Multiple promoter elements required for leukemia inhibitory factor-stimulated M2 muscarinic acetylcholine receptor promoter activity

Treatment of neuronal cells with leukemia inhibitory factor (LIF) results in increased M-2 muscarinic acetylcholine receptor promoter activity. We demonstrate here that multiple promoter elements mediate LIF stimulation of M-2 gene transcription. We identify a LIF inducible element (LIE) in the M-2 promoter with high homology to a cytokine-inducible ACTG-containing sequence in the vasoactive intestinal peptide promoter. Mutagenesis of both a STAT (signal transducers and activators of transcription) element and the LIE in the M-2 promoter is required to attenuate stimulation of M-2 promoter activity by LIF completely. Mobility shift assays indicate that a LIF-stimulated complex binds to a 70 base pair M-2 promoter fragment. Furthermore, a STAT element within this fragment can bind to LIF-stimulated nuclear STAT1 homodimers in vitro. Mutagenesis experiments show that cytokine-stimulated activation of M-2 promoter activity requires tyrosine residues on glycoprotein 130 (gp130) that are also required for both STAT1 and STAT3 activation. Dominant negative STAT1 or STAT3 can block LIF-stimulated M-2 promoter activity. Real-time RT-PCR analysis indicates that LIF-stimulated induction of M-2 mRNA is partially dependent on protein synthesis. These results show that regulation of M-2 gene transcription in neuronal cells by LIF occurs through a complex novel mechanism that is dependent on LIE, STAT and de novo protein synthesis.