Nuclear factor-kappaB mediates the inhibitory effects of tumor necrosis factor-alpha on growth hormone-inducible gene expression in liver.
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.
Buzzelli
Mark D
MD
Department of Surgery, The Pennsylvania State University College of Medicine, 500 University Drive, MCH070, Hershey, Pennsylvania 17033, USA.
Nagarajan
Murali
M
Radtka
John F
JF
Shumate
Margaret L
ML
Navaratnarajah
Maithili
M
Lang
Charles H
CH
Cooney
Robert N
RN
D000818:Animals; D002460:Cell Line; D015870:Gene Expression / Q000187:drug effects; D013006:Growth Hormone / Q000494:pharmacology*; D008025:Ligases / Q000235:genetics; D008099:Liver / Q000187:drug effects* / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D050799:STAT5 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D016328:NF-kappa B; D050799:STAT5 Transcription Factor; D014409:Tumor Necrosis Factor-alpha; D013006:Growth Hormone; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases