40S Ribosomal protein S6 kinase integrates daylength perception and growth regulation in Arabidopsis thaliana.

Plant growth occurs via the interconnection of cell growth and proliferation in each organ following specific developmental and environmental cues. Therefore, different photoperiods result in distinct growth patterns due to the integration of light and circadian perception with specific Carbon (C) partitioning strategies. In addition, the TARGET OF RAPAMYCIN (TOR) kinase pathway is an ancestral signaling pathway that integrates nutrient information with translational control and growth regulation. Recent findings in Arabidopsis (Arabidopsis thaliana) have shown a mutual connection between the TOR pathway and the circadian clock. However, the mechanistical network underlying this interaction is mostly unknown. Here, we show that the conserved TOR target, the 40S ribosomal protein S6 kinase (S6K) is under circadian and photoperiod regulation both at the transcriptional and post-translational level. Total S6K (S6K1 and S6K2) and TOR-dependent phosphorylated-S6K protein levels were higher during the light period and decreased at dusk especially under short day conditions. Using chemical and genetic approaches, we found that the diel pattern of S6K accumulation results from 26S proteasome-dependent degradation and is altered in mutants lacking the circadian F-box protein ZEITLUPE (ZTL), further strengthening our hypothesis that S6K could incorporate metabolic signals via TOR, which are also under circadian regulation. Moreover, under short days when C/energy levels are limiting, changes in S6K1 protein levels affected starch, sucrose and glucose accumulation and consequently impacted root and rosette growth responses. In summary, we propose that S6K1 constitutes a missing molecular link where day-length perception, nutrient availability and TOR pathway activity converge to coordinate growth responses with environmental conditions.

Ribosomal protein S6 kinase beta-1 gene variants cause hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a genetic heart muscle disease with preserved or increased ejection fraction in the absence of secondary causes. Mutations in the sarcomeric protein-encoding genes predominantly cause HCM. However, relatively little is known about the genetic impact of signalling proteins on HCM. METHODS AND RESULTS: Here, using exome and targeted sequencing methods, we analysed two independent cohorts comprising 401 Indian patients with HCM and 3521 Indian controls. We identified novel variants in ribosomal protein S6 kinase beta-1 (RPS6KB1 or S6K1) gene in two unrelated Indian families as a potential candidate gene for HCM. The two unrelated HCM families had the same heterozygous missense S6K1 variant (p.G47W). In a replication association study, we identified two S6K1 heterozygotes variants (p.Q49K and p.Y62H) in the UK Biobank cardiomyopathy cohort (n=190) compared with matched controls (n=16 479). These variants are neither detected in region-specific controls nor in the human population genome data. Additionally, we observed an S6K1 variant (p.P445S) in an Arab patient with HCM. Functional consequences were evaluated using representative S6K1 mutated proteins compared with wild type in cellular models. The mutated proteins activated the S6K1 and hyperphosphorylated the rpS6 and ERK1/2 signalling cascades, suggesting a gain-of-function effect. CONCLUSIONS: Our study demonstrates for the first time that the variants in the S6K1 gene are associated with HCM, and early detection of the S6K1 variant carriers can help to identify family members at risk and subsequent preventive measures. Further screening in patients with HCM with different ethnic populations will establish the specificity and frequency of S6K1 gene variants.

Associations of the Methylation Levels of NFAT5, PVT1, RPS6KA1, and MIB1 with Steroid-Resistant Asthma.

BACKGROUND: This study detected the methylation levels of nuclear factor-5 (NFAT5), PVT1, ribosomal protein S6 kinase A-1 (RPS6KA1), and MIB1 in patients with steroid-resistant asthma (SRA) and explored their associations with SRA. METHODS: In our pilot study, we found abnormal methylation of NFAT5, PVT1, RPS6KA1, and MIB1 in SRA patients according to genome-wide methylation screening. This study expanded the sample size to further validate the results of the pilot study. Twenty patients with SRA, 20 patients with bronchial asthma, and 20 healthy volunteers constituted the SRA group, asthma control group, and healthy control group, respectively. The clinical data of all the participants were collected. Peripheral blood was taken for DNA extraction. The methylation loci and levels of NFAT5, PVT1, RPS6KA1, and MIB1 were detected using the Sequenom MassARRAY Nanodispenser RS1000. Data were processed and analyzed with SPSS 22.0 software. RESULTS: There were 24 CpG loci detected in the NFAT5 segment 7 in the PVT1 segment, 4 in the RPS6KA1 segment, and 3 in the MIB1 segment. Among these genes, RPS6KA exhibited hypomethylation in the SRA group, which showed significant differences at the CpG_1, CpG_2, and CpG_3 loci compared with the other groups (p < 0.05). No significant differences in the methylation levels of NFAT5, PVT1, and MIB1 were observed among the groups (p > 0.05). CONCLUSIONS: RPS6KA1 is hypomethylated in SRA patients, which may play a role in the development of SRA via the MAPK signaling pathway. However, the influence of the methylation of NFAT5, PVT1, and MIB1 on SRA development remains to be explored.

IL-23 amplifies the epithelial-mesenchymal transition of mechanically conditioned alveolar epithelial cells in rheumatoid arthritis-associated interstitial lung disease through mTOR/S6 signaling.

Epithelial-mesenchymal transition (EMT) creates an environment facilitating fibrosis following alveolar epithelial cell injury. IL-23 has important roles in chronic autoimmune conditions like rheumatoid arthritis (RA), but its role in the interstitial lung disease that affects patients with RA is unclear. This study aimed to determine the profibrogenic role of IL-23 on somatic alveolar type I (ATI) epithelial cells. Primary ATI cells were isolated from rats and cultured on plastic dishes for 1-3 wk. After prolonged culture (≥14 days) on rigid culture dishes, primary ATI cells gradually acquired a mesenchymal phenotype, identified by decreased expression of caveolin-1, and reorganization of F-actin cytoskeleton, indicating the initiation of EMT by matrix stiffness. To determine how IL-23 promotes EMT in vitro, transitioning ATI cells, cultured on a stiff substrate for ≥14 days were stimulated with IL-23. The EMT phenotype was significantly enhanced by IL-23, which upregulated α-smooth muscle actin (α-SMA), collagen I/III protein, and decreased caveolin-1. Furthermore, IL-23 significantly promoted cell invasion, as well as apoptotic resistance on transitioning ATI cells. Mechanistically, IL-23-induced EMT was mammalian target of rapamycin/ribosomal protein S6 (mTOR/S6) signaling dependent and reversible by rapamycin. Transcriptional sequencing analysis of human lung fibrosis biopsy tissue revealed key roles for IL-23 in rheumatoid arthritis-associated interstitial lung disease (RA-ILD). This result was further validated by significantly upregulated IL-23 expression at the mRNA level in RA-ILD lung sections. Notably, transitioning ATI epithelial cells were abundantly detected in RA-ILD tissue. Taken together, these data support a role for IL-23 in the pathogenesis of RA lung fibrosis by promoting EMT in alveolar epithelial cells through mTOR/S6 signaling.

Somatodendritic accumulation of Tau in Alzheimer's disease is promoted by Fyn-mediated local protein translation.

The cause of protein accumulation in neurodegenerative disease is incompletely understood. In Alzheimer's disease (AD), the axonally enriched protein Tau forms hyperphosphorylated aggregates in the somatodendritic domain. Consequently, a process of subcellular relocalization driven by Tau phosphorylation and detachment from microtubules has been proposed. Here, we reveal an alternative mechanism of de novo protein synthesis of Tau and its hyperphosphorylation in the somatodendritic domain, induced by oligomeric amyloid-ß (Aß) and mediated by the kinase Fyn that activates the ERK/S6 signaling pathway. Activation of this pathway is demonstrated in a range of cellular systems, and in vivo in brains from Aß-depositing, Aß-injected, and Fyn-overexpressing mice with Tau accumulation. Both pharmacological inhibition and genetic deletion of Fyn abolish the Aß-induced Tau overexpression via ERK/S6 suppression. Together, these findings present a more cogent mechanism of Tau aggregation in disease. They identify a prominent role for neuronal Fyn in integrating signal transduction pathways that lead to the somatodendritic accumulation of Tau in AD.

Investigation of the hepatic mTOR/S6K1/SREBP1 signalling pathway in rats at different ages: from neonates to adults.

BACKGROUND: Dysfunctions in the lipogenic process controlled by the hepatic mTOR/S6K1/SREBP-1c signaling pathway may contribute to the pathogenesis of various chronic diseases. In the present study, we aimed to determine age-related changes in the mTOR/S6K1/SREBP1 pathway in rat liver tissues. METHODS AND RESULTS: We performed Western Blot analysis to determine age-related changes in the mTOR/S6K1/SREBP1 pathway in Sprague Dawley male rats liver tissues of six different age groups representing neonatal, infant, weaning, puberty, young adult, adult life periods, and Oil Red O staining to evaluate age-related lipid accumulation. We observed an increase in Akt and p-Akt levels with age in compared to the 0-day-old group. Total mTOR and SREBP1 expression increased from the 0-day-old to the 28-day-old group but decreased in the following age groups. p-mTOR and p-S6K1 levels in the 0-day-old group were higher than the other groups. S6K1 expression was lowest in the 0-day-old group and showed changes among the age groups. Lipid accumulation was seen in liver sections taken from the 12-month-old group. mTOR/S6K1/SREBP1 pathway expression showed changes with age during the neonatal-adult life cycle stages in rat liver tissues. CONCLUSION: We suggest that understanding the molecular mechanisms age-related changes of lipogenesis function is necessary to contribute to the development of therapeutic approaches.

PKD deletion promotes autophagy and inhibits hypertrophy in cardiomyocyte.

Protein kinase D (PKD) plays an important role in the development of cardiac hypertrophy induced by pressure overload. However, the mechanism involved is unclear. This study, using primary cardiomyocyte culture, PKD knockdown and overexpression, and other molecular techniques, tested our hypothesis that PKD pathway mediates cardiac hypertrophy by negatively regulating autophagy in cardiomyocyte. Neonatal cardiomyocytes were isolated from Wistar rats and cell hypertrophy was induced by norepinephrine treatment (PE, 10-4 mol/L), and divided into the following groups: (1) Vehicle; (2) PE; (3) PE + control siRNA; (4) PE + Rapamycin (100 nM); (5) PE + PKD-siRNA (2 × 108 U/0.1 ml); (6) PE + PKD siRNA + 3 MA (10 mM). The results showed that PE treatment induced cardiomyocyte hypertrophy, which were confirmed by cell size and biomarkers of cardiomyocyte hypertrophy including increased ANP and BNP mRNA. PKD knockdown or Rapamycin significantly inhibited PE-induced cardiomyocyte hypertrophy. In addition, PKD siRNA increased autophagy activity determined by electron microscopy, increased biomarkers of autophagy by Western blot, accompanied by down-regulated AKT/mTOR/S6K pathway. All the effects of PKD knockout were inhibited by co-treatment with 3-MA, an autophagy inhibitor. Oppositely, the autophagy in cardiomyocytes was inhibited by PKD overexpression. These results suggest that PKD participates in the development of cardiac hypertrophy by regulating autophagy via AKT/mTOR/S6K pathway.

Hypertrophy of Rat Skeletal Muscle Is Associated with Increased SIRT1/Akt/mTOR/S6 and Suppressed Sestrin2/SIRT3/FOXO1 Levels.

Despite the intensive investigation of the molecular mechanism of skeletal muscle hypertrophy, the underlying signaling processes are not completely understood. Therefore, we used an overload model, in which the main synergist muscles (gastrocnemius, soleus) of the plantaris muscle were surgically removed, to cause a significant overload in the remaining plantaris muscle of 8-month-old Wistar male rats. SIRT1-associated pro-anabolic, pro-catabolic molecular signaling pathways, NAD and H2S levels of this overload-induced hypertrophy were studied. Fourteen days of overload resulted in a significant 43% (p < 0.01) increase in the mass of plantaris muscle compared to sham operated animals. Cystathionine-ß-synthase (CBS) activities and bioavailable H2S levels were not modified by overload. On the other hand, overload-induced hypertrophy of skeletal muscle was associated with increased SIRT1 (p < 0.01), Akt (p < 0.01), mTOR, S6 (p < 0.01) and suppressed sestrin 2 levels (p < 0.01), which are mostly responsible for anabolic signaling. Decreased FOXO1 and SIRT3 signaling (p < 0.01) suggest downregulation of protein breakdown and mitophagy. Decreased levels of NAD+, sestrin2, OGG1 (p < 0.01) indicate that the redox milieu of skeletal muscle after 14 days of overloading is reduced. The present investigation revealed novel cellular interactions that regulate anabolic and catabolic processes in the hypertrophy of skeletal muscle.

Deacetylation of S6 kinase promotes high glucose-induced glomerular mesangial cell hypertrophy and matrix protein accumulation.

S6 kinase acts as a driver for renal hypertrophy and matrix accumulation, two key pathologic signatures of diabetic nephropathy. As a post-translational modification, S6 kinase undergoes acetylation at the C terminus. The role of this acetylation to regulate kidney glomerular cell hypertrophy and matrix expansion is not known. In mesangial cells, high glucose decreased the acetylation and enhanced phosphorylation of S6 kinase and its substrates rps6 and eEF2 kinase that lead to dephosphorylation of eEF2. To determine the mechanism of S6 kinase deacetylation, we found that trichostatin A, a pan-histone deacetylase (HDAC) inhibitor, blocked all high glucose-induced effects. Furthermore, high glucose increased the expression and association of HDAC1 with S6 kinase. HDAC1 decreased the acetylation of S6 kinase and mimicked the effects of high glucose, resulting in mesangial cell hypertrophy and expression of fibronectin and collagen I (α2). In contrast, siRNA against HDAC1 inhibited these effects by high glucose. A C-terminal acetylation-mimetic mutant of S6 kinase suppressed high glucose-stimulated phosphorylation of S6 kinase, rps6 and eEF2 kinase, and inhibited the dephosphorylation of eEF2. Also, the acetylation mimetic attenuated the mesangial cell hypertrophy and fibronectin and collagen I (α2) expression. Conversely, an S6 kinase acetylation-deficient mutant induced all the above effects of high glucose. Finally, in the renal glomeruli of diabetic rats, the acetylation of S6 kinase was significantly reduced concomitant with increased HDAC1 and S6 kinase activity. In aggregate, our data uncovered a previously unrecognized role of S6 kinase deacetylation in high glucose-induced mesangial cell hypertrophy and matrix protein expression.

RSK2 phosphorylates T-bet to attenuate colon cancer metastasis and growth.

Metastasis is a major cause of cancer-related deaths. Approximately 80% of patients with colorectal cancer develop liver metastasis and 20% develop lung metastasis. We found that at different stages of colon cancer, IFNγ secretion from peripheral blood mononuclear cells was decreased compared with healthy controls. The ribosomal S6 kinase (RSK) family of kinases has multiple cellular functions, and we examined their roles in this observed IFNγ decrease. Flow cytometry analysis of wild-type (WT) and RSK2 knockout (KO) mice revealed significantly lower levels of IFNγ in the RSK2 KO mice compared with the WT mice. Since IFNγ is a component of immunity, which contributes to protection against metastatic carcinomas, we conducted a colon cancer liver metastasis experiment. We found significantly greater metastasis in RSK2 KO mice compared with WT mice. Transcription factor T-bet can directly activate Ifnγ gene transcription. In vitro kinase assay results showed that RSK2 phosphorylated T-bet at serines 498 and 502. We show that phosphorylation of T-bet by RSK2 is required for IFNγ expression, because knockdown of RSK2 expression or overexpression of mutant T-bet reduces IFNγ mRNA expression. To verify the function of the phosphorylation sites, we overexpressed a constitutively active mutant T-bet (S498E/S502E) in bone marrow. Mutant T-bet restored the IFNγ mRNA levels and dramatically reduced the metastasis rate in these mice. Overall, these results indicate that phosphorylation of T-bet is required for the inhibition of colon cancer metastasis and growth through a positive regulation of RSK2/T-bet/IFNγ signaling.

Inhibition of Endothelial Notch Signaling Impairs Fatty Acid Transport and Leads to Metabolic and Vascular Remodeling of the Adult Heart.

BACKGROUND: Nutrients are transported through endothelial cells before being metabolized in muscle cells. However, little is known about the regulation of endothelial transport processes. Notch signaling is a critical regulator of metabolism and angiogenesis during development. Here, we studied how genetic and pharmacological manipulation of endothelial Notch signaling in adult mice affects endothelial fatty acid transport, cardiac angiogenesis, and heart function. METHODS: Endothelial-specific Notch inhibition was achieved by conditional genetic inactivation of Rbp-jκ in adult mice to analyze fatty acid metabolism and heart function. Wild-type mice were treated with neutralizing antibodies against the Notch ligand Delta-like 4. Fatty acid transport was studied in cultured endothelial cells and transgenic mice. RESULTS: Treatment of wild-type mice with Delta-like 4 neutralizing antibodies for 8 weeks impaired fractional shortening and ejection fraction in the majority of mice. Inhibition of Notch signaling specifically in the endothelium of adult mice by genetic ablation of Rbp-jκ caused heart hypertrophy and failure. Impaired heart function was preceded by alterations in fatty acid metabolism and an increase in cardiac blood vessel density. Endothelial Notch signaling controlled the expression of endothelial lipase, Angptl4, CD36, and Fabp4, which are all needed for fatty acid transport across the vessel wall. In endothelial-specific Rbp-jκ-mutant mice, lipase activity and transendothelial transport of long-chain fatty acids to muscle cells were impaired. In turn, lipids accumulated in the plasma and liver. The attenuated supply of cardiomyocytes with long-chain fatty acids was accompanied by higher glucose uptake, increased concentration of glycolysis intermediates, and mTOR-S6K signaling. Treatment with the mTOR inhibitor rapamycin or displacing glucose as cardiac substrate by feeding a ketogenic diet prolonged the survival of endothelial-specific Rbp-jκ-deficient mice. CONCLUSIONS: This study identifies Notch signaling as a novel regulator of fatty acid transport across the endothelium and as an essential repressor of angiogenesis in the adult heart. The data imply that the endothelium controls cardiomyocyte metabolism and function.

The polyproline-motif of S6K2: eIF5A translational dependence and importance for protein-protein interactions.

Ribosomal S6 kinase 1 (S6K1) and S6K2 proteins are effectors of the mammalian target of rapamycin complex 1 pathway, which control the process of protein synthesis in eukaryotes. S6K2 is associated with tumor progression and has a conserved C-terminus polyproline rich motif predicted to be important for S6K2 interactions. It is noteworthy that the translation of proteins containing sequential prolines has been proposed to be dependent of eukaryotic translation initiation factor 5A (eIF5A) translation factor. Therefore, we investigated the importance of polyproline-rich region of the S6K2 for its intrinsic phosphorylation activity, protein-protein interaction and eIF5A role in S6K2 translation. In HeLa cell line, replacing S6K2 polyproline by the homologous S6K1-sequence did not affect its kinase activity and the S6K2 endogenous content was maintained after eIF5A gene silencing, even after near complete depletion of eIF5A protein. Moreover, no changes in S6K2 transcript content was observed, ruling out the possibility of compensatory regulation by increasing the mRNA content. However, in the budding yeast model, we observed that S6K2 production was impaired when compared with S6K2∆Pro, after reduction of eIF5A protein content. These results suggest that although the polyproline region of S6K2 is capable of generating ribosomal stalling, the depletion of eIF5A in HeLa cells seems to be insufficient to cause an expressive decrease in the content of endogenous S6K2. Finally, coimmunoprecipitation assays revealed that the replacement of the polyproline motif of S6K2 alters its interactome and impairs its interaction with RPS6, a key modulator of ribosome activity. These results evidence the importance of S6K2 polyproline motif in the context of S6Ks function.

Genome-wide screen for cell growth regulators in fission yeast.

Cellular growth control is important for all living organisms, but experimental investigation into this problem is difficult because of the complex range of growth regulatory mechanisms. Here, we have used the fission yeast Schizosaccharomyces pombe to identify potential master regulators of growth. At the restrictive temperature, the S. pombe pat1ts mei4Δ strain enters the meiotic developmental program, but arrests in meiotic G2 phase as mei4+ is essential for meiotic progression. These cells do not grow, even in an abundance of nutrients. To identify regulators of growth that can reverse this growth arrest, we introduced an ORFeome plasmid library into the pat1tsmei4Δ strain. Overexpression of eight genes promoted cell growth; two of these were core RNA polymerase subunits, and one was sck2+ , an S6 kinase thought to contribute to TORC1 signalling. Sck2 had the greatest effect on cell growth, and we also show that it significantly increases the cellular transcription rate. These findings indicate, for the first time, that global transcriptional control mediated through S6 kinase signalling is central to cellular growth control.

A Drosophila genetic screen for suppressors of S6kinase-dependent growth identifies the F-box subunit Archipelago/FBXW7.

This study was designed to identify novel negative regulators of the Drosophila S6kinase (dS6K). S6K is a downstream effector of the growth-regulatory complex mTORC1 (mechanistic-Target-of-Rapamycin complex 1). Nutrients activate mTORC1, which in turn induces the phosphorylation of S6K to promote cell growth, whereas fasting represses mTORC1 activity. Here, we screened 11,000 RNA-interfering (RNAi) lines and retained those that enhanced a dS6K-dependent growth phenotype. Since RNAi induces gene knockdown, enhanced tissue growth supports the idea that the targeted gene acts as a growth suppressor. To validate the resulting candidate genes, we monitored dS6K phosphorylation and protein levels in double-stranded RNAi-treated S2 cells. We identified novel dS6K negative regulators, including gene products implicated in basal cellular functions, suggesting that feedback inputs modulate mTORC1/dS6K signaling. We also identified Archipelago (Ago), the Drosophila homologue of FBXW7, which is an E3-ubiquitin-ligase subunit that loads ubiquitin units onto target substrates for proteasome-mediated degradation. Despite a previous report showing an interaction between Ago/FBXW7 and dS6K in a yeast two-hybrid assay and the presence of an Ago/FBXW7-consensus motif in the dS6K polypeptide, we could not see a direct interaction in immunoprecipitation assay. Nevertheless, we observed that loss-of-ago/fbxw7 in larvae resulted in an increase in dS6K protein levels, but no change in the levels of phosphorylated dS6K or dS6K transcripts, suggesting that Ago/FBXW7 indirectly controls dS6K translation or stability. Through the identification of novel negative regulators of the downstream target, dS6K, our study may help deciphering the underlying mechanisms driving deregulations of mTORC1, which underlies several human diseases.

Molecular Characterization of Two Mitogen-Activated Protein Kinases: p38 MAP Kinase and Ribosomal S6 Kinase From Bombyx mori (Lepidoptera: Bombycidae), and Insight Into Their Roles in Response to BmNPV Infection.

Proteins p38 map kinase and ribosomal S6 kinase (S6K) as members of mitogen-activated protein kinases (MAPKs) play important roles against pathogens. In this study, Bmp38 and BmS6K were identified as differentially expressed proteins from iTRAQ database. Bmp38 and BmS6K were expressed, and recombinant proteins were purified. The bioinformatics analysis showed that both proteins have serine/threonine-protein kinases, catalytic domain (S_TKc) with 360 and 753 amino acids, respectively. The real-time quantitative polymerase chain reaction (RT-qPCR) results suggest that Bmp38 and BmS6K had high expression in the midgut and hemolymph. The comparative expression level of Bmp38 and BmS6K in BC9 was upregulated than in P50 in the midgut after Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Western bolt results showed a positive correlation between RT-qPCR and iTRAQ data for Bmp38, but BmS6K data showed partial correlation with iTRAQ. Injection of anti-Bmp38 and anti-BmS6K serum suggested that Bmp38 may be involved against BmNPV infection, whereas BmS6K may require phosphorylation modification to inhibit BmNPV infection. Taken together, our results suggest that Bmp38 and BmS6k might play an important role in innate immunity of silkworm against BmNPV.

Effects of Early Exposure of Isoflurane on Chronic Pain via the Mammalian Target of Rapamycin Signal Pathway.

Persistent post-surgical pain (PPSP) is a chronic pain condition, often with neuropathic features, that occurs in approximately 20% of children who undergo surgery. The biological basis of PPSP has not been elucidated. Anesthetic drugs can have lasting effects on the developing nervous system, although the clinical impact of this phenomenon is unknown. Here, we used a mouse model to test the hypothesis that early developmental exposure to isoflurane causes cellular and molecular alteration in the pain perception circuitry that causes a predisposition to chronic, neuropathic pain via a pathologic upregulation of the mammalian target of the rapamycin (mTOR) signaling pathway. Mice were exposed to isoflurane at postnatal day 7 and select cohorts were treated with rapamycin, an mTOR pathway inhibitor. Behavioral tests conducted 2 months later showed increased evidence of neuropathic pain, which did not occur in rapamycin-treated animals. Immunohistochemistry showed neuronal activity was chronically increased in the insular cortex, anterior cingulate cortex, and spinal dorsal horn, and activity was attenuated by rapamycin. Immunohistochemistry and western blotting (WB) showed a co-incident chronic, abnormal upregulation in mTOR activity. We conclude that early isoflurane exposure alters the development of pain circuits and has the potential to contribute to PPSP and/or other pain syndromes.

Regulation of the expression of key signalling molecules in mTOR pathway of skeletal muscle satellite cells in neonatal chicks: Effects of leucine and glycine-leucine peptide.

This study was conducted to analyse the effects of leucine (Leu) and glycine (Gly)-Leu peptide on expressions of key signalling molecules in mTOR pathway of skeletal muscle satellite cells in neonatal chicks. The 4-day-old male AA broilers with similar weight were selected to obtain the broiler skeletal muscle satellite cells with the two-step method of collagenase-I and trypsin digestion. The satellite cells were subjected to primary culture in vitro, and they were cultured in DMEM medium with the Leu concentration of 0.2 mM and 2 mM as well as with the Gly-Leu peptide concentration of 0.2 mM and 2 mM. The experiment lasted for 5 days. The results showed that TOR, S6K1 and 4E-BP1 mRNA expressions in the medium with Leu concentration of 2 mM were significantly higher than that in 0.2 mM group (p < 0.05). There was no difference between the medium with Gly-Leu concentration of 2 mM and 0.2 mM on the TOR, S6K1 and 4E-BP1 mRNA expressions (p > 0.05). In conclusion, Leu significantly increases TOR, S6K1 and 4E-BP1 mRNA expressions of skeletal muscle satellite cells, but Gly-Leu peptide has no effect on them.

6- and 8-Prenylnaringenin, Novel Natural Histone Deacetylase Inhibitors Found in Hops, Exert Antitumor Activity on Melanoma Cells.

BACKGROUND/AIMS: Prenylnaringenins are natural prenylflavonoids with anticancer properties. However, the underlying mechanisms have not been elucidated yet. Here we report a novel mode of action of 6- and 8-prenylnaringenin (PN) on human melanoma cells: Inhibition of cellular histone deacetylases (HDACs). METHODS: We performed in silico and in vitro analyses using 6-PN or 8-PN to study a possible interaction of 6-PN or 8-PN with HDAC as well as Western blot and FACS analyses, real-time cell proliferation and cell viability assays to assess the impact of 6-PN and 8-PN on human metastatic melanoma cells. RESULTS: In silico, 6-PN and 8-PN fit into the binding pocket of HDAC2, 4, 7 and 8, binding to the zinc ion of their catalytic center that is essential for enzymatic activity. In vitro, 100 µmol/L of 6-PN or 8-PN inhibited all 11 conserved human HDAC of class I, II and IV. In clinical oncology HDAC inhibitors are currently investigated as new anticancer compounds. In line, treatment of SK-MEL-28 cells with 6-PN or 8-PN induced a hyperacetylation of histone complex H3 within 2 h. Further, 6-PN or 8-PN mediated a prominent, dose-dependent reduction of cellular proliferation and viability of SK-MEL-28 and BLM melanoma cells. This effect was apoptosis-independent and accompanied by down-regulation of mTOR-specific pS6 protein via pERK/pP90 in SK-MEL-28 cells. CONCLUSION: The identification of a broad inhibitory capacity of 6-PN and 8-PN for HDAC enzymes with antiproliferative effects on melanoma cells opens the perspective for clinical application as novel anti-melanoma drugs and the usage as innovative lead structures for chemical modification to enhance pharmacology or inhibitory activities.

Transfer-RNA-mediated enhancement of ribosomal proteins S6 kinases signaling for cell proliferation.

While transfer-RNAs (tRNAs) are known to transport amino acids to ribosome, new functions are being unveiled from tRNAs and their fragments beyond protein synthesis. Here we show that phosphorylation of 90-kDa RPS6K (ribosomal proteins S6 kinase) was enhanced by tRNALeu overexpression under amino acids starvation condition. The phosphorylation of 90-kDa RPS6K was decreased by siRNA specific to tRNALeu and was independent to mTOR (mammalian target of rapamycin) signaling. Among the 90-kDa RPS6K family, RSK1 (ribosomal S6 kinase 1) and MSK2 (mitogen-and stress-activated protein kinase 2) were the major kinases phosphorylated by tRNALeu overexpression. Through SILAC (stable isotope labeling by/with amino acids in cell culture) and combined mass spectrometry analysis, we identified EBP1 (ErbB3-binding protein 1) as the tRNALeu-binding protein. We suspected that the overexpression of free tRNALeu would reinforce ErbB2/ErbB3 signaling pathway by disturbing the interaction between ErbB3 and EBP1, resulting in RSK1/MSK2 phosphorylation, improving cell proliferation and resistance to death. Analysis of samples from patients with breast cancer also indicated an association between tRNALeu overexpression and the ErbB2-positive population. Our results suggested a possible link between tRNALeu overexpression and RSK1/MSK2 activation and ErbB2/ErbB3 signaling.

Ribosomal protein S6 kinase 1 signaling in prefrontal cortex controls depressive behavior.

Current treatments for major depressive disorder (MDD) have a time lag and are ineffective for a large number of patients. Development of novel pharmacological therapies requires a comprehensive understanding of the molecular events that contribute to MDD pathophysiology. Recent evidence points toward aberrant activity of synaptic proteins as a critical contributing factor. In the present studies, we used viral-mediated gene transfer to target a key mediator of activity-dependent synaptic protein synthesis downstream of mechanistic target of rapamycin complex 1 (mTORC1) known as p70 S6 kinase 1 (S6K1). Targeted delivery of two mutants of S6K1, constitutively active or dominant-negative, to the medial prefrontal cortex (mPFC) of rats allowed control of the mTORC1/S6K1 translational pathway. Our results demonstrate that increased expression of S6K1 in the mPFC produces antidepressant effects in the forced swim test without altering locomotor activity. Moreover, expression of active S6K1 in the mPFC blocked the anhedonia caused by chronic stress, resulting in a state of stress resilience. This antidepressant response was associated with increased neuronal complexity caused by enhanced S6K1 activity. Conversely, expression of dominant-negative S6K1 in the mPFC resulted in prodepressive behavior in the forced swim test and was sufficient to cause anhedonia in the absence of chronic stress exposure. Together, these data demonstrate a critical role for S6K1 activity in depressive behaviors, and suggest that pathways downstream of mTORC1 may underlie the pathophysiology and treatment of MDD.