C/EBP-related sites in addition to a stat site are necessary for ciliary neurotrophic factor-leukemia inhibitory factor-dependent transcriptional activation by the vasoactive intestinal peptide cytokine response element

The neuropoietic cytokines ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) regulate VIP gene expression through a cytokine response element (CyRE) which interacts with members of the STAT transcription factor family. The CyRE STAT site is, however, insufficient to mediate full transcriptional activation by CNTF/LIF, suggesting that other sequences and nuclear proteins are also important. As C/EBP proteins participate in the transcriptional effects of the related cytokine, interleukin-6, we investigated the role of possible C/EBP-binding sites in the response of the VIP CyRE to CNTF/LIF. Using DNase I footprinting, transactivation studies, DNA mobility shift assays, and mutational analysis, three sites within the VIP CyRE were identified as C/EBP-related binding sites and shown to be important to CNTF/LIF-mediated transcriptional activation. The CyRE C/EBP-related sites interact with nuclear proteins from the human neuroblastoma cell line, NBFL, including a novel, protein synthesis-dependent, nuclear protein complex, induced by CNTF treatment. These nuclear proteins are not, however, recognized by antisera to known C/EBP proteins. Therefore, other nuclear proteins regulated by independent pathways act in concert with the JAKSTAT pathway to mediate CNTF/LIF regulation of VIP gene expression through the CyRE.