CD14-independent activation of cardiomyocyte signal transduction by bacterial endotoxin.
In the heart, lipopolysaccharide (LPS) induces the production of proinflammatory cytokines that cause myocardial dysfunction; however, the signaling pathways involved in cardiomyocyte responses are poorly understood. We studied LPS-induced signaling by treating cardiomyocyte cultures with 0.01-10 microgram/ml LPS for 0-24 h in the presence or absence of 2.5% serum. Cytosolic and nuclear proteins were analyzed for expression and activation of protein kinases. Members of the extracellular signal-regulated kinase (ERK) and signal transducer and activators of transcription (STAT) protein families were uniformly expressed and specifically phosphorylated in response to LPS. Activation was biphasic; peaking at 5-10 min and 24 h after treatment. Inhibitor experiments provided evidence that ERK proteins may regulate STAT activity. Serum did not augment endotoxin-induced phosphorylation. Although cardiomyocytes expressed low levels of CD14 and LPS-binding protein, specific enzymatic removal of glycosyl phosphatidylinositol-linked receptors or incubation with an anti-CD14 antibody had no effect on kinase activation. Treatment of cells with an excess of detoxified LPS attenuated endotoxin-induced signaling. In addition, endotoxin stimulated specific binding of nuclear factors to AP-1, nuclear factor-kappaB (NF-kappaB), STAT1 (SIE, sis-inducible element), and STAT3 consensus-binding sequences. Finally, inhibition of ERK phosphorylation reduced, and NF-kappaB nuclear translocation prevented, tumor necrosis factor-alpha production. Our results indicate that LPS-induced activation of signal transduction in cardiomyocytes occurs by a CD14-independent mechanism.
Cowan
D B
DB
Department of Anesthesia, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA. cowan_d@a1.tch.harvard.edu
Poutias
D N
DN
Del Nido
P J
PJ
McGowan
F X
FX
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D006321:Heart / Q000187:drug effects / Q000502:physiology*; D018950:Lipopolysaccharide Receptors / Q000502:physiology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009206:Myocardium / Q000166:cytology; D016328:NF-kappa B / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D012485:Salmonella typhi; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D015534:Trans-Activators / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism
D004268:DNA-Binding Proteins; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D016328:NF-kappa B; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; C494081:Stat1 protein, rat; C494088:Stat3 protein, rat; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D020928:Mitogen-Activated Protein Kinases