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

Nuclear factor κB (NF-κB) 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-κB and degradation of IκB-α, the cytosolic inhibitor of NF-κB 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-κB activity and Western blot analysis for IκB-α were performed. Both LPS and ET-1 led to activation of NF-κB 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-κB activation was attenuated and not different from control (1.7 ± 0.24 fold DU compared with negative control; P = NS). In addition, both LPS and ET-1 mediated NF-κB 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 IκB-α present in the THP-1 cytoplasmic extracts (2.1 ± 1.5% and 54 ± 15.7% of ADU vs negative control (P < 0.05). NF-κB 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.