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PubMed2005

Possible involvement of phosphatidylinositol 3-kinase/Akt signal pathway in vasopressin-induced HSP27 phosphorylation in aortic smooth muscle A10 cells.

We previously reported that p38 mitogen-activated protein (MAP) kinase takes a part in arginine vasopressin (AVP)-induced heat shock protein 27 (HSP27) phosphorylation in aortic smooth muscle A10 cells. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) is involved in the phosphorylation of HSP27 in these cells. AVP time-dependently induced the phosphorylation of PI3K and Akt. Akt inhibitor, 1l-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, partially suppressed the phosphorylation of HSP27. The AVP-induced HSP27 phosphorylation was attenuated by LY294002, a PI3K inhibitor. The combination of Akt inhibitor and SB203580, a p38 MAP kinase inhibitor, completely suppressed the AVP-induced phosphorylation of HSP27. Furthermore, LY294002 or Akt inhibitor did not affect the AVP-induced phosphorylation of p38 MAP kinase and SB203580 did not affect the phosphorylation of PI3K or Akt. These results suggest that PI3K/Akt plays a part in the AVP-induced phosphorylation of HSP27, maybe independently of p38 MAP kinase, in aortic smooth muscle A10 cells.

Animals↗
PubMed2004

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

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

Cell Differentiation↗
PubMed2006

A phosphorylation site in the Toll-like receptor 5 TIR domain is required for inflammatory signalling in response to flagellin.

Flagellin, the major structural subunit of bacterial flagella, potently induces inflammatory responses in mammalian cells by activating Toll-like receptor (TLR) 5. Like other TLRs, TLR5 recruits signalling molecules to its intracellular TIR domain, leading to inflammatory responses. Phosphatidylinositol 3-kinase (PI3K) has been reported to play a role in early TLR signalling. We identified a putative binding site for PI3K at tyrosine 798 in the TLR5 TIR domain, at a site analogous to the PI3K recruitment domain in the interleukin-1 receptor. Mutation of this residue did not affect homodimerization, but prevented inflammatory responses to flagellin. While we did not detect direct interaction of PI3K with TLR5, we demonstrated by mass spectrometry that Y798 is phosphorylated in flagellin-treated HEK 293T cells. Together, these results suggest that phosphorylation of Y798 in TLR5 is required for signalling, but not for TLR5 dimerization.

Amino Acid Sequence↗
PubMed2005

Signalling via the reperfusion injury signalling kinase (RISK) pathway links closure of the mitochondrial permeability transition pore to cardioprotection.

Post-ischemic interventions that activate phosphatidylinositol-3-OH kinase (PI3K)-Akt or ERK1/2 pro-survival kinases (the so-called "reperfusion injury signalling kinase (RISK) pathway") during the first few minutes of reperfusion protect against lethal reperfusion-induced injury. We have previously shown that insulin protects against reperfusion-induced injury via activation of the PI3K-Akt pathway. In addition, opening of the mitochondrial permeability transition pore (mPTP) at the time of reperfusion is a major determinant of lethal reperfusion-induced injury, and pharmacologically inhibiting it is cardioprotective. In this study, we examined the relationship between the pro-survival kinase pathways and mPTP opening. Specifically, we tested the hypothesis that activation of the pro-survival kinase pathway by insulin protects cardiomyocytes by reducing the probability of mPTP opening upon reperfusion. Laser illumination of the fluorophore, tetramethyl rhodamine methyl ester (TMRM), was used to induce oxidative stress in the preparation of adult rat ventricular cardiomyocytes. Maintained illumination ultimately induces mPTP opening, detected as a global mitochondrial depolarization, followed by ATP depletion and rigor contracture. Insulin significantly delayed mPTP opening by a factor of approximately 1.7-fold (P<0.001). The effect of insulin was prevented by Wortmannin and by LY-294002, inhibitors of the PI3K pathway, by SH-6, a selective inhibitor of Akt, and by L-NAME, an inhibitor of nitric oxide production. The expression of a dominant negative construct of Akt eliminated the effect of insulin in delaying mPTP opening in a cardiac cell line. Furthermore, the overexpression of constitutively active Akt was sufficient to maximally delay mPTP opening. These results indicate that activation of the PI3K-Akt pro-survival kinase pathway inhibits opening of the mPTP, and demonstrate an important link between the survival kinases and the mPTP.

Animals↗
PubMed2006

Parathyroid hormone activates phosphoinositide 3-kinase-Akt-Bad cascade in osteoblast-like cells.

To understand the molecular basis underlying the anabolic action of parathyroid hormone (PTH) on bone, the anti-apoptotic action of PTH on osteoblast-like cells was investigated. Since Akt is a key protein kinase for cell survival, we focused on a possible involvement of Akt in the anti-apoptotic action of PTH. Human osteoblast-like MG-63 cells cultured without serum were treated with PTH. Western blot analysis revealed that PTH rapidly phosphorylated Akt and induced its nuclear translocation. The phosphorylation of pro-apoptotic protein Bad was also increased by PTH, leading to its inactivation. The PTH-dependent activation of Akt was also detected in other osteoblastic cell lines, SaOS-2 and ROS 17/2.8. The pretreatment of MG-63 cells with either one of inhibitors for phosphoinositide 3-kinase (PI3K), wortmannin or LY294002 prevented Akt and Bad phosphorylation. Furthermore, co-immunoprecipitation analysis revealed that PTH receptor (PTH-1R) directly interacted with p85, a regulatory subunit of PI3K, in a PTH-dependent manner. Serum withdrawal induced the apoptosis of MG-63 cells, and PTH prevented the apoptosis, which was inhibited by PI3K inhibitors. These results demonstrate the presence of a novel PTH/PTH receptor signaling cascade consisting of PTH-1R, PI3K, Akt and Bad and that this cascade can work as an anti-apoptotic signaling pathway in osteoblast-like cells.

Androstadienes↗
PubMed2004

Involvement of phosphatidylinositol 3-kinase and insulin-like growth factor-I in YXLST-mediated neuroprotection.

In the present study, we examine the neuroprotective role of the external Qi of YXLST in cultured retinal neurons. Primary retinal neuronal cultures were grown from retinas of 0-2-day-old Sprague-Dawley rats. Cultures were treated directly with external Qi of YXLST 30 min prior to H(2)O(2) exposure in most experiments. Cell viability was measured by 3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) assay. Apoptotic cell death was evaluated by the TdT-mediated digoxigenin-dUTP nick-end labeling TUNEL assay, and by DNA laddering analysis. Northern blot analysis was performed to examine the level of insulin-like growth factor-I (IGF-I) gene expression. Phosphatidylinositol 3-kinase (PI3K) assay was performed to study the PI3K activity. The results showed that treatment of external Qi of YXLST significantly attenuated neuronal death that was induced by 24-h exposure to hydrogen peroxide, and greatly inhibited hydrogen peroxide-induced apoptosis. External Qi of YXLST also upregulated IGF-I gene expression and increased PI3K activity. These observations indicate that external Qi-mediated IGF-I expression and PI3K signaling could be one of the mechanisms in neuroprotection by YXLST.

Animals↗
PubMed2005

The neuroprotection of insulin on ischemic brain injury in rat hippocampus through negative regulation of JNK signaling pathway by PI3K/Akt activation.

Current studies demonstrated that cell survival is determined by a balance among signaling cascades, including those that recruit the Akt and JNK pathways. In our present work, the relationship between Akt1 and JNK1/2 was evaluated after cerebral ischemia-reperfusion in the hippocampus in a four-vessel occlusion model of Sprague-Dawley rats. This paper was based on our present and previous studies. Firstly, Akt1 had one active peak during reperfusion following 15 min ischemia. Secondly, two peaks of JNK1/2 activation occurred during reperfusion, respectively. Thirdly, the phosphorylation of JNK substrates c-Jun and Bcl-2, and the activation of a key protease of caspase-3 were detected. They only had one active peak, respectively, during reperfusion. To clarify the mechanism of Akt1 activation and further define whether JNK1/2 activation could be regulated by Akt1 through PI3K pathway, LY294002 and insulin were, respectively, administrated to the rats prior to ischemia. Our research indicated that LY294002, a PI3K inhibitor, significantly suppressed Akt1 activation. Furthermore, LY294002 significantly strengthened both peaks of JNK1/2 activation, c-Jun activation, Bcl-2 phosphorylation, and the activation of caspase-3 during reperfusion. In contrast, insulin, a PI3K agonist, not only obviously activated Akt1 during early and later reperfusion, but also inhibited phosphorylation of JNK1/2, c-Jun, and Bcl-2 and attenuated the activation of caspase-3. In addition, pretreatment of insulin significantly increased the number of the surviving CA1 pyramidal cells at 5 days of reperfusion. Consequently, our results indicated that the cross-talk between Akt1 and JNK1/2 could be mediated by insulin receptor through PI3K in rat hippocampus during reperfusion. This signaling pathway might play a neuroprotective role against ischemic insults via inhibition of the JNK pathway, involving the death effector of caspase-3.

Analysis of Variance↗
PubMed2004

Cross-talk between the survival kinases during early reperfusion: its contribution to ischemic preconditioning.

OBJECTIVES: Recruitment of the survival kinase cascades, PI3K-Akt and Raf-MEK1/2-Erk1/2, at the time of reperfusion, following a lethal ischemic insult, may mediate the protection associated with ischemic preconditioning (IPC). The exact interplay between these two kinase cascades in mediating this effect is not clear. We examine the 'cross-talk' between these kinase cascades in their contribution to IPC-induced protection. METHODS AND RESULTS: In isolated perfused rat hearts subjected to 35 min of lethal ischemia +/- ischemic preconditioning, the phosphorylation states of Akt, Erk1/2, p70S6K were determined after 15 min of reperfusion, and infarct size was measured after 120 min of reperfusion. IPC induced a threefold increase in Akt, Erk1/2, and p70S6K phosphorylation, at reperfusion. We found that inhibiting the PI3K-Akt (using LY294008) at reperfusion induced the phosphorylation of Erk1/2-p70S6K, and conversely, that inhibiting the MEK1/2-Erk1/2 pathway (using PD 98059) at reperfusion, induced the phosphorylation of Akt, suggesting 'cross-talk' between the two kinase pathways. However, this effect was not accompanied by a reduction in infarct size (43.1 +/- 7.2% with LY 294008 and 57.7 +/- 7.0% with PD 98059 vs. 46.3 +/- 5.8% in control; P = NS), suggesting that both the kinase cascades may need to be activated to mediate IPC-induced protection. IPC reduced the infarct-risk volume ratio to 17.8 +/- 2.3% from 46.3 +/- 5.8% in control (P < 0.01). Inhibiting p70S6K, a kinase situated downstream of both PI3K and Erk1/2, using rapamycin, abolished IPC-induced protection (46.0 +/- 7.7% with IPC+RAPA vs. 17.8 +/- 2.3% with IPC; P < 0.01). CONCLUSIONS: We report that, the survival kinase cascades PI3K-Akt and MEK1/2-Erk1/2, which are recruited at the time of reperfusion in response to ischemic preconditioning, exhibit 'cross-talk' such that inhibiting one cascade activates the other and vice versa. Furthermore, at the time of reperfusion, these kinase cascades mediate IPC-induced protection, by acting in concert via p70S6K.

Animals↗
PubMed2005

High glucose-induced apoptosis in human vascular endothelial cells is mediated through NF-kappaB and c-Jun NH2-terminal kinase pathway and prevented by PI3K/Akt/eNOS pathway.

Our previous studies demonstrated that high glucose-induced apoptosis in human umbilical vein endothelial cells (HUVECs) is mediated by sequential activation of c-Jun N-terminal kinase (JNK) and caspase, and prevented by exogenous nitric oxide (NO). In this study we further elucidated the roles of the transcriptional factor NF-kappaB, phosphatidylinositol 3'-kinase (PI3K), Akt and endothelial nitric oxide synthase (eNOS) in the apoptosis of HUVECs induced by high glucose. The results showed that high glucose-induced apoptosis was significantly enhanced by PI3K inhibitors (wortmannin and LY294002), NOS inhibitor (NG-nitro-arginine methyl ester) and eNOS antisense oligonucleotide. In contrast, apoptosis was markedly reduced by NF-kappaB inhibitor (pyrrolidine dithiocarbamate, PDTC), NF-kappaB antisense oligonucleotide, NO donor (sodium nitroprusside, SNP), and overexpression of Akt. The high glucose-induced NF-kappaB activation and transient Akt phosphorylation were prevented by the presence of vitamin C. Moreover, high glucose-induced increase in eNOS expression was attenuated by PI3K inhibitors and the negative mutant of PI3K. The activity of JNK induced by high glucose was suppressed by NF-kappaB-specific antisense oligonucleotide. Taken together our results demonstrated that high glucose-induced HUVECs apoptosis is through NF-kappaB-dependent JNK activation and reactive oxygen species (ROS)-dependent Akt dephosphorylation. Activation of the ROS/PI3K/Akt/eNOS signaling pathway in early phase exerts protective effects against the induction of apoptosis by high glucose.

Androstadienes↗
PubMed2006

Regulation of protein kinase B activity by PTEN and SHIP2 in human prostate-derived cell lines.

Protein Kinase B (PKB/Akt) is a key regulator of cell proliferation, motility and survival. The activation status of PKB is regulated by phosphatidylinositol 3-kinase (PI3K) via the synthesis of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3, PIP3). PTEN antagonises PI3K by degrading PIP3 to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Deregulation of PKB through loss of functional PTEN has frequently been implicated in the progression of tumours, including prostate cancer, and the PTEN-negative prostate cancer cell lines LNCaP and PC3 have been widely used as models for this mechanism of constitutive PKB activation. However, other enzymes in addition to PTEN can antagonise PI3K, including SHIP2, which degrades PIP3 to phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2). We investigated the role of PTEN and SHIP2 in the regulation of PKB phosphorylation in a panel of human prostate-derived epithelial cell lines. In the PTEN-positive prostate-derived cell lines PNT2, PNT1a and P4E6, PI3K inhibition by LY294002 caused rapid dephosphorylation of PKB at ser473 (T(1/2)<2 min), leading to its inactivation. In the PTEN-null line LNCaP, LY294002-induced PKB dephosphorylation was much slower (T(1/2)>20 min), but in PC3 cells (also PTEN-null) it was only slightly slower than in PTEN-positive cells (T(1/2)=3 min). PKB dephosphorylation paralleled loss of plasma membrane PIP3. PNT1a, P4E6 and PC3, but not PNT2 or LNCaP, expressed SHIP2. SiRNA-mediated knockdown of SHIP2 expression markedly slowed PKB inactivation in response to LY294002 in PC3 but not in other SHIP2-positive cells, whereas knockdown of PTEN expression in PNT2, PNT1a and P4E6 resulted in higher steady-state levels of PKB phosphorylation and slowed, but did not prevent, LY294002-induced PKB inactivation. Thus SHIP2 substitutes for PTEN in the acute regulation of PKB in PC3 cells but not other prostate cell lines, where PTEN may share this role with further PIP3-degrading mechanisms.

Cell Line↗
PubMed2006

Bruton's tyrosine kinase together with PI 3-kinase are part of Toll-like receptor 2 multiprotein complex and mediate LTA induced Toll-like receptor 2 responses in macrophages.

Lipoteichoic acid (LTA) of Gram-positive bacteria initiates innate immune responses via Toll-like receptor-2 (TLR2), resulting in the activation of intracellular signaling and production of inflammatory cytokines in macrophages. Although Bruton's tyrosine kinase (Btk) is biologically important molecule implicated in immune regulation and recently in TLR signaling its importance for LTA-TLR2 mediated responses has not been evaluated. In this study, we detected Btk in the LTA signaling complex with TLR2 and PI 3-kinase (PI3K). The constitutive interaction of these proteins was mediated via PI3K Src homology (SH3) -domain. Both Btk and PI3K were activated by LTA stimulation and the LTA induced cytokine expression was differentially modulated by these kinases. LTA induced the activation of nuclear factor kappaB (NFkappaB), however, only Btk inhibition affected the LTA induced Ser536 phosphorylation and DNA-binding of NFkappaB. In conclusion, our results demonstrate that Btk and PI3K occupy important roles in TLR2-induced activation of macrophages, resulting in selective regulation of cytokines.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2005

Leptin regulates insulin sensitivity via phosphatidylinositol-3-OH kinase signaling in mediobasal hypothalamic neurons.

To investigate whether phosphatidylinositol-3 kinase (PI3K) signaling mediates the metabolic effects of hypothalamic leptin action, adenoviral gene therapy was used to direct expression of leptin receptors to the area of the hypothalamic arcuate nucleus (ARC). This intervention markedly improved insulin sensitivity in genetically obese, leptin-receptor-deficient Koletsky (fa(k)/fa(k)) rats via a mechanism that was not dependent on reduced food intake but was attenuated by approximately 44% by third-ventricular infusion of the PI3K inhibitor LY294002. Conversely, ARC-directed expression of a constitutively active mutant of protein kinase B (PKB/Akt, an enzyme activated by PI3K) mimicked the insulin-sensitizing effect of restored hypothalamic leptin signaling in these animals, despite having no effect on food intake or body weight. These findings suggest that hypothalamic leptin signaling is an important determinant of glucose metabolism and that the underlying neuronal mechanism involves PI3K.

Adenoviridae↗
PubMed2003

Lipopolysaccharide activates Akt in vascular smooth muscle cells resulting in induction of inducible nitric oxide synthase through nuclear factor-kappa B activation.

Bacterial lipopolysaccharide and other immunostimulants induce gene expression of an isoform of nitric oxide synthase (iNOS) in vascular smooth muscle cells. This process is dependent on nuclear factor-kappa B (NF-kappa B) activation. The aim of this study was to investigate whether the NF-kappa B and Akt signaling pathways converge to induce iNOS in lipopolysaccharide-stimulated vascular smooth muscle cells. Treatment of vascular smooth muscle cells with lipopolysaccharide plus interferon-gamma (LPS/IFN) caused activation of Akt and NF-kappa B. LPS/IFN caused activation of the iNOS promoter and transcription of iNOS mRNA/protein, resulting in NO production. A pharmacological inhibitor of the phosphoinositide 3-kinase (PI3K)-Akt pathway, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), inhibited phosphorylation of Akt and suppressed activation of NF-kappa B by attenuating the phosphorylation and degradation of I kappa B. LY294002 thereby inhibited LPS/IFN-induced iNOS expression and NO production. Another inhibitor of the PI3K-Akt pathway, wortmannin, also inhibited NO production in VSMC. LY294002 similarly inhibited interleukin-1 beta- or tumor necrosis factor-alpha-induced NO production. The data indicate that lipopolysaccharide or cytokine stimulation of vascular smooth muscle cells leads to activation of the PI3K-Akt pathway, which then activates the NF-kappa B pathway. Thus, the PI3K-Akt pathway controls the expression of iNOS in lipopolysaccharide- and cytokine-stimulated vascular smooth muscle cells.

Animals↗
PubMed2007

Involvement of phosphatidylinositol-3-kinase and ERK pathways in the production of TGF-beta1 by macrophages treated with liposomes composed of phosphatidylserine.

We explored the involvement of phosphatidylinositol 3-kinase (PI3K) and ERK pathways in the production of TGF-beta1 by macrophages treated with liposomes composed of phosphatidylserine (PS-liposomes). PS-liposomes activated Akt, downstream of the PI3K signal cascade, and ERK which led to the expression of TGF-beta1. PI3K inhibitors, LY294002 and wortmannin, inhibited the activation of Akt and ERK following the treatment with PS-liposomes. These inhibitors also suppressed the production of TGF-beta1. Furthermore, PS-liposomes activated macrophages to induce TGF-beta1 expression through PS-specific receptors. These findings suggested that a PI3K-ERK signaling pathway via the PS-receptor is intimately involved in the production of TGF-beta1 which regulates macrophage functions.

Animals↗
PubMed2006

Regulation of heme oxygenase-1 gene expression through the phosphatidylinositol 3-kinase/PKC-zeta pathway and Sp1.

The molecular mechanisms involved in modulation of the antioxidant cell defence by survival signals remain largely unexplored. Here, we report a mechanistic connection between the survival signal elicited by nerve growth factor (NGF) and the antioxidant cell defence represented by heme oxygenase-1 (HO-1) at the level of a newly identified Sp1 site in the human ho1 proximal promoter. By using luciferase reporter constructs we identified a PI3K-responsive region containing a GC-box that resembled the response element for Sp1. Indeed, transfection of Sp1-deficient SL2 cells, electrophoretic mobility shift assays, the use of the GC-box binding drug mithramycin, and mutation of the GC-box provided evidence for a Sp1-like site in the PI3K-sensitive region. Then, we observed with the use of a Sp1-Gal4 chimera that PI3K regulates the transactivating capacity of Sp1. Cotransfection of active PI3K and PKC-zeta expression vectors resulted in substantial increase of Sp1 phosphorylation and in synergistic activation of both Sp1-Gal4 and endogenous Sp1. Moreover, these effects were mimicked by cotransfection of active MEK and ERK expression vectors and were blocked by the MEK inhibitor PD98059. Inhibition of HO-1 with Sn protoporphyrin IX and blockage of Sp-1-mediatied upregulation of HO-1 with mithramycin attenuated antioxidant and cytoprotective functions of NGF against hydrogen peroxide. This study elucidates how NGF contributes to protection of target cells against oxidative stress.

Animals↗
PubMed2004

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

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

Androstadienes↗
PubMed2004

Arrest of human lung fibroblasts in G2 phase after irradiation is regulated by converging phosphatidylinositol-3 kinase and beta1-integrin signaling in vitro.

PURPOSE: Cell-matrix interactions might confer cellular radioresistance in vitro. As a function of radiation, the impact of fibronectin (FN) and phosphatidylinositol-3 kinase (PI3K)-related signaling on survival, the cell cycle, and the beta1-integrin signaling kinases integrin-linked kinase (ILK), protein kinase Balpha/Akt (PKBalpha/Akt), and glycogen synthase kinase-3beta (GSK-3beta) was examined in normal lung fibroblasts in vitro. METHODS AND MATERIALS: Normal human CCD32 lung fibroblasts grown on polystyrene, FN, or poly-L-lysine were irradiated with 0-8 Gy. Colony forming assays, flow cytometric DNA analysis, immunoblotting (Chk1, Chk2, Cdc25C, Cdk1, 14-3-3, p53, p21), and protein kinase assays (ILK, PKBalpha/Akt, GSK-3beta) were performed with or without PI3K inhibition using LY294002 or wortmannin. RESULTS: FN significantly elevated clonogenic survival of CCD32 cells after irradiation compared with polystyrene or poly-L-lysine. FN improved accumulation of irradiated cells in G(2)/M (60%) compared with polystyrene (43%). LY294002 prevented radiation-dependent G(2) blockage on polystyrene; on FN, G(2) arrest was only slightly reduced. Radiation- and PI3K inhibition-related changes in expression and phosphorylation of the various cell cycle proteins tested correlated with the cell cycle data acquired. The kinase activities of ILK, PKBalpha/Akt, and GSK-3beta were strongly induced by irradiation on polystyrene, but not on FN, which was a result of a FN-mediated increase of basal kinase activities. In contrast to polystyrene, FN enabled radiation-dependent induction of ILK and GSK-3beta in a PI3K-independent manner. CONCLUSION: The data indicate a tight convergence of cell-matrix and cell-growth factor interactions that seem to optimize the cellular responsiveness to ionizing radiation in terms of survival and G(2) arrest. ILK, PKBalpha/Akt, and GSK-3beta involved in integrin signaling were uncovered as new molecular factors within the cellular radiation response. Our findings might also provide insight into normal tissue effects and cellular radioresistance.

Androstadienes↗
PubMed2005

Inhibition of phosphatidylinositol-3-kinase causes cell death through a protein kinase B (PKB)-dependent mechanism and growth arrest through a PKB-independent mechanism.

PURPOSE: To identify whether inhibition of phosphatidylinositol-3-kinase (PI3K) causes apoptosis through inhibition of protein kinase B (PKB), implicating PKB as an important therapeutic target in prostate cancer. METHODS AND MATERIALS: After treatment with the PI3K inhibitor, LY294002, proliferation and apoptosis of the prostate cancer cell line, LNCaP, were measured by cell cycle analysis and cleavage of poly (ADP-ribose) polymerase. To test the hypothesis that inhibition of PKB is responsible for the LY294002-induced apoptosis, LNCaP cells expressing a constitutively active form of PKB were generated. RESULTS: Treatment of LNCaP cells with the PI3K inhibitor, LY294002, caused inactivation of PKB, growth arrest, and apoptosis. LY294002-induced apoptosis was increased in the absence of serum. The G1 growth arrest was associated with an increase in p27(kip1) expression. Cells expressing constitutively active PKB were protected from apoptosis induced by LY294002, but not from the G1 growth arrest induced by PI3K inhibition. CONCLUSION: These data suggest that PKB activity regulates apoptosis, but not G1 arrest, and identify PKB as a potential critical target for cancer therapy. Targeted therapy against kinases might complement more conventional therapies, including androgen suppression for prostate cancer.

Apoptosis↗