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The mutant plasmacytoma cell line S107 allows the identification of distinct pathways leading to NF-kappaB activation
B Baumann1, B Kistler, A Kirillov
1Institut für Medizinische Strahlenkunde und Zellforschung, Universität Würzburg, Versbacher Strasse 5, 97078 Würzburg, Germany.
Abstract:
Studies on the mechanisms of inducible and constitutive activity of NF-kappaB transcription factors have been hampered by the lack of appropriate mutant cell lines. We have analyzed the defect in the murine S107 plasmacytoma cell line, which was previously found to lack both constitutive and inducible NF-kappaB activity. Our analysis shows that these cells bear a specific defect that interferes with NF-kappaB induction by many diverse stimuli, such as lipopolysaccharide, phorbol 12-myristate 13-acetate, UV light, x-rays, and H2O2. This does not however represent a general signal transduction defect, because AP-1 transcription factors are readily induced by the same stimuli. Phosphatase inhibitors such as okadaic acid as well as calyculin A can efficiently induce NF-kappaB in S107 cells via a pathway apparently insensitive to the radical scavenger pyrrolidine dithiocarbamate. Furthermore, MEKK1 a protein kinase supposedly induced by some of the above stimuli, is also capable of activating NF-kappaB. Interestingly, both the potent physiological inducer of NF-kappaB TNFalpha as well as endoplasmic reticulum overload can induce NF-kappaB via a PDTC sensitive pathway. In all cases, DNA-binding NF-kappaB complexes are comprised predominantly of p50-RelA heterodimers, and NF-kappaB activation results in the induction of transiently transfected or resident reporter genes. In summary, these results suggest that the pathways for many NF-kappaB-inducing stimuli converge at a specific junction, and this pivotal step is mutated in the S107 cell line. Yet there are alternative routes bypassing this critical step that also lead to NF-kappaB induction. These routes utilized by tumor necrosis factor alpha and endoplasmic reticulum overload are still intact in this cell line.
Insights
Researchers identified a specific defect in S107 plasmacytoma cells that blocks NF-kappaB (nuclear factor kappa B) activation by many stimuli. However, alternative pathways remain functional, allowing NF-kappaB induction via TNFalpha and ER stress.
Area of Science:
- Molecular Biology
- Immunology
- Cell Signaling
Background:
- Nuclear factor kappa B (NF-kappaB) transcription factors are crucial regulators of immune responses and cellular processes.
- Studying NF-kappaB mechanisms has been challenging due to the limited availability of suitable mutant cell lines.
- The murine S107 plasmacytoma cell line was previously noted for its lack of both constitutive and inducible NF-kappaB activity.
Purpose of the Study:
- To analyze the specific molecular defect responsible for the lack of NF-kappaB activity in the S107 cell line.
- To investigate the signaling pathways involved in NF-kappaB induction and identify potential alternative routes.
Main Methods:
- Analysis of NF-kappaB induction in S107 cells using various stimuli (LPS, PMA, UV, X-rays, H2O2).
- Assessment of AP-1 transcription factor induction to rule out general signal transduction defects.
- Investigation of NF-kappaB activation using phosphatase inhibitors (okadaic acid, calyculin A) and MEKK1.
- Examination of NF-kappaB induction pathways sensitive or insensitive to pyrrolidine dithiocarbamate (PDTC).
- Characterization of DNA-binding NF-kappaB complexes and reporter gene activity.
Main Results:
- S107 cells exhibit a specific defect impairing NF-kappaB induction by diverse stimuli, while AP-1 induction remains intact.
- NF-kappaB can be induced in S107 cells by phosphatase inhibitors and MEKK1, indicating pathway-specific defects.
- Tumor necrosis factor alpha (TNFalpha) and endoplasmic reticulum (ER) overload induce NF-kappaB via PDTC-sensitive pathways that are still functional in S107 cells.
- NF-kappaB complexes primarily consist of p50-RelA heterodimers, leading to reporter gene activation.
Conclusions:
- The S107 cell line harbors a mutation at a critical convergence point in multiple NF-kappaB signaling pathways.
- Alternative NF-kappaB induction routes, notably those activated by TNFalpha and ER stress, bypass this mutated step and remain functional.
- This study provides a valuable tool for dissecting the complex mechanisms of NF-kappaB regulation and signaling.