Tumor necrosis factor-alpha activates the human prolactin gene promoter via nuclear factor-kappaB signaling.
Pituitary function has been shown to be regulated by an increasing number of intrapituitary factors, including cytokines. Here we show that the important cytokine TNF-alpha 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-alpha 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-alpha, Western blot analysis showed rapid inhibitory protein kappaB (IkappaBalpha) degradation and phosphorylation of p65. Confocal microscopy of cells expressing fluorescence-labeled p65 and IkappaBalpha fusion proteins showed transient cytoplasmic-nuclear translocation and subsequent oscillations in p65 localization and confirmed IkappaBalpha degradation. This was associated with increased nuclear factor kappaB (NF-kappaB)-mediated transcription from an NF-kappaB-responsive luciferase reporter construct. Disruption of NF-kappaB signaling by expression of dominant-negative variants of IkappaB kinases or truncated IkappaBalpha abolished TNF-alpha activation of the prolactin promoter, suggesting that this effect was mediated by NF-kappaB. TNF-alpha 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.
Friedrichsen
Sönke
S
Endocrine Science Research Group School of Biological Sciences, University of Manchester, UK.
Harper
Claire V
CV
Semprini
Sabrina
S
Wilding
Michael
M
Adamson
Antony D
AD
Spiller
Dave G
DG
Nelson
Glyn
G
Mullins
John J
JJ
White
Michael R H
MR
Davis
Julian R E
JR
D000818:Animals; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation / Q000502:physiology; D017930:Genes, Reporter; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism*; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism*; D011388:Prolactin / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology*; D051381:Rats; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D011388:Prolactin