Synergistic signaling by corticotropin-releasing hormone and leukemia inhibitory factor bridged by phosphorylated 3',5'-cyclic adenosine monophosphate response element binding protein at the nur response element (NurRE)-signal transducers and activat

Leukemia inhibitory factor (LIF) cooperates with CRH at the pituitary level to induce POMC gene transcription, resulting in activation of the pituitary-adrenal axis. However, the underlying molecular mechanisms remain elusive. Here, we show that the NurRE-signal transducers and activators of transcription (STAT) composite element of the POMC promoter was the predominant target of the LIF-CRH synergy. Whereas NurRE or STAT sites alone conferred synergy, the maximal response was found with the NurRE-STAT reporter, suggesting that direct DNA binding of both transcription factors is required for an optimal synergy. During LIF-CRH stimulation, Nur77 and activated STAT1-3 were bound to the composite element, and the binding of each factor was abolished by appropriate mutations. CREB was also detected in this complex in a stimulation-dependent and DNA binding-independent manner. Nur77 and STAT1-3 bound to the NurRE-STAT site were each sufficient for CREB recruitment. Recombinant CREB directly interacted with recombinant Nur77 or STAT1-3. Moreover, CREB-Nur77 interaction was increased by CREB phosphorylation at Ser-133 and the dominant-negative mutant CREB-M1 efficiently inhibited the synergistic LIF-CRH response. This synergism was also inhibited after transfection of CREB-small interfering RNA. We conclude that both CREB phosphorylation at Ser-133 and level of CREB expression are crucial in LIF-CRH synergism where CREB, without direct DNA binding, could improve the stability of Nur77 and STAT1-3 binding to POMC promoter and facilitate the recruitment of coactivators. This novel intrapituitary signaling mechanism may have more general implications in cross talks between cAMP-protein kinase A and Janus kinase-STAT pathways.