Phosphorylation Of p70S6K Research Articles

Clinical spectrum of MTOR-related hypomelanosis of Ito with neurodevelopmental abnormalities

PurposeHypomelanosis of Ito (HI) is a skin marker of somatic mosaicism. Mosaic MTOR pathogenic variants have been reported in HI with brain overgrowth. We sought to delineate further the pigmentary skin phenotype and clinical spectrum of neurodevelopmental manifestations of MTOR-related HI. MethodsFrom two cohorts totaling 71 patients with pigmentary mosaicism, we identified 14 patients with Blaschko-linear and one with flag-like pigmentation abnormalities, psychomotor impairment or seizures, and a postzygotic MTOR variant in skin. Patient records, including brain magnetic resonance image (MRI) were reviewed. Immunostaining (n = 3) for melanocyte markers and ultrastructural studies (n = 2) were performed on skin biopsies. ResultsMTOR variants were present in skin, but absent from blood in half of cases. In a patient (p.[Glu2419Lys] variant), phosphorylation of p70S6K was constitutively increased. In hypopigmented skin of two patients, we found a decrease in stage 4 melanosomes in melanocytes and keratinocytes. Most patients (80%) had macrocephaly or (hemi)megalencephaly on MRI. ConclusionMTOR-related HI is a recognizable neurocutaneous phenotype of patterned dyspigmentation, epilepsy, intellectual deficiency, and brain overgrowth, and a distinct subtype of hypomelanosis related to somatic mosaicism. Hypopigmentation may be due to a defect in melanogenesis, through mTORC1 activation, similar to hypochromic patches in tuberous sclerosis complex. Graphical Abstract [Display omitted]

Akt3-mTOR regulates hippocampal neurogenesis in adult mouse.

Akt signaling has been associated with adult neurogenesis in the hippocampal dentate gyrus (DG). We reported cognitive dysfunction in Akt3 knockout (Akt3-KO) mice with the down-regulation of mTOR activation. However, little is known about the effects of Akt3 signaling on hippocampal neurogenesis. Herein, we show that progenitor cells, neuroblasts, and mature newborn neurons in hippocampal DG expressed Akt3 protein. The Akt3 phosphorylation in hippocampal DG was increased after voluntary wheel running for 7days in wild-type mice (running WT mice), but not in Akt3-KO mice (running Akt3-KO mice). Subsequently, we observed that the proliferation of progenitor cells was suppressed in Akt3-KO mice and the mTOR inhibitor rapamycin-treated mice, whereas enhanced in running WT mice rather than running Akt3-KO mice. Neurite growth of neuroblasts was impaired in Akt3-KO mice and rapamycin-treated mice. In contrast, neither differentiation of progenitor cells nor migrating of newly generated neurons was altered in Akt3-KO mice or running WT mice. The levels of p70S6K and 4EBP1 phosphorylation were declined in Akt3-KO mice and elevated in running WT mice depending on mTOR activation. Furthermore, telomerase activity, telomere length, and expression of telomerase reverse transcriptase (TERT) were decreased in Akt3-KO mice but increased in running WT mice rather than running Akt3-KO mice, which required the mTOR activation. The study provides in vivo evidence that Akt3-mTOR signaling plays an important role in the proliferation of progenitor cells and neurite growth through positive regulated TERT expression and activation of p70S6K and 4EBP1.

Pyraclostrobin induced AMPK/mTOR pathways mediated autophagy in RAW264.7 macrophages

Pyraclostrobin(PCT) is a highly effective and broad-spectrum strobilurin fungicide. The mode action of PCT is inhibiting mitochondrial respiration. With the widespread use of PCT in preventing and controlling crop diseases, its potential safety risks to mammals have gradually attracted attention. This paper focuses on the cytotoxicity of PCT and its molecular mechanism, RAW264.7 macrophages were selected as a research model and conducted systematic toxicology studies in vitro, including MTT assay, colony formation assay, alkaline comet assay, fluorescent staining, ATP assay and Western blotting. The results revealed that PCT decreased viability and inhibited the proliferation of RAW264.7 cells in a concentration- dependent manner. Interestingly, PCT induced DNA damage, the resulting autophagosome, the accumulation of Beclin-1, the reduction of p62, the translocation and the formation of LC3-II. Furthermore, the results showed that PCT-induced the production of excessive ROS, leading to mitochondrial permeability transition pore (mPTP) opening, ATP depletion, and the elimination of mitochondria by autophagy. Furthermore, PCT treatment group significantly enhanced the phosphorylation level of AMPK, decreased the mTOR and p70s6k phosphorylation levels and activated the AMPK/mTOR signaling pathway in RAW264.7 cells. In conclusion, these results showed that PCT induced autophagy in the RAW264.7 cells might potentially have risks to mammal safety.

Repeated bouts of resistance exercise in rats alter mechanistic target of rapamycin complex 1 activity and ribosomal capacity but not muscle protein synthesis.

What is the central question of this study? Is muscle protein synthesis (MPS) additionally activated following exercise when ribosomal capacity is increased after repeated bouts of resistance exercise (RE)? What is the main finding and its importance? Skeletal muscles with increased ribosome content through repeated RE bouts showed sufficient activation of MPS with lower mechanistic target of rapamycin complex 1 signalling. Thus, repeated bouts of RE possibly change the translational capacity and efficiency to optimize translation activation following RE. Resistance exercise (RE) activates ribosome biogenesis and increases ribosome content in skeletal muscles. However, it is unclear whether the increase in ribosome content subsequently causes an increase in RE-induced activation of muscle protein synthesis (MPS). Thus, this study aimed to investigate the relationship between ribosome content and MPS after exercise using a rat RE model. Male Sprague-Dawley rats were categorized into three groups (n=6 for each group): sedentary (SED) and RE trained with one bout (1B) or three bouts (3B). The RE stimulus was applied to the right gastrocnemius muscle by transcutaneous electrical stimulation under isoflurane anaesthesia. The 3B group underwent stimulation every other day. Our results revealed that 6 h after the last bout of RE, muscles in the 3B group showed an increase in total RNA and 18S+28S rRNA content per muscle weight compared with the SED and 1B groups. In both the 1B and 3B groups, MPS, estimated by puromycin incorporation in proteins, was higher than that in the SED group 6h after exercise; however, no significant difference was observed between the 1B and 3B groups. In the 1B and 3B groups, phosphorylated p70S6K at Thr-389 increased, indicating mechanistic target of rapamycin complex 1 (mTORC1) activity. p70S6K phosphorylation level was lower in the 3B group than in the 1B group. Finally, protein synthesis per ribosome (indicator of translation efficiency) was lower in the 3B group than in the 1B group. Thus, three bouts of RE changed the ribosome content and mTORC1 activation, but not the degree of RE-induced global MPS activation.

Functional analysis of LAT3 in prostate cancer: Its downstream target and relationship with androgen receptor.

L‐type amino acid transporter 3 (LAT3, SLC43A1) is abundantly expressed in prostate cancer (PC) and is thought to play an essential role in PC progression through the cellular uptake of essential amino acids. Here, we analyzed the expression, function, and downstream target of LAT3 in PC. LAT3 was highly expressed in PC cells expressing androgen receptor (AR), and its expression was increased by dihydrotestosterone treatment and decreased by bicalutamide treatment. In chromatin immunoprecipitation sequencing of AR, binding of AR to the SLC43A1 region was increased by dihydrotestosterone stimulation. Knockdown of LAT3 inhibited cell proliferation, migration, and invasion, and the phosphorylation of p70S6K and 4EBP‐1. Separase (ESPL1) was identified as a downstream target of LAT3 by RNA sequencing analysis. In addition, immunostaining of prostatectomy specimens was performed. In the multivariate analysis, high expression of LAT3 was an independent prognostic factor for recurrence‐free survival (hazard ratio: 3.24; P = .0018). High LAT3 expression was correlated with the pathological T stage and a high International Society of Urological Pathology grade. In summary, our results suggest that LAT3 plays an important role in the progression of PC.

Direct P70S6K1 inhibition to replace dexamethasone in synergistic combination with MCL-1 inhibition in multiple myeloma

AbstractNovel combination therapies have markedly improved the lifespan of patients with multiple myeloma (MM), but drug resistance and disease relapse remain major clinical problems. Dexamethasone and other glucocorticoids are a cornerstone of conventional and new combination therapies for MM, although their use is accompanied by serious side effects. We aimed to uncover drug combinations that act in synergy and, as such, allow reduced dosing while remaining effective. Dexamethasone and the myeloid cell leukemia 1 (MCL-1) inhibitor S63845 (MCL-1i) proved the most potent combination in our lethality screen and induced apoptosis of human myeloma cell lines (HMCLs) that was 50% higher compared with an additive drug effect. Kinome analysis of dexamethasone-treated HMCLs revealed a reduction in serine/threonine peptide phosphorylation, which was predicted to result from reduced Akt activity. Biochemical techniques showed no dexamethasone-induced effects on FOXO protein or GSK3 but did show a 50% reduction in P70S6K phosphorylation, downstream of the Akt-mTORC1 axis. Replacing dexamethasone by the P70S6K1 isoform-specific inhibitor PF-4708671 (S6K1i) revealed similar and statistically significant synergistic apoptosis of HMCLs in combination with MCL-1i. Interestingly, apoptosis induced by the P70S6K1i and MCL-1i combination was more-than-additive in all 9 primary MM samples tested; this effect was observed for 6 of 9 samples with the dexamethasone and MCL-1i combination. Toxicity on stem and progenitor cell subsets remained minimal. Combined, our results show a strong rationale for combination treatments using the P70S6K inhibitor in MM. Direct and specific inhibition of P70S6K may also provide a solution for patients ineligible or insensitive to dexamethasone or other glucocorticoids.

Nucleoprotein-enriched diet enhances protein synthesis pathway and satellite cell activation via ERK1/2 phosphorylation in unloaded rat muscles.

What is the central question of this study? The purpose of this study was to determine whether the nucleotides in a nucleoprotein-enriched diet could ameliorate the unloading-associated decrease in soleus muscle mass and fibre size. What is the main finding and its importance? The results indicate that the nucleotides in the nucleoprotein-enriched diet could ameliorate the unloading-associated decrease in typeI fibre size and muscle mass, most probably owing to the activation of protein synthesis pathways and satellite cell proliferation and differentiation via ERK1/2 phosphorylation. Thus, nucleotide supplementation appears to be an effective countermeasure for muscle atrophy. Hindlimb unloading decreases both the protein synthesis pathway and satellite cell activation and results in muscle atrophy. Nucleotides are included in nucleoprotein and provide the benefits of increasing extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. ERK1/2 phosphorylation is also important in the activation of satellite cells, especially for myoblast proliferation and stimulating protein synthesis pathways. Therefore, we hypothesized that nucleotides in the nucleoproteins would ameliorate muscle atrophy by increasing the protein synthesis pathways and satellite cell activation during hindlimb unloading in rat soleus muscle. Twenty-four female Wistar rats were divided into four groups: control rats fed a basal diet without nucleoprotein (CON), control rats fed a nucleoprotein-enriched diet (CON+NP), hindlimb-unloaded rats fed a basal diet (HU) or hindlimb-unloaded rats fed a nucleoprotein-enriched diet (HU+NP). HU for 2weeks resulted in reductions in phosphorylation of p70S6K and rpS6, the numbers of myoblast determination protein (MyoD)- and myogenin- positive nuclei, typeI muscle fibre size and muscle mass. Both CON+NP and HU+NP rats showed an increase in ERK1/2, phosphorylation of p70S6K and rpS6, and the numbers of MyoD- and myogenin-positive nuclei compared with their basal diet groups. The NP diet also ameliorated the unloading-associated decrease in typeI muscle fibre size and muscle mass. The results indicate that the nucleotides in the nucleoprotein-enriched diet could ameliorate the unloading-associated decrease in typeI fibre size and muscle mass, most probably owing to the activation of protein synthesis pathways and satellite cell proliferation and differentiation via ERK1/2 phosphorylation. Thus, nucleotide supplementation appears to be an effective countermeasure for muscle atrophy.

Glucagon Activates mTOR1/2 Signaling via an EPAC/Rap1 Signaling Axis in Hepatocyte Cultures

Activation of the protein kinase mTOR (mechanistic target of rapamycin) in complex 1/2 (mTORC1/2) in the liver is repressed during fasting and rapidly stimulated in response to feeding. The effect of a meal on hepatic mTORC1/2 is largely mediated by an increase in plasma levels of nutrients (i.e. amino acids) and growth factors (i.e. insulin). By contrast, fasting is associated with elevated plasma levels of glucagon, which is conventionally viewed as playing a counter-regulatory role to insulin. However, glucagon and insulin secretion are both rapidly increased by consumption of a protein meal. In fact, amino acid-induced glucagon secretion is many times greater than that seen with fasting. Herein we investigated the impact of insulin and glucagon on mTORC1/2 activation in rat H4IIE and human HepG2 cell cultures. As expected, insulin enhanced phosphorylation of the mTORC1 substrates p70S6K1 and 4E-BP1. Surprisingly, the effect of glucagon on mTORC1 signaling was biphasic, wherein there was an acute increase in phosphorylation of p70S6K1 and 4E-BP1 over the first hour of exposure, followed by latent suppression. Notably, the stimulatory effect of glucagon on mTORC1 was not additive with insulin, suggesting a convergent signaling axis. Glucagon-induced mTORC1 activation was associated with enhanced cAMP levels and required activation of the glucagon receptor, PI3K/Akt, and exchange protein activated by cAMP (EPAC). An EPAC-specific cAMP analog as well as PKA inhibition enhanced activation of mTORC1/2. EPAC acts as the guanine nucleotide exchange factor for the small GTPase Rap1, and in H4IIE cultures, transient expression of Rap1 was sufficient to stimulate activation of mTORC1/2. Finally, we found that glucagon addition to culture medium was sufficient to promote Rap1-GTP loading in H4IIE cells. Overall, the findings support that glucagon elicits biphasic regulation of hepatic mTORC1/2 by an EPAC-dependent increase in Rap1-GTP which is followed by a repression of mTORC1.

SLC7A5 serves as a prognostic factor of breast cancer and promotes cell proliferation through activating AKT/mTORC1 signaling pathway.

BackgroundThe purpose of our research was to determine if the clinical, pathological, and prognostic functions of SLC7A5 are the same as those of other molecular breast cancer (BC) subgroups.MethodsWe used the Oncomine and The Cancer Genome Atlas (TCGA) online databases to examine the expression and genetic changes of SLC7A5 in BC tissues. Immunohistochemical analysis was used to validate the SLC7A5 protein expression in subtypes of BC, while Kaplan-Meier figures and log-rank tests were used to evaluate the prognostic relevance of SLC7A5. Uni- and multivariate Cox regression models were adapted to analyze hazard ratios (HRs) and the independent prognostic factors. We analyzed the alterations of different malignancies of luminal cells by up-regulation of SLC7A5 in human luminal cell lines MCF-7. SLC7A5 was overexpressed in luminal cells, and then the AKT, mTOR, and p70-S6K phosphorylation and expression were analyzed by western blot analysis and real-time quantitative polymerase chain reaction (qPCR).ResultsOur results suggested that SLC7A5 was overexpressed in BC cell lines and in patients’ tissues. Elevated SLC7A5 messenger RNA (mRNA) and SLC7A5 protein expression was correlated to a worse clinical prognosis (P<0.001) in luminal subtypes of BC. The multivariate analysis suggested that high level of SLC7A5 expression could be an independent prognostic factor for decreased overall survival (OS). The study also demonstrated that SLC7A5 overexpression increased proliferation of MCF-7 cells by reducing the cell cycle arrest in G1 phase. Our mechanistic study further indicates that SLC7A5 enhances the proliferation of the MCF-7 cell by activation of AKT/mTORC1 pathway through phosphorylation.ConclusionsOur study demonstrated that SLC7A5 may have a vital function in the biology of BC cells, indicating that SLC7A5 is a potential prognostic biomarker and may be a valuable therapeutic target in BC patients.

The effect of repeated bouts of electrical stimulation-induced muscle contractions on proteolytic signaling in rat skeletal muscle.

Mechanistic target of rapamycin complex 1 (mTORC1) plays a central role in muscle protein synthesis and repeated bouts of resistance exercise (RE) blunt mTORC1 activation. However, the changes in the proteolytic signaling when recurrent RE bouts attenuate mTORC1 activation are unclear. Using a RE model of electrically stimulated rat skeletal muscle, this study aimed to clarify the effect of repeated RE bouts on acute proteolytic signaling, particularly the calpain, autophagy‐lysosome, and ubiquitin‐proteasome pathway. p70S6K and rpS6 phosphorylation, indicators of mTORC1 activity, were attenuated by repeated RE bouts. Calpain 3 protein was decreased at 6 h post‐RE in all exercised groups regardless of the bout number. Microtubule‐associated protein 1 light chain 3 beta‐II, an indicator of autophagosome formation, was increased at 3 h and repeated RE bouts increased at 6 h, post‐RE. Ubiquitinated proteins were increased following RE, but these increases were independent of the number of RE bouts. These results suggest that the magnitude of autophagosome formation was increased following RE when mTORC1 activity was attenuated with repeated bouts of RE.

The effect of young and old ex vivo human serum on cellular protein synthesis and growth in an in vitro model of aging.

In vitro models of muscle aging are useful for understanding mechanisms of age-related muscle loss and aiding the development of targeted therapies. To investigate mechanisms of age-related muscle loss in vitro utilizing ex vivo human serum, fasted blood samples were obtained from four old (72 ± 1 yr) and four young (26 ± 3 yr) men. Older individuals had elevated levels of plasma CRP, IL-6, HOMA-IR, and lower concentric peak torque and work-per-repetition compared with young participants (P < 0.05). C2C12 myotubes were serum and amino acid starved for 1 h and conditioned with human serum (10%) for 4 h or 24 h. After 4 h, C2C12 cells were treated with 5 mM leucine for 30 min. Muscle protein synthesis (MPS) was determined through the surface sensing of translation (SUnSET) technique and regulatory signaling pathways were measured via Western blot. Myotube diameter was significantly reduced in myotubes treated with serum from old, in comparison to young donors (84%, P < 0.001). MPS was reduced in myotubes treated with old donor serum, compared with young serum before leucine treatment (32%, P < 0.01). MPS and the phosphorylation of Akt, p70S6K, and eEF2 were increased in myotubes treated with young serum in response to leucine treatment, with a blunted response identified in cells treated with old serum (P < 0.05). Muscle protein breakdown signaling pathways did not differ between groups. In summary, we show that myotubes conditioned with serum from older individuals had decreased myotube diameter and MPS compared with younger individuals, potentially driven by low-grade systemic inflammation.

Phenformin and ataxia-telangiectasia mutated inhibitors synergistically co-suppress liver cancer cell growth by damaging mitochondria.

Inhibitors of ataxia–telangiectasia mutated (ATM), such as KU‐55933 (Ku), represent a promising class of novel anticancer drugs. In addition, the biguanide derivative phenformin exhibits antitumor activity superior to that of the AMPK activator metformin. Herein, we assessed the potential combinatorial therapeutic efficacy of phenformin and Ku when used to inhibit the growth of liver cancer cells, and we assessed the mechanisms underlying such efficacy. The Hep‐G2 and SMMC‐7721 liver cancer cell lines were treated with phenformin and Ku either alone or in combination, after which the impact of these drugs on cellular proliferation was assessed via 3‐(4,5‐dimethylthiazol) 2, 5‐diphenyltetrazolium and colony formation assays, whereas Transwell assays were used to gauge cell migratory activity. The potential synergy between these two drugs was assessed using the compusyn software, while flow cytometry was employed to evaluate cellular apoptosis. In addition, western blotting was utilized to measure p‐ATM, p‐AMPK, p‐mTOR, and p‐p70s6k expression, while mitochondrial functionality was monitored via morphological analyses, JC‐1 staining, and measurements of ATP levels. Phenformin and Ku synergistically impacted the proliferation, migration, and apoptotic death of liver cancer cells. Together, these compounds were able to enhance AMPK phosphorylation while inhibiting the phosphorylation of mTOR and p70s6k. These data also revealed that phenformin and Ku induced mitochondrial dysfunction as evidenced by impaired ATP synthesis, mitochondrial membrane potential, and abnormal mitochondrial morphology. These findings suggest that combination treatment with phenformin and Ku may be an effective approach to treating liver cancer via damaging mitochondria within these tumor cells.

Antidepressant-like effect of guanosine involves activation of AMPA receptor and BDNF/TrkB signaling.

Guanosine is a purine nucleoside that has been shown to exhibit antidepressant effects, but the mechanisms underlying its effect are not well established. We investigated if the antidepressant-like effect induced by guanosine in the tail suspension test (TST) in mice involves the modulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, voltage-dependent calcium channel (VDCC), and brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB) pathway. We also evaluated if the antidepressant-like effect of guanosine is accompanied by an acute increase in hippocampal and prefrontocortical BDNF levels. Additionally, we investigated if the ability of guanosine to elicit a fast behavioral response in the novelty suppressed feeding (NSF) test is associated with morphological changes related to hippocampal synaptogenesis. The antidepressant-like effect of guanosine (0.05 mg/kg, p.o.) in the TST was prevented by DNQX (AMPA receptor antagonist), verapamil (VDCC blocker), K-252a (TrkBantagonist), or BDNF antibody. Increased P70S6K phosphorylation and higher synapsin I immunocontent in the hippocampus, but not in the prefrontal cortex, were observed 1 h after guanosine administration. Guanosine exerted an antidepressant-like effect 1, 6, and 24 h after its administration, an effect accompanied by increased hippocampal BDNF level. In the prefrontal cortex, BDNF level was increased only 1 h after guanosine treatment. Finally, guanosine was effective in the NSF test (after 1 h) but caused no alterations in dendritic spine density and remodeling in the ventral dentate gyrus (DG). Altogether, the results indicate that guanosine modulates targets known to be implicated in fast antidepressant behavioral responses (AMPA receptor, VDCC, and TrkB/BDNF pathway).

MAPK, AKT/FoxO3a and mTOR pathways are involved in cadmium regulating the cell cycle, proliferation and apoptosis of chicken follicular granulosa cells

The occurrence of cadmium (Cd) in feed is a major problem in animal health and production. Studies have confirmed that Cd depresses egg production of laying hens, which is closely related to follicular atresia. This study aimed to assess the toxic impacts of Cd on the ovarian tissue, and to examine the mechanism of Cd-induced granulosa cell proliferation and apoptosis. Results from the nitric oxide (NO) and malondialdehyde (MDA) content, total superoxide dismutase (T-SOD), glutathione peroxide (GSH-Px), total nitric oxide synthase (T-NOS) and adenosine triphosphatase (ATPase) activities, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and hematoxylin-eosin (H & E) staining indicated that excess Cd induced oxidative stress, granulosa cell apoptosis and follicular atresia in the layer ovary. Low-dose Cd exposure (1 μM) induced the granulosa cell proliferation, upregulated the mRNA levels of RSK1 and RHEB, activated FoxO3a, AKT, ERK1/2, mTOR and p70S6K1 phosphorylation, and promoted cell cycle progression from phase G1 to S. However, high-dose Cd exposure (15 μM) induced reactive oxygen species (ROS) generation and cell apoptosis, upregulated the mRNA levels of the inflammatory factors, ASK1, JNK, p38 and TAK1, downregulated the expressions of RSK1 and RHEB genes, and inhibited the phosphorylation of ERK1/2, mTOR and p70S6K1 proteins, and the cell cycle progression. Rapamycin pre-treatment completely blocked the phosphorylation of mTOR and p70S6K1 proteins, and the cell cycle progression induced by 1 μM Cd, and accelerated 15 μM Cd-induced cell apoptosis and cell cycle arrest. The microRNA sequencing result showed that 15 μM Cd induced differential expression of microRNA genes, which may regulate AKT, ERK1/2 and mTOR signaling and cell cycle progression by regulating the activity of G proteins and cell cycle-related proteins. Conclusively, these results indicated that Cd can cause the ovarian damage and follicular atresia, and regulate cell cycle, cell proliferation or apoptosis of granulosa cells through MAPK, AKT/FoxO3a and mTOR pathways in laying hens.

Reactivations of Latent Viral Infections Are Associated with an Increased Thr389 p70S6k Phosphorylation in Peripheral Lymphocytes of Renal Transplant Recipients.

Reactivations of BK polyoma virus (BKPyV) and human cytomegalovirus (HCMV) frequently cause life- and graft-threatening complications after renal transplantation. Both viruses are dependent on the mTOR pathway for replication. In this study we investigated the association of viral replication with mTOR activity in peripheral lymphocytes of renal transplant recipients. A flow-cytometry based assay for the measurement of Thr389 p70S6k phosphorylation, a surrogate marker of the mTOR pathway was established. Forty-eight adult renal transplant recipients were recruited to measure p70S6k activity in their peripheral blood mononuclear cells. This data set in conjunction with information concerning previous replication of BKPyV and HCMV was examined for correlations. Episodes of BKPyV replication were significantly associated with increased p70S6k phosphorylation in CD4+ T lymphocytes (p = 0.0002) and CD19+ B lymphocytes (p = 0.0073). HCMV infection of patients with a high-risk HCMV constellation of donor and recipient (D+/R−) was associated with increased p70S6k phosphorylation in CD19+ B lymphocytes (p = 0.0325). These associations were found to be independent of the trough levels of the immunosuppressive drugs. Conclusion: P70S6k phosphorylation in peripheral lymphocytes is associated with BKPyV reactivations and to a lesser extent with HCMV infections in renal transplant recipients.

LPAR5 promotes thyroid carcinoma cell proliferation and migration by activating class IA PI3K catalytic subunit p110β.

Lysophosphatidic acid receptor 5 (LPAR5) is involved in mediating thyroid cancer progression, but the underlying mechanism needs to be further revealed. In this study, we confirmed that LPAR5 is upregulated in papillary thyroid carcinoma (PTC), especially in BRAF‐like PTC, by analyzing The Cancer Genome Atlas (TCGA) database and performing immunohistochemistry assay in human thyroid cancer tissues. LPAR5‐specific antagonist TC LPA5 4 treatment inhibited CGTH‐W3, TPC‐1, B‐CPAP, and BHT‐101 cell proliferation, CGTH‐W3 and TPC‐1 cell migration significantly. In vivo, TC LPA5 4 treatment could delay CGTH‐W3 xenograft growth in nude mice. We also found that LPAR5‐specific antagonist TC LPA5 4, PI3K inhibitor wortmannin, or mTOR inhibitor rapamycin pretreatment abrogated phosphorylation of Akt and p70S6K1 stimulated by LPA in CGTH‐W3 and TPC‐1 cells. Stimulating CGTH‐W3 cells transfected with pEGFPC1‐Grp1‐PH fusion protein with LPA resulted in the generation of phosphatidylinositol (3,4,5)‐triphosphate, which indicates that PI3K was activated by LPA directly. The p110β‐siRNA instead of p110α‐siRNA transfection abrogated the increase of levels of phosphorylated Akt and S6K1 stimulated by LPA. Furthermore, immunoprecipitation assay confirmed an interaction between LPAR5 and p110β. Overall, we provide new insights that the downregulation of LPAR5 decreased the proliferation and migration phenotype via the PI3K/Akt pathway. Inhibition of LPAR5 or the PI3K/Akt signal may be a novel therapeutic strategy for treating thyroid cancer.

TNFα enhances trovafloxacin-induced in vitro hepatotoxicity by inhibiting protective autophagy

Trovafloxacin (TVX) is associated with idiosyncratic drug-induced liver injury (iDILI) and inflammation-mediated hepatotoxicity. However, the inflammatory stress-regulated mechanisms in iDILI remain unclear. Herein, we elucidated the novel role of tumor-necrosis factor alpha (TNFα), an inflammatory stress factor, in TVX-induced in vitro hepatotoxicity and synergistic toxicity. TVX specifically induced synergistic toxicity in HepG2 cells with TNFα, which inhibits autophagy. TVX-treated HepG2 cells induced protective autophagy by inhibiting the expression of mTOR signaling proteins, while ATG5 knockdown in HepG2 cells, responsible for the impairment of autophagy, enhanced TVX-induced toxicity due to the increase in cytochrome C release and JNK pathway activation. Interestingly, the expression of mTOR signal proteins, which were suppressed by TVX, disrupted the negative feedback of the PI3K/AKT pathway and TNFα rebounded p70S6K phosphorylation. Co-treatment with TVX and TNFα inhibited protective autophagy by maintaining p70S6K activity, which enhanced TVX-induced cytotoxicity. Phosphorylation of p70S6K was inhibited by siRNA knockdown and rapamycin to restore TNFα-inhibited autophagy, which prevented the synergistic effect on TVX-induced cytotoxicity. These results indicate that TVX activates protective autophagy in HepG2 cells exposed to toxicity and an imbalance in negative feedback regulation of autophagy by TNFα synergistically enhanced the toxicity. The finding from this study may contribute to a better understanding of the mechanisms underlying iDILI associated with inflammatory stress.

A Fish-Derived Protein Hydrolysate Induces Postprandial Aminoacidaemia and Skeletal Muscle Anabolism in an In Vitro Cell Model Using Ex Vivo Human Serum.

Fish-derived proteins, particularly fish protein hydrolysates (FPH), offer potential as high-quality sources of dietary protein, whilst enhancing economic and environmental sustainability. This study investigated the impact of a blue whiting-derived protein hydrolysate (BWPH) on aminoacidaemia in vivo and skeletal muscle anabolism in vitro compared with whey protein isolate (WPI) and an isonitrogenous, non-essential amino acid (NEAA) control (0.33 g·kg−1·body mass−1) in an ex vivo, in vitro experimental design. Blood was obtained from seven healthy older adults (two males, five females; age: 72 ± 5 years, body mass index: 24.9 ± 1.6 kg·m2) in three separate trials in a randomised, counterbalanced, double-blind design. C2C12 myotubes were treated with ex vivo human serum-conditioned media (20%) for 4 h. Anabolic signalling (phosphorylation of mTOR, p70S6K, and 4E-BP1) and puromycin incorporation were determined by immunoblotting. Although BWPH and WPI both induced postprandial essential aminoacidaemia in older adults above the NEAA control, peak and area under the curve (AUC) leucine and essential amino acids were more pronounced following WPI ingestion. Insulin was elevated above baseline in WPI and BWPH only, a finding reinforced by higher peak and AUC values compared with NEAA. Muscle protein synthesis, as measured by puromycin incorporation, was greater after incubation with WPI-fed serum compared with fasted serum (P = 0.042), and delta change was greater in WPI (P = 0.028) and BWPH (P = 0.030) compared with NEAA. Myotube hypertrophy was greater in WPI and BWPH compared with NEAA (both P = 0.045), but was similar between bioactive conditions (P = 0.853). Taken together, these preliminary findings demonstrate the anabolic potential of BWPH in vivo and ex vivo, thus providing justification for larger studies in older adults using gold-standard measures of acute and chronic MPS in vivo.

MTOR Inhibitors as Radiosensitizers in Neuroendocrine Neoplasms.

Peptide receptor radioligand therapy (PRRT) has evolved as an important second-line treatment option in the management of inoperable and metastatic neuroendocrine neoplasms (NEN). Though high radiation doses can be delivered to the tumors, complete remission is still rare. Radiosensitization prior to PRRT is therefore considered to be a promising strategy to improve the treatment effect. In this study, effect and mechanism of mTOR inhibitors were investigated in a comprehensive panel of five NEN cell lines (BON, QGP-1, LCC-18, H727, UMC-11), employing assays for cellular proliferation, clonogenic survival, cell cycle modification and signaling. mTOR inhibition lead to growth arrest with a biphasic concentration-response pattern: a partial response at approximately 1 nM and full response at micromolar concentrations (8–48 µM). All cell lines demonstrated elevated p70S6K phosphorylation yet also increased phosphorylation of counterregulatory Akt. The pulmonary NEN cell line UMC-11 showed the lowest induction of phospho-Akt and strongest growth arrest by mTOR inhibitors. Radiation sensitivity of the cells (50% reduction versus control) was found to range between 4 and 8 Gy. Further, mTOR inhibition was employed together with irradiation to evaluate radiosensitizing effects of this combination treatment. mTOR inhibition was found to radiosensitize all five NEN cells in an additive manner with a moderate overall effect. The radiation-induced G2/M arrest was diminished under combination treatment, leading to an increased G1 arrest. Further investigation involving a suitable animal model as well as radioligand application such as 177Lu-DOTATATE or 177Lu-DOTATOC will have to demonstrate the full potential of this strategy for radiosensitization in NEN.

Fumonisin B1 induces nephrotoxicity via autophagy mediated by mTORC1 instead of mTORC2 in human renal tubule epithelial cells

Fumonisin B1 (FB1), a worldwide contaminating mycotoxin, can cause global food issue. It has been reported that FB1 is related to chronic kidney disease of unknown etiology. However, the study of FB1-induced nephrotoxicity in vitro is very limited and the mechanism is unknown. Human renal tubule epithelial (HK-2) cells were used in this study. The results showed that FB1 exposure could decrease cell viability, induce cell apoptosis and up-regulate the expression of Kim-1, collagen I, α-SMA and TGF-β1. In addition, autophagy was activated after FB1 exposure, including the conversion of LC3 and up-regulation of ATGs. Furthermore, autophagy inhibitor 3-MA could block FB1-induced abnormalities. And antioxidant enzymes (Gpx1 and Gpx4) were obviously down-regulated and intracellular ROS levels displayed an ascent trend as FB1 exposure concentrations increased. Employing of antioxidant NAC could suppress FB1-induced nephrotoxicity and autophagy. FB1 inhibited the phosphorylation of p70 S6k, a downstream protein of mTORC1. Also, oxidative stress, autophagy and phosphorylation of p70 S6k induced by FB1 was inhibited by MHY1485, an activator of mTOR. But the phosphorylation of AKT, a downstream protein of mTORC2 showed no change with or without MHY1485. Taken together, FB1 induced nephrotoxicity via autophagy mediated by mTORC1 instead of mTORC2 in HK-2 cells.