TNF-α activates the human prolactin gene promoter via NF-κB signaling

• Published in: Endocrinology (Philadelphia) 2005-10, Vol.147 (2), p.773-781
• Main Authors: Friedrichsen, Sönke, Harper, Claire V., Semprini, Sabrina, Wilding, Michael, Adamson, Antony D., Spiller, Dave G., Nelson, Glyn, Mullins, John J., White, Michael R.H., Davis, Julian R.E.
• Format: Article
• Language: English
• ISSN: 0013-7227
• DOI: 10.1210/en.2005-0967

Pituitary function has been shown to be regulated by an increasing number of intra-pituitary factors including cytokines. Here we show that the important cytokine TNF-α activates prolactin gene transcription in pituitary GH3 cells stably expressing luciferase under control of 5 kb of the human prolactin promoter. Similar regulation of the endogenous rat prolactin gene by TNF-α in GH3 cells was confirmed using real time PCR. Luminescence microscopy revealed heterogeneous dynamic response patterns of promoter activity in individual cells. In GH3 cells treated with TNF-α, western blot analysis showed rapid IκBα degradation and phosphorylation of p65. Confocal microscopy of cells expressing fluorescence labelled p65 and IκBα fusion proteins showed transient cytoplasmic-nuclear translocation and subsequent oscillations in p65 localisation and confirmed IκBα degradation. This was associated with increased NF-κB mediated transcription from an NF-κB responsive luciferase reporter construct. Disruption of NF-κB signaling by expression of dominant negative variants of IKKs or truncated IκBα abolished TNF-α activation of the prolactin promoter, suggesting that this effect was mediated by NF-κB. TNF-α signaling was found to interact with other endocrine signals to regulate prolactin gene expression, and is likely to be a major paracrine modulator of lactotroph function.