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PubMed2005

Phosphatidylinositol 3-kinase/Akt pathway mediates heme oxygenase-1 regulation by lipopolysaccharide.

The stress-inducible protein heme oxygenase-1 exerts potent antiinflammatory, antiapoptotic and cytoprotective effects in vitro and in vivo. Another important mediator of cytoprotection, the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway activates many proteins involved in the maintenance of cellular homeostasis. Since activation of heme oxygenase-1 and PI3K/Akt both protect the cellular environment, we postulated that PI3K/Akt can regulate the induction of heme oxygenase-1 by proinflammatory stress. The treatment of primary murine macrophage cells (RAW 264.7) with lipopolysaccharide induced heme oxygenase-1 protein and mRNA expression, and increased the phosphorylation of Akt and p38 mitogen activated protein kinase (p38 MAPK). These cellular effects of lipopolysaccharide were markedly diminished by pre-treatment with wortmannin, a specific inhibitor of PI3K. Furthermore, lipopolysaccharide-inducible heme oxygenase expression was blocked by SB203580, a specific inhibitor of p38 MAPK. Both wortmannin and SB203580 decreased lipopolysaccharide-inducible NF-E2-related factor (Nrf2) DNA binding activity. Transfection of macrophages with dominant negative mutants of PI3K, Akt and Nrf2, as well as wortmannin treatment, significantly reduced the transcriptional activity of a minimal heme oxygenase-1 promoter luciferase construct (D33HO-1luc). We demonstrate, to our knowledge for the first time, that upon proinflammatory stimulation heme oxygenase-1 gene expression in macrophages depends on PI3K/Akt and p38 MAPK acting upstream of Nrf2-dependent promoter activation.

Animals↗
PubMed2026

Network pharmacology-based prediction and experimental validation of the anti-hyperuricemic effects of oolong tea polyphenols.

OBJECTIVE: This study aimed to identify candidate therapeutic targets of oolong tea polyphenols (TP) against hyperuricemia (HUA) using network pharmacology and bioinformatics, and to validate the predicted molecular mechanism through in vivo experimentation. METHODS: Drug and disease targets were retrieved from public databases, and overlapping targets were identified by Venn diagram analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on the shared targets, and a protein-protein interaction (PPI) network was constructed to identify hub genes. For in vivo validation, an HUA mouse model was established by 15 days of oral potassium oxonate (PO) administration. Model mice then received TP by gavage at low (0.5 g⋅kg-1⋅d-1), medium (1 g⋅kg-1⋅d-1), or high (2 g⋅kg-1⋅d-1) doses for an additional 15 days. Serum biochemical markers, histopathological changes, and pathway-related protein expression were assessed by enzyme-linked immunosorbent assay (ELISA), hematoxylin and eosin (HE) staining, and western blot analysis, respectively. RESULTS: Network pharmacology analysis identified 59 overlapping targets between TP and HUA; GO and KEGG enrichment analyses revealed that these targets were primarily associated with hormone metabolism and the PI3K-AKT signaling pathway. In the animal experiment, TP dose-dependently reduced serum uric acid (SUA) levels in hyperuricemic mice. At the molecular level, low and medium doses of TP suppressed phosphorylation of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR), whereas the high dose paradoxically activated this pathway and concomitantly elevated interleukin-1β levels. These findings indicate that TP modulates uric acid metabolism through a non-monotonic, dose-dependent mechanism. CONCLUSION: By combining network pharmacology with animal experiments, this study identified the PI3K/AKT/mTOR signaling pathway as a likely mediator of the anti-hyperuricemic action of oolong tea polyphenols (TP). A medium dose of TP achieved the most balanced outcome, attenuating inflammation and preserving hepatic and renal architecture; the high dose, by contrast, paradoxically elevated interleukin-1β (IL-1β) and overactivated PI3K/AKT/mTOR signaling, underscoring the importance of dose calibration. These data suggest that a medium dose of TP may represent a feasible dietary strategy against hyperuricemia. Further work-including monomer identification, direct target validation, and clinical evaluation-is warranted to confirm and extend these preclinical findings.

PI3K/Akt/mTOR signaling pathway↗
PubMed2026

Puerarin Attenuates Binge Ethanol-Induced Cortical Neurotoxicity in Association with AKT/mTOR Signaling and Autophagy-Related Responses.

Puerarin (Pue), a major isoflavone derived from Pueraria lobata, has demonstrated neuroprotective potential in multiple neurological disorders; however, its effects on ethanol (EtOH)-induced cortical injury and the associated molecular responses remain incompletely understood. In the present study, network pharmacology was combined with in vivo and in vitro experiments to investigate molecular responses associated with the effects of Pue on EtOH-induced neurotoxicity. Public databases were used to predict targets of Pue and alcohol-related brain injury, followed by protein-protein interaction analysis, Gene Ontology annotation, and Kyoto Encyclopedia of Genes and Genomes pathway enrichment. A total of 101 overlapping targets were identified, among which TNF, AKT1, EGFR, TP53, and PPARG emerged as major hub targets, and PI3K-Akt signaling pathway was among the pathways that remained significantly enriched after FDR correction. In a 4-day binge EtOH rat model, Pue attenuated EtOH-associated increases in oxidative stress, neuronal degeneration, and apoptotic markers in cortical tissue. This was accompanied by attenuation of the EtOH-associated reductions in the p-AKT/AKT and p-mTOR/mTOR ratios, as well as an attenuation of EtOH-associated changes in LC3, ATG5, and Beclin-1 expression. In primary cortical neurons, Pue partially attenuated the EtOH-associated loss of neuronal viability and preserved neurite morphology. Bafilomycin A1 (BafA1)-based analysis of LC3-II and p62/SQSTM1 showed an overall BafA1-sensitive increase in LC3-II without a significant treatment-dependent difference in the BafA1 response. Collectively, these findings suggest that Pue attenuates binge EtOH-induced cortical neurotoxicity in association with changes in AKT/mTOR phosphorylation and autophagy-related responses.

AKT/mTOR signaling↗
PubMed2003

Tellurium compound AS101 induces PC12 differentiation and rescues the neurons from apoptotic death.

Parkinson's disease is characterized by the loss of dopaminergic neurons in the substantia nigra (SN). Studies show that anti-apoptotic and neurotrophic agents are suitable candidates to prevent delayed cell death and/or restore neural function. Here we present the nontoxic immunomodulating compound AS101, which has the ability to induce neurite outgrowth and neural differentiation in PC12 cells. The present study shows that components of the ras signaling pathway are crucial for AS101-induced PC12 differentiation. These include p21ras and its downstream effectors, c-raf-1 and MEK, as well as PI3K. Moreover, these components mediate AS101-induced upregulation of p21waf, which is obligatory for AS101-induced PC12 differentiation. Furthermore, nitric oxide plays a significant role in these AS101 activities. Finally, we show that AS101 prevents apoptosis of NGF-differentiated PC12 cells after NGF withdrawal. Taken together, these results suggest that AS101 induces PC12 cell differentiation and survival by activating the ras-ERK1/2 and ras-PI3K signal transduction pathways, as well as inducing NO production. Our findings may be important in understanding the regulation of survival/apoptosis of neurons deprived of neurotropic support. Futhermore the data propose that AS101 may have clinical potential in the treatment of neurodegenerative disorders like Parkinson's disease.

Animals↗
PubMed2005

Induction of KLF2 by fluid shear stress requires a novel promoter element activated by a phosphatidylinositol 3-kinase-dependent chromatin-remodeling pathway.

Fluid shear stress maintains vascular homeostasis by influencing endothelial gene expression. One mechanism by which shear stress achieves this is through the induction of transcription factors including Krüppel-like factor 2(KLF2). We have previously reported that a 62-bp region of the KLF2 promoter is responsible for its shear stress-induced expression via the binding of nuclear factors. In this study, we find that the 62-bp shear stress response region contains a 30-bp tripartite palindrome motif. Electrophoretic mobility supershift and chromatin immunoprecipitation assays demonstrate that PCAF (P-300/cAMP-response element-binding protein-binding protein-associated factor)) and heterogeneous nuclear ribonucleoprotein D bind this region as components of the shear stress regulatory complex. We have also characterized a PI3K-dependent/Akt-independent pathway responsible for shear stress-induced KLF2 nuclear binding, promoter activation, and mRNA expression. Furthermore, the shear stress response region of the KLF2 promoter was specifically immunoprecipitated by antibodies against acetylated histones H3 and H4 in shear-stressed but not static hemangioendothelioma cells. The acetylation of these histones was blocked by PI3K inhibition. Finally, we have found that KLF2 increases endothelial nitric-oxide synthase expression in murine endothelial cultures, an effect that is also blocked by PI3K inhibition. These results define the DNA regulatory element, signal transduction pathway, and molecular mechanism activating the flow-dependent expression of a vital endothelial transcription factor.

Acetyltransferases↗
PubMed2026

Transcriptomic characterization of the intestine in Stichopus monotuberculatus under gradient temperature stress and HSP gene family-mediated molecular adaptation.

The increasing frequency of extreme temperature events under climate change poses a growing threat to the stability of tropical sea cucumber aquaculture. To characterize the molecular responses of the tropical sea cucumber Stichopus monotuberculatus to acute temperature stress, juveniles were exposed for 96 h to 15 °C, 20 °C, 25 °C, 30 °C, and 35 °C, followed by transcriptomic profiling of the intestine. By transcriptomic analysis, 2258, 634, 1618, and 2980 differentially expressed genes (DEGs) were identified at 15, 20, 30, and 35 °C compared to control, respectively. More DEGs were generally detected at temperatures further from 25 °C, with the 35 °C group showing the largest transcriptional response. Although cold and heat stress both affected metabolism and protein homeostasis, their enrichment profiles differed. At 15 °C, DEGs were mainly enriched in the spliceosome and p53 signaling pathways, highlighting RNA processing and p53 signaling as prominent features of the cold-stress response. At 35 °C, DEGs were mainly enriched in the PI3K-Akt signaling pathway, ubiquitin-mediated proteolysis, and mitophagy, indicating enhanced regulation of cell survival, protein turnover, and mitochondrial quality control. HSP genes also responded differently to cold and heat stress. Most HSP70 and HSP90 family members were downregulated at low temperatures, whereas HSP70 genes and small heat shock proteins were markedly upregulated at high temperatures. Overall, the intestinal transcriptome showed distinct responses to cold and heat stress. These results identify pathways and HSP genes potentially involved in the temperature response of S. monotuberculatus and provide useful information for evaluating temperature tolerance and defining suitable temperatures for its aquaculture.

Heat shock protein↗
PubMed2002

BDNF-mediated signal transduction is modulated by GSK3beta and mood stabilizing agents.

Brain-derived neurotrophic factor (BDNF) is a major neurotrophin in the brain and abnormal regulation of BDNF may contribute to the pathophysiology of mood disorders. In the present study, we examined if alterations in the activity of glycogen synthase kinase-3-beta (GSK3beta) or treatment with mood stabilizers modulated BDNF-mediated signal transduction pathways in differentiated human neuroblastoma SH-SY5Y cells. BDNF increased the phosphorylation of the forkhead transcription factor FKHRL1 through activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, and the phosphorylation of the cyclic AMP response element binding protein (CREB) through activation of extracellular signal-regulated kinase1/2 (ERK1/2). BDNF also increased serine(9) -phosphorylation of GSK3beta, which inhibits GSK3beta activity. Overexpression of GSK3beta did not affect BDNF-induced phosphorylation of Akt, ERK1/2, or FKHRL1, but abolished CREB phosphorylation induced by BDNF. This inhibition of BDNF-induced CREB phosphorylation in GSK3beta-overexpressing SH-SY5Y cells was blocked by treatment with lithium. In contrast to lithium, sodium valproate and lamotrigine did not affect BDNF-mediated signaling, whereas carbamazepine induced a rapid and prolonged phosphorylation of ERK1/2 and CREB in the absence or the presence of BDNF. Therefore, increased GSK3beta selectively attenuates BDNF-induced CREB phosphorylation, and lithium and carbamazepine can facilitate activation of CREB.

Anticonvulsants↗
PubMed2005

A role for phosphatidylinositol 3-kinase in TCR-stimulated ERK activation leading to paxillin phosphorylation and CTL degranulation.

PI3K is an important regulator of a number of cellular processes. We examined the contribution of PI3K to mouse CTL signaling, leading to degranulation. We show that TCR-triggered, but not phorbol ester and calcium ionophore-induced, CTL degranulation is dependent on PI3K activity. Although PI3K activity is required for optimal LFA-1-mediated adhesion and cell spreading, this most likely does not account for its full contribution to degranulation. We demonstrate that PI3K is required for TCR-stimulated ERK activation in CTL, which we have shown previously to be required for CTL degranulation. We thus define a pathway through which PI3K most likely regulates degranulation and in which ERK appears to be a key signaling molecule. Furthermore, we identified the cytoskeletal adaptor paxillin as a target of ERK downstream of TCR stimulation. Consistent with a role in degranulation, we demonstrate that paxillin is localized to the microtubule organizing center in resting cells and upon target cell binding is recruited to the contact point with the target cell. These studies demonstrate that PI3K regulates ERK activity leading to CTL degranulation, and identify paxillin as a target of ERK downstream of the TCR. That paxillin is independently phosphorylated by both tyrosine kinase(s) and ERK downstream of the TCR and localized both at the microtubule organizing center and at the target cell contact point suggests an important role for paxillin in CTL-mediated killing.

Animals↗
PubMed2004

Inhibition of neuronal phenotype by PTEN in PC12 cells.

The mechanisms of neuronal differentiation in PC12 cells are still not completely understood. Here, we report that the tumor suppressor PTEN has a profound effect on differentiation by affecting several pathways involved in nerve growth factor (NGF) signaling. When overexpressed in PC12 cells, PTEN (phosphatase and tensin homologue deleted on chromosome ten) blocked neurite outgrowth induced by NGF. In addition, these cells failed to demonstrate the transient mitogenic response to NGF, as well as subsequent growth arrest. Consistent with these observations was a finding that PTEN significantly inhibits NGF-mediated activation of the members of mitogen-activated protein kinase kinase (MEK)/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT signaling pathways, crucial for these processes. While exploring possible mechanisms of PTEN effects on NGF signaling, we discovered a significant down-regulation of both high-affinity (TrkA) and low-affinity (p75) NGF receptors in PTEN-overexpressing clones. Subsequent microarray analysis of several independent clonal isolates revealed a myriad of neuronal genes to be affected by PTEN. All of these changes were validated by quantitative PCR. Of particular interest were the genes for the key enzymes of the dopamine synthesis pathway, receptors for different neurotransmitters, and neuron-specific cytoskeleton proteins, among others. Some, but not all effects could be reproduced by pharmacological inhibitors of PI3K and/or MAPK, suggesting that PTEN may influence some genes by mechanisms independent of these signaling pathways. Our findings may shed new light on the role of this tumor suppressor during normal brain development and suggest a previously uncharacterized mechanism of PTEN action in neuron-like cells.

Animals↗
PubMed2000

5' phospholipid phosphatase SHIP-2 causes protein kinase B inactivation and cell cycle arrest in glioblastoma cells.

The tumor suppressor protein PTEN is mutated in glioblastoma multiform brain tumors, resulting in deregulated signaling through the phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB) pathway, which is critical for maintaining proliferation and survival. We have examined the relative roles of the two major phospholipid products of PI3K activity, phosphatidylinositol 3,4-biphosphate [PtdIns(3,4)P2] and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], in the regulation of PKB activity in glioblastoma cells containing high levels of both of these lipids due to defective PTEN expression. Reexpression of PTEN or treatment with the PI3K inhibitor LY294002 abolished the levels of both PtdIns(3, 4)P2 and PtdIns(3,4,5)P3, reduced phosphorylation of PKB on Thr308 and Ser473, and inhibited PKB activity. Overexpression of SHIP-2 abolished the levels of PtdIns(3,4,5)P3, whereas PtdIns(3,4)P2 levels remained high. However, PKB phosphorylation and activity were reduced to the same extent as they were with PTEN expression. PTEN and SHIP-2 also significantly decreased the amount of PKB associated with cell membranes. Reduction of SHIP-2 levels using antisense oligonucleotides increased PKB activity. SHIP-2 became tyrosine phosphorylated following stimulation by growth factors, but this did not significantly alter its phosphatase activity or ability to antagonize PKB activation. Finally we found that SHIP-2, like PTEN, caused a potent cell cycle arrest in G(1) in glioblastoma cells, which is associated with an increase in the stability of expression of the cell cycle inhibitor p27(KIP1). Our results suggest that SHIP-2 plays a negative role in regulating the PI3K-PKB pathway.

3T3 Cells↗
PubMed2004

Anti-HLA class I antibody-mediated activation of the PI3K/Akt signaling pathway and induction of Bcl-2 and Bcl-xL expression in endothelial cells.

Anti-human leukocyte antigen (HLA) antibodies (Ab) have long been implicated in the process of acute and chronic allograft rejection, yet their mechanism(s) of action is not well understood. The aim of this study was to determine whether ligation of HLA class I molecules by anti-HLA Ab on the surface of human endothelial cells (EC) activates the PI3 Kinase (PI3K)/Akt signaling pathway and downstream target proteins of the cell death apparatus. We report that Ab ligation of major histocompatibility complex (MHC) class I molecules on the surface of EC triggers phosphorylation of Akt, PI3K, and recruitment of PI3K and Akt into a signaling unit with focal adhesion kinase. Signaling through class I also stimulated phosphorylation of Bad and upregulated expression of Bcl-2 and Bcl-xL. Pretreatment of EC with the PI3K inhibitor wortmannin blocked class I-mediated expression of Bcl-2, but not Bcl-xL, suggesting a role for the PI3K/Akt signaling pathway in regulation of class I-induced Bcl-2 expression. The intracellular events initiated by class I ligation were influenced by the concentration of the anti-HLA Ab with the lowest tested concentrations of Ab stimulating the highest level of Akt phosphorylation, Bcl-xL and Bcl-2 expression. Consistent with the in vitro experiments, analysis of biopsy samples from heart transplant recipients with evidence of Ab-mediated rejection exhibited increased Bcl-2 expression on the vascular endothelium. These results suggest that exposure of the graft endothelium to low concentrations of anti-HLA Ab may promote cell survival by transducing signals resulting in upregulation of cell survival genes.

Androstadienes↗
PubMed2004

The role of PTEN and its signalling pathways, including AKT, in breast cancer; an assessment of relationships with other prognostic factors and with outcome.

PTEN is a novel tumour suppressor gene located on chromosome 10. PTEN mutations are believed to exert their effects through the putative PI3K-AKT-mTOR signalling pathway. Specifically, loss of PTEN leads to activation of AKT, which in turn promotes anti-apoptotic and pro-cell cycle entry pathways believed to be essential in tumourigenesis. Whilst PTEN mutations are frequent in a variety of sporadic cancers and inherited cancer syndromes, it is not clear how frequently PTEN mutations and immunohistochemical loss of PTEN expression occur in sporadic breast cancer. This study used tissue microarrays (TMAs) to assess wild-type PTEN and pAKT immunohistochemical staining in 670 and 691 cases, respectively, of primary operable breast cancer. Scores of 0, 1, and 2 were given for negative, weakly positive, and strongly positive degrees of immunoreactivity, respectively. In addition, immunohistochemical assessment of epidermal growth factor receptor (EGFR), Her2, and proliferation by MIB1 expression was performed on the same TMAs and the scores were compared with those of PTEN and pAKT. Eight per cent of cases did not express wild-type PTEN. No correlation was observed between patient, tumour and outcome variables and PTEN. pAKT expression correlated inversely with adverse tumour variables such as tumour grade (p< 0.001) and correlated positively with ER status (p< 0.001). No correlation was seen between either PTEN or AKT and EGFR, Her2 or MIB1. No association of PTEN or pAKT was seen in Kaplan-Meier or multivariate analysis for overall survival. The results indicate that loss of PTEN expression is infrequent in breast cancer. PTEN and AKT do not appear to be prognostic markers. The study argues against the current model of a simple linear tumourigenic PTEN-PI3K-AKT-mTOR pathway in breast cancer. It also suggests that, in this group of breast cancers, the most common upstream regulator of AKT may be ER rather than PTEN, EGFR or Her2.

Adult↗
PubMed2002

Role of phosphatidylinositol 3-kinase/Akt pathway in angiotensin II and insulin-like growth factor-1 modulation of nitric oxide synthase in vascular smooth muscle cells.

The aim of this study was to examine the role of the phosphatidylinositol 3-kinase (PI3K)/serine/threonine kinase Akt signaling pathway in mediating interactions between angiotensin II (Ang II) and insulin-like growth factor-1 (IGF-1) in regulation of inducible nitric oxide synthase (iNOS) in vascular smooth muscle cells (VSMCs). Exposure to 100 nM IGF-1 for 10 min resulted in increased insulin-receptor substrate-1 associated PI3K activity and Akt kinase activity, whereas 100 nM Ang II pretreatment for 5 min strikingly decreased these IGF-1 effects. NOS activity was also increased in VSMCs following exposure to IGF-1 (10 min up to 24 h). Pretreatment with Ang II for 5 min reduced IGF-1-induced NOS activity. IGF-1 treatment for 24 hr increased iNOS gene transcription, and Ang II pretreatment reduced this stimulation of iNOS gene expression by attenuating PI3K/Akt signaling. These results implicate PI3K/ Akt pathways in Ang II/IGF-1 regulation of iNOS in VSMCs.

Angiotensin II↗
PubMed2006

Controlled activation of ErbB1/ErbB2 heterodimers promote invasion of three-dimensional organized epithelia in an ErbB1-dependent manner: implications for progression of ErbB2-overexpressing tumors.

Receptor tyrosine kinases of the ErbB family are implicated in a number of cancers, including that of the breast. ErbB receptors are activated by ligand-induced formation of homodimers and heterodimers. Receptor heterodimerization is thought to play a critical role in breast cancers overexpressing multiple members of the ErbB family. Although coexpression of ErbB receptors is associated with poor patient prognosis, the mechanisms by which receptor heterodimerization regulates tumor progression are not clear, due in part to a lack of methods that allow controlled activation of specific receptor heterodimers in mammary epithelial cells. Here, we report an approach to activate ErbB1-ErbB2 heterodimers in a nontumorigenic breast epithelial cell line, MCF-10A, without interference from endogenous ErbB receptors. Using such a method, we show that whereas both ErbB2 homodimers and ErbB1-ErbB2 heterodimers were equally potent in activating the Ras/mitogen-activated protein kinase pathway, the heterodimers were more potent in activating the phosphoinositide 3'-kinase (PI3K) and phospholipase Cgamma1 pathways than ErbB2 homodimers. We combined the dimerization system with a three-dimensional cell culture approach to show that whereas both ErbB2 homodimers and ErbB1-ErbB2 heterodimers induced disruption of three-dimensional acini-like structures, only heterodimers promoted invasion of cells through extracellular matrix. The ability of heterodimers to induce invasion required the ErbB1 kinase activity and required activation of PI3K, Ras/mitogen-activated protein kinase, and phospholipase Cgamma1 signaling pathways. Thus, we have identified cell invasion as a heterodimer-specific biological outcome and suggest that coexpression of ErbB1 may critically regulate invasive progression of ErbB2-positive breast cancers.

Breast↗
PubMed2006

Calcineurin activation is not necessary for Doxorubicin-induced hypertrophy in H9c2 embryonic rat cardiac cells: involvement of the phosphoinositide 3-kinase-Akt pathway.

The calcium/calmodulin-dependent phosphatase calcineurin has been shown to be both necessary and sufficient to induce cardiac hypertrophy in vivo and in vitro. Treatment with the antineoplastic agent doxorubicin (DOX) was shown to activate calcineurin signaling in H9c2 rat cardiac muscle cells; however, the effect of this activation on hypertrophy was not investigated. Therefore, the present study was undertaken to examine the involvement of calcineurin activation in DOX-induced cardiac cell hypertrophy. H9c2 cells were treated with 1 microM DOX for 2 h following pretreatment with and in the presence of calcineurin inhibitors cyclosporine A (CsA) or FK506 (tacrolimus). Subsequent analysis of calcineurin signaling and cellular hypertrophy was performed 8 to 48 h after the treatment. DOX treatment activated calcineurin signaling and resulted in cellular hypertrophy as assessed by an increase in cell volume and protein content per cell. Inhibition of calcineurin with CsA or FK506 blocked DOX-induced calcineurin signaling. However, this inhibition did not prevent the DOX-induced hypertrophic response in H9c2 cells. Further evaluation of the possible signaling pathways involved in DOX-induced H9c2 cellular hypertrophy revealed that DOX treatment resulted in phosphorylation of the serine/threonine protein kinase Akt, a downstream effector of phosphoinositide 3-kinase (PI3K). Moreover, the DOX-induced hypertrophic response was blunted by LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], a specific inhibitor for PI3K. These results demonstrate that, although calcineurin is activated by DOX treatment, it is not necessary for DOX-induced hypertrophy in H9c2 cells. Rather, the PI3K-Akt signaling pathway seems to be more critically involved in DOX-induced hypertrophy.

Animals↗
PubMed2003

Selenite-induced survival of HuH7 hepatoma cells involves activation of focal adhesion kinase-phosphatidylinositol 3-kinase-Akt pathway and Rac1.

Selenium has been shown to sustain the growth of selected human hepatocellular carcinoma cell lines under serum-free conditions, but the detailed mechanism remained undetermined. In the present study, the molecular mechanism(s) involving sodium selenite (Na2SO3, Se) as a survival agent were determined. Selenite not only protects HuH7 cells from serum deprivation-induced apoptosis, it also supports its long-term growth in sodium selenite (10(-7)m) supplemented serum-free medium. The anti-apoptotic effect correlates with activation of focal adhesion kinase and the phosphatidylinositol 3-kinase (PI3K)-Akt kinase pathway. Using HuH7 cells stably transfected with a constitutively active Akt kinase and PI3K inhibitor LY294002, selenite-induced cell survival was shown to be PI3K-Akt-dependent. Parallel changes included a significant reduction in the intracellular reactive oxygen species content, the reversal of DNA fragmentation, and the suppression of caspase and apoptosis signal-regulating kinase 1 activities. HuH7 cells stably expressing a Rac1 mutant N17 (Rac1N17-HuH7) are refractory to selenite treatment. In these cells selenite supplement neither triggers Akt activation nor supports cell proliferation. Participation of Rac1 activation in this event is supported by the fact that selenite treatment drastically enhanced activation of Rac1. The exact link between selenite treatment, Rac1 activation, and activation of the focal adhesion kinase-PI 3-kinase, however, remains to be characterized. The mitogenic signaling mediated by selenite may involve unconventional growth stimuli including higher glutathione peroxidase 1 activity and higher transcription levels of selenoprotein P. The selenium-HuH7 system we have established thus provides a unique tool that will allow the biological role of selenite in growth regulation of hepatocytes to be studied in detail.

Apoptosis↗
PubMed2005

Uncommon mutation, but common amplifications, of the PIK3CA gene in thyroid tumors.

CONTEXT: As in many other human cancers, overactivation of the phosphotidylinositol 3-kinase (PI3K)/Akt signaling pathway occurs frequently in thyroid cancer, but the mechanism is not completely clear. OBJECTIVE: Because activating mutations and genomic amplification of the PIK3CA gene, which encodes the p110a catalytic subunit of PI3K, are common in many cancers, we sought to investigate this phenomenon in thyroid tumors. DESIGN: To search for PIK3CA mutations, we isolated genomic DNA from primary thyroid tumors of various types and performed direct sequencing of the exons of PIK3CA gene that carry the most common mutations in other cancers. We used real-time quantitative PCR to investigate genomic amplification of the PIK3CA gene. RESULTS: We found no PIK3CA gene mutations in 37 benign thyroid adenomas, 52 papillary thyroid cancers, 25 follicular thyroid cancers, 13 anaplastic thyroid cancers, 13 medullary thyroid cancers, and seven thyroid tumor cell lines. We found a C3075T single-nucleotide polymorphism in exon 20 of this gene in two cases. With a copy number of 4 or more defined as amplification, we found PIK3CA gene amplification in four of 34 (12%) benign thyroid adenomas, three of 59 (5%) papillary thyroid cancer, five of 21 (24%) follicular thyroid cancer, none of 14 (0%) medullary thyroid cancer, and five of seven (71%) thyroid tumor cell lines. The PIK3CA gene amplification and consequent Akt activation were confirmed by fluorescence in situ hybridization and Western blotting studies using cell lines, respectively. CONCLUSION: These data suggest that mutation of the PIK3CA gene is not common, but its amplification is relatively common and may be a novel mechanism in activating the PI3K/Akt pathway in some thyroid tumors.

Class I Phosphatidylinositol 3-Kinases↗
PubMed2003

Glucocorticoid Receptor Counteracts Tumorigenic Activity of Akt in Skin through Interference with the Phosphatidylinositol 3-Kinase Signaling Pathway.

The skin-targeted overexpression of the glucocorticoid receptor (GR) in transgenic mice dramatically impairs the inflammatory responses to tumor promoter agents and suppresses skin tumor development. The antiinflammatory, rapid effects of corticosteroids are partially exerted through interference of GR with the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in several tissues, a highly relevant pathway in the mouse skin tumor progression process. In this work, we aimed to elucidate whether a cross-talk mechanism between GR and PI3K/Akt occurred in intact skin as well as the biological relevance of this interaction during skin tumorigenesis. We report that, in transgenic mice overexpressing the receptor, GR physically associated with p85 alpha/PI3K in skin, resulting in decreased Akt and I kappa B kinase activity. GR activation by dexamethasone in normal mouse skin also decreased Akt activity within minutes, whereas cotreatment with the GR antagonist RU486 abolished dexamethasone action. Indeed, GR exerted a nongenomic action because keratinocyte transfection with a transcriptionally defective receptor mutant still decreased PI3K and Akt activity. Moreover, GR coexpression greatly reduced the accelerated growth of malignant tumors and increased Akt activity induced by Akt-transfected keratinocytes, as shown by in vivo tumorigenic assays. Overall, our data strongly indicate that GR/PI3K-Akt cross-talk constitutes a major mechanism underlying the antitumor effect of glucocorticoids in skin.

Administration, Topical↗