Click to download
Mechanisms differentiating the selective regulation of iNOS expression in cultured vascular smooth muscle cells by AP-1 dominant negative constructs and the JNK inhibitor SP600125 Expression of the inducible nitric oxide synthase (iNOS) enzyme in vascular cells require the activation of a complex cascade of signalling events which are yet to be fully elucidated. One of the potential pathways may be that involving the c-Jun NH2-terminal kinases (JNKs). The critical role of the latter is however controversial (Finder et al., 2001; Ho et al., 2007). In this study we have determined whether the induction of iNOS require JNK activation by employing SP600125 (a pharmacological inhibitor of JNK), or transfecting cells with either TAM-67 or a-Fos which are both dominant negative constructs of the JNK downstream target AP-1. Furthermore, we have investigated the expression profile of AP-1 subunits, and determined how their activation is regulated by SP600125, TAM-67 or a-Fos. Rat aortic smooth muscle cells (RASMCs) in culture were treated with SP600125 (0.1-10 μM) prior to stimulation with bacterial lipopolysaccharide (LPS; 100 μg/ml) and interferon-γ (IFN-γ; 100 units/ml) for 24 h. Transfection of TAM-67 or a-Fos was carried out as described previously (Cui et al., 2005) . Production of NO was determined by the Griess assay. iNOS or JNK isoforms were detected by western blotting. Changes in the activated status of AP-1 subunits were determined using the AP-1 family TransAM kit as described in the manufacturer’s protocol. Statistical analysis was carried out using one way ANOVA followed by the Dunnett test or a two way ANOVA with multiple comparisons for changes in iNOS and NO production and for analysis of AP-1 subunit expression respectively. SP600125 had no significant effect on iNOS expression or NO production while TAM-67 inhibited both processes. In contrast, a-Fos caused a further increase in induced nitrite levels and iNOS expression (by 20%) . Western blotting confirmed the expression of both JNK1 and JNK2. Moreover, analysis of AP-1 subunits revealed that LPS and IFN-γ induced activation of c-Jun, JunD and Fra-1. More importantly, treatment with SP600125 blocked the activation of c-Jun as did TAM-67. However, in SP600125 treated cells, the inhibition of c-Jun was accompanied by a parallel upregulation in JunB and JunD activity which were not evident with TAM-67. a-Fos selectively suppressed c-Fos activation. Taken together, our data confirm a critical role for the JNK signalling pathway in the induction of iNOS in RASMCs but this is not evident with SP600125, presumably because of a potential compensatory effect of induced JunB and/or JunD which may regulate iNOS induction and thus NO production (Cho et al., 2001; Kristof et al., 2001). The effects with a-Fos suggest a negative coupling of c-Fos activation to the induction of iNOS. These findings highlight the complex regulation of iNOS expression by the JNK/AP-1 signalling cascade and demonstrate the need for caution in interpreting data obtained solely with pharmacological inhibitors.
Cho et al. (2002). Nitric Oxide, 6:319-332.
|
|
