HpSlyD inducing CDX2 and VIL1 expression mediated through TCTP protein may contribute to intestinal metaplasia in the stomach

Helicobacter pylori infection is the most important risk factor for gastric intestinal metaplasia (IM). Our previous study demonstrated that infection with H. pylori HpslyD-positive strains associated with IM. To further investigate the signalling pathway involved in HpSlyD-induced IM, CDX2 and VIL1 expressions were determined before and after HpSlyD application. TCTP was knocked down by siRNA or overexpressed by plasmid transfection. An HpSlyD binding protein was used to block HpSlyD’s enzymatic activity. The expression of CDX2 and TCTP in gastric diseases was measured by immunohistochemistry. Our results showed HpSlyD induced CDX2 and VIL1 expressions. TCTP protein expression was markedly increased after application of HpSlyD and in an HpSlyD-expressing stable cell line. Downregulation of TCTP protein led to decreased HpSlyD-induced CDX2 and VIL1. Overexpression of TCTP protein improved the expression of CDX2 and VIL1. Co-application of HpSlyD and FK506 led to significant reductions in CDX2, VIL1, and TCTP expression. Immunohistochemistry demonstrated that CDX2 and TCTP expression was higher in HpslyD-positive specimens compared with HpslyD-negative ones. Expression of CDX2 was positively correlated with TCTP in HpslyD-positive cells. Our study is the first to show that HpSlyD induction of CDX2 and VIL1 expression mediated through TCTP may contribute to IM in the stomach.

factors are associated with different histopathological changes of the gastric mucosa. For example, the strain carrying cagA and vacA can produce a stronger inflammatory response, which is related to the occurrence of precancerous lesions such as GIM 5 . In a previous study, we identified a novel peptidylproline cis-trans-isomerase (PPIases, EC number 5.2.1.8) associated with gastric carcinogenesis, which encodes the protein H. pylori SlyD (HpSlyD) 6 . HpSlyD has the ability to promote cell proliferation, malignant transformation and invasion, and to inhibit apoptosis 7,8 . Further study has shown that infection with HpslyD-positive strains may be associated with atrophic gastritis 9 . However, the signalling pathway involved in HpSlyD-induced intestinal metaplasia is not yet completely understood.
Caudal-related homeobox 2 (CDX2) is a molecular engine that regulates intestinal differentiation. It can directly promote the expression of a variety of intestinal cell-specific factors, while playing an irreplaceable role in maintaining intestinal cell proliferation, development and differentiation. Under normal conditions, CDX2 expression is restricted to the intestine, but it is ectopically expressed in IM lesions, not only of the stomach, but also of the oesophagus and gall bladder, among other locations. CDX2 activation plays a key role in the development of GIM 10 . Villin 1 (VIL1) is a structural protein involved in the formation of small intestinal microvilli and has upregulation of expression in IM. VIL1 is a known transcriptional target of CDX2 11 . Both CDX2 and VIL1 play a key role in the development of gastric metaplasia. It has been reported in the literature that H. pylori can affect CDX2 and VIL1 expression [12][13][14] . However, it is unclear whether HpSlyD affects CDX2 and VIL1 expression, and if it does, how it regulates CDX2 and VIL1 transcriptional expression is also unclear.
Translationally controlled tumor protein (TCTP), a highly conserved protein found in eukaryotic cells, is an important tumor-associated protein identified in a study of tumor reverse screening. In 2007, the journal Nature reported 15 that TCTP controls growth and differentiation in drosophila and TCTP overexpression occurs in many human cancers, such as breast cancer and liver cancer [16][17][18][19][20][21] . Recent studies have shown that TCTP is also pivotal in the cell reprogramming network, with a role as a checkpoint, and it regulates the transition points of cell phenotype under a variety of physiological and pathological states 22 . It is unclear whether TCTP is involved in the regulation of GIM. In our previous study, using differential proteomics, we screened for changes in protein expression associated with the expression of HpSlyD in a stable cell line. Among the 21 up-regulated proteins, the one elevated the most was TCTP, suggesting that TCTP may be involved in HpSlyD-mediated regulation (data not shown). However, this speculation needs to be further verified.
In this study, we investigated whether HpSlyD could induce CDX2 and VIL1 expression in vivo and in vitro and whether TCTP regulates CDX2 and VIL1 expression induced by HpSlyD, and we aimed to clarify the signalling pathway involved in HpSlyD-induced IM in the stomach.

Materials and Methods
Cell culture and treatment. The human gastric carcinoma cell lines AGS and N87 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). They were grown in Ham's F-12 medium (HyClone, USA) or Dulbecco's modified Eagle's medium (DMEM; HyClone, USA) supplemented with 10% foetal bovine serum (FBS, Gibco, Australia) in an atmosphere consisting of 5% CO 2 at 37 °C. AGS cells were transfected with either SlyD-GFP or GFP plasmids and stable cell lines were obtained using the methods described by Zhu et al. 8 . N-terminal His tagged SlyD was purified by Ni 2+ affinity chromatography as described earlier 7 . For all experiments, HpSlyD was used at a concentration of 200 ng/mL. The HpSlyD binding protein tacrolimus (FK506) was purchased from Astellas Ireland Co., Ltd., dissolved in ddH 2 O at a concentration of 18 mg/ml and stored at −20 °C until use. RNA extraction and Real-time quantitative RT-PCR (qPCR). Total RNA was extracted using TRI Reagent (Ambion, USA) and converted to cDNA using a PrimeScript RT reagent kit (Takara, Japan). Human CDX2 (forward 5′-TTCACTACAGTCGCTACATCACC-3′; reverse 5′-TTGTTGATTTTCCTCTCCTTTGC-3′) and VIL1 (forward 5′-GGCAAGAGGAACGTGGTAGC-3′; reverse 5′-CGGTCCATTCCACTGGATGA-3′) were amplified with SYBR Green (SYBR Premix Ex Taq II, Takara, USA) in a fluorescence reader ABI Prism 7500. The following PCR parameters were used: 95 °C for 30 seconds, 40 cycles of 95 °C for 15 seconds, 55 °C for 30 seconds and finally an elongation step at 72 °C for 30 seconds. Each reaction was performed in triplicate and normalized to GAPDH. Relative expression of the target genes was determined using the 2 −ΔΔCt method 23 . Thereafter, expression was expressed as fold difference relative to that of the untreated control cells. The results are expressed as mean ± SD of representative triplicates.
Protein extraction and western blot. Western blot analysis was performed using standard techniques.
Briefly, cells (2 × 10 6 /well) were treated with or without SlyD (200 ng/mL) for 40 hours. Total protein was extracted using a lysis buffer (2% mercaptoethanol, 20% glycerol, and 4% SDS, in 100 mM Tris-HCl buffer, pH 6.8). Equal amounts of total protein (60 µg/lane) were separated and transferred to PVDF membranes (Bio-Rad, Hercules, CA). The membranes were incubated with primary antibodies overnight at 4 °C: rabbit monoclonal anti-CDX2 Human tissue specimens and immunohistochemistry. Tissue samples were obtained from 84 individuals with gastritis (GS), 91 individuals with intestinal type atrophic gastritis (IM-GA) and 58 with gastric cancer (GC) who participated in the Zhuanghe Gastric Diseases Screening Program between 2008 and 2011, including 133 men and 100 women, 149 cases ≤ 60 years of age and 84 cases > 60 years of age. All subjects were histologically diagnosed based on the updated Sydney System for gastritis. This study was approved by the Ethics Committee of the First Affiliated Hospital of China Medical University Shenyang, China. Written informed consent was obtained from the participants.

Statistical analysis.
All analyses were carried out by using SPSS for Windows version 16.0. Data were presented as mean ± SD. Differences in the mRNA and protein expression levels of CDX2, VIL1 and TCTP between the treated and non-treated group were analysed by Student's t-test. The correlations between H. pylori infection in tissue samples with other factors were determined using the bilateral χ 2 test. Non-parametric tests were used to analyse the differences of CDX2 and TCTP protein detected by IHC. Correlation analysis was performed between TCTP and CDX2 expression. A value of P < 0.05 was defined as statistically significant.

Results
HpSlyD induces CDX2 and VIL1 expression in gastric epithelial cell lines. The occurrence of gastric IM during H. pylori infection has been reported to be dependent on induction of CDX2 expression in gastric epithelial cells 30 . Thus, in initial studies, we evaluated CDX2 expression and the expression of another epithelial cell differentiation marker, VIL1, in human gastric cancer cell lines before and after treatment with HpSlyD. AGS or N87 cells were incubated with 200 µg/ml HpSlyD for 40 hours. The level of CDX2 mRNA in the non-treated group was significantly lower than that of the treated group in both cell lines (Fig. 1A). Similarly, mRNAs encoding VIL1 were up-regulated in the treated cells compared with the non-treated cells (Fig. 1B). In addition, CDX2 protein (as well as VIL1 protein) was also expressed at this time point (Fig. 1C-E). CDX2 and VIL1 mRNA expression in AGS cells expressing SlyD-GFP were significantly higher than in control AGS cells and AGS cells expressing GFP alone ( Fig. 2A,B). The same differences were also found in the protein expression of CDX2 and VIL1 (Fig. 2C-E). Our results showed that in both gastric epithelial cell lines and HpSlyD stably expressing cell line, CDX2 and VIL1 expression was affected by the presence of HpSlyD.
HpSlyD induced TCTP expression in human gastric epithelial cells. In our previous study, we found that TCTP is a highly expressed protein in an HpslyD-GFP stable cell line, suggesting that TCTP may be involved in HpslyD-mediated biological effects. With this information in hand, we next addressed whether HpslyD can induce increased TCTP expression in AGS, N87, and the HpslyD-GFP stable cell line. As shown in Fig. 3, TCTP expression was markedly increased in AGS and N87 cells treated with 200 µg/ml HpSlyD for 40 hours and in the HpslyD-GFP stable cell line, suggesting that HpSlyD affects TCTP expression in gastric epithelial cells.

HpSlyD induction of CDX2 and VIL1 expression inhibited by knockdown of TCTP.
To further examine whether TCTP regulates CDX2 and VIL1 expression induced by HpSlyD, we conducted a series of studies addressing the role of TCTP in HpSlyD induction of CDX2 and VIL1. AGS, N87, and AGS HpslyD-GFP stably expressing cell lines were transfected with TCTP siRNA or nonspecific siRNA for 6 hr and then treated with HpSlyD for another 40 hr. As shown in Fig. 4, TCTP-specific siRNA strongly inhibited HpSlyD-induced upregulation of CDX2 and VIL1, suggesting the involvement of TCTP in H. pylori induced CDX2 signalling. The same result can also be seen in both N87 cells and the HpslyD-GFP stably expressing cell line (Fig. 4A-D). Our data demonstrate that TCTP has a promotion effect on HpSlyD-induced CDX2 and VIL1 expression. TCTP introduction upregulated the expression of CDX2 and VIL1. The above results showed that TCTP was involved in HpSlyD-induced upregulation of CDX2 and VIL1. Whether the introduction of TCTP gene to the cell lines has the same biological effects as HpSlyD? We then transfected a TCTP expression plasmid and a control plasmid (Origene, China) into AGS and N87 cells using Lipofectamine 2000 (Invitrogen, USA). TCTP overexpresion was evaluated 24 hours after transfection by western blot. As shown in Fig. 5, TCTP introduction upregulated the CDX2 and VIL1 expression both in AGS and N87 cells, suggesting the involvement of TCTP in inducing CDX2 signaling. Our data demonstrate that TCTP overexpression has a promotion effect on CDX2 and VIL1 expression, just as the same biological effects as HpSlyD has.
HpSlyD binding protein FK506 blocks HpSlyD-induced expression of CDX2, VIL1, and TCTP in AGS and N87 cells. FK506 can block the function of FK506-binding protein (FKBP) by binding to the immunophilin FKBP12 [31][32][33][34][35] . HpSlyD is a member of the FKBP family. First, we assessed whether FK506 could inhibit HpSlyD enzymatic activity. As shown in Fig. 6, with E. coli SlyD as a positive control, enzymatic activity analysis revealed that PPIase activity was substantially lower in cells treated with HpSlyD+FK506 than in those treated with HpSlyD alone. Therefore, our data suggest that FK506 can suppress PPIase activation of HpSlyD.
We next addressed the effect of FK506 on HpSlyD-induced expression of CDX2, VIL1 and TCTP. As shown in Fig. 7, co-treatment of cells with HpSlyD and FK506 led to significant reductions in CDX2, VIL1 and TCTP expression compared with cells treated with HpSlyD alone in both the AGS (Fig. 7A-D) and N87 (Fig. 7E-H

HpSlyD related to the expression of CDX2 and TCTP in different gastric diseases. The above in
vitro studies showed that HpSlyD induces CDX2 and VIL1 expression mediated through TCTP. To determine if a similar phenomenon occurs in vivo we immunostained human different gastric diseases tissue with or without HpSlyD infection. The information from the patients' included in this study is summarized in Supplement Table 1. There was no statistically significant difference in age and sex between groups.  In GS group, the IS of CDX2 expression was no statistical difference no matter in H. pylori positive cases than in the negative ones or in the HpslyD positive cases than in the negative ones (P > 0.05, Fig. 8A-E). The IS of TCTP expression was also no difference between H. pylori groups (P > 0.05, Fig. 8C,D,F). These results indicated that the HpslyD positive H. pylori strain doesn't promotes the expression of CDX2 and TCTP in GS.
In IM-GA group, the IS of CDX2 expression was higher not only in H. pylori positive cases than in the negative cases but also in the HpslyD positive cases than in the negative group (P < 0.001, Fig. 9A,B,E). The same expression trend can also be seen in the IS of TCTP expression (P < 0.001, Fig. 9C,D,F). These results show that HpslyD positive H. pylori strain promotes the expressions of CDX2 and TCTP in IM-GA. In GC group, the IS of CDX2 expression was higher in H. pylori positive specimens than in the negative specimens, and higher in HpslyD positive specimens than in the negative specimens (P < 0.05 and P < 0.01, Fig. 10A,B,E). The same expression trend can also be seen in the IS of TCTP expression (P < 0.001, Fig. 10C,D,F). These results show that HpslyD positive H. pylori strain promotes the expressions of CDX2 and TCTP in GC.
And then we compared TCTP and CDX2 expressions of different gastric diseases in HpslyD positive. As shown in Fig. 11, the IS of CDX2 and TCTP expressions are significantly higher in GC than that of IM-GA, which is also significantly higher in IM-GA than that of GS, indicating that the HpslyD positive H. pylori strain promotes the expression of CDX2 and TCTP with the development of gastric diseases.
TCTP is positively correlated with CDX2 in H. pylori slyD positive infection. We next evaluated the relationship between TCTP and CDX2 expression. As shown in Fig. 12, we identified a positive correlation between TCTP and CDX2 levels in HpslyD positive cases (Spearman's correlation coefficient, r = 0.3644, P < 0.01) but not in HpslyD negative cases (r = 0.1292, P = 0.4089) or H. pylori negative cases (r = 0.2585, P = 0.067).

Discussion
In a previous study, we identified HpslyD as a gastric cancer-associated gene 6 . Further study has shown that infection with slyD-positive H. pylori strains is associated with atrophic gastritis 9 . However, the mechanism by which HpslyD provokes metaplastic changes is poorly understood. In this study, we fill this gap with studies showing that HpSlyD induces CDX2 and VIL1 expression both in vitro and in vivo. In addition, this study is the first to confirm that the TCTP-mediated signalling pathway is involved in HpSlyD-induced IM in the stomach. These results provide novel information that contributes to understanding the molecular events that precede the development of gastric diseases caused by H. pylori infection. Metaplasia is a process whereby a completely differentiated cell transforms into another type of mature cell, and this process is stimulated by certain factors in response to environmental changes. IM refers to a series of phenotype changes from stomach epithelium to an intestinal phenotype during the process of changing from gastritis to atrophic gastritis and sometimes to intestinal-type gastric cancer. This change is caused by an integration of genetic factors expression, transcription factors, signalling pathways and growth factors. CDX2 is a homeobox transcription factor that is critical for intestinal differentiation 36,37 , and is a specific biomarker of the early steps of the gastric carcinogenic cascade, driving the development of IM 38,39 . The key role of CDX2 in the metaplastic transformation of the gastric mucosa was categorically demonstrated by the use of two transgenic mouse models with ectopic expression of CDX2 in the gastric epithelium and subsequent development of IM with absorptive, goblet and enteroendocrine cell types 40,41 . VIL1 is a structural protein involved in the formation of small intestinal microvilli and its expression is upregulated in IM. VIL1 is a known transcriptional target of CDX2 11 . Using two kinds of gastric epithelial cells in vitro we showed that HpSlyD induced CDX2 and VIL1 expression. Furthermore, a similar result was confirmed in an HpslyD stable cell line, which we constructed in previous studies. Therefore, our results indicate that the expression of CDX2 and VIL1 is associated with the presence of HpSlyD.
SlyD, as a multifaceted protein, belongs to the PPIase FKBP family and catalyses the intrinsically slow cistrans isomerization of peptidylprolyl bonds (Xaa-Pro) to facilitate the protein folding process 42,43 , but its role as a PPIase in vivo is not well understood. Previous functional and interactional studies have shown that HpSlyD is involved in nickel ion integration of urease and hydrogenase 42,44 . Our previous studies showed that HpslyD is a high-copy gene in gastric cancer patients by constructing a gastric cancer-related H. pylori differential gene (E) Boxplot shows that CDX2 expression is significantly higher in H. pylori positive and HpslyD positive cases than in negative ones. (F) Boxplot shows that TCTP expression is significantly higher in H. pylori positive and HpslyD positive cases than in negative ones. ***P < 0.001. library and that HpSlyD influences the gastric cell biological processes of cell proliferation, transformation and migration 7,8 . Recently, some researchers demonstrated an emerging role of mammalian PPIase in cell differentiation 45 and therefore bacterial-derived PPIase may also be involved in phenotype transitions. The present study suggests that HpSlyD regulates CDX2 and VIL1 to promote IM transition in gastric epithelial cells. This study broadens our understanding of bacterial-derived PPIase and provides a theoretical basis for understanding the function of HpSlyD and an in-depth exploration of the pathogenesis of H. pylori.
The molecular mechanism of H. pylori's regulation of CDX2 expression has been reported in the literature. Camilo 12 . Thus, CDX2 and VIL1 expression regulated by H. pylori is a relatively complex process involving the interaction of many signalling pathways. However, the signalling pathway involved in HpSlyD-induced CDX2 and VIL1 expression is not yet completely understood. TCTP is at the heart of the cell-reprogramming network, playing the role of a checkpoint, and is involved in regulating transition points of cell phenotypes under a variety of physiological or pathological states. In vitro, we found that TCTP expression was markedly increased in AGS and N87 cells treated with HpSlyD and in an HpslyD stable cell line, suggesting that HpSlyD also affects TCTP expression in gastric epithelial cells. Meanwhile, we observed that downregulation of TCTP protein led to decreased HpSlyD-induced CDX2 and VIL1 expression and overexpression of TCTP improved the levels of CDX2 and Villin. Co-treatment with HpSlyD and FK506 led to a significant reduction in CDX2, VIL1 and TCTP expression. Furthermore, IHC staining demonstrated that CDX2 and TCTP expression were higher in H. pylori positive specimens than in H. pylori negative specimens, and higher in HpslyD positive specimens than in HpslyD negative specimens. HpslyD positive H. pylori strain promotes the expression of CDX2 (E) Boxplot shows that CDX2 expression is significantly higher in H. pylori positive and HpslyD positive cases than in negative ones. (F) Boxplot shows that TCTP expression is significantly higher in H. pylori positive and HpslyD positive cases than in negative ones. *P < 0.05, **P < 0.01, ***P < 0.001. and TCTP with the development of gastric diseases. In HpslyD positive specimens, the expression of CDX2 was positively correlated with TCTP. Our results show that HpSlyD induces CDX2 and VIL1 expression mediated through TCTP and contributes to IM and the development of gastric diseases. We can further speculate that HpSlyD can activate cell differentiation mediated by transcriptional factors through TCTP, re-programming gastric epithelial cells from the gastric phenotype to the intestinal phenotype. This process may also be involved in the malignant transformation of gastric tissue harbouring this chronic and stable infection.
In conclusion, we demonstrated that H. pylori infection leads to increased expression of CDX2 and VIL1 and that TCTP enhances this expression, and these changes were associated with the development of IM and cancer in the gastric mucosa. The results presented in this study show that HpSlyD is a positive regulator of IM progression, and therefore, it may be a possible therapeutic target for inhibiting the formation of IM after H. pylori infection. Our results provide novel information for understanding the molecular events that precede the development of gastric IM, reinforcing the role of the HpSlyD-TCTP-CDX2 pathway in the whole process. Our study also provides an important molecular target for the clinical monitoring of H. pylori infection and 'type-based therapy' , and provides insight into ideas and strategies for blocking H. pylori-related IM formation and decreasing the risk of progression to gastric cancer.  Boxplot shows that CDX2 expression of HpslyD positive is significantly higher in GC than that in IM-GA, and also is significantly higher in IM-GA than that in GS. (B) Boxplot shows that TCTP expression of HpslyD positive is significantly higher in GC than that in IM-GA, and also is significantly higher in IM-GA than that in GS. *P < 0.05, **P < 0.01, ***P < 0.001.