Figure 2. High inorganic phosphate (Pi) concentrations increase AP-1 activation in transformation-sensitive JB6 cells.

(A) High Pi increases AP-1 transcriptional activity in JB6 cells: cells were transfected with a 4xAP-1 luciferase and Renilla reporter constructs, and after 24 hrs serum starved overnight. Medium was then supplemented to a final concentration of 1, 2, or 3mM Pi in the presence of DMSO (control) or 10ng/ml TPA in DMSO for 6 hrs. AP-1 transcriptional activity was measured using a luciferase reporter assay (6 replicates/treatment) and Renilla by Stop-and-Glo. Luciferase activity was then normalized to Renilla and averaged. Columns with different characters differ at p<0.05. (B) High Pi increases within 5 min AP-1 DNA binding in JB6 cells: cells were supplemented with 2mM Pi for 0, 5, 10, 15, 30, or 60 min and EMSA was performed using an oligo containing a consensus AP-1 binding element. (C) High Pi primarily increases within 5 min DNA binding of the AP-1 proteins Fra-2, JunB, and FosB: cells were supplemented with 2 mM Pi for 5 min and EMSA was performed using an oligo containing a consensus AP-1 element and antibodies specific to the 7 individual AP-1 family members (Fra-1, Fra-2, c-fos, FosB, JunB, c-jun, JunD) and CREB (representative of multiple experiments). (D) High Pi alters AP-1 protein forms: JB6 cells were serum starved (1mM Pi) followed by addition of 2mM Pi (3mM final) for the indicated times. Nuclear lysates were analyzed by Western blotting and probed as indicated. (E) High Pi increases Egr1 expression within 20 minutes. JB6 cells were treated as in D and cells harvested for Northern analysis. Results are representative of multiple experiments.