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Human selenium-dependent glutathione peroxidase (GP) is implicated as a mechanism of resistance against oxygen free radicals. The 5' flanking sequence upstream from the coding region of GP contained an oxygen-responsive element termed ORE1 that is responsive to hypoxia, as well as several copies of the activator protein-1 (AP-1)- and AP-1-like-binding sites. In this study, we sought to define the molecular events that lead to GP gene transcription in response to hyperoxia in human umbilical-vein endothelial cells, and asked whether such induction is mimicked and sustained by activation of protein kinase C (PKC) by phorbol esters. Treatment of cells with 100 nM phorbol 12,13-dibutyrate (PdBu) induced a delayed (24-48 h) but significant (2-fold) increase in steady-state GP mRNA levels. Steady-state GP mRNA levels also rose after exposure to 95% O2, again after considerable delay (48-72 h). For both PdBu and oxygen, induction was transcriptionally regulated, as demonstrated by nuclear run-on experiments. The simulations by PdBu and oxygen were additive. In contrast with PdBu, hyperoxia did not stimulate translocation of PKC from the cytosol to the particulate fraction, although the specific activity of both cytosolic and particulate-associated PKC was increased 2-fold in cells exposed to 95% O2 for 5 days. In addition, gel mobility-shift assays using double-stranded tumour-promoting-agent-responsive element (TRE) and nuclear extracts derived from phorbol- and oxygen-treated cells revealed that PdBu, but not hyperoxia, increased AP-1 DNA-binding activity. On the other hand, the up-regulation of GP expression by oxygen could not be accounted for by the ORE1 core sequence, since no specific protein-DNA binding activity could be detected using nuclear extracts from hyperoxic cells and ORE1. Taken together, these results suggest that there may be different molecular mechanisms controlling GP expression. After exposure to PdBu, GP undergoes transcriptional activation via a process that can be readily explained by a classic AP-1 interaction with the TRE sites in the GP promoter. During hyperoxia, GP also undergoes transcriptional activity, but via a process that appears to involve neither TRE nor ORE1.

Two tyrosine phosphoproteins in phorbol ester-sensitive EL4 (S-EL4) mouse thymoma cells have been identified as the p120 c-Cbl protooncogene product and the p85 subunit of phosphatidylinositol 3-kinase. Tyrosine phosphorylation of p120 and p85 increased rapidly after phorbol ester stimulation. Phorbol ester-resistant EL4 (R-EL4) cells expressed comparable amounts of c-Cbl and phosphatidylinositol 3-kinase protein but greatly diminished tyrosine phosphorylation. Co-immunoprecipitation experiments revealed complexes of c-Cbl with p85, and of p85 with the tyrosine kinase Lck in phorbol ester-stimulated S-EL4 but not in unstimulated S-EL4 or in R-EL4 cells. In vitro binding of c-Cbl with Lck SH2 or SH3 domains was detected in both S-EL4 and R-EL4 cells, suggesting that c-Cbl, p85, and Lck may form a ternary complex. In vitro kinase assays revealed phosphorylation of p85 by Lck only in phorbol ester-stimulated S-EL4 cells. Collectively, these results suggest that Cbl-p85 and Lck-p85 complexes may form in unstimulated S-EL4 and R-EL4 cells but were not detected due to absence of tyrosine phosphorylation of p85. Greatly decreased tyrosine phosphorylation of c-Cbl and p85 in the complexes may contribute to the failure of R-EL4 cells to respond to phorbol ester.

The T84 colonic cell line, a cultured Cl- secretory cell, elevates intracellular free Ca2+ [( Ca2+]i) in a concentration-dependent manner when exposed to carbachol or histamine. As determined with a fluorescence microscope imaging system, exposure of T84 cells to 100 microM carbachol or histamine resulted in an immediate [Ca2+]i rise of approximately 50-80 nM in all cells. Preincubation of monolayers for 1 h or longer with 0.4 microM phorbol 12,13-dibutyrate (PDB) reduced the number of cells which responded to histamine or carbachol and reduced the magnitude of the increase in the responding cells. This effect reached its maximum after 2 h and persisted for at least 24 h of PDB incubation. Binding of quinuclidinyl benzilate, a cholinergic receptor antagonist, indicated that down-regulation of external receptors was not an explanation for this effect. Examination of phospholipase C activity in T84 cell membranes showed increased basal activity in PDB-treated compared with control cells. Measurement of inositol phosphates generated by intact cells using myo-[3H]inositol incorporation or receptor binding assays showed that 2 h of incubation with PDB elevated basal levels of inositol 1,4,5-trisphosphate and prevented any further carbachol-induced generation of inositol trisphosphate. Probably as a consequence, both total cell calcium and Ca2+ ionophore-releasable calcium were decreased after 2 h of PDB incubation. Membrane-associated protein kinase C activity was elevated after a 2 h exposure to PDB but was below the level of detection after 24 h with PDB. Protein kinase C antagonists neither duplicated nor blocked the uncoupling of carbachol receptors induced by long term treatment with PDB. The results suggest that prolonged PDB incubation caused uncoupling and elevation of phospholipase C activity from cholinergic and histaminergic receptor regulation resulting in increased basal levels of inositol 1,4,5-trisphosphate. Protein kinase C apparently is not involved directly in the mechanism that leads to these effects.