Autoregulation of pituitary corticotroph SOCS-3 expression: Characterization of the murine SOCS-3 promoter

Pituitary corticotroph SOCS-3 is a novel intracellular regulator of leukemi a inhibitory factor (LIF)-mediated proopiomelanocortin gene expression and adrenocorticotropic hormone (ACTH) secretion, inhibiting LIF-activated Janu s kinase-signal transducers and activators of transcription (STAT) signalin g in a negative autoregulatory loop. We now demonstrate in corticotroph AtT -20 cells that LIF-stimulated endogenous SOCS-3 mRNA expression is blocked in stable transfectants of SOCS-3 wild type or in dominant negative STAT-3 mutants, respectively. We characterized approximate to 3.8-kb genomic 5' se quence of murine SOCS-3, including approximate to 2.9-kb sequence upstream of the transcription start site (+1), which was determined by 5' rapid ampl ification of cDNA ends and RNase protection assay. Different 5' constructs were cloned into the pGL3Basic vector, and luciferase activity was assayed in transiently transfected ACTH-secreting corticotroph AtT-20 cells. A STAT -1/STAT-3 binding element, located at nucleotides -72 to -64, was essential for LIF stimulation of SOCS-3 promoter activity. LIF induced 10-fold incre ased luciferase activity in a wild-type construct spanning -2757 to +929 ba ses. However, deletion or point mutation of the STAT-1/STAT-3 binding eleme nt abrogated LIF action (2- to 3-fold). Electrophoretic mobility-shift assa y analysis confirmed specific binding of STAT-1 and STAT-3 to this region. These results characterize the genomic 5' region of murine SOCS-3 and ident ify an important STAT-1/STAT-3 binding element therein. Thus, LIF-stimulate d SOCS-3 gene expression is at least in part mediated by STAT-3 and STAT-1. The cytokine inhibitor SOCS-3 acts in a negative loop to autoregulate its own gene expression, thus limiting its accumulation in the corticotroph cel l. These results demonstrate a mechanism for corticotroph plasticity with r apid "on" and "off' ACTH induction in response to neuro-immunoendocrine sti muli, such as LIF.