u-0126 and lysophosphatidic-acid

The transcription factor E2F4 controls proliferation of normal and cancerous intestinal epithelial cells. E2F4 localization in normal human intestinal epithelial cells (HIEC) is cell cycle-dependent, being cytoplasmic in quiescent differentiated cells but nuclear in proliferative cells. However, the intracellular signaling mechanisms regulating such E2F4 localization remain unknown.. Treatment of quiescent HIEC with serum induced ERK1/2 activation, E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition while inhibition of MEK/ERK signaling by U0126 prevented these events. Stimulation of HIEC with epidermal growth factor (EGF) also led to the activation of ERK1/2 but, in contrast to serum or lysophosphatidic acid (LPA), EGF failed to induce E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition. Furthermore, Akt and GSK3β phosphorylation levels were markedly enhanced in serum- or LPA-stimulated HIEC but not by EGF. Importantly, E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition were all observed in response to EGF when GSK3 activity was concomitantly inhibited by SB216763. Finally, E2F4 was found to be overexpressed, phosphorylated and nuclear localized in epithelial cells from human colorectal adenomas exhibiting mutations in APC and KRAS or BRAF genes, known to deregulate GSK3/β-catenin and MEK/ERK signaling, respectively.. The present results indicate that MEK/ERK activation and GSK3 inhibition are both required for E2F4 phosphorylation as well as its nuclear translocation and S phase entry in HIEC. This finding suggests that dysregulated E2F4 nuclear localization may be an instigating event leading to hyperproliferation and hence, of tumor initiation and promotion in the colon and rectum.

Topics: Adenoma; Butadienes; Cell Cycle; Cell Line; Cell Proliferation; Cells, Cultured; Colorectal Neoplasms; E2F4 Transcription Factor; Enzyme Inhibitors; Epidermal Growth Factor; Glycogen Synthase Kinase 3; Humans; Intestinal Mucosa; Lysophospholipids; MAP Kinase Signaling System; Mitogens; Nitriles; Phosphorylation; Transcription, Genetic

Transcriptional co-activator with PDZ-binding motif (TAZ), a downstream effector of the Hippo pathway, has been reported to regulate organ size, tissue homeostasis, and tumorigenesis by acting as a transcriptional co-activator. Lysophosphatidic acid (LPA) is a bioactive lipid implicated in tumorigenesis and metastasis of ovarian cancer through activation of G protein-coupled receptors. However, the involvement of TAZ in LPA-induced tumorigenesis of ovarian cancer has not been elucidated.. In order to demonstrate the role of TAZ in LPA-stimulated tumorigenesis, the effects of LPA on TAZ expression and cell migration were determined by Western blotting and chemotaxis analyses in R182 human epithelial ovarian cancer cells.. Treatment of R182 cells with the LPA receptor inhibitor Ki16425 blocked LPA-induced cell migration. In addition, transfection of R182 cells with small interfering RNA specific for LPA receptor 1 resulted in abrogation of LPA-stimulated cell migration. LPA induced phosphorylation of ERK and p38 MAP kinase in R182 cells and pretreatment of cells with the MEK-ERK pathway inhibitor U0126, but not the p38 MAPK inhibitor SB202190, resulted in abrogation of LPA-induced cell migration. Pretreatment of R182 cells with U0126 attenuated LPA-induced mRNA levels of TAZ and its transcriptional target genes, such as CTGF and CYR61, without affecting phosphorylation level of YAP. These results suggest that MEK-ERK pathway plays a key role in LPA-induced cell migration and mRNA expression of TAZ in R182 cells, without affecting stability of TAZ protein. In addition, small interfering RNA-mediated silencing of TAZ expression attenuated LPA-stimulated migration of R182 cells. These results suggest that TAZ plays a key role in LPA-stimulated migration of epithelial ovarian cancer cells.

Topics: Butadienes; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cells, Cultured; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Isoxazoles; Lysophospholipids; Neoplasms, Glandular and Epithelial; Nitriles; Ovarian Neoplasms; Propionates; Protein Stability; Trans-Activators; Transcription Factors; Transcriptional Coactivator with PDZ-Binding Motif Proteins

Na(+) absorption is a vital process present in all living organisms. We have reported previously that lysophosphatidic acid (LPA) acutely stimulates Na(+) and fluid absorption in human intestinal epithelial cells and mouse intestine by stimulation of Na(+)/H(+) exchanger 3 (NHE3) via LPA(5) receptor. In the current study, we investigated the mechanism of NHE3 activation by LPA(5) in Caco-2bbe cells. LPA(5)-dependent activation of NHE3 was blocked by mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 and U0126, but not by phosphatidylinositol 3-kinase inhibitor LY294002 or phospholipase C-β inhibitor U73122. We found that LPA(5) transactivated the epidermal growth factor receptor (EGFR) and that inhibition of EGFR blocked LPA(5)-dependent activation of NHE3, suggesting an obligatory role of EGFR in the NHE3 regulation. Confocal immunofluorescence and surface biotinylation analyses showed that LPA(5) was located mostly in the apical membrane. EGFR, on the other hand, showed higher expression in the basolateral membrane. However, inhibition of apical EGFR, but not basolateral EGFR, abrogated LPA-induced regulation of MEK and NHE3, indicating that LPA(5) selectively activates apical EGFR. Furthermore, transactivation of EGFR independently activated the MEK-ERK pathway and proline-rich tyrosine kinase 2 (Pyk2). Similarly to MEK inhibition, knockdown of Pyk2 blocked activation of NHE3 by LPA. Furthermore, we showed that RhoA and Rho-associated kinase (ROCK) are involved in activation of Pyk2. Interestingly, LPA(5) did not directly activate RhoA but was required for transactivation of EGFR. Together, these results unveil a pivotal role of apical EGFR in NHE3 regulation by LPA and show that the RhoA-ROCK-Pyk2 and MEK-ERK pathways converge onto NHE3.

Topics: Butadienes; Caco-2 Cells; Chromones; Enzyme Inhibitors; ErbB Receptors; Estrenes; Flavonoids; Focal Adhesion Kinase 2; Humans; Intestines; Lysophospholipids; Mitogen-Activated Protein Kinase Kinases; Morpholines; Nitriles; Phosphoinositide-3 Kinase Inhibitors; Phospholipase C beta; Pyrrolidinones; Receptors, Lysophosphatidic Acid; rho-Associated Kinases; Sodium-Hydrogen Exchanger 3; Sodium-Hydrogen Exchangers

1 Mitogen-activated protein kinases mediate hormone/neurotransmitter action on proliferation and differentiation and participate in receptor regulation. The effect of inhibitors of mitogen-activated kinase kinase (MEK) on alpha(1B)-adrenoceptor phosphorylation state and function was studied using different cell lines. It was observed that at nanomolar concentrations the MEK inhibitors, PD98059 (2'-amino-3'-methoxyflavone) and UO126 [1,4-(diamino-2,3-dicyano/1,4-bis-(2-aminophenylthio)-butadiene], increased alpha(1B)-adrenoceptor phosphorylation and diminished the functional response of this receptor to noradrenaline. These agents did not alter the action of lysophosphatidic acid. 2 Staurosporine (IC(50) approximately 0.8 nm) (a general protein kinase inhibitor) and bis-indolyl-maleimide I (IC(50) approximately 200 nm) (a selective protein kinase C inhibitor) inhibited PD98059-induced alpha(1B)-adrenoceptor phosphorylation. In contrast, neither wortmannin (phosphoinositide 3-kinase inhibitor) nor genistein (protein tyrosine kinase inhibitor) had any effect. The data suggest the possibility that MEK might exert control on the activity of the enzymes that regulate receptor phosphorylation, such as G-protein-coupled receptor kinases, protein kinase C or serine/threonine protein phosphatases. 3 Coimmunoprecipitation studies showed a constant association of total extracellular signal-regulated kinase 2 (ERK2) with alpha(1B)-adrenoceptors. Association of phospho-ERK 1/2 to alpha(1B)-adrenoceptors increased not only in response to agonist but also in response to agents that increase alpha(1B)-adrenoceptor and ERK1/2 phosphorylation [such as endothelin-1, phorbol 12-myristate-13-acetate (PMA) and epidermal growth factor (EGF)]; not surprisingly, PD98059 decreased this effect. 4 Our data show that blockade of MEK activity results in increased alpha(1B)-adrenoceptor phosphorylation, diminished adrenoceptor function and perturbation of receptor-ERK1/2 interaction.

Topics: Androstadienes; Animals; Butadienes; Calcium Signaling; Cell Line; Cricetinae; Endothelin-1; Epidermal Growth Factor; Flavonoids; Genistein; Humans; Indoles; Lysophospholipids; Maleimides; MAP Kinase Kinase 2; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Nitriles; Norepinephrine; Phosphorylation; Protein Kinase Inhibitors; Rats; Receptors, Adrenergic, alpha-1; RNA, Small Interfering; Staurosporine; Tetradecanoylphorbol Acetate; Wortmannin

Lysophosphatidic acid (LPA) is a bioactive phospholipids and involves in various cellular events, including tumor cell migration. In the present study, we investigated LPA receptor and its transactivation to EGFR for cyclooxygenase-2 (COX-2) expression and cell migration in CAOV-3 ovarian cancer cells. LPA induced COX-2 expression in a dose-dependent manner, and pretreatment of the cells with pharmacological inhibitors of Gi (pertussis toxin), Src (PP2), EGF receptor (EGFR) (AG1478), ERK (PD98059) significantly inhibited LPA- induced COX-2 expression. Consistent to these results, transfection of the cells with selective Src siRNA attenuated COX-2 expression by LPA. LPA stimulated CAOV-3 cell migration that was abrogated by pharmacological inhibitors and antibody of EP2. Higher expression of LPA2 mRNA was observed in CAOV-3 cells, and transfection of the cells with a selective LPA2 siRNA significantly inhibited LPA-induced activation of EGFR and ERK, as well as COX-2 expression. Importantly, LPA2 siRNA also blocked LPA-induced ovarian cancer cell migration. Collectively, our results clearly show the significance of LPA2 and Gi/Src pathway for LPA-induced COX-2 expression and cell migration that could be a promising drug target for ovarian cancer cell metastasis.

Topics: Butadienes; Cell Line, Tumor; Cell Movement; CSK Tyrosine-Protein Kinase; Cyclooxygenase 2; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Female; Flavonoids; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Lysophospholipids; Nitriles; Ovarian Neoplasms; Pertussis Toxin; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Pyrimidines; Quinazolines; Receptors, Lysophosphatidic Acid; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP2 Subtype; Signal Transduction; src-Family Kinases; Transcriptional Activation; Tyrphostins

Osteoblast maturation is partly controlled by the interaction of 1alpha,25(OH)(2)D(3) (D3), an active metabolite of Vitamin D, with other growth factors. The first reports describing the in vitro effect of D3 on human osteoblast differentiation performed experiments in the presence of serum. One potentially exciting candidate that might help explain the D3 responses observed for osteoblasts cultured with serum is lysophosphatidic acid (LPA). Drawn to the possibility that D3 and serum borne LPA might interact to induce osteoblast maturation we co-treated human cells with D3 and serum in the presence of Ki16425, an LPA receptor antagonist. Ki16425 inhibited osteoblast maturation as determined by markedly reduced alkaline phosphatase (ALP) expression. We subsequently found that LPA and D3 acted synergistically in generating mature osteoblasts and that this differentiation response could be inhibited using pertussis toxin, implying an important role of Galphai signal transduction. Furthermore, we found evidence for a dependency on both mitogen activated protein kinase kinase (MEK) and Rho associated coiled kinase (ROCK) for LPA and D3 stimulated maturation.

Topics: Alkaline Phosphatase; Butadienes; Calcitriol; Cell Differentiation; Cells, Cultured; Cyclooxygenase 2; Cytoskeleton; Drug Synergism; Humans; Intracellular Signaling Peptides and Proteins; Isoxazoles; Lysophospholipids; Mitogen-Activated Protein Kinase Kinases; Nitriles; Osteoblasts; Pertussis Toxin; Phosphorylation; Propionates; Protein Serine-Threonine Kinases; rho-Associated Kinases; Stress Fibers

The sodium hydrogen exchanger isoform 1 (NHE1) is present in nearly all cells. Regulation of proton flux via the exchanger is a permissive step in cell growth and tumorgenesis and is vital in control of cell volume. The regulation of NHE1 by growth factors involves the Ras-extracellular signal regulated kinase (ERK) pathway, however, the mechanism for G protein-coupled receptor (GPCR) activation of NHE1 is not well established. In this report, the relationship between GPCRs, ERK, and NHE1 in CCL39 cells is investigated. We give evidence that two agonists, the specific alpha(1)-adrenergic agonist, phenylephrine and the water-soluble lipid mitogen, lysophosphatidic acid (LPA) activate NHE1 in CCL39 cells. Activation of ERK by phenylephrine and LPA occurs in a dose- and time-dependent manner. Optimal ERK activation was observed at 10 min and displayed a maximum stimulation at 100 microM phenylephrine and 10 microM LPA. alpha(1)-Adrenergic stimulation also led to a rise in steady-state pH(i) of 0.16+/-0.02 pH units, and incubation with LPA induced a 0.43+/-0.06 pH unit increase in pH(i). Phenylephrine-induced activation of NHE1 transport and ERK activity was inhibited by pretreating the cells with the MEK inhibitor PD98059. While only half of the LPA activatable exchange activity was abolished by PD98059 and U0126. To further demonstrate the specificity of the phenylephrine and LPA regulation of NHE1 and ERK, CCL39 cells were transfected with a kinase inactive MEK. The data indicate that ERK activation is essential for phenylephrine stimulation of NHE1, and that ERK and RhoA are involved in LPA stimulation of NHE1 by more than one mechanism. In addition, evidence of the convergence of these two pathways is shown by the loss of NHE1 activity when both pathways are inhibited and by the partial additivity of the two agonists on ERK and NHE1 activity. These studies indicate a direct involvement of ERK in the alpha(1)-adrenergic activation of NHE1 and a significant role for both ERK and RhoA in LPA stimulation of NHE1 in CCL39 fibroblasts.

Topics: Amides; Animals; Butadienes; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Flavonoids; Hydrogen-Ion Concentration; Intracellular Signaling Peptides and Proteins; Lysophospholipids; MAP Kinase Kinase 1; Nitriles; Phenylephrine; Phosphorylation; Protein Isoforms; Protein Serine-Threonine Kinases; Pyridines; Receptors, Adrenergic, alpha; Receptors, G-Protein-Coupled; rho-Associated Kinases; Signal Transduction; Sodium-Hydrogen Exchangers; Transfection