calpain-inhibitor-iii and epoxomicin

The peptidyl-proline isomerase Protein Never in Mitosis Gene A Interacting-1 (PIN1) increases the level or activity of several transcription factors that can induce the inducible nitric oxide (NO) synthase (iNOS). PIN1 can also regulate mRNA and protein turnover. Here, the effect of depletion of PIN1 on induction of iNOS by Escherichia coli endotoxin (LPS) and interferon-gamma (IFNgamma) in murine aortic endothelial cells (MAEC) was determined. Suppression of PIN1 by 85% with small hairpin RNA enhanced the induction of NO and iNOS protein by LPS-IFNgamma. There was no effect on induction of iNOS mRNA, suggesting a posttranscriptional effect. The enhanced levels of iNOS protein were functionally significant since LPS-IFNgamma was cytotoxic to MAEC lacking PIN1 but not MAEC harboring an inactive control construct, and because cytotoxicity was blocked by the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester. Consistent with posttranscriptional action, knockdown of PIN1 increased the stability of iNOS protein in cycloheximide-treated cells. Furthermore, loss of iNOS was blocked by the calpain inhibitor carbobenzoxy-valinyl-phenylalaninal but not by the selective proteasome inhibitor epoxomicin. Immunoprecipitation indicated that PIN1 can interact with iNOS. Pull down of iNOS with a wild-type glutathione-S-transferase-PIN1 fusion protein, but not with a mutant of the amino terminal phospho-(serine/threonine)-proline binding WW domain of PIN1, indicated that this domain mediates interaction. The results suggest that PIN1 associates with iNOS and can limit its induction by facilitating calpain-mediated degradation in MAEC.

Topics: Animals; Calpain; Cell Survival; Cells, Cultured; Dipeptides; Endothelial Cells; Enzyme Inhibitors; Escherichia coli Proteins; Interferon-gamma; Lipopolysaccharides; Mice; NG-Nitroarginine Methyl Ester; NIMA-Interacting Peptidylprolyl Isomerase; Nitric Oxide; Nitric Oxide Synthase Type II; Oligopeptides; Peptidylprolyl Isomerase; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; Protein Processing, Post-Translational; Recombinant Proteins; RNA Interference; RNA, Messenger; RNA, Small Interfering; Time Factors

To firmly establish the pathway involved in ligand-induced degradation of the AHR, cell lines derived from mouse rat or human tissues were exposed to inhibitors specific to the proteasome or calpain proteases and exposed to TCDD. The level of endogenous AHR and CYP1A1 protein was then evaluated by quantitative Western blotting. Treatment of cells with the calpain inhibitors: calpeptin, calpain inhibitor III, or PD150606 either individually or in combinations up to 75 microM did not reduce TCDD-induced degradation of the AHR, the induction of endogenous CYP1A1 or the nuclear accumulation of the AHR. The activity of the inhibitors was verified with an in vivo calpain assay. In contrast, exposure of cells to the specific proteasome inhibitors: epoxomicin (1-5 microM), proteasome inhibitor I (5-10 microM) or lactacystin (5-15 microM) completely inhibited TCDD-induced degradation of the AHR. Inhibition of AHR degradation with these compounds did not reduce the induction of endogenous CYP1A1. In addition, exposure of the Hepa-1 line to the various proteasome inhibitors caused an accumulation of the AHR in the nucleus in the absence of TCDD exposure. Finally, Western blot analysis of the DNA bound AHR showed that its molecular mass was unchanged in comparison to the unliganded (cytoplasmic) AHR. Thus, these studies conclusively implicate the proteasome and not calpain proteases in the ligand-induced degradation of the mouse, rat and human AHR and suggest that the pharmacological use of proteasome inhibitors may impact the time course and magnitude of gene regulatory events mediated through the AHR.

Topics: Acetylcysteine; Acrylates; Animals; Cell Line, Tumor; Cell Nucleus; Cysteine Proteinase Inhibitors; Cytochrome P-450 CYP1A1; Dipeptides; Dose-Response Relationship, Drug; Down-Regulation; Drug Antagonism; Drug Combinations; Environmental Pollutants; Humans; Mice; Oligopeptides; Polychlorinated Dibenzodioxins; Rats; Receptors, Aryl Hydrocarbon