Gastrokine 1 inhibits gastrin-induced cell proliferation
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Gastrokine 1 (GKN1) acts as a gastric tumor suppressor. Here, we investigated whether GKN1 contributes to the maintenance of gastric mucosal homeostasis by regulating gastrin-induced gastric epithelial cell growth.
We assessed the effects of gastrin and GKN1 on cell proliferation in stable AGSGKN1 and MKN1GKN1 gastric cancer cell lines and HFE-145 nonneoplastic epithelial cells. Cell viability and proliferation were analyzed by MTT and BrdU incorporation assays, respectively. Cell cycle and expression of growth factor receptors were examined by flow cytometry and Western blot analyses.
Gastrin treatment stimulated a significant time-dependent increase in cell viability and proliferation in AGSmock and MKN1mock, but not in HFE-145, AGSGKN1, and MKN1GKN1, cells, which stably expressed GKN1. Additionally, gastrin markedly increased the S-phase cell population, whereas GKN1 significantly inhibited the effect of gastrin by regulating the expression of G1/S cell-cycle regulators. Furthermore, gastrin induced activation of the NF-kB and β-catenin signaling pathways and increased the expression of CCKBR, EGFR, and c-Met in AGS and MKN1 cells. However, GKN1 completely suppressed these effects of gastrin via downregulation of gastrin/CCKBR/growth factor receptor expression. Moreover, GKN1 reduced gastrin and CCKBR mRNA expression in AGS and MKN1 cells, and there was an inverse correlation between GKN1 and gastrin, as well as between GKN1 and CCKBR mRNA expression in noncancerous gastric mucosae.
These data suggest that GKN1 may contribute to the maintenance of gastric epithelial homeostasis and inhibit gastric carcinogenesis by downregulating the gastrin-CCKBR signaling pathway.
KeywordsGKN1 Gastrin Homeostasis Cell growth Stomach
Gastrin is a gastrointestinal peptide hormone that has a key role in the regulation of gastric acid secretion and in gastric epithelial organization and maintenance by a variety of endocrine and paracrine mediators [1, 2]. Gastrin also regulates several important cellular processes in the gastric epithelium, including cell proliferation, apoptosis, migration, and invasion [3, 4]. Insulin-gastrin transgenic hypergastrinemic INS-GAS mice showed an increased proliferation of gastric epithelium, with dysplasia in 100 % and frank malignancy in 75 % of mice . The observed malignant progression was significantly accelerated in Helicobacter felis-infected INS-GAS mice . The infusion of gastrin at supraphysiological levels in humans was reported to result in increased gastric cell proliferation, as demonstrated by 3H-thymidine labeling studies . Interestingly, hypergastrinemia associated with chronic Helicobacter pylori (H. pylori) infection may act as a co-factor during the development of gastric adenocarcinoma . Taken together, these findings suggest that gastrin not only plays an important role in gastric tumorigenesis but can also be a potential therapeutic target for gastric cancer.
Gastrokine 1 (GKN1) encodes an 18-kDa antral mucosal protein (AMP-18) that is highly expressed in the antrum of the stomach . GKN1 also protects gastric mucosa and promotes healing by facilitating restitution and proliferation after injury . Interestingly, differential expression studies identified GKN1 as a gene strongly downregulated in H. pylori-infected gastric mucosal epithelial cells and gastric cancer [8, 9, 10], considering GKN1 as a putative stomach-specific tumor suppressor gene. Recently, it was shown that GKN1 induces senescence through the p16/Rb pathway activation in gastric cancer cells , and that its overexpression induces Fas-mediated apoptosis , suggesting that, in the absence of GKN1, gastric epithelial cells continuously proliferate without undergoing apoptosis. We previously reported that GKN1 has tumor suppressor activity through the regulation of epigenetic alterations and epithelial-mesenchymal transition [13, 14], implying its potential utility in clinical prediction/diagnosis. Thus, we hypothesized that GKN1 may contribute to the homeostasis of gastric mucosal epithelial cells by regulating gastrin activity.
In this study, we examined the impacts of GKN1 on gastrin activity in gastric cancer cell lines and gastric mucosal epithelium and demonstrated the effects of both genes on cell viability and proliferation, cell-cycle progression, and expression of growth factor receptors. Overall, we found that GKN1 may contribute to the homeostasis of gastric epithelial cells and suppress gastric carcinogenesis by inhibiting gastrin-induced cell proliferation.
Materials and methods
Cell culture and transfection of GKN1
AGS and MKN1 gastric cancer cell lines and HFE-145 nonneoplastic gastric epithelial cells were obtained from the American Type Culture Collection and Dr. Hassan (Washington, DC, USA). These cells were cultured at 37 °C in 5 % CO2 in RPMI-1640 medium (Lonza, Basel, Switzerland) supplemented with 10 % heat-inactivated fetal bovine serum. GKN1 cDNA was cloned into the pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA, USA). We generated AGS and MKN1 cell lines, which stably expressed GKN1 (AGSGKN1 and MKN1GKN1 cells), as described previously . Briefly, the human GKN1 expression vector was transfected into AGS and MKN1 cells using Lipofectamine 2000 (Invitrogen). The medium was changed after 24 h, and G418 (Wako, Osaka, Japan) was added to the culture medium to a final concentration of 1 mg/ml. Thereafter, cells were cultured in the presence of G418 for 8 weeks. The marked expression of GKN1 was confirmed by immunoblot analysis in HFE-145 cells and stable GKN1 transformants, AGSGKN1 and MKN1GKN1, but not in the stable mock cells, AGSmock and MKN1mock .
Measurement of cell viability and proliferation
We investigated whether the recombinant gastrin protein (Sigma, St. Louis, MO, USA) is involved in regulation of cell viability by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assay in AGSmock, MKN1mock, AGSGKN1, and MKN1GKN1 cells at 24, 48, and 72 h after treatment with gastrin (100 nM). MTT assay was also performed in HFE-145 cells after silencing of GKN1 by shGKN1 transfection, to further examine whether cell viability was dependent on activity of the GKN1 protein. Absorbance was measured with a spectrophotometer at 540 nm, and cell viability was expressed relative to the mock control.
For cell proliferation analysis, a BrdU incorporation assay was performed in AGSmock, MKN1mock, AGSGKN1, MKN1GKN1, and HFE-145 cells at 24, 48, and 72 h after treatment with gastrin (100 nM), using the BrdU cell proliferation assay kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Absorbance was measured with a spectrophotometer at 450 nm, and proliferation was expressed relative to the mock control.
Cell-cycle analysis by flow cytometry
To investigate the molecular mechanisms of gastrin-induced cell proliferation, gastrin (100 nM)-treated AGS and MKN1 cells were collected and stained with propidium iodide (PI) for 45 min in the dark before analysis. The percentages of cells in different phases of the cell cycle were determined using a FACSCalibur Flow Cytometer with CellQuest 3.0 software (BD Biosciences, Heidelberg, Germany). Experiments were performed in triplicate, and the average values were used for quantification.
Expression of cell-cycle regulators and growth factor receptors
We next determined whether the effect of gastrin on cell-cycle progression is blocked by GKN1. Expression of the G0/G1-phase proteins, including p53, p21, CDK6, cyclin D1, and β-catenin, was examined in AGSmock, MKN1mock, AGSGKN1, and MKN1GKN1 cells at 48 h after treatment with gastrin (100 nM). In addition, we analyzed the expression of gastrin receptor, cholecystokinin-B receptor (CCKBR), and growth factor receptors, such as epidermal growth factor receptor (EGFR) and c-Met, in AGS, MKN1, and HFE-145 cells at 48 h after treatment with gastrin (100 nM) and transfection with GKN1 or shGKN1. Cell lysates were separated on a 10 % polyacrylamide gel and transferred onto a Hybond PVDF membrane (Amersham Pharmacia Biotech, Piscataway, NJ, USA). After blocking, the membrane was subsequently probed with antibodies against target proteins. Protein bands were detected using enhanced chemiluminescence reagents (Amersham Pharmacia Biotech).
Primer sequences for real-time RT-PCR
Expression of NF-κB pathway proteins
To determine whether gastrin is involved in regulation of the NF-κB signaling pathway, the expression of NF-κB-related proteins, including NF-κB p-p65, NF-κB p65, IKKα/β, and IκB, was examined in mock and GKN1 stable AGS and MKN1 cells at 48 h after treatment with gastrin (100 nM). Cell lysates were separated on a 10 % polyacrylamide gel and transferred onto a Hybond PVDF membrane (Amersham Pharmacia Biotech). After blocking, the membrane was subsequently probed with antibodies against NF-κB p-p65, NF-κB p65, IKKα/β, and IκB (Cell Signaling). Protein bands were detected using enhanced chemiluminescence reagents (Amersham Pharmacia Biotech).
Expression of GKN1, gastrin, and CCKBR in gastric cancer cell lines and gastric mucosae
Expression of gastrin and CCKBR mRNA transcripts was analyzed in stable AGSGKN1 and MKN1GKN1 cells by real-time RT-PCR. Also, the expression of gastrin and CCKBR was examined in 55 frozen noncancerous gastric mucosae by real-time RT-PCR and compared with the expression level of GKN1. Real-time RT-PCR was performed using SYBR Green Q-PCR Master Mix (Stratagene), according to the manufacturer’s instructions. Each reaction was run for 40 cycles. Gastrin, CCKBR, and GKN1 mRNAs were quantified by SYBR Green Q-PCR and normalized to mRNA of the housekeeping gene, GAPDH. The primer sequences are described in Table 1. The standard curve method was used for quantification of the relative amounts of gene expression products. This method provides unit-less normalized expression values that can be used for direct comparison of the relative amount of mRNA in different samples. All samples were tested in triplicate. Data are reported as relative quantities, according to an internal calibrator using the 2−△△CT method . Written informed consent was obtained from all participants in accordance with the Declaration of Helsinki. The study was approved by the Institutional Review Board of The Catholic University of Korea, College of Medicine (CUMC09U089).
The effects of gastrin and GKN1 on cell viability, proliferation, and cell-cycle progression were assessed by Student’s t test. Data are expressed as mean values ± SD from at least three independent experiments. Spearman’s correlation test was used to investigate relationships between GKN1, gastrin, and CCKBR mRNA expression in 55 noncancerous gastric mucosae. Statistical analyses were performed using Graphpad 5.0 (GraphPad software, San Diego, CA, USA). A P value less than 0.05 was considered statistically significant.
GKN1 inhibits gastrin-induced cell growth
In the BrdU incorporation assay, treatment with gastrin also led to a time-dependent augmentation of cell proliferation in AGSmock and MKN1mock cells, but not in HFE-145 and GKN1 stable AGSGKN1 and MKN1GKN1 cells (P < 0.05) (Fig. 1b). When GKN1 was silenced by specific shGKN1 in HFE-145 cells, gastrin treatment enhanced cell proliferation in a time-dependent manner (P < 0.05) (Fig. 1b).
GKN1 inhibits gastrin-induced cell-cycle progression
GKN1 downregulates the expression of CCKBR and growth factor receptors
GKN1 inhibits gastrin-induced activation of the NF-κB signaling pathway
Because treatment with gastrin is reported to result in activation of the NF-κB signaling pathway , we next investigated whether the NF-κB pathway is activated in response to gastrin via the canonical pathway involving IKKα/β and IκB in AGS and MKN1 cells. Gastrin enhanced the expression of p-p65 and slightly increased that of IKKα/β, although it did not affect the expression of p65 and IκB proteins. However, GKN1 reverted the expression of IκB and decreased the expression of IKKα/β, p-p65, and p65 proteins (Fig. 3d).
We next analyzed the mRNA expression of gastrin and CCKBR in AGSmock, MKN1mock, AGSGKN1, and MKN1GKN1 cells. Both gastrin and CCKBR mRNA levels were significantly diminished in AGS and MKN1 cells stably expressing the GKN1 protein (P = 0.015 and P = 0.026; P = 0.045 and P = 0.038; respectively) (Fig. 3e, f). Expectedly, CCKBR mRNA expression was markedly elevated in AGS and MKN1 cells treated with gastrin (P = 0.017 and P = 0.047, respectively) (Fig. 3f). Nevertheless, GKN1 totally inhibited gastrin-induced expression of CCKBR mRNA in AGS and MKN1 cells (P = 0.028 and P = 0.024, respectively) (Fig. 3f).
GKN1 inhibits the c-myc-induced expression of CCKBR
Similarly, gastrin and CCKBR mRNA levels were dramatically augmented in c-myc-transfected AGS and MKN1 cells (P = 0.029 and 0.045, respectively) (Fig. 4b, c). However, GKN1 significantly downregulated the mRNA expression of gastrin and CCKBR (P = 0.008 and 0.01) and abrogated the stimulating effect of c-myc on both genes (P = 0.02 and 0.031, respectively) (Fig. 4b, c).
GKN1 inversely correlates with the gastrin/CCKBR mRNA expression in noncancerous gastric mucosae
Generally, gastrointestinal epithelium is characterized by a very high cellular turnover rate, which leads to epithelial renewal every 3 to 5 days, and apoptosis is a key regulator of this turnover . Continuous processes of cell proliferation, differentiation, and self-renewal are counterbalanced by apoptosis, thus maintaining gastric epithelial homeostasis. A large body of evidence showed that gastrin, acting through the cholecystokinin-B receptor (CCKBR), plays a significant role in the proliferation of gastric epithelial cells and may be abnormally expressed in gastrointestinal carcinoma cells [19, 20]. It was reported that gastrin and CCKBR are co-expressed in human gastric carcinoma tissues  and that the exogenous gastrin exhibits a potent stimulatory effect on the proliferation of gastric cancer cells [22, 23]. In the present study, we similarly demonstrated that gastrin stimulates growth of gastric cancer cells (P < 0.05) (Fig. 1a, b). However, gastrin-induced cell growth was dramatically inhibited in GKN1 stably transfected AGS and MKN1 cells, and in HFE-145 cells expressing GKN1 (P < 0.05) (Fig. 1a, b). Additionally, treatment of HFE-145 cells with gastrin after GKN1 silencing by transfection with shGKN1 significantly enhanced cell proliferation (P < 0.05) (Fig. 1a, b). Thus, these data suggest that GKN1 may contribute to gastric mucosal homeostasis and gastric carcinogenesis by regulating gastrin-induced cell proliferation.
Previous studies have demonstrated that gastrin promotes cell proliferation as well as increasing the proportion of the S-phase cell population by upregulating cyclin D1 and CDK4 via activation of the β-catenin and CRE-binding protein pathways [24, 25, 26]. The fact that GKN1 suppresses gastrin-induced cell proliferation implies that GKN1 can modulate cell-cycle progression by regulating gastrin. We found a concomitant increase of the S-phase cell population in gastrin-treated AGS and MKN1 cells (P = 0.038 and 0.014, respectively), whereas no effect of gastrin on the G2/M-phase of the cell cycle was detected in flow cytometry analysis (Fig. 2a). Interestingly, GKN1 completely reverted the increase of the S-phase population in gastrin-treated AGS and MKN1 cells (P = 0.012 and 0.005, respectively) and induced G2/M arrest (P = 0.002 and 0.01, respectively) (Fig. 2a). These results suggest that GKN1 may inhibit gastrin-induced cell-cycle progression and corroborate the previous observation that GKN1 induces G0/G1 and G2/M arrests . In addition, gastrin treatment increased the expression of positive cell-cycle regulators, including cyclin D1, Cdk6, and β-catenin, whereas GKN1 suppressed the expression of these proteins and upregulated the expression of negative cell-cycle regulators, such as p53 and p21 (Fig. 2b). These findings suggest that GKN1 counteracts the proliferative effects of gastrin by modulating the expression of cell-cycle regulators, especially those related to the G1/S-phase transition.
Binding of gastrin to CCKBR in gastric epithelial cells induced the expression and release of heparin-binding epidermal growth factor-like growth factor, which subsequently transactivated epithermal growth factor receptor (EGFR) and its downstream signaling pathways [27, 28]. Also, it was reported that increase of epidermal growth factor (EGF) and EGFR expression was found in the gastric mucosae of patients with chronic gastritis and gastric cancer [29, 30]. In addition, the expression of gastrin and c-Met proteins was significantly increased in gastric cancer . Furthermore, gastrin markedly enhanced the endogenous expression of CCKBR, and overexpression of CCKBR protein was detected in the stomach of hypergastrinaemic animals . Here, to further clarify the mechanism underlying the inhibitory activity of GKN1 on gastrin-induced cell proliferation, we set out to determine if GKN1 inhibits the expression of the gastrin-specific receptor, CCKBR, and growth factor receptors. Gastrin treatment stimulated the increased expression of CCKBR, EGFR, and c-Met proteins, whereas GKN1 completely abrogated the expression of these genes in AGS and MKN1 cells (Fig. 3a, b). Conversely, GKN1 silencing with shGKN1 increased the expression of CCKBR, EGFR, and c-Met proteins in HFE-145 cells (Fig. 3c). Additionally, as it was previously reported that gastrin is capable of activating the NF-κB signaling pathway , we investigated whether the NF-κB pathway is activated in response to gastrin via the canonical pathway, involving IKKα/β and IκB, in AGS and MKN1 cells. Gastrin enhanced the expression of p-p65 and slightly increased IKKα/β, although it did not affect the expression of p65 and IκB proteins. However, GKN1 reverted the expression of IκB and inactivated the expression of IKKα/β, p-p65, and p65 proteins (Fig. 3d). Interestingly, GKN1 reduced the mRNA expression of both gastrin and CCKBR in AGS and MKN1 gastric cancer cells (P = 0.015 and P = 0.026; P = 0.045 and P = 0.038, respectively) (Fig. 3e, f). These results indicate that GKN1 may inhibit cell proliferation through suppression of the gastrin-CCKBR-growth factor receptor and NF-κB signaling pathways, thus contributing to gastric mucosal homeostasis.
It has been reported that gastrin activates β-catenin/Tcf-4 signaling and thereby induces the expression of CCKBR and early responsive genes, such as c-myc [32, 33, 34]. Previously, we found that GKN1 directly binds to the c-myc protein and downregulates its expression in immunoprecipitation assay . In this study, we examined whether GKN1 downregulates CCKBR by inactivating the c-myc-induced gastrin expression. A significantly increased level of the CCKBR protein was detected in AGSmock and MKN1mock cells transfected with c-myc, but the c-myc transfection had no effect on the CCKBR expression in both stable cell lines expressing GKN1 (Fig. 4a). Interestingly, the mRNA levels of gastrin and CCKBR were dramatically increased in c-myc-transfected AGS and MKN1 cells (P = 0.029 and 0.045, respectively), whereas GKN1 significantly diminished the mRNA expression of both genes (P = 0.008 and 0.01), even in cells transfected with c-myc (P = 0.02 and 0.031, respectively) (Fig. 4b, c). These observations suggest that GKN1 may inhibit the expression of CCKBR by downregulating c-myc activity.
It is well known that the main site of gastrin synthesis is the G cell within the antropyloric mucosa [20, 35], whereas the GKN1 protein and mRNA de novo synthesis is confirmed in surface mucous cells of the gastric antrum and fundus in the human [10, 36, 37, 38], mouse , and chicken . In this study, we examined 55 noncancerous gastric mucosa samples, and an inverse relationship between gastrin and GKN1 (P < 0.0001) (Fig. 5a), as well as between CCKBR and GKN1 mRNA expression (P < 0.0001) (Fig. 5b), was found. Additionally, a positive association between gastrin and CCKBR mRNA transcripts was observed (P < 0.0001) (Fig. 5c). These findings imply that the physiological synthesis of gastrin in G cells and its absence in gastric epithelial cells may be maintained by the abundant expression of GKN1, which inhibits the gastrin-induced cell proliferation and thereby is important in gastric mucosal homeostasis-preserving mechanisms.
In conclusion, the present study demonstrates a novel function of a gastric tumor suppressor gene, GKN1, in maintaining gastric mucosal homeostasis and inhibiting gastric carcinogenesis, arising through repression of gastrin-induced cell proliferation. Gastrin stimulated a significant time-dependant augmentation of gastric cancer cell viability and proliferation as well as inducing cell-cycle progression by increasing the S-phase cell population. Furthermore, gastrin facilitated the expression of CCKBR and growth factor receptors, including EGFR and c-Met, and activated the NF-κB signaling pathway in AGS and MKN1 gastric cancer cells. However, GKN1 completely restrained these impacts of gastrin on gastric cancer cell proliferation by downregulating the gastrin-CCKBR-growth factor receptor and NF-κB signaling pathways. Moreover, analysis of 55 noncancerous gastric mucosa samples showed an inverse relationship between GKN1 and gastrin, as well as between GKN1 and CCKBR mRNA expression. Expectedly, a positive association between gastrin and CCKBR mRNA transcripts was observed. Although the exact molecular mechanisms regulating the growth-promoting effect of gastrin remain to be fully elucidated, this is the first report that GKN1 may suppress gastrin-induced cell proliferation by downregulating the gastrin-CCKBR-growth factor receptor and NF-κB signaling pathways and thereby contribute to gastric mucosal homeostasis and gastric carcinogenesis. Additional functional and translational studies of GKN1 and gastrin will broaden our understanding of the physiological homeostasis of gastric mucosal cells and provide us with novel diagnostic and therapeutic modalities for preventing gastric cancer.
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A2A2A01002531).
Conflict of interest
The authors disclose no potential conflicts of interest.
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