ABSTRACT
Leptin is an adipocyte-derived hormone that not only regulates food intake and energy homeostasis but also induces growth hormone (GH) mRNA expression and release, thereby controlling growth and metabolism in mammals. The molecular mechanism of leptin-induced regulation of GH gene transcription is unclear. The current study investigated the effects of leptin on the chicken GH (cGH) promoter and the molecular mechanism underlying leptin-induced cGH gene expression in vitro. Leptin activated the cGH promoter in the presence of chPit-1α in CHO cells stably expressing the chicken leptin receptor. Promoter activation did not require STAT-binding elements in the cGH promoter or STAT3 activity. However, JAK2 activation was required for leptin-dependent activity. JAK2-dependent pathways include p42/44 MAPK and PI3K, and inhibition of these pathways partially blocked leptin-induced cGH gene transcription. Although CK2 directly activates JAK2, a CK2 inhibitor blocked leptin-dependent activation of the cGH gene without affecting JAK2 phosphorylation. The CK2 inhibitor suppressed Erk1/2 and Akt phosphorylation. Additional data implicate Src family kinases in leptin-dependent cGH gene activation. These results suggest that leptin activates the cGH gene in the presence of chPit-1α via several leptin-activated kinases. Although further study is required, we suggest that the leptin-induced JAK2/p42/44 MAPK and JAK2/PI3K cascades are activated by Src-meditated CK2, leading to CBP phosphorylation and interaction with chPit-1α, resulting in transactivation of the cGH promoter.
Subject(s)
Growth Hormone/genetics , Leptin/pharmacology , Promoter Regions, Genetic/drug effects , Animals , CHO Cells , Chickens , Cricetinae , Cricetulus , Enzyme Activation/drug effects , Janus Kinase 2/metabolism , Leptin/metabolism , Mice , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Receptors, Leptin/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effects , src-Family Kinases/antagonists & inhibitorsABSTRACT
Leptin is a multi-functional adipokine in vertebrates. The leptin gene and protein are found in many vertebrates; however, the existence of leptin in birds remains controversial. Here we detected leptin-like activity in avian blood using chicken leptin receptor (chLEPR) and green fluorescent protein (GFP)-fused chicken signal transducer and activator of transcription (chSTAT3) co-expressed in CHO-K1 cells (CHO-chLEPR/STAT3). We validated that rat serum specifically induces intranuclear migration of GFP-fused chSTAT3 (GFP-chSTAT3) in CHO-chLEPR/STAT3 cells, but not in CHO-K1 cells expressing GFP-STAT3 (CHO-chSTAT3) before testing the avian blood samples. Blood of chickens (Gallus gallus), wild jungle crows (Corvus macrorhynchos), and carrion crows (Corvus corone) accumulated the GFP signal into nuclei, and frequency varied in each blood sample. Western blotting showed that chicken and crow blood samples specifically phosphorylated GFP-chSTAT3 in the chLEPR-transfected cells. These results indicate that avian blood contains a leptin-like molecule that specifically binds to LEPR, suggesting that the leptin system is conserved across all vertebrate classes.
Subject(s)
Avian Proteins/metabolism , Cell Nucleus/metabolism , Receptors, Leptin/metabolism , STAT3 Transcription Factor/metabolism , Active Transport, Cell Nucleus , Animals , Avian Proteins/blood , CHO Cells , Chickens , Cricetulus , Crows , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Phosphorylation , Rats, Wistar , Receptors, Leptin/blood , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , STAT3 Transcription Factor/blood , STAT3 Transcription Factor/genetics , Species SpecificityABSTRACT
In the present study, we expressed chicken (ch) Pit-1α (chPit-1α) and chPit-1γin vitro to compare the roles of chPit-1s in the transcription of the chicken growth hormone (chGH) gene. Both green fluorescence protein (GFP)-fused chPit-1γ and GFP-fused chPit-1α were localized in the nuclei of COS-7 cells. In a luciferase reporter gene assay, both chPit-1α and chPit-1γ transactivated the chGH promoter, and chPit-1α showed a more potent effect than chPit-1γ. On the other hand, an increase of cellular cAMP induced by forskolin promoted transactivation of the chGH gene with chPit-1α and chPit-1γ to similar extents. These results suggest that chPit-1γ may modulate the basal promoter activity of the chGH gene to the same degree as chPit-1α; however, a structural difference observed at the N-terminus transactivation domains in chPit-1α and chPit-1γ could be associated with the efficiency of basal activation of the chGH promoter.