Use Of Wortmannin Research Articles

Restoration of keratocyte cell phenotype through epithelial‐stromal cell interactions in a 3D hydrogel model

AbstractPurpose In vivo, epithelial cells are connected both anatomically and functionally with stromal keratocytes. In vitro co‐culturing aims to recapture this cellular anatomy and functionality by bringing together two or more cell types within the same culture environment. The aim of this study was to investigate epithelial‐stromal cell signalling for the control of corneal stromal cell plasticity in 3D culture models.Methods Corneal stromal cells were activated to their injury phenotype (fibroblasts) before being encapsulated in 3D collagen hydrogels. 3 different epithelial‐stromal co‐culture methods were then examined; epithelial explant; transwell; the use of conditioned media. Using non‐destructive monitoring tools we revealed how epithelial co‐culturing affects stromal cell differentiation in terms of construct contraction and elastic modulus measurements in a 3D collagen hydrogel environment for prolonged culture periods. Cell viability, phenotype, morphology and protein expression was investigated to corroborate our mechanical findings.Results It was shown that activated stromal cells retain their plasticity in vitro. Activated corneal stromal cells that were fibroblastic in phenotype were successfully reverted to a non‐activated keratocyte cell lineage in terms of behaviour and biological properties; and then back again via TGF‐β1 media supplementation. It was then revealed that epithelial‐stromal interactions can be blocked via the use of wortmannin inhibition.Conclusion A greater understanding of stromal‐epithelial interactions and what mediates them offers great pharmacological potential in the regulation of corneal wound healing, with the potential to treat corneal diseases and injury whereby such interactions are vital.

Evidence for phosphatidylinositol-3-OH-kinase (PI3-kinase) involvement in Cd-mediated oxidative effects on hemocytes of mussels

This study investigated phosphatidylinositol-3-OH-kinase (PI3-kinase) involvement in the induction of cadmium-mediated oxidative effects on hemocytes of mussel Mytilus galloprovincialis. PI3-kinase was investigated with the use of wortmannin, a specific covalent inhibitor of PI3-kinase. Moreover, phorbol-myristate acetate (PMA), a well-known protein kinase C (PKC)-mediated NADPH oxidase and nitric oxide (NO) synthase stimulator, was also used for elucidating PI3-kinase involvement during the respiratory burst process in challenge hemocytes. According to the results, cells pre-treated with non-toxic concentrations of wortmannin (1 and/or 50nM, as revealed by neutral red retention assay) for 15min, showed a significant attenuation of cadmium ability (at concentration of 50μM) to promote cell death, superoxide anion (O2−) production, NO generation and lipid peroxidation (in terms of malondialdehyde equivalents). On the other hand, wortmannin-treated cells showed a significant attenuation of PMA ability to induce NO generation but not O2− production. These findings reveal that PI3-kinase could lead to a PKC-independent induction of NO synthase activity in cells faced with pro-oxidants, such as cadmium, while its activation could be fundamental for the regulation of NAPDH oxidase activity, probably through a PKC-dependent signaling pathway.

Akt associates with nuclear factor κB and plays an important role in chemoresistance of gastric cancer cells

The ubiquitously expressed serine-threonine kinase Akt and the transcription factor NF-kappaB both are involved in cell proliferation and apoptosis. Furthermore, the activation of Akt or NF-kappaB has been suggested to associate with chemo-resistance of human tumors. The exact mechanism and interreaction of Akt and NF-kappaB pathway on chemoresistance in gastric cancer is still unknown. We explored the function of Akt and NF-kappaB pathway on chemoresistance in human gastric cancer cells. MTT method was used to analyze the influence of chemotherapeutics and the combined use of wortmannin or MG-132 on the growth of SGC-7901 cells. Apoptosis of SGC-7901 was detected by TUNEL and Annexin V/PI methods. The protein level of NF-kappaB was analyzed by immunocytochemical staining. EMSA was used to confirm the increased nuclear translocation of RelA. The protein level of p-Akt and p-IkappaBalpha were analyzed by Western blotting. Etoposide and doxorubicin suppressed the growth of SGC-7901 time and dose-dependently. Combined use of wortmannin or MG-132 can suppress growth further. Chemotherapeutics induced apoptosis of SGC-7901 and activated Akt and NF-kappaB, combined use of wortmannin or MG-132 induced apoptosis further and attenuated the activation of NF-kappaB. The combined use of wortmannin attenuated the activation of Akt, but combined use of MG-132 did not attenuate the activation of Akt. The activation of NF-kappaB is a branch mechanism of Akt anti-apoptosis effects. The chemotherapeutics induced apoptosis and induced the activation of Akt and NF-kappaB in SGC-7901 cell, suppression the activation of Akt or NF-kappaB can increase the effects of chemotherapeutics. NF-kappaB is a downstream target of Akt.

Tetanus toxin H C fragment reduces neuronal MPP + toxicity

The non-toxic carboxi-terminal domain of the heavy chain of tetanus toxin (H C) elicits neuroprotection of cerebellar granule neurons against apoptotic death induced by potassium deprivation. In this study we sought to determine whether H C also prevents the apoptosis induced by the mitochondrial poison 1-methyl-4-phenylpyridinium (MPP +), which induces a form of Parkinsonism. Pre-treatment of cultures with H C prevented MPP +-induced cell death, as well as impaired the MPP +-triggered apoptotic cascade. Cytochrome c release from the mitochondria, procaspase-3 activation and chromatin condensation, were significantly reduced in H C-pre-treated neurons. Moreover, H C induced Ser 112 and Ser 136 BAD phosphorylation, which correlated with the detachment of BAD from Bcl-X L and its association to 14-3-3. In turn, Bcl-X L remained bound to Bax, impairing its translocation to mitochondria of stressed neurons. The use of Wortmannin or PD98059 demonstrated the involvement of the PI3K/Akt as well as the ERK1/2 transduction pathways in the H C fragment effect. Interestingly, the H C fragment also induces an increase in the DNA binding activity of NF-κB, a well-established transcription factor involved in the prevention of neuronal death. Taken together, our results strongly suggest that the recombinant H C fragment of tetanus toxin can act as a neuroprotector in a model of MPP +-triggered apoptosis.

Proteomics Exploration Reveals That Actin Is a Signaling Target of the Kinase Akt

The serine/threonine kinase Akt is a key mediator of cell survival and cell growth that is activated by most growth factors, but its downstream signaling largely remains to be elucidated. To identify signaling partners of Akt, we analyzed proteins co-immunoprecipitated with Akt in MCF-7 breast cancer cells. Mass spectrometry analysis (MALDI-TOF and MS-MS) of SDS-PAGE-separated Akt co-immunoprecipitates allowed the identification of 10 proteins: alpha -actinin, valosin-containing protein, inhibitor kappaB kinase, mortalin, tubulin beta, cytokeratin 8, actin, 14-3-3sigma, proliferating cell nuclear antigen, and heat shock protein HSP27. The identification of these putative Akt binding partners were validated with specific antibodies. Interestingly, the major protein band observed in Akt co-immunoprecipitates was found to be the cytoskeleton protein actin for which a 14-fold increase was observed in Akt-activated compared with non-activated conditions. The interaction between Akt and actin was further confirmed by reverse immunoprecipitation, and confocal microscopy demonstrated a co-localization specifically induced under growth factor stimulation. The use of wortmannin indicated a dependence on the phosphatidylinositol 3-kinase pathway. Using a phospho-Akt substrate antibody, the phosphorylation of actin on an Akt consensus site was detected upon growth factor stimulation, both in cellulo and in vitro, suggesting that actin is a substrate of Akt kinase activity. Interestingly, cortical remodeling of actin associated with cell migration was reversed by small interfering RNA directed against Akt, indicating the involvement of Akt in the dynamic reorganization of actin cytoskeleton germane to breast cancer cell migration. Together these data identify actin as a new functional target of Akt signaling.

Exercise training improves insulin-induced and insulin-like growth factor-1-induced vasorelaxation in rat aortas

Improved vasorelaxant response is one of the beneficial effects of exercise training on vascular function. The mechanism for this response is, however, poorly understood. The aim of this study was to investigate the effects of exercise training on insulin-induced and insulin-like growth factor-1 (IGF-1)-induced vasorelaxation. Fourteen 6-week-old male Wistar rats were randomly divided into sedentary control and exercise groups. For 12 weeks, the exercise group ran on a treadmill 60 min/day, 5 days/week. After exercise training, insulin-induced and IGF-1-induced vasorelaxant responses were evaluated by measuring the isometric tension of aortic rings. The vasorelaxant role of phosphatidylinositol 3-kinase (PI3K) and nitric oxide synthase (NOS) was examined by applying inhibitors, such as wortmannin (an inhibitor of PI3K) and N ω-nitro- l-arginine methyl ester ( l-NAME, a NOS inhibitor). In addition, we examined the vascular response to the NO donor, sodium nitroprusside (SNP). We found that: (1) exercise training significantly enhanced both insulin-induced and IGF-1-induced vasorelaxation in rat aortas; (2) this vasorelaxant effect disappeared after the use of wortmannin or l-NAME; (3) there was no significant difference in SNP-induced vasorelaxation between control and exercise groups. Our findings indicate that exercise training enhances insulin-induced and IGF-1-induced vasorelaxant responses which are mediated through the PI3K-NOS-dependent pathway.

Wortmannin inhibits the myofilament Ca 2+ sensitization induced by endothelin-1

Endothelin-1 induces a positive inotropic effect due to a combination of an increase in Ca 2+ transients and myofilament Ca 2+ sensitivity in rabbit ventricular myocardium. We carried out the experiments to examine the potential contribution of myosin light chain kinase to the Ca 2+ sensitization induced by endothelin-1 by use of wortmannin that inhibits myosin light chain kinase at high concentrations (IC 50=200 nM). Wortmannin at 3 μM suppressed the basal force of contraction, but did not affect the positive inotropic effect mediated by β-adrenoceptors. Wortmannin at 1 and 3 μM markedly inhibited the positive inotropic effect of endothelin-1, but did not affect the increase in Ca 2+ transients elicited by endothelin-1. The present findings imply that the increase in myofilament Ca 2+ sensitivity induced by endothelin-1 may be in part due to activation of myosin light chain kinase in rabbit ventricular myocardium.

Phosphatidyl-inositol-3 kinase-independent insulin action pathway(s) in the human ovary.

Hyperandrogenism observed in women with a variety of insulin-resistant states is thought to be due to a stimulatory effect of insulin on ovarian steroid hormone production. However, it is not known what mechanisms could allow the ovary to remain sensitive to insulin while classical target organs for insulin action (liver, fat, and muscle) exhibit insulin resistance. One hypothesis proposed to explain this paradox suggests that a postbinding divergence of insulin receptor signaling occurs in the ovary and that signaling pathways for steroid hormone synthesis and other ovarian effects of insulin may be distinct from classical glucose signaling pathways. We now report that activation of phosphatidyl-inositol-3 (PI-3) kinase, which is crucial for glucose transport, is not necessary for the insulin-induced stimulation of progesterone production or for the insulin-induced inhibition of insulin-like growth factor binding protein 1 (IGFBP-1) production in cultured human ovarian cells. Human granulosa cells obtained during in vitro fertilization procedures were cultured with 10, 10(2), 10(3), or 10(4) ng/mL insulin with or without preincubation with 100 nM wortmannin, a specific irreversible inhibitor of PI-3 kinase. IGFBP-1 concentration in the conditioned medium was measured using immunoradiometric assay or by Western blot analysis. Progesterone concentration was measured using RIA. Additional studies were carried out in cultures of human ovarian cells prepared from homogenized whole ovarian tissue of a woman with a family history of breast cancer and a mutation of BRCA-1 gene who underwent bilateral oophorectomy. These cells were cultured with 10(3) ng/mL insulin with or without preincubation with 100 nM wortmannin. Two-way ANOVA was used to compare mean values of IGFBP-1 and progesterone according to insulin dose and the use of wortmannin. In cultured granulosa cell medium, progesterone production was stimulated by insulin in a dose-related manner up to 175% of control (P < 0.0001). In tissue culture medium from ovarian cells obtained from a patient with BRCA-gene mutation, concentration of progesterone in the tissue culture medium increased from 2.5 +/- 0.2 ng/mL for control to 5.4 +/- 0.3 ng/mL for cells incubated with insulin (P < 0.001). IGFBP-1 production in tissue culture medium from human granulosa cells was inhibited by insulin to the nadir of 45% of control (P < 0.0001). Preincubation with wortmannin, despite complete inhibition of PI-3 kinase in both cell systems confirmed by Western blot analysis, failed to significantly alter these results. We conclude that inhibition of PI-3 kinase by wortmannin fails to abolish stimulatory effect of insulin on progesterone production or inhibitory effect of insulin on IGFBP-1 production in cultured human ovarian cells. These findings suggest that activation of PI-3 kinase, an enzyme crucial for insulin-stimulated glucose transport, is not necessary for the above effects of insulin in the ovary. These data provide evidence for the presence of PI-3 kinase-independent insulin signaling pathway(s) in human ovarian cells.

Cell cycle-dependent effects of wortmannin on radiation survival and mutation.

Wortmannin, a known radiation sensitizer, has been used in experiments with synchronized cells to compare its effect on radiation survival and mutation induction within the cell cycle. PL61 cells (CHO cells with an inactivated HPRT gene containing a single active copy of a bacterial gpt gene) were synchronized by mitotic selection. Wortmannin administered before gamma irradiation caused a greater sensitization in G(1)-phase cells relative to late S/G(2)-phase cells. Preferential radiosensitization of G(1)-phase cells by wortmannin sets a limit to the proposed use of wortmannin in radiation therapy, since, in contrast to normal tissues, tumors usually have high proportions of S-phase cells. Wortmannin increased mutation frequencies in both G(1)- and S/G(2)-phase cells. Interestingly, relative increases in radiation-induced mutations in G(1) and S/G(2) phases were comparable. The results are discussed in terms of the contributions of different repair modes in the production of mutations.

Study of the cytolethal distending toxin (CDT)-activated cell cycle checkpoint: Involvement of the CHK2 kinase

The bacterial cytolethal distending toxin (CDT) triggers a G2/M cell cycle arrest in eukaryotic cells by inhibiting the CDC25C phosphatase-dependent CDK1 dephosphorylation and activation. We report that upon CDT treatment CDC25C is fully sequestered in the cytoplasmic compartment, an effect that is reminiscent of DNA damage-dependent checkpoint activation. We show that the checkpoint kinase CHK2, an upstream regulator of CDC25C, is phosphorylated and activated after CDT treatment. In contrast to what is observed with other DNA damaging agents, we demonstrate that the activation of CHK2 can only take place during S-phase. Use of wortmannin and caffeine suggests that this effect is not dependent on ATM but rather on another as yet unidentified PI3 kinase family member. These results confirm that the CDT is therefore responsible for specific genomic injuries that block cell proliferation by activating a cell cycle checkpoint.

Wortmannin inhibits translation of tumor necrosis factor-alpha in superantigen-activated T cells.

The superantigen toxic shock syndrome toxin (TSST)-1 can induce tumor necrosis factor (TNF)-alpha expression in T cells and monocytes, through different signaling pathways. We have stimulated peripheral blood mononuclear cells with TSST-1 and found that the major cell producers of TNF-alpha as detected by cytofluorimetry and immunocytochemistry were CD4(+) T lymphocytes. The expression of TNF-alpha by CD4(+) T cells can be inhibited by either, wortmannin (WN) or LY 294002, two phosphatidylinositol 3-kinase (PI 3-K) inhibitors. The inhibitory effect is not transcriptional as WN does not change the mRNA steady state of TNF-alpha at any of the concentrations tested and LY 294002 when preincubated with mononuclear cells at its median inhibitory concentration (IC(50) = 1. 4 microM) significantly inhibited the expression of TNF-alpha but not its mRNA. Immunoprecipitation of pulse-labeled intracellular TNF-alpha showed a specific decrease in the synthesis of this cytokine on cells treated with PI 3-K inhibitors. The p38 mitogen-activated protein kinase (MAPK) is involved in control of TNF-alpha translation in human macrophages. In T cells, we have found that the p38 MAPK inhibitor SB 203580 significantly decreased the secretion of TNF-alpha but not its mRNA. In addition, the combined use of WN and SB 203580 had an additive inhibitory effect on secretion of TNF-alpha. Therefore, both PI 3-K and p38 MAPK signaling pathways control TNF-alpha production in T cells.

Differential Regulation of Muscarinic Acetylcholine Receptor-sensitive Polyphosphoinositide Pools and Consequences for Signaling in Human Neuroblastoma Cells

In this study we have quantitatively assessed the basal turnover of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and M3-muscarinic receptor-mediated changes in phosphoinositides in the human neuroblastoma cell line, SH-SY5Y. We demonstrate that the polyphosphoinositides represent a minor fraction of the total cellular phosphoinositide pool and that in addition to rapid, sustained increases in [3H]inositol phosphates dependent upon the extent of receptor activation by carbachol, there are equally rapid and sustained reductions in the levels of polyphosphoinositides. Compared with phosphatidylinositol 4-phosphate (PtdIns(4)P), PtdIns(4,5)P2 was reduced with less potency by carbachol and recovered faster following agonist removal suggesting protection of PtdIns(4,5)P2 at the expense of PtdIns(4)P and indicating specific regulatory mechanism(s). This does not involve a pertussis toxin-sensitive G-protein regulation of PtdIns(4)P 5-kinase. Using wortmannin to inhibit PtdIns 4-kinase activity, we demonstrate that the immediate consequence of blocking the supply of PtdIns(4)P (and therefore PtdIns(4,5)P2) is a failure of agonist-mediated phosphoinositide and Ca2+ signaling. The use of wortmannin also indicated that PtdIns is not a substrate for receptor-activated phospholipase C and that 15% of the basal level of PtdIns(4,5)P2 is in an agonist-insensitive pool. We estimate that the agonist-sensitive pool of PtdIns(4,5)P2 turns over every 5 s (0.23 fmol/cell/min) during sustained receptor activation by a maximally effective concentration of carbachol. Immediately following agonist addition, PtdIns(4,5)P2 is consumed >3 times faster (0.76 fmol/cell/min) than during sustained receptor activation which represents, therefore, utilization by a partially desensitized receptor. These data indicate that resynthesis of PtdIns(4,5)P2 is required to allow full early and sustained phases of receptor signaling. Despite the critical dependence of phosphoinositide and Ca2+ signaling on PtdIns(4,5)P2 resynthesis, we find no evidence that this rate resynthesis is limiting for agonist-mediated responses.

Blockage of chemotactic peptide-induced stimulation of neutrophils by wortmannin as a result of selective inhibition of phosphatidylinositol 3-kinase.

Wortmannin, a fungal metabolite, inhibited 32P labeling of phosphatidylinositol trisphosphate, a product of phosphatidylinositol 3-kinase (PI 3-kinase), selectively in formyl peptide-stimulated 32P-loaded guinea pig neutrophils. The inhibition was of the same concentration dependence (with the half-maximal inhibition around 50 nM) as was observed for the simultaneous inhibition of formyl peptide-induced superoxide anion production. Wortmannin inhibited all three of the PI 3-kinase activities found in the cytosol fraction of guinea pig neutrophils, with a similar dose dependence (the half-maximal effects at 5 nM). Wortmannin was also effective on an immunologically purified preparation of the enzyme. The inhibition was of a noncompetitive type with regard to ATP and was observed consistently when PI, PI monophosphate, or PI bisphosphate was used as substrate. PI 4-kinase activity was not affected. It is concluded, therefore, that wortmannin abolished the formyl peptide-induced stimulation of neutrophils as a result of the inhibition of PI 3-kinase. An essential role of PI 3-kinase in receptor-mediated signaling in neutrophils thus evidenced with the use of wortmannin will be expanded to other cellular signaling systems.