Glucose, dexamethasone, and the unfolded protein response regulate TRB3 mRNA expression in 3T3-L1 adipocytes and L6 myotubes

Tribbles 3 (TRB3) is a recently recognized atypical inactive kinase that negatively regulates Akt activity in hepatocytes, resulting in insulin resistance. Recent reports link TRB3 to nutrient sensing and regulation of cell survival under stressful conditions. We studied the regulation of TRB3 by glucose, insulin, dexamethasone (Dex), and the unfolded protein response (UPR) in 3T3-L1 adipocytes and in L6 myotubes. In 3T3-L1 adipocytes, incubation in high glucose with insulin did not increase TRB3 mRNA expression. Rather, TRB3 mRNA increased fourfold with glucose deprivation and two- to threefold after incubation with tunicamcyin (an inducer of the UPR). Incubation of cells in no glucose or in tunicamcyin stimulated the expression of CCAAT/enhancer-binding protein homologous protein. In L6 myotubes, absent or low glucose induced TRB3 mRNA expression by six- and twofold, respectively. The addition of Dex to 5 mM glucose increased TRB3 mRNA expression twofold in 3T3-L1 adipocytes but decreased it 16% in L6 cells. In conclusion, TRB3 is not the mediator of high glucose or glucocorticoid-induced insulin resistance in 3T3-L1 adipocytes or L6 myotubes. TRB3 is induced by glucose deprivation in both cell types as a part of the UPR, where it may be involved in regulation of cell survival in response to glucose depletion.     

PI3K activates negative and positive signals to regulate TRB3 expression in hepatic cells

TRB3 is a pseudokinase whose expression is regulated during stress response and changing of nutrient status. TRB3 negatively regulates Akt activation and noticeably, TRB3 expression is induced by insulin. Here, we sought to determine the dynamic relationship between TRB3 expression and Akt activation. We find that insulin induces TRB3 expression in cell type dependent manner such that in hepatic cells and adipocytes but not Beta cells and muscle cells. In Fao hepatoma cells, induction of TRB3 expression by insulin restrains Akt activation and renders Akt refractory to further activation. In addition, we have also analyzed the roles of PI3K and its downstream kinases Akt and atypical PKC in TRB3 expression. Induction of TRB3 expression by insulin requires PI3K. However, inactivation of Akt enhances TRB3 expression whereas inhibition of PKCzeta expression impairs TRB3 expression induced by insulin. Our data demonstrated that PI3K conveys both negative and positive signals to TRB3 expression. We suggest that insulin-induced TRB3 expression functions as an indicator how multiple insulin-induced signal transduction pathways are balanced.     

Phosphatidylinositol 3-kinase signaling controls levels of hypoxia-inducible factor 1.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway has inherent oncogenic potential. It is up-regulated in diverse human cancers by either a gain of function in PI3K itself or in its downstream target Akt or by a loss of function in the negative regulator PTEN. However, the complete consequences of this up-regulation are not known. Here we show that insulin and epidermal growth factor or an inactivating mutation in the tumor suppressor PTEN specifically increase the protein levels of hypoxia-inducible factor (HIF) 1alpha but not of HIF-1beta in human cancer cell lines. This specific elevation of HIF-1alpha protein expression requires PI3K signaling. In the prostate carcinoma-derived cell lines PC-3 and DU145, insulin- and epidermal growth factor-induced expression of HIF-1alpha was inhibited by the PI3K-specific inhibitors LY294002 and wortmannin in a dose-dependent manner. HIF-1beta expression was not affected by these inhibitors. Introduction of wild-type PTEN into the PTEN-negative PC-3 cell line specifically inhibited the expression of HIF-1alpha but not that of HIF-1beta. In contrast to the HIF-1alpha protein, the level of HIF-1alpha mRNA was not significantly affected by PI3K signaling. Vascular endothelial growth factor reporter gene activity was induced by insulin in PC-3 cells and was inhibited by the PI3K inhibitor LY294002 and by the coexpression of a HIF-1 dominant negative construct. Vascular endothelial growth factor reporter gene activity was also inhibited by expression of a dominant negative PI3K construct and by the tumor suppressor PTEN.     

Toll-like receptor 4 ligation confers chemoresistance to docetaxel on PC-3 human prostate cancer cells

Recent studies have indicated that Toll-like receptors (TLRs) are implicated in the development of chemoresistance in cancer cells. TLR4 has been shown to be highly expressed in prostate cancer cells and contributes to tumor cell survival and invasion. In this study, we aimed to investigate the role of TLR4 signaling in the chemoresistance of prostate cancer cells. We showed that ligation of TLR4 with lipopolysaccharide (LPS) abrogated docetaxel-induced growth suppression in PC-3 prostate cancer cells, with an increase in the half-maximal inhibitory concentration. Downregulation of TLR4 using small-interference RNA sensitized PC-3 cells to docetaxel-induced apoptosis as determined by annexin V staining and poly (ADP-ribose) polymerase cleavage, which was coupled with increased Bax expression and decreased Bcl-2. TLR4 ligation resulted in a marked increase in the phosphorylation of phosphatidylinositol 3-kinase (PI3-K) and Akt. The pretreatment with a PI3-K inhibitor LY294002 reduced LPS-mediated resistance to docetaxel, significantly decreasing the viability of PC-3 cells. Our data show that TLR4 ligation contributes to the chemosensitivity of prostate cancer cells, which at least partially involves the activation of the PI3-K/Akt pathway. Therefore, TLR4 signaling may represent a promising target for the improvement of chemotherapeutic efficacy in prostate cancer.     

H-ras Induces Glucose Uptake in Brown Adipocytes in an Insulin- and Phosphatidylinositol 3-Kinase-Independent Manner

Fetal brown adipocytes (parental cells) expressed mainly Glut4 mRNA glucose transporter, the expression of Glut1 mRNA being much lower. At physiological doses, insulin stimulation for 15 min increased 3-fold glucose uptake and doubled the amount of Glut4 protein located at the plasma membrane. Moreover, phosphatidylinositol (PI) 3-kinase activity was induced by the presence of insulin in those cells, glucose uptake being precluded by PI 3-kinase inhibitors such as wortmannin or LY294002. H-ras^lys12-transformed brown adipocytes showed a 10-fold higher expression of Glut1 mRNA and protein than parental cells, Glut4 gene expression being completely down-regulated. Glucose uptake increased by 10-fold in transformed cells compared to parental cells; this uptake was unaltered in the presence of insulin and/or wortmannin. Transient transfection of parental cells with a dominant form of active Ras increased basal glucose uptake by 5-fold, no further effects being observed in the presence of insulin. However, PI 3-kinase activity (immunoprecipitated with anti-αp85 subunit of PI 3-kinase) remained unaltered in H-ras permanent and transient transfectants. Our results indicate that activated Ras induces brown adipocyte glucose transport in an insulin-independent manner, this induction not involving PI 3-kinase activation.     

Bcl-xL mediates a survival mechanism independent of the phosphoinositide 3-kinase/Akt pathway in prostate cancer cells

Among various molecular strategies by which prostate cancer cells evade apoptosis, phosphoinositide 3-kinase (PI3K)/Akt signaling represents a dominant survival pathway. However, different prostate cancer cell lines such as LNCaP and PC-3 display differential sensitivity to the apoptotic effect of PI3K inhibition in serum-free media, reflecting the heterogeneous nature of prostate cancer in apoptosis regulation. Whereas both cell lines are equally susceptible to LY294002-mediated Akt dephosphorylation, only LNCaP cells default to apoptosis, as evidenced by DNA fragmentation and cytochrome c release. In PC-3 cells, Akt deactivation does not lead to cytochrome c release, suggesting that the intermediary signaling pathway is short-circuited by an antiapoptotic factor. This study presents evidence that Bcl-xL overexpression provides a distinct survival mechanism that protects PC-3 cells from apoptotic signals emanating from PI3K inhibition. First, the Bcl-xL/BAD ratio in PC-3 cells is at least an order of magnitude greater than that of LNCaP cells. Second, ectopic expression of Bcl-xL protects LNCaP cells against LY294002-induced apoptosis. Third, antisense down-regulation of Bcl-xL sensitizes PC-3 cells to the apoptotic effect of LY294002. The physiological relevance of this Bcl-xL-mediated survival mechanism is further underscored by the protective effect of serum on LY294002-induced cell death in LNCaP cells, which is correlated with a multifold increase in Bcl-xL expression. In contrast to Bcl-xL, Bcl-2 expression levels are similar in both cells lines, and do not respond to serum stimulation, suggesting that Bcl-2 may not play a physiological role in antagonizing apoptosis signals pertinent to BAD activation in prostate cancer cells.     

Alpha-tomatine synergises with paclitaxel to enhance apoptosis of androgen-independent human prostate cancer PC-3 cells in vitro and in vivo

Alpha (α)-tomatine, a major saponin found in tomato has been shown to inhibit the growth of androgen-independent prostate cancer PC-3 cells. The effects of α-tomatine in combination with the chemotherapeutic agent paclitaxel against PC-3 cells were investigated in the present study. Combined treatment with a sub-toxic dose of α-tomatine and paclitaxel significantly decreased cell viability with concomitant increase in the percentage of apoptotic PC-3 cells. The combined treatment, however, had no cytotoxic effect on the non-neoplastic prostate RWPE-1 cells. Apoptosis of PC-3 cells was accompanied by the inhibition of PI3K/Akt pro-survival signaling, an increase in the expression of the pro-apoptotic protein BAD but a decrease in the expressions of anti-apoptotic proteins, Bcl-2 and Bcl-xL. Results from a mouse xenograft model showed the combined treatment completely suppressed subcutaneous tumor growth without significant side effects. Consistent with its in vitro anti-cancer effects, tumor materials from mice showed increased apoptosis of tumor cells with reduced protein expression of activated PI3K/Akt. These results suggest that the synergistic anti-cancer effects of paclitaxel and α-tomatine may be beneficial for refractory prostate cancer treatment.     

Induced expression of MMP-9 in C6 glioma cells is inhibited by PDGF via a PI 3-kinase-dependent pathway

The involvement of phosphatidylinositol 3 (PI 3)-kinase in the signalling pathways leading to MMP-9 expression in glioma cells remains unclear. Here, we report that PI 3-kinase inhibits MMP-9 expression induced by either IL-1 or TNF-alpha in rat C6 glioma cells. Using zymography and semi-quantitative RT-PCR analysis, we showed that treatment of C6 cells with wortmannin, an inhibitor of PI 3-kinase activity, potentiated the expression of MMP-9 induced by both cytokines. In contrast, platelet-derived growth factor (PDGF), an inducer of PI 3-kinase activity in C6 cells, inhibited IL-1- or TNF-alpha-induced MMP-9 secretion. Accordingly, this inhibition by PDGF was prevented by wortmannin. Furthermore, stable C6 clones over-expressing the dominant-negative form the regulatory subunit of PI 3-kinase potentiated the expression of MMP-9 induced by IL-1 or TNF-alpha. Taken together, these results suggest that PI 3-kinase may act as a negative regulator of MMP-9 expression in C6 glioma cells.     

Phosphatidylinositol 3'-kinase, but not S6-kinase, is required for insulin-like growth factor-I and IL-4 to maintain expression of Bcl-2 and promote survival of myeloid progenitors

Phosphatidylinositol 3'-kinase (PI 3-kinase) catalyzes the formation of 3' phosphoinositides and has been implicated in an intracellular signaling pathway that inhibits apoptosis in both neuronal and hemopoietic cells. Here, we investigated two potential downstream mediators of PI 3-kinase, the serine/threonine p70 S6-kinase (S6-kinase) and the antiapoptotic protein B cell lymphoma-2 (Bcl-2). Stimulation of factor-dependent cell progenitor (FDCP) cells with either IL-4 or insulin-like growth factor (IGF)-I induced a 10-fold increase in the activity of both PI 3-kinase and S6-kinase. Rapamycin blocked 90% of the S6-kinase activity but did not affect PI 3-kinase, whereas wortmannin and LY294002 inhibited the activity of both S6-kinase and PI 3-kinase. However, wortmannin and LY294002, but not rapamycin, blocked the ability of IL-4 and IGF-I to promote cell survival. We next established that IL-3, IL-4, and IGF-I increase expression of Bcl-2 by >3-fold. Pretreatment with inhibitors of PI 3-kinase, but not rapamycin, abrogated expression of Bcl-2 caused by IL-4 and IGF-I, but not by IL-3. None of the cytokines affected expression of the proapoptotic protein Bax, suggesting that all three cytokines were specific for Bcl-2. These data establish that inhibition of PI 3-kinase, but not S6-kinase, blocks the ability of IL-4 and IGF-I to increase expression of Bcl-2 and protect promyeloid cells from apoptosis. The requirement for PI 3-kinase to maintain Bcl-2 expression depends upon the ligand that activates the cell survival pathway.     

PI3K/PTEN/AKT signaling regulates prostate tumor angiogenesis

PI3K pathway exerts its function through its downstream molecule AKT in regulating various cell functions including cell proliferation, cell transformation, cell apoptosis, tumor growth and angiogenesis. PTEN is an inhibitor of PI3K, and its loss or mutation is common in human prostate cancer. But the direct role and mechanism of PI3K/PTEN signaling in regulating angiogenesis and tumor growth in vivo remain to be elucidated. In this study, by using chicken chorioallantoic membrane (CAM) and in nude mice models, we demonstrated that inhibition of PI3K activity by LY294002 decreased PC-3 cells-induced angiogenesis. Reconstitution of PTEN, the molecular inhibitor of PI3K in PC-3 cells inhibited angiogenesis and tumor growth. Immunohistochemical staining indicated that PTEN expression suppressed HIF-1, VEGF and PCNA expression in the tumor xenographs. Similarly, expression of AKT dominant negative mutant also inhibited angiogenesis and tumor growth, and decreased the expression of HIF-1 and VEGF in the tumor xenographs. These results suggest that inhibition of PI3K signaling pathway by PTEN inhibits tumor angiogenesis and tumor growth. In addition, we found that AKT is the downstream target of PI3K in controlling angiogenesis and tumor growth, and PTEN could inhibit angiogenesis by regulating the expression of HIF-1 and VEGF expression through AKT activation in PC-3 cells.     

Matrix-independent activation of phosphatidylinositol 3-kinase,Stat3, and cyclin A-associated Cdk2 Is essential for anchorage-independent growth of v-Ros-transformed chicken embryo fibroblasts

The question remains open whether the signaling pathways shown to be important for growth and transformation in adherent cultures proceed similarly and play similar roles for cells grown under anchorage-independent conditions. Chicken embryo fibroblasts (CEF) infected with the avian sarcoma virus UR2, encoding the oncogenic receptor protein-tyrosine kinase (RPTK) v-Ros, or with two of its transformation-impaired mutants were grown in nonadherent conditions in methylcellulose (MC)-containing medium, and the signaling functions essential for Ros-induced anchorage-independent growth were analyzed. We found that the overall tyrosine phosphorylation of cellular proteins in CEF transformed by v-Ros or by two oncogenic nonreceptor protein-tyrosine kinases (PTKs), v-Src and v-Yes, was dramatically reduced in nonadherent conditions compared with that in adherent conditions, indicating that cell adhesion to the extracellular matrix plays an important role in efficient substrate phosphorylation by these constitutively activated PTKs. The UR2 transformation-defective mutants were differentially impaired compared with UR2 in the activation of phosphatidylinositol 3-kinase (PI 3-kinase) and Stat3 in nonadherent conditions. Consistently, the constitutively activated mutants of PI 3-kinase and Stat3 rescued the ability of the UR2 mutants to promote anchorage-independent growth. Conversely, dominant negative mutants of PI 3-kinase and Stat3 inhibited UR2-induced anchorage-independent growth. UR2-infected CEF grown in nonadherent conditions displayed faster cell cycle progression than the control or the UR2 mutant-infected cells, and this appeared to correlate with a PI 3-kinase-dependent increase in cyclin A-associated Cdk2 activity. Treatment of UR2-infected cells with Cdk2 inhibitors led to the loss of the anchorage-independent growth-promoting activity of UR2. In conclusion, we have adopted an experimental system enabling us to study the signaling pathways in cells grown under anchorage-independent conditions and have identified matrix-independent activation of PI 3-kinase and Stat3 signaling functions, as well as the PI 3-kinase-dependent increase of cyclin A-associated Cdk2 kinase activity, to be critical for the Ros-PTK-induced anchorage-independent growth.     

Cyclin D1 Expression Mediated by Phosphatidylinositol 3-Kinase through mTOR-p70S6K-Independent Signaling in Growth Factor-Stimulated NIH 3T3 Fibroblasts

Phosphatidylinositol (PI) 3-kinase is required for G₁ to S phase cell cycle progression stimulated by a variety of growth factors and is implicated in the activation of several downstream effectors, including p70^S6K. However, the molecular mechanisms by which PI 3-kinase is engaged in activation of the cell cycle machinery are not well understood. Here we report that the expression of a dominant negative (DN) form of either the p110α catalytic or the p85 regulatory subunit of heterodimeric PI 3-kinase strongly inhibited epidermal growth factor (EGF)-induced upregulation of cyclin D1 protein in NIH 3T3(M17) fibroblasts. The PI 3-kinase inhibitors LY294002 and wortmannin completely abrogated increases in both mRNA and protein levels of cyclin D1 and phosphorylation of pRb, inducing G₁ arrest in EGF-stimulated cells. By contrast, rapamycin, which potently suppressed p70^S6K activity throughout the G₁ phase, had little inhibitory effect, if any, on either of these events. PI 3-kinase, but not rapamycin-sensitive pathways, was also indispensable for upregulation of cyclin D1 mRNA and protein by other mitogens in NIH 3T3 (M17) cells and in wild-type NIH 3T3 cells as well. We also found that an enforced expression of wild-type p110 was sufficient to induce cyclin D1 protein expression in growth factor-deprived NIH 3T3(M17) cells. The p110 induction of cyclin D1 in quiescent cells was strongly inhibited by coexpression of either of the PI 3-kinase DN forms, and by LY294002, but was independent of the Ras-MEK-ERK pathway. Unlike mitogen stimulation, the p110 induction of cyclin D1 was sensitive to rapamycin. These results indicate that the catalytic activity of PI 3-kinase is necessary, and could also be sufficient, for upregulation of cyclin D1, with mTOR signaling being differentially required depending upon cellular conditions.     

Role of Translation Initiation Factor 2B in Control of Cell Survival by the Phosphatidylinositol 3-Kinase/Akt/Glycogen Synthase Kinase 3β Signaling Pathway

The phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signaling pathway is an important mediator of growth factor-dependent survival of mammalian cells. A variety of targets of the Akt protein kinase have been implicated in cell survival, including the protein kinase glycogen synthase kinase 3beta (GSK-3beta). One of the targets of GSK-3beta is translation initiation factor 2B (eIF2B), linking global regulation of protein synthesis to PI 3-kinase/Akt signaling. Because of the central role of protein synthesis, we have investigated the involvement of eIF2B, which is inhibited as a result of GSK-3beta phosphorylation, in programmed cell death. We demonstrate that expression of eIF2B mutants lacking the GSK-3beta phosphorylation or priming sites is sufficient to protect both Rat-1 and PC12 cells from apoptosis induced by overexpression of GSK-3beta, inhibition of PI 3-kinase, or growth factor deprivation. Consistent with these effects on cell survival, expression of nonphosphorylatable eIF2B prevented inhibition of protein synthesis following treatment of cells with the PI 3-kinase inhibitor LY294002. Conversely, cycloheximide induced apoptosis of PC12 and Rat-1 cells, further indicating that protein synthesis was required for cell survival. Inhibition of translation resulting from treatment with cycloheximide led to the release of cytochrome c from mitochondria, similar to the effects of inhibition of PI 3-kinase. Expression of nonphosphorylatable eIF2B prevented cytochrome c release resulting from PI 3-kinase inhibition but did not affect cytochrome c release or apoptosis induced by cycloheximide. Regulation of translation resulting from phosphorylation of eIF2B by GSK-3beta thus appears to contribute to the control of cell survival by the PI 3-kinase/Akt signaling pathway, acting upstream of mitochondrial cytochrome c release.     

Protein kinase C modulates negatively the hepatocyte growth factor-induced migration, integrin expression and phosphatidylinositol 3-kinase activation

Previously, we reported that, in hepatocyte growth factor (HGF)-induced HepG2 cells, protein kinase C (PKC) decreased the duration of intensive Erk1/Erk2 MAP kinase activation. This study shows that the inhibition of PKC enhanced significantly the HGF-induced integrin expression. Beside the prolonged activation of Erk1/Erk2, the activity of phosphatidylinositol 3-kinase (PI 3K) was required for growth factor-induced integrin expression. PI 3-kinase was activated to a higher extent in response to HGF than to epidermal growth factor (EGF), though the activation was transient in both cases. In EGF-induced cells, PI 3K activation was terminated by the loss of phosphotyrosine docking sites for PI 3K. To the contrary, the decrease of PI 3K activation, which followed the HGF-induced increase was not accompanied by the loss of phosphotyrosine docking sites and was prevented by the inhibition of PKC. The negative modulator effects of PKC on integrin expression and PI 3-kinase activation correlated with its ability to limit the HGF-induced motogen response.     

Immune-mediated signaling in intestinal goblet cells via PI3-kinase- and AKT-dependent pathways

In the intestinal epithelium, activation of phosphatidylinositol 3-kinase (PI3-kinase)/AKT pathways, via growth factor-mediated signaling, has been shown to regulate cell proliferation and inhibit apoptosis. An immune-activated receptor critical for Th2 immune responses, IL-4Ralpha can also activate PI3-kinase via insulin receptor substrate (IRS)-dependent signaling. Here, using the intestinal goblet cell-specific gene RELMbeta, we investigated the effect of PI3-kinase activation via Th2 immune responses on the goblet cell phenotype. IL-13 stimulation activated PI3-kinase and AKT signal transduction in LS174T cells. Not only did pharmacological inhibition of PI3-kinase and AKT1/2 inhibit RELMbeta induction by IL-13, but AKT inhibition also significantly reduced constitutive basal expression of RELMbeta, a response reproduced by the simultaneous pharmacological inhibition of both epidermal growth factor receptor and IGF-I receptor signaling. In vivo, the disruption of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), an inhibitor of PI3-kinase activation, led to the activation of RELMbeta expression in the small intestine. Furthermore, induction of an intestinal Th2 immune response by infection with a small intestinal nematode parasite, Heligmosomoides polygyrus, led to enhanced epithelial cell proliferation, activation of AKT as demonstrated by the loss of Foxo1 nuclear localization, and robust induction of RELMbeta expression in wild-type, but not IL-4Ralpha knockout, mice. These results demonstrate that Th2 immune responses can regulate goblet cell responses by activation of PI3-kinase and AKT pathways via IL-4Ralpha.     

Dynamin inhibits phosphatidylinositol 3-kinase in hematopoietic cells

Phosphatidylinositol 3-kinase (PI 3-kinase) plays a role in late stages of endocytosis as well as in cellular proliferation and transformation. The SH3 domain of its regulatory p85 subunit stimulates the GTPase activity of dynamin in vitro. Dynamin is a GTPase enzyme required for endocytosis of activated growth factor receptors. An interaction between these proteins has not been demonstrated in vivo. Here, we report that dynamin associates with PI 3-kinase in hematopoietic cells. We detected both p85 and PI 3-kinase activity in dynamin immune complexes from IL-3-dependent BaF3 cells. However, this association was significantly reduced in BaF3 cells transformed with the BCR/abl oncogene. After transformation only a 4-fold increase in PI 3-kinase activity was detected in dynamin immune complexes, whereas grb2 associated activity was elevated 20-fold. Furthermore, dynamin inhibited the activity of both purified recombinant and immunoprecipitated PI 3-kinase. In BaF3 cells expressing a temperature-sensitive mutant of BCR/abl, a significant decrease in p85 and dynamin association was observed 4 h after the induction of BCR/abl activity. In contrast, in IL-3-stimulated parental BaF3 cells, this association was increased. Our results demonstrate an in vivo association of PI 3-kinase with dynamin and this interaction regulates the activity of PI 3-kinase.