A variety of stress signals stimulate cardiac myocytes to undergo hypertrophy. Persistent cardiac hypertrophy is associated with elevated risk for the development of heart failure. Recently, we showed that class II histone deacetylases (HDACs) suppress cardiac hypertrophy and that stress signals neutralize this repressive function by triggering phosphorylation-and CRM1-dependent nuclear export of these chromatin-modifying enzymes. However, the identities of cardiac HDAC kinases have remained unclear. Here, we demonstrate that signaling by protein kinase C (PKC) is sufficient and, in some cases, necessary to drive nuclear export of class II HDAC5 in cardiomyocytes. Inhibition of PKC prevents nucleocytoplasmic shuttling of HDAC5 in response to a subset of hypertrophic agonists. Moreover, a nonphosphorylatable HDAC5 mutant is refractory to PKC signaling and blocks cardiomyocyte hypertrophy mediated by pharmacological activators of PKC. We also demonstrate that protein kinase D (PKD), a downstream effector of PKC, directly phosphorylates HDAC5 and stimulates its nuclear export. These findings reveal a novel function for the PKC/PKD axis in coupling extracellular cues to chromatin modifications that control cellular growth, and they suggest potential utility for small-molecule inhibitors of this pathway in the treatment of pathological cardiac gene expression.Coordinated changes in gene transcription during cell growth and differentiation require mechanisms for coupling intracellular signaling pathways with the genome. The acetylation of nucleosomal histones has emerged as a central mechanism in the control of gene transcription during such cellular transitions (20). Acetylation of histones by histone acetyltransferases promotes transcription by relaxing chromatin structure, whereas histone deacetylation by histone deacetylases (HDACs) reverses this process, resulting in transcriptional repression. How these chromatin-modifying enzymes are linked to, and controlled by, intracellular signaling is only beginning to be understood.There are two classes of HDACs that can be distinguished by their structures and expression patterns. Class I HDACs (HDAC1, HDAC2, and HDAC3) are expressed ubiquitously and are composed mainly of a catalytic domain (13). In contrast, class II HDACs (HDAC4, HDAC5, HDAC7, and HDAC9) display more restricted expression patterns and contain an N-terminal extension, which mediates interactions with other transcriptional cofactors and confers responsiveness to calcium-dependent signaling (12,25,33). Signaling by calcium/ calmodulin-dependent protein kinase (CaMK) results in phosphorylation of the N termini of class II HDACs, which govern their intracellular localization and interactions with other factors (29, 32). Phosphorylation of signal-responsive serine residues creates docking sites for the 14-3-3 family of chaperone proteins, which promote shuttling of HDACs from the nucleus to the cytoplasm in a CRM1-dependent fashion (14,21,30,31,48).CaMK signaling to class II HDACs governs the activity of th...

We investigated the potential of mouse embryonic stem (ES) cells to differentiate into hepatocytes in vitro. Differentiating ES cells expressed endodermal-specific genes, such as K K-fetoprotein, transthyretin, K K 1-anti-trypsin and albumin, when cultured without additional growth factors and late differential markers of hepatic development, such as tyrosine aminotransferase (TAT) and glucose-6-phosphatase (G6P), when cultured in the presence of growth factors critical for late embryonic liver development. Further, induction of TAT and G6P expression was induced regardless of expression of the functional SEK1 gene, which is thought to provide a survival signal for hepatocytes during an early stage of liver morphogenesis. The data indicate that the in vitro ES differentiation system has a potential to generate mature hepatocytes. The system has also been found useful in analyzing the role of growth factors and intracellular signaling molecules in hepatic development. ß 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

The calcium/calmodulin-dependent phosphatase calcineurin plays a central role in the control of cardiomyocyte hypertrophy in response to pathological stimuli. Although calcineurin is present at high levels in normal heart, its activity appears to be unaffected by calcium during the course of a cardiac cycle. The mechanism(s) whereby calcineurin is selectively activated by calcium under pathological conditions has remained unclear. Here, we demonstrate that diverse signals for cardiac hypertrophy stimulate expression of canonical transient receptor potential (TRPC) channels. TRPC consists of a family of seven membrane-spanning nonselective cation channels that have been implicated in the nonvoltage-gated influx of calcium in response to G protein-coupled receptor signaling, receptor tyrosine kinase signaling, and depletion of internal calcium stores. TRPC3 expression is up-regulated in multiple rodent models of pathological cardiac hypertrophy, whereas TRPC5 expression is induced in failing human heart. We demonstrate that TRPC promotes cardiomyocyte hypertrophy through activation of calcineurin and its downstream effector, the nuclear factor of activated T cells transcription factor. These results define a novel role for TRPC channels in the control of cardiac growth, and suggest that a TRPC-derived pool of calcium contributes to selective activation of calcineurin in diseased heart. Cardiac hypertrophy is an adaptive response of the heart to many forms of cardiac disease, including hypertension, mechanical load abnormalities, myocardial infarction, valvular dysfunction, cardiac arrhythmias, endocrine disorders, and genetic mutations in cardiac contractile protein genes. Whereas the hypertrophic response is thought to be an initially compensatory mechanism that augments cardiac performance, sustained hypertrophy is maladaptive and frequently leads to ventricular dilation and the clinical syndrome of heart failure. Accordingly, cardiac hypertrophy has been established as an independent risk factor for cardiac morbidity and mortality (1).Abnormal calcium handling, characterized by elevated intracellular diastolic calcium levels, is a hallmark of cardiac hypertrophy and heart failure. Elevated intracellular calcium not only impairs the contractile performance of the heart, but also activates calcium-dependent signaling pathways that mediate maladaptive cardiac remodeling (2). One such pathway is regulated by the calcium-calmodulin-dependent phosphatase calcineurin, which has been shown to be sufficient, and in some cases, necessary for pathological hypertrophy (3-5). Activated calcineurin dephosphorylates the transcription factor nuclear factor of activated T-cells (NFAT), 4 facilitating translocation of NFAT to the nucleus where it acts in concert with other proteins to mediate expression of prohypertrophic genes. The activity of calcineurin is tightly regulated in vivo by a negative feedback mechanism; one of the most highly sensitive NFAT target genes encodes a potent calcineurin inhibitor, modulatory calcineuri...

In response to pathological stresses such as hypertension or myocardial infarction, the heart undergoes a remodeling process that is associated with myocyte hypertrophy, myocyte death, and fibrosis. Histone deacetylase 5 (HDAC5) is a transcriptional repressor of cardiac remodeling that is subject to phosphorylationdependent neutralization in response to stress signaling. Recent studies have suggested a role for protein kinase C (PKC) and its downstream effector, protein kinase D1 (PKD1), in the control of HDAC5 phosphorylation. While PKCs are well-documented regulators of cardiac signaling, the function of PKD1 in heart muscle remains unclear. Here, we demonstrate that PKD1 catalytic activity is stimulated in cardiac myocytes by diverse hypertrophic agonists that signal through G protein-coupled receptors (GPCRs) and Rho GTPases. PKD1 activation in cardiomyocytes occurs through PKC-dependent and -independent mechanisms. In vivo, cardiac PKD1 is activated in multiple rodent models of pathological cardiac remodeling. PKD1 activation correlates with phosphorylation-dependent nuclear export of HDAC5, and reduction of endogenous PKD1 expression with small interfering RNA suppresses HDAC5 shuttling and associated cardiomyocyte growth. Conversely, ectopic overexpression of constitutively active PKD1 in mouse heart leads to dilated cardiomyopathy. These findings support a role for PKD1 in the control of pathological remodeling of the heart via its ability to phosphorylate and neutralize HDAC5.

Here, we disrupted the p70 S6 kinase (p70 s6k ) gene in murine embryonic stem cells to determine the role of this kinase in cell growth, protein synthesis, and rapamycin sensitivity. p70 s6k؊͞؊ cells proliferated at a slower rate than parental cells, suggesting that p70 s6k has a positive inf luence on cell proliferation but is not essential. In addition, rapamycin inhibited proliferation of p70 s6k؊͞؊ cells, indicating that other events inhibited by the drug, independent of p70 s6k , also are important for both cell proliferation and the action of rapamycin. In p70 s6k؊͞؊ cells, which exhibited no ribosomal S6 phosphorylation, translation of mRNA encoding ribosomal proteins was not increased by serum nor specifically inhibited by rapamycin. In contrast, rapamycin inhibited phosphorylation of initiation factor 4E-binding protein 1 (4E-BP1), general mRNA translation, and overall protein synthesis in p70 s6k؊͞؊ cells, indicating that these events proceed independently of p70 s6k activity. This study localizes the function of p70 s6k to ribosomal biogenesis by regulating ribosomal protein synthesis at the level of mRNA translation.The serine͞threonine kinase, p70 s6k