Helicobacter Pylori Cytotoxin‑Associated Gene a Activates Tumor
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MOLECULAR MEDICINE REPORTS 12: 6337-6345, 2015 Helicobacter pylori cytotoxin‑associated gene A activates tumor necrosis factor‑α and interleukin‑6 in gastric epithelial cells through P300/CBP‑associated factor‑mediated nuclear factor‑κB p65 acetylation QIONG LIN, HUI XU, XINTAO CHEN, GUORONG TANG, LAN GU and YEHONG WANG Department of Gastroenterology, Wuxi Children's Hospital Affiliated to Nanjing Medical University, Wuxi, Jiangsu 214023, P.R. China Received October 19, 2014; Accepted July 2, 2015 DOI: 10.3892/mmr.2015.4143 Abstract. Helicobacter pylori-initiated chronic gastritis Korea being higher than those in developed countries (1‑5). is characterized by the cytotoxin-associated gene (Cag) Helicobacter pylori colonizes the stomach and duodenum pathogenicity island‑dependent upregulation of pro‑inflam- areas, causing chronic inflammation of the gastric mucosa, matory cytokines in gastric epithelial cells, which is largely and the development of stomach ulcers and neoplasms mediated by the activation of nuclear factor (NF)‑κB as a (gastric cancer and mucosa‑associated lymphoid tissue) (6,7). transcription factor. However, the precise regulation of NF‑κB However, the mechanism by which Helicobacter pylori activation, particularly post‑translational modifications in the infection causes pathological changes of the gastric mucosa CagA‑induced inflammatory response, has remained elusive. remains to be fully elucidated. The present study showed that Helicobacter pylori CagA, Inflammatory responses have been known to have a key an important virulence factor, induced the expression of role in the pathogenesis of Helicobacter pylori infection P300/CBP‑associated factor (PCAF) in gastric epithelial cells. and contribute to chronic gastritis as well as gastric and Further study revealed that PCAF was able to physically asso- duodenal ulcers in the patients (8‑10). A growing body of ciate with the NF‑κB p65 sub-unit and enhance its acetylation. evidence has demonstrated that elevated levels of inflam- More importantly, PCAF‑induced p65 acetylation was shown matory factors, including tumor necrosis factor (TNF)‑α, to contribute to p65 phosphorylation and further upregulation interferon-γ, interleukin (IL)‑6, IL‑8 and IL‑32 were associ- of tumor necrosis factor (TNF)‑α and interleukin (IL)‑6 in ated with more serious pathogenesis of patients infected with gastric adenocarcinoma cells. In conclusion, the results of Helicobacter pylori (10‑15). However, the underlying regula- the present study indicated that Helicobacter pylori CagA tory mechanisms of the production of inflammatory factors enhanced TNF‑α and IL‑6 in gastric adenocarcinoma cells have largely remained elusive. through PCAF‑mediated NF‑κB p65 sub-unit acetylation. Nuclear factor (NF)‑κB has an important role in the regulation of inflammatory responses in mammals (16,17). Introduction It has been demonstrated that Helicobacter pylori infection activates NF‑κB and its target genes, particularly inflam- Infection with Helicobacter pylori is one of the most common matory factors in epithelial cells, which is thought to be types of bacterial infection. Helicobacter pylori infection critical for Helicobacter pylori‑initiated chronic inflam- mainly occurs in economically underdeveloped regions, with mation (9,18‑21). The virulence factor cytotoxin‑associated the infection rates of pediatric patients in China, Japan and gene A (CagA) encoded by the Helicobacter pylori Cag����������������� patho- genicity island, has an important role in the pathogenicity of Helicobacter pylori, including Helicobacter pylori-induced activation of NF‑κB and expression of NF‑κB target genes (22,23). However, the exact function of CagA in the Correspondence to: Dr Yehong Wang, Department of activation of NF‑κB and the NF‑κB‑dependent inflammatory Gastroenterology, Wuxi Children's Hospital Affiliated to Nanjing response has not been well characterized. Medical University, 299 Qingyang Road, Wuxi, Jiangsu 214023, P. R. Ch i na Emerging evidence indicated that post‑translational modi- E‑mail: [email protected] fication, such as acetylation, has important roles in various biological events, including inflammation (24‑26). It has been Key words: Helicobacter pylori, cytotoxin-associated gene A, reported that the acetylation of the NF‑κB p65 sub-unit has inflammation, nuclear factor‑κB p65, P300/CBP-associated factor, an important role in its activation (27‑29). In addition, recent acetylation studies showed that P300/CBP‑associated factor (PCAF), also frequently referred to as lysine (K) acetyltransferase 2B, is a transcription co‑activator that contains several nuclear 6338 LIN et al: NF‑κB p65 SUBUNIT ACETYLATION AND Helicobacter pylori INFECTION receptor‑interacting domains and can function as an acetyl Construction of overexpression plasmids. The open transferase (30‑33). PCAF has also been reported to be associ- reading frame of human PCAF mRNA (National Center ated with infection and inflammation (34‑37). Therefore, there of Biotechnology Information reference sequence, is a requirement to explore the roles of PCAF in mediating p65 NM_003884.4) containing a HA tag was amplified by poly- activation and the production of TNF‑α and IL-6 in gastric merase chain reaction (PCR) from cDNA of human AGS adenocarcinoma cells. cells. The PCR products and pcDNA3.1 vector were further The present study aimed to investigate the implication of digested with the two restriction enzymes of HindIII and XhoI, Helicobacter pylori CagA in the inflammatory response of and then ligated with each other by using T4 DNA ligase. The human gastric adenocarcinoma AGS cells in vitro, as well as recombinant plasmids were amplified in the Escherichia coli PCAF‑mediated p65 acetylation and its role in regulating the strain DH5α at 37˚C for 16 h. Subsequently, the plasmids production of TNF‑α and IL‑6 as two important inflammatory were extracted by QIAprep spin miniprep kit according to factors in AGS cells. the manufacturer's instructions. Finally, the constructed plas- mids (pcDNA3.1/PCAF‑HA) were sequenced to confirm the Materials and methods nucleotide sequence by GenScript (Nanjing, China). Reagents. Monoclonal rabbit antibodies against human total Construction of small hairpin (sh)RNA expression plasmids. p65 (cat. no. 8242), phospho (p‑)p65 (cat. no. 3031), as well Various shRNA sequences targeting human PCAF mRNA as monoclonal mouse antibodies against acetylated‑Lysine (NM_003884.4) were designed to silence the PCAF gene in (cat. no. 9681) and human β‑actin (cat. no. 3700) were from human AGS cells. The various DNA segments for the expres- Cell Signaling Technology (Danvers, MA, USA). Mouse mono- sion of PCAF shRNA were synthesized and inserted into the clonal antibodies against human PCAF (cat. no. sc‑13124) and shRNA pGCsi‑U6/neo/GFP plasmids by Shanghai Genkan Hemagglutinin (HA)‑tag (cat. no. sc‑7392) were purchased from Biotechnology Co., Ltd. The most effective shRNA expres- Santa Cruz Biotechnology (Dallas, TX, USA). For western blot sion plasmid to silence human PCAF gene was selected to be analysis, horseradish peroxidase‑conjugated anti‑mouse immu- used in subsequent experiments. The PCAF shRNA sequence noglobulin G (IgG) antibody (cat. no. 7076) and anti-rabbit IgG was as follows: AAG ATG GCC GTG TTA TTG GTG and the antibody (cat. no. 7074) were purchased from Cell Signaling Scrambled shRNA sequence was as follows: AAT GAC GGG Technology. Enhanced chemiluminescence (ECL) Western CTT GTT ATG GGT. Blotting Substrate, co‑immunoprecipitation (co‑IP) assay buffer, radioimmunoprecipitation assay (RIPA) lysis buffer, a bicincho- Cellular transfection. Plasmids were transfected into AGS ninic acid (BCA) protein assay kit and protein G‑Sepharose cells by using Lipofectamine 2000 according to the manu- beads were purchased from Thermo Fisher Scientific (Waltham, facturer's instructions. For transfection, 4 µg plasmid was MA, USA). Polyvinylidene difluoride (PVDF) membranes were mixed with 250 µl serum‑free RPMI‑1640. At the same time, obtained from Roche (Basel, Switzerland). TRIzol reagent was 10 µl Lipofectamine 2000 was mixed with 250 µl serum‑free purchased from Invitrogen Life Technologies (Carlsbad, CA, RPMI‑1640. The plasmids and Lipofectamine 2000 were USA). Moloney Murine Leukemia Virus reverse transcrip- further incubated with each other for 20 min at room tempera- tase and oligo (dT) 15 primer were purchased from Promega ture. Finally, the 500‑µl mixture was added into each well in (Madison, WI, USA). TaqMan® Fast Advanced Master Mix a 6‑well plate containing 4x105 AGS cells. The medium was was from Applied Biosystems (Thermo Fisher Scientific). replaced with serum containing RPMI‑1640 at 5 h after trans- The plasmids pGCsi‑U6/neo/green fluorescence protein (GFP) fection, and the cells were incubated sequentially. were obtained from Shanghai Genkan Biotechnology Co., Ltd (Shanghai, China). The pcDNA3.1 vector was purchased from Production of the CagA protein. Helicobacter pylori Invitrogen Life Technologies. The incision enzymes HindIII CagA‑His‑tag was constructed in the pET21a plasmid from and XhoI as well as T4 DNA ligase were purchased from Takara Novagen (Madison, WI, USA). The plasmid was then trans- Bio Inc. (Tokyo, Japan). The Escherichia coli strain DH5α formed into BL21(DE3) Singles™ Competent Cells (Novagen) was purchased from Molecular Cloning Laboratories (San for protein expression and purification according to the manu- Francisco, CA, USA). QIAprep spin miniprep kit was obtained facturer's instructions.