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PubMed2004

Neuregulin-1 induces branching morphogenesis in the developing lung through a P13K signal pathway.

Neuregulin-1 (NRG-1) induces signal transduction through the activation of its receptor, a heterodimer of human epidermal growth factor receptors 2 and 3 (HER2/HER3). Signal transduction through this receptor/ligand system plays a critical role in the developing heart, mammary gland, and nervous systems. Previous studies showed that NRG-1-induced HER2 activation resulted in pulmonary epithelial cell proliferation in the human fetal lung. The authors hypothesized that NRG-1 further contributes to lung development and maturation by inducing branching morphogenesis. In the present study, the authors show that NRG-1, HER2, and HER3, but not HER4, are expressed in the developing mouse lung. Addition of NRG-1 to fetal lung explants increased lung branching morphogenesis by 32% (P < .05). This increase in branching was blocked by 2C4, an antibody directed against HER2 that inhibits its dimerization and subsequent NRG-1-induced signal transduction. To gain an understanding of the intracellular signaling pathways involved in NRG-1-induced branching morphogenesis, the authors specifically blocked the phosphatidylinositol-3 kinase (PI3K) and mitogen activation protein kinase (MAPK) pathways. Inhibition of PI3K signaling significantly decreased NRG-1-induced branching morphogenesis (P < .05). Inhibition of NRG-1-induced MAPK activation had no effect on explant branching morphogenesis. These data suggest that NRG-1, binding to the HER2/HER3 heterodimer receptor complex, induces pulmonary branching morphogenesis through HER2 activation of the PI3K pathway.

Animals↗
PubMed2004

Differential requirements for DOCK2 and phosphoinositide-3-kinase gamma during T and B lymphocyte homing.

Chemokines guide lymphocytes from blood to secondary lymphoid organs by triggering integrin-dependent firm adhesion under vascular flow and directed migration of T and B lymphocytes within lymphoid tissue. Here, we analyze the roles of DOCK2, a mammalian homolog of Caenorhabditis elegans CED-5 and Drosophila melanogaster Myoblast City, and phosphoinositide-3-kinase (PI3K) during lymphocyte recirculation. DOCK2 mediated efficient lymphocyte migration in a largely PI3K-independent manner, although a minor, PI3K-dependent pathway for migration was observed in wild-type and DOCK2-deficient lymphocytes. In T cells, this residual migration depended mainly on PI3Kgamma, whereas other PI3K isoforms were implicated in B cells. In vitro adhesion assays and intravital microscopy of lymphoid organ vasculature uncovered an unexpected defect in integrin activation in DOCK2-/- B cells, whereas lack of DOCK2 did not affect chemokine-triggered integrin activation in T cells. DOCK2 and PI3Kgamma thus play distinct roles during T and B cell integrin activation and migration.

Actins↗
PubMed2006

Molecular basis for sensitivity and acquired resistance to gefitinib in HER2-overexpressing human gastric cancer cell lines derived from liver metastasis.

Gastric cancer metastasised to the liver was found to overexpress HER2 at a significantly higher incidence than primary gastric cancers. The purpose of the present study was to investigate the possibility of molecular therapy targeting HER2 overexpression in gastric cancer liver metastasis. We developed three new HER2-overexpressing gastric cancer cell lines (GLM-1, GLM-2, GLM-4) without epidermal growth factor receptor (EGFR) mutations derived from such liver metastasis, two of which had HER2 gene amplifications. All these GLM series of cell lines were highly sensitive to gefitinib in vitro, a specific inhibitor of EGFR tyrosine kinase (Iressa) rather than anti-HER2 antibody trastuzumab (Herceptin), whereas most of the HER2 low-expressing counterparts were not. In these HER2-overexpressing GLM series, protein kinase B (Akt), but not extracellular signal-regulated kinase 1/2 (ERK1/2), was constitutively phosphorylated, and gefitinib efficiently inhibited this Akt phosphorylation, induced strong apoptosis in vitro and exhibited antitumour activity in tumour xenografts in nude mice. This gefitinib-mediated antitumour effect in xenograft was significantly potentiated by trastuzumab treatment. On the other hand, gefitinib-resistant cells (GLM-1R) exhibited increased EGFR expression, followed by constitutive activation of mitogen-activated protein kinase (MAPK) pathway. These results suggest that the antitumour effect of gefitinib is due to the effective inhibition of HER2-driven constitutive activation of phosphatidylinositol-3-kinase (PI3K)/Akt pathway, and that the acquired resistance to gefitinib is due to the constitutive activation of Ras/MAPK pathway in compensation for PI3K/Akt pathway. Gastric cancer liver metastasis with HER2 overexpression would be a potential molecular target for gefitinib and trastuzumab.

Animals↗
PubMed2003

IL-2 activation of a PI3K-dependent STAT3 serine phosphorylation pathway in primary human T cells.

Interleukin-2 (IL-2) is the major growth factor of activated T lymphocytes. By inducing cell cycle progression and protection from apoptosis in these cells, IL-2 is involved in the successful execution of an immune response. Upon binding its receptor, IL-2 activates a variety of signal transduction pathways, including the Ras/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and Janus kinase (JAK)/STAT cascades. In addition, activation of phosphatidylinositol 3-kinase (PI3K) and several of its downstream targets has also been shown. However, the coupling of STAT3 serine phosphorylation to PI3K in response to IL-2 has yet to be shown in either T cell lines or primary human T cells. This report shows that the PI3K inhibitors LY294002 and wortmannin block activation of MEK and ERK by IL-2 in primary human T cells. Moreover, these inhibitors significantly reduce IL-2-triggered STAT3 serine phosphorylation without affecting STAT5 serine phosphorylation. Analysis of the effects of these inhibitors on cell cycle progression and apoptosis strongly suggests that PI3K-mediated events, which includes STAT3 activation, are involved in IL-2-mediated cell proliferation but not cell survival. Finally, results presented illustrate that in primary human T cells, activation of Akt is insufficient for IL-2-induced anti-apoptosis. Thus, these results demonstrate that IL-2 stimulates PI3K-dependent events that correlate with cell cycle progression, but not anti-apoptosis, in activated primary human T cells.

Apoptosis↗
PubMed2005

Suppression of phosphatidylinositol 3-kinase/Akt signaling pathway is a determinant of the sensitivity to a novel histone deacetylase inhibitor, FK228, in lung adenocarcinoma cells.

A novel histone deacetylase inhibitor, FK228, is a promising anticancer agent and has been proposed to modulate intracellular signaling, in addition to regulating gene transcription. We evaluated the effect of this agent on Akt-mediated signaling in relation to its cytotoxic activity using lung adenocarcinoma cell lines. Based on MTT assay and the appearance of cleaved poly (ADP-ribose) polymerase (PARP), we regarded A549 and PC14 cells as relatively sensitive and resistant cell lines, respectively. In A549 cells, FK228 suppressed the phosphorylation of Akt at Ser-473 and glycogen synthase kinase-3 without affecting these protein levels, indicating inhibition of the Akt-mediated signaling pathway. On the other hand, in PC14 cells, these biochemical reactions were not detected after treatment with FK228. The combination of FK228 and a phosphatidylinositol 3-kinase (PI3K)/Akt pathway inhibitor, LY294002, was determined to be synergistically cytotoxic in PC14 cells by isobologram analysis. This synergistic effect was attributable to the enhancement of apoptosis, as judged by flow cytometric analysis, and the appearance of cleaved PARP. The combination of FK228 with UCN-01, another PI3K/Akt pathway inhibitor, also exerted a synergistic effect. We concluded that FK228 suppresses the PI3K/Akt signaling pathway in a cell-specific manner, and this effect is a determinant of sensitivity to FK228.

Adenocarcinoma↗
PubMed2001

Growth factor withdrawal from primary human erythroid progenitors induces apoptosis through a pathway involving glycogen synthase kinase-3 and Bax.

The prevention of apoptosis is a key function of growth factors in the regulation of erythropoiesis. This study examined the role of the constitutively active serine/threonine kinase glycogen synthase kinase-3 (GSK3), a target of the phosphoinositide-3-kinase (PI3K)/Akt pathway, in the regulation of apoptosis in primary human erythroid progenitors. GSK3 phosphorylation at its key regulatory residues S21 (alpha isoform) and S9 (beta isoform) was high in steady-state culture, disappeared on growth factor withdrawal, and returned in response to treatment of cells with either erythropoietin or stem cell factor. Phosphorylation correlated with a PI3K-dependent reduction of 25% to 30% in measured GSK3 activity. LY294002, a specific inhibitor of PI3K, induced apoptosis in growth factor-replete erythroid cells to a degree similar to growth factor deprivation, whereas the Mek1 inhibitor U0126 had no effect, implicating PI3K and not mitogen-activated protein kinase in survival signaling. Growth factor-deprived erythroblasts, which undergo apoptosis rapidly, were protected from apoptosis by both lithium chloride, a GSK3 selective inhibitor, and inhibition of caspase activity. However, the clonogenic potential of single cells, which more accurately reflects cell survival, was maintained by lithium chloride, but not by caspase inhibition. Furthermore, lithium chloride, but not caspase inhibition, prevented the appearance of the conformational form of Bax associated with apoptosis induction. In summary, GSK3 activity is suppressed by erythropoietin and stem cell factor in human erythroid progenitor cells, and increased GSK3 activity, brought about by growth factor withdrawal, may regulate commitment to cell death through a caspase-independent pathway that results in a conformational change in Bax.

Amino Acid Chloromethyl Ketones↗
PubMed2002

Inhibition of phosphatidylinositol 3-kinase-Akt signaling blocks growth, promotes apoptosis, and enhances sensitivity of small cell lung cancer cells to chemotherapy.

A promising therapeutic alternative to inhibition of growth factor receptors is the inhibition of downstream signal transduction pathways. Such an approach may be especially important in tumors that can use signals from multiple growth factor receptors for growth and survival. Both stem cell factor (SCF) and insulin-like growth factor (IGF)-I, components of prominent small cell lung cancer (SCLC) autocrine loops, as well as FCS, can potently activate phosphatidylinositol 3-kinase (PI3K)-Akt signaling, albeit with different kinetics. SCF-induced PI3K-Akt activation occurs rapidly but fades within 60 min; IGF-I and FCS-induced activation persists for at least 6 h. SCF and IGF-I-mediated growth was potently inhibited by LY294002 in proportion to its ability to inhibit phosphatidylinositol 3-kinase (PI3K)-Akt signaling. A panel of six SCLC cell lines grown in 10% FCS was also very sensitive to LY294002, with average IC50 and LD50 of 5 and 25 microM, respectively. These drug concentrations suppressed the growth of the MRC-5 pulmonary fibroblast cell line and primary bronchial epithelial cells but did not induce significant cell death. Because LY294002 can also inhibit PI3K-related enzymes, we confirmed the role of the PI3K-Akt pathway in SCLC using doxycycline-regulated expression of a dominant-negative (kinase dead) and a constitutively active (CA; myristolated) Akt allele. Expression of dominant-negative Akt, which could only be achieved at relatively low levels, completely inhibited growth in the absence of exogenous growth factors and inhibited SCF-mediated growth but had no effect on IGF-I-mediated growth at the expression levels attained. Expression of CA Akt markedly augmented growth in the absence of exogenous growth factors but had minimal effect on growth in the presence of saturating concentrations SCF or IGF-I. Because PI3K-Akt signaling is known to promote survival under apoptotic stresses, we determined the effect of this pathway on SCLC sensitivity to etoposide. LY294002 potentiated the effect of low concentrations of etoposide in inhibiting growth and inducing apoptosis. The effect of low concentrations of LY294002 could largely be reversed by expression of CA Akt, suggesting that it was mediated by inhibition of Akt signaling. Expression of CA Akt by itself also induced resistance to etoposide-mediated apoptosis. Taken together, these data demonstrate that PI3K-Akt signaling promotes SCLC growth, survival, and chemotherapy resistance. Therefore, selective inhibitors of PI3K or Akt could potentially be useful as novel therapeutic agents in the treatment of SCLC.

Alleles↗
PubMed2005

Cross-talk between the two divergent insulin signaling pathways is revealed by the protein kinase B (Akt)-mediated phosphorylation of adapter protein APS on serine 588.

The APS adapter protein is recruited to the autophosphorylated kinase domain of the insulin receptor and initiates the phosphatidylinositol 3-kinase (PI3K)-independent pathway of insulin-stimulated glucose transport by recruiting CAP and c-Cbl. In this study, we have identified APS as a novel substrate for protein kinase B/Akt using an antibody that exhibits insulin-dependent immunoreactivity with a phosphospecific antibody raised against the protein kinase B substrate consensus sequence RXRXX(pS/pT) and a phosphospecific antibody that recognizes serine 21/9 of glycogen synthase kinase-3alpha/beta. This phosphorylation of APS is observed in both 3T3-L1 adipocytes and transfected cells. The insulin-stimulated serine phosphorylation of APS was inhibited by a PI3-kinase inhibitor, LY290004, a specific protein kinase B (PKB) inhibitor, deguelin, and knockdown of Akt. Serine 588 of APS is contained in a protein kinase B consensus sequence for phosphorylation conserved in APS across multiple species but not found in other members of this family, including SH2-B and Lnk. Mutation of serine 588 to alanine abolished the insulin-stimulated serine phosphorylation of APS and prevented the localization of APS to membrane ruffles. A glutathione S-transferase fusion protein containing amino acids 534-621 of APS was phosphorylated by purified PKB in vitro, and mutation of serine 588 abolished the PKB-mediated phosphorylation of APS in vitro. Taken together, this study identifies APS as a novel physiological substrate for PKB and the first serine phosphorylation site on APS. These data therefore reveal the molecular cross-talk between the insulin-activated PI3-kinase-dependent and -independent pathways previously thought to be distinct and divergent.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Semaphorin 4D/plexin-B1 induces endothelial cell migration through the activation of PYK2, Src, and the phosphatidylinositol 3-kinase-Akt pathway.

Semaphorins are cell surface and secreted proteins that provide axonal guidance in neuronal tissues and regulate cell motility in many cell types. They act by binding a family of transmembrane receptors known as plexins, which belong to the c-Met family of scatter factor receptors but lack an intrinsic tyrosine kinase domain. Interestingly, we have recently shown that Plexin-B1 is highly expressed in endothelial cells and that its activation by Semaphorin 4D elicits a potent proangiogenic response (J. R. Basile, A. Barac, T. Zhu, K. L. Guan, and J. S. Gutkind, Cancer Res. 64:5212-5224, 2004). In searches for the underlying molecular mechanism, we observed that Semaphorin 4D-stimulated endothelial cell migration requires the activation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Surprisingly, we found that Plexin-B1 stimulates PI3K-Akt through the activation of an intracellular tyrosine kinase cascade that involves the sequential activation of PYK2 and Src. This results in the tyrosine phosphorylation of Plexin-B1, the rapid recruitment of a multimeric signaling complex that includes PYK2, Src, and PI3K to Plexin-B1 and the activation of Akt. These findings suggest that Plexin-B1 may achieve its numerous physiological functions through the direct activation of intracellular tyrosine kinase cascades.

Animals↗
PubMed1997

Cripto-1 inhibits beta-casein expression in mammary epithelial cells through a p21ras-and phosphatidylinositol 3'-kinase-dependent pathway.

Cripto-1 (CR-1) is a recently discovered protein of the epidermal growth factor family that does not directly activate any of the known erbB type 1 tyrosine kinase receptors. Also, CR-1 stimulates the growth of HC-11 mouse mammary epithelial cells. We found that prior treatment of HC-11 cells with exogenous CR-1 induced a competency response to the lactogenic hormones dexamethasone, insulin, and prolactin (DIP) with respect to the induction of the milk protein beta-casein. In contrast, simultaneous treatment of mouse HC-11 cells with CR-1 in the presence of DIP inhibited beta-casein expression. The inhibitory effects of CR-1 on beta-casein expression in response to DIP were not unique to this mouse mammary epithelial cell line, because beta-casein and whey acidic protein expression in primary mouse mammary explant cultures established from midpregnant mice were also differentially inhibited by several epidermal growth factor-related peptides including CR-1. The mitogenic and differentiation effects of CR-1 are mediated by the binding of CR-1 to a cell surface receptor that is known to activate the ras/raf/mitogen-activated protein kinase (MAPK)/MAPK kinase pathway. The inhibitory response of CR-1 in HC-11 cells on beta-casein expression after treatment with DIP can be attenuated by B581, a peptidomimetic farnesyltransferase inhibitor that blocks p21ras farnesylation and activation, and by the phosphatidylinositol 3'-kinase (PI3k) inhibitor LY 294002 but not by PD 98059, a MAPK kinase inhibitor that blocks MAPK activation. These data suggest that the ability of CR-1 to block lactogenic hormone-induced expression of beta-casein is mediated through a p21ras-dependent, PI3k-mediated pathway. This is further substantiated by the observation that CR-1 is able to stimulate the tyrosine phosphorylation of the p85 PI3k regulatory subunit and to increase the activity of PI3k in HC-11 cells.

Animals↗
PubMed2004

Adenosine A3 receptor-mediated regulation of p38 and extracellular-regulated kinase ERK1/2 via phosphatidylinositol-3'-kinase.

The adenosine A(3) receptor generally couples to the G(i) class of heterotrimeric G proteins, thereby decreasing cAMP levels and also mediating signaling via release of betagamma subunits. Here we describe the central role of phosphatidylinositol-3'-kinase (PI3K) for adenosine A(3) receptor-induced intracellular signaling to the stress-activated protein kinase p38 and the extracellular signal-regulated protein kinases ERK1/2. We used Chinese hamster ovary cells expressing the human adenosine A(3) receptor, phospho-specific antibodies and different pharmacological tools to dissect the signaling pathways involving PI3K. The adenosine receptor agonist 5'N-ethylcarboxamidoadenosine induced a time- and dose-dependent increase in p38 and ERK1/2 phosphorylation, two signaling pathways that appeared also to be activated in the immortalized microglia cell line N13, which expressed endogenous adenosine A(3) receptors. The 5'N-ethylcarboxamidoadenosine-induced effects on p38 and ERK1/2 in CHO cells were blocked by pertussis toxin pretreatment and were sensitive to pharmacological inhibition of PI3K. In addition, inhibition of Rac/Cdc42, small GTPases of the Rho family, by clostridium toxin B, diminished p38 phosphorylation but did not affect ERK1/2. Furthermore, we identified the serine 727 site of signal transducer and activator of transcription STAT3 as a probable downstream target of ERK1/2, and thereby provide evidence that adenosine A(3) receptor mediated ERK1/2 activation has functional consequences.

Animals↗
PubMed2003

The phosphoinositide 3-kinase/AKT1 pathway involvement in drug and all-trans-retinoic acid resistance of leukemia cells.

Disruption of the apoptotic pathways may account for resistance to chemotherapy and treatment failures in human neoplastic disease. To further evaluate this issue, we isolated a HL-60 cell clone highly resistant to several drugs inducing apoptosis and to the differentiating chemical all-trans-retinoic acid (ATRA). The resistant clone displayed an activated phosphoinositide 3-kinase (PI3K)/AKT1 pathway, with levels of phosphatidylinositol (3,4,5) trisphosphate higher than the parental cells and increased levels of both Thr 308 and Ser 473 phosphorylated AKT1. In vitro AKT1 activity was elevated in resistant cells, whereas treatment of the resistant cell clone with two inhibitors of PI3K, wortmannin or Ly294002, strongly reduced phosphatidylinositol (3,4,5) trisphosphate levels and AKT1 activity. The inhibitors reversed resistance to drugs. Resistant cells overexpressing either dominant negative PI3K or dominant negative AKT1 became sensitive to drugs and ATRA. Conversely, if parental HL-60 cells were forced to overexpress an activated AKT1, they became resistant to apoptotic inducers and ATRA. There was a tight relationship between the activation of the PI3K/AKT1 axis and the expression of c-IAP1 and c-IAP2 proteins. Activation of the PI3K/AKT1 axis in resistant cells was dependent on enhanced tyrosine phosphorylation of the p85 regulatory subunit of PI3K, conceivably due to an autocrine insulin-like growth factor-I production. Our findings suggest that an up-regulation of the PI3K/AKT1 pathway might be one of the survival mechanisms responsible for the onset of resistance to chemotherapeutic and differentiating therapy in patients with acute leukemia.

Antineoplastic Agents↗
PubMed2004

The pivotal role of phosphatidylinositol 3-kinase-Akt signal transduction in virus survival.

Over the course of evolution, viruses have developed the ability to modulate a variety of host cell signalling pathways. Inhibition of apoptosis, in particular, has become recognized as an important contributory factor in virus survival. Apoptotic inhibition contributes to the establishment of latent and chronic infections and has been implicated in viral oncogenesis. The phosphatidylinositol 3-kinase (PI3K)-Akt pathway is utilized by many cell types for inhibition of apoptosis and cellular survival. Virus modulation of this pathway provides an alternative to the expression of viral oncogenes or the direct inhibition of pro-apoptotic proteins. It has become evident that many viruses require up-regulation of this pathway to sustain long-term infections and it is modulated, in some cases, by specific viral products to create an environment favourable for cellular transformation. In other cases, PI3K-Akt signalling simply helps to create an environment favourable for virus replication and virion assembly. This review details the modulation and function of PI3K-Akt signalling for virus survival.

Animals↗
PubMed2005

Hypothalamic phosphatidylinositol 3-kinase-phosphodiesterase 3B-cyclic AMP pathway of leptin signalling is impaired following chronic central leptin infusion.

Leptin signalling in the hypothalamus is critical for the maintenance of normal body weight. Although hyperleptinaemia in obese people suggests a state of leptin resistance, and diet-induced obesity in rodents is associated with central leptin resistance, the underlying mechanisms remain unclear. Recent evidence suggests that, in addition to the signal transducer and activator of the transcription-3 (STAT3) pathway, leptin action is critical for energy homeostasis through an insulin-like signalling pathway involving an increase in phosphatidylinositol 3-kinase (PI3K) and phosphodiesterase 3B (PDE3B) activities and reduction in cyclic AMP (cAMP) levels in the hypothalamus. Here, we show that chronic central leptin (160 ng/h) infusion, which resulted in the development of resistance to the satiety action of leptin, impaired the PI3K-PDE3B-cAMP pathway of leptin signalling in the hypothalamus in that PI3K and PDE3B activities were increased and cAMP levels were decreased in the hypothalamus on day 2 of leptin infusion but remained unchanged on day 16. Additionally, induction of tyrosyl phosphorylation of insulin receptor substrate-1 observed on day 2 was not evident on day 16 of leptin infusion. By contrast, signalling through the STAT3-pathway remained activated in the hypothalamus throughout 16 days of leptin infusion. These findings show a differential response in PI3K-PDE3B-cAMP (impaired) and STAT3 (up-regulated) pathways to chronic central leptin infusion, and suggest a selective resistance in the PI3K-PDE3B-cAMP pathway of leptin signalling following a chronic increase in hypothalamic leptin tone attained by central infusion of this peptide hormone.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2004

Rapid signalling pathway activation by androgens in epithelial and stromal cells.

Estradiol rapidly activates Src as well as the Src-dependent pathway in human mammary cancer-derived MCF-7 cells, in human prostate cancer-derived LNCaP cells and in Cos cells transiently expressing hERs [EMBO J. 15 (1996) 1292; EMBO J. 17 (1998) 2008]. In addition, estradiol immediately stimulates, yes, an ubiquitous member of the Src kinase family, in human colon carcinoma-derived Caco-2 cells [Cancer Res. 56 (1996) 4516]. Progestins and androgens activate the same pathway in human mammary and prostate cancer-derived cells [EMBO J. 17 (1998) 2008; EMBO J. 19 (2000) 5406]. We observed that estradiol also stimulates the phosphatidylinositol-3-kinase (PI3K)/AKT pathway in MCF-7 cells [EMBO J. 20 (2001) 6050]. In these cells, activation of the Src- and the PI3 K-dependent pathways is simultaneous and mediated by direct interactions of the two kinases with ERalpha. The signalling pathway activation by sex-steroid hormones leads to DNA synthesis and cell growth in human mammary and prostate cancer-derived cells [EMBO J. 19 (2000) 5406; EMBO J. 20 (2001) 6050; EMBO J. 18 (1999) 2500]. Furthermore, androgen stimulation of NIH3T3 fibroblasts activates the same pathways triggered by this hormone in LNCaP cells and promotes the S-phase entry or cytoskeleton changes in these cells [J. Cell Biol. 161 (2003) 547]. All the described effects are rapid and require classic steroid receptors, but, surprisingly, not their transcriptional activity. Indeed, a transcriptionally inactive mutant of hER mediates the estrogen-stimulated DNA synthesis of NIH3T3 fibroblasts [EMBO J. 18 (1999) 2500]. Furthermore, AR in NIH3T3 cells does not enter nuclei and is unable to respond to the hormone with transcription stimulation, whereas it activates signaling pathways and triggers important biological responses. Signaling pathway activation by steroids has also been described by other groups under different experimental conditions and/or in different cell types. In these cells, steroid stimulation triggers various effects, such as neuroprotection, vasorelaxation or bone protection [J. Neurosci. Res. 60 (2000) 321; Nature 407 (2000) 538; J. Cell Biochem. 76 (1999) 206]. Analysis of the mechanisms responsible for the hormone-dependent and steroid receptor-mediated pathway activation in epithelial as well as stromal cells reveals immediate association of steroid receptors with extranuclear signaling effectors [EMBO J. 17 (1998) 2008; Cancer Res. 56 (1996) 4516; EMBO J. 19 (2000) 5406; EMBO J. 20 (2001) 6050; J. Cell Biol. 161 (2003) 547]. These results further highlight the central role of the hormone-regulated protein-protein interactions in the steroid action. They also offer the possibility of interfering with important activities of hormones, such as proliferation or survival, cytoskeleton changes as well as invasiveness and vasorelaxation, without affecting the steroid effects that depend on receptor transcriptional activity.

Androgens↗
PubMed2004

VEGF regulates PCB 104-mediated stimulation of permeability and transmigration of breast cancer cells in human microvascular endothelial cells.

Polychlorinated biphenyl (PCB) congeners, a group of worldwide, persistent environmental contaminants, are known to cause carcinogenesis and tumor promotion, and may also affect the development of cancer metastasis. Because vascular endothelial cells create a selective barrier to the passage of cancer cells, we hypothesize that specific PCB congeners can disrupt endothelial integrity and increase the transendothelial migration of tumor cells. To examine this hypothesis, we elucidated the effects of 2,2',4,6,6'-pentachlorobiphenyl (PCB 104), a representative of highly ortho-substituted non-coplanar PCB congeners, on the endothelial permeability and transendothelial migration of MDA-MB-231 breast cancer cells. Exposure of human microvascular endothelial cell 1 (HMEC-1) to PCB 104 induced endothelial hyperpermeability and markedly increased transendothelial migration of MDA-MB-231 cells. These effects were associated with overexpression of vascular endothelial growth factor (VEGF). PCB 104-mediated elevation of VEGF expression was induced by phosphatidylinositol 3-kinase (PI3K) but not affected by co-treatments with antioxidants or the NF-kappaB inhibitor SN50. In addition, the PI3K-dependent pathway was involved in PCB 104-induced activation of AP-1, a transcription factor implicated in the regulation of VEGF gene expression. The VEGF receptor (KDR/Flk-1) antagonist SU1498 and the PI3K inhibitor LY294002 inhibited PCB 104-induced hyperpermeability. These results indicate that PCB 104 may contribute to tumor metastasis by inducing VEGF overexpression that stimulates endothelial hyperpermeability and transendothelial migration of cancer cells.

Breast Neoplasms↗
PubMed2005

Insulin and cholesterol pathways in neuronal function, memory and neurodegeneration.

Insulin and cholesterol play important roles in basic metabolic processes in peripheral tissues. Both insulin and cholesterol can also act as signalling molecules in the central nervous system that participate in neuronal function, memory and neurodegenerative diseases. A high-cholesterol diet improves spatial memory in experimental animals. beta-Amyloid, the toxic peptide in neurons of AD (Alzheimer's disease) patients, binds cholesterol and catalyses its oxidation to 7beta-hydroxycholesterol, a highly toxic oxysterol that is a potent inhibitor of alpha-PKC (alpha-protein kinase C), an enzyme critical in memory consolidation and synaptic plasticity and implicated in AD. Oxidized cholesterol also can act as a second messenger for insulin. Oxidized low-density lipoprotein inhibits insulin-dependent phosphorylation of the signalling kinases ERK (extracellular-signal-regulated kinase) and PKB/Akt. In sporadic AD patients, insulin levels are decreased, suggesting links between AD and diabetes. Insulin signalling is also important in synaptic plasticity. Insulin receptors are up-regulated and undergo translocation after spatial learning. Insulin modulates the activity of excitatory and inhibitory receptors including the glutamate and gamma-aminobutyric acid receptors and activates two biochemical pathways: the shc-ras-mitogen-activated protein kinase pathway and the PI3K (phosphoinositide 3-kinase)/PKC pathway, both of which are involved in memory processing. These findings point to a convergence at the biochemical level between pathways involved in AD and those important for normal memory.

Alzheimer Disease↗
PubMed2006

LMP1 strain variants: biological and molecular properties.

The ubiquitous herpesvirus Epstein-Barr virus (EBV) is linked to the development of several malignancies, including nasopharyngeal carcinoma. Latent membrane protein 1 (LMP1) is considered the EBV oncogene as it is necessary for EBV-induced transformation of B lymphocytes and is able to transform Rat-1 fibroblasts. LMP1 can activate a wide array of signaling pathways, including phosphatidylinositol 3-kinase (PI3K)-Akt and NF-kappaB. Six sequence variants of LMP1, termed Alaskan, China 1, China 2, Med+, Med-, and NC, have been identified, and individuals can be infected with multiple variants. The frequencies of detection of these variants differ for various EBV-associated malignancies from different geographic regions. In this study, the biological and signaling properties of the LMP1 variants have been characterized. All of the LMP1 variants transformed Rat-1 fibroblasts, induced increased motility of HFK cells, and induced increased homotypic adhesion of BJAB cells. While all the variants activated the PI3K-Akt signaling pathway to similar extents, the Alaskan, China 1, and Med+ variants had limited binding to the E3 ubiquitin ligase component homologue of Slimb and had slightly enhanced NF-kappaB signaling. These findings indicate that the signature amino acid changes of the LMP1 variants do not hinder or enhance their in vitro transforming potentials or affect their signaling properties.

Animals↗