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Leptin is an adipocyte-derived hormone involved in the regulation of feeding behavior and energy homeostasis in vertebrates. We recently reported that leptin activates the JAK-STAT signaling pathway through the chicken leptin receptor (chLEPR). However, the molecular inhibitory mechanism by suppressor of cytokine signaling 3 (SOCS3), observed in mammalian leptin signaling, has not been elucidated in avian species. Therefore, the role of chicken SOCS3 (chSOCS3) in signal transduction through the chLEPR was analyzed in this study. Leptin increases SOCS3 mRNA expression in chicken hepatoma cells, LMH, and also activates the chSOCS3 gene promoter in the chLEPRexpressing cells. Overexpression of chSOCS3 inhibited leptin-induced signaling by blocking phosphorylation of JAK2 and subsequent activation of STAT3 similar to that observed in mammals. Signaling inhibited by chSOCS3 was not restored in the chLEPR mutated docking site of SOCS3. In addition, mutation of Phe25 in the kinase inhibitory region of chSOCS3 abolished SOCS3 activity via the wild chLEPR. The present study indicates that SOCS3 is a negative feedback regulator of leptin signaling in chickens as well as in mammals. However, the inhibitory mechanism in chickens may differ slightly from that observed in mammals.

The chicken leptin receptor (chLEPR) activates Janus kinase (JAK) -signal transducers and activator of transcription (STAT) signaling pathway after leptin stimulation. We have previously developed a bioassay using leptin inducible reporter gene in cultured cells. However, we failed to detect leptin-like activity in chicken blood. In the present study, we expressed green fluorescent protein (GFP) fused chSTAT3 (GFP-chSTAT3) in cells expressing chLEPR and analyzed leptin dependent activation of the chSTAT3. Leptin phosphorylated GFP-chSTAT3 and by which fluorescent signal translocated into nuclei in COS-7 cells transiently expressing GFP-chSTAT3 with chLEPR. Furthermore, we established CHO-K1 cells stably expressing chLEPR and chSTAT3 (CHO-chLEPR/chSTAT3), and in which detected time-and dose-dependent activation of chSTAT3 by leptin. Therefore, the CHO-chLEPR/STAT3 cells would be an excellent tool to detect and monitor leptin-like activity in avian tissues.