Abstract
Cardiotrophin-1 (CT-1) was recently isolated by expression cloning based on its ability to induce an increase in cell size in neonatal rat ventricular cardiomyocytes. Sequence similarity data suggested that CT-1 is a novel member of a family of structurally related cytokines sharing the receptor component gp130. The present study documents that gp130 is required for CT-1 signaling in cardiomyocytes, by demonstrating that a monoclonal anti-gp130 antibody completely inhibits c-fos induction by CT-1. Similarly, a leukemia inhibitory factor receptor subunit beta (LIFRbeta) antagonist effectively blocks the CT-1 induction of c-fos, indicating a requirement for LIFRbeta in the hypertrophic response, as well. Upon stimulation with CT-1, both gpl30 and the LIFRbeta are tyrosine-phosphorylated, providing further evidence that CT-1 signals through the gp130/LIFRbeta heterodimer in cardiomyocytes. CT-1 induces a hypertrophic response in cardiomyocytes that is distinct from the phenotype seen after alpha-adrenergic stimulation, both with regard to cell morphology and gene expression pattern. Stimulation with CT-1 results in an increase in cardiac cell size that is characterized by an increase in cell length but no significant change in cell width. Confocal laser microscopy of CT-1 stimulated cells reveals the assembly of sarcomeric units in series rather than in parallel, as seen after alpha-adrenergic stimulation. CT-1 induces a distinct pattern of immediate early genes, and up-regulates the atrial natriuretic factor (ANF) gene, but does not affect skeletal alpha-actin or myosin light chain-2v expression. As evidenced by nuclear run-on transcription assays, both CT-1 and alpha-adrenergic stimulation lead to an increase in ANF gene transcription. Transient transfection analyses document that, in contrast to alpha-adrenergic stimulation, the CT-1 responsive cis-regulatory elements are located outside of the proximal 3 kilobase pairs of the ANF 5'-flanking region. These studies indicate that CT-1 can activate a distinct form of myocardial cell hypertrophy, characterized by the promotion of sarcomere assembly in series, via gpl30/LIFRbeta-dependent signaling pathways.
Highlights
Cardiotrophin-1 (CT-1) was recently isolated by expression cloning based on its ability to induce an increase in cell size in neonatal rat ventricular cardiomyocytes
The cloning of CT-1 based on its ability to induce an increase in cell size in cardiomyocyte culture [19] suggested that gp130dependent signaling pathways may be coupled to cardiomyocyte hypertrophy
CT-1 Signaling through the gp130/LIFR Heterodimer— Cardiotrophin-1 was recently isolated by expression cloning based on its ability to induce an increase in cell size in cardiomyocyte culture [19]
Summary
Murine LIF was produced at Genentech, Inc. Murine LIF was used throughout the study, except for the experiment employing the LIFR antagonist (hLIF-04), in which both murine LIF and human LIF were used. Following three phosphate-buffered saline washes, the chamber slides were incubated in 3% bovine serum albumin for 10 min to block nonspecific sites. Luciferase activities were determined in duplicate samples from each plate; 20 l of cell lysate were combined with 100 l of luciferase assay buffer (100 mM Tricine, pH 7.8, 10 mM MgSO4, 2 mM EDTA, 2 mM ATP, 1 mM dithiothreitol, 73 M -luciferin), and luciferase activities were measured using a Monolight 401 luminometer (Analytical Luminescence Laboratory). -Galactosidase activities were determined in duplicate samples from each plate; 20 l of cell lysate were added to 250 l of -galactosidase assay buffer (0.1 M sodium phosphate, pH 7.3, 1.2 mM MgCl2, 60 mM -mercaptoethanol, 1.8 mM chlorophenol red--D-galactopyranoside (Boehringer Mannheim)), and incubated at 37 °C for 15 min. Statistical Analysis—Data are presented as means Ϯ S.E. p values were determined using one-way analysis of variance
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