfected with different RTA promoter deletion reporter constructs for 42 h were treated with 20 ng/ml of TPA for 6 h, lysed, and assayed for luciferase activity. (B) Illustration of a wild type reporter (R-259) in the RTA promoter region (-15 to -259) responsive to TPA induction. R-259mut was a corresponding mutant reporter with the AP-1 site ablated. (C) Dominant negative constructs (DNs) of MAPK pathways inhibited TPA activation of the R-259 reporter while the mutant R-259mut reporter was not responsive to TPA treatment. Bjab cells transfected with R-259 or R-259mut reporter constructs together with a vector control (V) or DNs of the JNK, MEK and p38 MAPK pathways for 42 h were treated with TPA for 6 h, lysed and assayed for luciferase activity. (D) Inhibitors of MAPK pathways suppressed TPA activation of the R-259 reporter. Bjab cells transfected with R-259 or R-259mut reporter constructs for 42 h were treated with 20 ng/ml of TPA for 6 h with or without inhibitors of JNK, MEK and p38 pathways, lysed and assayed for luciferase activity. Both JNK inhibitor II (JNK inhibitor) and SB203580 (p38 inhibitor) were used at 50 μM while U0126 (MEK inhibitor) was used at 10 μM. (E) Overexpression of an active form (CA) of ERK, JNK or p38 was sufficient to activate the R-259 reporter but not the mutant R-259mut reporter. Bjab cells transfected with R-259 or R-259mut reporter constructs together with a vector control or a CA of ERK, JNK or p38 with or without their respective DNs for 48 h were lysed and assayed for luciferase activity. (F) DN of c-Fos or c-Jun inhibited TPA activation of the R-259 reporter. Bjab cells transfected with R-259 or R-259mut reporter constructs together with a vector control (V) or DN of c-Fos, c-Jun, or both for 42 h were treated with TPA for 6 h, lysed and assayed for luciferase activity.