Endocrine Abstracts (2005) 9 OC20

Transcriptional activation of the prolactin gene by TNF-alpha is mediated via NFkB

S Friedrichsen1, M Wilding1, C Harper2, G Nelson2, DS Spiller2, MRH White2 & JRE Davis1

1ESRG, University of Manchester, Manchester, UK; 2Centre for Cell Imaging, University of Liverpool, Liverpool, UK.

The neuroendocrine and immune systems are closely linked, and cytokines such as IL-1, IL-6 and tumour necrosis factor-alpha (TNF-alpha), secreted by immune cells or neighbouring pituicytes, may have important local effects on pituitary function to modulate endocrine mechanisms of regulation.

We have analysed the transcriptional regulation of the human prolactin (hPRL) gene promoter by TNF-alpha. Using stably transfected GH3 cells expressing luciferase under the control of the hPRL 5000bp exon 1b (pituitary-specific) promoter, we found a dose and time dependent increase in promoter activity after stimulation with TNF-alpha. Maximum expression levels were reached with 2 nanograms per millilitre TNF-alpha. There was a clear biphasic time-course of response, with a transient first peak after 6h and a second peak after 24h. Luminescence microscopy of living cells showed that individual cells responded with different temporal patterns. Real-time PCR analysis verified that endogenous prolactin transcription in GH3 cells is also increase after TNF-alpha treatment.

Activation of NFkB in GH3 cells after TNF-alpha stimulation was shown by DNA-protein complex formation in electromobility shift assays. Cotransfection of dominant negative components of the NFkB signalling pathway (dnIKK-alpha, -beta and -gamma) abolished TNF-alpha induced activation of the human prolactin promoter luciferase construct, whereas cotransfection of wildtype IKK-beta led to an increase in basal transcription levels.

In conclusion, these studies have shown that hPRL promoter activity is strongly induced by TNF-alpha, and that the response is most likely mediated via the NFkB signalling pathway. Gene activation follows a biphasic pattern, suggesting that TNF-alpha acts both directly on the promoter but might also have secondary, possibly indirect effects. These results, together with the fact that TNF-alpha is produced locally in the pituitary, suggest an important role in the paracrine regulation of lactotroph function, possibly as mediator of stress or immune challenges.

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