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Signal transduction events in Chinese hamster ovary cells expressing human CD14; effect of endotoxin desensitization
M Ferlito1, F Squadrito, P V Halushka
1Department of Physiology and Neuroscience, Medical University of South Carolina, Charleston 29425, USA.
Abstract:
Previous studies suggest that endotoxin (LPS) stimulation of CD14 receptors may be coupled to heterotrimeric G proteins. However, characterization of the G protein-coupled signaling pathways is incomplete. Also, specific changes in the transduction pathways occur in a phenomenon known as LPS tolerance or desensitization induced by prior exposure to LPS. In the present study, we examined potential CD14-dependent G protein-coupled signaling events in response to LPS, and changes in signaling in these pathways during LPS desensitization in Chinese Hamster Ovary (CHO) cells. LPS stimulated inhibitory kappa B alpha (IkappaB alpha) degradation and p38 phosphorylation in CHO cells transfected with human CD14 receptor (CHO-CD14), but not in CHO cells transfected with vector only. However, activation of these signaling events diverged early in the signal transduction pathways. Pretreatment with pertussis toxin, which inactivates inhibitor G protein (G alpha i) function, significantly inhibited LPS-induced p38 phosphorylation, but not LPS-induced IkappaB alpha degradation. Mastoparan, a putative G alpha i agonist, synergized with LPS to induce p38 phosphorylation. Thus, LPS stimulation of p38 phosphorylation is, in part, G alpha i coupled, whereas IkappaB alpha degradation is not. In subsequent studies, CHO-CD14 cells were desensitized by prior LPS exposure. LPS-desensitized cells exhibited augmented IkappaB alpha content and were refractory to LPS-induced IkappaB alpha degradation and p38 phosphorylation. Pretreatment with cycloheximide, a protein synthesis inhibitor, prevented the effect of LPS desensitization on augmenting cellular IkappaB alpha content and its refractoriness to LPS-induced degradation. However, cycloheximide pretreatment did not prevent impaired p38 phosphorylation in desensitized cells. IkappaB alpha upregulation in LPS tolerance may occur through increased synthesis and/or induction of protein that suppress IkappaB alpha degradation. The latter protein synthesis-dependent mechanisms may be distinct from mechanismis inhibiting p38 phosphorylation in tolerance. These findings suggest that LPS tolerance induces CD14-dependent signaling alterations in G alpha i-coupled pathways leading to mitogen-activated (MAP) kinase activation as well as G alpha i-independent pathways inducing IkappaB alpha degradation.
Insights
Lipopolysaccharide (LPS) triggers distinct signaling pathways involving G proteins and CD14 receptors. LPS tolerance impairs these pathways, affecting G protein-coupled signaling and mitogen-activated protein kinase activation.
Area of Science:
- Immunology
- Cell Signaling
- Molecular Biology
Background:
- Endotoxin (lipopolysaccharide, LPS) interaction with CD14 receptors is potentially linked to G protein signaling.
- The precise G protein-coupled signaling pathways and their modulation during LPS tolerance remain incompletely characterized.
- LPS tolerance, a state of desensitization after prior LPS exposure, involves specific changes in cellular signal transduction.
Purpose of the Study:
- To investigate CD14-dependent G protein-coupled signaling events initiated by LPS.
- To examine alterations in these signaling pathways during LPS-induced desensitization.
- To elucidate the roles of specific G proteins in LPS-mediated signaling and tolerance.
Main Methods:
- Utilized Chinese Hamster Ovary (CHO) cells transfected with human CD14 (CHO-CD14) and control vector-only cells.
- Assessed LPS-induced inhibitory kappa B alpha (IkappaB alpha) degradation and p38 phosphorylation.
- Employed pertussis toxin (G alpha i inhibitor) and mastoparan (G alpha i agonist) to probe G protein involvement.
- Investigated signaling in LPS-desensitized CHO-CD14 cells, with and without cycloheximide pretreatment.
Main Results:
- LPS stimulated IkappaB alpha degradation and p38 phosphorylation in CHO-CD14 cells, but not control cells.
- Pertussis toxin inhibited LPS-induced p38 phosphorylation, indicating G alpha i coupling, but did not affect IkappaB alpha degradation.
- Mastoparan synergized with LPS to enhance p38 phosphorylation.
- LPS-desensitized cells showed increased IkappaB alpha and were refractory to LPS-induced IkappaB alpha degradation and p38 phosphorylation.
- Cycloheximide prevented IkappaB alpha upregulation and refractoriness in desensitized cells, but not impaired p38 phosphorylation.
Conclusions:
- LPS-induced p38 phosphorylation is partially coupled to G alpha i, while IkappaB alpha degradation is not.
- LPS tolerance involves protein synthesis-dependent mechanisms that upregulate IkappaB alpha and inhibit its degradation.
- Distinct mechanisms underlie the inhibition of p38 phosphorylation and IkappaB alpha degradation during LPS tolerance.
- LPS tolerance induces CD14-dependent alterations in both G alpha i-coupled and G alpha i-independent signaling pathways.