Abstract 135: Subnanomolar Concentrations of Urocortin Induce Expression and Release of IL-6 Leading to STAT3 Activation
Background: We recently showed that short-term treatment with urocortin (Ucn) of HL-1 cells induces Src-mediated phosphorylation and subsequent nuclear translocation of Signal Transducer and Activator of Transcription (STAT)3. In breast cancer cells, STAT3 was found to be activated by IL-6, while Ucn was shown to stimulate IL-1β and IL-6 secretion from mononuclear inflammatory cells.
Aim: To verify whether cardiac myocytes can express and secrete IL-6, and, if so, to investigate the role of IL-6 in Ucn-mediated STAT3 activation.
Methods and Results: Immortalized mouse atrial HL-1 cardiomyocytes were cultured in Claycomb medium supplemented with 10% fetal bovine serum, before being serum-starved for 16 to 20 hours. IL-6 enzyme-linked immunosorbent assay (ELISA) of the conditioned medium from starved HL-1 cells showed that 16 and 24 hours treatment with 10 nM Ucn increased the secretion of IL-6 to 162.9±8.0% and 235.3±34.5%, respectively, as compared to control cells. Conversely, Ucn did not induce the release in the culture medium of IL-1β, as documented by IL-1β ELISA. Quantitative real-time PCR also confirmed the increased expression of IL-6 at the mRNA level. Interestingly, a concentration of Ucn as low as 0.01 nM, which was not sufficient to activate Src kinase, could indeed induce the expression and secretion of IL-6 by 275.7±9.4%. Moreover, transAM STAT3 transcription factor assay (ELISA) and Western blot analysis of HL-1 nuclear extracts showed that 100 nM Ucn increased the nuclear translocation of STAT3 by 132.2±10.9% and 2.6 folds, respectively. STAT3 nuclear translocation was significantly reduced, though not abrogated, by incubation of HL-1 cells with 2 μ g/ml of anti-IL-6 polyclonal antibody (109.1±10.2% and 1.1 folds, respectively).
Conclusions: We report for the first time that incubation of HL-1 cells with subnanomolar concentrations of Ucn induced the expression and secretion of IL-6, which resulted in STAT3 activation via an autocrine/paracrine mechanism, independent from Src activation.
- © 2011 by American Heart Association, Inc.