Signal transduction events in Chinese hamster ovary cells expressing human CD14; effect of endotoxin desensitization.
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.
Ferlito
M
M
Department of Physiology and Neuroscience, Medical University of South Carolina, Charleston 29425, USA.
Squadrito
F
F
Halushka
P V
PV
Cook
J A
JA
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003513:Cycloheximide / Q000494:pharmacology; D004361:Drug Tolerance; D004789:Enzyme Activation / Q000187:drug effects; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000037:antagonists & inhibitors* / Q000502:physiology; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D018950:Lipopolysaccharide Receptors / Q000502:physiology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D020935:MAP Kinase Signaling System / Q000187:drug effects* / Q000502:physiology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010455:Peptides; D037342:Pertussis Toxin; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021122:Protein Subunits; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D014862:Wasp Venoms / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases
D020672:I-kappa B Proteins; D036341:Intercellular Signaling Peptides and Proteins; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D010455:Peptides; D021122:Protein Subunits; D011500:Protein Synthesis Inhibitors; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D010566:Virulence Factors, Bordetella; D014862:Wasp Venoms; C037500:mastoparan; D003513:Cycloheximide; D037342:Pertussis Toxin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C498015:GNAI2 protein, human; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D013755:Tetradecanoylphorbol Acetate