Cells were transfected with each of the 5′-deletion constructs or with the promoterless pGL2-Basic control vector together with the 20-ng pRL-TK (Renilla luciferase expression vector, for normalization of transfection efficiencies). Cells were treated with vehicle (Me₂SO) or 150 nm PMA 12 h prior to harvesting and then assayed for luciferase activity. Data are the mean ± S.D. from three independent experiments with duplicates for each experiment.

A, time course of Egr-1 expression during PMA treatment. Western blot was performed using 50 µg of nuclear proteins from HepG2 cells treated with 150 nm PMA for various times. B, HepG2 cells were co-transfected with 1 µg of wild-type pB −246/−66Luc MAO B promoter construct and increasing amounts of an expression vector for Egr-1 under the control of a cytomegalovirus promoter. C, 1 µg of wild-type pB −246/−66 MAO B promoter construct or different mutants of pB −246/−66Luc were transfected with 1 µg of vector (pCMV) or Egr-1 expression plasmid. D, HepG2 cells were transfected with 1 µg of wild-type pB −246/−99 construct or different mutants of pB−246/−99Luc and treated with 150 nM PMA 16 h prior to harvesting or left untreated. E, effect of coexpression of Sp1, Sp3, and Egr-1 on promoter activity was shown. Five hundred nanograms of the expression plasmids were co-transfected with 1 µg of the proximal promoter construct pB−246/−99Luc into HepG2 cells. The amount of transfected plasmids was kept constant (2 µg) using a vector construct (pCMV). The endogenous MAO B activities in HepG2 cells that were cotransfected with the pB −246/−66 MAO B promoter construct and Sp1, Sp3, or Egr-1 or various combinations as indicated are shown at the bottom by Northern blot. Data are the mean ± S.D. from three independent experiments with duplicates for each experiment.

A, effect of calphostin C on the PMA-induced MAO B promoter activation. HepG2 cells were transiently transfected with the pB−246/−99 construct and preincubated with Calphostin C with increasing concentrations for 2 h. The cells were then treated with or without 150 nM PMA for 8 h before harvesting. B-E, HepG2 cells were transfected with 1 µg of pB−246/−99 construct in the presence of empty expression vector (pCMV3.1, control) or the dominant negative forms of kinases in the signaling pathway indicated in Fig. 7. The cells were harvested, and extracts were assayed for luciferase activity. For the PD90859 (a MEK1- and MEK2-specific inhibitor) treatment, PD90859 was added at 50 µm for 30 min prior to PMA addition. Data are the mean ± S.D. from three independent experiments with duplicates for each experiment. F, the specificities of dominant negative constructs were evaluated by Western blot. The protein levels of JNK1, ERK2, and p38 were detected in HepG2 cells that were transfected with their respective wild type (wt) and dominant negative forms (dn) by Western blot. Note that the expression of proteins were decreased after cotransfection with their respective dominant negative constructs.

A, Northern blot. Thirty micrograms of total RNA from HepG2 cells treated with 150 nM PMA for various times were hybridized to a human MAO A or MAO B cDNA probe. The same membrane was reprobed with the human β-actin probe as an internal loading control. The blot for MAO A or B was exposed for 48 h, whereas the blot for β-actin was exposed for 6 h. B, Western blot. Fifty micrograms of whole cell protein extracts from HepG2 cells treated with 150 nm PMA for various times were incubated with anti-MAO A or anti-MAO B antibodies. C, MAO A and MAO B activities. The catalytic activities of MAO A and MAO B were determined using 50 µg of whole cell protein extracts from HepG2 cells treated with 150 nm PMA for various times. Data are the mean ± S.D. from three independent experiments.

A, sequence of the −246/−255 MAO B promoter region. The potential overlapping cis-acting elements are indicated with boxes and identified by arrows. The sequence of wild-type and mutant oligonucleotides (m1, m2, and m3) are shown with mutations underlined. B, gel shift assay was performed using nuclear extracts from HepG2 cells (PMA-treated or not) and the radiolabeled promoter (−246/−225) fragment as a probe. Supershift assay was carried out using antibodies against Sp1, Sp3, and/or Egr-1. The DNA-protein complexes (I, II, and III) are indicated with arrows, and the identified protein components of the complexes are shown in parenthesis. The supershift complexes and free probes are also indicated. C, gel shift assay was performed using nuclear extracts from HepG2 cells (PMA-treated or not) and the radiolabeled wild-type (wt) or mutant promoter fragments (ml, m2, and m3). The protein components of the shifted bands are indicated with arrows.

A, expression of c-Jun and c-Fos during PMA treatment. Western blot was performed using HepG2 cells treated with 150 nM PMA for various times. B, effect of co-expression of Sp1, c-Jun, and c-Fos on promoter activity was shown. Five hundred nanograms of the expression plasmids were co-transfected with 1 µg of the proximal promoter construct pB−246/−99 into HepG2 cells. The amount of transfected plasmids was kept constant (2 µg) using vector construct (pCMV). The endogenous MAO B gene expression in HepG2 cells that were cotransfected with pB—246/—66 MAO B promoter construct and Sp1 or c-Jun or c-Fos or various combinations as indicated are shown at the bottom by Northern blot. C, 1 µg of wild-type pB−246/ −99Luc construct or different mutants of pB−246/−99Luc were transfected with 1 µg of vector (pCMV) alone or 500 ng of expression plasmid for c-Jun and 500 ng of expression plasmid. Data are the mean ± S.D. from three independent experiments with duplicates for each experiment.

The kinases indicated with an open box appear not to be involved. Solid lines connect individual kinases in the signal transduction pathway, whereas dotted lines appear not to be important for the PMA-induced MAO B activity.