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PubMed2004

Platelet-activating factor mediates Porphyromonas gingivalis lipopolysaccharide interference with salivary mucin synthesis via phosphatidylinositol 3-kinase-dependent constitutive nitric-oxide synthase activation.

Platelet -activating factor (PAF), a phospholipid-derived messenger molecule, is now recognized as the most proximal mediator of cellular events triggered by bacterial lipopolysaccharide (LPS) stimulation. In this study, we assessed the role of PAF in the disturbances in salivary mucin synthesis evoked by LPS of periodontopathic bacterium, P. gingivalis. Using primary culture of mucous acinar cells of sublingual salivary gland, we show that a specific PAF antagonist, BN52020, prevents in a dose-dependent fashion (up to 83.7%) the LPS-induced reduction in mucin synthesis, and the effect is reflected in a marked decrease in the LPS-induced apoptosis (74.8%), NO generation (82.6%), and the expression of TNF-alpha (76.1%). The impedance by BN52020 of the LPS inhibitory effect on mucin synthesis was blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), which also obviated the inhibitory effect of BN52020 on the LPS-induced upregulation in apoptosis, TNF-alpha, and NO. A potentiation in the impedance by BN52020 of the LPS detrimental effect on mucin synthesis was however attained with NOS-2 inhibitor, 1400W, while cNOS inhibitor, L-NNA caused a reduction in the impedance effect of BN52020. However, while 1400W and BN52020 countered the potentiating effect of wortmannin on the LPS-induced decrease in mucin synthesis, a further exacerbation of the effect of wortmannin occurred in the presence of L-NNA. The findings implicate PAF as a pivotal factor affecting the extent of pathological consequences of P. gingivalis infection on salivary glands capacity for mucin production, and suggest that its release in response to the LPS serves as a negative regulator of PI3K controlling the pathway of cNOS activation.

Amidines↗
PubMed2006

Mcl-1 is an anti-apoptotic factor for human hepatocellular carcinoma.

Defects in apoptosis signaling in hepatocytes contribute to tumorigenesis in hepatocellular carcinoma (HCC). In addition, treatment with chemotherapeutic drugs is often ineffective in HCC patients due to the apoptosis resistance of cancer cells. Anti-apoptotic members of the Bcl-2 family, including myeloid cell leukemia-1 (Mcl-1), which regulate intrinsic apoptosis induction at the mito-chondrial level, are often overexpressed in human cancer, and are implicated with disease grade and prognosis. Yet, little is known about the role of Mcl-1 in HCC. In this study, we analyzed the relevance of Mcl-1 expression for the apop-tosis resistance of human HCC. Mcl-1 protein expression was considerably enhanced in human HCC tissue compared to adjacent non-tumor tissue. In addition, Mcl-1 was prominently expressed in various HCC cell lines. Mcl-1 basal expression is dependent on a functional phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway; treatment of the cells with a specific PI3 kinase inhibitor led to both decreased Mcl-1 expression and a sensitization towards chemotherapeutic drug-induced apoptosis. Furthermore, the hepatocyte growth factor and epidermal growth factor induced Mcl-1 expression in an Akt- and ERK-dependent manner. Finally, specific upregulation of Mcl-1 in HCC cells inhibited chemotherapeutic drug-induced apoptosis. Our data suggest that Mcl-1 is an important factor for the apoptosis resistance of human HCC, and constitutes an interesting target for HCC therapy.

Apoptosis↗
PubMed2006

Osteopontin regulation by protein kinase B (Akt) in HepG2 cells.

AIM: The mechanism responsible for osteopontin regulation is not understood in HepG2 cells. The aim of this study was to investigate the relationship between protein kinase B (Akt), a key gene in PI3K signal transduction pathway, and osteopontin expression. METHODS: HepG2 cells were transfected with constitutively active Akt and dominant negative Akt using lipofectin. The Akt transfection was confirmed by Western blot analysis. Osteopontin expression was detected by both Northern blot and Western blot. RESULTS: Overexpression of exogenous Akt was detected in HepG2 cells by Western blot, indicating that HepG2 cells were successfully transfected with the Akt genes. In serum-free condition, the expression of osteopontin was either low or undetectable in HepG2 cells transfected with vector only, however, the expression increased after transfection of cells with constitutively active Akt. Osteopontin expression decreased when HepG2 cells were transfected with dominant negative Akt. CONCLUSION: Protein kinase B (Akt) gene regulated osteopontin expression in RNA level and protein level, suggesting that osteopontin synthesis can be blocked by inactivation of the Akt gene. This leads to a potential means of intervention for the inhibition of metastases in liver cancer.

Base Sequence↗
PubMed2006

Epithelial-mesenchymal transition induced by the stromal cell-derived factor-1/CXCR4 system in oral squamous cell carcinoma cells.

Epithelial-mesenchymal transition (EMT) refers to critical events occasionally observed during tumor progression, including invasion and metastasis, by which cancer cells acquire a fibroblast-like phenotype. Since the stromal cell-derived factor-1 (SDF-1)/CXCR4 system can facilitate lymph node metastasis in oral squamous cell carcinoma (SCC), we have explored the possibility that this system might be involved in EMT. Oral SCC cells, B88 and HNt, which have functional CXCR4 and lymph node metastatic potential, were found to lose their epithelial cell morphology due to SDF-1. In this context, the downregulation of epithelial markers, cytokeratin, E-cadherin and beta-catenin, and the upregulation of mesenchymal marker, vimentin and snail were detected. Furthermore, upregulation of vimentin by treatment with SDF-1 was impaired by phosphatidylinositol 3 kinase (PI3K) inhibitor Wortmannin, but not by mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor U0126. In the type I collagen embedding culture, SDF-1-treated B88 cells formed protruding extensions, but the effect was impaired by treatment with Wortmannin. These results suggested that EMT induced by the SDF-1/CXCR4 system might be involved in the lymph node metastasis of oral SCCs via activation of PI3K-Akt/PKB pathway.

Biomarkers, Tumor↗
PubMed2006

Involvement of PI3K/PKG/ERK1/2 signaling pathways in cortical neurons to trigger protection by cotreatment of acetyl-L-carnitine and alpha-lipoic acid against HNE-mediated oxidative stress and neurotoxicity: implications for Alzheimer's disease.

Oxidative stress has been shown to underlie neuropathological aspects of Alzheimer's disease (AD). 4-Hydroxy-2-nonenal (HNE) is a highly reactive product of lipid peroxidation of unsaturated lipids. HNE-induced oxidative toxicity is a well-described model of oxidative stress-induced neurodegeneration. GSH plays a key role in antioxidant defense, and HNE exposure causes an initial depletion of GSH that leads to gradual toxic accumulation of reactive oxygen species. In the current study, we investigated whether pretreatment of cortical neurons with acetyl-L-carnitine (ALCAR) and alpha-lipoic acid (LA) plays a protective role in cortical neuronal cells against HNE-mediated oxidative stress and neurotoxicity. Decreased cell survival of neurons treated with HNE correlated with increased protein oxidation (protein carbonyl, 3-nitrotyrosine) and lipid peroxidation (HNE) accumulation. Pretreatment of primary cortical neuronal cultures with ALCAR and LA significantly attenuated HNE-induced cytotoxicity, protein oxidation, lipid peroxidation, and apoptosis in a dose-dependent manner. Additionally, pretreatment of ALCAR and LA also led to elevated cellular GSH and heat shock protein (HSP) levels compared to untreated control cells. We have also determined that pretreatment of neurons with ALCAR and LA leads to the activation of phosphoinositol-3 kinase (PI3K), PKG, and ERK1/2 pathways, which play essential roles in neuronal cell survival. Thus, this study demonstrates a cross talk among the PI3K, PKG, and ERK1/2 pathways in cortical neuronal cultures that contributes to ALCAR and LA-mediated prosurvival signaling mechanisms. This evidence supports the pharmacological potential of cotreatment of ALCAR and LA in the management of neurodegenerative disorders associated with HNE-induced oxidative stress and neurotoxicity, including AD.

Acetylcarnitine↗
PubMed2003

TGF-beta1 modulates Fas (APO-1/CD95)-mediated apoptosis of human pre-B cell lines.

We have previously shown that Fas-induced apoptosis is markedly enhanced by IL-7 in human pre-B but not pro-B cell lines. In addition, pre-B cell receptor (pre-BCR) ligation significantly potentiates the IL-7 effects on Fas-triggered pre-B cell death. We show herein that transforming growth factor (TGF)-beta 1 sharply reduces Fas-induced death rate of pre-B but not pro-B cells. TGF-beta 1 causes inhibition of Fas-mediated disruption of mitochondrial transmembrane potential and cleavage of caspase 8, Bid and caspase 3. Bcl2 expression is markedly increased in TGF-beta 1-treated pre-B cells, whereas cellular FLICE-like inhibitory protein long (c-FLIPL), Bcl-XL, Bax, and Bad expression remains unchanged. TGF-beta 1 causes a selective growth arrest of pre-B cells in G0/G1 phase of the cell cycle and induces a partial down-modulation of both Fas and pre-BCR expression. All TGF-beta 1-mediated effects, but Bcl2 up-regulation, can be reproduced by the LY294002 phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor but not by inhibitors of the MAPK/ERK (MEK) and Janus kinase (Jak)/STAT pathways, which promote cell death. Akt phosphorylation is strongly inhibited by TGF-beta1 in pre-B but not pro-B cells and is not modified by Fas engagement. Altogether, our findings suggest that TGF-beta1 prevents Fas-induced apoptosis of pre-B lines by inhibiting PI3K pathway and by enhancing expression of Bcl2. They also suggest that the PI3K/Akt pathway is involved in the control of Fas and pre-BCR expression, a checkpoint in B cell development.

Apoptosis↗
PubMed2003

The c-Fes protein-tyrosine kinase accelerates NGF-induced differentiation of PC12 cells through a PI3K-dependent mechanism.

The c-fes protooncogene encodes a non-receptor protein-tyrosine kinase (Fes) that has been implicated in the differentiation of myeloid haematopoietic cells. Fes is also expressed in several neuronal cell types and the vascular endothelium, suggestive of a more general function in development. To examine the role of Fes in neuronal differentiation, we investigated the effect of Fes expression on process outgrowth in PC12 cells following stimulation with nerve growth factor (NGF). PC12 cells expressing wild-type and activated mutants of Fes extended processes faster and of greater length than control cells. In contrast, expression of kinase-inactive Fes was without effect, indicating that cooperation with NGF requires Fes kinase activity. Short-term treatment of PC12-Fes cells with NGF enhanced tyrosine phosphorylation of Fes, suggesting upstream regulation by the NGF receptor. Fes-mediated acceleration of neurite outgrowth was blocked by wortmannin and LY294002, implicating phosphatidylinositol 3-kinase (PI3K) activation in the Fes-induced response. In contrast, the MEK inhibitor PD98059 was without effect, suggesting that the Ras-Erk pathway is not involved. These data provide the first evidence that Fes may contribute to morphological differentiation of neuronal cells by enhancing NGF signalling through the PI3K pathway.

Animals↗
PubMed2006

The PI3K inhibitor LY294002 blocks drug export from resistant colon carcinoma cells overexpressing MRP1.

Multidrug resistance may be achieved by the activation of membrane transporters, detoxification, alterations in DNA repair or failure in apoptotic pathways. Recent data have suggested an involvement of mitogenic signalling pathways mediated by Ras and phosphoinositol-3-kinase (PI3K/Akt) in controlling multidrug resistance. Since these pathways are important targets for therapeutic interference, we sought to investigate whether blocking effectors kinases by specific inhibitors would result in a sensitization toward cytotoxic drugs. We found that cotreatment of drug-resistant HT29RDB colon cancer cells with the topoisomerase inhibitor doxorubicin and the PI3K-inhibitor LY294002 resulted in massive apoptosis, while cotreatment with the Mek inhibitors PD98059 or U0126 had no effect. This suggested that the PI3K-pathways controls cell survival and drug resistance in these cells. Besides blocking Akt phosphorylation, the PI3K-inibitor increased the intracellular doxorubicin concentration threefold. LY294002 inhibits drug export in a competitive manner as revealed by measuring drug efflux in the presence and the absence of inhibitor. The efficacy of drug efflux inhibition by LY294002 was similar to that achieved by the MRP1 inhibitors MK571 and genistein. We conclude that the PI3K inhibitor LY294002 may have therapeutic potential when combined with doxorubicin in the treatment of MRP1-mediated drug resistance.

Antibiotics, Antineoplastic↗
PubMed1999

p70 S6 kinase-mediated protein synthesis is a critical step for vascular endothelial cell proliferation.

In this work, we analyzed the role of the PI3K-p70 S6 kinase (S6K) signaling cascade in the stimulation of endothelial cell proliferation. We found that inhibitors of the p42/p44 MAPK pathway (PD98059) and the PI3K-p70 S6K pathway (wortmannin, Ly294002, and rapamycin) all block thymidine incorporation stimulated by fetal calf serum in the resting mouse endothelial cell line 1G11. The action of rapamycin can be generalized, since it completely inhibits the mitogenic effect of fetal calf serum in primary endothelial cell cultures (human umbilical vein endothelial cells) and another established capillary endothelial cell line (LIBE cells). The inhibitory effect of rapamycin is only observed when the inhibitor is added at the early stages of G(0)-G(1) progression, suggesting an inhibitory action early in G(1). Rapamycin completely inhibits growth factor stimulation of protein synthesis, which perfectly correlates with the inhibition of cell proliferation. In accordance with its inhibitory action on protein synthesis, activation of cyclin D1 and p21 proteins by growth factors is also blocked by preincubation with rapamycin. Expression of a p70 S6K mutant partially resistant to rapamycin reverses the inhibitory effect of the drug on DNA synthesis, indicating that rapamycin action is via p70 S6K. Thus, in vascular endothelial cells, activation of protein synthesis via p70 S6K is an essential step for cell cycle progression in response to growth factors.

Animals↗
PubMed2000

Stimulation of protein kinase C modulates insulin-like growth factor-1-induced akt activation in PC12 cells.

Activation of protein kinase C (PKC) plays an important role in the negative regulation of receptor signaling, but its effect on insulin-like growth factor-1 (IGF-1) receptor signaling remains unclear. In this study, we characterized the intracellular pathways involved in IGF-1-induced activation of Akt and evaluated the effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) on the Akt activation by IGF-1. IGF-1 induced a time- and concentration-dependent activation of Akt. The effect of IGF-1 was blocked by the phosphatidylinositide 3-kinase (PI3K) inhibitors LY294002 (50 micrometer) and wortmannin (0.5 micrometer), but not by the MEK inhibitor PD98059 (50 micrometer) or the p70 S6 kinase pathway inhibitor rapamycin (50 nm), suggesting that the stimulation of Akt by IGF-1 is mediated by the PI3K pathway. Interestingly, cotreatment with PMA (400 nm) attenuated IGF-1-induced activation of Akt. The attenuation was blocked completely by the PKC inhibitor GO6983 (0.5 micrometer), but only partially by the MEK inhibitor PD98059 (50 micrometer), indicating that MAPK-dependent and -independent pathways are involved. PMA induced the activation of PKC in PC12 cells, and this induction was blocked by GO6983. These data further support the role of PKC in the effect of PMA. Moreover, PKCdelta is likely involved in the action of PMA on the basis of data obtained using isoform-specific inhibitors such as rottlerin. PMA also decreased IGF-1-induced tyrosine phosphorylation of insulin receptor substrate-1 and its association with PI3K. Taken together, these results suggest, for the first time, that stimulation of PKC modulates IGF-1-induced activation of Akt.

Animals↗
PubMed2006

Therapeutic potential of phosphatidylinositol 3-kinase inhibitors in inflammatory respiratory disease.

The phosphoinositide 3-kinase(s) (PI3K) are a family of proteins that catalyze the phosphorylation of the 3-OH position of phosphoinositides and generate lipids that control a wide variety of intracellular signaling pathways. They are classified into three families according to their structure and substrate specificity and are thought to have distinct biological roles. Recent studies suggested that numerous components of the PI3K pathway play a crucial role in the expression and activation of inflammatory mediators, inflammatory cell recruitment, immune cell function, airway remodeling, and corticosteroid insensitivity in chronic inflammatory respiratory disease. Selective PI3K inhibitors have been developed that reduce inflammation and some characteristics of disease in experimental animal models. Targeting specific PI3K isoforms that may be overexpressed or overactive in disease should allow for selective treatment of respiratory diseases. Encouraging data from animal models, primary cells and clinical studies in other diseases suggest that inhibitors of PI3K/Akt may prove to be useful novel therapies in the treatment of asthma and chronic obstructive pulmonary disease.

Animals↗
PubMed2006

Phosphatidylinositol 3-kinase-dependent suppression of the human inducible nitric-oxide synthase promoter is mediated by FKHRL1.

The synthesis of nitric oxide by inducible nitric-oxide synthase (iNOS) plays an important role in the innate immune response by promoting microbial killing and cell damage. In response to inflammatory cytokines and bacterial products, the human iNOS (hiNOS) gene undergoes rapid transcriptional activation via binding of stimulatory transcription factors (e.g. AP-1 and NF-kappaB) to its 5'-flanking region. However, maximal hiNOS promoter induction was suppressed via an unknown phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. We hypothesized that inhibition of the transcription factor FKHRL1 by the PI3K/protein kinase B pathway attenuates hiNOS promoter induction by bacterial lipopolysaccharide and interferon-gamma (LPS/IFN-gamma). Human lung epithelial adenocarcinoma (A549) cells were transiently transfected with an 8.3-kb hiNOS promoter luciferase reporter construct. Co-expression of dominant-negative protein kinase B potentiated LPS/IFN-gamma-stimulated hiNOS promoter activity. In response to LPS/IFN-gamma, FKHRL1 was phosphorylated in a PI3K- and time-dependent fashion. Co-expression of constitutively active FKHRL1 increased hiNOS promoter activity and mRNA levels. Dominant-negative siRNA expression showed that FKHRL1 was necessary for the inhibitory effects of PI3K on hiNOS induction. The same effect was observed upon mutation of a consensus FKHRL1-binding site in the hiNOS promoter. By gel-shift analysis, the corresponding oligonucleotide probe bound endogenous FKHRL1 in an LPS/IFN-gamma- and PI3K-sensitive fashion. Regulation of the hiNOS promoter by FKHRL1 represents a potentially important molecular mechanism by which the PI3K pathway might suppress pro-inflammatory and proapoptotic responses to cytokines and bacterial products.

Androstadienes↗
PubMed2000

Cross-talk between phosphatidylinositol 3-kinase and sphingomyelinase pathways as a mechanism for cell survival/death decisions.

Peptide hormones act to regulate apoptosis through activation of multiple pro- and anti-apoptotic signaling cascades of which lipid signaling events represent an important facet of the cellular rheostat that determines survival and death decisions. Activation of sphingomyelinase, which generates ceramide, is an intermediate in cellular stress responses and induction of apoptosis in many systems. Conversely, phosphatidylinositol 3-kinase (PI3K) is a critical signaling molecule involved in regulating cell survival and proliferation pathways. In the present study, we investigate cross-talk between the PI3K and sphingomyelinase pathways as a mechanism for regulation of cell survival/death decisions. We show that phorbol ester, insulin-like growth factor 1, and a constitutively active PI3K suppress both tumor necrosis factor-induced apoptosis and ceramide generation. Conversely, inhibition of the PI3K pathway with expression of a kinase-dead PI3K both prevented survival signaling and enhanced tumor necrosis factor-induced ceramide generation. The ability of exogenous sphingomyelinase to induce ceramide generation was partially suppressed by expression of constitutively active PI3K and enhanced by inhibition of PI3K suggesting that cross-talk between PI3K and ceramide generation within cells is regulated subsequent to activation of sphingomyelinase.

Apoptosis↗
PubMed2006

[Bezafibrate up-regulate endothelial nitric oxide gene expressions via peroxisome proliferator-activated receptors alpha-dependent and independent pathways in cultured Bovine endothelial cells].

OBJECTIVE: To investigate the effects and related mechanisms of bezafibrate, a ligand of peroxisome proliferator-activated receptors alpha (PPARalpha), on endothelial nitric oxide synthase (eNOS). METHODS: Firstly, in cultured bovine aorta endothelial cells (BAEC), the effects of bezafibrate on eNOS mRNA and protein levels were investigated by Northern blot and Western blot. The half-life of eNOS mRNA and NO production determined from BAECs treated with bezafibrate were performed by real-time quantitative PCR and NO assay. Next, the effects of bezafibrate on signal pathways in BAEC, through inhibitors of PPARalpha, PI3 kinase and MAPK, were investigated by Western blot. Then luciferase reporter gene driven by human eNOS promoter were cloned and transfected endothelial cells to see the effects of bezafibrate on eNOS promoter-driven luciferase activity. RESULTS: In cultured BAEC, bezafibrate significantly upregulated eNOS expressions at protein and mRNA levels in a concentration-dependent fashion (50 - 200 micromol/L) (P < 0.05), and increased nitric oxide (NO) production, respectively[control (14.97 +/- 1.29) micromol/L, different concentration groups (25.12 +/- 1.25) micromol/L, (30.12 +/- 1.85) micromol/L, (33.47 +/- 1.22) micromol/L], and enhanced phosphorylation of eNOS-ser-1179 site (P < 0.05), but not eNOS-thr-497 site. Through real-time quantitative PCR, bezafibrate prolonged eNOS mRNA half-life from 3.1 hour to 6.1 hour were demonstrated. Further studies showed that bezafibrate treatments significantly enhanced dose-dependently luciferase activity (relative luciferase activity in 100 micromol/L and 200 micromol/L groups 4429.43 +/- 391.41 and 6187.5 +/- 307.53), compared with untreated luciferase reporter gene group (3361.81 +/- 316.85) (P < 0.05 and P < 0.01, respectively), and induced MAPK phosphorylation expression (P < 0.05 and P < 0.01, respectively). Then these effects of bezafibrate upregulated eNOS expressions could be blocked by PPARalpha antagonist, MAPK and PI3K inhibitor while not affected by PKC and MEK inhibitor (P < 0.01). CONCLUSIONS: Bezafibrate can upregulate eNOS expression, enhance eNOS-ser-1179 site phosphorylation, increase NO production and stabilize eNOS mRNA through PPARalpha dependent pathway and PPARalpha independent MAPK and PI3K pathways. This mechanism provides additional anti-atherosclerotic and anti-hypertension benefits of bezafibrate in addition of lipid-lowering effects.

Animals↗
PubMed2000

Stimulation of glycogen synthesis by insulin requires S6 kinase and phosphatidylinositol-3-kinase in HTC-IR cells.

In order to study the role of phosphatidylinositol-3-kinase (PI3K), PKB, FRAP, S6 kinase, and MAP kinase in insulin-stimulated glycogen synthesis, we used a specific inhibitor of PI3K, LY294002, the immunosuppressant inhibitor of FRAP, rapamycin, and the inhibitor of MAPK kinase (MEK)/MAPK, PD98059, in rat HTC hepatoma cells overexpressing human insulin receptors. The PI3K inhibitor LY294002 completely blocks insulin-stimulated glycogen synthesis by inhibiting glycogen synthase, PKB (Akt-1), and FRAP (RAFT) autophosphorylation, as well as p70 S6 kinase activation, whereas insulin receptor substrates tyrosine phosphorylation and MEK activity were not affected. However, rapamycin only partially blocks insulin-stimulated glycogen synthesis by partial inhibition of glycogen synthase, whereas it completely blocks S6 kinase activation and FRAP autophosphorylation, but does not affect either PKB autophosphorylation, MEK activity, or insulin receptor tyrosine phosphorylation. Insulin-stimulated glycogen synthesis and glycogen synthase were not affected by the MEK/MAPK inhibitor PD98059. These data suggest that the PI3K, and not the MAPK pathway plays an important role in the insulin-stimulated glycogen synthesis in the hepatocyte, partly mediated by FRAP and S6 kinase activation. However, the inhibition of FRAP and S6 kinase activation is not sufficient to block insulin-stimulated glycogen synthesis, suggesting an important role of a branching pathway upstream of S6 kinase and downstream of PI3K, which is probably mediated by PKB in the signaling of the insulin receptor in hepatoma HTC cells.

Animals↗
PubMed2006

Phosphatidylinositol 3-kinase/protein kinase Akt negatively regulates plasminogen activator inhibitor type 1 expression in vascular endothelial cells.

Plasminogen activator inhibitor type 1 (PAI-1) regulates fibrinolytic activity and mediates vascular atherothrombotic disease. Endothelial cells (ECs) synthesize and secrete PAI-1, but the intracellular signaling pathways that regulate PAI-1 expression are not entirely known. We hypothesize that the phosphatidylinositol 3-kinase (PI3K)/protein kinase Akt pathway, which regulates endothelial function, could modulate PAI-1 expression in ECs. Cultured bovine aortic and human saphenous vein ECs were stimulated with TNF-alpha, ANG II, insulin, or serum, and PAI-1 expression was determined by Northern and Western analyses. Inhibition of PI3K with wortmannin or LY-294002 enhanced PAI-1 expression induced by these extracellular stimuli. Similarly, overexpression of a dominant-negative mutant of PI3K or Akt increased TNF-alpha- and insulin-induced PAI-1 expression. The increase in PAI-1 was due to transcriptional and posttranscriptional mechanisms as PI3K inhibitors increased PAI-1 promoter activity and mRNA stability. The induction of PAI-1 by TNF-alpha and insulin is mediated, in part, by ERK and p38 MAPK. PI3K inhibitors augmented TNF-alpha- and insulin-induced phosphorylation of these MAPKs. Simvastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, which is known to activate PI3K/Akt, blocks TNF-alpha- and insulin-induced PAI-1 expression. Treatment with PI3K inhibitors reversed the inhibitor effects of simvastatin on TNF-alpha- and insulin-induced PAI-1 expression. These findings indicate that the PI3K/Akt pathway acts as a negative regulator of PAI-1 expression in ECs, in part, through the downregulation of MAPK pathways. These results suggest that factors that activate the PI3K/Akt pathway in ECs may have therapeutic benefits for atherothrombotic vascular disease.

Androstadienes↗
PubMed2002

c-erbB2-induced disruption of matrix adhesion and morphogenesis reveals a novel role for protein kinase B as a negative regulator of alpha(2)beta(1) integrin function.

Overexpression of the growth factor receptor subunit c-erbB2, leading to its ligand-independent homodimerization and activation, has been implicated in the pathogenesis of mammary carcinoma. Here, we have examined the effects of c-erbB2 on the adhesive properties of a mammary epithelial cell line, HB2/tnz34, in which c-erbB2 homodimerization can be induced by means of a transfected hybrid "trk-neu" construct. trk-neu consists of the extracellular domain of the trkA nerve growth factor (NGF) receptor fused to the transmembrane and cytoplasmic domains of c-erbB2, allowing NGF-induced c-erbB2 homodimer signaling. Both spreading and adhesion on collagen surfaces were impaired on c-erbB2 activation in HB2/tnz34 cells. Antibody-mediated stimulation of alpha(2)beta(1) integrin function restored adhesion, suggesting a direct role for c-erbB2 in integrin inactivation. Using pharmacological inhibitors and transient transfections, we identified signaling pathways required for suppression of integrin function by c-erbB2. Among these was the MEK-ERK pathway, previously implicated in integrin inactivation. However, we could also show that downstream of phosphoinositide-3-kinase (PI3K), protein kinase B (PKB) acted as a previously unknown, potent inhibitor of integrin function and mediator of the disruptive effects of c-erbB2 on adhesion and morphogenesis. The integrin-linked kinase, previously identified as a PKB coactivator, was also found to be required for integrin inactivation by c-erbB2. In addition, the PI3K-dependent mTOR/S6 kinase pathway was shown to mediate c-erbB2-induced inhibition of adhesion (but not spreading) independently of PKB. Overexpression of MEK1 or PKB suppressed adhesion without requirement for c-erbB2 activation, suggesting that these two pathways partake in integrin inhibition by targeting common downstream effectors. These results demonstrate a major novel role for PI3K and PKB in regulation of integrin function.

Animals↗
PubMed2002

TSH signaling and cell survival in 3T3-L1 preadipocytes.

Thyroid-stimulating hormone (TSH) action in adipose tissue remains largely unknown. Our previous work indicates that human preadipocytes express functional TSH receptor (TSHR) protein, demonstrated by TSH activation of p70 S6 kinase (p70 S6K). We have now studied murine 3T3-L1 preadipocytes to further characterize TSH signaling and cellular action. Western blot analysis of 3T3-L1 preadipocyte lysate revealed the 100-kDa mature processed form of TSHR. TSH activated p70 S6K and protein kinase B (PKB/Akt), as measured by immunoblot analysis. Preincubation with wortmannin or LY-294002 completely blocked TSH activation of p70 S6K and PKB/Akt, implicating phosphoinositide 3-kinase (PI3K) in their regulation. TSH increased phosphotyrosine protein(s) in the 125-kDa region and augmented the associated PI3K activity fourfold. TSH had no effect on cAMP levels in 3T3-L1 preadipocytes, suggesting that adenylyl cyclase is not involved in TSH activation of the PI3K-PKB/Akt-p70 S6K pathway. 3T3-L1 preadipocyte cell death was reduced by 29-76% in serum-deprived (6 h) preadipocytes treated with 1-20 microM TSH. In the presence of 20 microM TSH, an 88% reduction in terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)-positive cells was observed in serum-starved (3 h) 3T3-L1 preadipocytes as well as a 93% reduction in the level of cleaved activated caspase 3. In summary, TSH acts as a survival factor in 3T3-L1 preadipocytes. TSH does not stimulate cAMP accumulation in these cells but instead activates a PI3K-PKB/Akt-p70 S6K pathway.

3T3 Cells↗