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PubMed2006

Cartducin stimulates mesenchymal chondroprogenitor cell proliferation through both extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt pathways.

Cartducin, a paralog of Acrp30/adiponectin, is a secretory protein produced by both chondrogenic precursors and proliferating chondrocytes, and belongs to a novel C1q family of proteins. We have recently shown that cartducin promotes the growth of both mesenchymal chondroprogenitor cells and chondrosarcoma-derived chondrocytic cells in vitro. However, the cartducin-signaling pathways responsible for the regulation of cell proliferation have not been documented. In this study, we examined whether cartducin exists in serum and further investigated the intracellular signaling pathways stimulated by cartducin in mesenchymal chondroprogenitor cells. Western blot analysis showed that, unlike Acrp30/adiponectin, cartducin was undetectable in mouse serum. Next, mesenchymal chondroprogenitor N1511 cells were stimulated with cartducin, and three major groups of mitogen-activated protein kinase (MAPK) pathways and the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway were examined. Cartducin activated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt, but not c-jun N-terminal kinase (JNK) nor p38 MAPK. The MEK1/2 inhibitor, U0126, blocked cartducin-stimulated ERK1/2 phosphorylation and suppressed the DNA synthesis induced by cartducin in N1511 cells. The PI3K inhibitor, LY294002, blocked cartducin-stimulated Akt phosphorylation and a decrease in cartducin-induced DNA synthesis in N1511 cells was also observed. These data suggest that cartducin is a peripheral skeletal growth factor, and that the proliferation of mesenchymal chondroprogenitor cells stimulated by cartducin is associated with activations of the ERK1/2 and PI3K/Akt signaling pathways.

Adipokines↗
PubMed2006

EGF-induced cell migration is mediated by ERK and PI3K/AKT pathways in cultured human lens epithelial cells.

Cataract is considered as the most common cause of blindness, which is curable only by surgery. Postsurgery, however, many patients gradually develop the complication of posterior capsule opacification (PCO) or secondary cataract, arising from stimulated cell proliferation and cell migration within the lens capsule. The migration of human lens epithelial cells (HLECs) plays crucial roles in the remodeling of lens capsule and cataract formation, but less is known about the cell-signaling mechanism of migration. We observed that epithelial growth factor (EGF) induced cell migration in cultured human lens epithelial cells through the ERK and PI3K/AKT pathways. EGF induced cell migration in a dose-dependent manner; EGF-induced EGFR phosphorylation and downstream activation of c-Jun N-terminal protein kinase (JNK), p38 MAP kinase (p38), extracellular signal-regulated kinase (ERK1/2) and AKT, were inhibited by PD153035 (EGFR inhibitor), JNKi (JNK inhibitor), SB203580 (p38 inhibitor), U0126 (MEK/ERK inhibitor), and LY294002 (PI3K/AKT inhibitor), respectively. Furthermore, we found that EGF induced activity of matrix metalloproteinase-2 (MMP-2) in cultured HLECs. EGF-induced MMP-2 activity was significantly inhibited by treatment of PD153035, U0126, and LY294002, but not SB203580 and JNK inhibitor, suggesting that ERK and the phosphatidylinositol-3-kinase (PI3K)/AKT pathways selectively mediate EGF-stimulated MMP-2 activity and cell migration in cultured HLECs in vitro. Taken together, our results suggest that the cell-signaling pathways involved in EGF-stimulated cell migration may constitute potential therapeutic targets in the treatment of PCO.

Cell Line↗
PubMed2005

Fc receptor gamma-chain activation via hOSCAR induces survival and maturation of dendritic cells and modulates Toll-like receptor responses.

We previously reported the characterization of human osteoclast-associated receptor (hOSCAR), a novel Fc receptor gamma-chain (FcRgamma)-associated receptor expressed by myeloid cells. Here we show that ligation of hOSCAR by specific antibodies promotes dendritic cell (DC) survival by an extracellular signal-regulated kinase (ERK)- and phosphatidylinositol 3-kinase (PI3K)-dependent pathway, linked to expression of the Bcl-2 and Bcl-x(L) antiapoptotic molecules. Crosslinking of hOSCAR leads to maturation of DCs, as demonstrated by up-regulation of maturation markers, decrease in dextran uptake capacity, and secretion of immunesystem effectors such as interleukin-8 (IL-8)/CXC chemokine ligand 8 (CXCL8), IL-12 p40, monocyte chemoattractant protein-1 (MCP-1)/chemokine receptor ligand 2 (CCL2) and macrophage-derived chemokine (MDC)/CCL22. Stimulation of hOSCAR acts in conjunction with the Toll-like receptor (TLR) ligands, lipopolysaccharide (LPS), R-848, and polyinosinic-polycytidylic acid (poly(I:C)), to increase the expression of maturation markers, and to modulate cytokine release. A PI3K-dependent up-regulation of IL-10 release is observed with all the TLR ligands used, whereas regulation of IL-12 production is variable depending on the TLR stimulated. hOSCAR engagement on DCs did not significantly increase the proliferation of naive T cells; however, when co-incubated with TLR ligands, an enhanced proliferation was observed. The percentage of interferon (IFN)-gamma-producing T cells is decreased when hOSCAR engagement is combined with LPS stimulation. Altogether, these data suggest that hOSCAR may modulate the responses of both innate resistance and adaptive immunity.

Antibodies, Monoclonal↗
PubMed2006

Glucose regulates Foxo1 through insulin receptor signaling in the pancreatic islet beta-cell.

Glucose controls islet beta-cell mass and function at least in part through the phosphatidylinositol 3-kinase (PI3K)/Akt pathway downstream of insulin signaling. The Foxo proteins, transcription factors known in other tissues to be negatively regulated by Akt activation, affect proliferation and metabolism. In this study, we tested the hypothesis that glucose regulates Foxo1 activity in the beta-cell via an autocrine/paracrine effect of released insulin on its receptor. Mouse insulinoma cells (MIN6) were starved overnight for glucose (5 mmol/l) then refed with glucose (25 mmol/l), resulting in rapid Foxo1 phosphorylation (30 min, P < 0.05 vs. untreated). This glucose response was demonstrated to be time (0.5-2 h) and dose (5-30 mmol/l) dependent. The use of inhibitors demonstrated that glucose-induced Foxo1 phosphorylation was dependent upon depolarization, calcium influx, and PI3K signaling. Additionally, increases in glucose concentration over a physiological range (2.5-20 mmol/l) resulted in nuclear to cytoplasmic translocation of Foxo1. Phosphorylation and translocation of Foxo1 following glucose refeeding were eliminated in an insulin receptor knockdown cell line, indicating that the glucose effects are mediated primarily through the insulin receptor. Activity of Foxo1 was observed to increase with decreased glucose concentrations, assessed by an IGF binding protein-1 promoter luciferase assay. Starvation of MIN6 cells identified a putative Foxo1 target, Chop, and a Chop-promoter luciferase assay in the presence of cotransfected Foxo1 supported this hypothesis. The importance of these observations was that nutritional alterations in the beta-cell are associated with changes in Foxo1 transcriptional activity and that these changes are predominantly mediated through glucose-stimulated insulin secretion acting through its own receptor.

Animals↗
PubMed2002

Induction of human MDR1 gene expression by 2-acetylaminofluorene is mediated by effectors of the phosphoinositide 3-kinase pathway that activate NF-kappaB signaling.

The expression of P-glycoprotein encoded by the multidrug resistance (MDR1) gene is associated with the emergence of the MDR phenotype in cancer cells. Human MDR1 and its rodent homolog mdr1a and mdr1b are frequently overexpressed in liver cancers. However, the underlying mechanisms are largely unknown. The hepatocarcinogen 2-acetylaminofluorene (2-AAF) efficiently activates rat mdr1b expression in cultured cells and in Fisher 344 rats. We recently reported that activation of rat mdr1b in cultured cells by 2-AAF involves a cis-activating element containing a NF-kappaB binding site located -167 to -158 of the rat mdr1b promoter. 2-AAF activates IkappaB kinase (IKK), resulting in degradation of IkappaBbeta and activation of NF-kappaB. In this study, we report that 2-AAF could also activate the human MDR1 gene in human hepatoma and embryonic fibroblast 293 cells. Induction of MDR1 by AAF was mediated by DNA sequence located at -6092 which contains a NF-kappaB binding site. Treating hepatoma cells with 2-AAF activated phosphoinositide 3-kinase (PI3K) and its downstream effectors Rac1, and NAD(P)H oxidase. Transient transfection assays demonstrated that constitutively activated PI3K and Rac1 enhanced the activation of the MDR1 promoter by 2-AAF. Treatment of hepatoma cells with 2-AAF also activated another PI3K downstream effector Akt. Transfection of recombinant encoding a dominant activated Akt also enhanced the activation of MDR1 promoter activation by 2-AAF. These results demonstrated that 2-AAF up-regulates MDR1 expression is mediated by the multiple effectors of the PI3K signaling pathway.

2-Acetylaminofluorene↗
PubMed2003

Inactivation of the tuberous sclerosis complex-1 and -2 gene products occurs by phosphoinositide 3-kinase/Akt-dependent and -independent phosphorylation of tuberin.

The tuberous sclerosis complex (TSC) is a genetic disorder that is caused through mutations in either one of the two tumor suppressor genes, TSC1 and TSC2, that encode hamartin and tuberin, respectively. Interaction of hamartin with tuberin forms a heterodimer that inhibits signaling by the mammalian target of rapamycin to its downstream targets: eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). During mitogenic sufficiency, the phosphoinositide 3-kinase (PI3K)/Akt pathway phosphorylates tuberin on Ser-939 and Thr-1462 that inhibits the tumor suppressor function of the TSC complex. Here we show that tuberin-hamartin heterodimers block protein kinase C (PKC)/MAPK- and phosphatidic acid-mediated signaling toward mammalian target of rapamycin-dependent targets. We also show that two TSC2 mutants derived from TSC patients are defective in repressing phorbol 12-myristate 13-acetate-induced 4E-BP1 phosphorylation. PKC/MAPK signaling leads to phosphorylation of tuberin at sites that overlap with and are distinct from Akt phosphorylation sites. Phosphorylation of tuberin by phorbol 12-myristate 13-acetate was reduced by treatment of cells with either bisindolylmaleimide I or UO126, inhibitors of PKC and MAPK/MEK (MAPK/ERK kinase), respectively, but not by wortmannin (an inhibitor of PI3K). This work reveals that both PI3K-independent and -dependent mechanisms modulate tuberin phosphorylation in vivo.

Adaptor Proteins, Signal Transducing↗
PubMed2006

The selective protein kinase C beta inhibitor enzastaurin induces apoptosis in cutaneous T-cell lymphoma cell lines through the AKT pathway.

Enzastaurin displays pro-apoptotic properties against a spectrum of malignancies and is currently being investigated in clinical trials. We have investigated the effects of enzastaurin on the viability of the cutaneous T-cell lymphoma cell lines HuT-78 and HH by using 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, cell cycle analysis, propidium iodide and annexin-V staining, and caspase-3-mediated proteolytic activation. Enzastaurin-treatment decreased cell viability, increased annexin V-FITC-positive cells, and increased the proportion of sub-G1 populations in both cell lines that was not reversed by the T-cell growth stimulating cytokines IL-2, IL-7, IL-15. Enzastaurin-induced cell death involved caspase-3-activated cleavage of poly(ADP-ribose) polymerase that was inhibited by the pan-caspase inhibitor ZVAD-fmk, whereas the increase in sub-G1 population was only partially inhibited by ZVAD-fmk. Furthermore, enzastaurin downregulated AKT activity and its downstream effectors GSK3beta and ribosomal protein S6. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway has been implicated in the growth and survival of hematologic malignancies and inhibition of this pathway is considered as a therapeutic target. Protein kinase C activation contributes to PI3K/AKT activation, but it is unknown how enzastaurin may interfere with signaling through this pathway. These results demonstrate that enzastaurin, at clinically achievable concentrations, induces apoptosis and affects AKT signaling, and provide a rationale for further in vivo studies addressing the therapeutic efficacy in cutaneous T-cell lymphoma patients.

Apoptosis↗
PubMed2005

PTEN and p27 expression in mature T-cell and NK-cell neoplasms.

PTEN is a tumor suppressor gene located on chromosome 10q23 and is amongst the most commonly mutated genes in human cancers. The lipid phosphatase activity of Pten enables it to dephosphorylate PIP3, thereby antagonizing growth factor stimulated PI3-kinase signaling mediated by AKT/PKB. The growth inhibition effect of PTEN has been shown to be mediated by p27 which is one of the important effector molecules downstream of the AKT pathway. Recently the importance of the Pten and AKT pathway in the regulation of the immune system and development of hematological malignancies has been shown. Loss of Pten and p27 expressions were examined immunohistochemically in 45 patients with peripheral T- and NK-cell lymphoma. Partial or complete loss of Pten was detected in 66.7% of the cases of anaplastic large cell lymphoma (ALCL) compared to only 12.5% of all other mature T-/NK-cell lymphomas combined. Loss of p27 was identified in 64.9% of cases, which showed a positive correlation with Pten loss. In this study, we showed that loss of Pten is more frequent in ALCL as compared to other mature T-/NK-cell lymphomas, which strongly correlates with the loss of p27 expression. Our findings provide further evidence for the importance of the deregulation of the PI3K-AKT pathway in ALCL.

Adult↗
PubMed2005

Silibinin inhibits cell invasion through inactivation of both PI3K-Akt and MAPK signaling pathways.

Silibinin, isolated from Silybum marianum, has been known for its hepatoprotective properties and recent studies have revealed its antiproliferative and apoptotic effects on several cancer cells. An inhibitory effect of silibinin on tumor invasion and matrix metalloproteinase-2 (MMP-2) and urokinasetype plasminogen activator (u-PA) activities in culture medium has been observed in our previous study and the impacts of silibinin on enzyme activities of MMPs, u-PA, mitogen-activated protein kinase (MAPK) and Akt in A549 cells were continued to explore in this study. Our results showed that silibinin exerted an inhibitory effect on the phosphorylation of Akt, as well as extracellular signal-regulated kinases 1 and 2 (ERK1/2), which are the members of the MAPK family involved in the up-regulation of MMPs or u-PA, while no effects on the activities of p38(MAPK) and stress-activated protein kinase/c-Jun N-terminal kinase were observed. A treatment with silibinin to A549 cells also led to a dose-dependent inhibition on the activation of NF-kappaB, c-Jun and c-Fos. Additionally, the treatment of inhibitors specific for MEK (U0126) or PI3K (LY294002) to A549 cells could result in a reduced expression of MMP-2 and u-PA concomitantly with a marked inhibition on cell invasion. These findings suggested that the inhibition on MMP-2 and u-PA expression by silibinin may be through a suppression on ERK1/2 or Akt phosphorylation, which in turn led to the reduced invasiness of the cancer cells.

Anticarcinogenic Agents↗
PubMed2002

Enhanced phosphorylation of PTEN in rat brain after transient middle cerebral artery occlusion.

A phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumor suppressor gene that suppresses cell growth, inhibits cell migration, and induces apoptosis. Phosphorylated form of PTEN (p-PTEN) is a key survival factor relating PI3K-Akt pathway and their downstream effectors. A spatiotemporal profiles of PTEN and p-PTEN expression were immunohistochemically examined after 90 min of transient middle cerebral artery occlusion in rats. In the ischemic core, PTEN progressively decreased by 3 days, whereas a rapid but transient increase of p-PTEN was found with a peak at 1 h after the reperfusion. In contrast, in the ischemic penumbra, PTEN showed a minor change and a gradual but sustained p-PTEN expression was observed in the ischemic penumbra with a peak at 12 h. In addition, the balance of population among strongly, moderately, and weakly stained cells was different between the ischemic core and penumbra at their peak time points. These results suggest an important role of p-PTEN for cell survival after ischemia as an upstream regulator for PI3K-Akt.

Animals↗
PubMed2006

Modeling HER2 effects on cell behavior from mass spectrometry phosphotyrosine data.

Cellular behavior in response to stimulatory cues is governed by information encoded within a complex intracellular signaling network. An understanding of how phenotype is determined requires the distributed characterization of signaling processes (e.g., phosphorylation states and kinase activities) in parallel with measures of resulting cell function. We previously applied quantitative mass spectrometry methods to characterize the dynamics of tyrosine phosphorylation in human mammary epithelial cells with varying human epidermal growth factor receptor 2 (HER2) expression levels after treatment with epidermal growth factor (EGF) or heregulin (HRG). We sought to identify potential mechanisms by which changes in tyrosine phosphorylation govern changes in cell migration or proliferation, two behaviors that we measured in the same cell system. Here, we describe the use of a computational linear mapping technique, partial least squares regression (PLSR), to detail and characterize signaling mechanisms responsible for HER2-mediated effects on migration and proliferation. PLSR model analysis via principal component inner products identified phosphotyrosine signals most strongly associated with control of migration and proliferation, as HER2 expression or ligand treatment were individually varied. Inspection of these signals revealed both previously identified and novel pathways that correlate with cell behavior. Furthermore, we isolated elements of the signaling network that differentially give rise to migration and proliferation. Finally, model analysis identified nine especially informative phosphorylation sites on six proteins that recapitulated the predictive capability of the full model. A model based on these nine sites and trained solely on data from a low HER2-expressing cell line a priori predicted migration and proliferation in a HER2-overexpressing cell line. We identify the nine signals as a "network gauge," meaning that when interrogated together and integrated according to the quantitative rules of the model, these signals capture information content in the network sufficiently to predict cell migration and proliferation under diverse ligand treatments and receptor expression levels. Examination of the network gauge in the context of previous literature indicates that endocytosis and activation of phosphoinositide 3-kinase (PI3K)-mediated pathways together represent particularly strong loci for the integration of the multiple pathways mediating HER2's control of mammary epithelial cell proliferation and migration. Thus, a PLSR modeling approach reveals critical signaling processes regulating HER2-mediated cell behavior.

Cell Line↗
PubMed2003

Extracellular matrix enhances heregulin-dependent BRCA1 phosphorylation and suppresses BRCA1 expression through its C terminus.

Germ line mutations in the breast cancer susceptibility gene BRCA1 account for the increased risk of early onset of familial breast cancer, whereas overexpression of the ErbB family of receptor tyrosine kinases has been linked to the development of nonfamilial or sporadic breast cancer. To analyze whether there is a link between these two regulatory molecules, we studied the effects of ErbB-2 activation by heregulin (HRG) on BRCA1 function. It was previously demonstrated that HRG induced the phosphorylation of BRCA1, which was mediated by the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Since altered interaction between cells and the surrounding extracellular matrix (ECM) is a common feature in a variety of tumors and since ECM modulates intracellular signaling, we hypothesized that ECM may affect the expression and HRG-dependent phosphorylation of BRCA1. Following stimulation by HRG, a strong increase in [(3)H]thymidine incorporation was observed in human T47D breast cancer cells seeded on plastic (PL). When T47D cells were seeded on laminin (LAM) or Matrigel, HRG induced a significantly higher proliferation than it did in cells seeded on PL. T47D cells seeded on poly-L-lysine had an abrogated mitogenic response, indicating the involvement of integrins in this process. HRG treatment induced a transient phosphorylation of BRCA1 that was enhanced in T47D cells grown on LAM. LAM-enhanced BRCA1 phosphorylation was mediated through alpha(6) integrin upon HRG stimulation. Accordingly, T47D cells grown on LAM had the greatest increase in ErbB-2 activation, PI3K activity, and phosphorylation of Akt. A similar pattern of BRCA1 mRNA expression was observed when T47D cells were seeded on PL, LAM, or COL4. There was a significant decrease in the steady state of the BRCA1 mRNA level on both the LAM and COL4 matrices compared to that for cells seeded on PL. In addition, HRG stimulation caused a significant decrease in BRCA1 mRNA expression that was dependent on protein synthesis. Pretreatment with both the calpain inhibitor ALLN (N-acetyl-Leu-Leu-norleucinal) and the proteosome inhibitor lactacystin inhibited the HRG-induced down-regulation of BRCA1 mRNA expression. Likewise, there was a strong decrease in the protein level of BRCA1 in T47D cells 4 h after treatment with HRG compared to its level in control nontreated T47D cells. Pretreatment with the proteosome inhibitors ALLN, lactacystin, and PSI [N-benzyloxycarbonyl-Ile-Glu-(O-t-butyl)-Ala-leucinal] inhibited also the HRG-induced down-regulation of BRCA1 protein in breast cancer cells. Interestingly, BRCA1 mRNA expression in HCC-1937 breast cancer cells, which express C-terminally truncated BRCA1, was not affected by either LAM or CL4. No phosphorylation of BRCA1 from HCC-1937 cells was observed in response to HRG. While Cdk4 phosphorylated wild-type BRCA1 in response to HRG in T47D cells, Cdk4 failed to phosphorylate the truncated form of BRCA1 in HCC-1937 cells. Furthermore, overexpression of wild-type BRCA1 in HCC-1937 cells resulted in the phosphorylation of BRCA1 and decreased BRCA1 expression upon HRG stimulation while overexpression of truncated BRCA1 in T47D cells resulted in a lack of BRCA1 phosphorylation and restoration of BRCA1 expression. These findings suggest that ECM enhances HRG-dependent BRCA1 phosphorylation and that ECM and HRG down-regulate BRCA1 expression in breast cancer cells. Furthermore, ECM suppresses BRCA1 expression through the C terminus of BRCA1.

Acetylcysteine↗
PubMed2005

Up-regulation of CC chemokine ligand 20 expression in human airway epithelium by IL-17 through a JAK-independent but MEK/NF-kappaB-dependent signaling pathway.

CCL20, like human beta-defensin (hBD)-2, is a potent chemoattractant for CCR6-positive immature dendritic cells and T cells in addition to recently found antimicrobial activities. We previously demonstrated that IL-17 is the most potent cytokine to induce an apical secretion and expression of hBD-2 by human airway epithelial cells, and the induction is JAK/NF-kappaB-dependent. Similar to hBD-2, IL-17 also induced CCL20 expression, but the nature of the induction has not been elucidated. Compared with a panel of cytokines (IL-1alpha, 1beta, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 18, IFN-gamma, GM-CSF, and TNF-alpha), IL-17 was as potent as IL-1alpha, 1beta, and TNF-alpha, with a time- and dose-dependent phenomenon in stimulating CCL20 expression in both well-differentiated primary human and mouse airway epithelial cell culture systems. The stimulation was largely dependent on the treatment of polarized epithelial cultures from the basolateral side with IL-17, achieving an estimated 4- to 10-fold stimulation at both message and protein levels. More than 90% of induced CCL20 secretion was toward the basolateral compartment (23.02 +/- 1.11 ng/chamber/day/basolateral vs 1.82 +/- 0.82 ng/chamber/day/apical). Actinomycin D experiments revealed that enhanced expression did not occur at mRNA stability. Inhibitor studies showed that enhanced expression was insensitive to inhibitors of JAK/STAT, p38, JNK, and PI3K signaling pathways, but sensitive to inhibitors of MEK1/2 and NF-kappaB activation, suggesting a MEK/NF-kappaB-based mechanism. These results suggest that IL-17 can coordinately up-regulate both hBD-2 and CCL20 expressions in airways through differentially JAK-dependent and -independent activations of NF-kappaB-based transcriptional mechanisms, respectively.

Active Transport, Cell Nucleus↗
PubMed2006

The phosphatidylinositol-3 kinase/Akt pathway mediates VEGF's neuroprotective activity and induces blood brain barrier permeability after focal cerebral ischemia.

Based on its trophic influence on neurons and vascular cells, vascular endothelial growth factor (VEGF) is a promising candidate for stroke treatment. VEGF's survival-promoting effects are purchased at the expense of an increased blood brain barrier permeability, which potentially compromises tissue survival. The mechanisms via which VEGF protects the brain against ischemia remained unknown. We examined signaling pathways underlying VEGF's neuroprotective activity in our transgenic mouse line, which expresses human VEGF165 under a neuron-specific enolase (NSE) promoter. We show that VEGF receptor-2 (Flk-1) is expressed on ischemic neurons and astrocytes and is activated by VEGF. Following 90-min episodes of middle cerebral artery occlusion, VEGF increased phosphorylated (but not total) Akt and ERK-1/-2 and reduced phosphorylated mitogen activated protein kinase/p38 and c-Jun NH2-terminal kinase (JNK)-1/-2 levels, at the same time decreasing inducible NO synthase expression in ischemic neurons. Inhibition of Akt with Wortmannin reversed VEGF's neuroprotective properties, diminished brain swelling, and restored the vascular permeability induced by VEGF to below levels in WT animals. The aggravation of brain injury by Wortmannin was associated with the restitution of p38, but not of JNK-1/-2, ERK-1/-2, or inducible NOS (iNOS). Our data demonstrate that VEGF mediates both neuroprotection and blood brain barrier permeability via the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Based on our observation that VEGF neuroprotection and vascular leakage depend on PI3K/Akt, which is putatively regulated by VEGF receptor-2, we predict that it may not easily be possible to make use of VEGF's neuroprotective function without accepting its unfavorable consequence, the increased vascular permeability.

Androstadienes↗
PubMed2002

Anchorage-independent multi-cellular spheroids as an in vitro model of growth signaling in Ewing tumors.

Little is known about the growth-signaling pathways that govern the proliferation of Ewing tumor (ET) cells either in vitro or in vivo. We have studied signal transduction pathways in ET cell lines and compared kinase expression levels and proliferation rates with primary tumors. Cell lines were studied both as conventional adherent monolayers and as anchorage-independent multi-cellular spheroids. Importantly, we observed significant differences between these in vitro models and found that ET spheroids were more closely related to primary tumors with respect to cell morphology, cell-cell junctions, proliferative index and kinase activation. Monolayer ET cells demonstrated serum-dependent phosphorylation of ERK1/2 and AKT and constitutively high serum-independent cyclin D1 protein expression. However, when ET cells were placed in suspension culture, there was immediate serum-independent activation of ERK1/2 and AKT. In addition, cyclin D1 protein expression was completely blocked until stable multi-cellular spheroids had formed, indicating that cell-cell adhesion is necessary for the proliferation of anchorage independent ET cells. This reduction in cyclin D1 expression was post-transcriptional and could be mimicked in monolayer cells by treatment with phosphatidyl inositol-3 kinase (PI3K) inhibitors. Moreover, PI3K inhibition significantly reduced ET cell proliferation and, in primary ET samples, cyclin D1 expression correlated with expression of activated AKT. Thus, the PI3K-AKT pathway appears to be critical for the proliferation of ET cells both in vitro and in vivo and tumor cell growth in vivo may be better represented by the study of anchorage-independent multi-cellular spheroids.

Artificial Gene Fusion↗
PubMed1999

Participation of PI3K and atypical PKC in Na+-K+-pump stimulation by IGF-I in VSMC.

The activity of the Na+-K+-pump is intricately linked to the maintenance of vascular tone. Here we demonstrate that insulin-like growth factor I (IGF-I) increases Na+-K+-pump activity in the vascular smooth muscle cell (VSMC) clone A7r5 in a time- and dose-dependent manner. This stimulatory effect of IGF-I was prevented by the tyrosine kinase inhibitor genistein (5 microM) and by the specific phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin (100 nM) and LY-294002 (25 microM). IGF-I activated a wortmannin-sensitive PI3K and its purported effector, the atypical protein kinase C (PKC)-zeta. Stimulation of PKC-zeta was prevented by the generic PKC inhibitor GF109203x (bisindolylmaleimide, 10 microM). Downregulation of diacylglycerol-sensitive (conventional and novel) PKCs by 24-h pretreatment with 1 microM phorbol 12-myristate 13-acetate had no effect on IGF-I-stimulated Na+-K+-pump activity. Similarly, inhibition of only conventional and novel PKCs with GF109203x (1 microM) had no effect on IGF-I-stimulated Na+-K+-pump activity. In contrast, a concentration of GF109203x (10 microM) that also inhibits the atypical PKCs abolished Na+-K+-pump stimulation by IGF-I. Neither the Na+-K+-2Cl- cotransporter inhibitor bumetanide (100 microM) nor the Na+/H+ exchanger inhibitor HOE-694 (5 microM) affected the Na+-K+-pump stimulation by IGF-I, suggesting that a rise in intracellular Na+ concentration is not necessary for increased Na+-K+-pump activity. These results suggest that IGF-I directly stimulates the Na+-K+ pump via a signaling pathway involving PI3K and atypical PKC (zeta).

Cell Line↗
PubMed2000

Overexpression of protein-tyrosine phosphatase-1B in adipocytes inhibits insulin-stimulated phosphoinositide 3-kinase activity without altering glucose transport or Akt/Protein kinase B activation.

Previous studies suggested that protein-tyrosine phosphatase 1B (PTP1B) antagonizes insulin action by catalyzing dephosphorylation of the insulin receptor (IR) and/or other key proteins in the insulin signaling pathway. In adipose tissue and muscle of obese humans and rodents, PTP1B expression is increased, which led to the hypothesis that PTP1B plays a role in the pathogenesis of insulin resistance. Consistent with this, mice in which the PTP1B gene was disrupted exhibit increased insulin sensitivity. To test whether increased expression of PTP1B in an insulin-sensitive cell type could contribute to insulin resistance, we overexpressed wild-type PTP1B in 3T3L1 adipocytes using adenovirus-mediated gene delivery. PTP1B expression was increased approximately 3-5-fold above endogenous levels at 16 h, approximately 14-fold at 40 h, and approximately 20-fold at 72 h post-transduction. Total protein-tyrosine phosphatase activity was increased by 50% at 16 h, 3-4-fold at 40 h, and 5-6-fold at 72 h post-transduction. Compared with control cells, cells expressing high levels of PTP1B showed a 50-60% decrease in maximally insulin-stimulated tyrosyl phosphorylation of IR and insulin receptor substrate-1 (IRS-1) and phosphoinositide 3-kinase (PI3K) activity associated with IRS-1 or with phosphotyrosine. Akt phosphorylation and activity were unchanged. Phosphorylation of p42 and p44 MAP kinase (MAPK) was reduced approximately 32%. Overexpression of PTP1B had no effect on basal, submaximally or maximally (100 nm) insulin-stimulated glucose transport or on the EC(50) for transport. Our results suggest that: 1) insulin stimulation of glucose transport in adipocytes requires </=45% of maximal tyrosyl phosphorylation of IR or IRS-1 and <50% of maximal activation of PI3K, 2) a novel PI3K-independent pathway may play a role in insulin-induced glucose transport in adipocytes, and 3) overexpression of PTP1B alone in adipocytes does not impair glucose transport.

3T3 Cells↗
PubMed2005

The insulin-like growth factor-I receptor kinase inhibitor, NVP-ADW742, sensitizes small cell lung cancer cell lines to the effects of chemotherapy.

PURPOSE: Insulin-like growth factor-I (IGF-I) is a potent growth factor for small cell lung cancer (SCLC) in both the autocrine and endocrine context. It also inhibits chemotherapy-induced apoptosis through activation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway and we have previously shown that inhibition of this signaling pathway enhances sensitivity of SCLC cell lines to chemotherapy. The purpose of this study was to determine whether the novel IGF-I receptor (IGF-IR) kinase inhibitor, NVP-ADW742, sensitizes SCLC cell lines to etoposide and carboplatin, which are commonly used in the treatment of SCLC. EXPERIMENTAL DESIGN: Cell growth in the presence of various combinations of NVP-ADW742, imatinib (STI571; Gleevec/Glivec), and chemotherapeutic agents was monitored using a 3-(4,5 dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and analyzed using the Chou-Talalay multiple-drug-effect equation. Induction of apoptosis was assessed using terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) and Western blot analysis of procaspase 3 and poly(ADP-ribose)polymerase cleavage. IGF-I-induced vascular endothelial cell growth factor expression was monitored by Northern blot and ELISA. RESULTS: NVP-ADW742 synergistically enhanced sensitivity of multiple SCLC cell lines to etoposide and carboplatin. Maximal enhancement occurred at concentrations of NVP-ADW742 that eliminated basal PI3K-Akt activity in individual cell lines. In the WBA cell line, in which the c-Kit receptor tyrosine kinase is partly responsible for basal PI3K-Akt activity, the combination of NVP-ADW742 and imatinib was superior to NVP-ADW742 alone in sensitizing the cells to etoposide. Enhancement of the sensitivity of SCLC cell lines to etoposide, as determined by MTT assay, correlated closely with sensitization to the induction of apoptosis as measured by TUNEL and caspase activation assays. Treatment with NVP-ADW742 also eliminated IGF-I-mediated expression of vascular endothelial cell growth factor, suggesting that in addition to enhancing sensitivity of SCLC to chemotherapy, this kinase inhibitor could potentially inhibit angiogenesis in vivo. CONCLUSIONS: Inhibition of IGF-IR signaling synergistically enhances the sensitivity of SCLC to etoposide and carboplatin. This enhancement in sensitivity to chemotherapy tightly correlates with inhibition of PI3K-Akt activation. Future SCLC clinical trials incorporating IGF-IR inhibitors alone or in combination with other kinase inhibitors should include assessment of PI3K-Akt activity as a pharmacodynamic end-point.

Antineoplastic Agents↗