Moschella Phillip C, Rao Vijay U, McDermott Paul J, Kuppuswamy Dhandapani
Cardiology Division of the Department of Medicine, Gazes Cardiac Research Institute, Medical University of South Carolina, 114 Doughty Street, Charleston, SC 29425, USA.
J Mol Cell Cardiol. 2007 Dec;43(6):754-66. doi: 10.1016/j.yjmcc.2007.09.015. Epub 2007 Oct 4.
Activation of both mTOR and its downstream target, S6K1 (p70 S6 kinase) have been implicated to affect cardiac hypertrophy. Our earlier work, in a feline model of 1-48 h pressure overload, demonstrated that mTOR/S6K1 activation occurred primarily through a PKC/c-Raf pathway. To further delineate the role of specific PKC isoforms on mTOR/S6K1 activation, we utilized primary cultures of adult feline cardiomyocytes in vitro and stimulated with endothelin-1 (ET-1), phenylephrine (PE), TPA, or insulin. All agonist treatments resulted in S2248 phosphorylation of mTOR and T389 and S421/T424 phosphorylation of S6K1, however only ET-1 and TPA-stimulated mTOR/S6K1 activation was abolished with infection of a dominant negative adenoviral c-Raf (DN-Raf) construct. Expression of DN-PKC(epsilon) blocked ET-1-stimulated mTOR S2448 and S6K1 S421/T424 and T389 phosphorylation but had no effect on insulin-stimulated S6K1 phosphorylation. Expression of DN-PKC(delta) or pretreatment of cardiomyocytes with rottlerin, a PKC(delta) specific inhibitor, blocked both ET-1 and insulin stimulated mTOR S2448 and S6K1 T389 phosphorylation. However, treatment with Gö6976, a specific classical PKC (cPKC) inhibitor did not affect mTOR/S6K1 activation. These data indicate that: (i) PKC(epsilon) is required for ET-1-stimulated T421/S424 phosphorylation of S6K1, (ii) both PKC(epsilon) and PKC(delta) are required for ET-1-stimulated mTOR S2448 and S6K1 T389 phosphorylation, (iii) PKC(delta) is also required for insulin-stimulated mTOR S2448 and S6K1 T389 phosphorylation. Together, these data delineate both distinct and combinatorial roles of specific PKC isoforms on mTOR and S6K1 activation in adult cardiac myocytes following hypertrophic stimulation.
mTOR及其下游靶点S6K1（p70 S6激酶）的激活均被认为与心脏肥大有关。我们早期在1 - 48小时压力超负荷猫模型中的研究表明，mTOR/S6K1激活主要通过PKC/c - Raf途径发生。为了进一步阐明特定PKC亚型在mTOR/S6K1激活中的作用，我们在体外利用成年猫心肌细胞的原代培养物，并用内皮素 - 1（ET - 1）、去氧肾上腺素（PE）、佛波酯（TPA）或胰岛素进行刺激。所有激动剂处理均导致mTOR的S2248磷酸化以及S6K1的T389和S421/T424磷酸化，然而只有ET - 1和TPA刺激的mTOR/S6K1激活被显性负性腺病毒c - Raf（DN - Raf）构建体的感染所消除。DN - PKC(ε)的表达阻断了ET - 1刺激的mTOR S2448以及S6K1 S421/T424和T389磷酸化，但对胰岛素刺激的S6K1磷酸化没有影响。DN - PKC(δ)的表达或用PKC(δ)特异性抑制剂rottlerin预处理心肌细胞，阻断了ET - 1和胰岛素刺激的mTOR S2448以及S6K1 T389磷酸化。然而，用特异性经典PKC（cPKC）抑制剂Gö6976处理并不影响mTOR/S6K1激活。这些数据表明：（i）PKC(ε)是ET - 1刺激S6K1的T421/S424磷酸化所必需的；（ii）PKC(ε)和PKC(δ)都是ET - 1刺激mTOR S2448以及S6K1 T389磷酸化所必需的；（iii）PKC(δ)也是胰岛素刺激mTOR S2448以及S6K1 T389磷酸化所必需的。总之，这些数据阐明了特定PKC亚型在肥大刺激后成年心肌细胞中mTOR和S6K1激活方面的独特和组合作用。
