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Glycogen synthase kinase-3beta is a negative regulator of cardiomyocyte hypertrophy.

Haq S, Choukroun G, Kang ZB, Ranu H, Matsui T, Rosenzweig A, Molkentin JD, Alessandrini A, Woodgett J, Hajjar R, Michael A, Force T - J. Cell Biol. (2000)

Bottom Line: Hypertrophy is a basic cellular response to a variety of stressors and growth factors, and has been best characterized in myocytes.Pathologic hypertrophy of cardiac myocytes leads to heart failure, a major cause of death and disability in the developed world.Several cytosolic signaling pathways have been identified that transduce prohypertrophic signals, but to date, little work has focused on signaling pathways that might negatively regulate hypertrophy.

View Article: PubMed Central - PubMed

Affiliation: Cardiology Division, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02129-2060, USA.

ABSTRACT
Hypertrophy is a basic cellular response to a variety of stressors and growth factors, and has been best characterized in myocytes. Pathologic hypertrophy of cardiac myocytes leads to heart failure, a major cause of death and disability in the developed world. Several cytosolic signaling pathways have been identified that transduce prohypertrophic signals, but to date, little work has focused on signaling pathways that might negatively regulate hypertrophy. Herein, we report that glycogen synthase kinase-3beta (GSK-3beta), a protein kinase previously implicated in processes as diverse as development and tumorigenesis, is inactivated by hypertrophic stimuli via a phosphoinositide 3-kinase-dependent protein kinase that phosphorylates GSK-3beta on ser 9. Using adenovirus-mediated gene transfer of GSK-3beta containing a ser 9 to alanine mutation, which prevents inactivation by hypertrophic stimuli, we demonstrate that inactivation of GSK-3beta is required for cardiomyocytes to undergo hypertrophy. Furthermore, our data suggest that GSK-3beta regulates the hypertrophic response, at least in part, by modulating the nuclear/cytoplasmic partitioning of a member of the nuclear factor of activated T cells family of transcription factors. The identification of GSK-3beta as a transducer of antihypertrophic signals suggests that novel therapeutic strategies to treat hypertrophic diseases of the heart could be designed that target components of the GSK-3 pathway.

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Hypertrophic stimuli inhibit GSK-3β via phosphorylation of Ser 9 by a PI3-K-dependent protein kinase. A, ET-1 induces Ser 9 phosphorylation of GSK-3β by a PI3-K–dependent kinase. Cells were pretreated for 30 min with vehicle (DMSO), wortmannin (100 nM), or LY294002 (10 μM), and then were stimulated with vehicle (ET−) or ET-1 (ET+) for 40 min. Western blotting of whole cell lysates was performed with antiphospho Ser 9 GSK-3β and, to confirm that equivalent amounts of protein were loaded, with anti-GSK-3β. Experiment shown is representative of three. B, ET-1–induced inactivation of GSK-3β is PI3-K-dependent. Cardiomyocytes were pretreated for 30 min with wortmannin (100 nM) or vehicle (DMSO), and then were stimulated with ET-1 or vehicle for 40 min, followed by GSK-3β immune complex kinase assay. n = 3 experiments, assayed in duplicate. *P < 0.01 vs. control (ET−/WT−). #P < 0.01 vs. ET-1 alone (ET+/WT−). C, GSK-3βA9 is not inhibited by ET-1 in cardiomyocytes. Cells were transduced with AdGSK-3βA9 (A9) or AdGFP (GFP) at an MOI of 100 pfu/cell, or no virus (C). 36 hours later, cells were stimulated with vehicle or ET-1 for 30 min, lysates were prepared and anti-HA immune complex kinase assays were performed. Experiments were performed in parallel with the experiments presented in Fig. 1 A, demonstrating significant inhibition of GSK-3β by ET-1. *P < 0.01 vs. respective no virus control (C) and GFP virus control.
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Figure 2: Hypertrophic stimuli inhibit GSK-3β via phosphorylation of Ser 9 by a PI3-K-dependent protein kinase. A, ET-1 induces Ser 9 phosphorylation of GSK-3β by a PI3-K–dependent kinase. Cells were pretreated for 30 min with vehicle (DMSO), wortmannin (100 nM), or LY294002 (10 μM), and then were stimulated with vehicle (ET−) or ET-1 (ET+) for 40 min. Western blotting of whole cell lysates was performed with antiphospho Ser 9 GSK-3β and, to confirm that equivalent amounts of protein were loaded, with anti-GSK-3β. Experiment shown is representative of three. B, ET-1–induced inactivation of GSK-3β is PI3-K-dependent. Cardiomyocytes were pretreated for 30 min with wortmannin (100 nM) or vehicle (DMSO), and then were stimulated with ET-1 or vehicle for 40 min, followed by GSK-3β immune complex kinase assay. n = 3 experiments, assayed in duplicate. *P < 0.01 vs. control (ET−/WT−). #P < 0.01 vs. ET-1 alone (ET+/WT−). C, GSK-3βA9 is not inhibited by ET-1 in cardiomyocytes. Cells were transduced with AdGSK-3βA9 (A9) or AdGFP (GFP) at an MOI of 100 pfu/cell, or no virus (C). 36 hours later, cells were stimulated with vehicle or ET-1 for 30 min, lysates were prepared and anti-HA immune complex kinase assays were performed. Experiments were performed in parallel with the experiments presented in Fig. 1 A, demonstrating significant inhibition of GSK-3β by ET-1. *P < 0.01 vs. respective no virus control (C) and GFP virus control.

Mentions: Several mechanisms of inhibition of GSK-3 have been described. Insulin and IGF-1 inactivate GSK-3 via phosphorylation of a serine residue in the NH2-terminal region of the kinase (Ser 9 for GSK-3β and Ser 21 for GSK-3α; Stambolic and Woodgett 1994). This is mediated by a PI3-K-dependent kinase, possibly either PKB/Akt or the integrin-linked kinase (ILK; Cross et al. 1995; Delcommenne et al. 1998). Other mechanisms, including one mediated by Ca2+ and a Ca2+/calmodulin-dependent protein kinase kinase (Yano et al. 1998), and an ill-defined mechanism employed by the Wnt/wingless pathway, possibly involving protein kinase C (Cook et al. 1996), also inactivate GSK-3, but these pathways are not PI3-K-dependent and do not result in phosphorylation of Ser 9. Therefore, we determined the mechanism of inhibition of GSK-3β by hypertrophic stimuli. We found that ET-1 induced pronounced phosphorylation of GSK-3β on Ser 9, and that this phosphorylation was blocked by the PI3-K inhibitors, wortmannin or LY294002 (Fig. 2 A). The effect of the PI3-K inhibition on Ser 9 phosphorylation exactly correlated with the effect on GSK-3β kinase activity, since wortmannin prevented the ET-1–induced inactivation of GSK-3β (Fig. 2 B). These data strongly suggest that the ET-1–induced inhibition of GSK-3β is mediated via phosphorylation of Ser 9 by a PI3-K-dependent kinase.


Glycogen synthase kinase-3beta is a negative regulator of cardiomyocyte hypertrophy.

Haq S, Choukroun G, Kang ZB, Ranu H, Matsui T, Rosenzweig A, Molkentin JD, Alessandrini A, Woodgett J, Hajjar R, Michael A, Force T - J. Cell Biol. (2000)

Hypertrophic stimuli inhibit GSK-3β via phosphorylation of Ser 9 by a PI3-K-dependent protein kinase. A, ET-1 induces Ser 9 phosphorylation of GSK-3β by a PI3-K–dependent kinase. Cells were pretreated for 30 min with vehicle (DMSO), wortmannin (100 nM), or LY294002 (10 μM), and then were stimulated with vehicle (ET−) or ET-1 (ET+) for 40 min. Western blotting of whole cell lysates was performed with antiphospho Ser 9 GSK-3β and, to confirm that equivalent amounts of protein were loaded, with anti-GSK-3β. Experiment shown is representative of three. B, ET-1–induced inactivation of GSK-3β is PI3-K-dependent. Cardiomyocytes were pretreated for 30 min with wortmannin (100 nM) or vehicle (DMSO), and then were stimulated with ET-1 or vehicle for 40 min, followed by GSK-3β immune complex kinase assay. n = 3 experiments, assayed in duplicate. *P < 0.01 vs. control (ET−/WT−). #P < 0.01 vs. ET-1 alone (ET+/WT−). C, GSK-3βA9 is not inhibited by ET-1 in cardiomyocytes. Cells were transduced with AdGSK-3βA9 (A9) or AdGFP (GFP) at an MOI of 100 pfu/cell, or no virus (C). 36 hours later, cells were stimulated with vehicle or ET-1 for 30 min, lysates were prepared and anti-HA immune complex kinase assays were performed. Experiments were performed in parallel with the experiments presented in Fig. 1 A, demonstrating significant inhibition of GSK-3β by ET-1. *P < 0.01 vs. respective no virus control (C) and GFP virus control.
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Figure 2: Hypertrophic stimuli inhibit GSK-3β via phosphorylation of Ser 9 by a PI3-K-dependent protein kinase. A, ET-1 induces Ser 9 phosphorylation of GSK-3β by a PI3-K–dependent kinase. Cells were pretreated for 30 min with vehicle (DMSO), wortmannin (100 nM), or LY294002 (10 μM), and then were stimulated with vehicle (ET−) or ET-1 (ET+) for 40 min. Western blotting of whole cell lysates was performed with antiphospho Ser 9 GSK-3β and, to confirm that equivalent amounts of protein were loaded, with anti-GSK-3β. Experiment shown is representative of three. B, ET-1–induced inactivation of GSK-3β is PI3-K-dependent. Cardiomyocytes were pretreated for 30 min with wortmannin (100 nM) or vehicle (DMSO), and then were stimulated with ET-1 or vehicle for 40 min, followed by GSK-3β immune complex kinase assay. n = 3 experiments, assayed in duplicate. *P < 0.01 vs. control (ET−/WT−). #P < 0.01 vs. ET-1 alone (ET+/WT−). C, GSK-3βA9 is not inhibited by ET-1 in cardiomyocytes. Cells were transduced with AdGSK-3βA9 (A9) or AdGFP (GFP) at an MOI of 100 pfu/cell, or no virus (C). 36 hours later, cells were stimulated with vehicle or ET-1 for 30 min, lysates were prepared and anti-HA immune complex kinase assays were performed. Experiments were performed in parallel with the experiments presented in Fig. 1 A, demonstrating significant inhibition of GSK-3β by ET-1. *P < 0.01 vs. respective no virus control (C) and GFP virus control.
Mentions: Several mechanisms of inhibition of GSK-3 have been described. Insulin and IGF-1 inactivate GSK-3 via phosphorylation of a serine residue in the NH2-terminal region of the kinase (Ser 9 for GSK-3β and Ser 21 for GSK-3α; Stambolic and Woodgett 1994). This is mediated by a PI3-K-dependent kinase, possibly either PKB/Akt or the integrin-linked kinase (ILK; Cross et al. 1995; Delcommenne et al. 1998). Other mechanisms, including one mediated by Ca2+ and a Ca2+/calmodulin-dependent protein kinase kinase (Yano et al. 1998), and an ill-defined mechanism employed by the Wnt/wingless pathway, possibly involving protein kinase C (Cook et al. 1996), also inactivate GSK-3, but these pathways are not PI3-K-dependent and do not result in phosphorylation of Ser 9. Therefore, we determined the mechanism of inhibition of GSK-3β by hypertrophic stimuli. We found that ET-1 induced pronounced phosphorylation of GSK-3β on Ser 9, and that this phosphorylation was blocked by the PI3-K inhibitors, wortmannin or LY294002 (Fig. 2 A). The effect of the PI3-K inhibition on Ser 9 phosphorylation exactly correlated with the effect on GSK-3β kinase activity, since wortmannin prevented the ET-1–induced inactivation of GSK-3β (Fig. 2 B). These data strongly suggest that the ET-1–induced inhibition of GSK-3β is mediated via phosphorylation of Ser 9 by a PI3-K-dependent kinase.

Bottom Line: Hypertrophy is a basic cellular response to a variety of stressors and growth factors, and has been best characterized in myocytes.Pathologic hypertrophy of cardiac myocytes leads to heart failure, a major cause of death and disability in the developed world.Several cytosolic signaling pathways have been identified that transduce prohypertrophic signals, but to date, little work has focused on signaling pathways that might negatively regulate hypertrophy.

View Article: PubMed Central - PubMed

Affiliation: Cardiology Division, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02129-2060, USA.

ABSTRACT
Hypertrophy is a basic cellular response to a variety of stressors and growth factors, and has been best characterized in myocytes. Pathologic hypertrophy of cardiac myocytes leads to heart failure, a major cause of death and disability in the developed world. Several cytosolic signaling pathways have been identified that transduce prohypertrophic signals, but to date, little work has focused on signaling pathways that might negatively regulate hypertrophy. Herein, we report that glycogen synthase kinase-3beta (GSK-3beta), a protein kinase previously implicated in processes as diverse as development and tumorigenesis, is inactivated by hypertrophic stimuli via a phosphoinositide 3-kinase-dependent protein kinase that phosphorylates GSK-3beta on ser 9. Using adenovirus-mediated gene transfer of GSK-3beta containing a ser 9 to alanine mutation, which prevents inactivation by hypertrophic stimuli, we demonstrate that inactivation of GSK-3beta is required for cardiomyocytes to undergo hypertrophy. Furthermore, our data suggest that GSK-3beta regulates the hypertrophic response, at least in part, by modulating the nuclear/cytoplasmic partitioning of a member of the nuclear factor of activated T cells family of transcription factors. The identification of GSK-3beta as a transducer of antihypertrophic signals suggests that novel therapeutic strategies to treat hypertrophic diseases of the heart could be designed that target components of the GSK-3 pathway.

Show MeSH
Related in: MedlinePlus