Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “PI3K pathway”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources
PubMed2003

Role of phosphoinositide 3-kinase in the nonselective cation channel activation by endothelin-1/endothelinB receptor.

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) in Chinese hamster ovarian cells expressing endothelin(B) receptor (CHO-ET(B)R). These channels can be discriminated using the Ca(2+) channel blockers, LOE 908 and SK&F 96365. LOE 908 is a blocker of NSCC-1 and NSCC-2, whereas SK&F 96365 is a blocker of NSCC-2. In this study, we investigated the possible role of phosphoinositide 3-kinase (PI3K) in the ET-1-induced activation of NSCCs in CHO-ET(B)R using wortmannin and LY-294002, inhibitors of PI3K. ET-1-induced Ca(2+) influx was partially inhibited in CHO-ET(B)R pretreated with wortmannin or LY-294002. In contrast, addition of wortmannin or LY-294002 after stimulation with ET-1 did not suppress Ca(2+) influx. The Ca(2+) channels activated by ET-1 in wortmannin- or LY-294002-treated CHO-ET(B)R were sensitive to LOE 908 and resistant to SK&F 96365. In conclusion, NSCC-2 is stimulated by ET-1 via PI3K-dependent cascade, whereas NSCC-1 is stimulated independently of the PI3K pathway. Moreover, PI3K seems to be required for the initiation of the Ca(2+) entry through NSCC-2 but not for its maintenance.

Acetamides↗
PubMedDate not supplied

Impairment of TNF-alpha production and action by imidazo[1,2- alpha] quinoxalines, a derivative family which displays potential anti-inflammatory properties.

In a previous study, we analysed the synthesis and properties of a series of imidazo[1,2-alpha]quinoxalines designed in our laboratory as possible imiquimod analogues. We found that these imidazo[1,2-alpha]quinoxalines were in fact potent inhibitors of phosphodiesterase 4 enzymes (PDE4). PDE4 inhibition normally results in an increase in intracellular cAMP which, in PBMC, induces the suppression of TNF-alpha mRNA transcription and thus cytokine synthesis. Such an effect is antagonistic to that of imiquimod. Furthermore, some TNF-alpha-induced activity, such as cell apoptosis which is dependent on the intracellular cAMP levels might also be affected. Therefore, by counteracting the properties of TNF-alpha and/or its production, the imidazo[1,2-alpha]quinoxalines could be considered as potential anti-inflammatory drugs. The present study was performed to confirm or refute this hypothesis. For this, we characterized the effects of imidazo[1,2-alpha]quinoxalines both on TNF-alpha activity and synthesis in regard to their ability to act as inhibitors of PDE4 (IPDE4). We found that the imidazo[1,2-alpha]quinoxalines dose-dependently prevented the TNF-alpha-triggered death of L929 cells, with the 8-series (-NHCH3 in R4) being the most potent. Moreover, when the effect of the 8-series on TNF-alpha production was investigated using gamma9delta2 T cells, it was observed that these compounds impaired the TCR:CD3-triggered TNF-alpha production. Structure-activity analysis revealed that these properties of the drugs did not coincide with their IPDE4 properties. This prompted further exploration into other signalling mechanisms possibly involved in TNF-alpha action and production, notably the p38 MAPK and the PI3K pathway. We demonstrate here that the imidazo[1,2-alpha]quinoxalines targeted these pathways in a different way: they activated the p38 MAPK pathway whilst inhibiting the PI3K pathway. Such effects on cell signalling could account for the imidazo[1,2-alpha]quinoxalines effects on 1) action and 2) production of TNF-alpha, which define these drugs as potential anti-inflammatory agents.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2000

Cardiotrophin-1 phosphorylates akt and BAD, and prolongs cell survival via a PI3K-dependent pathway in cardiac myocytes.

Growth factors and cytokines trigger survival signaling in a wide variety of cell systems, including cardiac myocytes. Participation of the phosphatidylinositol 3-OH kinase (PI3K)/Akt pathway in survival signaling has already been described in some cell types, but its involvement in the survival of cardiac myocytes is as yet unknown. Recently, CT-1, an interleukin 6-related cytokine, was shown to have survival-promoting, anti-apoptotic effects on cultured cardiac myocytes. However, roles of PI3K-dependent pathways in this signaling have not been elucidated. In the present study, therefore, we examined the participation of the PI3K/Akt pathway in CT-1-induced, survival-promoting signaling in cultured ventricular myocytes. It was found that CT-1 phosphorylated and activated Akt, and the effect was blocked by the PI3K inhibitors LY294002 and wortmannin. CT-1 also phosphorylated the pro-apoptotic factor, BAD, and the BAD phosphorylation was inhibited by LY294002, suggesting that phosphorylation of BAD is one of the key events by which the PI3K/Akt pathway mediates CT-1-induced survival signaling. Further, CT-1 PI3K-dependently prolonged the survival of serum-starved ventricular myocytes by preventing apoptosis. In summary, our findings show that PI3K-dependent survival signals contribute to CT-1-mediated ventricular myocyte survival. In vivo, the death of ventricular myocytes leads to heart failure, and downregulation of survival signals and/or augmentation of pro-apoptotic signals are likely to be important components of disease processes. Thus, the extent to which CT-1 and the PI3K/Akt pathway mitigate such pathological processes, in vivo, is an important question for the future.

Animals↗
PubMed2005

The MAP kinase pathway is upstream of the activation of GSK3beta that enables it to phosphorylate MAP1B and contributes to the stimulation of axon growth.

In pheochromocytoma 12 (PC12) cells and sympathetic neurons, nerve growth factor (NGF) engagement with the tropomyosin-related tyrosine kinase (TrkA) receptor activates the serine/threonine kinase glycogen synthase kinase 3beta (GSK3beta), enabling it to phosphorylate the microtubule-associated protein 1B (MAP1B). GSK3beta phosphorylation of MAP1B acts as a molecular switch to regulate microtubule dynamics in growing axons, and hence the rate of axon growth. An important question relates to the identification of the upstream pathway linking the activation of GSK3beta with TrkA engagement. TrkA can utilise a number of intracellular signalling pathways, including the mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol-3 kinase (PI3K) pathway. We now show, using pharmacological inhibitor studies of PC12 cells and sympathetic neurons in culture and in vitro kinase and activation assays, that the MAPK pathway, and not the PI3K pathway, links NGF engagement with the TrkA receptor to GSK3beta activation in PC12 cells and sympathetic neurons. We also show that activated GSK3beta is a small fraction of the total GSK3beta present in developing brain and that it is not part of a multiprotein complex. Thus, NGF drives increased neurite growth rates partly by coupling the MAPK pathway to the activation of GSK3beta and thereby phosphorylation of MAP1B.

Animals↗
PubMed2005

Transforming acidic coiled coil 1 promotes transformation and mammary tumorigenesis.

Transforming acidic coiled coil 1 (TACC1) is a putative oncogene located within a breast cancer amplicon found on human chromosome 8p11. Although TACC1 has been reported to transform fibroblasts, it is also down-regulated in a subset of mammary tumors treated with anthracyclin. Here, we show that ectopic TACC1 overexpression can cooperate with Ras to induce focus formation in murine fibroblast cultures and prevent death caused by overexpression of Pten or a dominant-negative form of protein kinase B (PKB)/Akt. In transgenic mice carrying TACC1 under the control of the mouse mammary tumor virus promoter, TACC1 expression reduced apoptosis during mammary gland involution, increased the penetrance of mammary tumors in a pten+/- background, and decreased the average age of mammary tumor onset in a mouse model based on a phosphatidylinositol 3'-kinase (PI3K)-decoupled mutant of polyoma middle T. Elevated levels of both phospho-PKB and phospho-extracellular signal-regulated kinase were found in mammary tissue containing the TACC1 transgene. Thus, TACC1 positively regulates the Ras and PI3K pathways, promotes Ras-mediated transformation, and prevents apoptosis induced by PI3K pathway inhibition. TACC1 also cooperates with tumorigenic mutations in the PI3K pathway and thereby plays an oncogenic role in tumor formation in the murine mammary gland.

Animals↗
PubMed2006

RNA interference-mediated depletion of phosphoinositide 3-kinase activates forkhead box class O transcription factors and induces cell cycle arrest and apoptosis in breast carcinoma cells.

Breast cancer is one of the most common malignancies affecting women in the Western world and one in seven women is predicted to develop invasive breast cancer in their lifetime. Breast cancer arises following the accumulation of a series of somatic changes often including deregulation of key signal transduction pathways. The phosphoinositide 3-kinase (PI3K) pathway has been shown to be activated in breast cancer and overexpression of PI3K is sufficient to confer a malignant phenotype. Activation of the PI3K pathway serves to repress forkhead box class O (FoxO) transcription factor-mediated growth arrest and apoptosis. In this study, we used small interfering RNA (siRNA) to knockdown PI3K in three breast cancer cell lines representing different stages of cancer development. Transfection of PI3K siRNA in breast cancer cells resulted in a significant decrease in cell viability and induction of apoptosis irrespective of their estrogen receptor alpha (ERalpha) or ErbB2 status. PI3K depletion also resulted in a significant G(1) phase cell cycle arrest in ERalpha-positive breast cancer cells. Further, our data showed that PI3K knockdown resulted in a significant activation of FoxO; interestingly, a simultaneous knockdown of FoxO1a rescued the cells from apoptosis. Furthermore, the downstream effects of FoxO activation were found to be inhibition of cyclin-dependent kinase 4, cyclin-dependent kinase 6, and cyclin D1, and accumulation of p27/Kip1. Thus, we suggest that (a) PI3K plays a critical role in breast cancer development and (b) gene therapeutic approaches aimed at PI3K or the pharmacologic inhibitors of PI3K could be developed for the management of breast cancer.

Apoptosis↗
PubMed2003

The phosphatidylinositol 3-kinase/Akt pathway enhances Smad3-stimulated mesangial cell collagen I expression in response to transforming growth factor-beta1.

Transforming growth factor (TGF)-beta has been associated with renal glomerular matrix accumulation. We previously showed that Smad3 promotes COL1A2 gene activation by TGF-beta1 in human glomerular mesangial cells. Here, we report that the PI3K/Akt pathway also plays a role in TGF-beta1-increased collagen I expression. TGF-beta1 stimulates the activity of phosphoinositide-dependent kinase (PDK)-1, a downstream target of PI3K, starting at 1 min. Akt, a kinase downstream of PDK-1, is phosphorylated and concentrates in the membrane fraction within 5 min of TGF-beta1 treatment. The PI3K inhibitor LY294002 decreases TGF-beta1-stimulated alpha1(I) and alpha2(I) collagen mRNA expression. Similarly, LY294002 or an Akt dominant negative construct blocks TGF-beta1 induction of COL1A2 promoter activity. However, PI3K stimulation alone is not sufficient to increase collagen I expression, since neither a constitutively active p110 PI3K construct nor PDGF, which induces Akt phosphorylation, is able to stimulate COL1A2 promoter activity or mRNA expression, respectively. LY294002 inhibits stimulation of COL1A2 promoter activity by Smad3. In a Gal4-LUC assay system, blockade of the PI3K pathway significantly decreases TGF-beta1-induced transcriptional activity of Gal4-Smad3. Activity of SBE-LUC, a Smad3/4-responsive construct, is stimulated by over-expression of Smad3 or Smad3D, in which the three C-terminal serine phospho-acceptor residues are mutated. This induction is blocked by LY294002, suggesting that inhibition of the PI3K pathway decreases Smad3 transcriptional activity independently of C-terminal serine phosphorylation. However, TGF-beta1-induced total serine phosphorylation of Smad3 is decreased by LY294002, suggesting that Smad3 is phosphorylated by the PI3K pathway at serine residues other than the direct TGF-beta receptor I target site. Thus, although the PI3K-PDK1-Akt pathway alone is insufficient to stimulate COL1A2 gene transcription, its activation by TGF-beta1 enhances Smad3 transcriptional activity leading to increased collagen I expression in human mesangial cells. This cross-talk between the Smad and PI3K pathways likely contributes to TGF-beta1 induction of glomerular scarring.

Cells, Cultured↗
PubMed2003

Scaffolding protein Gab2 mediates fibroblast transformation by the SEA tyrosine kinase.

Transformation of fibroblasts by V-SEA involves activation of the ERK and phosphatidylinositol 3-kinase (PI3K) pathways. Effector proteins that are key mediators of the ERK and PI3K pathways, namely Grb2, the tyrosine phosphatase, SHP2 and PI3K, interact with the two phosphotyrosines found in the bidentate motif in the carboxy-terminal region of V-SEA. Genetic analysis demonstrated that while Y557 was a primary binding site and thus activator of the PI3K-Akt pathway, Y564 also contributed to the activation of this pathway. Y564 was located within a Grb2-binding motif, this raised the possibility that a protein that associated with Grb2 might be important for this PI3K activation. The scaffolding proteins Gab1 and/or Gab2 were candidates for this role. In this report, we demonstrate that V-SEA preferentially interacts with Gab2. Furthermore by using Gab2 null fibroblasts, we demonstrate that Gab2 is essential for fibroblast transformation by V-SEA. Using mutant forms of Gab2, we show that activation of the PI3K-Akt pathway via Gab2 is required for V-SEA-induced transformation. However, efficient fibroblast transformation also requires the SHP2 interaction site on Gab2.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Synchronous activation of ERK and phosphatidylinositol 3-kinase pathways is required for collagen and extracellular matrix production in keloids.

Keloid fibroproliferation appears to be influenced by epithelial-mesenchymal interactions between keloid keratinocytes (KKs) and keloid fibroblasts (KFs). Keloid and normal fibroblasts exhibit accelerated proliferation and collagen I and III production in co-culture with KKs compared with single cell culture or co-culture with normal keratinocytes. ERK and phosphatidylinositol 3-kinase (PI3K) pathway activation has been observed in excessively proliferating KFs in co-culture with KKs. We hypothesized that ERK and PI3K pathways might be involved in collagen and extracellular matrix production in KFs. To test our hypothesis, four samples of KFs were co-cultured in defined serum-free medium with KKs for 2-5 days. KF cell lysate was subjected to Western blot analysis. Compared with KF single cell culture, phospho-ERK1/2 and downstream phospho-Elk-1 showed up-regulation in the co-culture groups, as did phospho-PI3K and phospho-Akt-1, indicating ERK and PI3K pathway activation. Western blotting of the conditioned medium demonstrated increased collagen I-III, laminin beta2, and fibronectin levels. Addition of the MEK1/2-specific inhibitor U0126 or the PI3K-specific inhibitor LY294002 (but not p38 kinase and JNK inhibitors) completely nullified collagen I-III production and significantly decreased laminin beta2 and fibronectin secretion. In the presence of the MEK1/2 or PI3K inhibitor, fibronectin demonstrated changes in molecular mass reflected by faster in-gel migration. These data strongly suggest that synchronous activation of both the ERK and PI3K pathways is essential for collagen I-III and laminin beta2 production. These pathways additionally appear to affect the side chain attachments of fibronectin. Modulation of these pathways may suggest a direction for keloid therapy.

Blotting, Western↗
PubMed2003

Novel functional interactions between Trk kinase and p75 neurotrophin receptor in neuroblastoma cells.

To understand the functional interactions between the TrkA and p75 nerve growth factor (NGF) receptors, we stably transfected LAN5 neuroblastoma cells with an expression vector for ET-R, a chimeric receptor with the extracellular domain of the epidermal growth factor receptor (EGFR), and the TrkA transmembrane and intracellular domains. EGF activated the ET-R kinase and induced partial differentiation. NGF, which can bind to endogenous p75, did not induce differentiation but enhanced the EGF-induced response, leading to differentiation of almost all cells. A mutated NGF, 3T-NGF, that binds to TrkA but not to p75 did not synergize with EGF. Enhancement of EGF-induced differentiation required at least nanomolar concentrations of NGF, consistent with the low-affinity p75 binding site. EGF may induce a limited number of neuronal cells because it also enhanced apoptosis. Both NGF and a caspase inhibitor reduced apoptosis and, thereby, enhanced differentiation. NGF seems to enhance survival through the phosphatidylinositol-3 kinase (PI3K) pathway. Consistent with this hypothesis, Akt, a downstream effector of the PI3K pathway, was hyperphosphorylated in the presence of EGF+NGF. These results demonstrate that TrkA kinase initiates differentiation, and p75 enhances differentiation by rescuing differentiating cells from apoptosis via the PI3K pathway. Even though both EGF and NGF are required for differentiation of LAN5/ET-R cells, only NGF is required for survival of the differentiated cells. In the absence of NGF, the cells die by an apoptotic mechanism, involving caspase-3. An anti-p75 antibody blocked the survival effect of NGF. Brain-derived neurotrophic factor also enhanced cell survival, indicating that in differentiated cells, NGF acts through the p75 receptor to prevent apoptosis.

Animals↗
PubMed2006

Protein chip discovery of secreted proteins regulated by the phosphatidylinositol 3-kinase pathway in ovarian cancer cell lines.

Ovarian cancer has the highest mortality among the gynecologic malignancies. The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated, leading to increased cell survival. This study aimed to identify secreted proteins regulated by the PI3K pathway in ovarian cancer cell lines. Surface-enhanced laser desorption-ionization time-of-flight mass spectrometry with cation-exchange protein-chips was used to analyze secreted proteins from five ovarian cancer cell lines (SKOV-3, PE01, OVCAR-3, OV167, and OV207). To activate the PI3K pathway, cells were treated with 50 ng/mL epidermal growth factor (EGF) with or without 10 micromol/L LY294002, a PI3K inhibitor. Proteins induced by EGF and inhibited by LY294002, in the m/z range 7,500 to 9,500, were purified chromatographically, identified by peptide mass fingerprinting and NH(2)-terminal sequencing, and confirmed by immunodepletion. Two immunologically related proteins, m/z approximately 8,385 and 8,922, were identified as truncated and intact forms, respectively, of interleukin 8, a chemokine previously shown to be elevated in serum of ovarian cancer patients. Another protein, m/z 7,866, was identified as CXC chemokine ligand 1 (CXCL1) or GRO-alpha, a chemokine associated with melanoma formation and some epithelial cancers. EGF-stimulated CXCL1 levels were variably decreased by mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase and p38 MAPK inhibition in the five cell lines, but only LY294002 fully reversed the EGF effect in all cell lines. Immunoreactive CXCL1 levels in 160 conditioned media were highly correlated with corresponding peak intensities at m/z 7,866 by mass spectrometry, indicating the quantitative nature of these analyses. We conclude that proteomic analysis of cell models of human disease may facilitate the discovery of pathway-dependent proteins.

Blotting, Western↗
PubMed2004

PI3K-AKT pathway mediates growth and survival signals during development of fetal mouse lung.

We examined the roles of the PI3K-AKT signalling pathway in fetal lung development. By Western blotting, phosphorylated AKT (pAKT) was highly expressed in fetal days 12 and 14 with decreased expression thereafter. By immunohistochemistry, pAKT was expressed mainly in the respiratory epithelium of early fetal days. We examined the effects of fibroblast growth factor 1 (FGF1), PI3K inhibitors (LY294002 and wortmannin), MAPK inhibitor (PD98059) and both of FGF1 and each inhibitor on lung morphogenesis, BrdU incorporation and apoptosis. In the FGF1-treated explants, the number of terminal buds and BrdU-labelled cells increased significantly, while the LY294002-, wortmannin-, PD98059-treated explants demonstrated obvious decreases. The effects by FGF1 were inhibited by LY294002, wortmannin and PD98059. Regardless of the presence of FGF1, the LY294002-, wortmannin- and PD98059-treated explants increased apoptosis revealed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay in the mesenchyme of the explants. At the same time, the effect of LY294002, wortmannin, PD98059 on expression of surfactant apoprotein C (SPC) were also studied. The LY294002 and wortmannin treatments showed decreased expression of SPC. These findings suggest that the PI3K-AKT signalling pathway plays a pivotal role in mouse lung development through various biological processes.

Androstadienes↗
PubMed2006

Vitamin E protected cultured cortical neurons from oxidative stress-induced cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase.

The role of vitamin E in the CNS has not been fully elucidated. In the present study, we found that pre-treatment with vitamin E analogs including alphaT (alpha-tocopherol), alphaT3 (alpha -tocotrienol), gammaT, and gammaT3 for 24 h prevented the cultured cortical neurons from cell death in oxidative stress stimulated by H2O2, while Trolox, a cell-permeable analog of alphaT, did not. The preventive effect of alphaT was dependent on de novo protein synthesis. Furthermore, we found that alphaT exposure induced the activation of both the MAP kinase (MAPK) and PI3 kinase (PI3K) pathways and that the alphaT-dependent survival effect was blocked by the inhibitors, U0126 (an MAPK pathway inhibitor) or LY294002 (a PI3K pathway inhibitor). Interestingly, the up-regulation of Bcl-2 (survival promoting molecule) was induced by alphaT application. The up-regulation of Bcl-2 did not occur in the presence of U0126 or LY294002, suggesting that alphaT-up-regulated Bcl-2 is mediated by these kinase pathways. These observations suggest that vitamin E analogs play an essential role in neuronal maintenance and survival in the CNS.

Animals↗
PubMed2001

The role of genetic abnormalities of PTEN and the phosphatidylinositol 3-kinase pathway in breast and ovarian tumorigenesis, prognosis, and therapy.

Breast and ovarian cancers exhibit similar epidemiologic, genotypic, and phenotypic characteristics. Phosphatidylinositol 3-kinase (PI3K) and the PTEN tumor suppressor gene product phosphorylate and dephosphorylate the same 3' site in the inositol ring of membrane phosphatidylinositols. Germ-line mutations in the PTEN tumor suppressor gene are causative of Cowden's breast cancer predisposition syndrome, and PTEN is frequently mutated in sporadic breast cancers. In contrast, amplification of multiple components of the PI3K pathway is a hallmark of serous epithelial ovarian cancers. The resultant activation of the PI3K pathway in both breast and ovarian cancers contributes to cell-cycle progression, decreased apoptosis, and increased metastatic capabilities. Strikingly, both ovarian and breast cancer cells are selectively sensitive to pharmacologic and genetic manipulation of the PI3K pathway, making molecular therapeutics targeting this pathway particularly attractive approaches for these cancers.

Animals↗
PubMed2006

How a RING finger protein and a steroid hormone control autophagy.

Previous work in our laboratory has indicated that the steroid hormone ecdysone triggers programmed autophagy in the fat body of Drosophila larvae by downregulating the class I phosphoinositide 3-kinase (PI3K) pathway. We recently found evidence that Deep orange (Dor), a Drosophila RING finger protein implicated in late-endosomal trafficking, controls ecdysone signaling as well as autolysosome fusion, thus exerting a dual regulation of autophagy. We found that dor mutants are defective in programmed autophagy. The mutant larvae showed impaired upregulation of ecdysone signaling during development, accompanied by a failure to downregulate the PI3K pathway. Downregulation of the PI3K pathway could be restored by feeding the dor mutants with ecdysone. Even though ecdysone signaling and autophagy were impaired in the dor mutants, we detected an accumulation of autophagosomes in dor mutant fat bodies. This could probably be attributed to the failure of autophagosomes to fuse with lysosomes. In this Addendum we review these findings and provide some speculations about how Dor may control both ecdysone signalling and autolysosomal fusion.

Animals↗
PubMed2005

The insulin-PI3K/TOR pathway induces a HIF-dependent transcriptional response in Drosophila by promoting nuclear localization of HIF-alpha/Sima.

The hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of a constitutively expressed HIF-beta subunit and an oxygen-regulated HIF-alpha subunit. We have previously defined a hypoxia-inducible transcriptional response in Drosophila melanogaster that is homologous to the mammalian HIF-dependent response. In Drosophila, the bHLH-PAS proteins Similar (Sima) and Tango (Tgo) are the functional homologues of the mammalian HIF-alpha and HIF-beta subunits, respectively. HIF-alpha/Sima is regulated by oxygen at several different levels that include protein stability and subcellular localization. We show here for the first time that insulin can activate HIF-dependent transcription, both in Drosophila S2 cells and in living Drosophila embryos. Using a pharmacological approach as well as RNA interference, we determined that the effect of insulin on HIF-dependent transcriptional induction is mediated by PI3K-AKT and TOR pathways. We demonstrate that stimulation of the transcriptional response involves upregulation of Sima protein but not sima mRNA. Finally, we have analyzed in vivo the effect of the activation of the PI3K-AKT pathway on the subcellular localization of Sima protein. Overexpression of dAKT and dPDK1 in normoxic embryos provoked a major increase in Sima nuclear localization, mimicking the effect of a hypoxic treatment. A similar increase in Sima nuclear localization was observed in dPTEN homozygous mutant embryos, confirming that activation of the PI3K-AKT pathway promotes nuclear accumulation of Sima protein. We conclude that regulation of HIF-alpha/Sima by the PI3K-AKT-TOR pathway is a major conserved mode of regulation of the HIF-dependent transcriptional response in Drosophila.

Active Transport, Cell Nucleus↗
PubMed2006

Pten (phosphatase and tensin homologue gene) haploinsufficiency promotes insulin hypersensitivity.

AIMS/HYPOTHESIS: Insulin controls glucose metabolism via multiple signalling pathways, including the phosphatidylinositol 3-kinase (PI3K) pathway in muscle and adipose tissue. The protein/lipid phosphatase Pten (phosphatase and tensin homologue deleted on chromosome 10) attenuates PI3K signalling by dephosphorylating the phosphatidylinositol 3,4,5-trisphosphate generated by PI3K. The current study was aimed at investigating the effect of haploinsufficiency for Pten on insulin-stimulated glucose uptake. MATERIALS AND METHODS: Insulin sensitivity in Pten heterozygous (Pten(+/-)) mice was investigated in i.p. insulin challenge and glucose tolerance tests. Glucose uptake was monitored in vitro in primary cultures of myocytes from Pten(+/-) mice, and in vivo by positron emission tomography. The phosphorylation status of protein kinase B (PKB/Akt), a downstream signalling protein in the PI3K pathway, and glycogen synthase kinase 3beta (GSK3beta), a substrate of PKB/Akt, was determined by western immunoblotting. RESULTS: Following i.p. insulin challenge, blood glucose levels in Pten(+/-) mice remained depressed for up to 120 min, whereas glucose levels in wild-type mice began to recover after approximately 30 min. After glucose challenge, blood glucose returned to normal about twice as rapidly in Pten(+/-) mice. Enhanced glucose uptake was observed both in Pten(+/-) myocytes and in skeletal muscle of Pten(+/-) mice by PET. PKB and GSK3beta phosphorylation was enhanced and prolonged in Pten(+/-) myocytes. CONCLUSIONS/INTERPRETATION: Pten is a key negative regulator of insulin-stimulated glucose uptake in vitro and in vivo. The partial reduction of Pten due to Pten haploinsufficiency is enough to elicit enhanced insulin sensitivity and glucose tolerance in Pten(+/-) mice.

Animals↗
PubMed2006

Immortalization of primary endothelial cells by the K1 protein of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to three different human cancers: Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The Kaposi's sarcoma lesion expresses high levels of angiogenic factors and is comprised of a mixed cell population, including endothelial cells that are infected with KSHV. We find that the KSHV K1 protein is expressed in Kaposi's sarcoma lesions and can immortalize and extend the life span of primary human umbilical vein endothelial cells in culture. Vascular endothelial growth factor (VEGF) is critical for the survival of endothelial cells, and we show that expression of K1 in endothelial cells resulted in increased levels of secreted VEGF and the activation of key signaling pathways, including the VEGF/VEGF receptor and the phosphatidylinositol-3'-OH-kinase (PI3K) pathway. The SH2 binding motifs present in the cytoplasmic tail of K1 were critical for K1's ability to activate these pathways. Activation of PI3K by K1 results in activation of Akt kinase and mammalian target of rapamycin and inactivation of the proapoptotic proteins FKHR, glycogen synthase kinase-3, and Bad, which are events indicative of cell survival. Because activation of the PI3K pathway is critical for transformation of many human cells, we suggest that PI3K activation by K1 is involved in endothelial cell immortalization and contributes to KSHV-associated tumorigenesis. We also report that K1 enhances angiogenesis in vivo and increases tumor vasculature and tumor size.

Animals↗