Abstract
Inflammatory cytokines, interleukin 1β and tumor necrosis factor-α, potently stimulate rat mesangial cells to express and secrete group IIA phospholipase A2 (PLA2). Cytokine-induced up-regulation of PLA2 has been blocked by inhibitors (antioxidants) of the transcription factor, nuclear factor-κB (NF-κB), suggesting a role for NF-κB in the regulation of group IIA PLA2 expression. Reactive oxygen species such as H2O2, which are elevated in mesangial cells after cytokine activation, can mimic cytokine-induced NF-κB activation. However, the source of reactive oxygen species generation in mesangial cells, produced by cytokine stimulation, has yet to be clarified. Recently, tumor necrosis factor-α has been demonstrated to increase superoxide radical generation in mesangial cells. Therefore, we hypothesized that a selective NADPH oxidase inhibitor, diphenyleneiodium chloride (DPI), could block cytokine-induced group IIA PLA2 up-regulation by attenuating NF-κB binding. To test this hypothesis, we isolated rat mesangial cells and characterized them by ultrastructural and immunochemical methods. This homogeneous mesangial cell population was responsive to cytokine as evidenced by an increase in steady-state levels of group IIA PLA2 mRNA and extracellular enzymatic activity over time. DPI (0.02–20 μM), added 90 min before cytokine activation, inhibited both group IIA PLA2 mRNA and enzymatic activity in a concentration-dependent manner. By electrophoretic mobility shift analysis, cytokine activation also increased specific NF-κB binding to one of two NF-κB consensus elements in the rat group IIA PLA2 promoter and also was suppressed by DPI pretreatment. Antibodies to NF-κB p65 (Rel A) and p50 (but not normal rabbit IgG) supershifted this retardation signal and verified the type of NF-κB species as the classical p50/p65 heterodimer.
Footnotes
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Send reprint requests to: Glenn Dorsam, Ph.D., Division of Allergy and Immunology, University of California Medical School, 533 Parnassus Ave., Room UB8B, Box 0711, San Francisco, CA 94143-0711. E-mail: gdorsam{at}itsa.ucsf.edu
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↵1 Funded in part by Azimuth Pharmaceuticals and the Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond, VA 23298. The laboratory of Richard Franson was the principle location were this work was performed.
- Abbreviations:
- PLA2
- phospholipase A2
- IL-1β
- interleukin 1β
- TNF-α
- tumor necrosis factor-α
- NF-κB
- nuclear factor- κB
- ROS
- reactive oxygen species
- AA
- arachidonic acid
- DPI
- diphenyleneiodium chloride
- FBS
- fetal bovine serum
- GM
- growth media
- DMSO
- dimethyl sulfoxide
- RT
- room temperature
- LSM
- low-serum media
- ACM
- activation media
- LDH
- lactate dehydrogenase
- Received June 10, 1999.
- Accepted August 27, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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