JNK-mediated regulation of iNOS and CAT expression and function in j774 macrophages

Marzieh Zamani, Shori Thakur, Anwar Baydoun. Uuniversity of Hertfordshire, AL10 9AB Hatfield, United Kingdom.

The expression of the inducible L-arginine-nitric oxide pathway in cultured cell systems require the activation of a complex cascade of signalling events that include members of the mitogen activated kinase family. Of these, the c-Jun NH2-terminal kinases (JNK) appear less well defined and with controversial findings reported in the literature. We have therefore carried out studies aimed at establishing the critical role of this pathway, and its downstream target activator-protein-1 (AP-1), in the regulation of iNOS and L-arginine transport in J774 macrophages. In addition, we have also analysed changes in cationic amino acid transporter (CAT) gene expression following JNK inhibition using SP600125 (Anthra[1,9-cd]pyrazol 6(2H)-one 1,9-pyrazoloanthrone) and report for the first time the differential regulation of different CAT isoforms by this compound.

The experiments were carried out on murine J774 macrophages cultured in Dulbecco’s modified Eagles medium supplemented with penicillin/streptomycin (1%) and 10 % foetal bovine serum. Confluent monolayers of cells were activated with bacterial lipopolysaccharide (LPS; 1 μg ml-1) for 24 h in the absence and presence of SP600125 (0.1-10 μM). Controls were incubated in culture medium alone. Nitric oxide production was determined by the standard Griess assay and iNOS expression by western blotting. L-arginine transport was monitored using L-[3H]arginine and CAT expression analysed by qPCR as described by Baydoun et al. (1999). Activation of AP-1 subunits was determined using the TransAM kit following activation of cells with LPS (1 μg ml-1) for periods of 30 min and 2 hours.

Induction of J774 macrophages with LPS resulted in the expected increase in both NO production and L-arginine transport. Both processes were inhibited by SP600125 which concentration-dependently blocked iNOS expression, NO synthesis and at 10 μM suppressed induced L-arginine transport back to basal control levels. Transporter activity in non-activated cells was unaltered. PCR analysis of gene expression detected iNOS and transcripts for the high affinity CAT-1 and CAT-2B and for the low affinity CAT-2A carriers. Pre-treatment of cells with SP600125 concentration dependently inhibited iNOS mRNA expression (35%) and selectively suppressed that for CAT-2B (28%) whilst having little or no effect of either control or induced mRNA expression of CAT-1 and CAT-2A. Moreover, SP600125 also blocked activation of c-Jun which has often been implicated in the induction of iNOS in various cell systems. These results, taken together, indicate that induction of both NO production and L-arginine transport are critically regulated by the JNK\\AP-1 pathway. More importantly, regulation of L-arginine transport appears to be targeted at regulating expression of the inducible high affinity transporter, CAT-2B.