Rosiglitazone inhibits MMP-9 protein expression through NF-κB and AP-1 dependent mechanisms in human THP-1 cells
Altered expression of matrix metalloproteinases is associated with the development of atherosclerosis. Macrophages, a key component of the atherosclerotic plaque not only secrete MMP-9 but also express high levels of the peroxisome proliferators-activated receptor-γ (PPARγ) nuclear receptor. Interestingly, glitazones (PPARγ agonists) inhibit MMP-9 expression in macrophages and vascular smooth muscle cells. However, the underlying mechanism is less clear, with NFκB, AP-1 and Stat pathways being implicated using luciferase reporter assay (Ricote et al. 1998). The aims of the present study were to investigate the direct effects of rosiglitazone (RGZ) and troglitazone (TGZ) on NFκB, AP-1 and GSK-3B signalling with regard to the inhibitory effect of the glitazones on PMA-induced MMP-9 expression in THP-1 macrophages.
THP-1 cells were cultured in RPMI 1640 medium with 10% FBS and antibiotics. Cells were incubated in serum-free medium supplemented with medium (untreated control), or phorbol 12-myrisate 13-acetate (PMA; 50ng/ml) for 48 hrs. RGZ or troglitazone (TGZ) were added to the medium prior to stimulation with PMA; cells were pre-exposed to GW9662 (PPARγ antagonist) for 1hr where appropriate. MMP-9 expression was determined by ELISA and real time PCR. AP-1 transcription factors were analysed ELISA; NFκB, IKBα and GSK-3B were analysed by Western blot. Data were analysis by oneway-ANOVA with post hoc analysis (Bonferroni) and expressed as mean ± SE. A value of P<0.05 was taken to indicate statistical significance.
TGZ and RGZ inhibit PMA-induced MMP-9 mRNA and protein expression by 50 and 60% respectively. Furthermore, GW9662 reversed the inhibitory effect of RGZ (1 μM) on MMP-9 protein expression (0.37 ± 0.02 vs 0.58 ± 0.036), but not mRNA expression; no effect was observed at higher concentrations of RGZ (10 μM) or with TGZ. RGZ inhibited (P<0.05) PMA-induced NFκB phosphorylation (2.0 ± 0.15 vs 1.4 ± 0.09), but did not affect IκBα degradation after 48 hr exposure; no effect were observed at earlier time points. PMA alone or in combination with RGZ did not alter GSK-3B phosphorylation. RGZ inhibited PMA-induced expression of c-Jun, JunD, cFos, Fra1 and Fra2 members of the AP-1 transcripition family by between 40-75%.
In conclusion, we demonstrate that RGZ and TGZ regulate MMP-9 protein and mRNA expression in THP-1 cells. In the case of RGZ this effect could be partially reversed by inhibition of PPARγ. Furthermore, RGZ mediated inhibition of MMP-9 involved downregulation of NFκB and multiple members of the AP-1 transcription family.
Ricote, M. et al (1998) Natr. 391: 79-82.