Abstract 16547: RANKL Stimulates Expression of Proinflammatory Cytokines in Neonatal Rat Cardiomyocytes
Receptor activator of NF-kB ligand (RANKL) is a member of the TNF superfamily, best known for its role in regulating bone remodeling and the inflammatory response. Interest is growing in its role in cardiovascular pathophysiology. While enhanced myocardial RANKL levels have been reported in human heart failure, it remains unclear if RANKL directly regulates proinflammatory cytokines in cardiomyocytes. We have elucidated the signaling mechanism by which RANKL acts as a novel inducer of inflammatory cytokines in neonatal rat cardiomyocytes. Cardiomyocytes express the mRNA of the RANKL receptor, RANK. RANKL (0.1 -1 μg/ml) stimulated a 5- to 10-fold induction in TNFα, IL-1α and IL-1β mRNA expression in a concentration dependent manner (P <0.01) that corresponded with increased TNFα production by ELISA (P <0.01). These effects were significantly attenuated when the expression of TRAF6, the adaptor for RANK was silenced by siRNA. Inhibitors of PLC (10 μM U73122), PKC (5 μM chelerythrine chloride), or JNK (30 μM SP600125) also suppressed RANKL-induced expression of these cytokines but not inhibitors of PI3K (20 μM LY294002), MEK1/2 (10 μM PD98059), or p38 MAPK (10 μM SB203580). Moreover, RANKL induced IkB-α phosphorylation and RANKL-induced expression of cytokines was reduced to basal levels in the presence of IKK inhibition (30 μM). To confirm the involvement of AP1 and NF-kB in RANKL-induced TNFα, IL-1α, and IL-1β expression, we performed chromatin immunoprecipitation (ChIP) assay using anti-p65 and anti-c-jun antibodies, which clearly demonstrated that RANKL treatment increased AP1 and NF-kB binding to the TNFα, IL-1α and IL-1β promoters in cardiomyocytes. These data demonstrate that RANKL induces TNFα, IL-1α and IL-1β expression via JNK/PKC-mediated AP1/NF-kB activation in cardiomyocytes and reveal a novel mechanism by which RANKL may promote inflammation during heart failure.
- © 2010 by American Heart Association, Inc.