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Published on: January 15, 2011
Extracellular Tax1 protein stimulates tumor necrosis factor-beta and immunoglobulin kappa light chain expression in
P F Lindholm1, R L Reid, J N Brady
1Laboratory of Molecular Virology, National Cancer Institute, Bethesda, Maryland 20892.
Insights
The human T-cell leukemia virus type I (HTLV-I) Tax1 protein directly activates gene expression by binding to NF-kappa B sites. Extracellular Tax1 regulates TNF-beta and Ig kappa genes, independent of new protein synthesis.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- The human T-cell leukemia virus type I (HTLV-I) Tax1 protein is known to increase the expression of genes containing NF-kappa B regulatory sequences.
- Extracellular Tax1 protein has been shown to induce NF-kappa B binding activity in lymphoid cells.
- HTLV-I infection is associated with elevated levels of lymphotoxin tumor necrosis factor-beta (TNF-beta) and immunoglobulin secretion.
Purpose of the Study:
- To investigate the interaction of NF-kappa B proteins with TNF-beta and immunoglobulin kappa (Ig kappa) light chain genes in Tax1-stimulated cells.
- To determine if new synthesis of NF-kappa B proteins is required for Tax1-induced binding activity.
- To identify the specific Tax1-stimulated proteins that interact with NF-kappa B-binding sites in the Ig kappa and TNF-beta promoters.
Main Methods:
- Gel shift assays using the Ig kappa NF-kappa B-binding site as a probe to analyze NF-kappa B complexes.
- Treatment with cycloheximide to assess the requirement for new protein synthesis.
- Detergent treatment of cytoplasmic extracts to investigate inhibitor regulation.
- Reverse transcription and polymerase chain reaction (RT-PCR) analysis to measure mRNA levels of TNF-beta and Ig kappa.
Main Results:
- Tax1 protein induced two distinct NF-kappa B gel shift complexes (C1 and C2).
- Complex C1 was inhibited by I kappa B, while complex C2 was not and was enhanced by detergent treatment, suggesting inhibitor regulation.
- Distinct proteins (75-kDa for Ig kappa, 59-kDa for TNF-beta) preferentially associated with the respective NF-kappa B-binding sites.
- Tax1 stimulation led to increased TNF-beta and Ig kappa mRNA levels.
Conclusions:
- Extracellular Tax1 protein can directly regulate the expression of endogenous cellular genes, including TNF-beta and Ig kappa.
- The induction of NF-kappa B binding activity by Tax1 does not require the synthesis of new NF-kappa B proteins.
- Tax1-mediated gene regulation involves distinct protein interactions with specific NF-kappa B-binding sites.
Abstract:
The human T-cell leukemia virus type I tax1 gene product is responsible for the increased expression of several cytokine and cellular genes that contain NF-kappa B regulatory sequences. Our laboratory has previously demonstrated that purified, extracellular Tax1 protein induced the nuclear accumulation of NF-kappa B binding activity in lymphoid cells. Since HTLV-I infection causes increased levels of lymphotoxin tumor necrosis factor-beta [TNF-beta] and immunoglobulin secretion, we have studied the interaction of NF-kappa B proteins from Tax1-stimulated cells with the TNF-beta and immunoglobulin kappa (Ig kappa) light chain genes. Tax1 induction of NF-kappa B occurred in the presence of cycloheximide, and Tax1 stimulation did not result in increased levels of NF-kappa B or c-rel RNA. These results indicate that new synthesis of NF-kappa B proteins was not required for induction of NF-kappa B-binding activity. With use of the Ig kappa NF-kappa B-binding site as a probe, two distinct NF-kappa B gel shift complexes were induced by the Tax1 protein. A slower-migrating complex, C1, was inhibited by the addition of purified I kappa B. In contrast, the faster-migrating C2 complex was not inhibited by I kappa B, but C2 was increased by detergent treatment of cytoplasmic extracts, suggesting that its binding activity was also regulated by an inhibitor. The Tax1-stimulated proteins that interacted with the NF-kappa B-binding sites in the Ig kappa and TNF-beta promoters were distinct. A 75-kDa protein preferentially associated with the Ig kappa NF-kappa B-binding site. In contrast, a 59-kDa protein associated with the TNF-beta NF-kappa B-binding site. Tax1 stimulation led to increased levels of TNF-beta and Ig kappa mRNA, as measured by reverse transcription and polymerase chain reaction analysis. These results represent the first experimental evidence that extracellular Tax1 can regulate the expression of endogenous cellular genes.
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