Implication of Gαi proteins and Src tyrosine kinases in endotoxin-induced signal transduction events and mediator production

Previous studies have suggested that heterotrimeric G proteins and tyrosine kinases may be involved in lipopolysaccharide (LPS) signaling events. Signal transduction pathways activated by LPS were examined in human promonocytic THP-1 cells. We hypothesized that G(i) proteins and Src tyrosine kinase differentially affect mitogen-activated protein (MAP) kinases (MAPK) and nuclear factor kappa (NF-kappaB) activation. Post-receptor coupling to Galpha(i) proteins were examined using pertussis toxin (PTx), which inhibits Galpha(i) receptor-coupling. The involvement of the Src family of tyrosine kinases was examined using the selective Src tyrosine kinase inhibitor pyrazolopyrimidine-2 (PP2). Pretreatment of THP-1 cells with PTx attenuated LPS-induced activation of c-Jun-N-terminal kinase (JNK) and p38 kinase, and production of tumor necrosis factor-alpha (TNF-alpha) and thromboxane B-2 (TxB(2)). Pretreatment with PP2 inhibited TNF-alpha and TxB(2) production, but had no effect on p38 kinase or JNK signaling. Therefore, the Galpha(i)-coupled signaling pathways and Src tyrosine kinase-coupled signaling pathways are necessary for LPS-induced TNF-alpha and TxB(2) production, but differ in their effects on MAPK activation. Neither PTx nor PP2 inhibited LPS-induced activation of interleukin receptor activated kinase (IRAK) or inhibited translocation of NF-kappaB. However, PP2 inhibited LPS-induced NF-kappaB transactivation of a luciferase reporter gene construct in a concentration-dependent manner. Thus, LPS induction of Src tyrosine kinases may be essential in downstream NF-kappaB transactivation of genes following DNA binding. PTx had no effect on NF-kappaB activation of the reporter construct. These data suggest upstream divergence in signaling through Galpha(i) pathways leading to MAPK activation and other signaling events leading to IkappaBalpha degradation and NF-kappaB DNA binding.