Helicobacter-induced Intestinal Metaplasia in the Stomach Correlates with Elk-1 and Serum Response Factor Induction of Villin

Chronic Helicobacter pylori infection results in serious sequelae, including atrophy, intestinal metaplasia, and gastric cancer. Intestinal metaplasia in the stomach is defined by the presence of intestine-like cells expressing enterocyte-specific markers, such as villin. In this study, we demonstrate that villin is expressed in intestine-like cells that develop after chronic infection with H. pylori in both human stomach and in a mouse model. Transfection studies were used to identify specific regions of the villin promoter that are inducible by exposure of the cells to H. pylori. We demonstrated that induction of the villin promoter by H. pylori in a human gastric adenocarcinoma cell line (AGS) required activation of the Erk pathway. Elk-1 and the serum response factor (SRF) are downstream transcriptional targets of the Erk pathway. We observed inducible binding of Elk-1 and the SRF after 3 and 24 h of treatment with H. pylori, suggesting that the bacteria alone are sufficient to initiate a cascade of signaling events responsible for villin expression. Thus, H. pylori induction of villin in the stomach correlates with activation and cooperative binding of Elk-1 and the SRF to the proximal promoter of villin. Chronic Helicobacter pylori infection results in serious sequelae, including atrophy, intestinal metaplasia, and gastric cancer. Intestinal metaplasia in the stomach is defined by the presence of intestine-like cells expressing enterocyte-specific markers, such as villin. In this study, we demonstrate that villin is expressed in intestine-like cells that develop after chronic infection with H. pylori in both human stomach and in a mouse model. Transfection studies were used to identify specific regions of the villin promoter that are inducible by exposure of the cells to H. pylori. We demonstrated that induction of the villin promoter by H. pylori in a human gastric adenocarcinoma cell line (AGS) required activation of the Erk pathway. Elk-1 and the serum response factor (SRF) are downstream transcriptional targets of the Erk pathway. We observed inducible binding of Elk-1 and the SRF after 3 and 24 h of treatment with H. pylori, suggesting that the bacteria alone are sufficient to initiate a cascade of signaling events responsible for villin expression. Thus, H. pylori induction of villin in the stomach correlates with activation and cooperative binding of Elk-1 and the SRF to the proximal promoter of villin. Chronic inflammation of the gastric mucosa (chronic gastritis) develops in response to Helicobacter pylori infection or bacterial overgrowth in the hypochlorhydric stomach (1Blaser M.J. Parsonnet J. J. Clin. Investig. 1994; 94: 4-8Crossref PubMed Scopus (361) Google Scholar, 2Houben G.M. Stockbrugger R.W. Scand. J. Gastroenterol. Suppl. 1995; 212: 13-18Crossref PubMed Scopus (48) Google Scholar). Over time, the inflammatory process progresses, and alteration of the epithelial cell population occurs, which includes gradual loss of parietal cells coinciding with an increase in the number of mucous cells. Proliferation of mucous cell types with evidence of an intestinal phenotype (intestinal metaplasia) is a major precursor lesion in gastric cancer (3Correa P. Cancer Res. 1992; 52: 6735-6740PubMed Google Scholar). Moreover, intestinal metaplasia is a lesion that develops in a variety of cancers derived from organs of the forestomach (4Hampson S.J. Falzon M. Cowie A.G. Br. J. Urol. 1992; 69: 323-324Crossref PubMed Scopus (4) Google Scholar, 5Albores-Saavedra J. Nadji M. Henson D.E. Angeles-Angeles A. Pathol. Res. Pract. 1988; 183: 169-175Crossref PubMed Scopus (37) Google Scholar, 6Malhotra S.L. Med. Hypotheses. 1976; 2: 279-281Crossref PubMed Scopus (13) Google Scholar). Interestingly, intestinal metaplasia in different organs all express protein markers commonly found in normal intestinal enterocytes, e.g. TFF3, Cdx2, villin (7Moll R. Robine S. Dudouet B. Louvard D. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 1987; 54: 155-169Crossref PubMed Scopus (87) Google Scholar, 8Robine S. Huet C. Moll R. Sahuquillo-Merino C. Coudrier E. Zweibaum A. Louvard D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8488-8492Crossref PubMed Scopus (149) Google Scholar, 9Mizoshita T. Tsukamoto T. Nakanishi H. Inada K. Ogasawara N. Joh T. Itoh M. Yamamura Y. Tatematsu M. J. Cancer Res. Clin. Oncol. 2003; 129: 727-734Crossref PubMed Scopus (126) Google Scholar, 10Tsukamoto T. Inada K. Tanaka H. Mizoshita T. Mihara M. Ushijima T. Yamamura Y. Nakamura S. Tatematsu M. J. Cancer Res. Clin. Oncol. 2004; 130: 135-145Crossref PubMed Scopus (130) Google Scholar). The gastric epithelium develops from intestinal endoderm by 16 days postcoitum when the pyloric border is formed. At that time, fetal intestinal markers cease to be expressed in the gastric epithelium. Re-expression of intestine-specific genes in the adult stomach represents a shift to a metaplastic phenotype that correlates with increased gastric proliferation. Villin is expressed in primitive endoderm by embryonic day 5 and coalesces at the apical surface by day 8.5 postcoitum as the microvilli of the gut are being formed (11Ezzell R.M. Chafel M.M. Matsudaira P.T. Development (Camb.). 1989; 106: 407-419Crossref PubMed Google Scholar, 12Landry C. Huet C. Mangeat P. Sahuquet A. Louvard D. Crine P. Differentiation. 1994; 56: 55-65Crossref PubMed Scopus (14) Google Scholar, 13Maunoury R. Robine S. Pringault E. Leonard N. Gaillard J.A. Louvard D. Development (Camb.). 1992; 115: 717-728Crossref PubMed Google Scholar). By 16 days postcoitum, there is a one-cell distinction between villin expression in the duodenum and minimal expression in the antral glands (14Braunstein E.M. Qiao X.T. Madison B. Pinson K. Dunbar L. Gumucio D.L. Dev. Dyn. 2002; 224: 90-102Crossref PubMed Scopus (44) Google Scholar). By postnatal day 1, villin expression in the stomach has completely receded. Thus, there is a complete absence of villin expression in the corpus of the mouse stomach (15Pinto D. Robine S. Jaisser F. El Marjou F.E. Louvard D. J. Biol. Chem. 1999; 274: 6476-6482Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Villin is a structural protein regulated by increased intracellular calcium that in turn binds to actin and contributes to the formation of microvilli in the small bowel (16Friederich E. Pringault E. Arpin M. Louvard D. BioEssays. 1990; 12: 403-408Crossref PubMed Scopus (71) Google Scholar). Mice deficient for the villin gene are viable (17Pinson K.I. Dunbar L. Samuelson L. Gumucio D.L. Dev. Dyn. 1998; 211: 109-121Crossref PubMed Scopus (81) Google Scholar). Microvilli develop but do not respond normally to calcium-dependent signals, implicating the protein in the response to cell injury (17Pinson K.I. Dunbar L. Samuelson L. Gumucio D.L. Dev. Dyn. 1998; 211: 109-121Crossref PubMed Scopus (81) Google Scholar, 18Ferrary E. Cohen-Tannoudji M. Pehau-Arnaudet G. Lapillonne A. Athman R. Ruiz T. Boulouha L. Marjou ElF. Doye A. Fontaine J.J. Antony C. Babinet C. Louvard D. Jaisser F. Robine S. J. Cell Biol. 1999; 146: 819-830Crossref PubMed Scopus (129) Google Scholar). In addition, villin is expressed in intestinal metaplasia observed in Barrett's esophagus and in chronic atrophic gastritis (19MacLennan A.J. Orringer M.B. Beer D.G. Mol. Carcinog. 1999; 24: 137-143Crossref PubMed Scopus (24) Google Scholar). Therefore villin is an important marker of the pre-neoplastic cell type that forms in the gut in response to chronic injury (20Regalado S.P. Nambu Y. Iannettoni M.D. Orringer M.B. Beer D.G. Mol. Carcinog. 1998; 22: 182-189Crossref PubMed Scopus (40) Google Scholar). Whether true villin-positive intestinal metaplasia is a feature of the altered pattern of differentiation observed with Helicobacter colonization and inflammation has not been examined. In this report, we established that villin expression emerges in the infected stomachs of human subjects and a mouse model of Helicobacter infection. Further, we showed that the first 554 bp of the villin promoter contain elements capable of responding to H. pylori in culture. Within this proximal promoter region, we found that a serum response element (SRE) 1The abbreviations used are: SRE, serum response element; SRF, serum response factor; EBS, Ets binding site; YY1, YingYang1; DAPI, 4′,6-diamidino-2-phenylindole; m.o.i., multiplicity of infection. confers inducible regulation of the villin promoter by H. pylori and that Elk-1 and the serum response factor (SRF) form a ternary complex at this element. Bacteria—The mouse-adapted H. pylori SS1 strain was used to inoculate mice for up to 14 months and was a gift from Dr. K. Eaton, University of Michigan. The H. pylori J99 strain (American Type Culture Collection 700824) is a human isolate that was used in the cell culture experiments because of its ability to strongly induce human cell lines. The bacteria were cultivated on blood agar plates containing Campylobacter base agar (Difco) supplemented with 5% horse blood (Colorado Serum, Denver, CO), 5 μg/ml vancomycin, 10 μg/ml trimethoprim lactate, and 2 μg/ml nystatin (Sigma). The plates were incubated under microaerophilic conditions (CampyPak Plus, BBL; BD Biosciences) at 37 °C for 1-2 days. Human Tissue—Institutional Review Board approval from the University of Michigan was obtained prior to the acquisition of human tissue by Dr. Nguyen T. Vinh and Dr. Nguyen N. Thanh during endoscopy at Friendship Hospital and Tran Hung Dao Central Hospital, Hanoi, Vietnam. The biopsies were fixed in formalin and then paraffin-embedded. Plasmids—The -554 Villin P/Intron reporter construct was prepared as described previously (21Madison B.B. Dunbar L. Qiao X.T. Braunstein K. Braunstein E. Gumucio D.L. J. Biol. Chem. 2002; 277: 33275-33283Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar) and consists of 554 bp of the mouse 5′-flanking sequence, the first exon, and the entire first intron. Dominant negative Erk-1 and Erk-2 constructs were gifts from Dr. Melanie Cobb (University of Texas Southwestern, Dallas, TX) (22Frost J.A. Geppert T.D. Cobb M.H. Feramisco J.R. Proc. Natl. Acad. Sci., U. S. A. 1994; 91: 3844-3848Crossref PubMed Scopus (203) Google Scholar). Mutagenesis of the Ets and SRE sites within the villin reporter construct was accomplished using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol using the following primer: 5′-GGGCCCTATCTAACCTTATAAAGTGAAG-GAAGTAACT-3′. Deletion of the SRE/EBS binding site was created the same way using the following QuikChange primer: 5′-TCTGGGGCCCTATCTGGGGGGGGGGT-GGTGGTGAGGACC-3′. All constructs were confirmed by sequence analysis. The dominant negative Elk construct in was a gift from Dr. E. The Elk-1 sequence a the expressed protein H. M. C. C. Cobb M.H. J. 1995; PubMed Scopus Google Scholar, H. M. T. Mol. Biol. PubMed Scopus Google Scholar). and Helicobacter mice were from and in for up to 14 days all mice were for 3 days with in to the bacterial SS1 bacteria were in the was according to the at and a containing viable bacteria was used for infection. was with bacteria 5 days. All mice were with to analysis. The protocol used was by the University of Michigan and which an of and of by H. pylori was by for a of containing 10 of of of of and of at and as as by of the H. 16 according to P. C. Med. PubMed Scopus Google Scholar). of the human biopsies or mouse stomach were with and for the of inflammation and The presence of metaplasia was confirmed by a tissue of H. pylori and mice were were used for for 10 at and with villin expression in the stomach mouse the of the mouse and were in a of and and then incubated with the at for the in were using Texas from and from in a of at for was used to the were with an The cells were with an and with the H. pylori with Human Cell cells were to in supplemented with 5% fetal 5% horse μg/ml and μg/ml in 5% The cells were then for h in supplemented with μg/ml and μg/ml the cell line a variety of gastric J.R. M. P. D. J. PubMed Scopus (48) Google Scholar). regulation of villin protein was in cells using of of Campylobacter or J99 H. pylori for 24 to the of all and were using cells. and cells were to in as described The cells were on plates and using calcium or according to the manufacturer's after the cells were with and the with containing H. pylori J99 strain at Dominant negative expression constructs were and were h of the cells. were h after using a or was to the protein by the M.M. 1976; PubMed Scopus Google Scholar). cell were in 10 2 2 10 μg/ml μg/ml and The were in and on a The was for h with 5% or with 5% serum in for of The was to for 2 in was for The were by Erk-2 was from and were from protein were prepared by a E. P. M.M. Res. 1989; PubMed Scopus Google Scholar) supplemented with to The were in and on a The were a The was with for h in 5% in and then incubated with for 2 days at with the was incubated with a at for h at were with the and kit the cells were with the bacteria for 3 or 24 were then with and on cells were for h but H. pylori. protein were prepared by a E. P. M.M. Res. 1989; PubMed Scopus Google Scholar). was and then with using shift were at °C in a of containing 10 5 of of and the or the of the a of was to by the containing were by 2 of or the The were on a containing and The were and then used to results were by or of as using was Chronic Helicobacter infection in both human subjects and mice atrophy, and In human intestinal metaplasia is with the presence of cells and an increase in intestine-specific markers, e.g. villin S.J. A. R. Cell Res. 1994; PubMed Scopus Google Scholar). The metaplasia prior to the of gastric cancer (3Correa P. Cancer Res. 1992; 52: 6735-6740PubMed Google Scholar). the expression of villin as a marker for intestinal metaplasia in response to Helicobacter infection has not been we biopsies from human subjects infected with H. pylori. The gastric mucosa of infected showed cells with the presence of and cells we found that of intestinal metaplasia expressed villin protein villin with intestinal metaplastic in a H. mice were infected for 14 months with the mouse-adapted SS1 H. pylori we showed that the villin-positive cells were in the corpus from the same mouse was used as a for the villin normal corpus not express villin (14Braunstein E.M. Qiao X.T. Madison B. Pinson K. Dunbar L. Gumucio D.L. Dev. Dyn. 2002; 224: 90-102Crossref PubMed Scopus (44) Google this of the stomach was used as a negative for the The parietal cells were using an to the of the with the there was a in the number of parietal cells in the H. with an increase in villin-positive cells We that there was an increase in villin expression with intestinal metaplastic in both human and mouse H. of villin in mouse stomach after H. pylori infection. from a H. pylori was used to villin and a Texas was used to the of was used to of the mouse small used as a for villin villin expression was in the corpus of a is a of the corpus glands with parietal cells the corpus of a infected mouse increased villin expression and a in number of parietal cells of and H. villin expression with the expression of intestinal metaplasia in both human and mouse during chronic atrophic we villin was regulated by H. pylori in a human gastric cell The cell line gastric e.g. J.R. M. P. D. J. PubMed Scopus (48) Google Scholar). We found that cells express villin cells were with H. pylori and an not to induce C. The results demonstrated that villin protein increased with the J99 H. pylori strain but not with C. or the mouse-adapted SS1 the cells the cell line was used in the studies demonstrated that there are elements in the villin promoter between and -554 and between -554 and (21Madison B.B. Dunbar L. Qiao X.T. Braunstein K. Braunstein E. Gumucio D.L. J. Biol. Chem. 2002; 277: 33275-33283Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar). In addition, the first with the first 554 bp of the 5′-flanking We used the reporter construct containing 554 bp of the human villin promoter and the first to regulation by H. pylori. We induction of the villin promoter for 24 h by different bacterial H. pylori, and C. The results showed induction with the H. pylori J99 strain with C. or A. at C. is a that not A. induce gastritis Y. G. A. 2002; PubMed Scopus Google Scholar). We found that the with H. pylori the villin reporter at a bacterial of within h not activation of the villin promoter was Erk and the were used to villin induction by H. pylori. Dominant negative Erk-1 and constructs were with the villin reporter prior to with H. pylori with dominant negative villin induction by H. pylori The not villin induction that the of H. pylori on the villin promoter used the Erk the of the an was with Erk and confirmed that with H. pylori in an increase in Erk protein We found that H. pylori as previously S. M. R.M. J. 1999; Google Scholar). when with the that not induction by H. pylori, we that the was not of villin results were with prior studies implicating the in H. pylori induction of the T. A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Moreover, has been confirmed that H. pylori both the Erk-1 and but that the Erk-1 the factor and its downstream e.g. M.M. M. K. D. 2004; PubMed Scopus Google pylori Erk and cells were with H. pylori for up to 24 h or with of the or of the prior to the of cell for analysis. to the or forms of the and were We the villin promoter for targets of the and a of Elk-1 sites to an SRE at bp from the site elements of the and an Ets binding site form the SRE G. C. B. 2004; PubMed Scopus Google Scholar). The SRF forms a and binds the which in turn the binding of inducible Ets e.g. as ternary complex of the Ets the Ets site and with the SRF to form the ternary complex A. PubMed Scopus Google Scholar, R. Dev. 1994; PubMed Scopus Google Scholar). Elk-1 and SRF binding to the villin SRE was required for H. pylori we the element Dominant negative Elk-1 was used to the of Elk-1 to H. pylori induction of the The results showed that within the SRE/EBS site induction by H. pylori Moreover, with both and inducible activation of the promoter by H. pylori, that this factor is required for the is that Elk-1 binding is by of Erk to the and of this factor C. J.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to Elk-1 was during with H. pylori, an was The results showed that Elk-1 was at 3 and 24 h of treatment with H. pylori a for H. pylori we the induction of Elk-1 by In both induction of Erk Elk-1 protein that the increase was not to an increase in the form but because of an increase in the of Thus, we that the of H. pylori activation of the villin promoter was by activation of Erk in of Elk-1 its binding to and with SRF T. J.J. L. S.J. M.H. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). shift were to with H. pylori increased Elk-1 binding to the inducible villin element. were prepared after the cells with the bacteria for and 24 In there was binding of and SRF and binding of Elk-1 increase in binding to the inducible villin element was observed after 3 h of with the By 24 there was increased SRF binding in to the binding by was used as a and not shift of the The of the was confirmed in the and by with specific Moreover, the of the at was confirmed by with the which for all of the major the shift that and SRF to the element in exposure to bacteria for 3 Elk-1 binds and binding is is a in SRF, which correlates with of the element by the binding of By 24 both SRF and Elk-1 binding is and binding suggesting of this element by inducible pylori Elk-1 and SRE h of serum H. pylori J99 bacteria were with cells. shift were with of after treatment and 24 h of with the the from the cells was incubated with and YY1, SRF, or from cells with bacteria for 24 h was using the type YY1, SRF, and In this study, we showed that induction of villin in the stomach in atrophic human and mouse stomach in response to chronic H. pylori infection. results are with the that villin is an of this pre-neoplastic lesion in the stomach and esophagus T. Inada K. Tanaka H. Mizoshita T. Mihara M. Ushijima T. Yamamura Y. Nakamura S. Tatematsu M. J. Cancer Res. Clin. Oncol. 2004; 130: 135-145Crossref PubMed Scopus (130) Google Scholar). that villin expression develops in responding to chronic is the elements that its inducible expression. In the small villin is expressed during fetal transcriptional within the promoter are responsible for intestinal villin expression and prior to (21Madison B.B. Dunbar L. Qiao X.T. Braunstein K. Braunstein E. Gumucio D.L. J. Biol. Chem. 2002; 277: 33275-33283Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar). on the results this promoter the elements capable of regulation of villin expression in gastric cells. In this study, we found that villin was expressed in in both human subjects and in a mouse model infected with H. pylori. The expression with the of intestinal metaplasia in the human true cells are not observed in mouse of H. pylori we that villin expression was with the chronic infection. in studies between regulation of villin expression to the to or the atrophic epithelium. the of the on villin we a of studies that H. pylori was sufficient to villin expression and that the inducible expression required a results are with that regulation by the H. pylori protein Y. S. Y. K. A. M. H. T. Y. M. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the results are the first to regulation of an SRE by H. pylori within a promoter and a reporter that correlates with a pre-neoplastic both Elk-1 and SRF to the SRE of the element as as a not completely induction of the we expressed Elk-1 to of Elk to the was in that the dominant negative construct the of the -554 reporter construct and completely induction by H. pylori. inducible binding of SRF to this we that inducible activation and binding of Elk-1 a in the regulated expression of villin the intestinal metaplastic phenotype of the Interestingly, the binding of both Elk-1 and SRF was and not to the base line after 24 h of with the suggesting activation of the promoter or the for with in chronic expression of villin. be the of or In has been that the ternary complex with that and which G. C. B. 2004; PubMed Scopus Google Scholar). The of the villin SRE is by at which to the of and villin. that Elk-1 and SRF binding sites are to and the of the expression and binding studies that SRF as a is with cooperative binding between Elk-1 and SRF G. C. B. 2004; PubMed Scopus Google Scholar). as both a and the of transcriptional and M.J. E. 1999; PubMed Scopus Google Scholar). the in binding with H. pylori, that the of on the villin promoter is as a important of results is the between Elk-1 activation and the pre-neoplastic of the is that Elk-1 activation is regulated by the pathway. Interestingly, and H. E. R.M. Med. 1999; PubMed Scopus Google Scholar) showed that which Erk activation in the stomach H. E. R.M. Med. 1999; PubMed Scopus Google Scholar). is with the that in the formation and Moreover, a of the factor and the Erk-2 signaling in the R. B. M. D. Med. 2002; PubMed Scopus Google Scholar). Thus, is to that increased Erk-2 signaling in the stomach correlates with cell proliferation. the results demonstrate that induction of villin downstream of the its expression in the stomach is a marker of increased cell Helicobacter be of that increase epithelial the there is the for a variety of genes in the in the as a marker in the be in signaling and factor during the gastric studies of the human specific to be B. S. S. S. Y. C. K. K. Y. K. Cancer Res. 2003; Google Scholar). Thus, the signaling important to in the to gastric and specific