The effect of endothelin-1 on nuclear factor kappa B in macrophages

Nuclear factor kappaB (NF-kappaB) is a transcriptional factor which may be pivotal in the pathogenesis of atherosclerosis. Endothelin-1 (ET-1) is a peptide with proatherogenic properties. We hypothesized that ET-1 may act through activation of NF-kappaB and degradation of I kappaB-alpha, the cytosolic inhibitor of NF-kappaB activation, to create an atherogenic environment. The human monocytic cell line THP-1 was stimulated with ET-1 +/- the ET antagonist, BQ788 and the proteosome inhibitor, PSI. LPS was used as a positive control. Gel shift assays for NF-kappaB activity and Western blot analysis for I kappaB-alpha, were performed. Both LPS and ET-1 led to activation of NF-kappaB in nuclear extracts [3.4 +/-0.45 (LPS) and 2.9 +/-0.26 (ET-1) fold increase in Arbitrary Densitometric Units (ADU) compared with negative control (P<0.005 in both cases)]. In the presence of the ETB antagonist, BQ788, NF-<kappa>B activation was attenuated and not different from control (1.7 +/-0.24 fold ADU compared with negative control; P=NS). In addition, both LPS and ET-1 mediated NF-kappaB activation were attenuated by preincubation with the proteosome inhibitor, PSI (1.3 +/-0.58 and 1.1 +/-0.3 fold increase in ADU compared with negative control respectively). Both LPS and ET-1 led to a decrease in the amount of I kappaB-alpha present in the THP-1 cytoplasmic extracts (2.1 +/-1.5% and 54 +/- 15.7% of ADU vs negative control (P<0.05). NF-<kappa>B is activated by ET-1 in human THP-1 monocytes. This data supports a role for the ETs in the development of inflammation in the vessel wall in atherosclerosis. (C) 2001 Academic Press.