FIG. 8.

Effect of MEKK1 on transcriptional activity of TR3. (A) Inhibition of TR3 transcriptional activity by MEKK1. TR3 expression vector (25 ng), β-Gal expression vector (50 ng), and the indicated reporter gene (200 ng) were cotransfected into CV-1 cells in 24-well plates with or without the indicated amount (expressed in nanograms) of MEKK1-DA expression vector (5). Reporter gene activity was determined and normalized relative to cotransfected β-Gal gene activity. One of six (left panel) or three (right panel) similar experiments is shown. (B) TR3 transcriptional activity is unaffected by kinase-deficient MEKK1. TR3 expression vector (25 ng), (NurRE)₂-tk-CAT (100 ng), β-Gal expression vector (50 ng) with or without the indicated amount (in nanograms) of MEKK1-DN expression vector (5) was transfected, and reporter gene activity was measured as described for panel A. The bars are averages ± mean from two experiments. (C) TR3 expression is not altered by MEKK1.GFP-TR3 expression vector (5 μg) was transfected into H460 cells with or without MEKK1-DA (5 μg) expression vector. Forty-five hours after transfection, amounts of GFP-TR3-expressing cells (percent green cells) were determined by flow cytometry. Similar results were observed in Calu-6 cells. The bars represent the averages ± mean from two experiments. (D) MEKK1 expression does not alter TR3 nuclear localization. GFP-TR3 alone (1.0 μg) or together with MEKK1-DA (1.0 μg) was transiently expressed in H460 cells. Fourteen hours later, cells were immunostained with anti-Hsp60 antibody followed by Cy3-conjugated secondary antibody to detect mitochondria. GFP-TR3 was visualized using confocal microscopy, and the two images were overlaid (Overlay). (E) Effect of MEKK1 expression on transactivation of other nuclear receptors and AP-1. The expression vector for ER or TR (25 ng) and the corresponding reporter gene (ERE-tk-CAT or TREpal-tk-CAT, respectively) were cotransfected into CV-1 cells with or without MEKK1-DA expression vector (30 ng). Twenty hours after transfection, cells were treated with estradiol (10⁻⁸ M) or T₃ (10⁻⁷ M), and CAT activity was determined and normalized relative to cotransfected β-Gal gene activity. For measuring the effect of MEKK1 on AP-1 activity, the −73Col-CAT (250 ng) reporter (37) was transfected with or without MEKK1-DA expression vector (30 ng). Reporter gene activity was measured as described earlier. One of three similar experiments is shown. (F) MEKK1 does not affect the transrepression activity of TR3. TR3 expression vector (20 ng) with or without MEKK1-DA expression vector (30 ng) and/or GR expression vector (20 ng) was cotransfected into CV-1 cells with the GRE-tk-CAT reporter vector (50). Twenty hours after transfection, cells were treated with Dex (10⁻⁷ M), and CAT activity was determined and normalized relative to cotransfected β-Gal gene activity. One of two similar experiments is shown.