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Inhibition of peroxisome-proliferator-activated receptor (PPAR)alpha by MK886
1Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas, Austin, TX 78712, USA. kehrerjim@mail.utexas.edu
Abstract:
Although MK886 was originally identified as an inhibitor of 5-lipoxygenase activating protein (FLAP), recent data demonstrate that this activity does not underlie its ability to induce apoptosis [Datta, Biswal and Kehrer (1999) Biochem. J. 340, 371--375]. Since FLAP is a fatty-acid binding protein, it is conceivable that MK886 may affect other such proteins. A family of nuclear receptors that are activated by fatty acids and their metabolites, the peroxisome-proliferator-activated receptors (PPARs), have been implicated in apoptosis and may represent a target for MK886. The ability of MK886 to inhibit PPAR-alpha, -beta and -gamma activity was assessed using reporter assay systems (peroxisome-proliferator response element--luciferase). Using a transient transfection system in monkey kidney fibroblast CV-1 cells, mouse keratinocyte 308 cells and human lung adenocarcinoma A549 cells, 10--20 microM MK886 inhibited Wy14,643 activation of PPAR alpha by approximately 80%. Similar inhibition of PPAR alpha by MK886 was observed with a stable transfection reporter system in CV-1 cells. Only minimal inhibitory effects were seen on PPAR beta and PPAR gamma. MK886 inhibited PPAR alpha by a non-competitive mechanism as shown by its effects on the binding of arachidonic acid to PPAR alpha protein, and a dose-response study using a transient transfection reporter assay in COS-1 cells. An assay assessing PPAR ligand-receptor interactions showed that MK886 prevents the conformational change necessary for active-complex formation. The expression of keratin-1, a protein encoded by a PPAR alpha-responsive gene, was reduced by MK886 in a culture of mouse primary keratinocytes, suggesting that PPAR inhibition has functional consequences in normal cells. Although Jurkat cells express all PPAR isoforms, various PPAR alpha and PPAR gamma agonists were unable to prevent MK886-induced apoptosis. This is consistent with MK886 functioning as a non-competitive inhibitor of PPAR alpha, but may also indicate that PPAR alpha is not directly involved in MK886-induced apoptosis. Although numerous PPAR activators have been identified, the results show that MK886 can inhibit PPAR alpha, making it the first compound identified to have such an effect.
Insights
MK886, initially known as a 5-lipoxygenase activating protein (FLAP) inhibitor, is now identified as the first compound to non-competitively inhibit peroxisome-proliferator-activated receptor alpha (PPARα). This inhibition impacts gene expression and cellular function, though its direct role in MK886-induced apoptosis remains unclear.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- MK886 was initially identified as an inhibitor of 5-lipoxygenase activating protein (FLAP).
- Recent findings suggest MK886's apoptosis-inducing activity is independent of FLAP inhibition.
- Fatty-acid binding proteins, including nuclear receptors like peroxisome-proliferator-activated receptors (PPARs), are potential targets for MK886.
Purpose of the Study:
- To investigate MK886's potential to inhibit PPAR-alpha, -beta, and -gamma activity.
- To elucidate the mechanism of MK886's interaction with PPARs.
- To assess the functional consequences of PPAR inhibition by MK886 in cellular models.
Main Methods:
- Reporter assay systems (peroxisome-proliferator response element--luciferase) were used to assess PPAR activity.
- Transient and stable transfection systems in various cell lines (CV-1, 308, A549, COS-1) were employed.
- Assays for ligand-receptor interactions and analysis of keratin-1 expression were conducted.
Main Results:
- MK886 significantly inhibited Wy14,643 activation of PPAR alpha by approximately 80% in multiple cell lines.
- MK886 demonstrated minimal inhibitory effects on PPAR beta and PPAR gamma.
- The inhibition of PPAR alpha by MK886 was non-competitive and prevented necessary conformational changes for active-complex formation.
- MK886 reduced keratin-1 expression in primary mouse keratinocytes, indicating functional consequences of PPAR inhibition.
- PPAR agonists did not prevent MK886-induced apoptosis in Jurkat cells, suggesting PPAR alpha may not be directly involved.
Conclusions:
- MK886 is identified as the first compound to inhibit PPAR alpha activity.
- The mechanism of inhibition is non-competitive, affecting ligand-receptor interactions.
- While MK886 inhibits PPAR alpha, its direct role in MK886-induced apoptosis requires further investigation.