Phosphorylation of MNAR promotes estrogen activation of phosphatidylinositol 3-kinase

Estrogen actions are mediated by a complex interface of direct control of gene expression (the so-called "genomic action") and by regulation of cell signaling/phosphorylation cascades, referred to as the "nongenomic," or extranuclear, action. We have previously described the identification of MNAR (modulator of nongenomic action of estrogen receptor) as a novel scaffold protein that regulates estrogen receptor alpha (ERalpha) activation of cSrc. In this study, we have investigated the role of MNAR in 17beta-estradiol (E2)-induced activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Consistent with our previous results, a direct correlation was established between MNAR expression levels and E2-induced activation of PI3 and Akt kinases. Endogenous MNAR, ERalpha, cSrc, and p85, the regulatory subunit of PI3 kinase, interacted in MCF7 cells treated with E2. The interaction between p85 and MNAR required activation of cSrc and MNAR phosphorylation on Tyr 920. Consequently, the mutation of this tyrosine to alanine (Y920A) abrogated the interaction between MNAR and p85 and the E2-induced activation of the PI3K/Akt pathway, which was required for the E2-induced protection of MCF7 cells from apoptosis. Nonetheless, the Y920A mutant potentiated the E2-induced activation of the Src/MAPK pathway and MCF7 cell proliferation, as observed with the wild-type MNAR. These results provide new and important insights into the molecular mechanisms of E2-induced regulation of cell proliferation and apoptosis.     

Lipoarabinomannan from Mycobacterium tuberculosis promotes macrophage survival by phosphorylating Bad through a phosphatidylinositol 3-kinase/Akt pathway

Efforts in prevention and control of tuberculosis suffer from the lack of detailed knowledge of the mechanisms used by pathogenic mycobacteria for survival within host cell macrophages. The exploitation of host cell signaling pathways to the benefit of the pathogen is a phenomenon that deserves to be looked into in detail. We have tested the hypothesis that lipoarabinomannan (LAM) from the virulent species of Mycobacterium tuberculosis possesses the ability to modulate signaling pathways linked to cell survival. The Bcl-2 family member Bad is a proapoptotic protein. Phosphorylation of Bad promotes cell survival in many cell types. We demonstrate that man-LAM stimulates Bad phosphorylation in a phosphatidylinositol 3-kinase (PI-3K)-dependent pathway in THP-1 cells. Man-LAM activated PI-3K. LAM-stimulated phosphorylation of Bad was abrogated in cells transfected with a dominant-negative mutant of PI-3K (Delta p85), indicating that activation of PI-3K is sufficient to trigger phosphorylation of Bad by LAM. Since phosphorylation of Bad occurred at serine 136, the target of the serine/threonine kinase Akt, the effect of LAM on Akt kinase activity was tested. Man-LAM could activate Akt as evidenced from phosphorylation of Akt at Thr(308) and by the phosphorylation of the exogenous substrate histone 2B. Akt activation was abrogated in cells transfected with Deltap85. The phosphorylation of Bad by man-LAM was abrogated in cells transfected with a kinase-dead mutant of Akt. These results establish that LAM-mediated Bad phosphorylation occurs in a PI-3K/Akt-dependent manner. It is therefore the first demonstration of the ability of a mycobacterial virulence factor to up-regulate a signaling pathway involved in cell survival. This is likely to be one of a number of virulence-associated mechanisms by which bacilli control host cell apoptosis.     

CEACAM1 (CD66a) promotes human monocyte survival via a phosphatidylinositol 3-kinase- and AKT-dependent pathway

CEACAM1 (also known as CD66a) is a transmembrane glycoprotein that mediates homophilic intercellular interactions that influence cellular growth, immune cell activation, and tissue morphogenesis. Various studies have suggested a link between CEACAM1 and cellular apoptosis, including a recent demonstration that ERK1/2 signaling is triggered downstream of CEACAM1. In this study, we reveal that CEACAM1-long binding confers survival signals to human peripheral blood mononuclear cells. CEACAM-specific antibodies effectively protected peripheral blood mononuclear cells from apoptosis, with this effect being particularly dramatic for primary monocytes that undergo spontaneous apoptosis during in vitro culture. This protective effect was reiterated when using soluble CEACAM1, which binds to cell-surface CEACAM1 via homophilic interactions. Monocyte survival correlated with a CEACAM1-dependent up-regulation of the cellular inhibitor of apoptosis Bcl-2 and the abrogation of caspase-3 activation. CEACAM1 binding triggered a phosphatidylinositol 3-kinase-dependent activation of the protein kinase Akt without influencing the activity of extracellular signal-related kinase ERK, whereas the phosphatidylinositol 3-kinase-specific inhibitor LY294002 effectively blocked the protective effect of CEACAM1. Together, this work indicates that CEACAM1 confers a phosphatidylinositol 3-kinase- and Akt-dependent survival signal that inhibits mitochondrion-dependent apoptosis of monocytes. By controlling both ERK/MEK and PI3K/Akt pathways, CEACAM1 functions as a key regulator of contact-dependent control of cell survival, differentiation, and growth.     

Clusterin activates survival through the phosphatidylinositol 3-kinase/Akt pathway

Clusterin is, in its major form, a secreted heterodimeric disulfide-linked glycoprotein (75-80 kDa). It was first linked to cell death in the rat ventral prostate after androgen deprivation. Recent studies have demonstrated that overexpression of clusterin in prostatic cells protects them against tumor necrosis factor-alpha (TNFalpha)-induced apoptosis. However the details of this survival mechanism remain undefined. Here, we investigate how clusterin prevents cells from undergoing TNFalpha-induced apoptosis. We established a double-stable prostatic cell line for inducible clusterin by using the Tet-On gene expression system. We demonstrated that 50% of the cells overexpressing clusterin escaped from TNFalpha- and actinomycin D-induced cell death. Moreover we demonstrated that the incubation of MLL cells with conditioned medium containing the secreted clusterin or the supplementation of purified clusterin in the extracellular medium decreased the TNFalpha-induced apoptosis significantly. This extracellular action implicates megalin, the putative membrane receptor for clusterin to mediate survival. Indeed clusterin overexpression up-regulated the expression of megalin and induced its phosphorylation in a dose-dependent manner. We interestingly showed that clusterin overexpression is associated with the up-regulation of the phosphorylation of Akt. Activated Akt induced the phosphorylation of Bad and caused a decrease of cytochrome c release. These results enable us to pinpoint one mechanism by which secreted clusterin favors survival in androgen-independent prostate cancer cells, implicating its receptor megalin and Akt survival pathway.     

Molecular pharmacology of phosphatidylinositol 3-kinase inhibition in human glioma

Gliomas are primary brain tumors with poor prognosis that exhibit frequent abnormalities in phosphatidylinositol 3-kinase (PI3 kinase) signaling. We investigated the molecular mechanism of action of the isoform-selective Class I PI3 kinase and mTOR inhibitor PI-103 in human glioma cells. The potent inhibitory effects of PI-103 on the PI3 kinase pathway were quantified. PI-103 and the mTOR inhibitor rapamycin both inhibited RPS6 phosphorylation but there were clear differences in the response of upstream components of the PI3 kinase pathway, such as phosphorylation of Thr308-AKT, that were inhibited by PI-103 but not rapamycin. Gene expression profiling identified altered expression of genes encoding regulators of the cell cycle and cholesterol metabolism, and genes modulated by insulin or IGF1 signaling, rapamycin treatment or nutrient starvation. PI-103 decreased expression of positive regulators of G1/S phase progression and increased expression of the negative cell cycle regulator p27kip1. A reversible PI-103-mediated G1 cell cycle arrest occurred without significant apoptosis, consistent with the altered gene expression detected. PI-103 induced vacuolation and processing of LC-3i to LC-3ii, which are features of an autophagic response. In contrast to PI-103, LY294002 and PI-387 induced apoptosis, indicative of likely off-target effects. PI-103 interacted synergistically or additively with cytotoxic agents used in the treatment of glioma, namely vincristine, BCNU and temozolomide. Compared to individual treatments, the combination of PI-103 with temozolomide significantly improved the response of U87MG human glioma xenografts. Our results support the therapeutic potential for PI3 kinase inhibitors with PI-103-like profile as therapeutic agents for the treatment of glioma.     

Mammary epithelial-mesenchymal interaction regulates fibronectin alternative splicing via phosphatidylinositol 3-kinase

The way alternative splicing is regulated within tissues is not understood. A relevant model of this process is provided by fibronectin, an important extracellular matrix protein that plays a key role in cell adhesion and migration and contains three alternatively spliced regions known as EDI, EDII, and IIICS. We used a cell culture system to simulate mammary epithelial-stromal communication, a process that is crucial for patterning and function of the mammary gland, and studied the effects of extracellular signals on the regulation of fibronectin pre-mRNA alternative splicing. We found that soluble factors from a mammary mesenchymal cell-conditioned medium, as well as the growth factors HGF/SF (hepatocyte growth factor/scatter factor), KGF (keratinocyte growth factor), and aFGF (acidic fibroblast growth factor), stimulate EDI and IIICS but not EDII inclusion into fibronectin mRNA in the mammary epithelial cell line SCp2, favoring fibronectin isoforms associated with proliferation, migration, and tissue remodeling. We explored the signaling pathways involved in this regulation and found that the mammary mesenchymal cell-conditioned medium and HGF/SF act through a phosphatidylinositol 3-kinase-dependent cascade to alter fibronectin alternative splicing. This splicing regulation is independent from promoter structure and de novo protein synthesis but does require two exonic elements within EDI. These results shed light on how extracellular stimuli are converted into changes in splicing patterns.     

Differential effects of spermine on phosphatidylinositol 3-kinase and phosphatidylinositol phosphate 5-kinase

The metabolism of phosphoinositides plays an important role in the signal transduction pathways. We report here that naturally occuring polyamines affect the activities of phosphatidylinositol (PI) 3-kinase and PI 4-phosphate (PIP) 5-kinase differently. While polyamines inhibited the PI 3-kinase activity, they stimulated the activity of PIP 5-kinase in the order of spermine > spermidine > putrescine. Spermine inhibited the PI 3-kinase activity in a concentration-dependent manner with an IC₅₀ of 100 μM. On the other hand, spermine (5 mH) stimulated the activity of PIP 5-kinase 2–3 fold. Kinetic studies of spermine-mediated inhibition of PI 3-kinase revealed that it was noncompetitive with respect to ATP. The effect of Mg²⁺ and PIP, concentration on kinase activity was sigmoidal, with spermine inhibiting PI 3-kinase activity at all PIP₂ concentrations. While 1 mH calcium stimulated PI 3-kinase activity at submaximal concentrations of Mg²⁺ (1.25 mH), inhibition was observed at optimal concentration of Mg²⁺(2 mM). We propose that spermine may modulate the cellular signal by virtue of its differential effects on phosphoinositide kinases.     

Autotaxin promotes motility via G protein-coupled phosphoinositide 3-kinase gamma in human melanoma cells

Autotaxin (ATX), an exo-nucleotide pyrophosphatase and phosphodiesterase, stimulates tumor cell motility at sub-nanomolar levels and augments invasiveness and angiogenesis. We investigated the role of G protein-coupled phosphoinositide 3-kinase gamma (PI3Kgamma) in ATX-mediated tumor cell motility stimulation. Pretreatment of human melanoma cell line A2058 with wortmannin or LY294002 inhibited ATX-induced motility. ATX increased the PI3K activity in p110gamma, but not p85, immunoprecipitates. This effect was abrogated by PI3K inhibitors or inhibited by pertussis toxin. Furthermore, stimulation of tumor cell motility by ATX was inhibited by catalytically inactive form of PI3Kgamma, strongly indicating the crucial role of PI3Kgamma for ATX-mediated motility in human melanoma cells     

Regulation of phospholipase C-gamma 2 via phosphatidylinositol 3-kinase in macrophages.

Phospholipase C-gamma (PLC-gamma) isoforms are thought to be activated by both tyrosine phosphorylation and phosphatidylinositol 3,4,5 trisphosphate (PtdIns 3,4,5 P(3)), the product of phosphatidylinositol 3-kinase (PtdIns 3-kinase). In this study, we show that stimulation of mouse macrophages with either zymosan beads or bacteria (Prevotella intermedia) induced tyrosine phosphorylation of PLC-gamma 2. Zymosan stimulation also induced translocation to membrane and cytoskeleton fractions, which was inhibited by the PtdIns 3-kinase inhibitors wortmannin and LY 294002. However, the tyrosine phosphorylation of PLC-gamma 2 induced by zymosan was not affected by the inhibitors wortmannin and LY 294002. In contrast to zymosan and bacteria, PLC-gamma 2 was not phosphorylated by stimulation with lipopolysaccharide (LPS), phorbol ester or calcium ionophore. Moreover, the PLC-gamma 1 isoform was not detected in mouse macrophages. These data indicate that PtdIns 3-kinase is critical for the translocation but not for the tyrosine phosphorylation of PLC-gamma 2 in mouse macrophages and that the latter may be insufficient for enzyme activation.     

Cancer-specific mutations in phosphatidylinositol 3-kinase

Cancer-specific mutations in the catalytic subunit of phosphatidylinositol 3-kinase (PI3K) p110 alpha occur in diverse tumors in frequencies that can exceed 30%. The majority of these mutations map to one of three hot spots in the gene, and the rest are distributed over much of the PI3K coding sequence. Most of the cancer-specific mutations induce a gain of function that results in oncogenicity, elevated lipid kinase activity and constitutive signaling through the kinases Akt and TOR. The location of the mutations on a model structure of p110 alpha indicates several distinct mechanisms for the gain of function. The mutated p110 alpha proteins are promising cancer targets. Although identification of mutant-specific small-molecule inhibitors seems technically challenging, the therapeutic benefits from such inhibitors could be extremely important.     

PDGF stimulates DNA synthesis in human vascular smooth muscle cells via a novel wortmannin-insensitive phosphatidylinositol 3-kinase

The class 1(A) phosphatidylinositol 3-kinase enzymes consist of a number of heterodimeric complexes of regulatory and catalytic subunits and have been implicated in a number of cellular responses. While platelet-derived growth factor (PDGF)-induced chemotaxis of human vascular smooth muscle cells (HVSMC) is inhibited by both wortmannin and LY294002, DNA synthesis is only inhibited by LY294002. Serum-induced DNA synthesis however is inhibited by LY294002, wortmannin and rapamycin. Similarly PDGF-induced protein kinase B (PKB) activation is inhibited by LY294002 but not by wortmannin or rapamycin. In conclusion PDGF-induced DNA synthesis appears to occur through a phosphatidylinositol 3-kinase (PI3-K)-dependent, but wortmannin-insensitive, PKB/Akt pathway.     

Inhibition of phosphatidylinositol 3-kinase promotes tumor cell resistance to chemotherapeutic agents via a mechanism involving delay in cell cycle progression

Approaches to overcome chemoresistance in cancer cells have involved targeting specific signaling pathways such as the phosphatidylinositol 3-kinase (PI3K) pathway, a stress response pathway known to be involved in the regulation of cell survival, apoptosis and growth. The present study determined the effect of PI3K inhibition on the clonogenic survival of human cancer cells following exposure to various chemotherapeutic agents. Treatment with the PI3K inhibitors LY294002 or Compound 15e resulted in increased survival of MDA-MB-231 breast carcinoma cells after exposure to doxorubicin, etoposide, 5-fluorouracil, and vincristine. Increased survival following PI3K inhibition was also observed in DU-145 prostate, HCT-116 colon and A-549 lung carcinoma cell lines exposed to doxorubicin. Increased cell survival mediated by LY294002 was correlated with a decrease in cell proliferation, which was linked to an increase in the proportion of cells in the G₁ phase of the cell cycle. Inhibition of PI3K signaling also resulted in higher levels of the cyclin-dependent kinase inhibitors p21^Waf1/Cip1 and p27^Kip1; and knockdown of p27^kip1 with siRNA attenuated resistance to doxorubicin in cells treated with LY294002. Incubation in the presence of LY294002 after exposure to doxorubicin resulted in decreased cell survival. These findings provide evidence that PI3K inhibition leads to chemoresistance in human cancer cells by causing a delay in cell cycle; however, the timing of PI3K inhibition (either before or after exposure to anti-cancer agents) may be a critical determinant of chemosensitivity.     

Hck enhances the adherence of lipopolysaccharide-stimulated macrophages via Cbl and phosphatidylinositol 3-kinase

Src family tyrosine kinases have previously been proposed to mediate some of the biological effects of lipopolysaccharide on macrophages. Accordingly, we have sought to identify substrates of Src family kinases in lipopolysaccharide-stimulated macrophages. Stimulation of Bac1.2F5 macrophage cells with lipopolysaccharide was found to induce gradual and persistent tyrosine phosphorylation of Cbl in an Src family kinase-dependent manner. Immunoprecipitation experiments revealed that Cbl associates with Hck in Bac1.2F5 cells, while expression of an activated form of Hck in Bac1.2F5 cells induces tyrosine phosphorylation of Cbl in the absence of lipopolysaccharide stimulation. The Src homology 3 domain of Hck can directly bind Cbl, and this interaction is important for phosphorylation of Cbl. Association of the p85 subunit of phosphatidylinositol (PI) 3-kinase with Cbl is enhanced following lipopolysaccharide stimulation of Bac1.2F5 cells, and transient expression experiments indicate that phosphorylation of Cbl by Hck can facilitate the association of p85 with Cbl. Lipopolysaccharide treatment also stimulates the partial translocation of Hck to the cytoskeleton of Bac1.2F5 cells. Notably, lipopolysaccharide enhances the adherence of Bac1.2F5 cells, an effect that is dependent on the activity of Src family kinases and PI 3-kinase. Thus, we postulate that Hck enhances the adherence of lipopolysaccharide-stimulated macrophages, at least in part, via Cbl and PI 3-kinase.     

Group IB secretory phospholipase A2 promotes matrix metalloproteinase-2-mediated cell migration via the phosphatidylinositol 3-kinase and Akt pathway

Secretory phospholipase A(2) (sPLA(2)), abundantly expressed in various cells including fibroblasts, is able to promote proliferation and migration. Degradation of collagenous extracellular matrix by matrix metalloproteinase (MMP) plays a role in the pathogenesis of various destructive disorders, such as rheumatoid arthritis, tumor invasion, and metastasis. Here we show that group IB PLA(2) increased pro-MMP-2 activation in NIH3T3 fibroblasts. MMP-2 activity was stimulated by group IB PLA(2) in a dose- and time-dependent manner. Consistent with MMP-2 activation, sPLA(2) decreased expression of type IV collagen. These effects are due to the reduction of tissue inhibitor of metalloproteinase-2 (TIMP-2) and the activation of the membrane type1-MMP (MT1-MMP). The decrease of TIMP-2 levels in conditioned media and the increase of MT1-MMP levels in plasma membrane were observed. In addition, treatment of cells with decanoyl Arg-Val-Lys-Arg-chloromethyl ketone, an inhibitor of pro-MT1-MMP, suppressed sPLA(2)-mediated MMP-2 activation, whereas treatment with bafilomycin A1, an inhibitor of H(+)-ATPase, sustained MMP-2 activation by sPLA(2). The involvement of phosphatidylinositol 3-kinase (PI3K) and Akt in the regulation of MMP-2 activity was further suggested by the findings that PI3K and Akt were phosphorylated by sPLA(2). Expression of p85alpha and Akt mutants, or pretreatment of cells with LY294002, a PI3K inhibitor, attenuated sPLA(2)-induced MMP-2 activation and migration. Taken together, these results suggest that sPLA(2) increases the pro-MMP-2 activation and migration of fibroblasts via the PI3K and Akt-dependent pathway. Because MMP-2 is an important factor directly involved in the control of cell migration and the turnover of extracellular matrix, our study may provide a mechanism for sPLA(2)-promoted fibroblasts migration.     

Insulin activation of phosphatidylinositol 3-kinase in human skeletal muscle in vivo

Hickey, Matthew S., Charles J. Tanner, D. Sean O'Neill,Lydia J. Morgan, G. Lynis Dohm, and Joseph A. Houmard. Insulin activation of phosphatidylinositol 3-kinase in human skeletal muscle invivo. J. Appl. Physiol. 83(3):718-722, 1997.The purpose of this investigation was to determinewhether insulin-stimulated phosphatidylinositol 3-kinase (PI3-kinase)activity is detectable in needle biopsies of human skeletal muscle.Sixteen healthy nonobese males matched for age, percent fat, fastinginsulin, and fasting glucose participated in one of two experimentalprotocols. During an intravenous glucose tolerance test (IVGTT)protocol, insulin-stimulated PI3-kinase activity was determined frompercutaneous needle biopsies at 2, 5, and 15 min post-insulinadministration (0.025 U/kg). In the second group, a 2-h, 100 mU · m² · min¹euglycemic hyperinsulinemic clamp was performed, and biopsies wereobtained at 15, 60, and 120 min after insulin infusion was begun.Insulin stimulated PI3-kinase activity by 1.6 ± 0.2-, 2.2 ± 0.3-, and 2.2 ± 0.4-fold at 2, 5, and 15 min, respectively, duringthe IVGTT. During the clamp protocol, PI3-kinase was elevated by 5.3 ± 1.3-, 8.0 ± 2.6-, and 2.7 ± 1.4-fold abovebasal at 15, 60, and 120 min, respectively. Insulin-stimulatedPI3-kinase activity at 15 min post-insulin administration wassignificantly greater during the clamp protocol vs. the IVGTT(P < 0.05). These observations suggest that insulin-stimulated PI3-kinase activity is detectable inneedle biopsies of human skeletal muscle, and furthermore, that theeuglycemic, hyperinsulinemic clamp protocol may be a useful tool toassess insulin signaling in vivo.

     

Insulin-like growth factor-II regulates the 12-lipoxygenase gene expression and promotes cell proliferation in human keratinocytes via the extracellular regulatory kinase and phosphatidylinositol 3-kinase pathways

To study the relationship between insulin-like growth factor-II (IGF-II) and 12-lipoxygenase (12-LOX) that are upregulated in psoriasis, we monitored 12-lipoxygenase expression in the insulin-like growth factor-II treated human keratinocytes and explored the signaling pathways of 12-lipoxygenase expression. Insulin-like growth factor-II induced 12-lipoxygenase mRNA and protein levels in human keratinocytes through two major signal transduction pathways, namely, the extracellular signaling-regulated kinase (ERK)-mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. The IGF-II-induced upregulation of 12-lipoxygenase was attenuated by pretreating the cells with selective inhibitors or by overexpressing dominant-negative MEK. In addition, treatment of HaCaT cells with the 12-lipoxygenase metabolite 12 (S)-hydroxyeicosatetraenoic acid (12(S)-HETE) directly stimulated DNA synthesis and mitogenesis, and injection of insulin-like growth factor-II into the skin of hairless mice induced epidermal hyperplasia. These results suggest that insulin-like growth factor-II is involved in the pathogenesis of psoriasis as a paracrine inducer of 12-lipoxygenase.