O-047. Signal transduction by growth hormone through Stat 5 is modulated by arachidonic acid

GH stimulation of various cell lines induces binding of nuclear proteins to DNA elements present in the promoter region of Serine Protease Inhibitor (SPI) gene 2.1. GH-dependent activation of the latent transcription factor Stat 5 and of PLA₂ has been described. Arachidonic acid (M) has been implicated in GH signalling pathways affecting gene transcription. Here we have investigated the possibility of an interplay between Stat 5 and M signalling pathways. Nuclear extracts obtained from BRL-4 cells cultured in the presence or absence of GH in combination with various inhibitors of M metabolism or inhibitors of phospholipase activity were used in electromobility shift assays with the SPI 2.1 Stat 5 response element SPIGLE1. Inhibitor's of M lipoxygenase (LOX) metabolism reduced GH-activated Stat 5 binding to SPIGLE1 while inhibition of cyclooxygenase (COX) metabolism had no effect. Quantification of the relative amounts of GH induced Stat 5 binding activity in cellular extracts treated with SKF 525A, a monooxygenase (EPOX) inhibitor, showed a 2-fold amplification of the activation compared with GH treatment alone. ETYA, an inhibitor of COX-, LOX- and EPOX- catalysed metabolism of M, inhibited GH-induced Stat 5 binding by 50%. Addition of M to serum-starved cells induced Stat 5 binding to SPIGLE1 but less efficiently than GH. Furthermore, mepacrine, a non-selective inhibitor of PLA₂ reduced GH activated Stat 5 binding whereas inhibitors of PLC had no effect. These results suggest that a metabolite(s) of M can modulate GH induced Stat 5 activation.