Characterization of the promoter region of murine NNT-1/BSF-3– a novel paracrine modulator of corticotroph function

Novel neurotrophin-1/B-cell stimulating factor-3 (NNT-1/BSF-3) belongs to the gp130 cytokine family and acts as a paracrine stimulus of corticotroph function. In murine folliculostellate TtT/GF cells NNT-1/BSF-3 expression is induced by pituitary adenylate-cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) through PKC-, PKA- and Erk1/2 dependent pathways (Auernhammer et al. Endocrinology 2003; 144:1202–1210, Vlotides et al. Endocrinology 2004; 145:716–727). In the current study we characterized the promoter region of murine NNT-1/BSF-3. 5'-Race technique demonstrated the transcription start site to be located –128bp upstream the translation initiation codon,. We cloned a fragment of the 5'-region spanning -2643/+128 bp (Genebank AC108138). Additionally, we generated several 5'-truncated fragments. All fragments were cloned into pGL3 basic. Reporter gene activity (luc) was measured in TtT/GF cells transiently transfected with the respective constructs or mock-transfected with pGL3 basic alone. Basal luc expression of mock-transfected cells was set as 1. Relative luc levels of all other groups were calculated in comparison with this control group. Transfected cells were stimulated with the neuropeptides PACAP (50 nM) and VIP (100nM) as well as with fetal calf serum (10%). Fragments ranging from -2643/+128 bp to –758/+128 bp showed comparably high basal and stimulated luciferase activities (e.g. –758/+128 bp fragment: basal 13; PACAP 43; VIP 51; FCS 39). In contrast, further 5'-truncation caused a significant loss of basal and especially stimulated luciferase activities (e.g. –508/+128 bp fragment: basal 7; PACAP 15; VIP 15; FCS 10). In summary, we cloned and characterized the functional 5'-region of murine NNT-1/BSF-3. Our data indicate that essential promoter activity is harbored in the area between -758 bp and -508 bp upstream of the transcription start site. PACAP, VIP and FCS are potent stimuli of murine NNT-1/BSF-3 promoter activity. Further studies to understand the molecular mechanisms of NNT-1/BSF-3 gene regulation are underway.