CD14-independent activation of cardiomyocyte signal transduction by bacterial endotoxin

Departments of 1 Anesthesia and 2 Cardiac Surgery, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115 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 µg/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- B (NF- B), STAT1 (SIE, sis-inducible element), and STAT3 consensus-binding sequences. Finally, inhibition of ERK phosphorylation reduced, and NF- B nuclear translocation prevented, tumor necrosis factor- production. Our results indicate that LPS-induced activation of signal transduction in cardiomyocytes occurs by a CD14-independent mechanism. lipopolysaccharide; kinase; receptor; phosphorylation; myocyte