Abstract
Vasoactive intestinal peptide (VIP) has been shown to increase prolactin (PRL) gene expression and secretion in turkey primary anterior pituitary cells. To characterize cis-acting elements involved in stimulation of PRL gene expression by VIP, 5 ′-flanking deletions and/or mutations of the turkey PRL promoter fused to the luciferase (Luc) reporter gene have been constructed for use in transient transfection assays. Deletion analysis of the turkey PRL promoter (tPRLP) indicated that the VIP-stimulated tPRLP activity was controlled by three major positive regulatory regions and two negative regions. The −74/+40 Luc construct exhibited a 7- to 8-fold increase in promoter activity in response to VIP treatment. Deletion of the 35-bp segment (−74/−40) or fusion of this sequence to the SV40 promoter demonstrated that a VIP response element (VRE) was present in this region. Functional analysis of this VRE (−74/−40) was performed by mutation of core sequences (TGAATGTATGCA, −61/−50) or deletion of a 35-bp segment and a Decoy assay. Electrophoretic mobility shift assays revealed the presence of three DNA–protein complexes bound to the region −73 to −41. The results of the present study demonstrated that VRE (35-bp) in the proximal PRL promoter is an important cis-acting element for VIP-stimulated PRL gene expression in turkey primary anterior pituitary cells.
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