Related Experiment Videos
In vivo control of NF-kappa B activation by I kappa B alpha
1Laboratory of Molecular Virology and Carcinogenesis, NCI-Frederick Cancer Research and Development Center, Frederick, MD 21702-1201.
Insights
Lipopolysaccharide (LPS) activates B cells by causing the inhibitor protein I kappa B alpha to dissociate from NF-kappa B complexes, leading to active NF-kappa B in vivo. This process involves I kappa B alpha modification and ongoing cellular turnover.
Area of Science:
- Immunology
- Molecular Biology
- Cellular Signaling
Background:
- Nuclear Factor kappa B (NF-kappa B) is a transcription factor sequestered in the cytoplasm by inhibitor of kappa B alpha (I kappa B alpha).
- In vitro studies indicate that I kappa B alpha dissociation activates NF-kappa B.
Purpose of the Study:
- To investigate the in vivo mechanism of NF-kappa B activation by lipopolysaccharide (LPS) in B and pre-B cells.
- To elucidate the role of I kappa B alpha in LPS-induced NF-kappa B signaling.
Main Methods:
- In vivo studies using B and pre-B cells treated with LPS.
- Immunoblotting and DNA binding assays to detect NF-kappa B components and activity.
- Analysis of I kappa B alpha and NF-kappa B complex dynamics.
Main Results:
- LPS treatment caused I kappa B alpha dissociation from NF-kappa B complexes in B and pre-B cells.
- Translocated NF-kappa B dimers were detected in the nucleus, increasing c-rel, p65, and p50 levels.
- Newly synthesized I kappa B alpha retained some dissociated NF-kappa B in the cytoplasm, suggesting I kappa B alpha modification is key.
- A continuous turnover of complexed I kappa B alpha was observed in unstimulated cells.
Conclusions:
- LPS-induced NF-kappa B activation occurs via dissociation of I kappa B alpha, not by preventing complex formation.
- The dissociation is likely due to I kappa B alpha modification, not changes in NF-kappa B subunits.
- Ongoing I kappa B alpha turnover in unstimulated cells maintains basal NF-kappa B levels, while rapid synthesis allows prompt signal termination.
Abstract:
The transcription factor NF-kappa B is stored in the cytoplasm in complexes with the inhibitor protein I kappa B alpha. It has been shown in vitro that dissociation of I kappa B alpha from these complexes results in active NF-kappa B. In this report we show that lipopolysaccharide (LPS)-induced activation of B or pre-B cells results in loss of I kappa B alpha from NF-kappa B complexes in vivo. Many liberated NF-kappa B dimers reached the nucleus, where increased c-rel, p65 and p50 were detected by immunoblotting and by DNA binding assays. Some liberated dimers were retained in the cytoplasm, however, through binding to newly synthesized I kappa B alpha, a finding which strongly suggests (i) that the LPS-induced signal causes dissociation of complexes rather than preventing their association and (ii) that dissociation results from modification of I kappa B alpha and not of c-rel or p65. No effect of LPS treatment was detected on p105 or p100, which also retain rel family members in the cytoplasm. Quite unexpectedly, we also found that in unstimulated cells there is a constant ongoing process of degradation and replacement of complexed I kappa B alpha. We propose that this turnover results in the low level of active NF-kappa B presumably necessary even in the unstimulated cell, and that the high rate of synthesis of I kappa B alpha provides the ability to turn off NF-kappa B activity rapidly as soon as the activating signal ceases.