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Bacterial peptidoglycan induces CD14-dependent activation of transcription factors CREB/ATF and AP-1
1Northwest Center for Medical Education, Indiana University School of Medicine, Gary, Indiana 46408, USA. dgupta@meded.iun.indiana.edu
Abstract:
Peptidoglycan (PGN), the major cell wall component of Gram-positive bacteria, induces secretion of cytokines in macrophages through CD14, the pattern recognition receptor that binds lipopolysaccharide and other microbial products. To begin to elucidate the mechanisms that regulate the transcription of cytokine genes, we wanted to determine which transcription factors are activated by PGN in mouse RAW264.7 and human THP-1 macrophage cells. Our results demonstrated that: (i) PGN induced phosphorylation of the transcription factors ATF-1 and CREB; (ii) ATF-1 and CREB bound DNA as a dimer and induced transcriptional activation of a CRE reporter plasmid, which was inhibited by dominant negative CREB and ATF-1; (iii) PGN induced phosphorylation of c-Jun, protein synthesis of JunB and c-Fos, and transcriptional activation of the AP-1 reporter plasmid, which was inhibited by dominant negative c-Fos; and (iv) PGN-induced activation of CREB/ATF and AP-1 was mediated through CD14. This is the first study to demonstrate activation of CREB/ATF and AP-1 transcription factors by PGN or by any other component of Gram-positive bacteria.
Insights
Peptidoglycan (PGN) from Gram-positive bacteria activates transcription factors CREB/ATF and AP-1 in macrophages via CD14. This study reveals novel mechanisms of bacterial-induced immune responses.
Area of Science:
- Immunology
- Microbiology
- Molecular Biology
Background:
- Peptidoglycan (PGN) is a major bacterial cell wall component.
- Macrophages utilize CD14 to recognize microbial products like PGN, initiating immune responses.
- Understanding cytokine gene regulation is crucial for deciphering immune signaling pathways.
Purpose of the Study:
- To identify transcription factors activated by PGN in macrophages.
- To elucidate the role of CD14 in PGN-mediated transcription factor activation.
- To investigate the signaling pathways regulating cytokine gene expression upon PGN stimulation.
Main Methods:
- Utilized mouse RAW264.7 and human THP-1 macrophage cell lines.
- Assessed transcription factor phosphorylation and DNA binding activity.
- Employed reporter gene assays for CRE and AP-1 elements.
- Investigated the role of CD14 using specific inhibitors and dominant-negative constructs.
Main Results:
- PGN induced phosphorylation of ATF-1 and CREB, leading to CRE reporter activation.
- PGN triggered c-Jun phosphorylation and JunB/c-Fos synthesis, activating AP-1 reporter.
- Dominant-negative inhibitors blocked PGN-induced transcriptional activation.
- CD14 mediated PGN-induced activation of both CREB/ATF and AP-1 pathways.
Conclusions:
- This study demonstrates, for the first time, PGN-induced activation of CREB/ATF and AP-1 transcription factors.
- CD14 is essential for mediating PGN-induced activation of these key transcription factors.
- These findings provide new insights into the molecular mechanisms of Gram-positive bacterial recognition and immune signaling.