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Nitric oxide modulates expression of matrix metalloproteinase-9 in rat mesangial cells
W Eberhardt1, T Beeg, K F Beck
1Zentrum der Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
Unlabelled:
Nitric oxide modulates expression of matrix metalloproteinase-9 in rat mesangial cells.
Background:
High-output levels of nitric oxide (NO) are produced by rat mesangial cells (MCs) in response to proinflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) by the inducible isoform of NO synthase (iNOS). We tested modulatory effects of NO on the expression and activities of matrix metalloproteinases-9 and -2 (MMP-9 and MMP-2), respectively. Temporal and spatial expression of these MMPs and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), seems to be critical in the extensive extracellular matrix (ECM) remodeling that accompanies sclerotic processes of the mesangium. Methods and Results. Using the NO donors S-Nitroso-N-acetyl-D,L-penicillamine (SNAP) and DETA-NONOate, we found strong inhibitory effects of NO mainly on the IL-1beta-induced MMP-9 mRNA levels. NO on its own had only weak effects on the expression of MMP-9 and MMP-2. The addition of the NOS inhibitor NG-monomethyl L-arginine (L-NMMA) dose dependently increased steady-state mRNA levels of cytokine-induced MMP-9, suggesting that endogenously produced NO exerts tonic inhibition of MMP-9 expression. MMP-9 activity in conditioned media from MCs costimulated with IL-1beta and NO donor contained less gelatinolytic activity than media of cells treated with IL-1beta alone. Exogenously added NO did not alter gelatinolytic activity of MMP-9 in cell-free zymographs. The expression levels of TIMP-1 were affected by NO similarly to the expression of MMP-9.
Conclusion:
We conclude that NO modulates cytokine-mediated expression of MMP-9 and TIMP-1 in rat MCs in culture. Our results provide evidence that NO-mediated attenuation of MMP-9 gelatinolytic activity is primarily due to a reduced expression of MMP-9 mRNA, and not the result of direct inhibition of enzymatic activity.
Insights
Nitric oxide (NO) reduces the expression of matrix metalloproteinase-9 (MMP-9) in rat mesangial cells, primarily by decreasing MMP-9 mRNA levels. This suggests NO plays a role in regulating extracellular matrix remodeling.
Area of Science:
- Nephrology
- Molecular Biology
- Cell Biology
Background:
- Rat mesangial cells (MCs) produce high levels of nitric oxide (NO) in response to inflammatory cytokines via inducible NO synthase (iNOS).
- Matrix metalloproteinases (MMP-9 and MMP-2) and their inhibitors (TIMPs) are crucial for extracellular matrix (ECM) remodeling in mesangial sclerosis.
- The modulatory effects of NO on MMP expression and activity were investigated in this study.
Purpose of the Study:
- To investigate the modulatory effects of nitric oxide (NO) on the expression and activity of matrix metalloproteinase-9 (MMP-9) and MMP-2 in rat mesangial cells (MCs).
- To determine the mechanism by which NO affects MMP-9 activity, specifically whether it involves reduced mRNA expression or direct enzymatic inhibition.
Main Methods:
- Rat mesangial cells were treated with NO donors (SNAP, DETA-NONOate) and inflammatory cytokines (IL-1beta).
- The effects of NO on MMP-9 and MMP-2 mRNA levels were assessed using quantitative methods.
- MMP-9 activity was measured in conditioned media using zymography.
- The impact of a nitric oxide synthase (NOS) inhibitor (L-NMMA) on cytokine-induced MMP-9 expression was evaluated.
Main Results:
- Nitric oxide (NO) significantly inhibited interleukin-1beta (IL-1beta)-induced matrix metalloproteinase-9 (MMP-9) mRNA expression.
- NO alone had minimal effects on MMP-9 and MMP-2 expression, but endogenously produced NO appeared to tonically inhibit MMP-9 expression.
- MMP-9 activity in cell culture media was reduced when cells were co-stimulated with IL-1beta and an NO donor, correlating with decreased MMP-9 mRNA levels.
- NO did not directly inhibit the enzymatic activity of MMP-9 in cell-free assays.
- Expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) was modulated by NO in a manner similar to MMP-9.
Conclusions:
- Nitric oxide (NO) modulates the expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in rat mesangial cells (MCs) in response to cytokine stimulation.
- The reduction in MMP-9 gelatinolytic activity by NO is primarily attributed to decreased MMP-9 mRNA expression, not direct inhibition of the enzyme's activity.