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Updated: Aug 28, 2026
Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation
Published on: April 29, 2015
cAMP induces CD14 expression in murine macrophages via increased transcription
S Liu1, S M Morris, S Nie
1Department of Surgery, University of Pittsburgh School of Medicine, Pennsylvania, USA. shubing@pitt.edu
Abstract:
CD14, a glycoprotein that binds bacterial lipopolysaccharide, plays a critical role in the inflammatory response to infection by gram-negative bacteria. Studies were undertaken to determine whether cyclic adenosine monophosphate (cAMP) regulates CD14 expression in macrophages. Incubation of RAW 264.7 cells with 8-Br-cAMP resulted in a significant increase in steady-state CD14 mRNA levels. The increase in mRNA levels was also associated with both cell-associated and soluble CD14 protein. H89 completely blocked the 8-Br-cAMP-induced CD14 mRNA up-regulation. There was no change in CD 14 mRNA half-life in the presence of 8-Br-cAMP. The CD14 gene transcription rate was increased about twofold after exposure to 8-Br-cAMP. cAMP-dependent increases in CD14 mRNA were also observed in rat peritoneal macrophages, demonstrating that this is an authentic response of mature macrophages. This study provides evidence that cAMP and protein kinase A are important regulators of CD14 expression in macrophages.
Insights
Cyclic adenosine monophosphate (cAMP) significantly increases CD14 expression in macrophages by enhancing gene transcription. This finding reveals cAMP and protein kinase A as key regulators of CD14, crucial for inflammatory responses.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- CD14 is a glycoprotein vital for inflammatory responses to Gram-negative bacterial infections.
- CD14 binds bacterial lipopolysaccharide, initiating immune signaling pathways.
- Understanding CD14 regulation is crucial for managing infections and inflammatory diseases.
Purpose of the Study:
- To investigate the role of cyclic adenosine monophosphate (cAMP) in regulating CD14 expression in macrophages.
- To elucidate the molecular mechanisms by which cAMP influences CD14 levels.
- To determine if cAMP-mediated regulation of CD14 is conserved in primary macrophages.
Main Methods:
- RAW 264.7 macrophage cell line and primary rat peritoneal macrophages were used.
- Cells were treated with 8-Br-cAMP (a cAMP analog) and H89 (a protein kinase A inhibitor).
- Quantitative analysis of CD14 mRNA levels, CD14 protein expression, mRNA half-life, and gene transcription rates was performed.
Main Results:
- 8-Br-cAMP treatment significantly increased both CD14 mRNA and protein levels in RAW 264.7 cells.
- The protein kinase A inhibitor H89 blocked the cAMP-induced upregulation of CD14 mRNA.
- cAMP exposure led to a twofold increase in CD14 gene transcription rate, with no change in mRNA half-life.
- Similar cAMP-dependent increases in CD14 mRNA were observed in primary rat macrophages.
Conclusions:
- Cyclic adenosine monophosphate (cAMP) is a significant positive regulator of CD14 expression in macrophages.
- The cAMP-dependent regulation of CD14 involves the activation of protein kinase A and increased gene transcription.
- These findings highlight a novel pathway for controlling CD14 expression, relevant to inflammatory and infectious diseases.
