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Pik3ip1 modulates cardiac hypertrophy by inhibiting PI3K pathway.

Song HK, Kim J, Lee JS, Nho KJ, Jeong HC, Kim J, Ahn Y, Park WJ, Kim do H - PLoS ONE (2015)

Bottom Line: We found that the expression level of Pik3ip1 was markedly higher in cardiomyocytes than in fibroblasts.Pik3ip1 deficiency was shown to lead to activation of PI3K/protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) signaling pathway, increasing protein synthesis and cell size.Collectively, our results elucidate the mechanisms for the roles of Pik3ip1 in PI3K/AKT signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences and Systems Biology Research Center, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea.

ABSTRACT
Cardiac hypertrophy is an adaptive response to various physiological and pathological stimuli. Phosphoinositide-3 kinase (PI3K) is a highly conserved lipid kinase involved in physiological cardiac hypertrophy (PHH). PI3K interacting protein1 (Pik3ip1) shares homology with the p85 regulatory subunit of PI3K and is known to interact with the p110 catalytic subunit of PI3K, leading to attenuation of PI3K activity in liver and immune cells. However, the role of Pik3ip1 in the heart remains unknown. In the present study, the effects of Pik3ip1 on cardiac hypertrophy were examined. We found that the expression level of Pik3ip1 was markedly higher in cardiomyocytes than in fibroblasts. The interaction of Pik3ip1 with the p110a subunit of PI3K in the heart was identified by immunoprecipitation using neonatal rat cardiomyocytes (NRCM). Approximately 35% knockdown of Pik3ip1 was sufficient to induce myocardial hypertrophy. Pik3ip1 deficiency was shown to lead to activation of PI3K/protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) signaling pathway, increasing protein synthesis and cell size. However, adenovirus-mediated overexpression of Pik3ip1 attenuated PI3K-mediated cardiac hypertrophy. Pik3ip1 was upregulated by PHH due to swimming training, but not by pathological cardiac hypertrophy (PAH) due to pressure-overload, suggesting that Pik3ip1 plays a compensatory negative role for PHH. Collectively, our results elucidate the mechanisms for the roles of Pik3ip1 in PI3K/AKT signaling pathway.

No MeSH data available.


Related in: MedlinePlus

Pik3ip1 silencing-induced cardiomyocyte hypertrophy is dependent on PI3K activity.NRCMs were transfected with siNegative and siPik3ip1 for 24 h and subsequently treated with 0.05% DMSO or LY294002 for 24 h. (A) Representative images (left) of NRCMs stained with anti-α-actinin antibody in siNegative or siPik3ip1-transfected NRCMs with or without LY294002. Scale bar: 100 μm. Quantification of relative cell surface areas (right). (n = 5, * p < 0.05 compared with siNegative-transfected NRCMs treated with DMSO, and ## p < 0.01 compared with siPik3ip1-transfected NRCMs treated with DMSO, ANOVA). (B) All siRNA-transfected NRCMs were incubated with DMSO or LY294002, after which protein synthesis was assessed using a leucine incorporation assay. (n = 12, ** p < 0.01 compared with siNegative-transfected NRCMs treated with DMSO and ## p < 0.01 compared with siPik3ip1-transfected NRCMs treated with DMSO, ANOVA). (C) The signaling molecules involved in the PI3K pathway in the 4 different types of samples (siNegative, siPik3ip1, siNegative plus LY294002, and siPik3ip1 plus LY294002) were verified by the indicated antibodies.
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pone.0122251.g004: Pik3ip1 silencing-induced cardiomyocyte hypertrophy is dependent on PI3K activity.NRCMs were transfected with siNegative and siPik3ip1 for 24 h and subsequently treated with 0.05% DMSO or LY294002 for 24 h. (A) Representative images (left) of NRCMs stained with anti-α-actinin antibody in siNegative or siPik3ip1-transfected NRCMs with or without LY294002. Scale bar: 100 μm. Quantification of relative cell surface areas (right). (n = 5, * p < 0.05 compared with siNegative-transfected NRCMs treated with DMSO, and ## p < 0.01 compared with siPik3ip1-transfected NRCMs treated with DMSO, ANOVA). (B) All siRNA-transfected NRCMs were incubated with DMSO or LY294002, after which protein synthesis was assessed using a leucine incorporation assay. (n = 12, ** p < 0.01 compared with siNegative-transfected NRCMs treated with DMSO and ## p < 0.01 compared with siPik3ip1-transfected NRCMs treated with DMSO, ANOVA). (C) The signaling molecules involved in the PI3K pathway in the 4 different types of samples (siNegative, siPik3ip1, siNegative plus LY294002, and siPik3ip1 plus LY294002) were verified by the indicated antibodies.

Mentions: To test whether PI3K inactivation restores Pik3ip1-deficient NRCM phenotypes, we performed a study with LY294002, a specific inhibitor of PI3K, using Pik3ip1-deficient NRCMs [23]. First, we investigated the effect of LY294002 on cell size after transfection of each siRNA in NRCMs. Twenty-four hours after siRNA transfection, the medium, which included 0.05% DMSO or 10 μM LY294002, was changed. As shown in Fig 4A, inhibition of PI3K activity by LY294002 attenuated the Pik3ip1-deficiency-mediated increase in cell size in NRCMs. In parallel, we measured protein synthesis of 3H-leucine using siPik3ip1-transfected NRCMs. As expected, protein synthesis was significantly decreased in LY294002-treated NRCMs, compared to cells treated with DMSO only (Fig 4B). We then examined the activation state of the PI3K/AKT/mTOR pathway by measuring the phosphorylation levels of each protein in siPik3ip1-transfected NRCMs. Phosphorylated forms of AKT, mTOR, and p70s6k were decreased in LY294002-treated NRCMs, compared to DMSO-treated cells. In contrast, phosphorylated eEF2, which was dephosphorylated by siPik3ip1, was increased by LY294002 treatment (Fig 4C). Collectively, the results suggest that Pik3ip1 deficiency activates the PI3K pathway and induces cardiac hypertrophy.


Pik3ip1 modulates cardiac hypertrophy by inhibiting PI3K pathway.

Song HK, Kim J, Lee JS, Nho KJ, Jeong HC, Kim J, Ahn Y, Park WJ, Kim do H - PLoS ONE (2015)

Pik3ip1 silencing-induced cardiomyocyte hypertrophy is dependent on PI3K activity.NRCMs were transfected with siNegative and siPik3ip1 for 24 h and subsequently treated with 0.05% DMSO or LY294002 for 24 h. (A) Representative images (left) of NRCMs stained with anti-α-actinin antibody in siNegative or siPik3ip1-transfected NRCMs with or without LY294002. Scale bar: 100 μm. Quantification of relative cell surface areas (right). (n = 5, * p < 0.05 compared with siNegative-transfected NRCMs treated with DMSO, and ## p < 0.01 compared with siPik3ip1-transfected NRCMs treated with DMSO, ANOVA). (B) All siRNA-transfected NRCMs were incubated with DMSO or LY294002, after which protein synthesis was assessed using a leucine incorporation assay. (n = 12, ** p < 0.01 compared with siNegative-transfected NRCMs treated with DMSO and ## p < 0.01 compared with siPik3ip1-transfected NRCMs treated with DMSO, ANOVA). (C) The signaling molecules involved in the PI3K pathway in the 4 different types of samples (siNegative, siPik3ip1, siNegative plus LY294002, and siPik3ip1 plus LY294002) were verified by the indicated antibodies.
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pone.0122251.g004: Pik3ip1 silencing-induced cardiomyocyte hypertrophy is dependent on PI3K activity.NRCMs were transfected with siNegative and siPik3ip1 for 24 h and subsequently treated with 0.05% DMSO or LY294002 for 24 h. (A) Representative images (left) of NRCMs stained with anti-α-actinin antibody in siNegative or siPik3ip1-transfected NRCMs with or without LY294002. Scale bar: 100 μm. Quantification of relative cell surface areas (right). (n = 5, * p < 0.05 compared with siNegative-transfected NRCMs treated with DMSO, and ## p < 0.01 compared with siPik3ip1-transfected NRCMs treated with DMSO, ANOVA). (B) All siRNA-transfected NRCMs were incubated with DMSO or LY294002, after which protein synthesis was assessed using a leucine incorporation assay. (n = 12, ** p < 0.01 compared with siNegative-transfected NRCMs treated with DMSO and ## p < 0.01 compared with siPik3ip1-transfected NRCMs treated with DMSO, ANOVA). (C) The signaling molecules involved in the PI3K pathway in the 4 different types of samples (siNegative, siPik3ip1, siNegative plus LY294002, and siPik3ip1 plus LY294002) were verified by the indicated antibodies.
Mentions: To test whether PI3K inactivation restores Pik3ip1-deficient NRCM phenotypes, we performed a study with LY294002, a specific inhibitor of PI3K, using Pik3ip1-deficient NRCMs [23]. First, we investigated the effect of LY294002 on cell size after transfection of each siRNA in NRCMs. Twenty-four hours after siRNA transfection, the medium, which included 0.05% DMSO or 10 μM LY294002, was changed. As shown in Fig 4A, inhibition of PI3K activity by LY294002 attenuated the Pik3ip1-deficiency-mediated increase in cell size in NRCMs. In parallel, we measured protein synthesis of 3H-leucine using siPik3ip1-transfected NRCMs. As expected, protein synthesis was significantly decreased in LY294002-treated NRCMs, compared to cells treated with DMSO only (Fig 4B). We then examined the activation state of the PI3K/AKT/mTOR pathway by measuring the phosphorylation levels of each protein in siPik3ip1-transfected NRCMs. Phosphorylated forms of AKT, mTOR, and p70s6k were decreased in LY294002-treated NRCMs, compared to DMSO-treated cells. In contrast, phosphorylated eEF2, which was dephosphorylated by siPik3ip1, was increased by LY294002 treatment (Fig 4C). Collectively, the results suggest that Pik3ip1 deficiency activates the PI3K pathway and induces cardiac hypertrophy.

Bottom Line: We found that the expression level of Pik3ip1 was markedly higher in cardiomyocytes than in fibroblasts.Pik3ip1 deficiency was shown to lead to activation of PI3K/protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) signaling pathway, increasing protein synthesis and cell size.Collectively, our results elucidate the mechanisms for the roles of Pik3ip1 in PI3K/AKT signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences and Systems Biology Research Center, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea.

ABSTRACT
Cardiac hypertrophy is an adaptive response to various physiological and pathological stimuli. Phosphoinositide-3 kinase (PI3K) is a highly conserved lipid kinase involved in physiological cardiac hypertrophy (PHH). PI3K interacting protein1 (Pik3ip1) shares homology with the p85 regulatory subunit of PI3K and is known to interact with the p110 catalytic subunit of PI3K, leading to attenuation of PI3K activity in liver and immune cells. However, the role of Pik3ip1 in the heart remains unknown. In the present study, the effects of Pik3ip1 on cardiac hypertrophy were examined. We found that the expression level of Pik3ip1 was markedly higher in cardiomyocytes than in fibroblasts. The interaction of Pik3ip1 with the p110a subunit of PI3K in the heart was identified by immunoprecipitation using neonatal rat cardiomyocytes (NRCM). Approximately 35% knockdown of Pik3ip1 was sufficient to induce myocardial hypertrophy. Pik3ip1 deficiency was shown to lead to activation of PI3K/protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) signaling pathway, increasing protein synthesis and cell size. However, adenovirus-mediated overexpression of Pik3ip1 attenuated PI3K-mediated cardiac hypertrophy. Pik3ip1 was upregulated by PHH due to swimming training, but not by pathological cardiac hypertrophy (PAH) due to pressure-overload, suggesting that Pik3ip1 plays a compensatory negative role for PHH. Collectively, our results elucidate the mechanisms for the roles of Pik3ip1 in PI3K/AKT signaling pathway.

No MeSH data available.


Related in: MedlinePlus