Dominant-negative PI3K Research Articles

Pathways to precision medicine: deciphering the secrets of physiological and pathological atrial enlargement.

Cardiac functional, morphological, and histological analysis, coupled with liquid chromatography and mass spectrometry, of two transgenic mouse models with cardiomyocyte-specific overexpression of insulin-like growth factor 1 receptor (IGF1R) or a dominant-negative PI3K mutant (DCM-dnPI3K) revealed distinctive functional and molecular profiles during physiological (driven by IGF1R overexpression) and pathological (driven by dn-PI3K overexpression) atrial remodeling. The current study confirmed previously reported findings, including ventricular dilatation and enhanced systolic function with no evidence of arrhythmia in IGF1R model, as well as ventricular hypertrophy and decreased systolic function with intermittent atrial fibrillation in DCM-dnPI3K model. Novel findings obtained from the left atrial (LA) characterization of female mice revealed that physiological atrial enlargement resulted from increased atrial myocyte size and was associated with preserved atrial function, as determined by maintained LA ejection fraction (EF). The proteomic profile of IGF1R transgenic (Tg) mice was enriched for metabolic remodeling and showed a protein expression pattern similar to that of healthy human atria; on the other hand, pathological atrial enlargement resulted from increased atrial fibrosis with normal myocyte size and was associated with impaired atrial function due to a reduced LA EF. The proteomic profile of DCM-dnPI3K mice was enriched to both metabolic and structural remodeling and showed a protein expression pattern similar to that of human AF atria.

Therapeutic targeting of oxidative stress with coenzyme Q10 counteracts exaggerated diabetic cardiomyopathy in a mouse model of diabetes with diminished PI3K(p110α) signaling

Diabetes-induced cardiac complications include left ventricular (LV) dysfunction and heart failure. We previously demonstrated that LV phosphoinositide 3-kinase p110α (PI3K) protects the heart against diabetic cardiomyopathy, associated with reduced NADPH oxidase expression and activity. Conversely, in dominant negative PI3K(p110α) transgenic mice (dnPI3K), reduced cardiac PI3K signaling exaggerated diabetes-induced cardiomyopathy, associated with upregulated NADPH oxidase. The goal was to examine whether chronic supplementation with the antioxidant coenzyme Q10 (CoQ10) could attenuate LV superoxide and diabetic cardiomyopathy in a setting of impaired PI3K signaling. Diabetes was induced in 6-week-old nontransgenic and dnPI3K male mice via streptozotocin. After 4 weeks of diabetes, CoQ10 supplementation commenced (10mg/kg ip, 3 times/week, 8 weeks). At study end (12 weeks of diabetes), markers of LV function, cardiomyocyte hypertrophy, collagen deposition, NADPH oxidase, oxidative stress (3-nitrotyrosine), and concentrations of CoQ9 and CoQ10 were determined. LV NADPH oxidase (Nox2 gene expression and activity, and lucigenin-enhanced chemiluminescence), as well as oxidative stress, were increased by diabetes, exaggerated in diabetic dnPI3K mice, and attenuated by CoQ10. Diabetes-induced LV diastolic dysfunction (prolonged deceleration time, elevated end-diastolic pressure, impaired E/A ratio), cardiomyocyte hypertrophy and fibrosis, expression of atrial natriuretic peptide, connective tissue growth factor, and β-myosin heavy chain were all attenuated by CoQ10. Chronic CoQ10 supplementation attenuates aspects of diabetic cardiomyopathy, even in a setting of reduced cardiac PI3K protective signaling. Given that CoQ10 supplementation has been suggested to have positive outcomes in heart failure patients, chronic CoQ10 supplementation may be an attractive adjunct therapy for diabetic heart failure.

VEGF Increases Paracellular Permeability in Brain Endothelial Cells via Upregulation of EphA2

Neurological disorders are associated with an increase in the permeability of human brain microvascular endothelial cells (HBMEC). Our previous findings have indicated that EphA2 could increase the permeability of HBMEC. Recent evidence has linked EphA2 and vascular endothelial growth factor (VEGF) to abnormalities in the vascular response. However, it is unclear whether EphA2 is involved in the VEGF-induced changes in the permeability of HBMEC. Here, changes in permeability were determined by measuring transendothelial electrical resistance (TEER) and the flux of FITC-dextran. We found that knockdown of EphA2 in HBMEC abolished the VEGF-induced reduction in TEER and increase in flux of fluorescent dextran. Moreover, VEGF-induced redistribution of ZO-1 and the recruitment of detergent-soluble occludin and claudin-5 were also prevented. Further results showed that VEGF increased EphA2 expression in a time- and dose-dependent manner, which was inhibited by a neutralizing antibody against VEGFR2 or SU1498. VEGF-induced EphA2 expression was suppressed in the brain endothelium following treatments with the PI3K inhibitor LY294002, Akt inhibitor or transfection with the dominant-negative PI3K mutants (Δp110). Similar results were obtained when ERK1/2 activation was inhibited by PD98059 or ERK1/2 siRNA transfection. Our data suggest that VEGF upregulates the expression of EphA2 in HBMEC through binding to VEGFR2 and subsequently activating the intracellular PI3K/Akt and ERK1/2 signaling pathways, which contribute to an increase in paracellular permeability. These data reveal a novel role for VEGF as a regulator of EphA2 expression in the brain endothelial cells and provide insights into the molecular mechanisms of VEGF-mediated changes in paracellular permeability.

PI3K-dependent host cell actin rearrangements are required for Cronobacter sakazakii invasion of human brain microvascular endothelial cells

Cronobacter sakazakii (C. sakazakii) is an opportunistic pathogen that can cause neonatal sepsis and meningitis. The mechanism involved in the pathogenesis of C. sakazakii meningitis remains largely unknown. Previous studies indicated that bacterial invasion of brain microvascular endothelial cells is required for penetration into the central nervous system. In this study, we found that C. sakazakii invasion of human brain microvascular endothelial cells (HBMEC) was significantly inhibited by cytochalasin D, a disrupting agent of actin microfilaments. Disassembly of actin stress fibers and cortical actin fibers was observed in HBMEC infected with C. sakazakii. C. sakazakii infection leads to increased Akt phosphorylation in HBMEC, which was blocked by treatment with PI3K inhibitors. Meanwhile, PI3K and Akt inhibitors significantly inhibited C. sakazakii invasion of HBMEC. Our further results illustrated that the C. sakazakii-induced Akt activation and C. sakazakii invasion were attenuated in HBMEC transfected with dominant-negative PI3K (Δp110). More importantly, the actin filaments rearrangements in HBMEC induced by C. sakazakii were effectively blocked by PI3K inhibitors treatment and transfection with Δp110. Taken together, our findings demonstrated that PI3K-mediated actin rearrangements are required for C. sakazakii invasion of HBMEC.

An Extensive Invasive Intracranial Human Glioblastoma Xenograft Model: Role of High Level Matrix Metalloproteinase 9

The lack of an intracranial human glioma model that recapitulates the extensive invasive and hypervascular features of glioblastoma (GBM) is a major hurdle for testing novel therapeutic approaches against GBM and studying the mechanism of GBM invasive growth. We characterized a high matrix metalloproteinase-9 (MMP-9) expressing U1242 MG intracranial xenograft mouse model that exhibited extensive individual cells and cell clusters in a perivascular and subpial cellular infiltrative pattern, geographic necrosis and infiltrating tumor-induced vascular proliferation closely resembling the human GBM phenotype. MMP-9 silencing cells with short hairpin RNA dramatically blocked the cellular infiltrative pattern, hypervascularity, and cell proliferation in vivo, and decreased cell invasion, colony formation, and cell motility in vitro, indicating that a high level of MMP-9 plays an essential role in extensive infiltration and hypervascularity in the xenograft model. Moreover, epidermal growth factor (EGF) failed to stimulate MMP-9 expression, cell invasion, and colony formation in MMP-9-silenced clones. An EGF receptor (EGFR) kinase inhibitor, a RasN17 dominant-negative construct, MEK and PI3K inhibitors significantly blocked EGF/EGFR-stimulated MMP-9, cell invasion, and colony formation in U1242 MG cells, suggesting that MMP-9 is involved in EGFR/Ras/MEK and PI3K/AKT signaling pathway-mediated cell invasion and anchorage-independent growth in U1242 MG cells. Our data indicate that the U1242 MG xenograft model is valuable for studying GBM extensive invasion and angiogenesis as well as testing anti-invasive and anti-angiogenic therapeutic approaches.

Abstract 3198: Growth factors protect intestinal stem cells from radiation-induced apoptosis by suppressing PUMA through the PI3K/AKT/p53 axis

Abstract Gastrointestinal toxicity is the primary limiting factor in abdominal and pelvic radiotherapy, but has no effective treatment currently. We recently showed a critical role of the BH3-only protein PUMA in acute radiation-induced GI damage and GI syndrome in mice. Growth factors such as IGF-I and bFGF have been shown to protect against radiation-induced intestinal injury, though the underlying mechanisms remain to be identified. We report here the suppression of PUMA through the PI3K/AKT/p53 axis in the intestinal stem cells (ISCs) as a novel molecular mechanism of growth factor-mediated intestinal radioprotection. IGF-I or bFGF impaired radiation-induced apoptosis, and the expression of PUMA and p53 in the crypt cells and intestinal stem cells. Using colonic epithelial cells that undergo PUMA-dependent and radiation-induced apoptosis, we found that a PI3K inhibitor, dominant-negative PI3K, or Mdm2 antagonist restored the induction of PUMA, p53 and apoptosis in the presence of growth factors. In contrast, overexpression of AKT suppressed the induction of PUMA and p53 by radiation. Furthermore, inhibiting PI3K or activating p53 abrogated growth factor-mediated suppression of apoptosis and PUMA expression in the intestinal crypts and stem cells following radiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3198.

Abstract 4864: EGF-induced RNA pol III transcription mediated by epigenetic modification of histone H3

Abstract Cancer cells have a consistent cytological feature of nucleolar hypertrophy, where RNA Pol I and Pol III genes are transcripted. This implies that transformation in situ is closely linked to the deregulation of rRNA transcription, because the size of the nucleolus reflects the levels of rRNA synthesis. RNA Pol III genes encode a variety of untranslated RNAs, including tRNAs and 5S rRNAs. Deregulation of RNA Pol III transcription enhances cellular tRNAs and 5S rRNA production, leading to an increase in translational capacity to promote cell proliferation, transformation, and tumor formation. tRNA gene promoters require the transcription factor complexes, TFIIIB (TBP, Brf1 and Bdp1) and TFIIIC in addition to RNA pol III, to specify accurate and efficient transcription. The TFIIIB subunits, Brf1 and Bdp1, are specifically used in the RNA pol III transcription. Recent study demonstrated that the induction of Brf1 caused an increase in cell proliferation and oncogenic transformation, whereas depletion of Brf1 impeded transformation. These results demonstrate that increases in RNA Pol III transcription are necessary for cellular transformation. Our previous study showed that EGF induces TBP expression through MAP kinases, mediating RNA Pol III transcription. We had identified that MAK kinases mediated phosphorylation of histone H3 at serine 10 and 28. However, it is not clear which upstream of MAP kinases involves in EGF-induced RNA Pol III transcription and whether epigenetic modifications of H3 mediate the induction genes. Our recent studies showed that EGF strongly induces phosphorylation of H3 serine 28 (H3S28). Furhermore, we found that EGFR inhibitor AG1517 reduced EGF-induced both RNA Pol III transcription and phosphorylation of H3S28, but PI3K inhibitor LY 294002 did not decrease the inductions. EGFR deficient blocked the induction of RNA Pol III genes and phosphorylation of H3S28. In contrast, dominant negative mutant PI3K, Δp85 did not repress the inductions by EGF. To further explore the mechanism underlying EGF-induced RNA Pol III transcription, we found that EGF increases TFIIIB subunit, Brf1 expression. To investigate whether the epigenetic modification phosphor-H3S28 mediates RNA Pol III transcription, we determined phosphorylated H3S28 occupancy on Brf1 promoter by using CHIP assay. The result indicates that phospho-H3S28 specifically occupant on Elk-1 binding site of mouse Brf1 promoter at −507/−346, but not at −71/+98, regulating RNA Pol III transcription. Taken together, these results indicate that EGF activates EGFR and induces phosphorylation of H3S28, which mediate Brf1 expression to upregulate RNA Pol III transcription. This project was supported by NIH grant AA017288 to S.Z. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4864.

Abstract 3123: Insulin-like Growth Factor-I Suppresses Bone Morphogenetic Protein Signaling through a Phosphatidylinositol 3-Kinase, Akt and mTOR-Dependant Mechanism

Abstract Bone morphogenetic proteins (BMPs) and insulin-like growth factor-I (IGF-I) function as critical regulators of prostate tumor cell growth. BMPs have recently been shown to be pivotal in controlling prostate tumorigenesis, and loss of BMP receptor function has been correlated to a higher Gleason grade in prostate cancer patients. The mechanism of such loss of BMP function in prostate cancer is unclear, and is likely to occur through multiple mechanisms. One likely candidate we are investigating is the tumor suppressor PTEN, which is lost up to 50% of patients with advanced prostate cancer. Loss of PTEN leads to enhanced activation of PI3K, Akt signaling and suppression of apoptosis. We provide the first evidence that IGF-I intercepts BMP4-induced apoptosis in prostate epithelial cell lines, as shown by changes in cell number, Hoechst 33342 staining, cell viability and flow cytometry. We show that IGF-I suppresses BMP4-induced activation of Smads 1, 5 and/or 8, and that such suppression is reversed by chemical inhibitors of PI3K, Akt or mTOR, or by wild-type PTEN or a dominant-negative PI3K expression construct efficiently delivered to cells by transduction adenoviral. We further show that IGF-I represses BMP4-induced transcriptional activation of the BMP targets, Id-1, Id-2, and Id-3. These results suggest that IGF-I signaling via a PI3K, Akt and mTOR-dependent mechanism inhibits transcriptional activity and apoptosis by BMP4 through suppressing the activation of Smads 1, 5 and/or 8 in prostate epithelial cells. All in all, our findings have important implications in the therapeutic intervention of prostate cancer by restoring BMP-mediated tumor suppressive capabilities by blocking IGF-I signaling pathway's oncogenic activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3123.

PI3Kinase/Akt activation trigger ROS production in a model of pulmonary ischemia

We reported earlier that ischemia or loss of shear in the lung endothelium causes endothelial membrane depolarization (via KATP channel closure) that leads to NADPH oxidase activation and reactive oxygen species (ROS) production. ROS production was abolished in the presence of PI3Kinase/Akt inhibitor, wortmanin. We thus hypothesized that PI3K/Akt activation is the trigger for ROS production with pulmonary ischemia. We used pulmonary microvascular endothelial cells, either wild type (WT), KATP null (KIR6.2−/−), NADPH oxidase null (gp91phox−/−) or WT infected with dominant negative (DN) PI3K, and isolated WT and Akt‐1 null lungs to study signal transduction with ischemia. In WT lungs, the PI3K inhibitor wortmannin had no effect on ischemia mediated depolarization. Cells with DN PI3K and lungs with Akt1−/− depolarized with ischemia but failed to show NADPH oxidase assembly and ROS generation. Depolarization in WT and Akt−/− lungs was blocked upon addition of cromakalim (KATP channel agonist). In WT cells, Akt phosphorylation that was observed with ischemia and was abolished in KIR6.2−/− but not in gp91phox−/− cells. These results indicate that KATP induced depolarization leads to PI3K/Akt activation that triggers NADPH oxidase assembly and eventual ROS generation. Thus, ROS which has hitherto been shown to activate PI3K/Akt in other systems lies downstream of PI3K/Akt in our model of pulmonary ischemia.

Amyloid β Interaction with Receptor for Advanced Glycation End Products Up-Regulates Brain Endothelial CCR5 Expression and Promotes T Cells Crossing the Blood-Brain Barrier

How circulating T cells infiltrate into the brain in Alzheimer disease (AD) remains unclear. We previously reported that amyloid beta (Abeta)-dependent CCR5 expression in brain endothelial cells is involved in T cell transendothelial migration. In this study, we explored the signaling pathway of CCR5 up-regulation by Abeta. We showed that inhibitors of JNK, ERK, and PI3K significantly decreased Abeta-induced CCR5 expression in human brain microvascular endothelial cells (HBMECs). Chromatin immunoprecipitation assay revealed that Abeta-activated JNK, ERK, and PI3K promoted brain endothelial CCR5 expression via transcription factor Egr-1. Furthermore, neutralization Ab of receptor for advanced glycation end products (RAGE; an Abeta receptor) effectively blocked Abeta-induced JNK, ERK, and PI3K activation, contributing to CCR5 expression in HBMECs. Abeta fails to induce CCR5 expression when truncated RAGE was overexpressed in HBMECs. Transendothelial migration assay showed that the migration of MIP-1alpha (a CCR5 ligand)-expressing AD patients' T cells through in vitro blood-brain barrier model was effectively blocked by anti-RAGE Ab, overexpression of truncated RAGE, and dominant-negative PI3K, JNK/ERK, or Egr-1 RNA interference in HBMECs, respectively. Importantly, blockage of intracerebral RAGE abolished the up-regulation of CCR5 on brain endothelial cells and the increased T cell infiltration in the brain induced by Abeta injection in rat hippocampus. Our results suggest that intracerebral Abeta interaction with RAGE at BBB up-regulates endothelial CCR5 expression and causes circulating T cell infiltration in the brain in AD. This study may provide a new insight into the understanding of inflammation in the progress of AD.

Depolarization is the trigger for PI3K/Akt activation and leads to ROS production in a model of pulmonary ischemia

We reported that ischemia or loss of shear in the lung endothelium causes endothelial plasma membrane depolarization (via KATP channel closure) that leads to NADPH oxidase activation and reactive oxygen species (ROS) production. Both high K+ induced depolarization and ischemia resulted in activation of PI3K; its inhibitor (wortmanin) prevented NADPH oxidase assembly and ROS production with ischemia. We used pulmonary microvascular endothelial cells, either wild type (WT), KATP−/−, gp91phox−/− or WT infected with dominant negative (DN) PI3K, and isolated Akt‐1 null lungs to study signal transduction with ischemia. Cells with DN PI3K and lungs with Akt1−/−, both showed the depolarization response but failed to show NADPH oxidase assembly and ROS generation. In WT cells, Akt activation (as monitored by the expression of p‐Akt) that was observed with ischemia was abolished in KATP−/− but not in gp91phox−/− cells. Blocking the ischemic depolarization by addition of cromakalim (KATP channel agonist) to gp91phox−/− cells prevented Akt phosphorylation. These results indicate that KATP induced depolarization leads to NADPH oxidase assembly and ROS generation and precedes PI3K/Akt activation. Thus, ROS which has hitherto been shown to activate PI3K/Akt in other systems lies downstream of PI3K/Akt in our model of pulmonary ischemia.

Induction of serum‐ and glucocorticoid‐induced kinase‐1 (SGK1) by cAMP regulates increases in α‐ENaC

Alpha-ENaC expression and activity is regulated by a variety of hormones including beta-adrenergic agonists via the second messenger cAMP. We evaluated the early intermediate pathways involved in the up-regulation of SGK1 by DbcAMP and whether SGK1 is a prerequisite for induction of alpha-ENaC expression. Submandibular gland epithelial (SMG-C6) cells treated with DbcAMP (1 mM) induced both SGK1 mRNA and protein expression. DbcAMP-stimulated SGK1 mRNA expression was decreased by actinomycin D and mRNA and protein expressions were attenuated by PKA inhibitors (H-89 and KT5720). Inhibition of PI3-K with either LY294002 or dominant negative PI3-K reduced DbcAMP-stimulated SGK1 protein and mRNA levels, attenuated the phosphorylation of CREB (a cAMP-activated transcription factor) and decreased alpha-ENaC protein levels and Na(+) transport. In addition, the combination of PKA inhibitors with dominant negative PI3-K synergistically inhibited DbcAMP-induced Na(+) transport. Inhibition of SGK1 expression by siRNA decreased but did not obliterate DbcAMP-induced alpha-ENaC expression. Thus, in a cell line which endogenously exhibits minimal alpha-ENaC expression, induction of SGK1 by DbcAMP occurs via the PI3-K and PKA pathways. Increased alpha-ENaC levels and function are partly dependent upon the early induction of SGK1 expression.

Role of PI3Kinase/Akt and Rac in activation of the endothelial NADPH oxidase with Ischemia

We have shown previously that ischemia or abrupt cessation of flow in the lung endothelia in situ and in pulmonary microvascular endothelial cells (PMVEC) in vitro causes ROS production. This occured via NADPH oxidase as knockout of gp91phox prevented the response. We also reported that the trigger for NADPH oxidase activation is depolarization of the endothelial cell plasma membrane (via KATP channels) as preventing the depolarization with a channel agonist prevented the ROS generation. In phagocytes, the small G protein, rac (Rac1) is known to participate in NADPH assembly and activation. To investigate if Rac plays a similar role in endothelial NADPH oxidase assembly, we examined the link between endothelial cell membrane depolarization, Rac activation and NADPH oxidase assembly. Using PMVECs transfected with RacGFP, Rac translocation was monitored with membrane depolarization (achieved either by high external K+ or by ischemia). The significant increase in Rac translocation that was observed with depolarization was attenuated by C difficile Toxin B (Rac inhibitor) as also by wortmannin and LY294002 (PI3K inhibitors). PMVECs infected with dominant negative PI3K (mutant PI3K p85 subunit) showed no translocation of Rac to plasma membrane upon depolarization of the cells. These results indicate that PI3K/Akt may be upstream of Rac and that Rac 1 translocation is required for NADPH oxidase activation.

Insulin-like growth factor-I induces cyclooxygenase-2 expression via PI3K, MAPK and PKC signaling pathways in human ovarian cancer cells

Elevated levels of insulin-like growth factor-I (IGF-I) are associated with ovarian carcinogenesis and progression. However, the molecular mechanisms by which IGF-I contributes to ovarian cancer development remain to be elucidated. Cyclooxygenase-2 (COX-2) is a crucial player in the pathogenesis of human malignancies. Herein we showed that IGF-I efficiently induced COX-2 expression and PGE 2 biosynthesis at physiologically relevant concentrations in human ovarian cancer cells. IGF-I treatment significantly increased COX-2 transcriptional activation. IGF-I also stabilized COX-2 mRNA through the COX-2 3′-untranslated region (3′-UTR), which appeared independent of the conserved AU-rich elements. We next investigated the signaling pathways involved in IGF-I-induced COX-2 expression. We found that PI3K inhibitor wortmannin or LY294002 blocked COX-2 expression induced by IGF-I. Wortmannin treatment or a dominant negative PI3K mutant significantly inhibited IGF-I-induced COX-2 mRNA stabilization, but only slightly decreased COX-2 transcriptional activation. We showed that ERK1/2 and p38 MAPKs were required for IGF-I-induced COX-2 expression and that activation of both pathways by IGF-I increased COX-2 transcriptional activation and its mRNA stability. IGF-I stimulated PKC activation in the cells and pretreatment with PKC inhibitor bisindolylmaleimide prevented IGF-I-induced COX-2 transcriptional activation and mRNA stabilization, and inhibited COX-2 mRNA and protein expression. Taken together, our data demonstrate that IGF-I induces COX-2 expression in human ovarian cancer cells, which is mediated by three parallel signaling cascades — PI3K, MAPK, and PKC pathways that differentially regulate COX-2 expression at transcriptional and post-transcriptional levels.

Rapid up-regulation of c-FLIP expression by BCR signaling through the PI3K/Akt pathway inhibits simultaneously induced Fas-mediated apoptosis in murine B lymphocytes

Cross-linking of BCR rapidly induces protection of B cells from Fas-mediated apoptosis, which has been assumed one of the important survival mechanisms of B cells during antigen stimulation. In the mouse B cell line A20, which is sensitive to Fas-mediated apoptosis, stimulation of BCR inhibited apoptosis induced via Fas upstream of caspase-8 activation with an associated rapid increase in the expression of both short and long forms of cellular caspase-8/FLICE-inhibitory protein (c-FLIP). The c-FLIP competitively inhibited the recruitment of caspase-8 to the death-inducing signaling complex (DISC), which took as long as 3 h to form after the stimulation of Fas in A20 cells. Knockdown of c-FLIP by a short hairpin RNA-expressing method rendered BCR-stimulated A20 cells sensitive to Fas-mediated apoptosis. The BCR-induced rapid expression of c-FLIP was not affected by inactivation of NF-κB, but was inhibited by either treatment with a PI3K inhibitor, LY294002, or expression of a dominant negative PI3K p85 subunit, both of which suppressed phosphorylation of Akt and sensitized BCR-stimulated A20 cells to Fas-mediated apoptosis. Overexpression of constitutively active Akt was shown not only to up-regulate c-FLIP expression but also to render A20 cells resistant to Fas-mediated apoptosis. Moreover, treatment with LY294002 also suppressed BCR-induced up-regulation of c-FLIP expression in spleen B cells. Taken together, BCR-stimulation was shown to rapidly trigger a survival signal against simultaneously or ongoingly stimulated Fas-mediated apoptosis by promoting a PI3K/Akt signaling pathway-mediated up-regulation of c-FLIP expression.

Effects of Polycyclic Aromatic Hydrocarbons (PAHs) on Vascular Endothelial Growth Factor Induction through Phosphatidylinositol 3-Kinase/AP-1-dependent, HIF-1α-independent Pathway

Previous studies have demonstrated that exposure to polycyclic aromatic hydrocarbons (PAHs) and its derivatives is associated with an increased risk of skin cancers, and the carcinogenic effect of PAHs is thought to involve both tumor initiation and promotion. Whereas PAH tumor initiation is well characterized, the mechanisms involved in the tumor promotion of PAHs remain elusive. In the present study, we investigated the effects of PAHs on vascular endothelial growth factor (VEGF) expression by comparison of its induction between the active metabolite and its parent compound (B[a]PDE versus B[a]P) or between active compound and its relatively inactive analog (5-MCDE versus CDE). We found that exposure of cells to (+/-)-anti-benzo-[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE) or (+/-)-anti-5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE) led to marked induction of VEGF in Cl41 cells, whereas benzo[a]pyrene (B[a]P) or chrysene-1,2-diol-3,4-epoxide (CDE) did not exhibit significant inductive effects. Exposure of cells to B[a]PDE and 5-MCDE did not induce HIF-1alpha activation, whereas AP-1 was significantly activated. Moreover, overexpression of TAM67 (a dominant-negative mutant c-Jun) dramatically blocked that VEGF induction. Electrophoretic mobility shift assay showed that AP-1 was only able to specifically recognize and bind to its AP-1 potential binding site within -1136 and -1115 of the VEGF promoter region. Site-directed mutation of this AP-1 binding site eliminated the VEGF transcriptional activity induced by B[a]PDE, suggesting that the AP-1 binding site between -1136 and -1115 in the VEGF promoter region is critical for VEGF induction by B[a]PDE. In addition, overexpression of Deltap85 (a dominant-negative mutant PI-3K) impaired B[a]PDE- and 5-MCDE-induced VEGF induction. Considering our previous findings that PI-3K is an upstream mediator for c-Jun/AP-1 activation, we conclude that the VEGF induction by B[a]PDE and 5-MCDE is through PI-3K/AP-1-dependent and HIF-1alpha-independent pathways. These findings may help us to understand the mechanisms involved in PAH carcinogenic effects.

ERKs activation and calcium signaling are both required for VEGF induction by vanadium in mouse epidermal Cl41 cells

The previous studies have demonstrated that vanadium exposure can cause a variety of biological effects. However, the mechanisms involved in the biological effects caused by vanadium are not well understood. Our previous studies have shown that exposure of mouse epidermal Cl 41 cells to vanadate stimulated the phosphorylation of both ERKs and p38K, and calcium signaling leading NFAT activation. In view of the evidence that ERKs and p38 kinase contribute to VEGF induction, we investigated in the present study the potential roles of ERKs, p38K, and calcium signaling in VEGF induction caused by vanadium exposure. Exposure of Cl 41 cells to vanadium led to VEGF induction in both time- and dose-dependent manners. Pre-treatment of Cl 41 cells with PD98059, an inhibitor of MEK1/2-ERKs pathway, but not SB202190, an inhibitor for p38K pathway, resulted in a dramatic inhibition of VEGF induction by vanadium. More interesting, pre-treatment of Cl 41 cells with intracellular calcium chelator, but not calcium channel blocker, resulted in a dramatic decrease in VEGF induction by vanadium. However, both PI-3K inhibitors and overexpression of Deltap85, a dominant negative PI-3K mutant, resulted in only a marginal decrease in VEGF induction by vanadium. Moreover, mTOR, as a downstream molecule of PI-3K, did not attribute to VEGF induction by vanadium because rapamycin pre-treatment did not show any inhibitory effect on VEGF induction. These results indicate that ERKs and intracellular stored calcium release play a critical role in VEGF induction by vanadium. PI-3K is partially involved in VEGF induction by vanadium, while p38K and mTOR are not involved. Those results will help us to understand the molecular mechanisms involved in vanadium-induced biological effects.

Integrin-linked kinase function is required for transforming growth factor β-mediated epithelial to mesenchymal transition

The role of integrin-linked kinase (ILK) in transforming growth factor β (TGFβ)-mediated epithelial to mesenchymal transition was investigated. A stable transfection of dominant-negative ILK results in the prevention of TGFβ-mediated E-cadherin delocalization. TGFβ-mediated phosphorylation of Akt at Ser-473 was inhibited by dominant-negative ILK and PI3K inhibitors, LY294002 and wortmannin. Treatment with TGFβ stimulated induction of Akt and ILK kinase activity in HaCat control cells. This increased ILK activity by TGFβ was lowered by PI3K inhibitor, LY294002. In addition, PI3K inhibitor, dominant-negative Akt, and dominant-negative ILK could not block TGFβ-mediated C-terminal phosphorylation of Smad2. Taken together, these data suggest that PI3K–ILK–Akt pathway that is independent of the TGFβ-induced Smad pathway is required for TGFβ-mediated epithelial to mesenchymal transition.

CXCL16 Signals via Gi, Phosphatidylinositol 3-Kinase, Akt, IκB Kinase, and Nuclear Factor-κB and Induces Cell-Cell Adhesion and Aortic Smooth Muscle Cell Proliferation

CXCL16, a recently discovered transmembrane chemokine, is expressed in human aortic smooth muscle cell (ASMC). It facilitates uptake of low density lipoproteins by macrophages, resulting in foam cell formation. However, it is not known whether ASMC express CXCR6, the receptor for CXCL16, or whether CXCL16 affects ASMC biology. To dissect the biological and signal transduction pathways elicited by CXCL16, human aortic smooth muscle cells (HASMC) were treated with pharmacological inhibitors or transiently transfected with pathway-specific dominant-negative or kinase-dead expression vectors prior to the addition of CXCL16. HASMC expressed CXCR6 at basal conditions. Exposure of HASMC to CXCL16 increased NF-kappa B DNA binding activity, induced kappa B-driven luciferase activity, and up-regulated tumor necrosis factor-alpha expression in an NF-kappa B-dependent manner. However, treatment with pertussis toxin (G(i) inhibitor), wortmannin or LY294002 (phosphatidylinositol 3-kinase (PI3K inhibitors)), or Akt inhibitor or overexpression of dominant-negative (dn) PI3K gamma, dnPDK-1, kinase-dead (kd) Akt, kdIKK-beta, dnIKK-gamma, dnI kappa B-alpha, or dnI kappa B-beta significantly attenuated CXCL16-induced NF-kappa B activation. Furthermore, CXCL16 increased cell-cell adhesion and induced cellular proliferation in an NF-kappa B-dependent manner. In conclusion, CXCL16 is a potent and direct activator of NF-kappaB and induces kappa B-dependent proinflammatory gene transcription. CXCL16-mediated NF-kappa B activation occurred via heterotrimeric G proteins, PI3K, PDK-1, Akt, and I kappa B kinase (IKK). CXCL16 induced I kappa B phosphorylation and degradation. Most importantly, CXCL16 increased cell-cell adhesion and induced kappa B-dependent ASMC proliferation, indicating that CXCL16 may play an important role in the development and progression of atherosclerotic vascular disease.

Necessity of tyrosine 719 and phosphatidylinositol 3′-kinase–mediated signal pathway in constitutive activation and oncogenic potential of c-kit receptor tyrosine kinase with the Asp814Val mutation

AbstractSubstitution of valine (Val) for aspartic acid (Asp) at codon 814 constitutively activates murine c-kit receptor tyrosine kinase (KIT), and Asp816Val mutation, corresponding to murine Asp814Val mutation, is found in patients with mastocytosis and acute myelocytic leukemia. However, the signal transduction pathways responsible for oncogenesis by the Asp814Val mutant (KITVal814) are not fully understood. To examine the oncogenic signal transduction of KITVal814, we converted 20 tyrosine (Tyr) residues to phenylalanine (Phe) in the cytoplasmic domain of KITVal814 or deleted the C-terminal region containing 2 other tyrosine residues (Del). Among various KITVal814- derived mutants, KITVal814-Tyr719Phe and KITVal814-Delseverely impaired receptor tyrosine phosphorylation and association with the p85 subunit of phosphatidylinositol 3′-kinase (p85PI3-K). Moreover, KITVal814-Tyr719Pheand KITVal814-Del failed to induce ligand-independent growth in Ba/F3 cells, indicating that Tyr719, the binding site for p85PI3-K, and the C-terminal region are indispensable for factor-independent growth by KITVal814. Although the C-terminal region was also required for ligand-dependent growth by wild-type KIT (KITWT), the Tyr719Phe substitution had negligible effects on ligand-dependent growth by KITWT. Furthermore, dominant-negative PI3-K significantly inhibited ligand-independent growth by KITVal814. These results demonstrate that Tyr719 is crucial for constitutive activation of KITVal814, but not for the ligand-induced activation of KITWT, and that the downstream signaling of PI3-K plays an important role in ligand-independent growth and tumorigenicity by KITVal814, thereby suggesting that KITVal814 is a unique activating mutation that leads to a distinguishable function from the effects of KITWT.