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Regulation of the processing and release of tumor necrosis factor alpha in a human macrophage cell line
K Nakada-Tsukui1, N Watanabe, Y Kobayashi
1Department of Biomolecular Science, Faculty of Science, Toho University, Chiba, Japan.
Abstract:
The proteolytic cleavage of a variety of membrane proteins, including pro-tumor necrosis factor alpha (pro-TNF-alpha), is induced by various reagents such as phorbol 12-myristate 13-acetate (PMA). In this study we generated a human macrophage cell line that constitutively produces TNF-alpha, and examined how the processing and release of TNF-alpha are regulated. The processing and release of TNF-alpha were enhanced by PMA through a protein kinase C-dependent pathway. Although only hydroxamate matrix metalloproteinase (MMP) inhibitors inhibited the basal processing and release of TNF-alpha, the PMA-induced processing and release of TNF-alpha were inhibited not only by MMP inhibitors but also by 1,10-phenanthroline, 3,4-dichloroisocoumarin (3,4-DCI), iodoacetamide, and Nalpha-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK). Hydroxamate MMP inhibitors and 1,10-phenanthroline inhibited the processing of TNF-alpha on the cell surface, whereas 3,4-DCI, iodoacetamide, and TPCK inhibited the transport of TNF-alpha to the cell surface. These results suggest that serine and/or cysteine protease(s) may be involved in PMA-induced processing and/or transport to the cell surface.
Insights
Phorbol 12-myristate 13-acetate (PMA) enhances tumor necrosis factor alpha (TNF-alpha) release via protein kinase C. Inhibitors reveal distinct roles for proteases in TNF-alpha processing and cell surface transport.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Tumor necrosis factor alpha (TNF-alpha) is a key cytokine involved in inflammation and immunity.
- Pro-TNF-alpha processing and release are regulated by proteolytic cleavage.
- Phorbol 12-myristate 13-acetate (PMA) is known to induce proteolytic cleavage of membrane proteins.
Purpose of the Study:
- To investigate the regulation of TNF-alpha processing and release in a human macrophage cell line.
- To identify the specific proteases involved in PMA-induced TNF-alpha release.
- To elucidate the mechanisms by which inhibitors affect TNF-alpha transport and processing.
Main Methods:
- Generation of a human macrophage cell line constitutively producing TNF-alpha.
- Treatment with PMA and various protease inhibitors (hydroxamate MMP inhibitors, 1,10-phenanthroline, 3,4-DCI, iodoacetamide, TPCK).
- Analysis of TNF-alpha processing, release, and cell surface transport.
Main Results:
- PMA enhanced TNF-alpha processing and release through a protein kinase C-dependent pathway.
- Hydroxamate MMP inhibitors and 1,10-phenanthroline inhibited cell surface TNF-alpha processing.
- 3,4-DCI, iodoacetamide, and TPCK inhibited TNF-alpha transport to the cell surface.
Conclusions:
- Serine and/or cysteine proteases are implicated in PMA-induced TNF-alpha processing and/or cell surface transport.
- Distinct protease classes differentially regulate TNF-alpha processing and transport.
- Understanding these pathways offers insights into TNF-alpha-mediated inflammatory responses.