Related Experiment Videos
Lipopolysaccharide induces Egr-1 mRNA and protein in murine peritoneal macrophages
D L Coleman1, A H Bartiss, V P Sukhatme
1Infectious Disease Section, Yale University School of Medicine, West Haven, CT.
Abstract:
Bacterial LPS has diverse effects on the function of immune cells, in general, and macrophages, in particular. The intracellular molecular events that mediate the effects of LPS are unclear. We undertook a series of studies in thioglycollate-elicited murine peritoneal macrophages to evaluate the effect of LPS on expression of Egr-1, a member of the immediate early response gene family. Egr-1 may function as an intranuclear "third messenger" because it is rapidly induced in a variety of cell types and encodes a 75- to 80-kDa nuclear phosphoprotein that activates transcription of genes containing the DNA consensus sequence GCGGGGGCG. LPS from Salmonella minnesota Re595 induced a maximal increase in Egr-1 mRNA in macrophages after 30 to 60 min of incubation that returned to baseline level by 120 min. LPS increased Egr-1 mRNA at 0.01 to 0.1 ng/ml with a maximal effect at 10 to 100 ng/ml. LPS markedly increased the transcription rate of Egr-1 by 10 min of incubation using nuclear run on analysis. Using a polyclonal anti-Egr-1 antibody, nuclear staining for Egr-1 protein was prominent after 1 to 2 h of incubation with LPS and declined to baseline by 4 h. Inasmuch as protein kinase C (PKC) has been implicated in mediating the effects of LPS, we determined whether PKC was required for LPS to increase Egr-1 mRNA. Two pharmacologic approaches were used to deplete PKC, PMA pretreatment, and H-7. The induction of Egr-1 mRNA by LPS was markedly reduced in PKC-depleted macrophages. These data reveal that LPS induces transcriptional activation of Egr-1 and increases Egr-1 protein in peritoneal macrophages. In addition, these findings support further study of the potential role of Egr-1 in mediating the effects of LPS in peritoneal macrophages.
Insights
Bacterial lipopolysaccharide (LPS) increases early growth response gene (Egr-1) expression in macrophages. Protein kinase C (PKC) is required for LPS-induced Egr-1 mRNA, suggesting Egr-1
Area of Science:
- Immunology
- Molecular Biology
- Cellular Biology
Background:
- Bacterial lipopolysaccharide (LPS) significantly impacts immune cell function, particularly macrophages.
- The precise intracellular mechanisms by which LPS exerts its effects remain incompletely understood.
- Early growth response gene 1 (Egr-1) is an immediate early response gene implicated as a nuclear "third messenger".
Purpose of the Study:
- To investigate the effect of LPS on Egr-1 expression in murine peritoneal macrophages.
- To elucidate the role of protein kinase C (PKC) in LPS-mediated Egr-1 induction.
Main Methods:
- Murine peritoneal macrophages were treated with Salmonella minnesota Re595 LPS.
- Egr-1 mRNA and protein levels were assessed using techniques including nuclear run-on assays and Western blotting.
- Protein kinase C (PKC) activity was modulated using pharmacologic agents (PMA pretreatment and H-7) to assess its role.
Main Results:
- LPS induced a rapid and transient increase in Egr-1 mRNA levels in macrophages.
- LPS markedly enhanced Egr-1 gene transcription and increased nuclear Egr-1 protein levels.
- Depletion of PKC significantly reduced LPS-induced Egr-1 mRNA expression.
Conclusions:
- LPS induces transcriptional activation of the Egr-1 gene and subsequent protein synthesis in peritoneal macrophages.
- Protein kinase C (PKC) plays a crucial role in mediating LPS-induced Egr-1 expression.
- Egr-1 may be a key mediator of LPS effects in macrophages, warranting further investigation.