Effect Of MTOR/p～(70)S6 Kinase Signal Pathway On The Protein Synthesis In CHL Cells And On The Cardiomyocytes Hypertrophy

Regulation of protein synthesis in eukaryotes plays a critical role in cell growth. Protein synthesis is regulated by a variety of stimuli including insulin and phorbol esters. Recent findings reveal that the target of rapamycin TOR controls an unusually abundant and diverse set of readouts all of which are important for cell growth, suggesting that this conserved kinase is such a central regulator. Protein kinase B (PKB) , a kinase which lies downstream of phosphatidylinositol 3 - kinase ( PI 3 - kinase) and is thought to be important in the signal transduc-tion pathway which leads to the phosphorylation of both p S6 kinase (p S6K) and eIF4E -binding protein 1(4E - BP1). Most studies on the signaling pathways involved in the regulation of protein synthesis have focused on insulin. It has been demonstrated that phorbol esters can also lead to increases in protein synthesis. In this report we characterize in parallel the signaling pathways by which both insulin and phorbol esters activate p S6K.Cardiac hypertrophy occurs in response to stresses such as increased after-load and ischemia or as a mechanism to compensate for heart dysfunction. Understanding the mechanisms of cardiac hypertrophy is important because severe heart dysfunction and arrhythmia can result. Cardiac hypertrophy is evoked by growth factors, neurohumoral factors, or by mechanical stress, which subsequently activate several intracellular signal transduction pathways. Recent studies demonstrated that p70S6K is a key factor for protein synthesis in a variety of cell types and it has also been reported that rapamycin, a specific inhibitor of the mammalian target of rapamycin (mTOR) , which is an upstream signaling of p70S6K,inhibits angiotensin E and bradykinin - mediated hypertrophy of cardiac myocytes. Thus, mTOR/p70S6K signal transduction may play a vital regulation in the hypertrophy growth of cardiac myocytes. Hypertrophy of cardiac myocytesis characterized by increased cell volume, increased protein synthesis. In this report we measured the expression of mTOR ( mammalian target of rapamycin) and p70S6 kinase(p70S6K) , the activity of p70S6K in cardiac hypertrophy of spontaneously hypertensive rats(SHRs). In addition, we show that mTOR/p70 S6K signal transduction mediates cardiomyocytes volume and protein synthesis stimulated by fetal bovine serum in the cultured cardiomyocytes.MethodsChinese hamster lung fibroblasts ( CHL) CultureCHL cells were maintained in Dulbecco 's modified Eagle 's medium (DMEM) supplemented with 10% fetal calf serum. Prior to treatment, cells were grown to 70% confluence before being serum starved for 18h.Neonatal Rat Ventricular Myocytes ( NRVMs) CultureNRVMs were cultured from 1 ~ 3 - day - old Sprague - Dawley rats. Briefly, 1 - mm3 cubes of ventricles were placed in ice - cold dissociation buffer ( pH 7.5) containing 0.25% trypsin. After mechanical dissociation of the tissue by several rounds of alternate gently pipetting and centrifugation at 1500 x g for 10 min at 4 , cells were plated in Dulbecco s modified Eagles medium (DMEM) with 10% calf serum at 37 in 5% CO2 for 48h and then were serum - starved for 48h.SDS - PAGE and ImmunoblottingTo analyze extracts, equal amounts of protein were resolved by SDS -PAGE and transferred to polyvinylidene difluoride membrane (Millipore). The resulting membranes were probed with anti -PKB,p70 S6K and mTOR polyclonal antibody and revealed using the ECL system.Vitro Kinase AssaysThe anti - p70S6K immunocomplex and the anti - PKB immunocomplex were each prepared using separate supernatants incubated 2h at 4 with the specific antibodies. p70S6K activity assays: The immunocomplexes were further washed twice with kinase buffer. The kinase reaction was carried out at 37 for15 min in kinase buffer containing 100 M ATP, 1 Ci [ -32P] ATP, and 5 M S6 kinase peptide substrate ( sequence: RRRLSSLRA). The reactions were stopped and blotted onto p81 papers and washed in 75mmol/L phosphoric acid, and radioactivity was measured by scintillation...