Gastric mucosal cell homeostatic physiome. Critical role of ER-initiated membranes restitution in the fidelity of cell function renewal.

Homeostatic cell physiology is preserved through the fidelity of the cell membranes restitution. The task is accomplished through the assembly of the precisely duplicated segments of the cell membranes, and transport to the site of their function. Here we examined the mechanism that initiates and directs the restitution of the intra- and extracellular membranes of gastric mucosal cell. The homeostatic restitution of gastrointestinal epithelial cell membrane components was investigated by studying the lipidomic processes in endoplasmic reticulum (ER) and Golgi. The biomembrane lipid synthesis during the formation of transport vesicles in the systems containing isolated organelle and the cell-specific cytosol (Cyt) from rat gastric mucosal epithelial cells was assessed. The results revealed that lipids of ER transport vesicle and the transmembrane and intravesicular cargo are delivered en bloc to the point of destination. En bloc delivery of proteins, incorporated into predetermined in ER lipid environment, ensures fidelity of the membrane modification in Golgi and the restitution of the lipid and protein elements that are consistent with the organelle and the cell function. The mechanism that maintains apical membrane restitution is mediated through the synthesis of membrane segments containing ceramide (Cer). The Cer-containing membranes and protein cargo are further specialized in Golgi. The portion of the vesicles destined for apical membrane renewal contains glycosphingolipids and phosphatidylinositol 3-phosphate. The vesicles containing phosphatidylinositol 4-phosphate are directed to endosomes. Our findings revealed that the preservation of the physiological equilibrium in cell structure and function is attributed to (1) a complete membrane segment synthesis in ER, (2) its transport in the form of ER-transport vesicle to Golgi, (3) the membrane components-defined maturation of lipids and proteins in Golgi, and (4) en bloc transfer of the new segment of the membrane to the cell apical membrane or intracellular organelle.     

Role of phosphatidylinositol 3-kinase in anti-IgM- and anti-IgD-induced apoptosis in B cell lymphomas

Cross-linking of surface Ig receptors with anti IgM (anti-mu heavy chain, anti-mu), but not anti-IgD (anti-delta heavy chain, anti-delta), Abs leads to growth arrest and apoptosis in several extensively characterized B cell lymphomas. By poorly understood mechanisms, both Igs transiently stimulate c-Myc protein expression. However, ultimately, only anti-mu causes a severe loss in c-Myc and a large induction of p27(Kip1) protein expression. Because phosphatidylinositol 3-kinase (PI3K) has been established as a major modulator of cellular growth and survival, we investigated its role in mediating anti-Ig-stimulated outcomes. Herein, we show that PI3K pathways regulate cell cycle progression and apoptosis in the ECH408 B cell lymphoma. Anti-mu and anti-delta driven c-Myc protein changes precisely follow their effects on the PI3K effector, p70(S6K). Upstream of p70(S6K), signaling through both Ig receptors depresses PI3K pathway phospholipids below control with time, which is followed by p27(Kip1) induction. Conversely, anti-delta, but not anti-mu stimulated PI3K-dependent phospholipid return to control levels by 4-8 h. Abrogation of the PI3K pathway with specific inhibitors mimics anti-mu action, potentiates anti-mu-induced cell death and, importantly, converts anti-delta to a death signal. Transfection with active PI3K kinase construct induces anti-mu resistance, whereas transfection with dominant negative PI3K augments anti-mu sensitivity. Our results show that prolonged disengagement of PI3K or down-regulation of its products by anti-mu (and not anti-delta) determines B cell fate.     

Inhibition of MG-63 cell proliferation and PDGF-stimulated cellular processes by inhibitors of phosphatidylinositol 3-kinase

Studies on a platelet-derived growth factor (PDGF) responsive osteosarcoma cell line, MG-63, were initiated to determine the effects of phosphatidylinositol (Ptdlns) 3-kinase inhibitors on serum-stimulated cell proliferation and PDGF-stimulated DNA replication, actin rearrangements, or Ptdlns 3-kinase activity. In a dose-dependent manner, the fungal metabolite wortmannin and a quercetin derivative, LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), inhibited serum-stimulated MG-63 cell proliferation. The mitogenic effects of PDGF on MG-63 cells, as determined by incorporation of [³H]-thymidine, were also substantially inhibited in the presence of 0.10 μM wortmannin or 10 μM LY294002. Furthermore, MG-63 cells stimulated by PDGF form distinct actin-rich, finger-like membrane projections which are completely inhibited by either 0.10 μM wortmannin or 10 μM LY294002. At these same concentrations, wortmannin and LY294002 were also effective at reducing levels of phosphatidylinositol 3-phosphate in PDGF-stimulated MG-63 cells. Treatment of these cells with increasing concentrations of wortmannin reduced the level of PDGF stimulated tyrosine phosphorylation of the PDGF receptor but did not significantly affect the amount of the Ptdlns 3-kinase regulatory subunit, p85, associated with the receptor. Additionally, pretreatment of cells with 0.250 μM wortmannin followed by stimulation with PDGF resulted in a slightly reduced level of receptor autokinase activity; however, similar treatment with 50 μM LY294002 did not affect the level of autokinase activity. These results demonstrate the effects of two different Ptdlns 3-kinase inhibitors on serum- and PDGF-stimulated MG-63 cell proliferation and PDGF-stimulated morphological changes and suggest a greater role for Ptdlns 3-kinase in these processes. J. Cell. Biochem. 64:182–195. © 1997 Wiley-Liss, Inc.     

Glial cell line-derived neurotrophic factor increases intracellular calcium concentration. Role of calcium/calmodulin in the activation of the phosphatidylinositol 3-kinase pathway

Moderate increases of intracellular Ca2+ concentration ([Ca2+]i), induced by either the activation of tropomyosin receptor kinase (Trk) receptors for neurotrophins or by neuronal activity, regulate different intracellular pathways and neuronal survival. In the present report we demonstrate that glial cell line-derived neurotrophic factor (GDNF) treatment also induces [Ca2+]i elevation by mobilizing this cation from internal stores. The effects of [Ca2+]i increase after membrane depolarization are mainly mediated by calmodulin (CaM). However, the way in which CaM exerts its effects after tyrosine kinase receptor activation remains poorly characterized. It has been reported that phosphatidylinositol 3-kinase (PI 3-kinase) and its downstream target protein kinase B (PKB) play a central role in cell survival induced by neurotrophic factors; in fact, GDNF promotes neuronal survival through the activation of the PI 3-kinase/PKB pathway. We show that CaM antagonists inhibit PI 3-kinase and PKB activation as well as motoneuron survival induced by GDNF. We also demonstrate that endogenous Ca2+/CaM associates with the 85-kDa regulatory subunit of PI 3-kinase (p85). We conclude that changes of [Ca2+]i, induced by GDNF, promote neuronal survival through a mechanism that involves a direct regulation of PI 3-kinase activation by CaM thus suggesting a central role for Ca2+ and CaM in the signaling cascade for neuronal survival mediated by neurotrophic factors.     

Negative regulation of phosphatidylinositol 3-kinase and Akt signalling pathway by PKC

Although substantial studies have begun to explore the regulation of phosphatidylinositol 3-kinase/Akt cascade by different signalling pathways, whether protein kinase C (PKC) activity plays a crucial role remains as yet unclear. In this study, we found that in A549 and HEK293 cells non-selective PKC inhibitors Ro 31-8220 and bisindolylmaleimide VIII, and PKCbeta inhibitor LY 379196, caused Akt/PKB phosphorylation at Ser 473 and increased the upstream activator, integrin-linked kinase (ILK) activity. The increased Akt phosphorylation was blocked by phosphatidylinositol 3-kinase inhibitor wortmannin and the newly identified PIP(3)-dependent kinases (PDK) inhibitor SB 203580. In contrast to the Akt stimulation caused by PKC inhibitors, PMA attenuated Akt/PKB phosphorylation. We also found that this stimulating effect on Akt phosphorylation by PKC inhibitors was not the result of phosphatase inhibition, since treatment with PP2A, PP2B and tyrosine phosphatase inhibitors (okadaic acid, FK506 and sodium orthovanadate, respectively) had no effect. We conclude that phosphatidylinositol 3-kinase/Akt signalling pathway is regulated by PKC in a negative manner.     

Insulin-dependent phosphatidylinositol 3-kinase/Akt and ERK signaling pathways inhibit TLR3-mediated human bronchial epithelial cell apoptosis

TLR3, one of the TLRs involved in the recognition of infectious pathogens for innate and adaptive immunity, primarily recognizes viral-associated dsRNA. Recognition of dsRNA byproducts released from apoptotic and necrotic cells is a recently proposed mechanism for the amplification of toxicity, suggesting a pivotal participation of TLR3 in viral infection, as well as in lung diseases where apoptosis plays a critical role, such as asthma and chronic obstructive pulmonary disease. In addition to metabolic control, insulin signaling was postulated to be protective by inhibiting apoptosis. Therefore, we explored the role of insulin signaling in protecting against TLR3-mediated apoptosis of human bronchial epithelial cells. Significant TLR3-mediated apoptosis was induced by polyinosinic-polycytidylic acid, a dsRNA analog, via caspase-8-dependent mechanisms. However, insulin efficiently inhibited TLR3/polyinosinic-polycytidylic acid-induced human bronchial epithelial cell apoptosis via PI3K/Akt and ERK pathways, at least in part, via upregulation of cellular FLIPs and through protein synthesis-independent mechanisms. These results indicate the significance of TLR3-mediated dsRNA-induced apoptosis in the pathogenesis of apoptosis-driven lung disease and provide evidence for a novel protective role of insulin.     

Differential membrane localization of ERas and Rheb, two Ras-related proteins involved in the phosphatidylinositol 3-kinase/mTOR pathway

Two Ras-related proteins, ERas and Rheb, which are involved in the phosphatidylinositol 3-kinase pathway, display high GTP affinity and have atypical CAAX motifs. The factors governing the intracellular localization of ERas and Rheb are incompletely understood. In the current study, we show by confocal microscopy that ERas is localized to the plasma membrane, whereas Rheb is confined to the endomembranes. Membrane localization of the two proteins was abolished by mutation of the cysteine of the CAAX motif. Membrane targeting was also abolished by a farnesyltransferase inhibitor but not by a geranylgeranyltransferase inhibitor. In mouse fibroblasts deficient in either Rce1 (Ras converting enzyme 1) or Icmt (isoprenylcysteine carboxyl methyltransferase), ERas was mislocalized mainly to the Golgi apparatus, whereas Rheb showed diffuse localization. Mutation of cysteines in the hypervariable region of ERas prevented the plasma membrane localization of ERas, very strongly suggesting that palmitoylation of the cysteines is essential for membrane targeting. The hypervariable region of Rheb does not contain cysteines or polybasic residues, and when it was replaced with the hypervariable region of H-Ras, Rheb displayed plasma membrane localization. These data indicate that ERas shares the same posttranslational modifications with H-Ras and N-Ras and is localized at the plasma membrane. Rheb also shares the same membrane-targeting pathway but because of the absence of palmitoylation is located on endomembranes.     

Role of phosphatidylinositol 3-kinase in oxidative stress-induced disruption of tight junctions

A recent study (Nusrat, A., Chen, J. A., Foley, C. S., Liang, T. W., Tom, J., Cromwell, M., Quan, C., and Mrsny, R. J. (2000) J. Biol. Chem. 275, 29816-29822) suggested that phosphatidylinositol 3-kinase (PI 3-kinase) may interact with occludin; however, there exists no evidence of direct interaction of PI 3-kinase with the tight junctions. Activation of PI 3-kinase by oxidative stress and its role in disruption of tight junctions was examined in Caco-2 cell monolayer. The oxidative stress-induced decrease in electrical resistance, increase in inulin permeability, and redistribution of occludin and ZO-1 were reduced by a PI 3-kinase inhibitor, LY294002. Oxidative stress-induced tyrosine phosphorylation and dissociation from the actin cytoskeleton of occludin and ZO-1 were reduced by LY294002. The regulatory subunit of PI 3-kinase, p85, and the PI 3-kinase activity were co-immunoprecipitated with occludin, which were rapidly increased by oxidative stress. Oxidative stress resulted in increased translocation of p85 from the intracellular compartment into the intercellular junctions. Pair-wise glutathione S-transferase pull-down assay showed that glutathione S-transferase-occludin (C-terminal tail) binds to recombinant p85. This study shows that oxidative stress increases the association of PI 3-kinase with the occludin, and that PI 3-kinase activity is involved in oxidative stress-induced disruption of tight junction.     

Eph B4 receptor signaling mediates endothelial cell migration and proliferation via the phosphatidylinositol 3-kinase pathway

The goals of this study were 2-fold: 1) to determine whether stimulation of Eph B4 receptors promotes microvascular endothelial cell migration and/or proliferation, and 2) to elucidate signaling pathways involved in these responses. The human endothelial cells used possessed abundant Eph B4 receptors with no endogenous ephrin B2 expression. Stimulation of these receptors with ephrin B2/Fc chimera resulted in dose- and time-dependent phosphorylation of Akt. These responses were inhibited by LY294002 and ML-9, blockers of phosphatidylinositol 3-kinase (PI3K) and Akt, respectively. Eph B4 receptor activation increased proliferation by 38%, which was prevented by prior blockade with LY294002, ML-9, and inhibitors of protein kinase G (KT5823) and MEK (PD98059). Nitrite levels increased over 170% after Eph B4 stimulation, indicating increased nitric oxide production. Signaling of endothelial cell proliferation appears to be mediated by a PI3K/Akt/endothelial nitric-oxide synthase/protein kinase G/mitogen-activated protein kinase cascade. Stimulation with ephrin B2 also increased migration by 63% versus controls. This effect was inhibited by blockade with PP2 (Src inhibitor), LY294002 or ML-9 but was unaffected by the PKG and MEK blockers. Eph B4 receptor stimulation increased activation of both matrix metalloproteinase-2 and -9. The results from these studies indicate that Eph B4 stimulates migration and proliferation and may play a role in angiogenesis.     

Requirement of phosphatidylinositol 3-kinase in focal adhesion kinase-promoted cell migration

We have previously shown that overexpression of focal adhesion kinase (FAK) in Chinese hamster ovary (CHO) cells promoted their migration on fibronectin. This effect was dependent on the phosphorylation of FAK at Tyr-397. This residue was known to serve as a binding site for both Src and phosphatidylinositol 3-kinase (PI3K), implying that either one or both are required for FAK to promote cell migration. In this study, we have examined the role of PI3K in FAK-promoted cell migration. We have demonstrated that the PI3K inhibitors, wortmannin and LY294002, were able to inhibit FAK-promoted migration in a dose-dependent manner. Furthermore, a FAK mutant capable of binding Src but not PI3K was generated by a substitution of Asp residue 395 with Ala. When overexpressed in CHO cells, this differential binding mutant failed to promote cell migration although its association with Src was retained. Together, these results strongly suggest that PI3K binding is required for FAK to promote cell migration and that the binding of Src and p130(Cas) to FAK may not be sufficient for this event.     

Role of MyD88 in phosphatidylinositol 3-kinase activation by flagellin/toll-like receptor 5 engagement in colonic epithelial cells

Bacterial flagellin, recognized by Toll-like receptor (TLR) 5, is suggested to be involved in colonic inflammation. However, the detailed signaling mechanisms mediated by flagellin/TLR5 engagement are not clear. Here we dissected the biochemical mechanism by which TLR5 engagement mediates phosphatidylinositol 3-kinase (PI3K) activation in colonic epithelial cells. We demonstrate that silencing TLR5 expression in nontransformed human colonic epithelial cells blocks flagellin-induced PI3K activation, indicating specific activation of PI3K by flagellin/TLR5 engagement. Moreover, we determine that TLR5 recruits the p85 regulatory subunit of PI3K to its cytoplasmic TIR domain in response to flagellin. However, the Src homology binding "YXXM" motif in the cytoplasmic TIR domain of TLR5 is not involved in p85 recruitment, implying that TLR5 indirectly recruits p85. Indeed, we demonstrate that the adaptor molecule MyD88 associates with TLR5 and silencing MyD88 expression blocks PI3K activation by disrupting the association between TLR5 and p85. Furthermore, we show that MyD88 associates with p85 in response to flagellin. Additionally, we determine that blocking PI3K activation reduces interleukin-8 production induced by flagellin in human colonic epithelial cells. Together, MyD88 bridges TLR5 engagement to PI3K activation in response to flagellin.     

CD40-dependent activation of phosphatidylinositol 3-kinase/Akt pathway mediates endothelial cell survival and in vitro angiogenesis

CD40 has been involved in tumor and inflammatory neoangiogenesis. In this study we determined that stimulation of endothelial CD40 with sCD154 induced resistance to apoptosis and in vitro vessel-like formation by human microvascular endothelial cells (HMEC). These effects were determined to be mediated by CD40-dependent signaling because they were inhibited by a soluble CD40-muIg fusion protein. Moreover, apoptosis of HMEC was associated with an impairment of Akt phosphorylation, which was restored by stimulation with sCD154. The anti-apoptotic effect as well as in vitro vessel-like formation and Akt phosphorylation were inhibited by treatment of HMEC with two unrelated pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002. CD40 stimulation induced a rapid increase in Akt enzymatic activity that was not prevented by cycloheximide, an inhibitor of protein synthesis. The enhanced Akt activity induced by stimulation of endothelial CD40 was temporarily correlated with the association of CD40 with TRAF6, c-Cbl, and the p85 subunit of PI3K. Expression of negative-dominant Akt inhibited the activation of endogenous Akt through CD40 stimulation, despite the observation that association of CD40 with TRAF6, c-Cbl, and PI3K was intact. The defective activation of Akt abrogated not only the anti-apoptotic effect of CD40 stimulation but also the proliferative response, the enhanced motility, and the in vitro formation of vessel-like tubular structures by CD40-stimulated HMEC. In conclusion, these results suggest that endothelial CD40, through activation of the PI3K/Akt signaling pathway, regulates cell survival, proliferation, migration, and vessel-like structure formation, all steps considered critical for angiogenesis.     

Role of phosphatidylinositol 3-kinase in platelet aggregation in type 1 diabetes mellitus

It was found that incubation of platelet rich plasma with wortmannin, an irreversible selective inhibitor of phosphoinositide 3-kinase (PI3K), leads to sharp drop in platelet aggregation ability in healthy donors, whereas in type 1 diabetes mellitus patients this effect was less manifested or not quite determined. Translocation dynamics of PI3K regulatory subunit into cytoskeleton fraction under induction of platelet aggregation by various ADP concentrations and after wortmannin treatment was studied. Reciprocal interaction of endothelial constitutive NO synthase with PI3K in mechanisms of platelet functional state regulation under studied pathological and normal conditions have been analyzed.     

A late, prolonged activation of the phosphatidylinositol 3-kinase pathway is required for T cell proliferation

Activation of the phosphatidylinositol-3 kinase (PI 3-K) pathway is associated with the proliferation of many cell types, including T lymphocytes. However, recent studies in cell lines stably expressing deletion mutants of IL-2R that fail to activate PI 3-K have questioned the requirement for this pathway in cell cycle regulation. In this study with IL-2 and IL-7, we show in primary T cells that, unlike IL-2, IL-7 fails to induce the early activation of PI 3-K seen within minutes and normally associated with cytokine signaling. However, kinetic experiments showed that both of these T cell growth factors induce a distinct and sustained phase of PI 3-K activity several hours after stimulation. This delayed activation correlates with cell cycle induction and from studies using inhibitors of PI 3-K signaling, we show that this later phase, unlike the early activation within minutes, is required for cell cycle induction. The data presented here will have major implications for our understanding of the mechanism of T cell proliferation as well as the regulation of PI 3-K activity.     

PTEN interactions with focal adhesion kinase and suppression of the extracellular matrix-dependent phosphatidylinositol 3-kinase/Akt cell survival pathway.

The tumor suppressor PTEN is a phosphatase with sequence homology to tensin. PTEN dephosphorylates phosphatidylinositol 3,4, 5-trisphosphate (PIP3) and focal adhesion kinase (FAK), and it can inhibit cell growth, invasion, migration, and focal adhesions. We investigated molecular interactions of PTEN and FAK in glioblastoma and breast cancer cells lacking PTEN. The PTEN trapping mutant D92A bound wild-type FAK, requiring FAK autophosphorylation site Tyr397. In PTEN-mutated cancer cells, FAK phosphorylation was retained even in suspension after detachment from extracellular matrix, accompanied by enhanced PI 3-K association with FAK and sustained PI 3-K activity, PIP3 levels, and Akt phosphorylation; expression of exogenous PTEN suppressed all five properties. PTEN-mutated cells were resistant to apoptosis in suspension, but most of the cells entered apoptosis after expression of exogenous PTEN or wortmannin treatment. Moreover, overexpression of FAK in PTEN-transfected cells reversed the decreased FAK phosphorylation and PI 3-K activity, and it partially rescued PIP3 levels, Akt phosphorylation, and PTEN-induced apoptosis. Our results show that FAK Tyr397 is important in PTEN interactions with FAK, that PTEN regulates FAK phosphorylation and molecular associations after detachment from matrix, and that PTEN negatively regulates the extracellular matrix-dependent PI 3-K/Akt cell survival pathway in a process that can include FAK.     

3-Deoxy-3-substituted-D-myo-inositol imidazolyl ether lipid phosphates and carbonate as inhibitors of the phosphatidylinositol 3-kinase pathway and cancer cell growth

3-Modified D-myo-inositol imidazolyl ether lipid phosphates and a carbonate were synthesized and evaluated as inhibitors of P13-K and Akt. These data are presented along with IC50 values for the inhibition of the growth of three cancer cell lines.     

Epidermal growth factor stimulates Rac activation through Src and phosphatidylinositol 3-kinase to promote colonic epithelial cell migration

Regulated intestinal epithelial cell migration plays a key role in wound healing and maintenance of a healthy gastrointestinal tract. Epidermal growth factor (EGF) stimulates cell migration and wound closure in intestinal epithelial cells through incompletely understood mechanisms. In this study we investigated the role of the small GTPase Rac in EGF-induced cell migration using an in vitro wound-healing assay. In mouse colonic epithelial (MCE) cell lines, EGF-stimulated wound closure was accompanied by a doubling of the number of cells containing lamellipodial extensions at the wound margin, increased Rac membrane translocation in cells at the wound margin, and rapid Rac activation. Either Rac1 small interfering (si)RNA or a Rac1 inhibitor completely blocked EGF-stimulated wound closure. Whereas EGF failed to activate Rac in colon cells from EGF receptor (EGFR) knockout mice, stable expression of wild-type EGFR restored EGF-stimulated Rac activation and migration. Pharmacological inhibition of either phosphatidylinositol 3-kinase (PI3K) or Src family kinases reduced EGF-stimulated Rac activation. Cotreatment of cells with both inhibitors completely blocked EGF-stimulated Rac activation and localization to the leading edge of cells and lamellipodial extension. Our results present a novel mechanism by which the PI3K and Src signaling cascades cooperate to activate Rac and promote intestinal epithelial cell migration downstream of EGFR.