Helicobacter pylori Induced Phosphatidylinositol-3-OH Kinase/mTOR Activation Increases Hypoxia Inducible Factor-1α to Promote Loss of Cyclin D1 and G0/G1 Cell Cycle Arrest in Human Gastric Cells

Abstract

Helicobacter pylori (H. pylori) is a human gastric pathogen that has been linked to the development of several gastric pathologies, such as gastritis, peptic ulcer, and gastric cancer. In the gastric epithelium, the bacterium modifies many signaling pathways, resulting in contradictory responses that favor both proliferation and apoptosis. Consistent with such observations, H. pylori activates routes associated with cell cycle progression and cell cycle arrest. H. pylori infection also induces the hypoxia-induced factor HIF-1α, a transcription factor known to promote expression of genes that permit metabolic adaptation to the hypoxic environment in tumors and angiogenesis. Recently, however, also roles for HIF-1α in the repair of damaged DNA and inhibition of gene expression were described. Here, we investigated signaling pathways induced by H. pylori in gastric cells that favor HIF-1α expression and the consequences thereof in infected cells. Our results revealed that H. pylori promoted PI3K/mTOR-dependent HIF-1α induction, HIF-1α translocation to the nucleus, and activity as a transcription factor as evidenced using a reporter assay. Surprisingly, however, transcription of known HIF-1α effector genes evaluated by qPCR analysis, revealed either no change (LDHA and GAPDH), statistically insignificant increases SLC2A1 (GLUT-1) or greatly enhance transcription (VEGFA), but in an HIF-1α-independent manner, as quantified by PCR analysis in cells with shRNA-mediated silencing of HIF-1α. Instead, HIF-1α knockdown facilitated G1/S progression and increased Cyclin D1 protein half-life, via a post-translational pathway. Taken together, these findings link H. pylori-induced PI3K-mTOR activation to HIF-1α induced G0/G1 cell cycle arrest by a Cyclin D1-dependent mechanism. Thus, HIF-1α is identified here as a mediator between survival and cell cycle arrest signaling activated by H. pylori infection.

Keywords: Cyclin D1; HIF-1α; Helicobacter pylori; PI3K/mTOR; cell cycle arrest.

Figure 1

H. pylori promotes transient HIF-1α induction, nuclear localization and transcriptional activity. (A) AGS cells were infected with H. pylori 26695 at MOI 100 for time periods indicated. The levels of HIF-1α and β-actin were analyzed by western blotting. The relative levels of HIF-1α were determined (mean ± SEM; n = 4, ^*p < 0.05; ^**p < 0.01). (B) AGS cells were infected with H. pylori 26695 at MOI 100 for time periods indicated and then cytoplasmic and nuclear fractions were obtained. The levels of HIF-1α, HSP90 (cytoplasmic marker) and LAP2 (nuclear marker) were analyzed by western blotting. The relative nuclear levels of HIF-1α were determined (mean ± SEM, n = 4; ^**p < 0.01). (C) AGS cells were co-transfected with the pGL3-HRE-luciferase and pON-β-galactosidase plasmids and then were infected with H. pylori at the indicated Multiplicity of Infection (MOI) for 8 h. The cells were lysed and the luciferase and β-galactosidase activities were determined. The luciferase activity was standarized to the β-galactosidase activity (mean ± SEM; n = 4; ^*p < 0.05; ^***p < 0.001).

Figure 2

The inhibition of PI3K precludes the H. pylori-promoted HIF-1α induction. (A) AGS cells were infected with H. pylori 26695 at MOI 100 for time periods indicated. The levels of p-S473-Akt, total Akt and β-actin were analyzed by western blotting. The relative levels of p-S473-Akt were determined (mean ± SEM; n = 4; ^*p < 0.05; ^**p < 0.01). (B,C) AGS cells were pre-treated with LY294002 (1, 5, and 10 μM) for 30 min and then infected with H. pylori 26695 at MOI 100 for 4 h (B) or 8 h (C). The levels of HIF-1α, p-S473-Akt, Akt, and β-actin were analyzed by western blotting. The relative levels of HIF-1α/β-actin were determined (mean ± SEM; n = 3–4, ^***p < 0.001).

Figure 3

The inhibition of mTOR precludes the H. pylori-promoted HIF-1α induction while Akt inhibition is only partially effective. (A,B) AGS cells were pre-treated with Rapamycin (50 nM) for 30 min and then infected with H. pylori 26695 at MOI 100 for 4 h (A) or 8 h (B). The levels of HIF-1α, p-S235/236-S6, S6, and β-actin were analyzed by western blotting. The relative levels of HIF-1α were determined (mean ± SEM; n = 3–4; ^***p < 0.001). (C,D) AGS cells were pre-treated with Akt inhibitor (1 μM) for 30 min and then infected with H. pylori 26695 at MOI 100 for 4 h (C) or 8 h (D). The levels of HIF-1α, p-S473-Akt, total Akt, and β-actin were analyzed by western blotting. The relative levels of HIF-1α were determined (mean ± SEM; n = 3.4, ^*p < 0.05; ^***p < 0.001).

Figure 4

Effect of H. pylori infection on HIF-1α target gene expression. AGS cells were infected with H. pylori 26695 at MOI 100 for 8 h and 24 h. mRNA levels of GLUT-1 (A), LDHA (B), GAPDH (C), Bcl-xL (D), and VEGFA (E) were evaluated by RT-qPCR. The levels of each mRNA were normalized to RNA of the RPS13 housekeeping gene. All data were expressed relative to values obtained for time 0 h of infection (mean ±SEM; n = 3–4; ^*p < 0.05).

Figure 5

Effect of HIF-1α silencing on gene induction in response to H. pylori infection. AGS sh-Scr C2 and sh-HIF-1α C7 cells were infected with H. pylori 26695 at MOI 100 for 8 and 24 h. mRNA levels of GLUT-1 (A), LDHA (B), VEGFA (C), and HIF-1α (D) were evaluated by RT-qPCR. The levels of each mRNA were normalized to RNA of the RPS13 housekeeping gene. All data were expressed relative to values obtained for time 0 h of infection in sh-Scr cells (mean ± SEM; n = 3–4, ^*p < 0.05; ^**p < 0.01; ^***p < 0.001).

Figure 6

H. pylori infection increases cells in G0/G1 phase and decreases cells in G2/M phase of cell cycle. AGS cells were infected with H. pylori 26695 at MOI 100 for 24 h and cell cycle distribution was determined by flow cytometry. (A) Representative profiles of the cell cycle distribution are shown for the indicated conditions. (B) Percentage of cells in each phase of the cell cycle phase are summarized (mean ± SEM, n = 4; ^*p < 0.05).

Figure 7

The lack of HIF-1α promotes the transition from G1 to S phase of the cell cycle in gastric cells infected with H. pylori. Stably transfected AGS sh-Scr C2 and AGS sh-HIF-1α C7 cells were infected with H. pylori 26695 at MOI 100 for 24 h and cell cycle distribution was determined by flow cytometry. (A) Representative cell cycle distribution profiles for the indicated conditions. (B) Percentage of cells in each phase of the cell cycle are summarized (mean ± SEM, n = 4; ^*p < 0.05; ^**p < 0.01) (C) Changes following infection in the percentage of cells in each phase of the cell cycle are summarized graphically (mean ± SEM, n = 4; ^*p < 0.05).

Figure 8

The lack of HIF-1α partially attenuates H. pylori-enhanced Cyclin D1 decline by a post-translational mechanism. (A) Stably transfected AGS sh-Scr C2 and AGS sh- HIF-1α C7 cells were infected with H. pylori 26695 at MOI 100 for 8 and 24 h. The levels of Cyclin D1, HIF-1α, and β-actin were analyzed by western blotting. The relative levels of Cyclin D1 were determined (mean ±SEM, n = 4; ^*p < 0.05; ^**p < 0.01). (B) Stably transfected AGS sh-Scr C2 and AGS sh- HIF-1α C7 cells were infected with H. pylori 26695 at MOI 100 for 8 and 24 h. mRNA levels of Cyclin D1 were evaluated by RT-qPCR. The levels of Cyclin D1 mRNA were normalized to RNA levels of RPS13, employed here as a housekeeping gene. Data were expressed relative to values obtained for time 0 h of infection (mean ± SEM, n = 4; ^**p < 0.01; ^***p < 0.001). (C) Stably transfected AGS sh-Scr C2 and AGS sh-HIF-1α C7 cells were infected with H. pylori 26695 at MOI 100 for 8 h and then treated with cycloheximide (100 μg/ml) for the time points indicated. The levels of Cyclin D1 and β-actin were analyzed by western blotting. The relative levels of Cyclin D1 were determined (mean ± SEM, n = 3). (D) Stably transfected AGS sh-Scr C2 and AGS sh-HIF-1α C7 cells were infected with H. pylori 26695 at MOI 100 for 8 h and then cycloheximide (CHX) (100 μg/ml) and/or MG132 (20 μM) were added. Cells extracts were collected after 15 min of these treatments. Cyclin D1 and β-actin levels were evaluated by western blotting. The relative levels of Cyclin D1 were determined (mean ± SEM, n = 4; ^*p < 0.05; ^**p < 0.01; ^***p < 0.001).

Figure 9

Proposed working model. H. pylori infection promotes the activation of PI3K and subsequently, of mTOR. The activation of this pathway increases the levels of HIF-1α, a protein mainly present in the nucleus. H. pylori-induced HIF-1α decreases the Cyclin D1 levels by a post-translational mechanism. Cyclin D1 is a protein necessary for G1 phase progression. Therefore, its reduction affects the normal progression of the cell cycle, promoting an arrest in G1 phase. Gray boxes: Inactive protein kinases. Green boxes: Active protein kinases associated with cell cycle progression. Blue boxes: effects linked to cell cycle arrest. Red boxes: HIF-1α as a switch between cell cycle progression and cell cycle arrest. Dashed arrows: steps ocurring by mechanism(s) that were not defined in this study.