Signal transduction events in Chinese hamster ovary cells expressing humanCD14; 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 chan ges in the transduction pathways occur in a phenomenon known as LPS toleran ce or desensitization induced by prior exposure to LPS, In the present stud y, we examined potential CD14-dependent G protein-coupled signaling events in response to LPS, and changes in signaling in these pathways during LPS d esensitization in Chinese Hamster Ovary (CHO) cells. LPS stimulated inhibit ory kappa B alpha (I kappaB alpha) degradation and p38 phosphorylation in C HO cells transfected with human CD14 receptor (CHO-CD14), but not in CHO ce lls transfected with vector only. However, activation of these signaling ev ents diverged early in the signal transduction pathways. Pretreatment with pertussis toxin, which inactivates inhibitor G protein (G alphai) function, significantly inhibited LPS-induced p38 phosphorylation, but not LPS-induc ed I kappaB alpha degradation. Mastoparan, a putative G alphai aganist, syn ergized with LPS to induce p38 phosphorylation. Thus, LPS stimulation of p3 8 phosphorylation is, in part, G alphai coupled, whereas I kappaB alpha deg radation is not. In subsequent studies, CHO-CD14 cells were desensitized by prior LPS exposure. LPS-desensitized cells exhibited augmented I kappaB al pha content and were refractory to LPS-induced I kappaB alpha degradation a nd p38 phosphorylation. Pretreatment with cycloheximide, a protein synthesi s inhibitor, prevented the effect of LPS desensitization on augmenting cell ular I kappaB alpha content and its refractoriness to LPS-induced degradati on. However, cycloheximide pretreatment did not prevent impaired p38 phosph orylation in desensitized cells. I kappaB alpha upregulation in LPS toleran ce may occur through increased synthesis and/or induction of protein that s uppress I kappaB 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-depend ent signaling alterations in G alphai-coupled pathways leading to mitogen-a ctivated (MAP) kinase activation as well as G alphai-independent pathways i nducing I kappaB alpha degradation.