Endocrine Abstracts

Oestrogen is an important regulator of the pituitary hormone prolactin (PRL) in vivo, although the regulation mechanism is poorly understood. Liganded oestrogen receptor (ER) synergises with the pituitary specific factor Pit-1 on the rat PRL promoter to facilitate chromatin looping and increased transcription; however, no such mechanism has been reported for the human PRL promoter, in which the ERE sequence is altered.

In pituitary GH3 cells stably transfected with a hPRL5000bp-luciferase construct, 1 nanomolar oestradiol for 24-48h effected a 1.6 plus/minus 0.15-fold induction of luciferase expression. 10 nanograms per millilitre Tumour Necrosis Factor alpha (TNFalpha) caused a 2.8 plus/minus 0.3-fold induction of luciferase activity at 24h. Combined treatment with both oestradiol and TNFalpha gave a synergistic 5.3 plus/minus 0.3-fold activation of hPRL promoter activity.

The oestrogen antagonists 4-hydroxy-tamoxifen and ICI 182,780 inhibited the effect of oestradiol alone, had little effect on stimulation by TNFalpha but blocked the synergistic interaction with combined treatment, suggesting liganded ER is essential for this effect on prolactin. Pituitary GH3 cells were transfected with a consensus ERE-luciferase reporter construct: strong induction by oestradiol was seen but there was no synergistic induction on addition of TNFalpha. These data suggest that oestrogen may affect hPRL gene transcription primarily through combinatorial interactions between ER and other transcription factors, such as Nuclear Factor kappaB (NFkappaB), specific to the hPRL promoter. We are currently assessing this using DNA-protein binding assays. In the hPRL promoter a potential NFkappaB binding site is found adjacent to the ERE at -1207bp, which in turn is adjacent to a Pit-1 site. We propose that the three transcription factors interact at the promoter to increase gene transcription, probably involving chromatin structure alterations.

This work was funded by a Medical Research Council PhD studentship to ADA, and by the Wellcome Trust.