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Nuclear Factor-B Mediates the Inhibitory Effects of Tumor Necrosis Factor- on Growth Hormone-Inducible Gene Expression in Liver

Departments of Surgery (M.D.B., M.N., J.F.R., M.L.S., M.N., R.N.C.) and Cellular and Molecular Physiology (C.H.L., R.N.C.), The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

Address all correspondence and requests for reprints to: Robert N. Cooney, M.D., Department of Surgery, The Pennsylvania State University College of Medicine, 500 University Drive, MCH070, Hershey, Pennsylvania 17033. E-mail: rcooney{at}psu.edu.

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-B (NFB) 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-B (IB) expression vectors (IBS/A or IBTrunc), p65 and p50 expression vectors, and p65 deletion constructs were used to investigate the NFB pathway. IBS/A and IBTrunc 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 NFB, especially p65, by a mechanism that does not require protein synthesis.