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Requirement for the orphan steroid receptor Nur77 in apoptosis of T-cell hybridomas
J D Woronicz1, B Calnan, V Ngo
1Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Abstract:
Apoptosis is a phenomenon observed during development of many cell types in many organisms. It is an internal, programmed cell death characterized by DNA fragmentation into nucleosome-size pieces. Anti-CD3-induced apoptosis in T-cell hybridomas and immature thymocytes requires new gene transcription and may be related to negative selection during T-cell development. Using subtractive hybridization, we isolated a complementary DNA clone encoding the orphan steroid receptor Nur77 (refs 7-9). It shows different patterns of messenger RNA induction between apoptotic and stimulated T cells. We report here the use of gel shift analysis to demonstrate that the Nur77 protein is present at high levels in apoptotic T-cell hybridomas and apoptotic thymocytes, but not in growing T cells or stimulated splenocytes. A Nur77 dominant negative protected T-cell hybridomas from activation-induced apoptosis. Hence Nur77 is necessary for induced apoptosis in T-cell hybridomas.
Insights
The orphan steroid receptor Nur77 is crucial for programmed cell death (apoptosis) in T-cells. Nur77 protein levels increase during T-cell apoptosis, and blocking it prevents this cell death process.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Apoptosis, or programmed cell death, is vital for T-cell development and function.
- Activation-induced cell death in T-cells requires new gene transcription.
Purpose of the Study:
- To investigate the role of the orphan steroid receptor Nur77 in T-cell apoptosis.
- To determine if Nur77 is necessary for anti-CD3-induced apoptosis in T-cell hybridomas.
Main Methods:
- Subtractive hybridization to isolate Nur77 cDNA.
- Gel shift analysis to detect Nur77 protein levels.
- Using a Nur77 dominant negative to assess its function in apoptosis.
Main Results:
- Nur77 mRNA is differentially induced in apoptotic versus stimulated T-cells.
- Nur77 protein is highly expressed in apoptotic T-cell hybridomas and thymocytes.
- A Nur77 dominant negative construct protected T-cell hybridomas from activation-induced apoptosis.
Conclusions:
- Nur77 protein is upregulated during T-cell apoptosis.
- Nur77 is a necessary factor for T-cell hybridoma apoptosis induced by anti-CD3 stimulation.