sirolimus and arginyl-glycyl-aspartic-acid

Cardiac fibroblasts are activated by mechanical stress, but the underlying mechanisms involved remain poorly understood. In this study, we investigated whether focal adhesion kinase (FAK) plays a role in the activation of cardiac fibroblasts in response to cyclic stretch.. Neonatal (NF-P3/80--third passage, 80% confluence) and adult (AF-P1/80--first passage, 80% confluence) rat cardiac fibroblasts were exposed to cyclic stretch (biaxial, 1 Hz), which enhanced FAK phosphorylation at Tyr397. Proliferation (anti-5-bromo-2'-deoxyuridine and anti-Ki67 nuclear labelling), differentiation into myofibroblasts (expression of alpha-smooth muscle actin--alpha-SMA), and the activity of matrix metalloproteinase-2 were equally enhanced in stretched NF-P3/80 and AF-P1/80. Treatment with the integrin inhibitor RGD peptide impaired FAK phosphorylation and increased apoptosis (TUNEL) in non-stretched and stretched NF-P3/80, whereas FAK silencing induced by small interfering RNA modestly enhanced apoptosis only in stretched cells. RGD peptide or FAK silencing suppressed the activation of NF-P3/80 invoked by cyclic stretch. In addition, NF-P3/80 depleted of FAK were defective in AKT Ser473, TSC-2 Thr1462, and S6 kinase Thr389 phosphorylation induced by cyclic stretch. The activation of NF-P3/80 invoked by cyclic stretch was prevented by pre-treatment with the mammalian target of rapamycin (mTOR) inhibitor rapamycin, whereas supplementation with the amino acid, leucine, activated S6K and rescued the stretch-induced activation of NF-P3/80 depleted of FAK.. These findings demonstrate a critical role for the mTOR complex, downstream from FAK, in mediating the activation of cardiac fibroblasts in response to mechanical stress.

Topics: Actins; Animals; Animals, Newborn; Apoptosis; Cell Differentiation; Cell Proliferation; Cells, Cultured; Collagen Type I; Enzyme Activation; Fibroblasts; Focal Adhesion Kinase 1; Intracellular Signaling Peptides and Proteins; Leucine; Matrix Metalloproteinase 2; Mechanotransduction, Cellular; Myocardium; Oligopeptides; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Ribosomal Protein S6 Kinases; RNA Interference; Serine; Sirolimus; Stress, Mechanical; Threonine; Time Factors; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins; Tyrosine

The present study was conducted to determine the magnitude and duration of ribosomal protein translation in response to pressure overload and determine if additional, paracrine events associated with mechanical transduction, such as integrin activation using a bioactive peptide ligand, RGD or endothelin stimulation lead to ribosomal protein translation. Polysome analysis of ventricular tissue samples obtained from an in vivo model of right-ventricular pressure overload (RVPO) showed a significant shift in the proportion of a 5'-terminal oligopyrimidine (5'-TOP) mRNA, rpL32, associated with the polysomal fraction when compared with non-5'-TOP mRNAs, beta-actin and beta-myosin heavy chain (beta-MHC), in the early stages of the hypertrophic response (24-48 h). Furthermore, this increase in polysome-bound rpL32 mRNA was accompanied by the phosphorylation of mammalian target of rapamycin (mTOR), p70 S6 kinase (S6K1), and S6 ribosomal protein. In our in vitro studies, treatment of primary cultures of adult feline cardiomyocytes (cardiocytes) with 100 nM endothelin, 9 mM RGD, 100 nM insulin, or 100 nM TPA activated mTOR via distinct signaling pathways and resulted in an increased proportion of polysome-bound rpL32 mRNA. Pre-treatment of cardiocytes with the mTOR inhibitor rapamycin blocked the agonist-induced rpL32 mRNA mobilization to polysomes. These results show that mechanisms that regulate ribosomal biogenesis in the myocardium are dynamically sensitive to pressure overload. Furthermore, our in vitro studies indicate that distinct pathways are operational during the early course of hypertrophic growth and converge to activate mTOR resulting in the translational activation of 5'-TOP mRNA.

Topics: Actins; Analysis of Variance; Animals; Blotting, Western; Cats; Cells, Cultured; Disease Models, Animal; Endothelins; Hypertrophy, Right Ventricular; Insulin; Myocytes, Cardiac; Myosin Heavy Chains; Nonmuscle Myosin Type IIB; Oligopeptides; Phosphorylation; Polyribosomes; Protein Biosynthesis; Protein Kinases; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal Protein S6; Ribosomal Protein S6 Kinases; RNA 5' Terminal Oligopyrimidine Sequence; RNA, Messenger; Signal Transduction; Sirolimus; Tetradecanoylphorbol Acetate; TOR Serine-Threonine Kinases

Chemotherapeutic agents induce apoptosis in cancer cells through effects on multiple intracellular targets. Recent observations suggest that a consistent cellular response to chemotherapeutic agents of disparate classes is down-regulation of glycolytic metabolism. Inhibition of glycolytic activity has been linked to apoptotic induction in several models. The serine/threonine kinase Akt (protein kinase B) promotes both glycolytic metabolism and survival, and these functions have been shown to be linked. Because of its key role in both glycolysis and survival, we examined the function of Akt in the cellular response to cytotoxic agents. Following exposure to any of several chemotherapeutic agents, an initial up-regulation in endogenous Akt activity is rapidly suppressed. Using cells containing constitutively active myristoylated Akt, dominant-negative kinase-dead Akt, or an empty vector control, we show here that Akt activation markedly increases resistance to microtubule-directed agents, including vincristine, colchicine, and paclitaxel. Akt also maintains increased glycolytic rate in response to antimicrotubule treatment. Rapamycin inhibits Akt-mediated maintenance of glycolysis and therapeutic resistance, indicating that these effects are dependent on mammalian target of rapamycin (mTOR). Furthermore, an activated mTOR mutant confers resistance to antimicrotubule agents. Taken together, these observations suggest that activation of the Akt-mTOR signaling pathway can augment glucose utilization and promote resistance to chemotherapeutic agents that do not directly target metabolic regulation. These data provide insight into potentially synergistic combinations of anticancer therapies.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-X Protein; Cell Cycle; Cells, Cultured; Colchicine; Drug Resistance, Neoplasm; Drug Synergism; Gene Expression Regulation; Genes, Dominant; Genetic Vectors; Glycolysis; Hematopoietic Stem Cells; Membrane Potentials; Mice; Microtubules; Mitochondria; Mutation; Myristic Acid; Oligopeptides; Paclitaxel; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Vincristine