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PubMed2000

Drosophila PTEN regulates cell growth and proliferation through PI3K-dependent and -independent pathways.

The control of cell and organ growth is fundamental to the development of multicellular organisms. Here, we show that dPTEN, a Drosophila homolog of the mammalian PTEN tumor suppressor gene, plays an essential role in the control of cell size, cell number, and organ size. In mosaic animals, dPTEN(-) cells proliferate faster than their heterozygous siblings, show an autonomous increase in cell size, and form organs of increased size, whereas overexpression of dPTEN results in opposite phenotypes. The loss-of-function phenotypes of dPTEN are suppressed by mutations in the PI3K target Dakt1 and the translational initiation factor eif4A, suggesting that dPTEN acts through the PI3K signaling pathway to regulate translation. Although activation of PI3K and Akt has been reported to increase rates of cellular growth but not proliferation, loss of dPTEN stimulates both of these processes, suggesting that PTEN regulates overall growth through PI3K/Akt-dependent and -independent pathways. Furthermore, we show that dPTEN does not play a major role in cell survival during Drosophila development. Our results provide a potential explanation for the high frequency of PTEN mutation in human cancer.

Amino Acid Sequence↗
PubMed2005

PWT-458, a novel pegylated-17-hydroxywortmannin, inhibits phosphatidylinositol 3-kinase signaling and suppresses growth of solid tumors.

Deregulated phosphatidylinositol 3-kinase (PI3K) signaling pathway is widely implicated in tumor growth and resistance to chemotherapy. While a strong rationale exists for pharmacological targeting of PI3K, only a few proof-of-principle in vivo efficacy studies are currently available. PWT-458, pegylated-17-hydroxywortmannin, is a novel and highly potent inhibitor of PI3K in animal models. Upon in vivo cleavage of its poly(ethyleneglycol) (PEG), PWT-458 releases its active moiety 17-hydroxywortmannin (17-HWT), the most potent inhibitor in its class. Here we show that a single intravenous injection of PWT-458 rapidly inhibited PI3K signaling, as measured by a complete loss of AKT (Ser-473) phosphorylation in xenograft tumors grown in nude mice. Following a daily X5 dosing regimen, PWT-458 demonstrated single-agent antitumor activity in nude mouse xenograft models of U87MG glioma, nonsmall cell lung cancer (NSCLC) A549, and renal cell carcinoma (RCC) A498. Efficacious doses ranged from 0.5 mg/kg to 10 mg/kg, achieving a superior therapeutic index over 17-HWT. PWT-458 augmented anticancer efficacy of a suboptimal dose of paclitaxel against A549 and U87MG tumors. Combination treatment of PWT-458 and an mTOR inhibitor, Pegylated-Rapamycin (Peg-Rapa), resulted in an enhanced antitumor efficacy in U87MG. Finally, PWT-458 in combination with interferon-alpha (Intron-A) caused a dramatic regression of RCC A498, which was not achieved by either agent alone. These studies identify PWT-458 as an effective anticancer agent and provide strong proof-of-principle for targeting the PI3K pathway as novel anticancer therapy.

Androstadienes↗
PubMed2006

Akt1 activation can augment hypoxia-inducible factor-1alpha expression by increasing protein translation through a mammalian target of rapamycin-independent pathway.

The phosphoinositide 3-kinase (PI3K)/Akt pathway is commonly activated in cancer; therefore, we investigated its role in hypoxia-inducible factor-1alpha (HIF-1alpha) regulation. Inhibition of PI3K in U87MG glioblastoma cells, which have activated PI3K/Akt activity secondary to phosphatase and tensin homologue deleted on chromosome 10 (PTEN) mutation, with LY294002 blunted the induction of HIF-1alpha protein and its targets vascular endothelial growth factor and glut1 mRNA in response to hypoxia. Introduction of wild-type PTEN into these cells also blunted HIF-1alpha induction in response to hypoxia and decreased HIF-1alpha accumulation in the presence of the proteasomal inhibitor MG132. Akt small interfering RNA (siRNA) also decreased HIF-1alpha induction under hypoxia and its accumulation in normoxia in the presence of dimethyloxallyl glycine, a prolyl hydroxylase inhibitor that prevents HIF-1alpha degradation. Metabolic labeling studies showed that Akt siRNA decreased HIF-1alpha translation in normoxia in the presence of dimethyloxallyl glycine and in hypoxia. Inhibition of mammalian target of rapamycin (mTOR) with rapamycin (10-100 nmol/L) had no significant effect on HIF-1alpha induction in a variety of cell lines, a finding that was confirmed using mTOR siRNA. Furthermore, neither mTOR siRNA nor rapamycin decreased HIF-1alpha translation as determined by metabolic labeling studies. Therefore, our results indicate that Akt can augment HIF-1alpha expression by increasing its translation under both normoxic and hypoxic conditions; however, the pathway we are investigating seems to be rapamycin insensitive and mTOR independent. These observations, which were made on cells grown in standard tissue culture medium (10% serum), were confirmed in PC3 prostate carcinoma cells. We did find that rapamycin could decrease HIF-1alpha expression when cells were cultured in low serum, but this seems to represent a different pathway.

Cell Line, Tumor↗
PubMed2000

Cell swelling-induced translocation of rat liver Na(+)/taurocholate cotransport polypeptide is mediated via the phosphoinositide 3-kinase signaling pathway.

Cell swelling stimulates phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) in hepatocytes, and the PI3K signaling pathway is involved in cAMP-mediated translocation of sinusoidal Na(+)/taurocholate (TC) cotransporter (Ntcp) to the plasma membrane. We determined whether cell swelling also stimulates TC uptake and Ntcp translocation via the PI3K and/or MAPK signaling pathway. All studies were conducted in isolated rat hepatocytes. Hepatocyte swelling induced by hypotonic media resulted in: 1) time- and medium osmolarity-dependent increases in TC uptake, 2) an increase in the V(max) of Na(+)/TC cotransport, and 3) wortmannin-sensitive increases in TC uptake and plasma membrane Ntcp mass. Hepatocyte swelling also induced wortmannin-sensitive activation of PI3K, protein kinase B, and p70(S6K). Rapamycin, an inhibitor of p70(S6K), inhibited cell swelling-induced activation of p70(S6K) but failed to inhibit cell swelling-induced stimulation of TC uptake. Because PD98095, an inhibitor of MAPK, did not inhibit cell swelling-induced increases in TC uptake, it is unlikely that the effect of cell swelling on TC uptake is mediated via the MAPK signaling pathway. Taken together, these results indicate that 1) cell swelling stimulates TC uptake by translocating Ntcp to the plasma membrane, 2) this effect is mediated via the PI3K, but not MAPK, signaling pathway, and 3) protein kinase B, but not p70(S6K), is a likely downstream effector of PI3K.

Animals↗
PubMed2006

Activation of Akt independent of PTEN and CTMP tumor-suppressor gene mutations in epilepsy-associated Taylor-type focal cortical dysplasias.

Focal cortical dysplasias (FCD) with Taylor-type balloon cells (FCD(IIb)) are frequently observed in biopsy specimens of patients with pharmacoresistant focal epilepsies. The molecular pathogenesis of FCD(IIb), which lack familial inheritance, is only poorly understood. Due to their highly differentiated, malformative nature and glioneuronal phenotype, FCD(IIb) share neuropathological characteristics with lesions observed in familial disorders such as cortical tubers present in patients with autosomal dominant tuberous sclerosis complex (TSC), related to mutations in the TSC1 or TSC2 genes, and dysplastic gangliocytomas of the cerebellum found in Cowden disease. Current data have indicated distinct allelic variants of TSC1 to accumulate in FCD(IIb). TSC1 represents a tumor suppressor operating in the phosphatidylinositol 3-kinase (PI3K)/insulin pathway. The tumor-suppressor gene PTEN is mutated in Cowden disease. Like PTEN, also carboxyl-terminal modulator protein (CTMP) modulates PI3K-pathway signaling, both via inhibition of Akt/PKB, a kinase inactivating the TSC1/TSC2 complex. Here, we have analyzed alterations of Akt, PTEN and CTMP relevant for insulin signaling upstream of TSC1/TSC2 in FCD(IIb). Immunohistochemistry with antibodies against phosphorylated Akt (phospho-Akt; Ser 473) in FCD(IIb) (n=23) showed strong phospho-Akt expression in dysplastic FCD(IIb) components. We have further studied sequence alterations of PTEN (n=34 FCD(IIb)) and CTMP (n=20 FCD(IIb)) by laser microdissection/single-strand conformation polymorphism analysis. We observed a somatic mutation in an FCD(IIb), i.e., amino-acid exchange at nucleotide position 834 (PTEN cDNA, GenBank AH007803.1) in exon 8 with replacement of phenylalanine by leucine (F278L). We also found several silent polymorphisms of PTEN in exon 2 and exon 8 as well as silent and coding polymorphisms but no mutations in CTMP. No loss of heterozygosity in FCD(IIb) (n=6) at 10q23 was observed. To our knowledge, we here report on the first somatic mutation of a tumor-suppressor gene, i.e., PTEN, in FCD(IIb). However, our study also demonstrates that mutational alterations of PTEN and CTMP do not play major pathogenetic roles for activation of Akt in FCD(IIb). Future studies need to determine the origin of insulin pathway activation upstream of TSC1/TSC2 in FCD(IIb).

Adaptor Proteins, Signal Transducing↗
PubMed2001

Role of phosphoinositide 3-kinase in monocyte recruitment under flow conditions.

Chemokines such as the monocyte chemol attractant protein-1 (MCP-1) convert monocyte rolling to firm arrest under physiological flow conditions via integrin activation and simultaneously activate phosphoinositide 3-kinase (PI3K). Here we used adenoviral gene transfer and biochemical inhibitors to manipulate PI3K-dependent pathways in human monocytes. In in vitro lipid kinase assays from purified human monocytes, we showed that MCP-1 activates the "classical" PI3Kalpha pathway and not PI3Kgamma, a PI3K isoform thought to be activated only by the betagamma complex of heterotrimeric G proteins. The activity of PI3Kalpha in purified human monocytes was evident within 30 s. MCP-1-induced monocyte arrest was significantly inhibited both by wortmannin (n = 4; p < 0.01) and LY294002 (n = 4; p < 0.01) with restoration of the rolling phenotype (p < 0.05 for both inhibitors, compared with rolling of control monocytes after MCP-1 treatment). To test the hypothesis that activation of PI3K is sufficient to induce monocyte adhesion, we transduced the monocytic THP-1 cell line with a recombinant adenovirus (Ad) carrying a constitutively active mutant of PI3K (Ad.BD110). We examined the ability of these cells to adhere to human vascular endothelium (HUVEC) transduced with adenoviruses carrying E-selectin, intercellular adhesion molecule-1 (ICAM-1), and VCAM-1. Under flow conditions, ICAM-1- and VCAM-1-dependent firm adhesion of Ad.BD110-transduced THP-1 cells was enhanced compared with THP-1 cells infected with control Ad (n = 4; p < 0.01 for both). Adhesion augmented by constitutive PI3K activation was entirely abrogated by pretreatment with wortmannin (n = 3; p < 0.01). In contrast, a constitutively active Akt construct had no effect on THP-1 adhesion (n = 3; p = NS). We conclude that PI3K activation is necessary and sufficient to enhance monocytic adhesion under physiological flow conditions. BD110-expressing THP-1 cells should provide a useful tool for identifying the signaling pathways downstream of PI3K that are necessary for monocyte recruitment relevant to a variety of human vascular pathologies.

Cell Line↗
PubMed2001

Interactions between the PI3K and Raf signaling pathways can result in the transformation of hematopoietic cells.

The PI3K/Akt and Raf/MEK/ERK signal transduction cascades are pivotal in transmitting signals from membrane receptors to downstream targets that regulate apoptosis, gene expression, and cell growth. The abilities of activated PI3K, Akt, Raf, and MEK proteins to abrogate the cytokine dependence of three different hematopoietic cell lines were determined. Activated PI3K or Akt expression by themselves did not efficiently annul cytokine dependence. Raf and MEK could abrogate the cytokine dependence of murine FDC-PI and human TF-1 cells; however, the frequency of transformation was dependent on the particular oncogene examined, as more factor-independent cells were isolated after infection with activated retroviruses encoding A-Raf or Raf-1 than were with MEK1 or B-Raf. Cytokine-independent deltaRaf-1-infected cells formed tumors on injection into immunocompromised mice, whereas cytokine-dependent cell lines did not, demonstrating the oncogenic effects of activation of the Raf/MEK/ERK pathway. Overexpression of the antiapoptotic Bcl-2 protein synergized with activation of the Raf/MEK/ERK cascade and increased the efficiency of transformation of FDC-PI and TF-1 cells. In contrast to the results observed with FDC-P1 and TF-I cells, the activated Raf genes did not relieve the cytokine dependence of murine FL5.12 cells. The abilities of the Raf and PI3K pathways to interact and annul the cytokine dependence of FL5.12 cells were determined. The combination of Raf and either PI3K or Akt expression relieved cytokine dependence of some FL5.12 cells, and the efficiency of transformation could be enhanced further by Bcl-2 or Bcl-XL overexpression. Thus, the antiapoptotic PI3K/Akt and Bcl-2/Bcl-XL proteins can interact with the growth-promoting Raf/MEK/ERK pathway and annul the cytokine dependence of certain hematopoietic cells.

Animals↗
PubMed2004

Polarity and proliferation are controlled by distinct signaling pathways downstream of PI3-kinase in breast epithelial tumor cells.

Loss of tissue polarity and increased proliferation are the characteristic alterations of the breast tumor phenotype. To investigate these processes, we used a three-dimensional (3D) culture system in which malignant human breast cells can be reverted to a normal phenotype by exposure to inhibitors of phosphatidylinositol 3-kinase (PI3K). Using this assay, we find that Akt and Rac1 act as downstream effectors of PI3K and function as control points of cellular proliferation and tissue polarity, respectively. Our results also demonstrate that the PI3K signaling pathway is an integral component of the overall signaling network induced by growth in 3D, as reversion affected by inhibition of PI3K signaling also down-modulates the endogenous levels of beta1 integrin and epidermal growth factor receptor, the upstream modulators of PI3K, and up-regulates PTEN, the antagonist of PI3K. These findings reveal key events of the PI3K pathway that play distinct roles to maintain tissue polarity and that when disrupted are instrumental in the malignant phenotype.

Actins↗
PubMed2005

Stable activation of phosphatidylinositol 3-kinase in the T cell immunological synapse stimulates Akt signaling to FoxO1 nuclear exclusion and cell growth control.

We have previously reported at the single cell level that PI3K is activated after conjugate formation between T lymphocytes and APCs. However, in contrast to cells exposed to an asymmetrical signal that usually increase 3'-phosphoinositides (3'-PI) transiently in the region of the activated receptors, T cells contacting APC accumulate 3'-PI across their whole plasma membrane far beyond the region of the immunological synapse (IS). Importantly, this effect is maintained over time, for hours, and although PI3K-dependent pathways translate in various cell types extracellular stimuli into a wide range of biological events, in primary T cells this stability is mostly required for cell division induced by Ag. Using imaging methodologies, the present article elucidates the molecular mechanisms responsible for this particular functioning of the PI3K pathway in primary human T lymphocytes interacting with APCs, especially with dendritic cells. The results reveal that the IS unremittingly recruits PI3K to maintain high 3'-PI levels in T cells through phosphotyrosine-dependent mechanisms, suggesting a major participation of class Ia PI3K. This persistent activation of PI3K results in the Akt-dependent sequestration of the FoxO transcription factor, FoxO1, outside the nucleus of T cells interacting with APCs. Using an active form of FoxO1, we demonstrate that this compartmentalization process can affect T cell growth after Ag recognition. We conclude that the need for sustained PI3K signaling within the consolidated IS is probably an undemanding tactic used by primary T cells critical for initiating cell cycle progression through the prolonged inactivation of FoxO1, one important factor that can control cell quiescence.

Active Transport, Cell Nucleus↗
PubMed2006

Oncogenic PI3K and its role in cancer.

PURPOSE OF REVIEW: The purpose of this review is to examine the contribution of the PI3K signaling pathway to the development of human tumors and to propose further studies to elucidate how to develop therapeutics for patients with mutations in this pathway. RECENT FINDINGS: More than 30% of various solid tumor types were recently found to contain mutations in PIK3CA, the catalytic subunit of PI3K. Further analysis of key genes in this pathway identified an additional eight genes altered in tumors. These were generally found to be mutated in a mutually exclusive manner, thus increasing the mutation frequency of the pathway to 40% in colorectal cancers and emphasizing the importance of the PI3K pathway in tumorigenesis. Functional analyses of PIK3CA mutations revealed that they increase its enzymatic activity, stimulate AKT signaling, allow growth factor-independent growth as well as increasing cell invasion and metastasis. SUMMARY: The PI3K signaling pathway is dysregulated by a variety of mechanisms in a large fraction of human tumors. Both mutational and functional analyses have shown that PIK3CA is an oncogene that plays an important role in tumor progression. Mutant members of the PI3K pathway, including PIK3CA, are good targets for therapeutic intervention because most of them are kinases, making them attractive for drug development. Gaining further insights into PIK3CA oncogenic mechanisms may produce new biomarkers and help the development of targeted therapeutics.

Cell Transformation, Neoplastic↗
PubMed2006

A Phosphatidylinositol 3-kinase-regulated Akt-independent signaling promotes cigarette smoke-induced FRA-1 expression.

The FRA-1 proto-oncogene is overexpressed in a variety of human tumors and is known to up-regulate the expression of genes involved in tumor progression and invasion. The phosphatidylinositol 3-kinase (PI3K)-Akt pathway is also known to regulate these cellular processes. More importantly, respiratory toxicants and carcinogens activate both the PI3K-Akt pathway and FRA-1 expression in human bronchial epithelial (HBE) cells. In this study we investigated a potential link between the PI3K-Akt pathway and the cigarette smoke (CS)-stimulated epidermal growth factor receptor-mediated FRA-1 induction in non-oncogenic HBE cells. Treatment of cells with LY294002, an inhibitor of the PI3K-Akt pathway, completely blocked CS-induced FRA-1 expression. Surprisingly pharmacological inhibition of Akt had no significant effect on CS-induced FRA-1 expression. Likewise the inhibition of protein kinase C zeta, which is a known downstream effector of PI3K, did not alter FRA-1 expression. We found that the PI3K through p21-activated kinase 1 regulates FRA-1 proto-oncogene induction by CS and the subsequent activation of the Elk1 and cAMP-response element-binding protein transcription factors that are bound to the promoter in HBE cells.

Carcinogens↗
PubMed2006

Emodin negatively affects the phosphoinositide 3-kinase/AKT signalling pathway: a study on its mechanism of action.

The development of selective cell-permeable inhibitors of protein kinases whose aberrant activation contributes to cell transformation is a promising approach in cancer treatment. Emodin is a natural anthraquinone derivative that exhibits anti-proliferative effects in various cancer cell lines by efficient induction of apoptosis. The phosphoinositide 3-kinase (PI3K)/AKT pathway has been shown to be central in the promotion of cell survival since the alteration of this signalling cascade is a frequent event in human malignancies. Previous published results indicated that treatment of cells with inhibitors of protein kinase CK2, such as emodin, induces apoptosis and that the anti-apoptotic effect of CK2 is partially mediated by target phosphorylation and up-regulation of AKT by CK2. In the present study, a screening with selected CK2 inhibitors induced a variable response with respect to AKT down-regulation, emodin being the most effective, suggesting that other mechanisms other than the inhibition of CK2 were responsible for the emodin-mediated modulation of AKT. We found that emodin does not directly affect AKT kinase. Furthermore, we show that the down-regulation of AKT is due to the emodin-mediated target inhibition of components of the PI3K pathway, which directly or indirectly affect AKT activity, i.e. the mammalian target of rapamycin and the phosphatase and tensin homolog deleted on chromosome 10, but not the phosphoinositide-dependent kinase 1. Taken together, our results highlight a new mechanism by which emodin exerts anti-cancer activity and suggest the further investigation of plant polyphenols, such as emodin, as therapeutic and preventive agents for cancer therapy.

Antineoplastic Agents, Phytogenic↗
PubMed2005

Heat shock protein 90 inhibitor 17-allylamino-17-demethoxygeldanamycin potentiates the radiation response of tumor cells grown as monolayer cultures and spheroids by inducing apoptosis.

Activation of the PI3K-Akt pathway is known to induce tumor radioresistance. In the current study, we examined the ability of 17AAG, which decreases the levels of Hsp90 client proteins including components of the PI3K-Akt pathway, to sensitize radioresistant human squamous cell carcinoma cells to X-irradiation. Human squamous cell carcinoma cell lines (SQ20B, SCC61 and SCC13) were incubated for 16 h at 37 degrees C in medium containing 17AAG. Radiation sensitivity was determined by clonogenic assays, and protein levels were examined by western blotting. Apoptosis was determined in monolayer cells by AO/EB double staining and in spheroids using the TdT-mediated dUTP nick end labeling assay. 17AAG (0.2 microM) enhanced the radiosensitivity more effectively in radioresistant SQ20B and SCC13 cells than in radiosensitive SCC61 cells. However, in all three cell lines, 17AAG increased radiation-induced apoptosis by reducing the expression of EGFR and ErbB-2 and inhibiting the phosphorylation of Akt. Furthermore, 17AAG (1 microM) sensitized SQ20B spheroids to radiation, and inhibition of Akt activation by 17AAG increased radiation-induced apoptosis in spheroids. The findings suggest that 17AAG effectively sensitizes radioresistant cells to radiation by inhibiting the PI3K-Akt pathway. Targeting the PI3K-Akt pathway with 17AAG could be a useful strategy for radiosensitization of carcinomas.

Apoptosis↗
PubMed2006

Phosphatidylinositol-3-phosphate kinase pathway activation protects leukemic large granular lymphocytes from undergoing homeostatic apoptosis.

T-cell large granular lymphocytic leukemia (T-LGL) is characterized by chronic clonal lymphoproliferation of cytotoxic T lymphocytes (CTLs). Despite exhibiting phenotypic properties of antigen-activated cells, including expression of Fas and FasL, T-LGL cells accumulate and demonstrate resistance to apoptosis. We propose that increased activity of a prosurvival signaling pathway in T-LGL is responsible for attenuated apoptosis in T-LGL. Given the importance of the phosphatidylinositol-3 kinase (PI3K)-AKT pathway in regulating the balance between survival and apoptosis, we analyzed AKT activity in T-LGL cells. Compared with resting CTLs from healthy donors, patients' T-LGL cells showed higher levels of phosphorylated AKT. We demonstrate that phospho-AKT induction is dependent on the upstream activity of a Src family kinase. Since the PI3K-AKT pathway can antagonize the ability of Fas to initiate apoptosis, we hypothesized that inhibition of PI3K would lead to reacquisition of Fas sensitivity in T-LGL. Inhibition of the PI3K-AKT pathway alone led to brisk spontaneous apoptosis of T-LGL. These results suggest that T-LGL pathogenesis is dependent on activity of the PI3K-AKT pathway, without which the leukemic cells will begin to undergo spontaneous apoptosis. We propose that novel therapeutics inhibiting the PI3K-AKT axis may provide effective treatment for T-LGL.

Apoptosis↗
PubMed2005

Survivin contributes to the anti-apoptotic activities of transforming growth factor alpha in mouse blastocysts through phosphatidylinositol 3'-kinase pathway.

Transforming growth factor alpha (TGFA) is produced by epithelial cells in the oviducts and uteri and has the potential to act as an anti-apoptotic factor on preimplantation embryos expressing its receptor. Previously, we demonstrated that survivin (also known as BIRC5), an anti-apoptotic gene expressed in mouse preimplantation embryos, protects embryos from apoptosis. In this study, we investigated the role of survivin on TGFA-mediated inhibition of apoptosis in mouse blastocysts. Under the suboptimal conditions produced by single embryo culture, blastocysts showed an increase of apoptosis that correlated with a decrease of survivin expression. TGFA treatment significantly decreased apoptosis and increased the levels of survivin mRNA in a dose-dependent manner in blastocyst, and conversely, these activities were neutralized by an anti-TGFA antibody. Antibody treatment alone exerted little effect on either the occurrence of apoptosis or the levels of survivin mRNA. Upregulation of survivin expression by TGFA treatment was insignificant before the blastocyst stage. Using an antisense approach, we examined whether upregulation of survivin is responsible for the anti-apoptotic effect of TGFA in blastocysts. Apoptosis was inhibited by TGFA treatment in blastocysts, but the effect was abrogated by cotreatment with antisense oligonucleotides directed against survivin. These data suggest that survivin contributes to the anti-apoptotic activities of TGFA in blastocysts. We also found that the upregulation of survivin expression was mediated by activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Thus, TGFA inhibits apoptosis in mouse blastocysts through upregulation of survivin expression via the PI3K pathway.

Animals↗
PubMed2006

Induction of Krox-24 by endogenous cannabinoid type 1 receptors in Neuro2A cells is mediated by the MEK-ERK MAPK pathway and is suppressed by the phosphatidylinositol 3-kinase pathway.

Neuro2a cells endogenously express cannabinoid type 1 (CB1) receptors. CB1 stimulation with HU210 activated ERK and induced the transcription factor Krox-24. A functional MEK-ERK pathway is an important requirement for CB1-mediated Krox-24 induction as blockade of MEK signaling by UO126 reduces both basal and CB1-mediated activation of Krox-24. CB1 receptor stimulation did not activate either JNK or p38 MAPK pathways or the pro-proliferation phosphatidylinositol 3-kinase (PI3K)-Akt pathway. However, serum removal or blockade of PI3K signaling by LY294002 transiently stimulated basal Krox-24 expression and increased CB1-mediated induction of Krox-24. This was consistent with a transient increase in pMEK, pERK, and pCREB levels following PI3K blockade. These data demonstrate that CB1-mediated activation of the Krox-24 transcription factor is negatively regulated through the PI3K-Akt pathway and reveals several points of signaling cross-talk between these two important kinase pathways.

Animals↗
PubMed2004

Importance of Akt signaling pathway for apoptosis in SARS-CoV-infected Vero E6 cells.

Severe acute respiratory syndrome (SARS) is an acute respiratory tract infectious disease that is associated with a new coronavirus (SARS-CoV). Our recent study indicated that SARS-CoV infection induces activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway and the p38 MAPK inhibitor partially inhibited its cytopathic effect in Vero E6 cells. The results of the present study indicated that before cell death, Akt, which is an inhibitor of apoptosis, was also activated in response to viral replication. Phosphorylation of a serine residue on Akt was detected at least 8 h postinfection (hpi), which declined after 18 hpi. Thus, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is activated in virus-infected Vero E6 cells. However, a threonine residue was not phosphorylated. A downstream target of Akt, glycogen synthase kinase 3beta (GSK-3beta), was slightly phosphorylated, indicating that the level of activation of Akt was very low. PKCzeta, which is downstream of the PI3K pathway, was also phosphorylated in virus-infected cells. These results suggested that weak activation of Akt cannot prevent apoptosis induced by SARS-CoV infection in Vero E6 cells.

Animals↗
PubMed2003

Up-regulation of interleukin-8 by novel small cytoplasmic molecules of nontypeable Haemophilus influenzae via p38 and extracellular signal-regulated kinase pathways.

Nontypeable Haemophilus influenzae (NTHI) is an important etiological agent of otitis media (OM) and of exacerbated chronic obstructive pulmonary diseases (COPD). Inflammation is a hallmark of both diseases. Interleukin-8 (IL-8), one of the important inflammatory mediators, is induced by NTHI and may play a significant role in the pathogenesis of these diseases. Our studies demonstrated that a soluble cytoplasmic fraction (SCF) from NTHI induced much greater IL-8 expression by human epithelial cells than did NTHI lipooligosaccharides and envelope proteins. The IL-8-inducing activity was associated with molecules of < or =3 kDa from SCF and was peptidase and lipase sensitive, suggesting that small lipopeptides are responsible for the strong IL-8 induction. Moreover, multiple intracellular signaling pathways were activated in response to cytoplasmic molecules. The results indicated that the p38 mitogen-activated protein kinase (MAPK) and Src-dependent Raf-1-Mek1/2-extracellular signal-regulated kinase mitogen-activated protein kinase (ERK MAPK) pathways are required for NTHI-induced IL-8 production. In contrast, the phosphatidylinositol 3-kinase (PI3K)-Akt pathway did not affect IL-8 expression, although this pathway was concomitantly activated upon exposure to NTHI SCF. The PI3K-Akt pathway was also directly activated by IL-8 and significantly inhibited by an antagonist of IL-8 receptors during NTHI stimulation. These results indicated that the PI3K-Akt pathway is activated in response to IL-8 that is induced by NTHI and may lead to other important epithelial cell responses. This work provides insight into essential molecular and cellular events that may impact on the pathogenesis of OM and COPD and identifies rational targets for anti-inflammatory intervention.

Base Sequence↗