Analysis of human neuropeptide FF gene expression

As an initial step to study the function of the gene encoding the human neuropeptide FF (NPFF), we cloned a 4.7-kb sequence from the promoter region. Primer extension and 5'-rapid amplification of cDNA ends revealed multiple transcription initiation sites. Northern blot analysis of the mRNA expression revealed a specific signal only in poly(A) + RNA from medulla and spinal cord. Chimeric luciferase reporter gene constructs were transiently transfected in A549, U-251 MG, SK-N-SH, SK-N-AS and PC12 cells. The promoter activity was directly comparable with the level of endogenous NPFF mRNA as determined by real-time quantitative RT-PCR. The highest promoter activity was measured when a region from - 552 to - 830 bp of the 5'-flanking region was fused to the constructs, and a potential silencer element was localized between nucleotides -220 and -551. A twofold increase in NPFF mRNA was observed after 72 h of nerve growth factor stimulation of PC12 cells and the region between - 61 and - 214 bp of the 5'-flanking region was found to be responsive to this stimulation. We postulate that control of human NPFF gene expression is the result of both positive and negative regulatory elements and the use of multiple transcription initiation sites.