DESENSITIZATION OF THE GROWTH HORMONE-INDUCED JANUS KINASE 2 (JAK-2) SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 5 (STAT5)-SIGNALING PATHWAY REQUIRES PROTEIN-SYNTHESIS AND PHOSPHOLIPASE-C

Signal transducers and activators of transcription (Stat) proteins are lai;ent cytoplasmic transcription factors that are tyrosine phosphory lated by Janus kinases (Jak) in response to GH and other cytokines. GH activates Stat6 by a mechanism that involves tyrosine phosphorylation and nuclear translocation. However, the mechanisms that turn off the GH-activated Jak2/Stat5 pathway are unknown. Continuous exposure to GH of BRL-4 cells, a rat hepatoma cell line st;ably transfected with rat GH receptor, induces a rapid but transient activation of Jak2 and Sta t5. GH-induced Stat5 DNA-binding activity was detected after 2 min and reached a maximum at 10 min. Continued exposure to GH resulted in a d esensitization characterized by 1) a rapid decrease in Stat5 DNA-bindi ng activity. The rate of decrease of activity was rapid up to 1 h of G H treatment, and the remaining activity declined slowly thereafter. Th e activity of Stat5 present after 5 h is still higher than the control levels and almost 10-20% with respect to maximal activity at 10 min; and 2) the inability of further GH treatment to reinduce activation of Stat5. In contrast, with transient exposures of BRL-4 cells to GH, St at5 DNA-binding activity could repeatedly be induced. GH-induced Jak2 and Stat5 activities were independent of ongoing protein synthesis. Ho wever, Jak2 tyrosine phosphorylation and Stat5 DNA-binding activity we re prolonged for at least 4 h in the presence of cycloheximide, which suggests that the maintenance of desensitization requires ongoing prot ein synthesis. Furthermore, inhibition of protein synthesis potentiate d GH-induced transcriptional activity in BRL-4 cells transiently trans fected with SPIGLE1CAT, a reporter plasmid activated by Stat5. GH-indu ced Jak2 and Stat5 activation were not affected by D609 or mepacrine, both inhibitors of phospholipase C. However, in the presence of D609 a nd mepacrine, GH maintained prolonged Jak2 and Stat5 activation. Trans activation of SPIGLE1 by GH was potentiated by mepacrine and D609 but not by the phospholipase A(2) inhibitor AACOCF(3). Thus, a regulatory circuit of GH-induced transcription through the Jak2/Stat5-signaling p athway includes a prompt GH-induced activation of Jak2/Stat5 followed by a negative regulatory response; ongoing protein synthesis and intra cellular signaling pathways, where phospholipase C activity is involve d, play a critical role to desensitize the GH-activated Jak2/Stat5-sig naling pathway.