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PubMed2006

Glycine-extended gastrin activates two independent tyrosine-kinases in upstream of p85/p110 phosphatidylinositol 3-kinase in human colonic tumour cells.

AIM: To investigate whether Src, JAK2 and phosphatidylinositol 3-kinase (PI3K) pathways are involved in the proliferation of human colonic tumour cells induced by glycine-extended gastrin (G-gly), the precursor of the mature amidated gastrin and to elucidate the molecular interaction between these three kinases in response to this peptide. METHODS: Using the human colonic tumour cell line HCT116 as a model, we first measured the activation of PI3K, p60-Src and JAK2 in response to G-gly by in vitro kinase assays. Then we investigated the involvement of these kinases in G-gly-induced cell proliferation by MTT test. RESULTS: G-gly stimulation induced p60-Src, JAK2 and PI3K activation in HCT116. The different pathways were involved in proliferation of human colon cancer cells induced by G-gly. Furthermore, we found that both Src and JAK2 were necessary to PI3K regulation by this peptide. However, we did not find any cross-talk between the two tyrosine kinases. CONCLUSION: Our results suggest that the p60-Src/PI3K and JAK2/PI3K pathways act independently to mediate G-gly proliferative effect on human colonic tumour cells.

Cell Proliferation↗
PubMed2001

Ceramide mediates tumor-induced dendritic cell apoptosis.

Induction of apoptosis in dendritic cells (DC) is one of the escape mechanisms of tumor cells from the immune surveillance system. This study aimed to clarify the underlying mechanisms of tumor-induced DC apoptosis. The supernatants (SN) of murine tumor cell lines B16 (melanoma), MCA207, and MCA102 (fibrosarcoma) increased C16 and C24 ceramide as determined by electrospray mass spectrometry and induced apoptosis in bone marrow-derived DC. N-oleoylethanolamine or D-L-threo 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), which inhibits acid ceramidase or glucosylceramide synthase and then increases endogenous ceramide, enhanced DC apoptosis and ceramide levels in the presence of tumor SN. Pretreatment with L-cycloserine, an inhibitor of de novo ceramide synthesis, or phorbol ester, 12-O-tetradecanoylphorbol-13-acetate reduced endogenous ceramide levels and protected DC from tumor-induced apoptosis. However, other DC survival factors, including LPS and TNF-alpha, failed to do so. The protective activity of 12-O-tetradecanoylphorbol-13-acetate is abrogated by pretreatment with phosphoinositide 3-kinase (PI3K) inhibitor, LY294002. Therefore, down-regulation of PI3K is the major facet of tumor-induced DC apoptosis. Tumor SN, N-oleoylethanolamine, or PDMP suppressed Akt, NF-kappaB, and bcl-x(L) in DC, suggesting that the accumulation of ceramide impedes PI3K-mediated survival signals. Taken together, ceramide mediates tumor-induced DC apoptosis by down-regulation of the PI3K pathway.

Animals↗
PubMed2002

The forkhead transcription factor FoxO regulates transcription of p27Kip1 and Bim in response to IL-2.

The cytokine IL-2 plays a very important role in the proliferation and survival of activated T cells. These effects of IL-2 are dependent on signaling through the phosphatidylinositol 3-kinase (PI3K) pathway. We and others have shown that PI3K, through activation of protein kinase B/Akt, inhibits transcriptional activation by a number of forkhead transcription factors (FoxO1, FoxO3, and FoxO4). In this study we have investigated the role of these forkhead transcription factors in the IL-2-induced T cell proliferation and survival. We show that IL-2 regulates phosphorylation of FoxO3 in a PI3K-dependent fashion. Phosphorylation and inactivation of FoxO3 appears to play an important role in IL-2-mediated T cell survival, because mere activation of FoxO3 is sufficient to trigger apoptosis in T cells. Indeed, active FoxO3 can induce expression of IL-2-regulated genes, such as the cdk inhibitor p27(Kip1) and the proapoptotic Bcl-2 family member Bim. Furthermore, we show that IL-2 triggers a rapid, PI3K-dependent, phosphorylation of FoxO1a in primary T cells. Thus, we propose that inactivation of FoxO transcription factors by IL-2 plays a critical role in T cell proliferation and survival.

Animals↗
PubMed2004

The role of the p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and phosphoinositide-3-OH kinase signal transduction pathways in CD40 ligand-induced dendritic cell activation and expansion of virus-specific CD8+ T cell memory responses.

Mature dendritic cells (DCs) are central to the development of optimal T cell immune responses. CD40 ligand (CD40L, CD154) is one of the most potent maturation stimuli for immature DCs. We studied the role of three signaling pathways, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and phosphoinositide-3-OH kinase (PI3K), in CD40L-induced monocyte-derived DC activation, survival, and expansion of virus-specific CD8(+) T cell responses. p38 MAPK pathway was critical for CD40L-mediated up-regulation of CD83, a marker of DC maturation. CD40L-induced monocyte-derived DC IL-12 production was mediated by both the p38 MAPK and PI3K pathways. CD40L-mediated DC survival was mostly mediated by the PI3K pathway, with smaller contributions by p38 MAPK and ERK pathways. Finally, the p38 MAPK pathway was most important in mediating CD40L-stimulated DCs to induce strong allogeneic responses as well as expanding virus-specific memory CD8(+) T cell responses. Thus, although the p38 MAPK, PI3K, and ERK pathways independently affect various parameters of DC maturation induced by CD40L, the p38 MAPK pathway within CD40L-conditioned DCs is the most important pathway to maximally elicit T cell immune responses. This pathway should be exploited in vivo to either completely suppress or enhance CD8(+) T cell immune responses.

CD40 Ligand↗
PubMed2004

The CD40-induced signaling pathway in endothelial cells resulting in the overexpression of vascular endothelial growth factor involves Ras and phosphatidylinositol 3-kinase.

Ligation of endothelial cell (EC) CD40 induces the expression of several proinflammatory cytokines as well as angiogenesis factors, including vascular endothelial growth factor (VEGF). Moreover, despite the reported importance of CD40 in cell-mediated immunity, little is known of the CD40-induced signaling pathways in EC. In this study, we have investigated the function of the Ras signaling pathway(s) for CD40-induced overexpression of VEGF. EC were transiently transfected with a full-length VEGF promoter-luciferase construct and a dominant-inhibitory mutant of Ras (Ras17N). Following transfection, ligation of CD40 with soluble CD40 ligand resulted in a significant increase in VEGF transcriptional activation, and the inhibitory mutant of Ras blocked this CD40-induced VEGF overexpression. Using EMSA and Western blot analysis, we demonstrated that CD40-dependent binding of nuclear protein(s) to the VEGF promoter and CD40-induced VEGF protein expression in EC were also inhibited by the Ras mutant. Immunoprecipitation studies revealed that ligation of CD40 on EC promoted an increased association of Ras with its effector molecules Raf, Rho, and phosphatidylinositol 3-kinase (PI3K). But, cotransfection of effector-loop mutants of Ras determined that only PI3K was functional for Ras-induced VEGF transcription. Also, wortmanin and a dominant-inhibitory mutant of PI3K inhibited CD40-induced overexpression of VEGF. Together these findings demonstrate that both Ras and PI3K are intermediaries in CD40-induced regulation of VEGF in EC. We believe our findings are of importance in several chronic inflammatory diseases, including atherosclerosis and allograft rejection associated with both CD40-CD40 ligand signaling as well as VEGF expression and function.

CD40 Antigens↗
PubMed2004

Reciprocal control of Forkhead box O 3a and c-Myc via the phosphatidylinositol 3-kinase pathway coordinately regulates p27Kip1 levels.

B cell receptor (BCR) engagement of murine WEHI 231 immature B lymphoma cells leads sequentially to a drop in NF-kappa B and c-Myc, and induction of the p27(Kip1) cyclin-dependent kinase inhibitor, which promotes growth arrest and apoptosis. BCR engagement was recently shown to induce a drop in phosphatidylinositol 3-kinase (PI3K)/Akt signaling, preceding the increase in p27. As induction of p27 is due to an increase in gene transcription, we investigated the role of the Forkhead box O (FOXO) transcription factor family, which has been shown to potently induce p27 promoter activity. We demonstrate that pharmacologic inhibitors of PI3K or BCR engagement lead to decreased inactive cytoplasmic levels and increased active functional nuclear FOXO3a. In contrast, inhibition of PI3K/Akt signaling decreased the levels of NF-kappa B and c-Myc, which has been shown to repress p27 promoter activity. To test the effects of ectopic c-Myc on endogenous p27 levels, WEHI 231 cells stably expressing c-Myc or empty vector DNA were prepared. Ectopic c-Myc blocked the induction of p27 expression upon either inhibition of PI3K or BCR engagement. Thus, p27(Kip1) is coordinately regulated via two arms of a signaling pathway that are inversely controlled upon inhibition of PI3K: induction of the activator FOXO3a and down-regulation of the repressor c-Myc.

Animals↗
PubMed2005

Phosphatidylinositol 3-kinase regulates thymic exit.

To understand the role of PI3K during T cell development, we generated transgenic mice expressing the N terminus of the PI3K catalytic subunit (p110(ABD); ABD, adaptor binding domain) in thymocytes. Expression of p110(ABD) activates endogenous p110 and results in the accumulation of mature single-positive CD3(high)heat-stable Ag(low) thymocytes. This is mostly due to a defect in emigration of those cells, as shown by the delayed appearance of peripheral T cells in neonatal transgenic mice and by competitive adoptive transfer experiments. Although the mechanisms underlying these effects of PI3K are not yet clear, our results show an important role for PI3K activity in the regulation of mature thymocyte exit to the periphery.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Prolonged TCR/CD28 engagement drives IL-2-independent T cell clonal expansion through signaling mediated by the mammalian target of rapamycin.

Proliferation of Ag-specific T cells is central to the development of protective immunity. The concomitant stimulation of the TCR and CD28 programs resting T cells to IL-2-driven clonal expansion. We report that a prolonged occupancy of the TCR and CD28 bypasses the need for autocrine IL-2 secretion and sustains IL-2-independent lymphocyte proliferation. In contrast, a short engagement of the TCR and CD28 only drives the expansion of cells capable of IL-2 production. TCR/CD28- and IL-2-driven proliferation revealed a different requirement for PI3K and for the mammalian target of rapamycin (mTOR). Thus, both PI3K and mTOR activities were needed for T cells to proliferate to TCR/CD28-initiated stimuli and for optimal cyclin E expression. In contrast, either PI3K or mTOR were sufficient for IL-2-driven cell proliferation as they independently mediated cyclin E induction. Interestingly, rapamycin delayed cell cycle entry of IL-2-sufficient T cells, but did not prevent their expansion. Together, our findings indicate that the TCR, CD28, and IL-2 independently control T cell proliferation via distinct signaling pathways involving PI3K and mTOR. These data suggest that Ag persistence and the availability of costimulatory signals and of autocrine and paracrine growth factors individually shape T lymphocyte expansion in vivo.

Animals↗
PubMed2006

Macrophage's proinflammatory response to a mycobacterial infection is dependent on sphingosine kinase-mediated activation of phosphatidylinositol phospholipase C, protein kinase C, ERK1/2, and phosphatidylinositol 3-kinase.

Previous studies have shown that the ability of Mycobacterium tuberculosis to block a Ca(2+) flux is an important step in its capacity to halt phagosome maturation. This affect on Ca(2+) release results from M. tuberculosis inhibition of sphingosine kinase (SPK) activity. However, these studies did not address the potential role of SPK and Ca(2+) in other aspects of macrophage activation including production of proinflammatory mediators. We previously showed that nonpathogenic Mycobacterium smegmatis and to a lesser extent pathogenic Mycobacterium avium, activate Ca(2+)-dependent calmodulin/calmodulin kinase and MAPK pathways in murine macrophages leading to TNF-alpha production. However, whether SPK functions in promoting MAPK activation upon mycobacterial infection was not defined in these studies. In the present work we found that SPK is required for ERK1/2 activation in murine macrophages infected with either M. avium or M. smegmatis. Phosphoinositide-specific phospholipase C (PI-PLC) and conventional protein kinase C (cPKC) were also important for ERK1/2 activation. Moreover, there was increased activation of cPKC and PI3K in macrophages infected with M. smegmatis compared with M. avium. This cPKC and PI3K activation was dependent on SPK and PI-PLC. Finally, in macrophages infected with M. smegmatis compared with M. avium, we observed enhanced secretion of TNF-alpha, IL-6, RANTES, and G-CSF and found production of these inflammatory mediators to be dependent on SPK, PI-PLC, cPKC, and PI3K. These studies are the first to show that the macrophage proinflammatory response following a mycobacterial infection is regulated by SPK/PI-PLC/PKC activation of ERK1/2 and PI3K pathways.

Animals↗
PubMed2007

The IREM-1 (CD300f) inhibitory receptor associates with the p85alpha subunit of phosphoinositide 3-kinase.

The immune receptor expressed by myeloid cell 1 (IREM-1) (CD300f) inhibitory receptor displays five cytoplasmic tyrosine residues, two of them (Y205 and Y249) fit with ITIMs, whereas Y236 and Y263 constitute putative binding sites for PI3K. In the present study, immunoprecipitation analysis revealed that both the p85alpha subunit of PI3K and Src homology region 2 domain-containing phosphatase-1 could be recruited by IREM-1 in transfected cells as well as in the U937 monocytic leukemia cells, which constitutively express the receptor. By assaying the ability of different IREM-1 mutants to regulate the secretion of beta-hexosaminidase induced via FcRepsilonI in rat basophilic leukemia cells, both Y205 and Y249 appeared crucial for IREM-1-mediated inhibition. Remarkably, engagement of an IREM-1 mutant (Y(205,249,284)F), which did not recruit Src homology region 2 domain-containing phosphatase-1 and lost its inhibitory function, induced rat basophilic leukemia cell degranulation. This effect was dependent on the recruitment of PI3K, requiring the integrity of Y236 and Y263, and was blocked by PI3K inhibitors (i.e., wortmannin and LY-294002). Altogether, these data reveal a putative functional duality of the IREM-1 myeloid cell receptor.

Animals↗
PubMed1999

Retinoic acid induces selective expression of phosphoinositide 3-kinase gamma in myelomonocytic U937 cells.

Retinoic acid provokes growth inhibition and differentiation of the human leukemic cell line U937 to macrophage-like cells. We report that treatment of U937 cells with all-trans retinoic acid (ATRA), but not the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, results in an increased gene expression of the phosphoinositide 3-kinase (PI3K) species PI3Kgamma. PI3Kgamma expression was transcriptionally elevated, indicating that the PI3Kgamma gene may be a direct target for ATRA. In contrast to its effect on PI3Kgamma expression, ATRA did neither affect the levels of the PI3K species beta and delta nor the adapter proteins p85 and p101. Enhanced expression by ATRA of PI3Kgamma correlated with an increase of PI3K lipid kinase activity. Additionally, ATRA induced significant and lasting stimulation of mitogen-activated protein kinase/Erk2 activity. This effect was sensitive to the PI3K inhibitors wortmannin or LY294002 and, therefore, attributed to the upregulation of PI3Kgamma expression. Our findings suggest that sustained MAPK activation via PI3Kgamma precedes ATRA -dependent differentiation or growth inhibition.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1999

Overexpression of Akt (protein kinase B) confers protection against apoptosis and prevents formation of ceramide in response to pro-apoptotic stimuli.

An immortalized dorsal root ganglion cell line F-11 exhibits many properties of spinal cord neurons and undergoes apoptosis in response to growth factor withdrawal and the exogenous addition of inhibitors of phosphatidylinositol-3-kinase (PI3K). To elucidate the mechanism of apoptosis we generated F-11 clones which overexpressed either the p110 subunit of PI3K, a constitutively active form of protein kinase B/Akt (Myristoylated Akt), or a dominant-negative form (c-Akt). The first two constructs were protective against apoptosis induced by PI3K inhibitors such as wortmannin and LY294002. Caspase-3 (CPP32) levels peaked at 4 hr to 6 hr in response to pro-apoptotic drugs, and this increase was attenuated by 50% in F-11 with constitutively active Akt. The Akt protection was confirmed by DNA fragmentation studies. Both neo-transfected and the c-Akt dominant-negative transfected F-11 cells showed increased ceramide formation (twofold) in response to staurosporine, wortmannin, or LY294002; whereas cells with a constitutively active Akt (Myr-Akt) showed no increase in ceramide when treated with staurosporine, wortmannin, or LY294002. Ceramide was a more potent activator of CPP32 and an inducer of apoptosis when added as the native form (hydroxy- or nonhydroxy-), rather than the more water-soluble C(2)-ceramide. Overexpression of PI3K (p110) and Akt protected cells against ceramide-induced apoptosis, suggesting that Ceramide action is upstream of Akt in these cells and suggesting that Akt might be a target for inhibition by ceramide. Both staurosporine and C(2)-ceramide activated the Jun kinase (JNK) cascade and C(2)-ceramide increased caspase-3 (CPP32) activity in cells expressing wild-type c-Jun, but not dominant-negative (TAM-67) c-Jun. We suggest that this pathway is also involved in apoptosis, consistent with the idea that ceramide has multiple kinase and kinase-modulating targets in the apoptotic pathway of neurons. J. Neurosci. Sci. 57:884-893, 1999.

Animals↗
PubMed1999

Cripto-1 induces phosphatidylinositol 3'-kinase-dependent phosphorylation of AKT and glycogen synthase kinase 3beta in human cervical carcinoma cells.

Cripto-1 (CR-1), a member of the epidermal growth factor-CFC peptide family, activates the ras/raf/mitogen-activated protein/extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. In the present study, the role of CR-1 in the phosphatidylinositol 3'-kinase (PI3K)/AKT (protein kinase B)/glycogen synthase kinase 3beta (GSK-3beta)-dependent signaling pathway was evaluated in human SiHa cervical carcinoma cells. Our data demonstrate that CR-1 can enhance the tyrosine phosphorylation of the p85 regulatory subunit of PI3K and transiently induce the phosphorylation of AKT in a time- and dose-dependent manner. In addition, CR-1 was found to induce the phosphorylation of GSK-3beta. Phosphorylation of AKT and GSK-3beta by CR-1 can be blocked by LY294002, a specific inhibitor of PI3K, thus leading to apoptosis. Finally, the apoptotic effect of LY294002 can be partially rescued by exogenous CR-1. In summary, our data suggest that human CR-1 may function as a survival factor through a PI3K-dependent signaling pathway involving AKT and GSK-3beta.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed2000

Modulation of hypoxia-inducible factor 1alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics.

Dysregulated signal transduction from receptor tyrosine kinases to phosphatidylinositol 3-kinase (PI3K), AKT (protein kinase B), and its effector FKBP-rapamycin-associated protein (FRAP) occurs via autocrine stimulation or inactivation of the tumor suppressor PTEN in many cancers. Here we demonstrate that in human prostate cancer cells, basal-, growth factor-, and mitogen-induced expression of hypoxia-inducible factor 1 (HIF-1) alpha, the regulated subunit of the transcription factor HIF-1, is blocked by LY294002 and rapamycin, inhibitors of PI3K and FRAP, respectively. HIF-1-dependent gene transcription is blocked by dominant-negative AKT or PI3K and by wild-type PTEN, whereas transcription is stimulated by constitutively active AKT or dominant-negative PTEN. LY294002 and rapamycin also inhibit growth factor- and mitogen-induced secretion of vascular endothelial growth factor, the product of a known HIF-1 target gene, thus linking the PI3K/PTEN/AKT/FRAP pathway, HIF-1, and tumor angiogenesis. These data indicate that pharmacological agents that target PI3K, AKT, or FRAP in tumor cells inhibit HIF-1alpha expression and that such inhibition may contribute to therapeutic efficacy.

Carrier Proteins↗
PubMed2000

A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells.

The microbially derived antiproliferative agent rapamycin inhibits cell growth by interfering with the signaling functions of the mammalian target of rapamycin (mTOR). In this study, we demonstrate that interleukin-3 stimulation induces a wortmannin-sensitive increase in mTOR kinase activity in a myeloid progenitor cell line. The involvement of phosphoinositide 3'-kinase (PI3K) in the regulation of mTOR activity was further suggested by findings that mTOR was phosphorylated in vitro and in vivo by the PI3K-regulated protein kinase, AKT/PKB. Although AKT phosphorylated mTOR at two COOH-terminal sites (Thr2446 and Ser2448) in vitro, Ser2448 was the major phosphorylation site in insulin-stimulated or -activated AKT-expressing human embryonic kidney cells. Transient transfection assays with mTOR mutants bearing Ala substitutions at Ser2448 and/or Thr2446 indicated that AKT-dependent mTOR phosphorylation was not essential for either PHAS-I phosphorylation or p70S6K activation in HEK cells. However, a deletion of amino acids 2430-2450 in mTOR, which includes the potential AKT phosphorylation sites, significantly increased both the basal protein kinase activity and in vivo signaling functions of mTOR. These results demonstrate that mTOR is a direct target of the PI3K-AKT signaling pathway in mitogen-stimulated cells, and that the identified AKT phosphorylation sites are nested within a "repressor domain" that negatively regulates the catalytic activity of mTOR. Furthermore, the activation status of the PI3K-AKT pathway in cancer cells may be an important determinant of cellular sensitivity to the cytostatic effect of rapamycin.

Androstadienes↗
PubMed2000

Tyrosine 1062 of RET-MEN2A mediates activation of Akt (protein kinase B) and mitogen-activated protein kinase pathways leading to PC12 cell survival.

The RET tyrosine kinase is a functional receptor for neurotrophic ligands of the glial cell line-derived neurotrophic factor (GDNF) family. Loss of function of RET is associated with congenital megacolon or Hirschsprung's disease, whereas germ-line point mutations causing RET activation are responsible for multiple endocrine neoplasia type 2 (MEN2A, MEN2B, and familial medullary thyroid carcinoma) syndromes. Here we show that the expression of a constitutively active RET-MEN2A oncogene promotes survival of rat pheochromocytoma PC12 cells upon growth factor withdrawal. Moreover, we show that the RET-MEN2A-mediated survival depends on signals transduced by the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) cascades. Thus, in PC12 cells, RET-MEN2A associates with the PI3K regulatory subunit p85 and promotes activation of Akt (also referred to as protein kinase B) in a PI3K-dependent fashion; in addition, RET-MEN2A promotes MAPK activation. PI3K recruitment and Akt activation as well as MAPK activation depend on RET-MEN2A tyrosine residue 1062. As a result, tyrosine 1062 of RET-MEN2A is essential for RET-MEN2A-mediated survival of PC12 cells cultured in growth factor-depleted media.

Animals↗
PubMed2001

Interleukin 6 inhibits proliferation and, in cooperation with an epidermal growth factor receptor autocrine loop, increases migration of T47D breast cancer cells.

Interleukin (IL)-6, a multifunctional regulator of immune response, hematopoiesis, and acute phase reactions, has also been shown to regulate cancer cell proliferation. We have investigated IL-6 signaling pathways and cellular responses in the T47D breast carcinoma cell line. The IL-6-type cytokines, IL-6 and oncostatin M, simultaneously inhibited cell proliferation and increased cell migration. In T47D cells, IL-6 stimulated the activation of Janus-activated kinase 1 tyrosine kinase and signal transducers and activators of transcription (STAT) 1 and STAT3 transcription factors. Expression of dominant negative STAT3 in the cells strongly reduced IL-6-mediated growth inhibition but did not prevent IL-6-induced cell migration. IL-6 treatment led to activation of the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3'-kinase (PI3K) pathways. Inhibition of MAPK or PI3K activity reversed IL-6- and oncostatin M-stimulated migration. Because cross-talk between cytokine receptors and members of the ErbB family of receptor tyrosine kinases has been described previously, we have examined their interaction in T47D cells. Down-regulation of ErbB receptor activity, through the use of specific pharmacological inhibitors or dominant negative receptor constructs, revealed that IL-6-induced MAPK activation was largely dependent on epidermal growth factor (EGF) receptor activity, but not on ErbB-2 activity. Using a monoclonal antibody that interferes with EGF receptor-ligand interaction, we have shown that in T47D cells, IL-6 cooperates with an EGF receptor autocrine activity loop for signaling through the MAPK and PI3K pathways and for cell migration. Both the tyrosine phosphatase SHP-2 and the multisubstrate docking molecule Gab1, which are potential links between IL-6 and the MAPK/PI3K pathways, were constitutively associated with the active EGF receptor. On IL-6 stimulation, SHP-2 and Gab1 were recruited to the gp130 subunit of the IL-6 receptor and tyrosine phosphorylated, allowing downstream signaling to the MAPK and PI3K pathways. Thus, in T47D breast carcinoma cells, IL-6 acts in synergy with EGF receptor autocrine activity to signal through the MAPK/PI3K pathways. Cooperation between IL-6 and the EGF receptor in T47D breast carcinoma cells illustrates how a combination of multiple stimuli, either exogenous or endogenous, may result in synergistic cellular responses.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Effects of ras and von Hippel-Lindau (VHL) gene mutations on hypoxia-inducible factor (HIF)-1alpha, HIF-2alpha, and vascular endothelial growth factor expression and their regulation by the phosphatidylinositol 3'-kinase/Akt signaling pathway.

Many oncogenes induce expression of vascular endothelial growth factor (VEGF), a key factor in tumor angiogenesis. Phosphatidylinositol 3'-kinase (PI3K)/Akt is a common signaling pathway for oncogenes and tumor suppressor genes and is involved in VEGF regulation. Because hypoxia is a major stimulus for VEGF production, we examined the effects of LY294002, a selective PI3K inhibitor, on hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha expression and on endogenous VEGF responses to hypoxia. A panel of breast cancer cell lines reflecting the different genetic changes occurring in human breast cancer was analyzed. LY294002 inhibited HIF-1alpha induction and phosphorylation under hypoxia. However, HIF-2alpha expression was not affected. Basal and hypoxia-inducible VEGF expression was reduced at both mRNA and protein levels by 50%. V12-ras overexpression resulted in an increase in hypoxia-induced HIF-1alpha and HIF-2alpha expression. This effect was blocked by PI3K inhibitor, demonstrating one mechanism for ras synergy with hypoxia-mediated induction of genes. The decreased HIF-1alpha expression was not dependent on VHL interaction because a renal carcinoma cell line with VHL mutation and constitutive high HIF-1alpha expression also showed down-regulation of HIF-1alpha after treatment with LY294002. These results have implications for the use of PI3K inhibitors to inhibit synergistic effects of hypoxia with a wide range of common oncogenes.

Basic Helix-Loop-Helix Proteins↗