Nuclear factor-κB mediates the inhibitory effects of tumor necrosis factor-α on growth hormone-inducible gene expression in liver

Mark D. Buzzelli, Murali Nagarajan, John F. Radtka, Margaret L. Shumate, Maithili Navaratnarajah, Charles H. Lang, Robert N. Cooney

Research output: Contribution to journalArticle

7 Citations (Scopus)

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-κB (NFκB) 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-κBα (IκBα) expression vectors (IκBαS/A or IκBαTrunc), p65 and p50 expression vectors, and p65 deletion constructs were used to investigate the NFκB pathway. IκBαS/A and IκBαTrunc 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 NFκB, especially p65, by a mechanism that does not require protein synthesis.

Original languageEnglish (US)
Pages (from-to)6378-6388
Number of pages11
JournalEndocrinology
Volume149
Issue number12
DOIs
StatePublished - Dec 1 2008

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Insulin-Like Growth Factor I
Growth Hormone
Serine Proteinase Inhibitors
Tumor Necrosis Factor-alpha
Gene Expression
Liver
Sphingomyelin Phosphodiesterase
JNK Mitogen-Activated Protein Kinases
Cycloheximide
Luciferases
Hepatocytes
Proteins

All Science Journal Classification (ASJC) codes

  • Endocrinology

Cite this

Buzzelli, Mark D. ; Nagarajan, Murali ; Radtka, John F. ; Shumate, Margaret L. ; Navaratnarajah, Maithili ; Lang, Charles H. ; Cooney, Robert N. / Nuclear factor-κB mediates the inhibitory effects of tumor necrosis factor-α on growth hormone-inducible gene expression in liver. In: Endocrinology. 2008 ; Vol. 149, No. 12. pp. 6378-6388.
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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-κB (NFκB) 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-κBα (IκBα) expression vectors (IκBαS/A or IκBαTrunc), p65 and p50 expression vectors, and p65 deletion constructs were used to investigate the NFκB pathway. IκBαS/A and IκBαTrunc 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 NFκB, especially p65, by a mechanism that does not require protein synthesis.",
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Nuclear factor-κB mediates the inhibitory effects of tumor necrosis factor-α on growth hormone-inducible gene expression in liver. / Buzzelli, Mark D.; Nagarajan, Murali; Radtka, John F.; Shumate, Margaret L.; Navaratnarajah, Maithili; Lang, Charles H.; Cooney, Robert N.

In: Endocrinology, Vol. 149, No. 12, 01.12.2008, p. 6378-6388.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Nuclear factor-κB mediates the inhibitory effects of tumor necrosis factor-α on growth hormone-inducible gene expression in liver

AU - Buzzelli, Mark D.

AU - Nagarajan, Murali

AU - Radtka, John F.

AU - Shumate, Margaret L.

AU - Navaratnarajah, Maithili

AU - Lang, Charles H.

AU - Cooney, Robert N.

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N2 - 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-κB (NFκB) 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-κBα (IκBα) expression vectors (IκBαS/A or IκBαTrunc), p65 and p50 expression vectors, and p65 deletion constructs were used to investigate the NFκB pathway. IκBαS/A and IκBαTrunc 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 NFκB, especially p65, by a mechanism that does not require protein synthesis.

AB - 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-κB (NFκB) 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-κBα (IκBα) expression vectors (IκBαS/A or IκBαTrunc), p65 and p50 expression vectors, and p65 deletion constructs were used to investigate the NFκB pathway. IκBαS/A and IκBαTrunc 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 NFκB, especially p65, by a mechanism that does not require protein synthesis.

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