Akt-dependent Phosphorylation Research Articles

PI 3-kinase/AKT/FOXO1 signaling in smooth muscle cells protects against abdominal aortic aneurysm

Abstract Background Abdominal aortic aneurysms (AAA) are characterized by loss of vascular smooth muscle cells (SMCs). Growth factors induce proliferation and cell survival of SMCs. PI 3-kinase (PI3K) is an important downstream mediator in growth factor signaling. Objective This project is based on the hypothesis that PI3K-mediated SMC proliferation can compensate for the loss of SMCs in AAA and thus inhibit AAA progression. Therefore, we determined effects of reduced PI3K activity in SMCs on AAA progression, cell proliferation as well as vascular remodeling. Methods To investigate the role of PI3K, we analyzed mice and aortic SMCs harboring a smooth muscle-specific deficiency of the PI3K catalytic subunit p110α (SM-p110α-/-). AAA development in SM-p110α-/- mice and wild-type (WT) littermates was induced by perfusion of the infrarenal aortic segment with porcine pancreatic elastase (PPE). AAA diameter was determined by echocardiography. Structural changes in the aorta were identified by (immuno)histochemical staining. SMC proliferation was measured in vitro by BrdU incorporation or real-time cell analysis. p110α induced signal transduction pathways were characterized by Western blot and transcriptome analysis. Results PPE treatment led to a massive loss of SMCs in the compromised aortic segment after 3 days in both SM-p110α-/- and WT mice. The aortic diameter was enlarged after 28 days in all PPE-treated mice. However, the increase was significantly higher in SM-p110α-/- mice (70.2±10.8%, n=9) compared to WT animals (42.4±6.0%, n=10) (p<0.05). A similar effect was obtained in aged (untreated) mice: In 24-month-old SM-p110α-/- mice, abdominal aortic lumen diameter was increased by 42% compared to 3-month-old animals, but only by 15% in corresponding WT mice. PPE-treated SM-p110α-/- mice were characterized by reduced vascular remodeling with less PCNA-positive proliferating cells compared to WT animals. In addition, proliferation of aortic p110α-/- SMCs in serum-containing medium was impaired. Mechanistic analyses showed that PDGF- and insulin-induced activation of AKT as well as AKT-dependent phosphorylation and inactivation of the transcription factors FOXO-1, -3 and -4 were significantly reduced in p110α-/- SMCs compared to WT cells. Further analyses revealed that specific inhibition of FOXO1 by AS1842856 rescued PDGF or serum stimulated proliferation of p110α-/- SMCs. In addition, expression analysis of FOXOs in patients revealed significantly increased FOXO1 expression in AAA compared to aortic samples from healthy individuals. Conclusion Deficiency of the catalytic PI3K isoform p110α in SMCs promotes the development and progression of AAA. p110α/AKT-dependent inactivation of FOXO1 induces SMC proliferation, which can ameliorate aortic wall damage. As FOXO1 inhibitors and a recently developed p110α activator are available, the PI3K/AKT/FOXO1 signaling cascade may provide a therapeutic target to influence the stability of the aortic wall in AAA.

Phospholipase Cδ-4 (PLCδ4) Acts as a Nuclear Player to Influence Cyclin B Expression in the Embryonal Rhabdomyosarcoma Cell Lines RD and A204.

Rhabdomyosarcoma (RMS), the most common form of sarcoma typical of pediatric age, arises from the malignant transformation of the mesenchymal precursors that fail to differentiate into skeletal muscle cells. Here, we investigated whether the protein phospholipase C δ4 (PLCδ4), a member of the PLC family involved in proliferation and senescence mechanisms of mesenchymal stromal stem cells, may play a role in RMS. Our molecular and morpho-functional data reveal that PLCδ4 is highly expressed in the fusion-negative, p53-positive, SMARCB1 heterozygous mutated embryonal RMS (ERMS) cell line A204, while it is poorly expressed in the ERMS cell lines RD (fusion-negative, MYC amplification, N-RAS (Q61H), homozygous mutated p53) and Hs729 (homozygous mutated p53) and the alveolar rhabdosarcoma (ARMS) cell line SJCRH30 (RH30; fusion positive, heterozygous mutated RARA, polyheterozygous mutated p53). To characterize the role of PLCδ4, the RD cell line was stably transfected with wild-type PLCδ4 (RD/PLCδ4). Overexpressed PLCδ4 mainly localized to the nucleus in RD cells and contributed to the phosphorylation of PRAS40 (T246), Chk2(T68), WNK1(T60), and Akt 1/273 (S473), as revealed by proteome profiler array analysis. Overexpression of PLCδ4 in RD cells enhanced cyclin B1 expression and resulted in G2/M-phase cell cycle arrest. In contrast, siRNA-mediated knockdown of PLCδ4 in A204 cells resulted in reduced cyclin B1 expression. Our study identifies a novel role for nuclear PLCδ4 as a regulator of cyclin B1 via Akt-dependent phosphorylation. The modulation of PLCδ4 expression and its downstream targets could represent a crucial signaling pathway to block embryonal RMS cell proliferation.

Hyperfunction of post-synaptic density protein 95 promotes seizure response in early-stage aβ pathology

Accumulation of amyloid-beta (Aβ) can lead to the formation of aggregates that contribute to neurodegeneration in Alzheimer’s disease (AD). Despite globally reduced neural activity during AD onset, recent studies have suggested that Aβ induces hyperexcitability and seizure-like activity during the early stages of the disease that ultimately exacerbate cognitive decline. However, the underlying mechanism is unknown. Here, we reveal an Aβ-induced elevation of postsynaptic density protein 95 (PSD-95) in cultured neurons in vitro and in an in vivo AD model using APP/PS1 mice at 8 weeks of age. Elevation of PSD-95 occurs as a result of reduced ubiquitination caused by Akt-dependent phosphorylation of E3 ubiquitin ligase murine-double-minute 2 (Mdm2). The elevation of PSD-95 is consistent with the facilitation of excitatory synapses and the surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors induced by Aβ. Inhibition of PSD-95 corrects these Aβ-induced synaptic defects and reduces seizure activity in APP/PS1 mice. Our results demonstrate a mechanism underlying elevated seizure activity during early-stage Aβ pathology and suggest that PSD-95 could be an early biomarker and novel therapeutic target for AD.

NUAK1 coordinates growth factor-dependent activation of mTORC2 and Akt signaling

BackgroundmTORC2 is a critical regulator of cytoskeleton organization, cell proliferation, and cancer cell survival. Activated mTORC2 induces maximal activation of Akt by phosphorylation of Ser-473, but regulation of Akt activity and signaling crosstalk upon growth factor stimulation are still unclear.ResultsWe identified that NUAK1 regulates growth factor-dependent activation of Akt by two mechanisms. NUAK1 interacts with mTORC2 components and regulates mTORC2-dependent activation of Akt by controlling lysosome positioning and mTOR association with this organelle. A second mechanism involves NUAK1 directly phosphorylating Akt at Ser-473. The effect of NUAK1 correlated with a growth factor-dependent activation of specific Akt substrates. NUAK1 induced the Akt-dependent phosphorylation of FOXO1/3a (Thr-24/Thr-32) but not of TSC2 (Thr-1462). According to a subcellular compartmentalization that could explain NUAK1’s differential effect on the Akt substrates, we found that NUAK1 is associated with early endosomes but not with plasma membrane, late endosomes, or lysosomes. NUAK1 was required for the Akt/FOXO1/3a axis, regulating p21CIP1, p27KIP1, and FoxM1 expression and cancer cell survival upon EGFR stimulation. Pharmacological inhibition of NUAK1 potentiated the cell death effect induced by Akt or mTOR pharmacological blockage. Analysis of human tissue data revealed that NUAK1 expression positively correlates with EGFR expression and Akt Ser-473 phosphorylation in several human cancers.ConclusionsOur results showed that NUAK1 kinase controls mTOR subcellular localization and induces Akt phosphorylation, demonstrating that NUAK1 regulates the growth factor-dependent activation of Akt signaling. Therefore, targeting NUAK1, or co-targeting it with Akt or mTOR inhibitors, may be effective in cancers with hyperactivated Akt signaling.

Formyl-peptide receptor 2 signalling triggers aerobic metabolism of glucose through Nox2-dependent modulation of pyruvate dehydrogenase activity.

The human formyl-peptide receptor 2 (FPR2) is activated by an array of ligands. By phospho-proteomic analysis we proved that FPR2 stimulation induces redox-regulated phosphorylation of many proteins involved in cellular metabolic processes. In this study, we investigated metabolic pathways activated in FPR2-stimulated CaLu-6 cells. The results showed an increased concentration of metabolites involved in glucose metabolism, and an enhanced uptake of glucose mediated by GLUT4, the insulin-regulated member of GLUT family. Accordingly, we observed that FPR2 transactivated IGF-IRβ/IRβ through a molecular mechanism that requires Nox2 activity. Since cancer cells support their metabolism via glycolysis, we analysed glucose oxidation and proved that FPR2 signalling promoted kinase activity of the bifunctional enzyme PFKFB2 through FGFR1/FRS2- and Akt-dependent phosphorylation. Furthermore, FPR2 stimulation induced IGF-IRβ/IRβ-, PI3K/Akt- and Nox-dependent inhibition of pyruvate dehydrogenase activity, thus preventing the entry of pyruvate in the tricarboxylic acid cycle. Consequently, we observed an enhanced FGFR-dependent lactate dehydrogenase (LDH) activity and lactate production in FPR2-stimulated cells. As LDH expression is transcriptionally regulated by c-Myc and HIF-1, we demonstrated that FPR2 signalling promoted c-Myc phosphorylation and Nox-dependent HIF-1α stabilization. These results strongly indicate that FPR2-dependent signalling can be explored as a new therapeutic target in treatment of human cancers.

Toxoplasma gondii inhibits the expression of autophagy-related genes through AKT-dependent inactivation of the transcription factor FOXO3a.

The intracellular parasite Toxoplasma gondii induces host AKT activation to prevent autophagy-mediated clearance; however, the molecular underpinnings are not fully understood. Autophagy can be negatively regulated through AKT-sensitive phosphorylation and nuclear export of the transcription factor Forkhead box O3a (FOXO3a). Using a combination of pharmacological and genetic approaches, herein we investigated whether T. gondii hinders host autophagy through AKT-dependent inactivation of FOXO3a. We found that infection by type I and II strains of T. gondii promotes gradual and sustained AKT-dependent phosphorylation of FOXO3a at residues S253 and T32 in human foreskin fibroblasts (HFF) and murine 3T3 fibroblasts. Mechanistically, AKT-sensitive phosphorylation of FOXO3a by T. gondii required live infection and the activity of PI3K but was independent of the plasma membrane receptor EGFR and the kinase PKCα. Phosphorylation of FOXO3a at AKT-sensitive residues was paralleled by its nuclear exclusion in T. gondii-infected HFF. Importantly, the parasite was unable to drive cytoplasmic localization of FOXO3a upon pharmacological blockade of AKT or overexpression of an AKT-insensitive mutant form of FOXO3a. Transcription of a subset of bona fide autophagy-related targets of FOXO3a was reduced during T. gondii infection in an AKT-dependent fashion. However, parasite-directed repression of autophagy-related genes was AKT-resistant in cells deficient in FOXO3a. Consistent with this, T. gondii failed to inhibit the recruitment of acidic organelles and LC3, an autophagy marker, to the parasitophorous vacuole upon chemically or genetically induced nuclear retention of FOXO3a. In all, we provide evidence that T. gondii suppresses FOXO3a-regulated transcriptional programs to prevent autophagy-mediated killing. IMPORTANCE The parasite Toxoplasma gondii is the etiological agent of toxoplasmosis, an opportunistic infection commonly transmitted by ingestion of contaminated food or water. To date, no effective vaccines in humans have been developed and no promising drugs are available to treat chronic infection or prevent congenital infection. T. gondii targets numerous host cell processes to establish a favorable replicative niche. Of note, T. gondii activates the host AKT signaling pathway to prevent autophagy-mediated killing. Herein, we report that T. gondii inhibits FOXO3a, a transcription factor that regulates the expression of autophagy-related genes, through AKT-dependent phosphorylation. The parasite's ability to block the recruitment of the autophagy machinery to the parasitophorous vacuole is impeded upon pharmacological inhibition of AKT or overexpression of an AKT-insensitive form of FOXO3a. Thus, our study provides greater granularity in the role of FOXO3a during infection and reinforces the potential of targeting autophagy as a therapeutic strategy against T. gondii.

An optogenetic-phosphoproteomic study reveals dynamic Akt1 signaling profiles in endothelial cells

The serine/threonine kinase AKT is a central node in cell signaling. While aberrant AKT activation underlies the development of a variety of human diseases, how different patterns of AKT-dependent phosphorylation dictate downstream signaling and phenotypic outcomes remains largely enigmatic. Herein, we perform a systems-level analysis that integrates methodological advances in optogenetics, mass spectrometry-based phosphoproteomics, and bioinformatics to elucidate how different intensity, duration, and pattern of Akt1 stimulation lead to distinct temporal phosphorylation profiles in vascular endothelial cells. Through the analysis of ~35,000 phosphorylation sites across multiple conditions precisely controlled by light stimulation, we identify a series of signaling circuits activated downstream of Akt1 and interrogate how Akt1 signaling integrates with growth factor signaling in endothelial cells. Furthermore, our results categorize kinase substrates that are preferably activated by oscillating, transient, and sustained Akt1 signals. We validate a list of phosphorylation sites that covaried with Akt1 phosphorylation across experimental conditions as potential Akt1 substrates. Our resulting dataset provides a rich resource for future studies on AKT signaling and dynamics.

Electronic Cigarette Exposure Induces Vascular Endothelial Dysfunction with NADPH Oxidase Activation and eNOS Uncoupling

BackgroundWe recently reported a mouse model of chronic electronic cigarette (e‐cig) exposure‐induced cardiovascular pathology, where long‐term exposure to e‐cig vape (ECV) induces cardiac abnormalities, impairment of endothelial function, and systemic hypertension. Using this model, we performed studies to delineate the underlying mechanisms of ECV‐induced vascular endothelial dysfunction (VED), which is a central trigger of cardiovascular disease.MethodsC57/BL6 male mice were exposed 2 hours/day, 5 days/week to ECV generated from e‐cig liquid containing 0, 6, or 24 mg/ml nicotine for 16 and 60 weeks. Blood pressure was measured by tail cuff and endothelial‐dependent and independent relaxation was measured on aortic segments using wire myography. Western blotting, immunofluorescence and ELISA were used to analyze the pathways involved in the mechanism of ECV‐induced VED.ResultsTime‐dependent elevation in blood pressure and systemic vascular resistance were observed, along with an impairment of acetylcholine‐induced aortic relaxation in ECV‐exposed mice, compared to air‐exposed control. Decreased intravascular nitric oxide (NO) levels and increased superoxide generation with elevated 3‐nitrotyrosine levels in the aorta of ECV‐exposed mice were observed, indicating that ECV‐induced superoxide reacts with NO to generate cytotoxic peroxynitrite. Exposure increased NADPH oxidase expression, supporting its role in ECV‐induced superoxide generation. Downregulation of endothelial nitric oxide synthase (eNOS) expression and Akt‐dependent eNOS phosphorylation occurred in the aorta of ECV‐exposed mice, indicating that exposure inhibited de novo NO synthesis. Following ECV exposure, the critical NOS cofactor tetrahydrobiopterin was decreased, with a concomitant loss of its salvage enzyme, dihydrofolate reductase. NADPH oxidase and NOS inhibitors abrogated ECV‐induced superoxide generation in the aorta of ECV exposed mice.ConclusionOur data demonstrate that ECV exposure activates NADPH oxidase and uncouples eNOS, causing a vicious cycle of superoxide generation and vascular oxidant stress that triggers VED and hypertension with predisposition to other cardiovascular disease.

Tongue Leptin Decreases Oro-Sensory Perception of Dietary Fatty Acids.

Leptin, an anorectic hormone, regulates food intake, energy expenditure and body weight. We assessed the implication of tongue leptin in the modulation of oro-sensory detection of dietary fatty acids in mice. The RT-PCR analysis showed that mRNA encoding leptin and leptin receptor (Ob-Rb) was expressed in mice taste bud cells (TBC). Confocal microscopic studies showed that the lipid sensor CD36 was co-expressed with leptin in mice TBC. Silencing of leptin or Ob-Rb mRNA in tongue papillae upregulated preference for a long-chain fatty acid (LCFA), i.e., linoleic acid (LA), in a two-bottle paradigm in mice. Furthermore, tongue leptin application decreased the preference for the LCFA. These results suggest that tongue leptin exerts an inhibitory action on fatty acid preference. In isolated mice TBC, leptin decreased LCFA-induced increases in free intracellular calcium concentrations, [Ca2+]i. Leptin and LCFA induced the phosphorylation of ERK1/2 and STAT-3 and there were no additive or opposite effects of the two agents on the degree of phosphorylation. However, leptin, but not the LCFA, induced phosphoinositide-3-kinase (PI-3-K)-dependent Akt phosphorylation in TBC. Furthermore, leptin induced hyperpolarization, whereas LCFA induced depolarization in TBC. Our study demonstrates that tongue leptin exerts an inhibitory action on oro-sensory detection of a dietary fatty acid by interfering with Ca2+ signaling and membrane potential in mice TBC.

Akt phosphorylates insulin receptor substrate to limit PI3K-mediated PIP3 synthesis.

The phosphoinositide 3-kinase (PI3K)-Akt network is tightly controlled by feedback mechanisms that regulate signal flow and ensure signal fidelity. A rapid overshoot in insulin-stimulated recruitment of Akt to the plasma membrane has previously been reported, which is indicative of negative feedback operating on acute timescales. Here, we show that Akt itself engages this negative feedback by phosphorylating insulin receptor substrate (IRS) 1 and 2 on a number of residues. Phosphorylation results in the depletion of plasma membrane-localised IRS1/2, reducing the pool available for interaction with the insulin receptor. Together these events limit plasma membrane-associated PI3K and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) synthesis. We identified two Akt-dependent phosphorylation sites in IRS2 at S306 (S303 in mouse) and S577 (S573 in mouse) that are key drivers of this negative feedback. These findings establish a novel mechanism by which the kinase Akt acutely controls PIP3 abundance, through post-translational modification of the IRS scaffold.

TRIB2 Stimulates Cancer Stem-Like Properties through Activating the AKT-GSK3β-β-Catenin Signaling Axis.

Tribbles homolog 2 (TRIB2) is implicated in tumorigenesis and drug resistance in various types of cancers. However, the role of TRIB2 in the regulation of tumorigenesis and drug resistance of cancer stem cells (CSCs) is still elusive. In the present study, we showed increased expression of TRIB2 in spheroid-forming and aldehyde dehydrogenase-positive CSC populations of A2780 epithelial ovarian cancer cells. Short hairpin RNA-mediated silencing of TRIB2 expression attenuates the spheroid-forming, migratory, tumorigenic, and drug-resistant properties of A2780 cells, whereas overexpression of TRIB2 increases the CSC-like characteristics. TRIB2 overexpression induced GSK3β inactivation by augmenting AKT-dependent phosphorylation of GSK3β at Ser9, followed by increasing β-catenin level via reducing the GSK3β-mediated phosphorylation of β-catenin. Treatment of TRIB2-ovexpressed A2780 cells with the phosphoinositide-3-kinase inhibitor LY294002 abrogated TRIB2-stimulated proliferation, migration, drug resistance of A2780 cells. These results suggest a critical role for TRIB2 in the regulation of CSC-like properties by increasing the stability of β-catenin protein via the AKT-GSK3β-dependent pathways.

Targeting therapy-resistant lung cancer stem cells via disruption of the AKT/TSPYL5/PTEN positive-feedback loop

Cancer stem cells (CSCs) are regarded as essential targets to overcome tumor progression and therapeutic resistance; however, practical targeting approaches are limited. Here, we identify testis-specific Y-like protein 5 (TSPYL5) as an upstream regulator of CSC-associated genes in non-small cell lung cancer cells, and suggest as a therapeutic target for CSC elimination. TSPYL5 elevation is driven by AKT-dependent TSPYL5 phosphorylation at threonine-120 and stabilization via inhibiting its ubiquitination. TSPYL5-pT120 also induces nuclear translocation and functions as a transcriptional activator of CSC-associated genes, ALDH1 and CD44. Also, nuclear TSPYL5 suppresses the transcription of PTEN, a negative regulator of PI3K signaling. TSPYL5-pT120 maintains persistent CSC-like characteristics via transcriptional activation of CSC-associated genes and a positive feedback loop consisting of AKT/TSPYL5/PTEN signaling pathway. Accordingly, elimination of TSPYL5 by inhibiting TSPYL5-pT120 can block aberrant AKT/TSPYL5/PTEN cyclic signaling and TSPYL5-mediated cancer stemness regulation. Our study suggests TSPYL5 be an effective target for therapy-resistant cancer.

Upregulated PD-L1 delays human neutrophil apoptosis and promotes lung injury in an experimental mouse model of sepsis

AbstractPD-L1 is a ligand for PD-1, and its expression has been shown to be upregulated in neutrophils harvested from septic patients. However, the effect of PD-L1 on neutrophil survival and sepsis-induced lung injury remains largely unknown. In this study, PD-L1 expression correlated negatively with rates of apoptosis in human neutrophils harvested from patients with sepsis. Coimmunoprecipitation assays on control neutrophils challenged with interferon-γ and LPS showed that PD-L1 complexes with the p85 subunit of phosphatidyl 3-kinase (PI3K) to activate AKT-dependent survival signaling. Conditional CRE/LoxP deletion of neutrophil PD-L1 in vivo further protected against lung injury and reduced neutrophil lung infiltration in a cecal ligation and puncture (CLP) experimental sepsis animal model. Compared with wild-type animals, PD-L1–deficient animals presented lower levels of plasma tumor necrosis factor-α and interleukin-6 (IL-6) and higher levels of IL-10 after CLP, and reduced 7-day mortality in CLP PD-L1–knockout animals. Taken together, our data suggest that increased PD-L1 expression on human neutrophils delays cellular apoptosis by triggering PI3K–dependent AKT phosphorylation to drive lung injury and increase mortality during clinical and experimental sepsis.

AKT-dependent signaling of extracellular cues through telomeres impact on tumorigenesis.

The telomere-bound shelterin complex is essential for chromosome-end protection and genomic stability. Little is known on the regulation of shelterin components by extracellular signals including developmental and environmental cues. Here, we show that human TRF1 is subjected to AKT-dependent regulation. To study the importance of this modification in vivo, we generate knock-in human cell lines carrying non-phosphorylatable mutants of the AKT-dependent TRF1 phosphorylation sites by CRISPR-Cas9. We find that TRF1 mutant cells show decreased TRF1 binding to telomeres and increased global and telomeric DNA damage. Human cells carrying non-phosphorylatable mutant TRF1 alleles show accelerated telomere shortening, demonstrating that AKT-dependent TRF1 phosphorylation regulates telomere maintenance in vivo. TRF1 mutant cells show an impaired response to proliferative extracellular signals as well as a decreased tumorigenesis potential. These findings indicate that telomere protection and telomere length can be regulated by extracellular signals upstream of PI3K/AKT activation, such as growth factors, nutrients or immune regulators, and this has an impact on tumorigenesis potential.

The miR-193a-5p/NCX2/AKT axis promotes invasion and metastasis of osteosarcoma.

MiR-193a-5p has been observed to have oncogenic or tumor suppressive functions in different kinds of cancers, but its role and molecular mechanism in osteosarcoma are elusive. Na+/Ca2+ exchangers (NCX1, NCX2 and NCX3) normally extrude Ca2+ from the cell, and deregulation of the intracellular Ca2+ homeostasis is related to several kinds of diseases, including cancer. The present study demonstrated that miR-193a-5p was upregulated in osteosarcoma tissues compared with the corresponding adjacent noncancerous tissues, and promoted colony formation, migration, invasion and epithelial-mesenchymal transition (EMT) in osteosarcoma cells (SaOS-2 and U-2OS), as well as metastasis in a murine xenograft model. Tandem mass tag-based quantitative proteomics analysis identified NCX2 as a potential target of miR-193a-5p. Luciferase activity assays and Western blotting further confirmed that miR-193a-5p recognized the 3′-untranslated region of NCX2 mRNA, and negatively regulated NCX2 expression. NCX2 was downregulated in osteosarcoma tissues, and its expression was negatively correlated with miR-193a-5p levels. Ectopic expression of NCX2 in osteosarcoma cells could reverse the oncogenicity of miR-193a-5p, indicating that miR-193a-5p exerted its effects by targeting NCX2. Further study demonstrated that NCX2 suppresses Ca2+-dependent Akt phosphorylation by decreasing intracellular Ca2+ concentration, and then inhibited EMT process. Treatment with the antagomir against miR-193a-5p sensitized osteosarcoma to the Akt inhibitor afuresertib in a murine xenograft model. In conclusion, a miR-193a-5p/NCX2/AKT signaling axis contributes to the progression of osteosarcoma, which may provide a new therapeutic target for osteosarcoma treatment.

Triterpenoid saponins from Ilex cornuta protect H9c2 cardiomyocytes against H2O2-induced apoptosis by modulating Ezh2 phosphorylation

Ethnopharmacological relevanceIlex cornuta Lindl. et Paxt. (Aquifoliaceae family) belongs to the Ilex genus. The leaves of this plant are used for the popular herbal tea “Ku-Ding-Cha” in China due to their health benefits for sore throat, obesity and hypertension. Our previous studies have shown that the extract of Ilex cornuta root exerts cardioprotective effects in rat models of myocardial ischaemic injury, and several new kinds of triterpenoid saponins from Ilex cornuta (TSIC) have protective effects against hydrogen peroxide (H2O2)-induced cardiomyocyte injury. Aim of the studyThe aim of this study was to clarify the underlying mechanisms by which TSIC protect against H2O2-induced cardiomyocyte injury. Materials and methodsAn H2O2-treated H9c2 cardiomyocyte line was used as an in vitro model of oxidation-damaged cardiomyocytes to evaluate the effects of TSIC. Apoptosis was detected with CCK-8 and annexin V assays and via analysis of the levels of apoptosis-associated proteins or genes. The underlying mechanisms related to Akt signalling, Ezh2 expression and activity, and ROS were clarified by Western blotting, quantitative PCR, flow cytometry and rescue experiments. ResultsTSIC protected H9c2 cells from H2O2-induced apoptosis. This effect of TSIC was attributable to inhibition of Ezh2 activity, as exhibited by attenuation of H2O2-induced Akt signalling-dependent phosphorylation of Ezh2 at serine 21 (pEzh2S21) upon TSIC pretreatment. In addition, feedback pathway between Akt-dependent Ezh2 phosphorylation and ROS was involved in TSIC-mediated protection of H9c2 cells from apoptosis. ConclusionsOur findings indicate a pivotal role of the pEzh2S21 network in TSIC-mediated protection against cardiomyocyte apoptosis, potentially providing evidence of the mechanism of TSIC in the treatment and prevention of cardiovascular diseases.

Inhibition of class I PI3K enhances chaperone-mediated autophagy.

Chaperone-mediated autophagy (CMA) is the most selective form of lysosomal proteolysis, where individual peptides, recognized by a consensus motif, are translocated directly across the lysosomal membrane. CMA regulates the abundance of many disease-related proteins, with causative roles in neoplasia, neurodegeneration, hepatosteatosis, and other pathologies relevant to human health and aging. At the lysosomal membrane, CMA is inhibited by Akt-dependent phosphorylation of the CMA regulator GFAP. The INS-PI3K-PDPK1 pathway regulates Akt, but its role in CMA is unclear. Here, we report that inhibition of class I PI3K or PDPK1 activates CMA. In contrast, selective inhibition of class III PI3Ks does not activate CMA. Isolated liver lysosomes from mice treated with either of two orally bioavailable class I PI3K inhibitors, pictilisib or buparlisib, display elevated CMA activity, and decreased phosphorylation of lysosomal GFAP, with no change in macroautophagy. The findings of this study represent an important first step in repurposing class I PI3K inhibitors to modulate CMA in vivo.

Neurogranin regulates eNOS function and endothelial activation

Endothelial nitric oxide (NO) is a critical mediator of vascular function and vascular remodeling. NO is produced by endothelial nitric oxide synthase (eNOS), which is activated by calcium (Ca2+)-dependent and Ca2+-independent pathways. Here, we report that neurogranin (Ng), which regulates Ca2+-calmodulin (CaM) signaling in the brain, is uniquely expressed in endothelial cells (EC) of human and mouse vasculature, and is also required for eNOS regulation. To test the role of Ng in eNOS activation, Ng knockdown in human aortic endothelial cells (HAEC) was performed using Ng SiRNA along with Ng knockout (Ng −/−) in mice. Depletion of Ng expression decreased eNOS activity in HAEC and NO production in mice. We show that Ng expression was decreased by short-term laminar flow and long-them oscillating flow shear stress, and that Ng siRNA with shear stress decreased eNOS expression as well as eNOS phosphorylation at S1177. We further reveled that lack of Ng expression decreases both AKT-dependent eNOS phosphorylation, NF-κB-mediated eNOS expression, and promotes endothelial activation. Our findings also indicate that Ng modulates Ca2+-dependent calcineurin (CaN) activity, which suppresses Ca2+-independent AKT-dependent eNOS signaling. Moreover, deletion of Ng in mice also reduced eNOS activity and caused endothelial dysfunction in flow-mediated dilation experiments. Our results demonstrate that Ng plays a crucial role in Ca2+-CaM-dependent eNOS regulation and contributes to vascular remodeling, which is important for the pathophysiology of cardiovascular disease.

PDK1 is important lipid kinase for RANKL-induced osteoclast formation and function via the regulation of the Akt-GSK3β-NFATc1 signaling cascade.

Perturbations in the balanced process of osteoblast-mediated bone formation and osteoclast-mediated bone resorption leading to excessive osteoclast formation and/or activity is the cause of many pathological bone conditions such as osteoporosis. The osteoclast is the only cell in the body capable of resorbing and degrading the mineralized bone matrix. Osteoclast formation from monocytic precursors is governed by the actions of two key cytokines macrophage-colony-stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL). Binding of RANKL binding to receptor RANK initiates a series of downstream signaling responses leading to monocytic cell differentiation and fusion, and subsequent mature osteoclast bone resorption and survival. The phosphoinositide-3-kinase (PI3K)-protein kinase B (Akt) signaling cascade is one such pathway activated in response to RANKL. The 3-phosphoinositide-dependent protein kinase 1 (PDK1), is considered the master upstream lipid kinase of the PI3K-Akt cascade. PDK1 functions to phosphorylate and partially activate Akt, triggering the activation of downstream effectors. However, the role of PDK1 in osteoclasts has yet to be clearly defined. In this study, we specifically deleted the PDK1 gene in osteoclasts using the cathepsin-K promoter driven Cre-LoxP system. We found that the specific genetic ablation of PDK1 in osteoclasts leads to an osteoclast-poor osteopetrotic phenotype in mice. In vitro cellular assays further confirmed the impairment of osteoclast formation in response to RANKL by PDK1-deficient bone marrow macrophage (BMM) precursor cells. PDK1-deficient BMMs exhibited reduced ability to reorganize actin cytoskeleton to form a podosomal actin belt as a result of diminished capacity to fuse into giant multinucleated osteoclasts. Notably, biochemical analyses showed that PDK1 deficiency attenuated the phosphorylation of Akt and downstream effector GSK3β, and reduced induction of NFATc1. GSK3β is a reported negative regulator of NFATc1. GSK3β activity is inhibited by Akt-dependent phosphorylation. Thus, our data provide clear genetic and mechanistic insights into the important role for PDK1 in osteoclasts.

AKT-Dependent Phosphorylation of ADAR1p110 and ADAR2 Represents a New and Important Link Between Cell Signaling and RNA Editing.

RNA editing is a process by which nascent RNA transcripts are covalently modified, thus enhancing the complexity of the transcriptome. The most common modifications are deaminations of adenosine to inosine at sites of complex RNA secondary structure, a process that is carried out by the adenosine deaminase acting on double-strand RNA (ADAR) family of RNA editases. Although much has been learned about the ADAR family members since their discovery, very little information on their post-transcriptional regulation has been reported. Similar to most proteins, the ADAR family members are post-translationally modified at multiple sites. We recently reported that members of the AKT kinase family directly phosphorylate ADAR1p110 and ADAR2 on a conserved threonine within the catalytic domain of the protein. Phosphorylation was observed to differentially inhibit the enzymatic activity of the ADAR proteins toward known RNA substrates. The direct downstream involvement of the AKT kinases in multiple major signaling pathways associated with cell survival, growth, glucose metabolism (insulin signaling), and differentiation is well established; thus, the AKT kinases represent a link between ADAR-dependent A-to-I editing and major signal transduction pathways that are necessary for cell maintenance and development.