Helicobacter pylori-associated regulation of forkhead transcription factors FoxO1/3a in human gastric cells.

BACKGROUND: Interaction of Helicobacter pylori with gastric mucosa leads to marked cellular and humoral host immunologic responses. The signaling pathways initiated by bacteria-host interaction that result in perturbations in cell structure and function remain unclear. Forkhead transcription factors of class O (FoxO) are implicated in the regulation of apoptosis, cell survival, and pathogenesis. H. pylori infection of gastric epithelial cells induces phosphoinositide-3 kinase (PI3K)-dependent Akt activation and cell survival signaling. We investigated the role of H. pylori-activated PI3K/Akt in the regulation of FoxO1/3a in gastric cells. METHODS: Immunoblot, immunoprecipitation, and fluorescence microscopy were used to assess the effect of infection of gastric epithelial cells with wild-type H. pylori and their isogenic cag pathogenicity island (PAI) or oipA mutants on the FoxO1/3a signaling pathways. Interleukin-8 release was determined by enzyme-linked immunosorbent assays. RESULTS: H. pylori infection resulted in activation of the PI3K p85 subunit and inactivation of FoxO1 and FoxO3a by their phosphorylation and translocation of from the nucleus to the cytoplasm. Inhibition of PI3K or Akt kinase activity reduced FoxO1/3a phosphorylation. Akt, FoxO1, or FoxO3a siRNA reduced H. pylori-induced interleukin-8 production. Infection with oipA mutants reduced PI3K/Akt activation and inhibited FoxO1/3a phosphorylation, whereas infection with cag PAI mutants reduced PI3K/Akt activity but did not inhibit FoxO1/3a activation. CONCLUSIONS: FoxO1 and FoxO3a are novel nuclear substrates of H. pylori-induced PI3K/Akt cell survival signaling pathways that partially control interleukin-8 production. OipA-regulated interleukin-8 release through PI3K/Akt is dependent on FoxO1/3a inactivation, whereas cag PAI-mediated interleukin-8 production employs FoxO1/3-independent signaling.

Paxillin is a novel cellular target for converging Helicobacter pylori-induced cellular signaling.

Paxillin is involved in the regulation of Helicobacter pylori-mediated gastric epithelial cell motility. We investigated the signaling pathways regulating H. pylori-induced paxillin phosphorylation and the effect of the H. pylori virulence factors cag pathogenicity island (PAI) and outer inflammatory protein (OipA) on actin stress fiber formation, cell phenotype, and IL-8 production. Gastric cell infection with live H. pylori induced site-specific phosphorylation of paxillin tyrosine (Y) 31 and Y118 in a time- and concentration-dependent manner. Activated paxillin localized in the cytoplasm at the tips of H. pylori-induced actin stress fibers. Isogenic oipA mutants significantly reduced paxillin phosphorylation at Y31 and Y118 and reduced actin stress fiber formation. In contrast, cag PAI mutants only inhibited paxillin Y118 phosphorylation. Silencing of epidermal growth factor receptor (EGFR), focal adhesion kinase (FAK), or protein kinase B (Akt) expression by small-interfering RNAs or inhibiting kinase activity of EGFR, Src, or phosphatidylinositol 3-kinase (PI3K) markedly reduced H. pylori-induced paxillin phosphorylation and morphologic alterations. Reduced FAK expression or lack of Src kinase activity suppressed H. pylori-induced IL-8 production. Compared with infection with the wild type, infection with the cag PAI mutant and oipA mutant reduced IL-8 production by nearly 80 and 50%. OipA-induced IL-8 production was FAK- and Src-dependent, although a FAK/Src-independent pathway for IL-8 production also exists, and the cag PAI may be mainly involved in this pathway. We propose paxillin as a novel cellular target for converging H. pylori-induced EGFR, FAK/Src, and PI3K/Akt signaling to regulate cytoskeletal reorganization and IL-8 production in part, thus contributing to the H. pylori-induced diseases.

Helicobacter pylori activate epidermal growth factor receptor- and phosphatidylinositol 3-OH kinase-dependent Akt and glycogen synthase kinase 3beta phosphorylation.

The signalling pathways leading to the development of Helicobacter pylori-induced gastric cancer remain poorly understood. We tested the hypothesis that H. pylori infections involve the activation of Akt signalling in human gastric epithelial cancer cells. Immunoblot, immunofluorescence and kinase assays show that H. pylori infection of gastric epithelial cells induced phosphorylation of Akt at Ser 473 and Thr 308. Mutations in the H. pylori virulence factor OipA dramatically reduced phosphorylation of Ser 473, while the cag pathogenicity island mutants predominantly inhibited phosphorylation of Thr 308. As the downstream of Akt activation, H. pylori infection inactivated the inactivation of glycogen synthase kinase 3beta at Ser 9 by its phosphorylation. As the upstream of Akt activation, H. pylori infection activated epidermal growth factor receptor (EGFR) at Tyr 992, phosphatidylinositol 3-OH kinase (PI3K) p85 subunit and PI3K-dependent kinase 1 at Ser 241. Pharmacologic inhibitors of PI3K or mitogen-activated protein kinase kinase (MEK), Akt knock-down and EGFR knock-down showed that H. pylori infection induced the activation of EGFR-->PI3K-->PI3K-dependent kinase 1-->Akt-->extracellular signal-regulated kinase signalling pathways, the inactivation of glycogen synthase kinase 3beta and interleukin-8 production. The combined functions of cag pathogenicity island and OipA were necessary and sufficient for full activation of signalling at each level. We propose activation of these pathways as a novel mechanism for H. pylori-mediated carcinogenesis.

OipA plays a role in Helicobacter pylori-induced focal adhesion kinase activation and cytoskeletal re-organization.

The initial signalling events leading to Helicobacter pylori infection associated changes in motility, cytoskeletal reorganization and elongation of gastric epithelial cells remain poorly understood. Because focal adhesion kinase (FAK) is known to play important roles in regulating actin cytoskeletal organization and cell motility we examined the effect of H. pylori in gastric epithelial cells co-cultured with H. pylori or its isogenic cag pathogenicity island (PAI) or oipA mutants. H. pylori induced FAK phosphorylation at distinct tyrosine residues in a dose- and time-dependent manner. Autophosphorylation of FAK Y397 was followed by phosphorylation of Src Y418 and resulted in phosphorylation of the five remaining FAK tyrosine sites. Phosphorylated FAK and Src activated Erk and induced actin stress fibre formation. FAK knock-down by FAK-siRNA inhibited H. pylori-mediated Erk phosphorylation and abolished stress fibre formation. Infection with oipA mutants reduced phosphorylation of Y397, Y576, Y577, Y861 and Y925, inhibited stress fibre formation and altered cell morphology. cag PAI mutants reduced phosphorylation of only FAK Y407 and had less effect on stress fibre formation than oipA mutants. We propose that activation of FAK and Src are responsible for H. pylori-induced induction of signalling pathways resulting in the changes in cell phenotype important for pathogenesis.

Lysophosphatidic acid induction of urokinase plasminogen activator secretion requires activation of the p38MAPK pathway.

Lysophosphatidic acid (LPA) is an important intercellular signaling molecule involved in a myriad of biological responses. Elevated concentrations of LPA are present in the ascites and plasma of ovarian cancer patients suggesting a role for LPA in the pathophysiology of ovarian cancer. We have demonstrated previously that oleoyl (18:1) LPA at concentrations present in ascites induces the secretion of urokinase plasminogen activator (uPA) from ovarian cancer cells, possibly linking LPA to cellular invasion. In this study we sought to elucidate which signaling pathway(s) are involved in LPA-mediated secretion of uPA from ovarian cancer cells. Specific inhibitors were utilized to determine if interference with the p38(MAPK), p42/44(MAPK), and PI3K pathways functionally blocked LPA-mediated uPA secretion. LPA stimulation of ovarian cancer cells markedly increased the phosphorylation and activity of p38(MAPK), p42/p44(MAPK), and PI3K. Both tyrosine phosphorylation and Src kinase activity were required for optimal activation of signaling by LPA including phosphorylation of p38(MAPK). Inhibition of p38(MAPK) signaling by SB202190 completely abrogated LPA-induced uPA secretion, while inhibition of the p42/44(MAPK) or PI3K pathways with PD98059 or wortmannin and LY294002, respectively, decreased but did not completely block uPA secretion. In contrast, inhibitors of phospholipase D or the p70S6 kinase pathway did not alter LPA-induced uPA secretion. Further, tyrosine phosphorylation and functional Src were required for optimal uPA secretion. Finally, LPA induces uPA secretion from ovarian cancer cells predominantly through the LPA2 receptor, with LPA3 contributing to this process. These results indicate that the p38(MAPK) signaling pathway is required for optimal LPA-dependent uPA secretion from ovarian cancer cells.

Nonclassical action of retinoic acid on the activation of the cAMP response element-binding protein in normal human bronchial epithelial cells.

Vitamin A (retinol) is essential for normal regulation of cell growth and differentiation. We have shown that the retinol metabolite retinoic acid (RA) induces mucous cell differentiation of normal human tracheobronchial epithelial (NHTBE) cells. However, early biological effects of RA in the differentiation of bronchial epithelia are largely unknown. Here, we showed that RA rapidly activated cAMP response element-binding protein (CREB). However, RA did not use the conventional retinoic acid receptor (RAR)/retinoid X receptor (RXR) to activate CREB. RA activated CREB in NHTBE and H1734 cells in which RARs/RXR were silenced with small interfering RNA (siRNA) targeting RAR/RXR expression or deactivated by antagonist. Inhibition of protein kinase C (PKC) or extracellular regulated kinase (ERK1/2) blocked the RA-mediated activation of CREB. In addition, depletion of p90 ribosomal S6 kinase (RSK) via siRSK1/2 completely abolished the activation, suggesting that PKC, ERK, and RSK are required for the activation. Altogether, this study provides the first evidence that RA rapidly activates CREB transcription factor via PKC, ERK, and RSK in a retinoid receptor-independent manner in normal bronchial epithelial cells. This noncanonical RA signaling pathway may play an important role in mediating early biological effects in the mucociliary differentiation of bronchial epithelia.

Identification of tissue- and cancer-selective promoters for the introduction of genes into human ovarian cancer cells.

OBJECTIVE: One potential limitation of gene therapy for epithelial tumors is the lack of tissue or tumor specificity of treatment. Tumor-selective expression of gene therapies may avoid deleterious side effects and improve the efficacy of the treatment. The aim of this study was to evaluate the tissue and tumor specificity of four different potential gene therapy promoters, to determine their usefulness in tissue-specific gene therapy of epithelial ovarian carcinomas. METHODS: Three potential epithelial cell-selective (hESE1, SLP1, OSP1) and one potential tumor-selective (hTERT) promoter were placed upstream of a luciferase construct to determine relative activity in a wide variety of normal and malignant cell lines. Transient transfection and luciferase assays were carried out in 12 epithelial ovarian (3 SV40 T antigen-transfected normal and 9 malignant) and 8 control cell lines. RESULTS: Luciferase assays revealed that the hTERT promoter presented the highest tumor selectivity. hESE1 and SLP1 promoters showed strong epithelial cell selectivity (hESE1, 16/17; SLP1, 15/17), with the OSP1 (11/17) promoter exhibiting lower epithelial selectivity. Of the potential promoters for gene therapy, hTERT promoter exhibited the strongest transcriptional activity in most of the tumor cell lines. None of the promoters exhibited strict ovarian epithelium selectivity. CONCLUSION: The hTERT promoter may be an optimal promoter for a univector gene therapy approach based on its high tumor selectivity. Utilization of multiple epithelial cell-specific promoters may result in a more tissue-selective gene therapy approach. Using a combination of promoters may prevent potential problems due to expression in nonepithelial stem cells that may constitutively express hTERT.