Induction of relA(p65) and I kappa B alpha subunit expression during differentiation of human peripheral blood monocytes to macrophages
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L Conti1, J Hiscott, M Papacchini
1Laboratory of Virology, Istituto Superiore di Sanità, Rome, Italy.
Abstract:
We evaluated the expression and DNA binding activity of nuclear factor (NF)-kappa B subunits in human peripheral blood monocytes and in monocyte-derived macrophages (MDMs). Constitutive DNA binding activity consisting of p50 homodimers was detected in nuclear extracts from both cell types. An additional complex composed of p50/RelA(p65) heterodimers appeared only in nuclear extracts from 7-day MDMs. Immunoblot analysis showed that the p50 subunit was constitutively expressed in monocytes and MDMs. In contrast, the RelA(p65) subunit was barely detectable in monocytes, but its level increased markedly in MDMs. Analysis of RelA(p65) mRNA revealed that the stability of RelA(p65) mRNA was significantly higher in MDMs, compared with monocytes. In MDMs, an upregulation of I kappa B alpha synthesis as well as the appearance of a novel M(r) 40,000 form of I kappa B alpha were also observed. These results suggest that macrophage differentiation results in the expression of active p50/RelA(p65) heterodimers with the capacity to activate target gene expression. The parallel induction of I kappa B alpha synthesis may allow for the continuous presence of a cytoplasmic reservoir of p50/RelA(p65) complexes that are readily available for inducer-mediated stimulation.
Insights
Macrophage differentiation increases nuclear factor (NF)-kappa B activity by upregulating RelA(p65) expression and stabilizing its mRNA. This leads to active NF-kappa B heterodimers ready for immune responses.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Nuclear factor (NF)-kappa B is a key transcription factor regulating immune responses.
- Monocytes differentiate into macrophages, undergoing significant cellular changes.
- Understanding NF-kappa B dynamics during differentiation is crucial for immune regulation.
Purpose of the Study:
- To investigate the expression and DNA binding activity of NF-kappa B subunits during monocyte to macrophage differentiation.
- To elucidate the molecular mechanisms underlying changes in NF-kappa B activity in macrophages.
Main Methods:
- Analysis of NF-kappa B subunit expression using immunoblotting.
- Assessment of DNA binding activity via nuclear extracts.
- Quantification of RelA(p65) mRNA stability.
- Evaluation of I kappa B alpha synthesis and forms.
Main Results:
- Constitutive p50 homodimer activity was found in both monocytes and macrophages.
- p50/RelA(p65) heterodimer activity emerged in 7-day macrophages.
- RelA(p65) subunit expression and mRNA stability significantly increased in macrophages.
- Macrophage differentiation induced I kappa B alpha synthesis and a novel M(r) 40,000 form.
Conclusions:
- Macrophage differentiation promotes the expression of active p50/RelA(p65) heterodimers.
- These activated heterodimers possess the capacity for target gene activation.
- Induced I kappa B alpha synthesis may maintain readily available cytoplasmic NF-kappa B complexes for stimulation.