Loss of PTEN expression does not contribute to PDK-1 activity and PKC activation-loop phosphorylation in Jurkat leukaemic T cells

Unopposed PI3-kinase activity and 3'-phosphoinositide production in Jurkat T cells, due to a mutation in the PTEN tumour suppressor protein, results in deregulation of PH domain-containing proteins including the serine/threonine kinase PKB/Akt. In Jurkat cells, PKB/Akt is constitutively active and phosphorylated at the activation-loop residue (Thr308). 3'-phosphoinositide-dependent protein kinase-1 (PDK-1), an enzyme that also contains a PH domain, is thought to catalyse Thr308 phosphorylation of PKB/Akt in addition to other kinase families such as PKC isoforms. It is unknown however if the loss of PTEN in Jurkat cells also results in unregulated PDK-1 activity and whether such loss impacts on activation-loop phosphorylation of other putative PDK-1 substrates such as PKC. In this study we have addressed if loss of PTEN in Jurkat T cells affects PDK-1 catalytic activity and intracellular localisation. We demonstrate that reducing the level of 3'-phosphoinositides in Jurkat cells with pharmacological inhibitors of PI3-kinase or expression of PTEN does not affect PDK-1 activity, Ser241 phosphorylation or intracellular localisation. In support of this finding, we show that the levels of PKC activation-loop phosphorylation are unaffected by reductions in the levels of 3'-phosphoinositides. Instead, the dephosphorylation that occurs on PKB/Akt at Thr308 following reductions in 3'-phosphoinositides is dependent on PP2A-like phosphatase activity. Our finding that PDK-1 functions independently of 3'-phosphoinositides in T cells is also confirmed by studies in HuT-78 T cells, a PTEN-expressing cell line with undetectable levels of 3'-phosphoinositides. We conclude therefore that loss of PTEN expression in Jurkat T cells does not impact on the PDK-1/PKC pathway and that only a subset of kinases, such as PKB/Akt, are perturbed as a consequence PTEN loss.     

Altering PI3K—Akt signalling in zebrafish embryos affects PTEN phosphorylation and gastrulation

Background information. PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a negative regulator of the PI3K (phosphoinositide 3‐kinase)–Akt (also called protein kinase B) signalling pathway and is essential for embryogenesis, but its function in early vertebrate embryos is unclear. Results. To address how PTEN functions in early embryos, we overexpressed one of the four zebrafish PTEN isoforms at the 1–2‐cell stage. Overexpression of Ptena454 alters phospho‐Akt levels and impairs cell movements associated with gastrulation. Heat shocking embryos increases phospho‐Akt levels and lowers phospho‐Ptena454 levels. Inhibiting CK2 (protein kinase CK2) activity reduces phospho‐Pten levels and augments the effects due to Ptena454 overexpression. Low phospho‐Akt and corresponding low phospho‐GSK‐3 (glycogen synthase kinase‐3) and high phospho‐Pten levels accompany wortmannin or LY294002 treatment, which inhibit PI3K activity. Conclusions. These results suggest that Ptena454 regulation is correlated to changes in phospho‐Akt levels. We propose a model in which homoeostasis in rapidly dividing and migrating embryonic cells depends on a counterbalance between pro‐survival signalling employing CK2 and GSK‐3 and the pro‐apoptotic activity of Ptena454.     

Suppression of Akt1 phosphorylation by adenoviral transfer of the PTEN gene inhibits hypoxia-induced proliferation of rat pulmonary arterial smooth muscle cells

Recent findings identify the role of proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. Phosphoinositide 3 kinase (PI3K) and serine/threonine kinase (Akt) proteins are expressed in vascular smooth muscle cells. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been identified as a negative regulator of cytokine signaling that inhibits the PI3K-Akt pathway. However, little is known about the role of PTEN/Akt signaling in hypoxia-associated vascular remodeling. In this study, we found that hypoxia-induced the expression of Akt1 mRNA and phosphorylated protein by at least twofold in rat PASMCs. Phospho-PTEN significantly decreased in the nuclei of PASMCs after hypoxic stimulation. After forcing over-expression of PTEN by adenovirus-mediated PTEN (Ad-PTEN) transfection, the expression of phospho-Akt1 was significantly suppressed in PASMCs at all time-points measured. Additionally, we showed here that hypoxia increased proliferation of PASMCs by nearly twofold and over-expression of PTEN significantly inhibited hypoxia-induced PASMCs proliferation. These findings suggest that phospho-PTEN loss in the nuclei of PASMCs under hypoxic conditions may be the major cause of aberrant activation of Akt1 and may, therefore, play an important role in hypoxia-associated pulmonary arterial remodeling. Finally, the fact that transfection with Ad-PTEN inhibits the phosphorylation of Akt1 in PASMCs suggests a potential therapeutic effect on hypoxia-associated pulmonary arterial remodeling.     

Phosphorylation of PTEN and Akt in astrocytes of the rat hippocampus following transient forebrain ischemia

To ascertain whether the PTEN (phosphatase and tensin homolog deleted on chromosome 10)/Akt signaling pathway is activated during ischemic brain injury, we investigated the expression and phosphorylation of PTEN and Akt by immunohistochemistry in the rat hippocampus after transient forebrain ischemia. Weak immunoreactivity for PTEN and its phosphorylated form (p-PTEN) was constitutively expressed in hippocampal neurons and astrocytes of the control rats, but their upregulation was detected mainly in reactive astrocytes in the ischemic hippocampus. Increased immunoreactivity for PTEN and p-PTEN occurred specifically in astrocytes by day 1 and was sustained for more than 2 weeks. The spatiotemporal activation of Akt in the ischemic hippocampus mirrored that of p-PTEN expression. Post-ischemic activation of Akt, revealed by phosphorylated Akt (p-Akt) immunoreactivity, was first detected at day 1 and was maintained for at least 2 weeks. Double-labeling experiments revealed that the cells expressing PTEN, p-PTEN, or p-Akt were reactive astrocytes expressing glial fibrillary acidic protein. These results demonstrate the increased phosphorylation of PTEN and Akt in reactive astrocytes of the post-ischemic hippocampus, suggesting that the PTEN/Akt pathway is involved in the astroglial reaction in the rat hippocampus after transient forebrain ischemia.This research was supported by Korea Science and Engineering Foundation (R01-2002-000-00334-0(2002)).     

Activation of Akt/PKB, increased phosphorylation of Akt substrates and loss and altered distribution of Akt and PTEN are features of Alzheimer's disease pathology

Studies suggest that activation of phosphoinositide 3-kinase-Akt may protect against neuronal cell death in Alzheimer's disease (AD). Here, however, we provide evidence of increased Akt activation, and hyperphosphorylation of critical Akt substrates in AD brain, which link to AD pathogenesis, suggesting that treatments aiming to activate the pathway in AD need to be considered carefully. A different distribution of Akt and phospho-Akt was detected in AD temporal cortex neurons compared with control neurons, with increased levels of active phosphorylated-Akt in particulate fractions, and significant decreases in Akt levels in AD cytosolic fractions, causing increased activation of Akt (phosphorylated-Akt/total Akt ratio) in AD. In concordance, significant increases in the levels of phosphorylation of total Akt substrates, including: GSK3beta(Ser9), tau(Ser214), mTOR(Ser2448), and decreased levels of the Akt target, p27(kip1), were found in AD temporal cortex compared with controls. A significant loss and altered distribution of the major negative regulator of Akt, PTEN (phosphatase and tensin homologue deleted on chromosome 10), was also detected in AD neurons. Loss of phosphorylated-Akt and PTEN-containing neurons were found in hippocampal CA1 at end stages of AD. Taken together, these results support a potential role for aberrant control of Akt and PTEN signalling in AD.     

Inhibition of PTEN by peroxynitrite activates the phosphoinositide-3-kinase/Akt neuroprotective signaling pathway

Peroxynitrite is usually considered as a neurotoxic nitric oxide-derivative. However, an increasing body of evidence suggests that, at low concentrations, peroxynitrite affords transient cytoprotection, both in vitro and in vivo. Here, we addressed the signaling mechanism responsible for this effect, and found that rat cortical neurons in primary culture acutely exposed to peroxynitrite (0.1 mmol/L) rapidly elicited Akt-Ser(473) phosphorylation. Inhibition of phosphoinositide-3-kinase (PI3K)/Akt pathway with wortmannin or Akt small hairpin RNA (shRNA) abolished the ability of peroxynitrite to prevent etoposide-induced apoptotic death. Endogenous peroxynitrite formation by short-term incubation of neurons with glutamate stimulated Akt-Ser(473) phosphorylation, whereas Akt shRNA enhanced the vulnerability of neurons against glutamate. We further show that Akt-Ser(473) phosphorylation was consequence of the oxidizing, but not the nitrating properties of peroxynitrite. Peroxynitrite failed to nitrate or phosphorylate neurotrophin tyrosine kinase receptors (Trks), and it did not modify the ability of brain-derived neurotrophic factor (BDNF), to phosphorylate its cognate receptor, TrkB; however, peroxynitrite enhanced BDNF-mediated Akt-Ser(473) phosphorylation. Finally, we found that peroxynitrite-stimulated Akt-Ser(473) phosphorylation was associated with an increased proportion of oxidized phosphoinositide phosphatase, PTEN, in neurons. Moreover, peroxynitrite prevented the increase of apoptotic neuronal death caused by over-expression of PTEN. Thus, peroxynitrite exerts neuroprotection by inhibiting PTEN, hence activating the anti-apoptotic PI3K/Akt pathway in primary neurons.     

Caspase-3-mediated cleavage of Akt: involvement of non-consensus sites and influence of phosphorylation

Here, we show for the first time that Akt1 is cleaved in vitro at the caspase-3 consensus site DQDD(456) downward arrow SM. Our data suggest QEEE(116) downward arrow E(117) downward arrow MD, EEMD(119) downward arrow, TPPD(453) downward arrow QD and DAKE(398) downward arrow IM as novel non-consensus caspase-3 cleavage sites. More importantly, we demonstrate that phosphorylation of Akt1 modulates its cleavage in a site-specific manner: Resistance to cleavage at site DAKE(398) (within the kinase domain) in response to phosphorylation suggests a possible mechanism by which the anti-apoptotic role of Akt1 is regulated. Our result is important in biological models which rely on Akt1 for cell survival.     

Heparanase induces Akt phosphorylation via a lipid raft receptor

The endoglycosidase heparanase is the predominant enzyme that degrades heparan sulfate side chains of heparan sulfate proteoglycans, activity that is strongly implicated in tumor metastasis. Apart of its well characterized enzymatic activity, heparanase was noted to exert also enzymatic-independent functions. Among these is the induction of Akt/PKB phosphorylation noted in endothelial- and tumor-derived cells. Protein domains of heparanase required for signaling were not identified to date, nor were identified heparanase binding proteins/receptors capable of transmitting heparanase signals. Here, we examined the possible function of mannose 6-phosphate receptor (MPR) and low-density lipoprotein-receptor related protein (LRP), recently implicated in cellular uptake of heparanase, as heparanase receptors mediating Akt phosphorylation. We found that heparanase addition to MPR- and LRP-deficient fibroblasts elicited Akt activation indistinguishable from control fibroblasts. In contrast, disruption of lipid rafts abrogated Akt/PKB phosphorylation following heparanase addition. These results suggest that lipid raft-resident receptor mediates heparanase signaling.     

Critical role of PTEN in the coupling between PI3K/Akt and JNK1/2 signaling in ischemic brain injury

JNK pathway is an important pro-apoptotic kinase cascade mediating cell death in response to a variety of extracellular stimuli including excitotoxicity, which results in selective and delayed neuronal death in the hippocampal CA1. On the contrary, activation of the protein kinase Akt, which is controlled by the opposing actions of PI3K and PTEN, contributes to enhanced resistance to apoptosis through multiple mechanisms. We here demonstrate that the temporal pattern of Akt activation reversely correlates with JNK1/2 activation following various time points of ischemic reperfusion. However, the activation of JNK1/2 could be decreased by the elevation of Akt activation via increasing the tyrosine phosphorylation of PTEN by bpv(pic), a potent PTPases inhibitor for PTEN, or by intracerebroventricular infusion of PTEN antisense oligodeoxynucleotides (AS-ODNs). In contrast, JNK1/2 activation was significantly increased by preventing PTEN degradation after pretreatment with proteasome inhibitor. The neuroprotective effects of bpv(pic) and PTEN AS-ODNs were significant in the CA1 subfield after transient global ischemia. In conclusion, the present results clearly show that PTEN plays a key regulatory role in the cross-talk between cell survival PI3K/Akt pathway and pro-death JNK pathway, and raise a new possibility that agents targeting phosphatase PTEN may offer a great promise to expand the therapeutic options in protecting neurons form ischemic brain damage.     

Akt phosphorylation is essential for nuclear translocation and retention in NGF-stimulated PC12 cells

Nerve growth factor (NGF) elicits Akt translocation into the nucleus, where it phosphorylates nuclear targets. Here, we describe that Akt phosphorylation can promote the nuclear translocation of Akt and is necessary for its nuclear retention. Overexpression of Akt-K179A, T308A, S473A-mutant failed to show either nuclear translocation or nuclear Akt phosphorylation, whereas expression of wild-type counterpart elicited profound Akt phosphorylation and induced nuclear translocation under NGF stimulation. Employing the PI3K inhibitor and a variety of mutants PI3K, we showed that nuclear translocation of Akt was mediated by activation of PI3K, and Akt phosphorylation status in the nucleus required PI3K activity. Thus the activity of PI3K might contribute to the nuclear translocation of Akt, and that Akt phosphorylation is essential for its nuclear retention under NGF stimulation conditions.     

Pyrrolidine dithiocarbamate induces cyclooxygenase-2 expression in NIH 3T3 fibroblast cells

Pyrrolidine dithiocarbamate (PDTC) is a metal chelating compound that can exert either pro-oxidant or antioxidant effects in different situations. Several studies demonstrate that it can inhibit cyclooxygenase-2 (COX-2) expression, which may be due to its antioxidant activity. Here, we found that PDTC rather increased COX-2 expression in NIH 3T3. The increase of COX-2 expression was inhibited by adding bathocuproline disulfonic acid, a non-permeable specific copper chelator, in the incubation medium. This result suggests that PDTC exerts its effect by transporting redox-active copper ions into the cells. In support of this observation, PDTC did not induce COX-2 expression in a serum-free environment. When PDTC was added with copper in the serum-free medium, it acted as the inducer of COX-2 expression. In addition, pretreatment of N-acetyl-L-cystein or dithiothreitol, other antioxidants, inhibited the PDTC-induced COX-2 expression. Our data indicate that PDTC induces COX-2 expression in NIH 3T3 cells, which may be due to its activities as a copper chelator and a pro-oxidant.     

LKB1IP promotes pathological cardiac hypertrophy by targeting PTEN/Akt signalling pathway

Pathological cardiac hypertrophy represents a leading cause of morbidity and mortality worldwide. Liver kinase B1 interacting protein 1 (LKB1IP) was identified as the binding protein of tumour suppressor LKB1. However, the role of LKB1IP in the development of pathological cardiac hypertrophy has not been explored. The aim of this study was to investigate the function of LKB1IP in cardiac hypertrophy in response to hypertrophic stimuli. We investigated the cardiac level of LKB1IP in samples from patients with heart failure and mice with cardiac hypertrophy induced by isoproterenol (ISO) or transverse aortic constriction (TAC). LKB1IP knockout mice were generated and challenged with ISO injection or TAC surgery. Cardiac function, hypertrophy and fibrosis were then examined. LKB1IP expression was significantly up-regulated on hypertrophic stimuli in both human and mouse cardiac samples. LKB1IP knockout markedly protected mouse hearts against ISO- or TAC-induced cardiac hypertrophy and fibrosis. LKB1IP overexpression aggravated ISO-induced cardiomyocyte hypertrophy, and its inhibition attenuated hypertrophy in vitro. Mechanistically, LKB1IP activated Akt signalling by directly targeting PTEN and then inhibiting its phosphatase activity. In conclusion, LKB1IP may be a potential target for pathological cardiac hypertrophy.     

PTEN overexpression promotes glioblastoma death through triggering mitochondrial division and inactivating the Akt pathway

Abstract

Objective: PTEN has been acknowledged as an anticancer factor in the progression of glioblastoma. Mitochondrial division has been found to be associated with cancer cell death.

Objective: The aim of our study is to explore whether PTEN attenuates the development of glioblastoma by modulating mitochondrial division.

Materials and methods: PTEN adenovirus was used to overexpress PTEN in U87 cells. Mitochondrial function was detected via western blot and immunofluorescence. Pathway blocker was used to inhibit the Akt activation.

Results: The results of our study demonstrated that PTEN overexpression reduced cell viability by increasing cell apoptosis. At the molecular level, PTEN overexpression activated mitochondrial apoptosis by mediating mitochondrial dysfunction. Furthermore, we found that Drp1-related mitochondrial division was required for PTEN-mediated mitochondrial dysfunction and cell death. Finally, we found that PTEN modulated Drp1-related mitochondrial division via the Akt pathway; inactivation of Akt induced cell death, and mitochondrial damage, similar to the results obtained via PTEN overexpression.

Conclusions: Taken together, our results clarify that the anticancer mechanism of PTEN in glioblastoma is dependent on the activation of Drp1-related mitochondrial division via Akt pathway modulation. This finding might provide new insight into the tumor-suppressive role played by PTEN in glioblastoma.     

PTBP1 promotes the growth of breast cancer cells through the PTEN/Akt pathway and autophagy

Invasion and migration is the hallmark of malignant tumors as well as the major cause for breast cancer death. The polypyrimidine tract binding, PTB, protein serves as an important model for understanding how RNA binding proteins affect proliferation and invasion and how changes in the expression of these proteins can control complex programs of tumorigenesis. We have investigated some roles of polypyrimidine tract binding protein 1 (PTBP1) in human breast cancer. We found that PTBP1 was upregulated in breast cancer tissues compared with normal tissues and the same result was confirmed in breast cancer cell lines. Knockdown of PTBP1 substantially inhibited tumor cell growth, migration, and invasion. These results suggest that PTBP1 is associated with breast tumorigenesis and appears to be required for tumor cell growth and maintenance of metastasis. We further analyzed the relationship between PTBP1 and clinicopathological parameters and found that PTBP1 was correlated with her‐2 expression, lymph node metastasis, and pathological stage. This will be a novel target for her‐2(⁺) breast cancer. PTBP1 exerts these effects, in part, by regulating the phosphatase and tensin homolog‐phosphatidylinositol‐4,5‐bisphosphate 3‐kinase/protein kinase B (PTEN‐PI3K/Akt) pathway and autophagy, and consequently alters cell growth and contributes to the invasion and metastasis.     

PTEN Overexpression Cooperates With Lithium to Reduce the Malignancy and to Increase Cell Death by Apoptosis via PI3K/Akt Suppression in Colorectal Cancer Cells

Lithium is a well‐established non‐competitive inhibitor of glycogen synthase kinase‐3β (GSK‐3β), a kinase that is involved in several cellular processes related to cancer progression. GSK‐3β is regulated upstream by PI3K/Akt, which is negatively modulated by PTEN. The role that lithium plays in cancer is controversial because lithium can activate or inhibit survival signaling pathways depending on the cell type. In this study, we analyzed the mechanisms by which lithium can modulate events related to colorectal cancer (CRC) progression and evaluated the role that survival signaling pathways such as PI3K/Akt and PTEN play in this context. We show that the administration of lithium decreased the proliferative potential of CRC cells in a GSK‐3β‐independent manner but induced the accumulation of cells in G2/M phase. Furthermore, high doses of lithium increased apoptosis, which was accompanied by decreased proteins levels of Akt and PTEN. Then, cells that were induced to overexpress PTEN were treated with lithium; we observed that low doses of lithium strongly increased apoptosis. Additionally, PTEN overexpression reduced proliferation, but this effect was minor compared with that in cells treated with lithium alone. Furthermore, we demonstrated that PTEN overexpression and lithium treatment separately reduced cell migration, colony formation, and invasion, and these effects were enhanced when lithium treatment and PTEN overexpression were combined. In conclusion, our findings indicate that PTEN overexpression and lithium treatment cooperate to reduce the malignancy of CRC cells and highlight lithium and PTEN as potential candidates for studies to identify new therapeutic approaches for CRC treatment. J. Cell. Biochem. 117: 458–469, 2016. © 2015 Wiley Periodicals, Inc.     

PTEN通过抑制PI3K-AKT信号通路上调Rad51基因的表达

前期研究发现pten缺陷细胞的自发DNA双链断裂损伤水平显著增加.本研究探讨了抑癌基因pten对参与DNA同源重组修复的rad51基因表达的影响和机制．用实时定量PCR技术检测了PTEN野生型和缺陷型细胞rad51的表达水平.结果发现,PTEN缺失会导致rad51的表达降低．PI3K激酶为PTEN的下游负调节靶分子,使用PI3K激酶抑制剂LY294002处理缺陷型细胞后,其rad51表达升高．在PTEN野生型细胞中分别转染Flag-Akt WT（野生型）和Flag-Akt AC（组成型激活）,或在PTEN缺陷型细胞中分别转染野生型PTEN和Akt-DN（失去激酶活性的Akt）. 利用RT-PCR技术检测上述细胞rad51的表达水平,同时利用Western印迹检测上述细胞RAD51蛋白的表达水平.结果发现,转染Flag-Akt WT和Flag-Akt AC后,均能促使PTEN野生型细胞中rad51在mRNA和蛋白水平降低;在PTEN缺陷型细胞中转染野生型PTEN或Akt-DN后,rad51在mRNA和蛋白水平均升高．在PTEN缺陷型细胞中使用siRNA沉默akt后,同样导致RAD51表达升高．结果提示,PTEN可以正向调节RAD51基因表达,PI3K/Akt是其信号通路机制之一．     

ATM-mediated PTEN phosphorylation promotes PTEN nuclear translocation and autophagy in response to DNA-damaging agents in cancer cells

PTEN (phosphatase and tensin homolog), a tumor suppressor frequently mutated in human cancer, has various cytoplasmic and nuclear functions. PTEN translocates to the nucleus from the cytoplasm in response to oxidative stress. However, the mechanism and function of the translocation are not completely understood. In this study, topotecan (TPT), a topoisomerase I inhibitor, and cisplatin (CDDP) were employed to induce DNA damage. The results indicate that TPT or CDDP activates ATM (ATM serine/threonine kinase), which phosphorylates PTEN at serine 113 and further regulates PTEN nuclear translocation in A549 and HeLa cells. After nuclear translocation, PTEN induces autophagy, in association with the activation of the p-JUN-SESN2/AMPK pathway, in response to TPT. These results identify PTEN phosphorylation by ATM as essential for PTEN nuclear translocation and the subsequent induction of autophagy in response to DNA damage.     

MicroRNA-10 Family Promotes the Epithelial-to-Mesenchymal Transition in Renal Fibrosis by the PTEN/Akt Pathway

Renal fibrosis (RF) is a common reason for renal failure, and epithelial-mesenchymal transition (EMT) is a vital mechanism that promotes the development of RF. It is known that microRNA-10 (miR-10) plays an important role in cancer EMT; however, whether it takes part in the EMT process of RF remains unclear. Therefore, we established an in vivo model of unilateral ureteral obstruction (UUO), and an in vitro model using TGF-β1, to investigate whether and how miR-10a and miR-10b take part in the EMT of RF. In addition, the combinatorial effects of miR-10a and miR-10b were assessed. We discovered that miR-10a and miR-10b are overexpressed in UUO mice, and miR-10a, miR-10b, and miRs-10a/10b knockout attenuated RF and EMT in UUO-treated mouse kidneys. Moreover, miR-10a and miR-10b overexpression combinatorially promoted RF and EMT in TGF-β1-treated HK-2 cells. Inhibiting miR-10a and miR-10b attenuated RF and EMT induced by TGF-β1. Mechanistically, miR-10a and miR-10b suppressed PTEN expression by binding to its mRNA3′-UTR and promoting the Akt pathway. Moreover, PTEN overexpression reduced miR-10a and miR-10b effects on Akt phosphorylation (p-Akt), RF, and EMT in HK-2 cells treated with TGF-β1. Taken together, miR-10a and miR-10b act combinatorially to negatively regulate PTEN, thereby activating the Akt pathway and promoting the EMT process, which exacerbates RF progression.     

Metformin sensitizes insulin signaling through AMPK-mediated PTEN down-regulation in preadipocyte 3T3-L1 cells

Insulin resistance is the primary cause responsible for type 2 diabetes. Phosphatase and tensin homolog (PTEN) plays a negative role in insulin signaling and its inhibition improves insulin sensitivity. Metformin is a widely used insulin-sensitizing drug; however, the mechanism by which metformin acts is poorly understood. To gain insight into the role of PTEN, we examined the effect of metformin on PTEN expression. Metformin suppressed the expression of PTEN in an AMP-activated protein kinase (AMPK)-dependent manner in preadipocyte 3T3-L1 cells. Knock-down of PTEN potentiated the increase in insulin-mediated phosphorylation of Akt/ERK. Metformin also increased the phosphorylation of c-Jun N-terminal kinase (JNK)-c-Jun and mammalian target of rapamycin (mTOR)-p70S6 kinase pathways. Both pharmacologic inhibition and knock-down of AMPK blocked metformin-induced phosphorylation of JNK and mTOR. Knock-down of AMPK recovered the metformin-induced PTEN down-regulation, suggesting the involvement of AMPK in PTEN regulation. PTEN promoter activity was suppressed by metformin and inhibition of mTOR and JNK by pharmacologic inhibitors blocked metformin-induced PTEN promoter activity suppression. These findings provide evidence for a novel role of AMPK on PTEN expression and thus suggest a possible mechanism by which metformin may contribute to its beneficial effects on insulin signaling.