Implication of Galpha i 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 lipopolysacchaide (LPS) signaling events. Signal transduction pathways activated by LPS we examined in human pomonocytic THP-l cells. We hypothesized that Gi proteins and Src tyrosine kinase differentially affect mitogen-activated protein (MAP) kinases (MAPK) and nuclear factor kappa(NF-kappaB) activation. Post-receptor coupling to Ga, 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 (TN-alpha) and thromboxane B2 (TXB2). Pretreatment with PP2 inhibited TNF-alpha and TxB2 production, but had no effect on p38 kinase or JNK signaling. Therefore, the Ga i-coupled signaling pathways and Src tyrosine kinase-coupled signaling pathways are necessary for LPS-induced TNF-alpha and TxB2 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 tansactivation of genes following DNA binding. PTx had no effect on NF-kaapaB 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-kaapaB DNA binding.
Ferlito
Marcella
M
Department of Physiology and Neuroscience, Medical University of South Carolina, Charleston 29425, USA.
Romanenko
Olga G
OG
Guyton
Kelly
K
Ashton
Sarah
S
Squadrito
Francesco
F
Halushka
Perry V
PV
Cook
James A
JA
D018929:Cell Culture Techniques; D004247:DNA / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000494:pharmacology*; D006801:Humans; D008070:Lipopolysaccharides / Q000009:adverse effects / Q000378:metabolism / Q000494:pharmacology*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000494:pharmacology; D009000:Monocytes; D016328:NF-kappa B / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction; D013929:Thromboxane B2 / Q000096:biosynthesis; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D019061:src-Family Kinases / Q000494:pharmacology*
D008070:Lipopolysaccharides; D016328:NF-kappa B; D011720:Pyrazoles; D011725:Pyridines; D014409:Tumor Necrosis Factor-alpha; C118531:pyrazolopyridine; D013929:Thromboxane B2; D004247:DNA; D019061:src-Family Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go