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Induction of unresponsiveness to tumor necrosis factor (TNF) after autocrine TNF expression requires TNF membrane
E Decoster1, B Vanhaesebroeck, E Boone
1Laboratory of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Ghent, B-9000 Ghent, Belgium.
Abstract:
Tumor necrosis factor (TNF) has a specific gene-inducing activity on many cell types and exerts a cytotoxic effect on a number of tumor cell lines. However, several tumor cell types are resistant to TNF-induced effects, and some of these produce TNF. We previously demonstrated that introduction of an exogenous TNF gene in the TNF-sensitive cell line L929sA induced autocrine TNF production and unresponsiveness to the cytotoxic activity of TNF. This resistance required biologically active TNF and was correlated with complete down-modulation of the TNF receptors on the cell surface. We have now characterized this process in more detail. The role of expression of the membrane-bound TNF proform and its subsequent proteolytic processing in the induction of TNF unresponsiveness was investigated. Exchange of the TNF presequence for the signal sequence of interleukin-6 resulted in production of secreted TNF, but not in induction of TNF resistance. On the other hand, expression of non-secretable, membrane-bound TNF generated complete TNF unresponsiveness. To explore whether the requirement for anchoring reflected a specific functional role of the TNF presequence, the latter was replaced by the membrane anchor of trimeric chicken hepatic lectin. Expression of this construct induced complete TNF unresponsiveness. Hence, the role of the TNF presequence in the induction of TNF unresponsiveness only involves its function as a membrane anchor, which permits oligomerization of the TNF molecule into a biologically active homotrimer.
Insights
Tumor necrosis factor (TNF) resistance in tumor cells is linked to membrane-bound TNF. Anchoring the TNF molecule to the cell surface, not its presequence, is crucial for inducing this unresponsiveness.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Tumor necrosis factor (TNF) exhibits gene-inducing and cytotoxic activities.
- Some tumor cells are resistant to TNF, with some producing TNF themselves.
- Previous work showed exogenous TNF gene introduction induced resistance and TNF receptor downregulation.
Purpose of the Study:
- To investigate the role of membrane-bound TNF proform processing in TNF unresponsiveness.
- To determine if TNF anchoring or its presequence is essential for inducing resistance.
Main Methods:
- Exchanging the TNF presequence with interleukin-6 signal sequence to produce secreted TNF.
- Expressing non-secretable, membrane-bound TNF.
- Replacing the TNF presequence with a membrane anchor from chicken hepatic lectin.
Main Results:
- Secreted TNF production did not induce TNF resistance.
- Expression of non-secretable, membrane-bound TNF led to complete resistance.
- Replacing the presequence with a membrane anchor also induced complete resistance.
Conclusions:
- TNF unresponsiveness requires biologically active, membrane-bound TNF.
- The TNF presequence's role is solely as a membrane anchor, enabling homotrimerization.
- Anchoring facilitates the formation of a functional TNF homotrimer, inducing resistance.