MiR-28 inhibits cardiomyocyte survival through suppressing PDK1/Akt/mTOR signaling

MicroRNAs play critical roles in regulating cell survival under multiple pathological conditions of heart diseases. Oxidative stress-induced apoptosis contributes greatly to heart ischemia-reperfusion injury. Herein, we describe a novel regulatory role of miR-28 on the survival of cardiomyocytes. We show that miR-28 was upregulated in cardiomyocytes treated with hydrogen peroxide (H₂O₂). MiR-28 gain of function sensitized cell apoptosis, whereas miR-28 loss of function partially rescued cell apoptosis induced by H₂O₂. Importantly, we observed a significant reduction in Akt/mammalian target of rapamycin (mTOR) signaling activity after miR-28 treatment. Luciferase activity assay and western blot analysis both revealed that, phosphoinositide-dependent kinase-1 (PDK1), which is critical for Akt activation, was directly and negatively modulated by miR-28. Our results therefore indicate that miR-28 regulates oxidative stress-induced cell apoptosis in heart muscle cells, which possibly involves a PDK1/Akt/mTOR-dependent mechanism. MIR-28 could serve as a critical therapeutic target to diminish oxidative stress-induced cell death in the heart.     

Overexpression of Akt1 upregulates glycogen synthase activity and phosphorylation of mTOR in IRS-1 knockdown HepG2 cells

Insulin receptor substrate (IRS) proteins are important docking proteins in mediating the insulin signaling cascade. We have investigated the effect of short interfering RNA (siRNA) mediated knockdown of IRS-1 on insulin signaling cascade in primary human hepatocellular carcinoma HepG2 cell line and HepG2 cells overexpressing Akt1/PKB-alpha (HepG2-CA-Akt/PKB). IRS-1 knockdown in both cell lines resulted in reduction of insulin stimulated Akt1 phosphorylation at Ser 473. In parental HepG2 cells, IRS-1 knockdown resulted in reduction (ca. 50%) in the basal level of phosphorylated mTOR (Ser 2448) irrespective of insulin treatment. In contrast, HepG2-CA-Akt/PKB cells showed an upregulation in the basal level of phosphorylated mTOR (Ser 2448) (ca. 40%). Insulin mediated phosphorylation of mTOR was reduced. IRS-1 knockdown also reduced the cell proliferation of parental HepG2 cells by ca. 30% in the presence/absence of insulin, whereas in HepG2-CA-Akt/PKB the cell proliferation was reduced by 15% and treatment of insulin further reduced it to ca. 50% (vs. control). IRS-1 knockdown also reduced the glycogen synthase (GS) activity in parental HepG2 cells, however, it was upregulated in HepG2-CA-Akt/PKB cells. These results suggest that knockdown of IRS-1 abolished basal as well as insulin mediated phosphorylation/activity of proteins involved in cell proliferation or glycogen metabolism in the parental Hep2 cells. IRS-1 knockdown in cells overexpressing constitutively active Akt1/PKB-alpha either did not change or upregulated the basal levels of phosphorylated/active proteins. However, insulin mediated response was either not altered or downregulated in these cells.     

Gax suppresses chemerin/CMKLR1‐induced preadipocyte biofunctions through the inhibition of Akt/mTOR and ERK signaling pathways

Adipose tissue is closely associated with angiogenesis and vascular remodeling. Chemerin is involved in inflammatory reaction and vascular dysfunction. However, the mechanisms of chemerin participating in vascular remodeling and whether Growth arrest‐specific homeobox (Gax) can effectively intervene it remain obscured. Here, 3T3‐F442A preadipocytes were cultured, injected into athymic mice to model fat pads, and treated respectively with Ad‐chemerin, Ad‐Gax, or specific inhibitors in vitro and in vivo. MTT, flow cytometry, Western blotting, and imunohisto(cyto)‐chemistry analyses showed that chemerin enhanced the expression of FABP4 and VEGF, activated Akt/mTOR and ERK pathways, increased the cell percent of S phase, decreased the percent of G0‐G1 phase and apoptotic cells, and augmented neovascular density in fat pads. Inversely, Gax suppressed the expression of these adipogenic and vasifactive markers and these signaling proteins, decreased the percent of S phase cells, and increased those of G0‐G1 phase and apoptotic cells, and reduced the neovascular density. Our results indicate that chemerin‐CMKLR1 activates Akt/mTOR and ERK pathways and facilitates preadipocyte proliferation, adipogenesis, and angiogenesis. Contrarily, Gax weakens the effect of chemerin on preadipocyte biofunctions.     

IGF1 regulates PKM2 function through Akt phosphorylation

Pyruvate kinase M2 (PKM2) acts at the crossroad of growth and metabolism pathways in cells. PKM2 regulation by growth factors can redirect glycolytic intermediates into key biosynthetic pathway. Here we show that IGF1 can regulate glycolysis rate, stimulate PKM2 Ser/Thr phosphorylation and decrease cellular pyruvate kinase activity. Upon IGF1 treatment we found an increase of the dimeric form of PKM2 and the enrichment of PKM2 in the nucleus. This effect was associated to a reduction of pyruvate kinase enzymatic activity and was reversed using metformin, which decreases Akt phosphorylation. IGF1 induced an increased nuclear localization of PKM2 and STAT3, which correlated with an increased HIF1α, HK2, and GLUT1 expression and glucose entrapment. Metformin inhibited HK2, GLUT1, HIF-1α expression and glucose consumption. These findings suggest a role of IGFIR/Akt axis in regulating glycolysis by Ser/Thr PKM2 phosphorylation in cancer cells.     

AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1

Autophagy is a process by which components of the cell are degraded to maintain essential activity and viability in response to nutrient limitation. Extensive genetic studies have shown that the yeast ATG1 kinase has an essential role in autophagy induction. Furthermore, autophagy is promoted by AMP activated protein kinase (AMPK), which is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cell-growth regulator that integrates growth factor and nutrient signals. Here we demonstrate a molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1. Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser 317 and Ser 777. Under nutrient sufficiency, high mTOR activity prevents Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction between Ulk1 and AMPK. This coordinated phosphorylation is important for Ulk1 in autophagy induction. Our study has revealed a signalling mechanism for Ulk1 regulation and autophagy induction in response to nutrient signalling.     

Plk1 inhibition enhances the efficacy of gemcitabine in human pancreatic cancer

Gemcitabine is the standard-of-care for chemotherapy in patients with pancreatic adenocarcinoma and it can directly incorporate into DNA or inhibit ribonucleotide reductase to prevent DNA replication and, thus, tumor cell growth. Most pancreatic tumors, however, develop resistance to gemcitabine. Polo-like kinase 1 (Plk1), a critical regulator in many cell cycle events, is significantly elevated in human pancreatic cancer. In this study, we show that Plk1 is required for the G1/S transition and that inhibition of Plk1 significantly reduces the DNA synthesis rate in human pancreatic cancer cells. Furthermore, the combined effect of a specific Plk1 inhibitor GSK461364A with gemcitabine was examined. We show that inhibition of Plk1 significantly potentiates the anti-neoplastic activity of gemcitabine in both cultured pancreatic cancer cells and Panc1-derived orthotopic pancreatic cancer xenograft tumors. Overall, our study demonstrates that co-targeting Plk1 can significantly enhance the efficacy of gemcitabine, offering a promising new therapeutic option for the treatment of gemcitabine-resistant human pancreatic cancer.     

miR-152/TNS1 axis inhibits non-small cell lung cancer progression through Akt/mTOR/RhoA pathway

Aim: The purpose of the present study was to explore the function and mechanism of tensin 1 (TNS1) in non-small cell lung cancer (NSCLC) progression.Methods: The expression of TNS1 in NSCLC cells and tissues was assessed by RT-PCR and Western blot. Besides, Kaplan–Meier survival analysis was recruited to explore the association between TNS1 and NSCLC. Cell growth was analyzed by MTT and flow cytometry assay, while cell metastasis was determined by wound healing and transwell assays. The targeting relationship between TNS1 and miR-152 was assessed by luciferase activity assays. And Western blot was employed to determine the expression of related proteins of Akt/mTOR/RhoA pathway.Results: TNS1 level was boosted in NSCLC cells and tissues, related to the prognosis of NSCLC patients. Furthermore, it was proved that TNS1 promoted the growth and metastasis of NSCLC cells via Akt/mTOR/RhoA pathway. And miR-152 targeted TNS1 to affect the progression of NSCLC.Conclusion: miR-152/TNS1 axis inhibits the progression of NSCLC by Akt/mTOR/RhoA pathway.     

GLUT1 enhances mTOR activity independently of TSC2 and AMPK

Enhanced GLUT1 expression in mesangial cells plays an important role in the development of diabetic nephropathy by stimulating signaling through several pathways resulting in increased glomerular matrix accumulation. Similarly, enhanced mammalian target of rapamycin (mTOR) activation has been implicated in mesangial matrix expansion and glomerular hypertrophy in diabetes. We sought to examine whether enhanced GLUT1 expression increased mTOR activity and, if so, to identify the mechanism. We found that levels of GLUT1 expression and mTOR activation, as evidenced by S6 kinase (S6K) and 4E-BP-1 phosphorylation, changed in tandem in cell lines exposed to elevated levels of extracellular glucose. We then showed that increased GLUT1 expression enhanced S6K phosphorylation by 1.7- to 2.9-fold in cultured mesangial cells and in glomeruli from GLUT1 transgenic mice. Treatment with the mTOR inhibitor, rapamycin, eliminated the GLUT1 effect on S6K phosphorylation. In cells lacking functional tuberous sclerosis complex (TSC) 2, GLUT1 effects on mTOR activity persisted, indicating that GLUT1 effects were not mediated by TSC. Similarly, AMP kinase activity was not altered by enhanced GLUT1 expression. Conversely, enhanced GLUT1 expression led to a 2.4-fold increase in binding of mTOR to its activator, Rheb, and a commensurate 2.1-fold decrease in binding of Rheb to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) consistent with mediation of GLUT1 effects by a metabolic effect on GAPDH. Thus, GLUT1 expression appears to augment mesangial cell growth and matrix protein accumulation via effects on glycolysis and decreased GAPDH interaction with Rheb.     

Oxymatrine synergistically enhances antitumor activity of oxaliplatin in colon carcinoma through PI3K/AKT/mTOR pathway

Oxymatrine (OMT), one of the main active components of extracts from the dry roots of Sophora flavescens, has been reported to possess many pharmacological properties including cancer-preventive and anti-cancer effects. The aim of the present study is to explore the efficiency of combination therapy with OMT and oxaliplatin (OXA) and identify the in vitro and in vivo cytotoxicity on colon cancer lines (HT29 and SW480) and mice model. Cells were treated with OMT and/or OXA and subjected to cell viability, colony formation, apoptosis, cell cycle, western blotting, xenograft tumorigenicity assay and immunohistochemistry. The results demonstrated that OMT and OXA inhibited the proliferation of colon cancer cells, and combination therapy of OMT and OXA resulted in a combination index?<?1, indicating a synergistic effect. Co-treatment with OMT and OXA caused G0/G1 phase arrest by upregulating P21, P27 and downregulating cyclin D, and induced apoptosis through decreasing the expression of p-PI3K, p-AKT, p-mTOR, p-p70S6K. In addition, pretreatment with a specific PI3K/AKT activator (IGF-1) significantly neutralized the pro-apoptotic activity of OXA?+?OMT, demonstrating the important role of PI3K/AKT in this process. Moreover, in nude mice model, co-treatment displayed more efficient inhibition of tumor weight and volume on SW480 xenograft mouse model than single-agent treatment with OXA or OMT. Immunohistochemistry analysis suggests the combinations greatly suppressed tumor proliferation, which consistent with our in vitro results. In conclusion, our findings highlight that the combination therapy with OMT and OXA exerted synergistic antitumor effects in colon cancer cells through PI3K/AKT/mTOR pathway and combination treatment with OMT and OXA would be a promising therapeutic strategy for colon carcinoma treatment.     

Akt attenuation of the serine protease activity of HtrA2/Omi through phosphorylation of serine 212

The serine protease HtrA2/Omi is released from the mitochondria into the cytosol following apoptosis stimuli, leading to the programmed cell death in caspase-dependent and -independent manners. The function of HtrA2/Omi closely relates to its protease activity, which is required for cleavage of its substrate such as the members of the X-linked inhibitor of apoptotic protein family. However, the regulation of HtrA2/Omi by signaling molecule has not been documented. Here we report that serine/threonine kinases Akt1 and Akt2 phosphorylate mitochondria-released HtrA2/Omi on serine 212 in vivo and in vitro, which results in attenuation of its serine protease activity and pro-apoptotic function. Abolishing HtrA2/Omi phosphorylation by Akt through mutation of serine 212 to alanine (HtrA2/Omi-S212A) retains its serine protease activity and induces more apoptosis as compared with wild-type HtrA2/Omi. Conversely, HtrA2/Omi-S212D, a mutant mimicking phosphorylation, lost the protease activity and failed to induce the programmed cell death. Furthermore, the phosphorylated HtrA2/Omi fails to cleave X-linked inhibitor of apoptotic protein without interfering with their complex formation. In addition, Akt inhibits the release of HtrA2/Omi from the mitochondria into the cytoplasm in response to cisplatin treatment. These data reveal for the first time that HtrA2/Omi is directly regulated by Akt and provide a mechanism by which Akt induces cell survival at post-mitochondrial level.     

hnRNP A1 promotes keratinocyte cell survival post UVB radiation through PI3K/Akt/mTOR pathway

hnRNP A1 acts as a critical splicing factor in regulating many alternative splicing events in various physiological and pathophysiological progressions. hnRNP A1 is capable of regulating UVB-induced hdm2 gene alternative splicing according to our previous study. However, the biological function and underlying molecular mechanism of hnRNP A1 in cell survival and cell cycle in response to UVB irradiation are still unclear. In this study, silencing hnRNP A1 expression by siRNA transfection led to decreased cell survival after UVB treatment, while promoting hnRNP A1 by lentiviruse vector resulted in increased cell survival. hnRNP A1 remarkably enhanced PI3K/Akt/mTOR signaling pathway by increasing phosphorylation of Akt, mTOR and P70S6 protein. Inhibition of PI3K/Akt signaling by LY294002 suppressed the expression of hnRNP A1. While mTOR signaling inhibitors, rapamycin and AZD8055, did not influence hnRNP A1 expression in HaCaT cells, suggesting that hnRNP A1 may be an upstream mediator of mTOR signaling. Furthermore, hnRNP A1 could alleviate UVB-provoked cell cycle arrest at G0/G1 phase and promoted cell cycle progression at G2/M phase. Our results indicate that hnRNP A1 promotes cell survival and cell cycle progression following UVB radiation.     

Vertical inhibition of the PI3K/Akt/mTOR pathway for the treatment of osteoarthritis

Osteoarthritis is characterized by degenerative alterations of articular cartilage including both the degradation of extracellular matrix and the death of chondrocytes. The PI3K/Akt pathway has been demonstrated to involve in both processes. Inhibition of its downstream target NF‐kB reduces the degradation of extracellular matrix via decreased production of matrix metalloproteinases while inhibition of mTOR increased autophagy to reduce chondrocyte death. However, mTOR feedback inhibits the activity of the PI3K/Akt pathway and inhibition of mTOR could result in increased activity of the PI3K/Akt/NF‐kB pathway. We proposed that the use of dual inhibitors of PI3K and mTOR could be a promising approach to more efficiently inhibit the PI3K/Akt pathway than rapamycin or PI3K inhibitor alone and produce better treatment outcome. J. Cell. Biochem. 114: 245–249, 2013. © 2012 Wiley Periodicals, Inc.     

缺氧诱导因子1与PI3K/Akt/mTOR信号转导通路

缺氧诱导因子1（HIF-1）是参与缺氧调节的核心因子,可调控一系列缺氧诱导基因的表达,与机体许多生理和病理过程也密切相关。尽管一些研究显示缺氧和非缺氧性刺激可通过PI3K/Akt/mTOR信号途径诱导HIF-1的表达和活性,PI3K信号途径是否参与对HIF-1的调节仍然是个有争议的研究热点。明确HIF-1和PI3K的相互作用关系,能进一步为肿瘤等相关疾病的防治提供新的思路和方法。本文主要就HIF-1和PI3K/Akt/mTOR关系作一简要综述。     

FKBPs and the Akt/mTOR pathway

FK506-binding proteins (FKBP) belong to the immunophilin family and are best known for their ability to enable the immunosuppressive properties of FK506 and rapamycin. For rapamycin, this is achieved by inducing inhibitory ternary complexes with the kinase mTOR. The essential accessory protein for this gain-of-function was thought to be FKBP12. We recently showed that this view might be too restricted, since larger FK506-binding proteins can functionally substitute for FKBP12 in mammalian cells. Recent studies have also shown that FK506-binding proteins can modulate Akt-mTOR signaling in the absence of rapamycin. Here we discuss the role of FK506-binding proteins for the mechanism of rapamycin as well as their intrinsic actions on the Akt/mTOR pathway.