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Antioxidant PDTC induces stromelysin expression in mesangial cells via a tyrosine kinase-AP-1 pathway
1Department of Medicine, University College London Medical School, United Kingdom.
Abstract:
We found that pyrrolidine dithiocarbamate (PDTC) induces the matrix metalloproteinase stromelysin in cultured glomerular mesangial cells. Although PDTC is a well-known inhibitor of nuclear factor-kappa B (NF-kappa B), this effect was independent of the NF-kappa B activity, since overexpression of a dominant negative mutant of p50 NF-kappa B subunit repressed activity of the kappa B site, whereas it failed to induce stromelysin. To elucidate the intracellular mechanisms involved, we focused on the role of activator protein 1 (AP-1), since its binding site, the 12-O-tetradecanoylphorbol 13-acetate (TPA) response element (TRE), is located in the 5'-flanking region of the stromelysin gene. Northern blot analysis revealed that PDTC upregulated expression of c-jun and c-fos before the expression of stromelysin. Transient transfection studies using a TRE-LacZ reporter plasmid elucidated that activity of AP-1 was significantly increased by PDTC. Stable transfection with a c-jun antisense cDNA or pretreatment with curcumin, a pharmacological inhibitor of c-Jun/AP-1, revealed that inactivation of AP-1 diminished the induction of stromelysin by PDTC. To identify the machinery involved upstream of AP-1 activation, the role of tyrosine kinases was investigated. Western blot analysis showed that PDTC induced phosphorylation of tyrosine kinases. Treatment of mesangial cells with tyrosine kinase inhibitors suppressed activation of AP-1 as well as induction of stromelysin by PDTC. These findings demonstrate that the antioxidant PDTC induces stromelysin expression via stimulation of the tyrosine kinase-AP-1 pathway independent of its suppressive action on NF-kappa B.
Insights
Pyrrolidine dithiocarbamate (PDTC) induces stromelysin in kidney cells. This occurs through the tyrosine kinase-activator protein 1 (AP-1) pathway, independent of nuclear factor-kappa B (NF-kappa B) inhibition.
Area of Science:
- Molecular biology
- Cell biology
- Renal physiology
Background:
- Pyrrolidine dithiocarbamate (PDTC) is recognized for inhibiting nuclear factor-kappa B (NF-kappa B).
- Stromelysin, a matrix metalloproteinase, plays roles in cellular processes within the kidney.
- Understanding the signaling pathways regulating gene expression in mesangial cells is crucial for renal research.
Purpose of the Study:
- To investigate the molecular mechanisms by which PDTC induces stromelysin expression in cultured glomerular mesangial cells.
- To determine the role of nuclear factor-kappa B (NF-kappa B) and activator protein 1 (AP-1) in PDTC-mediated stromelysin induction.
- To elucidate the upstream signaling events, specifically the involvement of tyrosine kinases, in this process.
Main Methods:
- Northern blot analysis to assess gene expression changes.
- Transient and stable transfection studies using reporter plasmids and antisense cDNA.
- Western blot analysis to detect protein phosphorylation.
- Pharmacological inhibition of signaling pathways using curcumin and tyrosine kinase inhibitors.
Main Results:
- PDTC upregulated c-jun and c-fos expression, preceding stromelysin induction.
- PDTC significantly increased activator protein 1 (AP-1) activity.
- Inhibition of AP-1 or tyrosine kinases abolished PDTC-induced stromelysin expression.
- PDTC-induced stromelysin expression was independent of NF-kappa B activity.
Conclusions:
- PDTC induces stromelysin expression in mesangial cells via a pathway involving tyrosine kinase activation and subsequent AP-1 stimulation.
- This induction mechanism operates independently of PDTC's known inhibitory effects on NF-kappa B.
- The findings highlight a novel signaling cascade regulating matrix metalloproteinase expression in renal cells.