Helicobacter pylori and mitogen-activated protein kinases mediate activator protein-1 (AP-1) subcomponent protein expression and DNA-binding activity in gastric epithelial cells

Emerging evidence has suggested a critical role for activator protein-1 (AP)-1 in regulating various cellular functions. The goal of this study was to investigate the effects of Helicobacter pylori and mitogen-activated protein kinases (MAPK) on AP-1 subcomponents expression and AP-1 DNA-binding activity in gastric epithelial cells. We found that H. pylori infection resulted in a time- and dose-dependent increase in the expression of the proteins c-Jun, JunB, JunD, Fra-1, and c-Fos, which make up the major AP-1 DNA-binding proteins in AGS and MKN45 cells, while the expression levels of Fra-2 and FosB remained unchanged. Helicobacter pylori infection and MAPK inhibition altered AP-1 subcomponent protein expression and AP-1 DNA-binding activity, but did not change the overall subcomponent composition. Different clinical isolates of H. pylori showed various abilities to induce AP-1 DNA binding. Mutation of cagA, cagPAI, or vacA, and the nonphosphorylateable CagA mutant (cagA(EPISA)) resulted in less H. pylori-induced AP-1 DNA-binding activity, while mutation of the H. pylori flagella had no effect. extracellular signal-related kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) each selectively regulated AP-1 subcomponent expression and DNA-binding activity. These results provide more insight into how H. pylori and MAPK modulate AP-1 subcomponents in gastric epithelial cells to alter the expression of downstream target genes and affect cellular functions.     

Reduced intracellular survival of Helicobacter pylori vacA mutants in comparison with their wild-types indicates the role of VacA in pathogenesis

The vacuolating cytotoxin VacA of Helicobacter pylori plays an important but yet unknown role in pathogenesis. We studied the impact of the vacuolating cytotoxin on H. pylori invasion of and survival within AGS cells (human gastric cell line derived from an antral adenocarcinoma). Isogenic vacA and cagA mutants were constructed in a wild-type clinical isolate H. pylori, AF4. An H. pylori VacA-deficient mutant, AF4(vacA::kan), was cultured in significantly lower numbers from AGS cells after 24 h incubation with gentamicin added to the culture medium than were the type I wild-type strain AF4 (P<0.03) and an isogenic cagA mutant (P<0.01). Complementation of the AF4 vacA mutant with broth culture supernatant from wild-type AF4 improved the intracellular survival of the vacA mutant. We conclude that H. pylori's vacuolating cytotoxin improves the intracellular survival of H. pylori within AGS cells, suggesting the role of the vacuolating cytotoxin in H. pylori pathogenesis.     

Roles of specific isoforms of protein kinase C in the transcriptional control of cyclin D1 and related genes

Although protein kinase C (PKC) has been implicated in cell cycle progression, cell proliferation, and tumor promotion, the precise roles of specific isoforms in these processes is not clear. Therefore, we constructed and analyzed a series of expression vectors that encode hemagglutinin-tagged wild type (WT), constitutively active mutants (Delta NPS and CAT), and dominant negative mutants of PKCs alpha, beta 1, beta 2, gamma, delta, epsilon, eta, zeta, and iota. Cyclin D1 promoter reporter assays done in serum-starved NIH3T3 cells indicated that the constitutively active mutants of PKC-alpha and PKC-epsilon were the most potent activators of this reporter, whereas the constitutively active mutant of PKC-delta inhibited its activity. Transient transfection studies with a series of 5'-deleted cyclin D1 promoter constructs showed that the proximal 964-base region, which contains AP-1, SP1, and CRE enhancer elements, is required for activation of the cyclin D1 promoter by PKC-alpha. Deletion of the AP-1 enhancer element located at position -954 upstream from the initiation site abolished PKC-alpha-dependent activation of cyclin D1 expression. Deletion of the SP1 or CRE enhancer elements did not have any effect. A dominant negative mutant of c-Jun inhibited activation of the cyclin D1 promoter in a concentration-dependent manner, providing further evidence that AP-1 activity is required for activation of the cyclin D1 promoter by PKC-alpha and PKC-epsilon. The constitutively active mutants of PKC-alpha and PKC-epsilon also activated c-fos, c-jun, and cyclin E promoter activity. Furthermore, NIH3T3 cells that stably express the constitutively active mutants of PKC-alpha or PKC-epsilon displayed increased expression of endogenous cyclins D1 and E and faster growth rates. These results provide evidence that the activation of PKC-alpha or PKC-epsilon in mouse fibroblasts can play an important role in enhancing cell cycle progression and cell proliferation.     

The size of cagA based on repeat sequence has the responsibility of the location of Helicobacter pylori in the gastric mucus and the degree of gastric mucosal inflammation

The aim of this study was to examine whether there is a relationship between cagA size of Japanese Helicobacter pylori strains and the location of these strains in the mucous layer, the degree of gastric inflammation and acid survival. Upper gastrointestinal endoscopies were done to 144 patients with dyspeptic symptom with informed consent, sera, biopsy specimens and H. pylori strains were obtained, and gastric histology and susceptibility to pH 3 of the strains were evaluated. To determine cagA size of Japanese strains using PCR, cagA of strain CPY3401 was sequenced. 74 H. pylori samples (72 cagA+) were obtained from the body and 56 samples (56 cagA +) obtained from the antrum. cagA size of 72 H. pylori strains from the body was mainly classified into 3 groups (short (48), middle (8), long (9), and others (7)) by PCR and all of that of 56 strains from the antrum except 2 was short. The size of cagA of isolated strains from the body is associated with enhanced gastritis, acid survival, and the location in the mucus. The long size cagA of which strain is acid sensitive, may be a strong selective pressure on strain that colonizes close to the host, which enhanced gastritis.     

Pathogenicity island-dependent activation of Rho GTPases Rac1 and Cdc42 in Helicobacter pylori infection

Helicobacter pylori has been identified as the major aetiological agent in the development of chronic gastritis and duodenal ulcer, and it plays a role in the development of gastric carcinoma. Attachment of H. pylori to gastric epithelial cells leads to nuclear and cytoskeletal responses in host cells. Here, we show that Rho GTPases Rac1 and Cdc42 were activated during infection of gastric epithelial cells with either the wild-type H. pylori or the mutant strain cagA. In contrast, no activation of Rho GTPases was observed when H. pylori mutant strains (virB7 and PAI) were used that lack functional type IV secretion apparatus. We demonstrated that H. pylori-induced activation of Rac1 and Cdc42 led to the activation of p21-activated kinase 1 (PAK1) mediating nuclear responses, whereas the mutant strain PAI had no effect on PAK1 activity. Activation of Rac1, Cdc42 and PAK1 represented a very early event in colonization of gastric epithelial cells by H. pylori. Rac1 and Cdc42 were recruited to the sites of bacterial attachment and are therefore probably involved in the regulation of local and overall cytoskeleton rearrangement in host cells. Finally, actin rearrangement and epithelial cell motility in H. pylori infection depended on the presence of a functional type IV secretion system encoded by the cag pathogenicity island (PAI).     

Helicobacter pylori induces apoptosis of rat gastric parietal cells

Gastric Helicobacter pylori infection may lead to multifocal atrophic corpus gastritis associated with loss of epithelial cells as well as glandular structures. The current work investigated H. pylori effects on cell death of isolated, nontransformed rat parietal cells (PC). Highly enriched rat PC (>97%) were isolated from gastric mucosa and cultured in serum-free medium over 24 h. The cells were cocultured over 8 h with cytotoxin-associated immunodominant protein (cagA)(+)/vacuolating toxin (vacA)(+) or with cagA(-)/vacA(-) H. pylori laboratory strains and also with H. pylori mutants deleted in several genes of the cag pathogenicity island. Staphylococcus aureus or Campylobacter jejuni were used as controls. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and electron microscopy. Interleukin (IL)-8 and cytokine-induced neutrophil chemoattractant (CINC)-1 secretion was measured by ELISA. Activation of nuclear factor-kappaB (NF-kappaB) was studied in nuclear extracts of PC by electrophoretic mobility shift assay. Apoptosis of PC was induced in a concentration- and time-dependent manner by cagA(+)/vacA(+) H. pylori strains but not by cagA(-)/vacA(-) negative strains or by the cagE knockout mutant. S. aureus and C. jejuni had no effect. PC showed no IL-8 or CINC-1 secretion on exposure to cagA(+)/vacA(+) H. pylori. cagA(+)/vacA(+) strains induced activation of NF-kappaB complexes in nuclear extracts of PC, which were composed of p65 and p50 subunits. No significant stimulation of NF-kappaB activation was detected by incubation of PC with the cagE knockout mutant. Preincubation of PC with antisense but not missense oligodeoxynucleotides against the p65 subunit significantly reduced DNA binding to the kappaB recognition sequence. The p65 oligonucleotides as well as the proteasome inhibitor N-CBZ-isoleucin-glutamin-(o-t-butyl-)-alanin-leucin and the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine completely prevented PC apoptosis induced by cagA(+)/vacA(+) strains. In summary, cagE presence appears to be essential for H. pylori-induced apoptosis of gastric parietal cells, and this effect is dependent on the activation of NF-kappaB and production of nitric oxide.     

Helicobacter pylori stimulates gastric epithelial cell MMP-1 secretion via CagA-dependent and -independent ERK activation

Because the mechanisms of Helicobacter pylori-induced gastric injury are incompletely understood, we examined the hypothesis that H. pylori induces matrix metalloproteinase-1 (MMP-1) secretion, with potential to disrupt gastric stroma. We further tested the role of CagA, an H. pylori virulence factor, in MMP-1 secretion. Co-incubation of AGS cells with Tx30a, an H. pylori strain lacking the cagA virulence gene, stimulated MMP-1 secretion, confirming cagA-independent secretion. Co-incubation with strain 147C (cagA(+)) resulted in CagA translocation into AGS cells and increased MMP-1 secretion relative to Tx30a. Transfection of cells with the recombinant 147C cagA gene also induced MMP-1 secretion, indicating that CagA can independently stimulate MMP-1 secretion. Co-incubation with strain 147A, containing a cagA gene that lacks an EPIYA tyrosine phosphorylation motif, as well as transfection with 147A cagA, yielded an MMP-1 secretion intermediate between no treatment and 147C, indicating that CagA tyrosine phosphorylation regulates cellular signaling in this model system. H. pylori induced activation of the MAP kinase ERK, with CagA-independent (early) and dependent (later) components. MEK inhibitors UO126 and PD98059 inhibited both CagA-independent and -dependent MMP-1 secretion, whereas p38 inhibition enhanced MMP-1 secretion and ERK activation, suggesting p38 negative regulation of MMP-1 and ERK. These data indicate H. pylori effects on host epithelial MMP-1 expression via ERK, with p38 playing a potential regulatory role.     

幽门螺杆菌通过激活磷脂酰肌醇3-激酶信号来调节细胞迁移

目的幽门螺杆菌被认为是诱发胃癌的最强的风险因素。幽门螺旋杆菌的毒性成分是可以增加癌症危险的cag分泌系统,它可以使cagA和肽聚糖易位进入宿主细胞,进而激活信号转导通路。AKT是磷脂酰肌醇3。激酶（PI3K）的目的蛋白,并在胃癌中被激活,但PI3K-AKT和具有潜在致癌性的幽门螺旋杆菌诱导的细胞反应之间的关系尚不清楚。方法我们揭示了介导幽门螺旋杆菌刺激的AKT活化和胃上皮细胞的这些生物学结果之间的分子通路。结果幽门螺旋杆菌以Scr和表皮生长因子受体依赖性方式增加PI3K-AKT的信号,是幽门螺旋杆菌诱导的细胞迁移不可或缺的。结论这些结果表明,PI3K-AKT信号调节幽门螺旋杆菌诱发的病理生理反应,从而降低癌变门槛。     

Helicobacter pylori infection interferes with epithelial Stat6-mediated interleukin-4 signal transduction independent of cagA,cagE, or VacA

Helicobacter pylori is a bacterial pathogen evolved to chronically colonize the gastric epithelium, evade immune clearance by the host, and cause gastritis, peptic ulcers, and even gastric malignancies in some infected humans. In view of the known ability of this bacterium to manipulate gastric epithelial cell signal transduction cascades, we determined the effects of H. pylori infection on epithelial IL-4-Stat6 signal transduction. HEp-2 and MKN45 epithelial cells were infected with H. pylori strains LC11 or 8823 (type 1; cagA(+)/cagE(+)/VacA(+)), LC20 (type 2; cagA(-), cagE(-), VacA(-)), and cagA, cagE, and vacA isogenic mutants of strain 8823, with some cells receiving subsequent treatment with the Th2 cytokine IL-4, a known Stat6 activator. Immunofluorescence showed a disruption of Stat6-induced nuclear translocation by IL-4 in LC11-infected HEp-2 cells. IL-4-inducible Stat6 DNA binding in HEp-2 and MKN45 cells was abrogated by infection, but MKN45 cell viability was unaffected. A decrease in IL-4-mediated Stat6 tyrosine phosphorylation in nuclear and whole cell lysates was also observed following infection with strains LC11 and LC20, while neither strain altered IL-4 receptor chain alpha or Janus kinase 1 protein expression. Furthermore, parental strain 8823 and its isogenic cagA, cagE, and vacA mutants also suppressed IL-4-induced Stat6 tyrosine phosphorylation to comparable degrees. Thus, H. pylori did not directly activate Stat6, but blocked the IL-4-induced activation of epithelial Stat6. This may represent an evolutionarily conserved strategy to disrupt a Th2 response and evade the host immune system, allowing for successful chronic infection.     

Cyclin D1 promotes mitogen-independent cell cycle progression in hepatocytes.

Cyclin D1 is widely believed to regulate progression through G1 phase of the cell cycle, and previous studies have shown that this protein is induced during hepatocyte proliferation in culture and in vivo. In this study, the role of cyclin D1 in the cell cycle of primary rat hepatocytes was further examined. Following epidermal growth factor stimulation, cyclin D1 was upregulated at time points corresponding to the mitogen restriction point, and this was associated with enhanced cyclin D1-associated kinase activity. To test whether cyclin D1 expression was sufficient to promote mitogen-independent progression through the G1-S transition, we constructed a replication-defective adenovirus that overexpressed human cyclin D1. Transfection with the cyclin D1 vector but not a control vector resulted in hepatocyte DNA synthesis in the absence of growth factor that was similar to that seen in mitogen-treated cells. Furthermore, cyclin D1 transfection led to activation of downstream biochemical events, including cyclin A and proliferating cell nuclear antigen expression and cyclin E- and cyclin A-associated kinase activation. These results suggest that cyclin D1 expression is sufficient to promote progression of hepatocytes through the G1 restriction point.     

Phosphorylation of CREB, a cyclic AMP responsive element binding protein, contributes partially to lysophosphatidic acid-induced fibroblast cell proliferation

Lysophospholipids regulate a wide array of biological processes including cell survival and proliferation. In our previous studies, we found that in addition to SRE, CRE is required for maximal c-fos promoter activation triggered by lysophosphatidic acid (LPA). c-fos is an early indicator of various cells into the cell cycle after mitogenic stimulation. However, role of CREB activation in LPA-stimulated proliferation has not been elucidated yet. Here, we investigate how LPA induces proliferation in Rat-2 fibroblast cell via CREB activation. We found that total cell number and BrdU-positive cells were increased by LPA. Moreover, levels of c-fos mRNA and cyclin D1 protein were increased via LPA-induced CREB phosphorylation. Furthermore, LPA-induced Rat-2 cell proliferation was decreased markedly by ERK inhibitor (U0126) and partially by MSK inhibitor (H89). Taken together, these results suggest that CREB activation could partially up-regulate accumulation of cyclin D1 protein level and proliferation of LPA-stimulated Rat-2 fibroblast cells.     

Cyclin A overexpression induces chromosomal double-strand breaks in mammalian cells

Cyclin A is a major regulator in vertebrate cell cycle, associated with cyclin-dependent kinase (Cdk), and involved in S-phase progression and entry into mitosis. It has been known that cyclin A overexpression not only causes premature S-phase entry but also induces prolongation of S phase. Here we show that ectopic expression of cyclin A leads to extensive γ?H2AX focus formation, which is indicative of DNA double-strand breaks. Likewise, cyclin E, but not cyclin B1 and cyclin D1, also induced the γ?H2AX focus formation, suggesting that these DNA lesions may be induced via aberrant DNA replication process. Moreover, the γ?H2AX focus formation was suppressed by co-expressing p21Cip1/Waf1 or dominant-negative Cdk2 mutant, suggesting that aberrant cyclin A-Cdk2 activation induces the chromosomal double-strand breaks.     

CCL21/CCR7 promotes G2/M phase progression via the ERK pathway in human non-small cell lung cancer cells

C-C chemokine receptor 7 (CCR7) contributes to the survival of certain cancer cell lines, but its role in the proliferation of human non-small cell lung cancer (NSCLC) cells remains vague. Proliferation assays performed on A549 and H460 NSCLC cells using Cell Counting Kit-8 indicated that activation of CCR7 by its specific ligand, exogenous chemokine ligand 21 (CCL21), was associated with a significant linear increase in cell proliferation with duration of exposure to CCL21. The CCL21/CCR7 interaction significantly increased the fraction of cells in the G(2)/M phase of the cell cycle as measured by flow cytometry. In contrast, CCL21/CCR7 had no significant influence on the G(0)/G(1) and S phases. Western blot and real-time PCR indicated that CCL21/CCR7 significantly upregulated expression of cyclin A, cyclin B1, and cyclin-dependent kinase 1 (CDK1), which are related to the G(2)/M phase transition. The expression of cyclin D1 and cyclin E, which are related to the G(0)/G(1) and G(1)/S transitions, was not altered. The CCL21/CCR7 interaction significantly enhanced phosphorylation of extracellular signal-regulated kinase (P-ERK) but not Akt, as measured by Western blot. LY294002, a selective inhibitor of PI3K that prevents activation of the downstream Akt, did not weaken the effect of CCL21/CCR7 on P-ERK. Coimmunoprecipitation further confirmed that there was an interaction between P-ERK and cyclin A, cyclin B1, or CDK1, particularly in the presence of CCL21. CCR7 small interfering RNA or PD98059, a selective inhibitor of MEK that disrupts the activation of downstream ERK, significantly abolished the effects of exogenous CCL21. These results suggest that CCL21/CCR7 contributes to the time-dependent proliferation of human NSCLC cells by upregulating cyclin A, cyclin B1, and CDK1 potentially via the ERK pathway.