Single-cell sequencing of the retina shows that LDHA regulates pathogenesis of autoimmune uveitis.

Autoimmune uveitis (AU) is a severe disorder causing poor vision and blindness. However, the cellular dynamics and pathogenic mechanisms underlying retinal injury in uveitis remain unclear. In this study, single-cell RNA sequencing of the retina and cervical draining lymph nodes in experimental autoimmune uveitis mice was conducted to identify the cellular spatiotemporal dynamics and upregulation of the glycolysis-related gene LDHA. Suppression of LDHA can rescue the imbalance of T effector (Teff) cells/T regulator (Treg) cells under inflammation via downregulation of the glycolysis-PI3K signaling circuit and inhibition of the migration of CXCR4+ Teff cells towards retinal tissue. Furthermore, LDHA and CXCR4 are upregulated in the peripheral blood mononuclear cells of Vogt-Koyanagi-Harada patients. The LDHA inhibitor suppresses CD4+ T cell proliferation in humans. Therefore, our data indicate that the autoimmune environment of uveitis regulates Teff cell accumulation in the retina via glycolysis-associated LDHA. Modulation of this target may provide a novel therapeutic strategy for treating AU.

Pasteurella multocida activates apoptosis via the FAK-AKT-FOXO1 axis to cause pulmonary integrity loss, bacteremia, and eventually a cytokine storm.

Pasteurella multocida is an important zoonotic respiratory pathogen capable of infecting a diverse range of hosts, including humans, farm animals, and wild animals. However, the precise mechanisms by which P. multocida compromises the pulmonary integrity of mammals and subsequently induces systemic infection remain largely unexplored. In this study, based on mouse and rabbit models, we found that P. multocida causes not only lung damage but also bacteremia due to the loss of lung integrity. Furthermore, we demonstrated that bacteremia is an important aspect of P. multocida pathogenesis, as evidenced by the observed multiorgan damage and systemic inflammation, and ultimately found that this systemic infection leads to a cytokine storm that can be mitigated by IL-6-neutralizing antibodies. As a result, we divided the pathogenesis of P. multocida into two phases: the pulmonary infection phase and the systemic infection phase. Based on unbiased RNA-seq data, we discovered that P. multocida-induced apoptosis leads to the loss of pulmonary epithelial integrity. These findings have been validated in both TC-1 murine lung epithelial cells and the lungs of model mice. Conversely, the administration of Ac-DEVD-CHO, an apoptosis inhibitor, effectively restored pulmonary epithelial integrity, significantly mitigated lung damage, inhibited bacteremia, attenuated the cytokine storm, and reduced mortality in mouse models. At the molecular level, we demonstrated that the FAK-AKT-FOXO1 axis is involved in P. multocida-induced lung epithelial cell apoptosis in both cells and animals. Thus, our research provides crucial information with regard to the pathogenesis of P. multocida as well as potential treatment options for this and other respiratory bacterial diseases.

Treponema pallidum membrane protein Tp47 induced autophagy and inhibited cell migration in HMC3 cells via the PI3K/AKT/FOXO1 pathway.

The migratory ability of microglia facilitates their rapid transport to a site of injury to kill and remove pathogens. However, the effect of Treponema pallidum membrane proteins on microglia migration remains unclear. The effect of Tp47 on the migration ability and autophagy and related mechanisms were investigated using the human microglial clone 3 cell line. Tp47 inhibited microglia migration, the expression of autophagy-associated protein P62 decreased, the expression of Beclin-1 and LC3-II/LC3-I increased, and the autophagic flux increased in this process. Furthermore, autophagy was significantly inhibited, and microglial cell migration was significantly increased after neutralisation with an anti-Tp47 antibody. In addition, Tp47 significantly inhibited the expression of p-PI3K, p-AKT, and p-mTOR proteins, and the sequential activation of steps in the PI3K/AKT/mTOR pathways effectively prevented Tp47-induced autophagy. Moreover, Tp47 significantly inhibited the expression of p-FOXO1 protein and promoted FOXO1 nuclear translocation. Inhibition of FOXO1 effectively suppressed Tp47-induced activation of autophagy and inhibition of migration. Treponema pallidum membrane protein Tp47-induced autophagy and inhibited cell migration in HMC3 Cells via the PI3K/AKT/FOXO1 pathway. These data will contribute to understanding the mechanism by which T. pallidum escapes immune killing and clearance after invasion into the central nervous system.

Majoon chobchini reinstates PDL-1 expression and blocks dendritic cell -T helper 17 pathogenic axis in rheumatoid arthritis animal model.

Dendritic cells (DCs) are the critical players in the puzzle of rheumatoid arthritis (RA) disease pathogenesis. Blockade of DC activation has been shown to curtail Th17 cell differentiation and its aberrant function in RA. Recent studies have pointed to the role of the PI3K/AKT signaling axis in the maturation and activation of DCs. However, it is yet to be established how PI3K/AKT inhibition would lead to the abolishment of DC activation and Th17 cell plasticity in RA. Herein, our study decoded whether and how majoon chobchini, an unani compound, abated dendritic cell maturation and regulated the Th17/Treg paradigm in RA. Given our results, majoon chobchini conspicuously restrained MHC II, CD86 expression and, subsequently elevated PDL-1 levels in DCs in-vivo. Of note, inhibition of DC maturation by majoon chobchini, in turn, favoured suppression of the Th17 cell population while driving Treg cell development in adjuvant induced arthritic (AA) rats. Concurrently, majoon chobchini decreased the catabolic effects of IL-17 (Th17 associated cytokine) via a reciprocal increase in IL-10 (Treg associated cytokine) levels in AA rats. Mechanistically, majoon chobchini sustained FoxO1 nuclear localization signaled through dampened PI3K/AKT phosphorylation in-vitro. In concert, PDL-1 expression was heightened in majoon chobchini treated activated DCs that provides a framework for ablation of the DC-Th17 cell pathogenic axis in RA. Notwithstanding, the PI3K inhibitor LY294002 exhibited similar inhibitory effects. In essence, majoon chobchini enhanced PDL-1 expression that abolished DC maturation via regulation of the PI3K/AKT/FoxO1 axis, thereby hindering Th17 differentiation in an animal model of RA. This further warrants a clinical investigation that could validate majoon chobchini as a prospective therapeutic drug in the treatment of RA.

Phenolipid JE improves metabolic profile and inhibits gluconeogenesis via modulating AKT-mediated insulin signaling in STZ-induced diabetic mice.

Phenolipids are characteristic phytochemicals of Syzygium genus. However, the antidiabetic potential and underlying molecular mechanism of these components are not fully elucidated. Herein, we studied the anti-diabetic effects of jambone E (JE), a phenolipid from S. cumini, with in vitro and in vivo models. Data from current study showed that JE enhanced glucose consumption and uptake, promoted glycogen synthesis, and suppressed gluconeogenesis in insulin resistant (IR)-HepG2 cells and primary mouse hepatocytes. JE also attenuated streptozotocin-induced hyperglycemia and hyperlipidemia in type 1 diabetic (T1D) mice. Eleven metabolites (e.g. trimethylamine n-oxide, 4-pyridoxic acid, phosphatidylinositol 39:4, phenaceturic acid, and hippuric acid) were identified as potential serum biomarkers for JE's antidiabetic effects by an untargeted metabolomics approach. The further molecular mechanistic study revealed that JE up-regulated phosphorylation levels of protein kinase B (AKT), glycogen synthase kinase 3 beta, and forkhead box O1 (FoxO1), promoted nuclear exclusion of FoxO1 whilst decreased gene expression levels of peroxisome proliferator-activated receptor gamma coactivator-1 alpha, phosphoenolpyruvate carboxykinase and glucose 6-phosphatase in IR-HepG2 cells and T1D mice. Our data suggested that JE might be a potent activator for AKT-mediated insulin signaling pathway, which was confirmed by the usage of AKT inhibitor and AKT-target siRNA interference, as well as the cellular thermal shift assay. Findings from the current study shed light on the anti-diabetic effects of phenolipids in the Syzygium species, which supports the use of medicinal plants in the Syzygium genus for potential pharmaceutical applications.

Hexavalent chromium caused DNA damage repair and apoptosis via the PI3K/AKT/FOXO1 pathway triggered by oxidative stress in the lung of rat.

Hexavalent chromium [Cr(VI)] is an occupational carcinogen that accumulates in the lungs and causes lung injury and even lung cancer. 36 SD male rats received inhalable intratracheal instillation of Cr(VI) (0.05, 0.25 mg Cr/kg) or the same volume (3 ml/kg) of normal saline weekly for 28 days (total 5 times). After 28 days of exposure, half of the rats in each group were sacrificed for investigation, and the rest stopped exposure and began to be self-repaired for two weeks. Histopathology analyses revealed that Cr(VI) induced slight dilatation and hemorrhage of perialveolar capillaries, pulmonary bronchodilation, and congestion with peripheral flaky-like necrosis accompanied by inflammatory cell infiltration, especially the 0.25 mg Cr/kg group. Cr(VI) exposure caused the increase of blood Cr, urinary Cr, MDA, urinary 8-hydroxy-2' -deoxyguanosine (8-OHdG), and the decrease of GSH and MDA, while two-week repair only reduced urinary Cr. Exposure to Cr(VI) significantly upregulated FOXO1 and downregulated p-AKT and p-FOXO1 for two weeks. PI3K in the 0.25 mg Cr/kg group was inhibited after two weeks of repair. Cr(VI) exposure mainly promoted GADD45a and CHK2 in the exposure group, promoted Bim, Bax/Bcl-2, and suppressed Bcl-2 and Bcl-xL in the repair group. These results demonstrate that Cr(VI) may induce DNA damage repair and apoptosis in the lung by activating the PI3K/AKT/FOXO1 pathway. Two-week repair may alleviate oxidative stress and DNA damage induced by Cr(VI) exposure but couldn't eliminate its effects. This study provides a new perspective for exploring the Cr(VI) induced lung cancer mechanism.

Kinsenoside attenuates liver fibro-inflammation by suppressing dendritic cells via the PI3K-AKT-FoxO1 pathway.

Kinsenoside (KD) exhibits anti-inflammatory and immunosuppressive effects. Dendritic cells (DCs) are critical regulators of the pathologic inflammatory milieu in liver fibrosis (LF). Herein, we explored whether and how KD repressed development of LF via DC regulation and verified the pathway involved in the process. Given our analysis, both KD and adoptive transfer of KD-conditioned DCs conspicuously reduced hepatic histopathological damage, proinflammatory cytokine release and extracellular matrix deposition in CCl4-induced LF mice. Of note, KD restrained the LF-driven rise in CD86, MHC-II, and CCR7 levels and, simultaneously, upregulated PD-L1 expression on DCs specifically, which blocked CD8+T cell activation. Additionally, KD reduced DC glycolysis, maintained DCs immature, accompanied by IL-12 decrease in DCs. Inhibiting DC function by KD disturbed the communication of DCs and HSCs with the expression or secretion of α-SMA and Col-I declined in the liver. Mechanistically, KD suppressed the phosphorylation of PI3K-AKT driven by LF or PI3K agonist, followed by enhanced nuclear transport of FoxO1 and upregulated interaction of FoxO1 with the PD-L1 promoter in DCs. PI3K inhibitor or si-IL-12 acting on DC could relieve LF, HSC activation and diminish the effect of KD. In conclusion, KD suppressed DC maturation with promoted PD-L1 expression via PI3K-AKT-FoxO1 and decreased IL-12 secretion, which blocked activation of CD8+T cells and HSCs, thereby alleviating liver injury and fibro-inflammation in LF.

IGF-1 ameliorates streptozotocin-induced pancreatic ß cell dysfunction and apoptosis via activating IRS1/PI3K/Akt/FOXO1 pathway.

BACKGROUND AND OBJECTIVE: Type 2 diabetes mellitus (T2DM) is an endocrine disorder with pancreatic ß cell dysfunction and/or reduced insulin sensitivity. IGF-1 is critically involved in pancreatic ß cell growth, differentiation, and insulin secretion. Insulin-mediated IRS1/PI3K/Akt/FOXO1 signaling has been proved to be closely associated with pancreatic ß cell function, hepatic glucose metabolism, and the development of T2DM. This present work was designed to demonstrate the protective role of IGF-1 against pancreatic ß cell dysfunction and to probe into the underlying mechanisms. METHODS: Herein, cell viability, cell apoptosis, insulin secretion, oxidative stress, and glycolysis in STZ-treated INS-1 cells were measured, so as to determine the biological function of IGF-1 against pancreatic ß cell dysfunction in T2DM. Additionally, whether IGF-1 could activate IRS1/PI3K/Akt/FOXO1 signaling pathway to manipulate the progression of T2DM was also investigated. RESULTS: It was discovered that IGF-1 treatment enhanced the viability and suppressed the apoptosis of STZ-treated INS-1 cells. Besides, IGF-1 treatment augmented insulin secretion of INS-1 cells in response to STZ. Moreover, IGF-1 exerted protective role against oxidative damage and displayed inhibitory effect on glycolysis in STZ-treated INS-1 cells. Mechanistically, IGF-1 treatment markedly boosted the activation of IRS1/PI3K/Akt/FOXO1 pathway. Furthermore, treatment with AG1024 (an inhibitor of IGF-1R) partially abolished the actions of IGF-1 on cell viability, cell apoptosis, insulin secretion, oxidative stress, and glycolysis in STZ-treated INS-1 cells. CONCLUSION: To conclude, IGF-1 could improve the viability and inhibit the apoptosis of STZ-treated pancreatic ß cells, induce insulin secretion, alleviate oxidative damage, as well as arrest glycolysis by activating IRS1/PI3K/Akt/FOXO1 pathway.

Activating BK channels ameliorates vascular smooth muscle calcification through Akt signaling.

Vascular calcification (VC) is characterized by pathological depositions of calcium and phosphate in the arteries and veins via an active cell-regulated process, in which vascular smooth muscle cells (VSMCs) transform into osteoblast/chondrocyte-like cells as in bone formation. VC is associated with significant morbidity and mortality in chronic kidney disease (CKD) and cardiovascular disease, but the underlying mechanisms remain unclear. In this study we investigated the role of large-conductance calcium-activated potassium (BK) channels in 3 experimental VC models. VC was induced in vascular smooth muscle cells (VSMCs) by ß-glycerophosphate (ß-GP), or in rats by subtotal nephrectomy, or in mice by high-dosage vitamin D3. We showed that the expression of BK channels in the artery of CKD rats with VC and in ß-GP-treated VSMCs was significantly decreased, which was functionally confirmed by patch-clamp recording. In ß-GP-treated VSMCs, BK channel opener NS1619 (20 µM) significantly alleviated VC by decreasing calcium content and alkaline phosphatase activity. Furthermore, NS1619 decreased mRNA expression of ostoegenic genes OCN and OPN, as well as Runx2 (a key transcription factor involved in preosteoblast to osteoblast differentiation), and increased the expression of α-SMA protein, whereas BK channel inhibitor paxilline (10 µM) caused the opposite effects. In primary cultured VSMCs from BK-/- mice, BK deficiency aggravated calcification as did BK channel inhibitor in normal VSMCs. Moreover, calcification was more severe in thoracic aorta rings of BK-/- mice than in those of wild-type littermates. Administration of BK channel activator BMS191011 (10 mg· kg-1 ·d-1) in high-dosage vitamin D3-treated mice significantly ameliorated calcification. Finally, co-treatment with Akt inhibitor MK2206 (1 µM) or FoxO1 inhibitor AS1842856 (3 µM) in calcified VSMCs abrogated the effects of BK channel opener NS1619. Taken together, activation of BK channels ameliorates VC via Akt/FoxO1 signaling pathways. Strategies to activate BK channels and/or enhance BK channel expression may offer therapeutic avenues to control VC.

PPARα agonist fenofibrate relieves acquired resistance to gefitinib in non-small cell lung cancer by promoting apoptosis via PPARα/AMPK/AKT/FoxO1 pathway.

Recent studies show that intracellular accumulation of cholesterol leads to acquired resistance to gefitinib in non-small cell lung cancer (NSCLC) cells. In this study we investigated how to regulate the cholesterol levels in gefitinib-resistant NSCLC cells. We showed that intracellular cholesterol levels in gefitinib-resistant cell lines (PC-9/GR, H1975, H1650, and A549) were significantly higher than that in gefitinib-sensitive cell line (PC-9). Treatment with gefitinib (5 µM) significantly increased intracellular cholesterol levels in PC-9/GR, H1975, and H1650 cells. Gefitinib treatment downregulated the expression of PPARα, LXRα, and ABCA1, leading to dysregulation of cholesterol efflux pathway. We found that a lipid-lowering drug fenofibrate (20, 40 µM) dose-dependently increased the expression of PPARα, LXRα, and ABCA1, decreased the intracellular cholesterol levels, and enhanced the antiproliferative effects of gefitinib in PC-9/GR, H1975, and H1650 cells. We revealed that fenofibrate increased the gefitinib-induced apoptosis via regulating the key proteins involved in the intrinsic apoptosis pathway. In PC-9/GR, H1975 and H1650 cells, fenofibrate dose-dependently increased the expression of AMPK, FoxO1, and decreased the expression of AKT, which were remarkably weakened by knockdown of PPARα. In PC-9/GR cell xenograft mice, combined administration of gefitinib (25 mg · kg-1 · d-1) and fenofibrate (100 mg · kg-1 · d-1) caused remarkable inhibition on tumor growth as compared to treatment with either drug alone. All the results suggest that fenofibrate relieves acquired resistance to gefitinib in NSCLC by promoting apoptosis via regulating PPARα/AMPK/AKT/FoxO1 pathway. We propose that combination of gefitinib and fenofibrate is a potential strategy for overcoming the gefitinib resistance in NSCLC.

Recombinant CCL17-dependent CCR4 activation alleviates neuroinflammation and neuronal apoptosis through the PI3K/AKT/Foxo1 signaling pathway after ICH in mice.

BACKGROUND: Intracerebral hemorrhage (ICH), a devastating subtype of stroke, is associated with high mortality and morbidity. Neuroinflammation is an important factor leading to ICH-induced neurological injuries. C-C Chemokine Receptor 4 (CCR4) plays an important role in enhancing hematoma clearance after ICH. However, it is unclear whether CCR4 activation can ameliorate neuroinflammation and apoptosis of neurons following ICH. The aim of the present study was to examine the effects of recombinant CCL17 (rCCL17)-dependent CCR4 activation on neuroinflammation and neuronal apoptosis in an intrastriatal autologous blood injection ICH model, and to determine whether the PI3K/AKT/Foxo1 signaling pathway was involved. METHODS: Two hundred twenty-six adult (8-week-old) male CD1 mice were randomly assigned to sham and ICH surgery groups. An intrastriatal autologous blood injection ICH model was used. rCCL17, a CCR4 ligand, was delivered by intranasal administration at 1 h, 3 h, and 6 h post-ICH. CCL17 antibody was administrated by intraventricular injection at 1 h post-ICH. C021, a specific inhibitor of CCR4 and GDC0068, an AKT inhibitor were delivered intraperitoneally 1 h prior to ICH induction. Brain edema, neurobehavioral assessments, western blotting, Fluoro-Jade C staining, terminal deoxynucleotidyl transferase dUTP nick end labeling, and immunofluorescence staining were conducted. RESULTS: Endogenous expression of CCL17 and CCR4 were increased following ICH, peaking at 5 days post-induction. CCR4 was found to co-localize with microglia, neurons, and astrocytes. rCCL17 treatment decreased brain water content, attenuated short- and long-term neurological deficits, deceased activation of microglia/macrophages and infiltration of neutrophils, and inhibited neuronal apoptosis in the perihematomal region post-ICH. Moreover, rCCL17 treatment post-ICH significantly increased the expression of CCR4, PI3K, phosphorylated AKT, and Bcl-2, while Foxo1, IL-1ß, TNF-α, and Bax expression were decreased. The neuroprotective effects of rCCL17 were reversed with the administration of C021 or GDC0068. CONCLUSIONS: rCCL17-dependent CCR4 activation ameliorated neurological deficits, reduced brain edema, and ameliorated neuroinflammation and neuronal apoptosis, at least in part, through the PI3K/AKT/Foxo1 signaling pathway after ICH. Thus, activation of CCR4 may provide a promising therapeutic approach for the early management of ICH.

Dexmedetomidine ameliorates lipopolysaccharide-induced acute lung injury by inhibiting the PI3K/Akt/FoxO1 signaling pathway.

PURPOSE: Dexmedetomidine (DEX) has been associated with inflammation, oxidative stress, and apoptosis, but its effects on lipopolysaccharide (LPS)-induced lung injury remain uncertain. The present study explored the effects of DEX on LPS-induced lung injury and studied the possible molecular mechanisms by testing the effects of the phosphoinositide-3 kinase (PI3K) inhibitor LY294002 and BEZ235. METHODS: Seventy C57BL/6 mice were randomly divided into the control, LPS, LPS + DEX, LPS + LY294002, LPS + BEZ235, LPS + DEX + LY294002, and LPS + DEX + BEZ235groups. Lung samples were collected 48 h after LPS treatment. RESULTS: DEX significantly inhibited LPS-induced increases in the lung weight/body weight ratio and lung wet/dry weight ratio, decreased inflammatory cell infiltration, and decreased the production of proinflammatory factors, such as interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor α (TNF-α)in the lungs. DEX also markedly attenuated the increases in malondialdehyde 5 (MDA 5) and inositol-dependent enzyme a (IRE-a), attenuated the decrease in superoxide dismutase 1(SOD-1), reversed the low expression of B-cell lymphoma-2 (Bcl-2), and the high expressions of Bax and Caspase-3. DEX also decreased the expression of phosphorylated PI3K and phosphorylated Akt and increased the expression of phosphorylated forkhead box-O transcription factor 1 (FoxO1). More interestingly, LY294002 or BEZ235 pretreatment significantly abolished the inhibitory effects of DEX on LPS-induced lung inflammation, oxidative stress, and apoptosis. CONCLUSIONS: These data suggest that DEX ameliorates LPS-induced acute lung injury partly through the PI3K/Akt/FoxO1 signaling pathway.

Screening active components from Rubus amabilis for pancreatic ß-cells protection.

CONTEXT: Rubus species (Rosaceae) have been used in folk medicine to treat diabetes due to their hypoglycaemic activity. OBJECTIVE: To screen the active components that act as hypoglycaemic agents in Rubus amabilis Focke and the underlying mechanisms. MATERIALS AND METHODS: Aqueous stem extract of R. amabilis was incubated with MIN6 ß-cells, PBS was used as the blank control. Then the cells were washed, cell membrane-bound components were dissociated and identified by UPLC/MS. Total procyanidins (PCs) in R. amabilis was enriched and the cytotoxicity and anti-proliferation on ß-cell were evaluated by MTT assay. PCs at 25, 50, and 75 µg/mL was applied for 24 h to determine its effects on palmitate (PA)-induced apoptosis and GSIS. Western blotting was employed to detect the protein expression of PI3K/Akt/FoxO1 signalling. The antioxidant indices were also measured. RESULTS: ß-Cell membrane-bound components were identified as three procyanidin B dimers and a C trimer. PCs showed no significant cytotoxicity up to a concentrations of 100 µg/mL. PCs treatment reversed the elevated apoptosis rate and impaired GSIS induced by PA. PCs markedly decreased the intracellular ROS and MDA production and increased the SOD activity. Moreover, PCs promoted the phosphorylation of Akt and FoxO1, and regulated Pdx-1 and Bax expression in MIN6 cells. Discussion and conclusion: The active components that act as hypoglycaemic agents in R. amabilis are procyanidins, which protected MIN6 cells against PA-induced apoptosis by activating PI3K/Akt/FoxO1 signalling. These results indicate that ß-cell extraction, combined with UPLC/MS, is a valid method for screening antidiabetic components from herbal medicines.

SPTLC1 inhibits cell growth via modulating Akt/FOXO1 pathway in renal cell carcinoma cells.

Serine palmitoyltransferase long chain-1 (SPTLC1), which is the rate-limiting enzyme for sphingolipid biosynthesis, has been indicated to be essential for carcinoma cell survival and proliferation in recent, but its role in the regulation of renal cell carcinoma (RCC) remains unknown. In the present study, we found that SPTLC1 expression was significantly decreased in RCC tissues compared to non-tumor tissues, and low SPTLC1 expression was associated with poor overall survival of RCC patients. In addition, our results revealed that forced expression of SPTLC1 could significantly inhibit cell growth in vitro and in vivo via, at least in part, modulating Akt/FOXO1 signaling pathway, thus representing a novel role of SPTLC1 in the regulation of tumor growth in RCC for the first time.

Uev1A promotes breast cancer cell survival and chemoresistance through the AKT-FOXO1-BIM pathway.

BACKGROUND: Ubiquitin-conjugating enzyme variant UEV1A is required for Ubc13-catalyzed K63-linked poly-ubiquitination that regulates several signaling pathways including NF-κB, MAPK and PI3K/AKT. Previous reports implicate UEV1A as a potential proto-oncogene and have shown that UEV1A promotes breast cancer metastasis through constitutive NF-кB activation. Ubc13-Uev1A along with TARF6 can also ubiquitinate AKT but its downstream events are unclear. METHODS: In this study, we experimentally manipulated UEV1 expression in two typical breast cancer cell lines MDA-MB-231 and MCF7 under serum starvation conditions and monitored AKT activation and its downstream protein levels, as well as cellular sensitivity to chemotherapeutic agents. RESULTS: We found that overexpression of UEV1A is sufficient to activate the AKT signaling pathway that in turn inhibits FOXO1 and BIM expression to promote cell survival under serum starvation conditions and enhances cellular resistance to chemotherapy. Consistently, experimental depletion of Uev1 in breast cancer cells inhibits AKT signaling and promotes FOXO1 and BIM expression to reduce cell survival under serum starvation stress and enhance chemosensitivity. CONCLUSIONS: Uev1A promotes cell survival under serum starvation stress through the AKT-FOXO1-BIM axis in breast cancer cells, which unveals a potential therapeutic target in the treatment of breast cancers.

Adenovirus type 36 regulates adipose stem cell differentiation and glucolipid metabolism through the PI3K/Akt/FoxO1/PPARγ signaling pathway.

BACKGROUND: This study aims to investigate the molecular mechanism of Adenovirus type 36 (Ad36) in adipocyte differentiation and glucolipid metabolism. METHODS: Rat obesity model was established by Ad36 infection and high-fat diet, respectively. Comparison of the body weight, clinical biochemical indicators, insulin sensitivity and lipid heterotopic deposition between these two models was performed. Ad36-induced adipocyte in vitro model was also established. The binding rate of FoxO1, PPARγ and its target gene promoter was detected using ChIP. The mRNA and protein expression levels of PPARγ and downstream target genes were detected by RT-PCR and Western blot, respectively. Oil red O staining was used to measure differentiation into adipocyte. Wortmannin (WM), inhibitor of PI3K, was used to act on Ad36-induced hADSCs. RESULTS: Ad36-induced obese rats did not exhibit disorders in blood glucose and blood TG, insulin resistance and lipid ectopic deposition. The expression of Adipoq, Lpin1 and Glut4 in the adipose tissue increased. Oil red O staining showed that Ad36 induced the differentiation of hAMSCs into human adipocytes in vitro. During this process, the binding rate of FoxO1 and PPARγ promoter regions was weakened. However, the binding rate of the transcription factor PPARγ to its target genes Acc, Adipoq, Lpin1 and Glut4 was enhanced, and thus increased the protein expression of P-FoxO1, PPARγ2, ACC, LPIN1, GLUT4 and ADIPOQ. The PI3K inhibitor Wortmannin reduced the expression of P-Akt, P-FoxO1 and PPARγ2, thereby inhibiting adipogenesis of hADSC. CONCLUSION: Ad36 may promote fatty acid and triglyceride synthesis, and improve insulin sensitivity by affecting the PI3K/Akt/FoxO1/PPARγ signaling pathway.

Effects of MicroRNA-27a on Myogenin Expression and Akt/FoxO1 Signal Pathway during Porcine Myoblast Differentiation.

Skeletal myoblast differentiation is controlled by a multitude of transcription factors and signal pathways. Myogenin is a critical transcriptional regulator in the initiation and maintenance of myoblast differentiation. The Akt/FoxO1 signal pathway plays an important role in myoblast differentiation. MicroRNAs are a kind of small noncoding RNAs that have been regarded as important regulators in skeletal muscle cell proliferation and differentiation. The objective of this study was to investigate the effects of microRNA-27a (miR-27a) on myogenin expression and Akt/FoxO1 signal pathway during porcine myoblast differentiation. Here, we found that the expression of miR-27a was gradually diminished at the early differentiation stage and then rebounded. Overexpression of miR-27a suppressed the mRNA and protein expression levels of myogenin during porcine myoblast differentiation, whereas inhibition of miR-27a promoted the mRNA and protein expression levels of myogenin. In addition, overexpression of miR-27a decreased the level of P-Akt/Akt and increased the protein level of FoxO1; however, inhibition of miR-27a increased the level of P-Akt/Akt and decreased the protein level of FoxO1. The present study demonstrated that miR-27a could inhibit myogenin expression and Akt/FoxO1 signal pathway during porcine myoblast differentiation.

Palmitic acid triggers cell apoptosis in RGC-5 retinal ganglion cells through the Akt/FoxO1 signaling pathway.

Hallmarks of the pathophysiology of glaucoma are oxidative stress and apoptotic death of retinal ganglion cells (RGCs). Lipotoxicity, involving a series of pathological cellular responses after exposure to elevated levels of fatty acids, leads to oxidative stress and cell death in various cell types. The phosphatidylinositol-3-kinase/protein kinase B/Forkhead box O1 (PI3K/Akt/FoxO1) pathway is crucial for cell survival and apoptosis. More importantly, FoxO1 gene has been reported to confer relatively higher risks for eye diseases including glaucoma. However, little information is available regarding the interaction between FoxO1 and RGC apoptosis, much less a precise mechanism. In the present study, immortalized rat retinal ganglion cell line 5 (RGC-5) was used as a model to study the toxicity of palmitic acid (PA), as well as underlying mechanisms. We found that PA exposure significantly decreased cell viability by enhancing apoptosis in RGC-5 cells, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. PA also induced a remarkable increase in reactive oxygen species and malondialdehyde. Moreover, PA significantly decreased the level of phospho-Akt and phospho-FoxO1 in cells. Finally, shRNA knockdown and plasmid overexpression studies displayed that downregulation of Akt protein or upregulation of FoxO1 protein augmented cell death, while knockdown of FoxO1 or overexpression of Akt1 abolished PA-induced cell death. Collectively, our results indicated that PA-induced cell death is mediated through modulation of Akt/FoxO1 pathway activity.

[Chinese medicinal compound CFF-1 induces the apoptosis and cycle-arrest of prostate cancer cells via the PI3K/AKT/FOXO1 signaling pathway].

OBJECTIVE: To explore the apoptosis-inducing effect of the Chinese medicinal compound CFF-1 on prostate cancer cells and its related molecular mechanisms. METHODS: Normal prostate WPMY-1 cells and prostate cancer LNCaP, CWR22Rv1, PC3 and DU145 cells were treated in dehydrated alcohol with CFF-1 at 0, 2, 5, or 10 mg/ml for 24 hours. Then the viability of the prostate cells was detected by morphological observation, MTT and CCK-8 assay, nuclear condensation and disruption measured by DAPI staining, the cell cycle and apoptosis calculated by flow cytometry, the activity of the PI3K/AKT/FOXO1 signaling pathway and the expressions of its downstream apoptosis- and cycle-related proteins determined by Western blot. RESULTS: CFF-1 significantly arrested the cell cycle in the G1 phase, decreased the cell viability and increased the nuclear condensation and disruption in a dose-dependent manner, and elevated the apoptosis rate of prostate cancer cells. At the molecular level, CFF-1 dose-dependently reduced the activity of the PI3K/AKT signaling pathway and phosphorylation of the FOXO1 protein, increased the transcription activity of FOXO1, and eventually regulated the expressions of cell apoptosis- and cycle-related genes. CONCLUSIONS: The Chinese medicinal compound CFF-1 can significantly inhibit the growth, arrest the cycle, and induce the apoptosis of prostate cancer cells by decreasing the activity of the PI3K/AKT/FOXO1 signaling pathway, which suggests its potential clinical application value in the treatment of prostate cancer.

Hyperglycemia-induced Sirt3 downregulation increases microglial aerobic glycolysis and inflammation in diabetic neuropathic pain pathogenesis.

BACKGROUND: Hyperglycemia-induced neuroinflammation significantly contributes to diabetic neuropathic pain (DNP), but the underlying mechanisms remain unclear. OBJECTIVE: To investigate the role of Sirt3, a mitochondrial deacetylase, in hyperglycemia-induced neuroinflammation and DNP and to explore potential therapeutic interventions. METHOD AND RESULTS: Here, we found that Sirt3 was downregulated in spinal dorsal horn (SDH) of diabetic mice by RNA-sequencing, which was further confirmed at the mRNA and protein level. Sirt3 deficiency exacerbated hyperglycemia-induced neuroinflammation and DNP by enhancing microglial aerobic glycolysis in vivo and in vitro. Overexpression of Sirt3 in microglia alleviated inflammation by reducing aerobic glycolysis. Mechanistically, high-glucose stimulation activated Akt, which phosphorylates and inactivates FoxO1. The inactivation of FoxO1 diminished the transcription of Sirt3. Besides that, we also found that hyperglycemia induced Sirt3 degradation via the mitophagy-lysosomal pathway. Blocking Akt activation by GSK69093 or metformin rescued the degradation of Sirt3 protein and transcription inhibition of Sirt3 mRNA, which substantially diminished hyperglycemia-induced inflammation. Metformin in vivo treatment alleviated neuroinflammation and diabetic neuropathic pain by rescuing hyperglycemia-induced Sirt3 downregulation. CONCLUSION: Hyperglycemia induces metabolic reprogramming and inflammatory activation in microglia through the regulation of Sirt3 transcription and degradation. This novel mechanism identifies Sirt3 as a potential drug target for treating DNP.