Molecular mechanisms of reactive oxygen species production: Role of activator protein-1 mediated transcription ofgp91phox, a subunit of the superoxide anion producing phagocyte NADPH oxidase

Abstract

Exposures to a variety of agents, including the tumor-promoting agent 12-O-Tetradecanoylphorbol-13-acetate (TPA), elicit inflammatory responses, which culminate with the release of reactive oxygen species (ROS) by phagocytes. Current hypotheses regarding tumor promotion include that ROS produced by phagocytic and epithelial cells may cause alterations in proliferation and/or differentiation of initiated cells within promoted tissues. Therefore, ROS production in response to TPA was investigated in HL60 phagocytes and MCF-7 mammary epithelial cells. The exposure of HL60 cells to TPA (0.1 μM) induced superoxide production and accumulation of gp91phox mRNA, which encodes for the superoxide-producing subunit of NADPH oxidase. Inhibiting transcription indicated TPA did not increase gp91phox mRNA stability. Transient transfection assays revealed that TPA-enhanced luciferase activity driven from a gp91phox promoter-reporter (p91phox). Inhibition of Activator Protein-1 (AP-1) activity by a transactivation mutant (TAM67) variant of c-Jun abrogated the TPA-responsiveness of p91phox. DNA binding assays with oligonucleotides derived from the gp91phox promoter led to the identification of a non-consensus AP-1 binding sequence (5'-TGAGTAA-3 ' = phox-TRE) located at -1090 of gp91phox. Members of the Jun and Fos families contributed to the formation of an AP-1 complex at the phox-TRE. Furthermore, point mutations in the phox-TRE abrogated AP-1 binding and the responsiveness of the 1.5-kb gp91phox promoter to TPA. In MCF-7 cells, TPA (0.1 μM) induced oxidation of the intracellular probe, 2', 7'-dichlorofluorescin-diacetate (DCFH-DA), an indication of an increased oxidative state. Since gp91phox mRNA was not detected in MCF-7 cells in the presence or absence of TPA, ROS emitted by gp91phox were ruled out as the source of oxidation. Due to its known peroxidase activity, the TPA-inducible cyclooxygenase-2 (COX-2) enzyme was investigated as the source of the increased DCFH-DA oxidation induced by TPA. Data supported that DCFH and DCFH-DA detected COX-2 activity in enzymatic and cellular assays, respectively. In vitro assays using recombinant human COX-2 yielded increased DCFH oxidation in the presence and absence of arachidonic acid. In cellular assays, the time-dependent TPA-induced DCFH-DA oxidation paralleled COX-2 expression and activity induced by TPA. Furthermore, the COX-2 specific inhibitor NS398 (50 and 100 muM) inhibited the TPA-induced oxidation of DCFH-DA in MCF-7 cells. COX-1 and -2 inhibitors, indomethacin and ibuprofen provided similar effects, but sulindac was less effective. In conclusion, gp91phox was regulated transcriptionally in response to TPA and the phox-TRE, which interacted with AP-1, was required for TPA-induced gp91phox promoter activity. These data support that increased ROS levels in TPA-promoted tissues may be due to ROS production by phagocytes. Additionally, the production of ROS by phagocytes may be mediated by AP-1 dependent up-regulation of gp91phox. The increased oxidative state detected in MCF-7 epithelial cells following TPA exposure was attributed to increased COX-2 activity. Therefore, DCFH-DA may be useful to screen for drugs that may have potential to induce or inhibit COX-2.