1. Nuclear factor-kappaB mediates the inhibitory effects of tumor necrosis factor-alpha on growth hormone-inducible gene expression in liver.
- Author
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Buzzelli MD, Nagarajan M, Radtka JF, Shumate ML, Navaratnarajah M, Lang CH, and Cooney RN
- Subjects
- Animals, Cell Line, Gene Expression drug effects, Ligases genetics, Promoter Regions, Genetic genetics, Protein Binding drug effects, Rats, STAT5 Transcription Factor metabolism, Signal Transduction drug effects, Signal Transduction genetics, Signal Transduction physiology, Growth Hormone pharmacology, Liver drug effects, Liver metabolism, NF-kappa B metabolism, Tumor Necrosis Factor-alpha pharmacology
- Abstract
TNF inhibits serine protease inhibitor 2.1 (Spi 2.1) and IGF-I gene expression by GH in CWSV-1 hepatocytes. The current study describes construction of a GH-inducible IGF-I promoter construct and investigates mechanisms by which TNF and nuclear factor-kappaB (NFkappaB) inhibit GH-inducible gene expression. CWSV-1 cells were transfected with GH-inducible Spi 2.1 or IGF-I promoter luciferase constructs, incubated with TNF signaling inhibitors (fumonisin B1 for sphingomyelinase and SP600125 for c-Jun N-terminal kinase), treated with or without TNF, and then stimulated with recombinant human GH. The 5- to 6-fold induction of Spi 2.1 and IGF-I promoter activity by GH was inhibited by TNF. Neither fumonisin B1 nor SP600125 prevented the inhibitory effects of TNF on GH-inducible promoter activity. Dominant-negative inhibitor-kappaBalpha (IkappaBalpha) expression vectors (IkappaBalphaS/A or IkappaBalphaTrunc), p65 and p50 expression vectors, and p65 deletion constructs were used to investigate the NFkappaB pathway. IkappaBalphaS/A and IkappaBalphaTrunc ameliorated the inhibitory effects of TNF on GH-inducible Spi 2.1 and IGF-I promoter activity. Cotransfection of CWSV-1 cells with expression vectors for p65 alone or p50 and p65 together inhibited GH-inducible Spi 2.1 and IGF-I promoter activity. Cotransfection with a C-terminal p65 deletion (1-450) enhanced GH-inducible promoter activity, whereas the N-terminal deletion (31-551) was inhibitory for IGF-I but not Spi 2.1. Cycloheximide did not antagonize the inhibitory effects of TNF on GH-inducible IGF-I expression. We conclude the inhibitory effects of TNF on GH-inducible promoter activity are mediated by NFkappaB, especially p65, by a mechanism that does not require protein synthesis.
- Published
- 2008
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