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Helicobacter pylori VacA, acting through receptor protein tyrosinephosphatase , is crucial for CagA phosphorylation in humanduodenum carcinoma cell line AZ-521Masayuki Nakano1,2,*, Kinnosuke Yahiro3, Eiki Yamasaki4, Hisao Kurazono4, Junko Akada5, Yoshio Yamaoka5,6,Takuro Niidome7, Masanori Hatakeyama8, Hidekazu Suzuki9, Taro Yamamoto2, Joel Moss10, Hajime Isomoto11
and Toshiya Hirayama1
ABSTRACTHelicobacter pylori, a major cause of gastroduodenal diseases,produces vacuolating cytotoxin (VacA) and cytotoxin-associatedgene A (CagA), which seem to be involved in virulence. VacAexhibits pleiotropic actions in gastroduodenal disorders via its specificreceptors. Recently, we found that VacA induced the phosphorylationof cellular Src kinase (Src) at Tyr418 in AZ-521 cells. Silencing ofreceptor protein tyrosine phosphatase (RPTP), a VacA receptor,reduced VacA-induced Src phosphorylation. Src is responsible fortyrosine phosphorylation of CagA at its Glu-Pro-Ile-Tyr-Ala (EPIYA)variant C (EPIYA-C) motif in Helicobacter pylori-infected gastricepithelial cells, resulting in binding of CagA to SHP-2 phosphatase.Challenging AZ-521 cells with wild-type H. pylori inducedphosphorylation of CagA, but this did not occur when challengedwith a vacA gene-disrupted mutant strain. CagA phosphorylation wasobserved in cells infected with a vacA gene-disrupted mutant strainafter addition of purified VacA, suggesting that VacA is required for H.pylori-induced CagA phosphorylation. Following siRNA-mediatedRPTP knockdown in AZ-521 cells, infection with wild-type H. pyloriand treatment with VacA did not induce CagA phosphorylation. Takentogether, these results support our conclusion that VacA mediatesCagA phosphorylation through RPTP in AZ-521 cells. These dataindicate the possibility that Src phosphorylation induced by VacA is
mediated through RPTP, resulting in activation of Src, leading toCagA phosphorylation at Tyr972 in AZ-521 cells.
KEY WORDS: Helicobacter pylori, VacA, CagA
INTRODUCTIONHelicobacter pylori is a major causative agent for the developmentof gastroduodenal diseases, including chronic gastritis, peptic ulcerand gastric cancers (Blaser and Atherton, 2004; Peek and Blaser,2002). It has been proposed that about 50% of the worldspopulation is infected with H. pylori, but only a small number ofthe infected individuals develop severe clinical manifestations suchas gastric adenocarcinoma (Wroblewski et al., 2010). Although anumber of virulence factors have been found in H. pylori,vacuolating cytotoxin (VacA) and cytotoxin-associated gene A(CagA) are considered to be the major factors in H. pylori-induceddiseases (Blaser and Atherton, 2004; Peek and Blaser, 2002).
VacA is a potent cytotoxin secreted by most clinical isolates ofH. pylori, and shows pleiotropic actions in cultured gastric epithelialcells, including generation of vacuoles in the cytoplasm,mitochondrial damage leading to apoptosis, and modulation ofsignal transduction pathways associated with immune responses(Boncristiano et al., 2003; Hisatsune et al., 2007; Isomoto et al.,2010; Nakayama et al., 2004, 2009; Yamasaki et al., 2006). Tofacilitate their biological actions in host cells, VacA binds to specificsurface receptors. We have identified three different cell surfaceproteins as VacA receptors: receptor protein tyrosine phosphatase and (RPTP and RPTP) and low-density lipoprotein receptor-related protein-1 (LRP1) (Yahiro et al., 1999, 2003, 2012). Inaddition, other molecules, including sphingomyelin, have beenreported to serve as VacA receptors (Gupta et al., 2010). Of theseVacA receptors, during H. pylori infection, RPTP is associatedwith the development of gastric ulcers in experimental animalmodels and LRP1 is involved in VacA-dependent autophagy,followed by CagA degradation in infected host cells (Fujikawa et al.,2003; Tsugawa et al., 2012; Yahiro et al., 2012). These data suggestthat both receptors are involved in intoxication by VacA. Therefore,we speculate that both receptors, RPTP and LRP1, are associatedwith the development of gastric disorders in H. pylori infection.However, the role of RPTP in intoxication with VacA is unclear.
Previous studies have shown that RPTP contributes to activationof cellular Src kinase (Src) and other Src family kinase (Su et al.,1999). It has been shown that Src activity is elevated in RPTP-overexpressing cultured cells, whereas the opposite was observed inRPTP-deficient cells (den Hertog et al., 1993; Harder et al., 1998;Su et al., 1999; Zeng et al., 2003; Zheng et al., 1992). Furthermore,it has been reported that Src kinase activity is reduced in RPTP-Received 11 March 2016; Accepted 11 October 2016
1Department of Bacteriology, Institute of Tropical Medicine, Nagasaki University,1-12-4, Sakamoto, Nagasaki 852-8523, Japan. 2Department of International Health,Institute of Tropical Medicine, Nagasaki University, 1-12-4, Sakamoto, Nagasaki852-8523, Japan. 3Department of Molecular Infectiology, Graduate School ofMedicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan.4Division of Food Hygiene, Department of Animal and Food Hygiene, ObihiroUniversity of Agriculture and Veterinary Medicine, Nishi 2-11, Inada-cho, Obihiro,Hokkaido 080-8555, Japan. 5Department of Environmental and PreventiveMedicine, Oita University Faculty of Medicine, Idaigaoka 1-1, Yufu, Oita 879-5593,Japan. 6Department of Medicine, Gastroenterology and Hepatology Section,Baylor College of Medicine, Houston, TX 77030, USA. 7Department of AppliedChemistry and Biochemistry, Graduate School of Science and Technology,Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 860-8555, Japan.8Division of Microbiology, Graduate School of Medicine, The University of Tokyo,7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-0033, Japan. 9Medical Education Center,Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo160-8582, Japan. 10Cardiovascular and Pulmonary Branch, NHLBI, NationalInstitutes of Health, Bethesda, MD 20892-1590, USA. 11Division of Medicine andClinical Science, Tottori University Faculty of Medicine, 86 Nishi-cho, Yonago,Tottori 683-8503, Japan.
*Author for correspondence (email@example.com)
This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.
2016. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2016) 9, 1473-1481 doi:10.1242/dmm.025361
knockout mice (Harder et al., 1998; Ponniah et al., 1999). Therefore,RPTP is an important physiological regulator of Src. RPTP candephosphorylate both phosphorylated tyrosine residues, pTyr530and pTyr418 (human Src numbering throughout; the inhibitoryphosphorylation site and active site of Src, respectively), therebycausing Src activation following autophosphorylation of Tyr418(Boggon and Eck, 2004; Vacaru and den Hertog, 2010; Zheng et al.,2000). In addition, based on immunohistochemistry using humangastric cancer tissues, it has been suggested that RPTP isassociated with the progression of gastric cancer (Wu et al., 2006).In the present study, we show the role of RPTP in VacA
intoxication and also demonstrate that VacA is associated withCagA phosphorylation in AZ-521 cells during H. pylori infection.We propose the possibility that VacA induces CagAphosphorylation through RPTP in AZ-521 cells.
RESULTSVacA induces Src phosphorylation in vitroPrevious studies have shown that VacA modulates signaltransduction pathways in AZ-521 cells (Hisatsune et al., 2007;Nakayama et al., 2009). In this study, we found that VacA enhancedphosphorylation at Tyr418 in Src, but heat-inactivated VacA(iVacA) did not have similar effects (Fig. 1A). We also examinedthe effect of VacA on Src phosphorylation at Tyr418 using othergastric epithelial cells: AGS cells (a human gastric adenocarcinomacell line) and NUGC3 cells (a gastric cancer cell line). AlthoughVacA stimulated Src phosphorylation at Tyr418 in NUGC3 cells aswell as in AZ-521 cells, we did not find any effects of VacA in AGScells (Fig. 1B), suggesting that Src phosphorylation induced byVacA is dependent on cell type.
VacA-induced Src phosphorylation is mediated by RPTPVacA intoxicates host cells through specific surface receptors. Wepreviously identified three molecules (RPTP, RPTP and LRP1)as VacA receptors (Yahiro et al., 1999, 2003, 2012). To evaluatewhich of these VacA receptors contributes to Src phosphorylation,we used small-interfering RNA (siRNA)-mediated knockdowns,which targeted each of the VacA receptors, in AZ-521 cells.Although VacA induced phosphorylation at Tyr418 of Src in thepresence of control siRNA, VacA did not induce phosphorylation atTyr418 of Src in siRNA-mediated RPTP knockdownAZ-521 cells(Fig. 2A). On the other hand, in AZ-521 cells, VacA enhancedphosphorylation at Tyr418 in Src after RPTP or LRP1 silencing(Fig. 2B,C). To verify these results, we also examined Srcphosphorylation induced by VacA using the RPTP constitutive-knockdown AZ-521 cells constructed by a shRNA lentiviralexpression system (Yahiro et al., 2012). We found that VacA didnot enhance phosphorylation at Tyr418 in Src in RPTPconstitutive-knockdown AZ-521 cells (Fig. 3), consistent with theresults using the siRNA-mediated RPTP knockdown AZ-521 cells(Fig. 2A). Taken together, we speculate that RPTP, but not RPTPor LRP1, is involved in VacA-dependent phosphorylation at Tyr418in Src.
VacA production by H. pylori induces CagA phosphorylationand