Combined blockade of α1 and β-adrenoceptors is essential for inhibition of TNFα-induced MMP-9 expression and activity in NIH3T3 fibroblasts

Anne Rietz1, Martina Hennessy1, Anthony Davies2, Yuri Volkov2, J. Paul Spiers1. 1Department of Pharmacology & Therapeutics, School of Medicine, Trinity College Dublin, Dublin, Ireland, 2Department of Clinical Medicine, School of Medicine, Trinity College Dublin, Dublin, Ireland.

Fibroblasts, an important source of matrix metalloproteinases (MMP), are involved in inflammatory and fibrotic remodelling in disease. Interestingly, both TNFα and MMP-9 expression have been shown to be reduced in serum from patients with idiopathic dilated cardiomyopathy following treatment with carvedilol (Ohtsuka et al., 2003). Indeed carvedilol reduces TNFα-induced MMP-9 activity in human aortic smooth muscle cells. However, the mechanism underpinning this remains unclear. The aim of the present study was to investigate the effects of α1 and β-adrenoceptor antagonism on TNFα mediated activation of MMP-9 and the role of NFκB in fibroblasts.

NIH3T3 fibroblasts were cultured in DMEM with 10% calf serum. Cells were transfected with MMP-9 or NFκB luciferase reporter constructs and an internal standard (pRL-CMV). Cells were pre-incubated in medium supplemented with vehicle (DMSO), prazosin (10μM), propranolol (10μM), prazosin+propranolol (P+P, 10μM), carvedilol (10μM), Vitamin C (VitC, 100μM), N-acetyl-L-cysteine (NAC, 100μM) or medium (control), prior to addition of TNFα (40ng) for 24 hrs. Transcriptional activation was measured by a dual-luciferase reporter assay, and data normalised to internal standard and expressed relative to control. MMP-9 expression and MMP-9 activity were measure by ELISA and gelatin zymography. Data were analysed by one-way ANOVA with post hoc analysis (Bonferroni), and expressed as mean±SEM. P<0.05 indicates statistical significance.

TNFα increased MMP-9 activity (3 fold, P<0.05) and expression compared to control (26.8±4.7 vs 3.1±0.9 ng/ml, P<0.05). This effect was inhibited by carvedilol and P+P (&ap;60%, P<0.05) but not by prazosin or propranolol. Carvedilol also attenuated the TNFα induced activation of the MMP-9 promoter by 60% (P<0.05). Interestingly, TNFα induced activation of the NFκB promoter (30-fold, P<0.05) was reduced by carvedilol, and both prazosin and propranolol (P<0.05). However, pre-treatment with antioxidants (VitC, NAC) did not alter TNFα-induced MMP-9 activity.

In conclusion, we demonstrate that combined blockade of α1 and β-adrenoceptors is a prerequisite for inhibition of TNFα-induced expression of MMP-9 in fibroblasts, and that this is independent of effects on NFκB. These data expand our understanding of how adrenergic antagonism is beneficial in diseases with an inflammatory component.

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