Biochemical and Biophysical Research Communications
β-Catenin/TCF/LEF1 can directly regulate phenylephrine-induced cell hypertrophy and Anf transcription in cardiomyocytes
Introduction
In response to physiological or pathological stimuli, cardiomyocytes undergo hypertrophy, characterized by increased cell size, fetal gene re-expression and enhancement of protein synthesis [1], [2]. In cardiomyocyte hypertrophy induced by different stimuli, the transcription of the fetal gene Anf is universally up-regulated. Anf transcription is thus a useful tool for understanding the transcriptional regulation of myocardial genes following hypertrophic stimulation, and for comparing the different signaling pathways and molecules involved in hypertrophy induced by different stimuli [1], [2], [3], [4], [5].
β-Catenin has been extensively studied in the Wnt/β-catenin signaling pathway [6]. The content of β-catenin in the cytosol is tightly regulated by a destruction complex consisting of Axin, the adenomatous polyposis coli gene product (APC), casein kinase I (CKI) and GSK-3β[7]. GSK-3β phosphorylates β-catenin, leading to its degradation by the ubiquitin-proteasome system. Inhibition of GSK-3β activity by Wnt or other pathways results in stabilization of β-catenin and its translocation from the cytosol into the nucleus where it partners with T-cell factor (TCF) and lymphoid enhancer factor (LEF) family members to induce transactivation of genes containing TCF/LEF1 binding elements in their promoters [8].
Recent studies showed the involvement of β-catenin in the development of cardiomyocyte hypertrophy [8], [9], [10], [11]. It could be stabilized by stimulation with phenylephrine (PE), functioning as a positive regulator for cardiomyocyte hypertrophy dependent on its transcriptional activity [9]. However, how β-catenin exerts its activity and whether hypertrophic responsive genes can be regulated by the β-catenin/TCF/LEF1 complex is largely unknown.
In this study, we evaluated the effects of knockdown of β-catenin on PE-induced cardiomyocyte hypertrophy and found that the up-regulation of Anf transcription was attenuated. We went on to explore the mechanism of how β-catenin regulates the transcription of Anf under hypertrophic stimulation.
Section snippets
Materials and methods
Cell culture. Neonatal rat ventricular myocytes (NRVMs) were prepared from 1–2-day-old Sprague–Dawley as previously described rats [12]. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL) supplemented with 15% fetal bovine serum for 48 h before the culture media were changed to serum-free medium and kept for another 24 h before further experiments.
HeLa cells were maintained in DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100
Knockdown of β-catenin attenuates PE-induced cardiomyocyte hypertrophy and Anf transcription
Several lines of evidence suggest a positive role for β-catenin in the process of cardiac hypertrophy [8], [9], [10], [11]. To further document the role of β-catenin, knockdown of β-catenin in NRVMs was employed. With transfection of siRNAs against β-catenin, the mRNA and protein levels of this molecule were both dramatically reduced (Fig. 1A and B). With knockdown of β-catenin, protein synthesis in response to PE stimulation was significantly inhibited (Fig. 1C). Cell area increase was also
Discussion
In this study, we investigated the effects of knockdown of β-catenin on PE-induced cardiomyocyte hypertrophy and found that protein synthesis and cell area increase were greatly attenuated. Meanwhile, up-regulation of the Anf gene was inhibited. As a transactivator, β-catenin generally exerts its function via TCF/LEF1 family members binding to a consensus sequence on the promoters of target genes [18], [19], [20]. This observation prompted us to investigate whether Anf could be a target of
Acknowledgments
We thank Drs. Nemer M., Wolff K., and Nusse R. for providing the corresponding plasmid constructs. We are grateful to Dr. Wong J., University of Cambridge, UK for his kind help in the preparation of this manuscript. This work was supported by the National Natural Sciences Foundation of China (30871253, 90919022) and the 111 Project of Ministry of Education of China (B07001).
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Chen-Guang Zhang and Zhu-Qing Jia contributed equally to this work.