Abstract 39: Identification of a Novel Tyrosine Kinase Family Involved in Urocortin-Mediated Cardioprotection
Background: Urocortin (Ucn) is a 40 amino acid peptide member of the corticortrophin-releasing hormone family. Endogenous cardiac Ucn expression is enhanced by ischemia/reperfusion injury in vitro, and addition of exogenous urocortin reduces cell death caused by ischemia/reperfusion in vitro, ex vivo and in vivo. Studies have also shown that a subfamily of MAP kinase, p42/p44 (also known as ERK1/2), is rapidly phosphorylated by Ucn and that activation of ERK1/2 is required for Ucn-mediated cardioprotective effects in vitro and ex vivo.
Aim: To investigate the molecular mechanism by means of which Ucn induces activation of ERK1/2.
Methods and Results: We first analyzed global changes in gene expression in cardiac cells exposed to 24 hours treatment with Ucn (10nM), using Affymetrix gene chip technology. Significantly, mRNA expression of Fyn, a member of Src tyrosine kinase family, was 2-fold up-regulated. Western blot analysis using antibody against total and active (phosphorylated) Src was subsequently performed. Incubation of 10 nM Ucn for 1, 5 and 15 minutes with immortalized mouse atrial HL-1 cells was sufficient to activate Src kinase, though not to induce its overexpression. Src phosphorylation, which occurs at the level of a specific tyrosine residue of Src, corresponding to Tyr416 of human Src, started after 1 min, peaked after 5 mins and declined after 15 mins incubation. Likewise, incubation of HL-1 cells with Ucn (10 nM) also induced phosphorylation, though not upregulation, of ERK1/2, whose activation, in accordance with the previous findings, started after 1 min and kept stable following 5 and 15 mins incubation. Importantly, a specific Src family kinase inhibitor, PP2, significantly reduced Ucn-induced phosphorylation of ERK1/2, suggesting that Src is an upstream modulator of ERK1/2 activation.
Conclusions: We report for the first time that short-term treatment with Ucn induces acute phosphorylation of Src tyrosine kinase family. This kinase family acts as an upstream modulator of ERK1/2, whose activation is involved in Ucn-mediated cardioprotection. Further studies are required to investigate the role of Src in Ucn-mediated cardioprotection in animal models of ischemia/reperfusion injury.