Myogenic signaling of phosphatidylinositol 3-kinase requires the serine-threonine kinase Akt/protein kinase B

The oncogene p3k, coding for a constitutively active form of phosphatidylinositol 3-kinase (PI 3-kinase), strongly activates myogenic differentiation. Inhibition of endogenous PI 3-kinase activity with the specific inhibitor LY294002, or with dominant-negative mutants of PI 3-kinase, interferes with myotube formation and with the expression of muscle-specific proteins. Here we demonstrate that a downstream target of PI 3-kinase, serine-threonine kinase Akt, plays an important role in myogenic differentiation. Expression of constitutively active forms of Akt dramatically enhances myotube formation and expression of the muscle-specific proteins MyoD, creatine kinase, myosin heavy chain, and desmin. Transdominant negative forms of Akt inhibit myotube formation and the expression of muscle-specific proteins. The inhibition of myotube formation and the reduced expression of muscle-specific proteins caused by the PI 3-kinase inhibitor LY294002 are completely reversed by constitutively active forms of Akt. Wild-type cellular Akt effects a partial reversal of LY294002-induced inhibition of myogenic differentiation. This result suggests that Akt can substitute for PI 3-kinase in the stimulation of myogenesis; Akt may be an essential downstream component of PI 3-kinase-induced muscle differentiation.     

Apoptosis and inhibition of the phosphatidylinositol 3-kinase/Akt signaling pathway in the anti-proliferative actions of dehydroepiandrosterone

Background Dehydroepiandrosterone (DHEA) is an endogenous steroid that is synthesized mainly in the adrenal cortex; it is found in plasma as the sulfate-conjugated form (DHEA-S). Pharmacological doses of DHEA exhibit anti-proliferative effects on malignant cell lines and some tumors in experimental animals. The purpose of this study was to evaluate the effect of these steroids on proliferation in human cancer cell lines.Methods HepG2 and HT-29 cell lines were treated with DHEA or DHEA-S at 0-200µM for 24h or at 100µM for 8–72h, and then effects on cell growth, and the cell cycle and on apoptosis, were evaluated by 3-[4,5-dimethylthiazol]-2yl-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry, respectively. Also, the effect of DHEA on phosphatidylinositol 3-kinase (PI3K)/Akt signaling was investigated in HepG2 cells by Western blotting.Results The growth of HepG2 and HT-29 cells was significantly inhibited by DHEA, in a dose- and time-dependent manner. This inhibition was greater in HepG2 than in HT-29 cells. Accumulation at G0/G1 phase in both cell lines was observed with DHEA treatment. However, apoptosis increased significantly only in HepG2 cells. In contrast, DHEA-S exhibited much weaker growth inhibitory and cytostatic effects on both cell lines, and apoptosis was not detected. In HepG2 cells treated with DHEA, apoptosis was associated with markedly reduced Akt phosphorylation (Thr³⁰⁸ and Ser⁴⁷³), suggesting that DHEA inhibited the PI3K/Akt signaling to induce apoptosis in these cells.Conclusions These results suggest that the induction of apoptosis through the inhibition of the PI3K/Akt signaling pathway is one of the anti-proliferative mechanisms of DHEA in certain tumors, but that DHEA also promotes cell-cycle arrest without the induction of apoptosis.     

Anti-apoptotic signaling by hepatocyte growth factor/Met via the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways

Hepatocyte growth factor (HGF) is a ligand of the receptor tyrosine kinase encoded by the c-Met protooncogene. HGF/Met signaling has multifunctional effects on various cell types. We sought to determine the role of HGF/Met in apoptosis and identify signal transducers involved in this process. In experiments with human SK-LMS-1 leiomyosarcoma cells, we show that the Akt kinase is activated by HGF in a time- and dose-dependent manner by phosphatidylinositol 3-kinase (PI3-kinase). Akt is also activated by active tumorigenic forms of Met, i.e., ligand-independent Tpr-Met, a truncated and constitutively dimerized form of Met, and a mutationally activated version of Met corresponding to that found in human hereditary papillary renal carcinoma. In NIH 3T3 cells transfected with wild-type Met, HGF inhibits apoptosis induced by serum starvation and UV irradiation. HGF-induced survival correlates with Akt activity and is inhibited by the specific PI3-kinase inhibitor LY294002, indicating that HGF inhibits cell death through the PI3-kinase/Akt signal transduction pathway. Furthermore, transiently transfected Tpr-Met activates Akt (both Akt1 and Akt2) and protects cells from apoptosis. Mitogen-activated protein kinase (MAPK) also is activated by HGF and rescues cells from apoptosis, although the cytoprotective effect is less marked than for PI3-kinase/Akt. Blocking MAPK with the specific MAPK kinase inhibitor PD098059 impairs the ability of HGF to promote cell survival. Similar results were obtained with NIH 3T3 cells expressing the fusion protein Trk-Met and stimulated with nerve growth factor, the Trk ligand. These results demonstrate that HGF/Met is capable of protecting cells from apoptosis by using both PI3-kinase/Akt and, to a lesser extent, MAPK pathways.     

Nucleophosmin-anaplastic lymphoma kinase associated with anaplastic large-cell lymphoma activates the phosphatidylinositol 3-kinase/Akt antiapoptotic signaling pathway

More than half of anaplastic large-cell lymphomas (ALCLs) have a chromosomal translocation t(2;5) that leads to the expression of a hybrid protein composed of the nucleolar phosphoprotein nucleophosmin (NPM) and the anaplastic lymphoma kinase (ALK) that exhibits an unregulated tyrosine kinase activity. We have previously identified PLC-gamma as a crucial downstream signaling molecule of NPM-ALK that contributes to its mitogenic potential. Here, we show that NPM-ALK recruits the C-terminal SH2 domain of the phosphatidylinositol 3-kinase (PI 3kinase) p85 subunit. PI 3-kinase assays revealed that the kinase is activated by NPM-ALK in vivo, in turn activating PKB/Akt in NPM-ALK-expressing cells. The use of 2 specific PI 3-kinase inhibitors, wortmannin and LY294002, demonstrated the requirement of PI 3-kinase for the growth of NPM-ALK-transformed cell lines, as well as a cell line established from a patient with ALCL. Primary murine bone marrow retrovirally transduced with NPM-ALK showed a transformed phenotype that was reversible on treatment with PI 3-kinase inhibitors. Flow cytometric analysis revealed that wortmannin-treated NPM-ALK-transformed cell lines underwent apoptosis. Furthermore, apoptosis induced by overexpression of the proapoptotic molecule Bad could be partially blocked by the overexpression of NPM-ALK. Thus, NPM-ALK activates the antiapoptotic PI 3-kinase/Akt pathway, which likely contributes to the molecular pathogenesis of ALCL. (Blood. 2000;96:4319-4327)     

A phosphatidylinositol 3-kinase/Akt/mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1

Tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) by the insulin receptor permits this docking protein to interact with signaling proteins that promote insulin action. Serine phosphorylation uncouples IRS-1 from the insulin receptor, thereby inhibiting its tyrosine phosphorylation and insulin signaling. For this reason, there is great interest in identifying serine/threonine kinases for which IRS-1 is a substrate. Tumor necrosis factor (TNF) inhibited insulin-promoted tyrosine phosphorylation of IRS-1 and activated the Akt/protein kinase B serine-threonine kinase, a downstream target for phosphatidylinositol 3-kinase (PI 3-kinase). The effect of TNF on insulin-promoted tyrosine phosphorylation of IRS-1 was blocked by inhibition of PI 3-kinase and the PTEN tumor suppressor, which dephosphorylates the lipids that mediate PI 3-kinase functions, whereas constitutively active Akt impaired insulin-promoted IRS-1 tyrosine phosphorylation. Conversely, TNF inhibition of IRS-1 tyrosine phosphorylation was blocked by kinase dead Akt. Inhibition of IRS-1 tyrosine phosphorylation by TNF was blocked by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a downstream target of Akt. mTOR induced the serine phosphorylation of IRS-1 (Ser-636/639), and such phosphorylation was inhibited by rapamycin. These results suggest that TNF impairs insulin signaling through IRS-1 by activation of a PI 3-kinase/Akt/mTOR pathway, which is antagonized by PTEN.     

Early involvement of the phosphatidylinositol 3-kinase/Akt pathway in lung cancer progression

Signaling through the phosphatidylinositol 3-kinase (PI3-kinase) pathway has been associated with lung tumorigenesis. We examined the association between gene copy number of the PI3-kinase catalytic subunit alpha (PIK3CA) and phosphorylated Akt expression in invasive and preinvasive lung cancers. We sought to determine at what stage of tumor development gene copy number increase or phosphorylated Akt overexpression might affect tumor development. We assessed PIK3CA gene copy number by fluorescence in situ hybridization and expression of phosphorylated Akt by immunohistochemistry in 242 invasive and 43 preinvasive lung cancers and correlated our findings with clinical outcome. The PIK3CA was amplified in 70% of squamous carcinomas, 38% of large cell carcinomas, 19% of adenocarcinomas, and 67% of small cell lung cancers. Phosphorylated Akt overexpression was frequently observed, and strongly so in 12 to 17% of lung cancers depending on nuclear or cytoplasmic localization. Neither PIK3CA gene copy number nor phosphorylated Akt protein expression had prognostic significance. In preinvasive lesions, amplification of the PIK3CA and overexpression of phosphorylated Akt were associated with severe dysplasia and each other. These observations suggest frequent and early involvement of the PI3-kinase pathway in lung cancer.     

Angiopoietin 1 directly induces destruction of the rheumatoid joint by cooperative, but independent, signaling via ERK/MAPK and phosphatidylinositol 3-kinase/Akt

OBJECTIVE: To determine whether angiopoietin 1 (Ang-1) potentiates overgrowth of the synovium and joint degradation in rheumatoid arthritis (RA), and to clarify the cell-signaling mechanisms of Ang-1 in the rheumatoid joint. METHODS: Expression of Ang-1, TIE-2 (a receptor for Ang-1), and matrix metalloproteinase 3 (MMP-3) was studied by immunohistochemistry. Activation of the ERK/MAPK and phosphatidylinositol (PI) 3-kinase/Akt pathways and of NF-kappaB was determined by Western blotting and an NF-kappaB p65 DNA binding activity assay, respectively. Induction of apoptosis was evaluated by nuclear staining, cell viability assay, and Western blotting of caspases. Synovial cell migration was evaluated by actin polymerization, Western blotting of Rho family proteins, and affinity purification with Rhotekin-Rho and p21-activated kinase 1. Matrix degradation was examined by induction of proMMP-3 secretion from synovial cells followed by in vitro cartilaginous matrix degradation assay. RESULTS: Ang-1 stimulated the ERK/MAPK and PI 3-kinase/Akt pathways in a cooperative but independent manner, which enhanced rheumatoid synovium overgrowth and joint destruction. In addition, Ang-1 activated NF-kappaB via Akt to promote cell growth, but also inhibited cell apoptosis via ERK and Akt. Ang-1 directly potentiated the extension of synovial cells in an ERK- and Akt-dependent manner by up-regulating Rho family proteins, which attenuated Rac signaling and led to membrane ruffling. Ang-1 induced proMMP-3 secretion from synovial cells, which resulted in direct degradation of the cartilaginous matrix. CONCLUSION: Ang-1 stimulates the ERK/MAPK and PI 3-kinase/Akt pathways cooperatively, but in a manner independent of each other, to directly potentiate synovium overgrowth and joint destruction in RA. In addition to inflammatory cytokines, Ang-1/TIE-2 signaling appears to be an independent factor that contributes to the destruction of the rheumatoid joint.     

Modulation of caveolin-1 expression can affect signalling through the phosphatidylinositol 3-kinase/Akt pathway and cellular proliferation in response to insulin-like growth factor I

The IGFs mediate their effects on cell function through the type I IGF receptor and numerous intracellular signalling molecules, including the phosphatidylinositol 3-kinase (PI-3K)/Akt pathway. The type I IGF receptor also binds to the caveolae protein caveolin-1, but the impact of caveolae on IGF/PI-3K/Akt signalling remains controversial. We have examined the effect of complete (knockout) and partial (knockdown) caveolin-1 deficiency on cellular IGF effects mediated via the PI-3K/Akt pathway. Under basal conditions, caveolin-1-deficient mouse embryonic fibroblast cells [MF(-/-)] incorporated significantly more [3H]thymidine than wild-type mouse embryonic fibroblast cells [MF(+/+)]; however, small hairpin RNA-mediated knockdown of caveolin-1 (80% reduction) in 3T3L1 fibroblasts had no effect on basal proliferation. Interestingly, IGF-I induced proliferation was similar in MF(-/-) and MF(+/+) cells, whereas caveolin-1 knockdown promoted a hyperproliferative response to IGF-I [pkDCav3T3L1(80) 12.4+/-0.4-fold; pkDShuffle3T3L1 4.3+/-0.2-fold induction; P<0.01]. Immunoblot analysis showed that caveolin-1 knockdown had no affect on Akt expression or activation. However, in MF(-/-) cells, IGF-I-stimulated phosphorylation of Akt was reduced despite up-regulated Akt levels. Further investigation demonstrated that caveolin knockout up-regulated Akt-2 and Akt-3 isoform expression, but Akt-1 expression was down-regulated; interestingly, coimmunoprecipitation studies revealed Akt-1 as the predominant isoform to be phosphorylated in response to IGF-I. In summary, caveolin-1 deficiency promotes a hyperproliferative response to IGF-I that is unrelated to Akt expression/activation. However, cells that lack caveolin are able to respond appropriately to IGF-I through compensatory changes in Akt isoform expression. These data posit caveolin-1 as a component of the IGF/PI-3K/Akt signalling modulus regulating cellular proliferation with implications for diseases, including cancers, which have altered caveolin expression.     

Embryonic mesoderm cells spread in response to platelet-derived growth factor and signaling by phosphatidylinositol 3-kinase.

Abnormal mesoderm movement, leading to defects in axial organization, is observed in mouse and Xenopus laevis embryos deprived of platelet-derived growth factor (PDGF) AA signaling. However, neither the cellular response to PDGF nor the signaling pathways involved are understood. Herein we describe an in vitro assay to examine the direct effect of PDGF AA on aggregates of Xenopus embryonic mesoderm cells. We find that PDGF AA stimulates aggregates to spread on fibronectin. This behavior is similar to that of migrating mesoderm cells in vivo that spread and form lamellipodia and filipodia on contact with fibronectin-rich extracellular matrix. We go on to show two lines of evidence that implicate phosphatidylinositol 3-kinase (PI3K) as an important component of PDGF-induced mesoderm cell spreading. (i) The fungal metabolite wortmannin, which inhibits signaling by PI3K, blocks mesoderm spreading in response to PDGF AA. (ii) Activation of a series of receptors with specific tyrosine-to-phenylalanine mutations revealed PDGF-induced spreading of mesoderm cells depends on PI3K but not on other signaling molecules that interact with PDGF receptors including phospholipase C gamma, Ras GTPase-activating protein, and phosphotyrosine phosphatase SHPTP2. These results indicate that a PDGF signal, medicated by PI3K, can facilitate embryonic mesoderm cell spreading on fibronectin. We propose that PDGF, produced by the ectoderm, influences the adhesive properties of the adjacent mesoderm cells during gastrulation.     

Insulin down-regulates insulin receptor substrate-2 expression through the phosphatidylinositol 3-kinase/Akt pathway

Insulin receptor substrate (IRS)-1 and IRS-2 are the major substrates that mediate insulin action. Insulin itself regulates the expression of the IRS protein in the liver, but the underlying mechanisms of IRS-1 and IRS-2 regulation are not fully understood. Here we report that insulin suppressed the expression of both IRS-1 and IRS-2 proteins in Fao hepatoma cells. The decrease in IRS-1 protein occurred via proteasomal degradation without any change in IRS-1 mRNA, whereas the insulin-induced suppression of IRS-2 protein was associated with a parallel decrease in IRS-2 mRNA without changing IRS-2 mRNA half-life. The insulin-induced suppression of IRS-2 mRNA and protein was blocked by the phosphatidylinositol (PI) 3-kinase inhibitor, LY294002, but not by the MAP kinase-ERK kinase (MEK) inhibitor, PD098059. Inhibition of Akt by overexpression of dominant-negative Akt also caused complete attenuation of the insulin-induced decrease in IRS-2 protein and partial attenuation of its mRNA down-regulation. Some nuclear proteins bound to the insulin response element (IRE) sequence on the IRS-2 gene in an insulin-dependent manner in vitro, and the binding was also blocked by the PI 3-kinase inhibitor. Reporter gene assay showed that insulin suppressed the activity of both human and rat IRS-2 gene promoters through the IRE in a PI 3-kinase-dependent manner. Our results indicate that insulin regulates IRS-1 and IRS-2 through different mechanisms and that insulin represses IRS-2 gene expression via a PI 3-kinase/Akt pathway.     

Aging is associated with decreased pancreatic acinar cell regeneration and phosphatidylinositol 3-kinase/Akt activation

BACKGROUND & AIMS: The effects of aging on pancreatic acinar cell proliferation have not been clearly defined. Phosphatidylinositol 3-kinase (PI3K)-mediated phosphorylation of Akt is a critical step for proliferation of various cell types and insulin secretion from pancreatic endocrine cells; however, its role in acinar cell proliferation is not known. The purpose of this study was to (1) delineate the effects of aging on pancreatic regeneration after partial pancreatectomy (Px) and (2) define the involvement of the PI3K/Akt pathway in pancreatic regeneration. METHODS: Following partial Px, pancreatic regeneration and activation of the PI3K pathway were compared in young and aged mice. Activation of the PI3K/Akt pathway was evaluated by Akt phosphorylation (pAkt). The role of the PI3K pathway in pancreatic regeneration after partial Px was assessed by effects of a pharmacologic PI3K inhibitor wortmannin or small interfering RNA (siRNA) to the p85alpha regulatory subunit. To confirm further the critical role of the PI3K/Akt pathway in pancreatic acinar cell proliferation, IGF-1-mediated cell proliferation was determined in cultured acinar cells pretreated with wortmannin or p85alpha siRNA. RESULTS: Pancreatic regeneration and pAkt expression after partial Px were significantly decreased with aging. Treatment with wortmannin or p85alpha siRNA reduced pancreatic regeneration after partial Px. The IGF-1-mediated cell proliferation in vitro was completely blocked by wortmannin or p85alpha siRNA but not by the MEK/ERK inhibitor PD98059. CONCLUSIONS: PI3K/Akt activation plays a critical role in the regeneration of pancreatic acini after resection. Furthermore, pancreatic regeneration is markedly attenuated in the aged pancreas most likely because of decreased PI3K/Akt activation.     

Trypanosoma cruzi trans-sialidase: a potent and specific survival factor for human Schwann cells by means of phosphatidylinositol 3-kinase/Akt signaling

Patients infected with Trypanosoma cruzi may remain asymptomatic for decades and show signs of neuroregeneration in the peripheral nervous system (PNS). In the absence of such neuroregeneration, patients may die in part by extensive neuronal destruction in the gastrointestinal tract. Thus, T. cruzi may, despite their invasion of the PNS, directly prevent cell death to keep nerve destruction in check. Indeed, T. cruzi invasion of Schwann cells, their prime target in PNS, suppressed host-cell apoptosis caused by growth-factor deprivation. The trans-sialidase (TS) of T. cruzi and the Cys-rich domain of TS reproduced the antiapoptotic activity of the parasites at doses (> or =3.0 nM) comparable or lower than those of bona fide mammalian growth factors. This effect was blocked by LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K). TS also activated Akt, a downstream effector of PI3K. Ectopic expression of TS in an unrelated parasite, Leishmania major, turned those parasites into activators of Akt in Schwann cells. In contrast, the Cys-rich domain of TS did not block apoptosis in Schwann cells overexpressing dominant-negative Akt or constitutively active PTEN, a negative regulator of PI3K/Akt signaling. The results demonstrate that T. cruzi, through its TS, triggers the survival of host Schwann cells via the PI3K/Akt pathway, suggesting a role for PI3K/Akt in the pathogenesis of Chagas' disease.     

Febrile temperatures control antineutrophil cytoplasmic autoantibody-induced neutrophil activation via inhibition of phosphatidylinositol 3-kinase/Akt

OBJECTIVE: Neutrophil activation by antineutrophil cytoplasmic autoantibodies (ANCAs) is central to the pathogenesis of the ANCA-associated vasculitides. Febrile infections occur frequently during these diseases, often in the context of immunosuppressive treatment. Heat exposure may affect the underlying pathophysiologic processes of the vasculitis. In this study we tested the hypothesis that short-term exposure to heat inhibits ANCA-induced neutrophil activation. METHODS: After exposure to temperatures from 37 degrees C to 42 degrees C, human neutrophils were primed with either tumor necrosis factor alpha (TNFalpha) or granulocyte-macrophage colony-stimulating factor (GM-CSF) and stimulated with monoclonal antibodies to myeloperoxidase or to proteinase 3. Respiratory burst activity was assayed using rhodamine and a nitroblue tetrazolium reduction assay. Specific inhibition experiments against p38 MAPK, ERK, and phosphatidylinositol 3-kinase (PI 3-kinase)/Akt, and Western blotting with phospho-specific antibodies were used to identify key components in the antibody-induced respiratory burst. RESULTS: A temperature-dependent reduction in ANCA-induced respiratory burst was observed over a range of heat exposures from 37 degrees C to 42 degrees C. Inhibition of human ANCA-induced neutrophil stimulation was significant at 40 degrees C (after priming with 2 ng/ml TNFalpha, mean [+/- SEM] fluorescence intensity [MFI] 114 +/- 12 at 37 degrees C versus 53 +/- 6 at 40 degrees C; after priming with 20 ng/ml GM-CSF, MFI 92 +/- 16 at 37 degrees C versus 35 +/- 6 at 40 degrees C; both P < 0.01). In the priming phase, the transient activation of the p38 MAPK, ERK, and PI 3-kinase/Akt pathways by TNFalpha was blocked by prior exposure of the neutrophils to heat, but GM-CSF-induced activation was unaltered by heat. However, in the second, antibody-induced wave of kinase activation, exposure to heat inhibited only the PI 3-kinase/Akt pathway, and these effects were independent of the priming agent used. CONCLUSION: Short-term spikes of modest heat abrogate ANCA-induced activation of neutrophils via inhibition of PI 3-kinase/Akt signaling. Febrile responses in ANCA-mediated diseases may therefore have a physiologic purpose.     

Axl/phosphatidylinositol 3-kinase signaling inhibits mineral deposition by vascular smooth muscle cells

The calcification of blood vessels correlates with increased morbidity and mortality in patients with atherosclerosis, diabetes, and end-stage kidney disease. The receptor tyrosine kinase Axl is emerging as an important regulator of adult mammalian physiology and pathology. This study tests the hypothesis that Axl prevents the deposition of a calcified matrix by vascular smooth muscle cells (VSMCs) and that this occurs via the phosphatidylinositol 3-kinase (PI3K) signaling pathway. First, we demonstrate that Axl is expressed and phosphorylated in confluent VSMCs and that its expression is markedly downregulated as these cells calcify their matrix. Second, we demonstrate that overexpression of wild-type Axl, using recombinant adenoviruses, enhances Axl phosphorylation and downstream signaling via PI3K and Akt. Furthermore, overexpression of Axl significantly inhibits mineral deposition by VSMCs, as assessed by alizarin red staining and (45)Ca accumulation. Third, the addition of a PI3K inhibitor, wortmannin, negates the inhibition of mineralization by overexpression of wild-type Axl, suggesting that activation of downstream signaling via PI3K is crucial for its inhibitory activity. In contrast, Axl-mediated signaling is not enhanced by overexpression of kinase-dead Axl and mineralization is accelerated, although beta-glycerophosphate is still required for this effect. Finally, the caspase inhibitor zVAD.fmk attenuates the increased mineralization induced by kinase-dead Axl, suggesting that kinase-dead Axl stimulates mineralization by inhibiting the antiapoptotic effect of endogenous Axl. Together, these results demonstrate that signaling through Axl inhibits vascular calcification in vitro and suggest that therapeutics targeting this receptor may open up new avenues for the prevention of vascular calcification in vivo.     

Regulation of G1 progression by the PTEN tumor suppressor protein is linked to inhibition of the phosphatidylinositol 3-kinase/Akt pathway

PTEN/MMAC1 is a tumor suppressor gene located on chromosome 10q23. Inherited PTEN/MMAC1 mutations are associated with a cancer predisposition syndrome known as Cowden's disease. Somatic mutation of PTEN has been found in a number of malignancies, including glioblastoma, melanoma, and carcinoma of the prostate and endometrium. The protein product (PTEN) encodes a dual-specificity protein phosphatase and in addition can dephosphorylate certain lipid substrates. Herein, we show that PTEN protein induces a G1 block when reconstituted in PTEN-null cells. A PTEN mutant associated with Cowden's disease (PTEN;G129E) has protein phosphatase activity yet is defective in dephosphorylating inositol 1,3,4,5-tetrakisphosphate in vitro and fails to arrest cells in G1. These data suggest a link between induction of a cell-cycle block by PTEN and its ability to dephosphorylate, in vivo, phosphatidylinositol 3,4,5-trisphosphate. In keeping with this notion, PTEN can inhibit the phosphatidylinositol 3,4, 5-trisphosphate-dependent Akt kinase, a downstream target of phosphatidylinositol 3-kinase, and constitutively active, but not wild-type, Akt overrides a PTEN G1 arrest. Finally, tumor cells lacking PTEN contain high levels of activated Akt, suggesting that PTEN is necessary for the appropriate regulation of the phosphatidylinositol 3-kinase/Akt pathway.     

The preventive effect of uncarboxylated osteocalcin against free fatty acid-induced endothelial apoptosis through the activation of phosphatidylinositol 3-kinase/Akt signaling pathway

Objective

Increasing evidence suggests that osteocalcin (OC), one of the osteoblast-specific proteins, has been associated with atherosclerosis, but results are conflicting. The aim of this study was to elucidate the independent effect of uncarboxylated osteocalcin (ucOC), an active form of osteocalcin which has been suggested to have an insulin sensitizing effect, on vascular endothelial cells.

Materials and Methods

We used human aortic endothelial cells and treated them with ucOC. Linoleic acid (LA) was used as a representative free fatty acid. Apoptosis was evaluated using various methods including a terminal deoxyribonucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling analysis kit and Western blotting for cleaved caspase 3, cleaved poly (ADP-ribose) polymerase and Bcl-xL. The phosphorylations of Akt and endothelial nitric oxide synthase (eNOS) as well as the level of NO were measured to confirm the effect of ucOC on insulin signaling pathway.

Results

Pretreatment of ucOC (30 ng/ml) prevented LA-induced apoptosis in insulin-stimulated endothelial cells; effects were abolished by pretreatment with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, wortmannin. Treatment of ucOC (ranged from 0.3 to 30 ng/ml) significantly increased the phosphorylation of Akt and eNOS and nitric oxide secretion from endothelial cells in a PI3-kinase dependent manner.

Conclusions

Our study is the first to demonstrate the independent effect of ucOC on vascular endothelial cells. Our results further suggest that ucOC could have beneficial effects on atherosclerosis.     

Cyclin G1-mediated epithelial-mesenchymal transition via phosphoinositide 3-kinase/Akt signaling facilitates liver cancer progression

Cyclin G1 deficiency is associated with reduced incidence of carcinogen-induced hepatocellular carcinoma (HCC), but its function in HCC progression remains obscure. We report a critical role of cyclin G1 in HCC metastasis. Elevated expression of cyclin G1 was detected in HCCs (60.6%), and its expression levels were even higher in portal vein tumor thrombus. Clinicopathological analysis revealed a close correlation of cyclin G1 expression with distant metastasis and poor prognosis of HCC. Forced expression of cyclin G1 promoted epithelial-mesenchymal transition (EMT) and metastasis of HCC cells in vitro and in vivo. Cyclin G1 overexpression enhanced Akt activation through interaction with p85 (regulatory subunit of phosphoinositide 3-kinase [PI3K]), which led to subsequent phosphorylation of glycogen synthase kinase-3β (GSK-3β) and stabilization of Snail, a critical EMT mediator. These results suggest that elevated cyclin G1 facilitates HCC metastasis by promoting EMT via PI3K/Akt/GSK-3β/Snail-dependent pathway. Consistently, we have observed a significant correlation between cyclin G1 expression and p-Akt levels in a cohort of HCC patients, and found that combination of these two parameters is a more powerful predictor of poor prognosis. CONCLUSIONS: Cyclin G1 plays a pivotal role in HCC metastasis and may serve as a novel prognostic biomarker and therapeutic target.