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. 2012 May 16;153(7):3006–3016. doi: 10.1210/en.2011-2069

Induction of Gastrin Expression in Gastrointestinal Cells by Hypoxia or Cobalt Is Independent of Hypoxia-Inducible Factor (HIF)

Lin Xiao 1, Suzana Kovac 1, Mike Chang 1, Arthur Shulkes 1, Graham S Baldwin 1, Oneel Patel 1,✉
PMCID: PMC3380302  PMID: 22593272

Abstract

Gastrin and its precursors have been shown to promote mitogenesis and angiogenesis in gastrointestinal tumors. Hypoxia stimulates tumor growth, but its effect on gastrin gene regulation has not been examined in detail. Here we have investigated the effect of hypoxia on the transcription of the gastrin gene in human gastric cancer (AGS) cells. Gastrin mRNA was measured by real-time PCR, gastrin peptides were measured by RIA, and gastrin promoter activity was measured by dual-luciferase reporter assay. Exposure to a low oxygen concentration (1%) increased gastrin mRNA concentrations in wild-type AGS cells (AGS) and in AGS cells overexpressing the gastrin receptor (AGS-cholecystokinin receptor 2) by 2.1 ± 0.4- and 4.1 ± 0.3-fold (P < 0.05), respectively. The hypoxia mimetic, cobalt chloride (300 μM), increased gastrin promoter activity in AGS cells by 2.4 ± 0.3-fold (P < 0.05), and in AGS-cholecystokinin receptor 2 cells by 4.0 ± 0.3-fold (P < 0.05), respectively. The observations that either deletion from the gastrin promoter of the putative binding sites for the transcription factor hypoxia-inducible factor 1 (HIF-1) or knockdown of either the HIF-1α or HIF-1β subunit did not affect gastrin promoter inducibility under hypoxia indicated that the hypoxic activation of the gastrin gene is likely HIF independent. Mutational analysis of previously identified Sp1 regulatory elements in the gastrin promoter also failed to abrogate the induction of promoter activity by hypoxia. The observations that hypoxia up-regulates the gastrin gene in AGS cells by HIF-independent mechanisms, and that this effect is enhanced by the presence of gastrin receptors, provide potential targets for gastrointestinal cancer therapy.

Gastrin is a gastrointestinal peptide hormone and growth factor primarily secreted by the G cells within the antral mucosa of the stomach. The different forms of gastrin are active in different tissues, with amidated gastrin (Gamide) acting in the stomach and gastrin precursors such as glycine-extended gastrin (Ggly) acting in the colon (1). Up-regulation of the gastrin gene contributes to gastrointestinal tumorigenesis, and increased expression of gastrin has been shown in colonic adenomatous polyps (2), as well as in colonic and gastric adenocarcinomas (3, 4). The Gamide receptor, cholecystokinin receptor 2 (CCK2R) is also expressed in colonic adenomatous polyps (2), but most gastric and colorectal carcinomas do not express CCK2R (5). Recently gastrin, acting via the CCK2R, has been shown to up-regulate its own expression in the gastric cancer cell line AGS-CCK2R (20). Up-regulation of the gastrin gene accelerates the formation of gastrointestinal tumors and promotes tumor growth, antiapoptosis, angiogenesis, and tissue remodeling (reviewed in Ref. 6).

Hypoxia is a frequent feature of many solid tumors because of rapid expansion and poor vasculature (7). In tumor cells hypoxia increases transcription of approximately 1.5% of genomic genes (8, 9). The pivotal element in hypoxia-induced cellular changes is the formation of the hypoxia-inducible factor 1 (HIF-1), which is a heterodimeric transcription factor consisting of HIF-1α and HIF-1β subunits, first identified by Wang and Semenza (10) more than a decade ago. Synthesis of HIF-1α occurs via oxygen-independent mechanisms but HIF-1α is targeted for degradation by the proteasomal system by an oxygen-dependent process that involves 2-oxoglutarate- and iron-dependent prolyl hydroxylase, asparaginyl hydroxylase and the Von Hippel-Lindau protein (11). Cobalt ions reduce the degradation of HIF-1α by replacing the non-heme iron in the prolyl hydroxylase active site and thereby inhibiting its activity (12). HIF-1 regulates hypoxia-inducible genes by directly binding to the core sequence of the hypoxia-responsive element (HRE) within the regulatory sequences of target genes. Previous research has revealed that HIF-1 increases the expression of several important growth factors, including vascular endothelial growth factor (VEGF), TNF-α, and IGF-2, and hence gives tumor cells a growth advantage under hypoxia (13).

Gastrins have been shown to play a role in angiogenesis. Both Gamide and Ggly increased tubule formation in human endothelial cells, and the effect was mediated via heparin binding-epidermal growth factor (14). The observation that elevated fasting serum Gamide concentrations were correlated with increased heparin binding-epidermal growth factor expression in the normal mucosa at the margin of human colorectal tumors, even though a significant increase was not seen within the tumor itself, suggested that gastrin may increase angiogenic activity close to the tumor (14). Stimulation of human colorectal cancer cell lines with Ggly increased the expression of the proangiogenic factor VEGF at the mRNA and protein levels in the absence of HIF-1 accumulation (15). Grabowska et al. (16) have shown that an internal ribosome binding site in the 5′-untranslated region of the gastrin gene can maintain translation of gastrin peptides under hypoxic conditions even when normal translational mechanisms are inactive.

Although circulating gastrin concentrations are increased after hypoxia in rats (17) and newborn calves (18), to our knowledge there has been no systematic investigation of the effects of hypoxia on the regulation of gastrin in gastrointestinal cancers. In the present study, we investigated regulation of the gastrin gene by hypoxia and by the hypoxia mimetic cobalt chloride (CoCl2) at both the transcriptional and translational levels in gastric and colorectal cancer cell lines. The regulatory sequences within the gastrin promoter were further defined by deletional and mutational analysis.

Materials and Methods

Cell culture

Human gastric adenocarcinoma (AGS) cells were cultured in RPMI 1640 medium (Invitrogen, Mulgrave, Australia). AGS cells stably transfected with the gastrin-CCK2 receptor (AGS-CCK2R cells) were described previously (19). Colon adenocarcinoma cells were cultured in DMEM (Invitrogen). The media were supplemented with 8% fetal bovine serum, 100 U/ml penicillin, 100 pg/ml streptomycin, and 10 mm HEPES (Invitrogen). All cells were maintained at 37 C in a humidified incubator with 95% air and 5% CO2. The hypoxia-treated cells were cultured in the same way as the controls except that the gas phase contained 94% nitrogen (N2), 5% CO2, and 1% O2, with oxygen concentrations monitored and automatically adjusted by an electronic oxygen controller (ProOx Model 110, Biospherix, Redfield, NY). Chemically induced hypoxia was achieved with CoCl2 (Sigma-Aldrich, Sydney, Australia).

RNA preparation and quantitative PCR

Cells were seeded at a density of 2.5 × 105/well in growth media 1 d before the treatment. After treatment total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized from isolated RNA with the SuperscriptTM II First Strand Synthesis system (Invitrogen) and was then used for real time PCR amplification using an ABI PRISM 7700 Sequence Detector (Applied Biosystems, Melbourne, Australia) and TaqMan chemistry. The following primers were used: hGastrin forward, 5′-CCGCAGTGCTGAGGATGAG-3′; hGastrin reverse, 5′-GGAGGTGGCTAGGCTCTGAA-3′, hGastrin MGB probe, 5′-CTAACAATCCTAGAACCAAG-3′. Gastrin gene expression was quantitated relative to 18S RNA expression.

RIA

Amidated gastrin (antiserum 1296) and its precursors glycine-extended gastrin (antiserum 7270) and progastrin (antiserum 1137) were measured with region-specific gastrin antisera using RIA as described previously (20). The cross-reactivity of antisera 7270 and 1137 for amidated gastrin is less than 0.1%.

Plasmid constructs

The gastrin-promoter luciferase vector (1300pGASLuc), which contains 1300 bp upstream promoter region and the first exon of the human gastrin gene, was a kind gift from Professor J. Merchant (University of Michigan, Ann Arbor, MI). The deletional and mutational constructs of the gastrin promoter were generated as described previously (21).

Transfection

Cells were transfected using Lipofectamine LTX or Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Briefly, cells were seeded at a density of 0.5–1.0 × 105 cells/ml 16 h before transfection to obtain an approximately 70–80% cell confluency at the time of transfection. Cells were transfected in 500 μl Opti-MEM, which contained 2 μl of Lipofectamine reagent, 0.5–1 μg of the test gastrin plasmid, and 0.05–0.1 μg Renilla vector pRL-SV40 (Promega). Cells were then incubated for 6 h before the medium was changed to 1 ml/well RPMI supplemented with 8% fetal bovine serum. Cells were incubated at 37 C for a further 24–48 h before treatment.

Reporter gene assay

Promoter activity was determined using a Luciferase Assay kit (Promega, Hawthorn, Australia). Briefly, cells were lysed with 60–70 μl/well 1 × Reporter Lysis Buffer, and firefly luciferase activity was measured with a MicroLumi XS luminometer (Harta Instruments, Gaithersburg, MA). Relative luciferase activity was normalized to the total protein content of each well determined with a Bradford protein assay kit (Bio-Rad, Gladesville, Australia).

Western blot analysis

Cells were washed once with ice-cold PBS and lysed with 0.1–0.2 ml preboiled sodium dodecyl sulfate lysis buffer, followed by protein separation by polyacrylamide gel electrophoresis. Proteins were transferred onto Hybond-C Extra nitrocellulose membrane (GE Healthcare, Rydalmere, Australia). HIF-1α protein was probed with a monoclonal mouse antihuman HIF-1α antibody (1:1000; BD Biosciences, North Ryde, Australia) followed by a secondary goat antimouse antibody (1:5000, Bio-Rad). As a loading control, blots were incubated with a horseradish peroxidase-conjugated rabbit anti-ß-actin or antiglyceraldehyde-3-phosphate dehydrogenase antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Bands were visualized in a LAS 3000 Image Reader (Fujifilm, Brookvale, Australia), with an ECL Advance Western Blotting Detection Kit (GE Healthcare). Protein bands were analyzed densitometrically with MultiGauge software (Fujifilm).

Stable HIF-1α knockdown in AGS cells

Predesigned and validated Mission short hairpin RNA (shRNA) plasmids for human HIF-1α [clone numbers TRCN0000003810 and TRCN0000010819, which each encode a hairpin-type small interfering RNA (siRNA)] and negative control (SHC002) were purchased from Sigma-Aldrich. The cells were transfected with either Mission HIF-1α shRNA plasmid or the negative control plasmid using the Neon transfection method (Invitrogen). Briefly, 1 × 106 cells were trypsinized, washed with PBS, and pelleted before resuspension in 100 μl Neon Resuspension Buffer. HIF-1α (5 μg) or control shRNA plasmid was added and well mixed into the cell suspension before transfection. The transfected cells were seeded in complete medium and selected with 1.0 μg/ml puromycin (Sigma-Aldrich) for 7 d before further assays.

Transient HIF-1β knockdown in AGS cells

Cells were transfected with siRNA targeting two different regions within the HIF-1β coding sequence (SASI_Hs01_00166998 and SASI_Hs01_00167000, Sigma-Aldrich) using the Neon transfection method (Invitrogen). Briefly, 1 × 106 cells were trypsinized, washed with PBS, and pelleted before resuspension in 100 μl Neon resuspension buffer. Five micrograms of 365pGASLuc and 500 ng of Renilla vector pRL-SV40 together with either 100 nm of each of HIF-1β siRNA or 200 nm of control lamin A/C siRNA were transfected into the AGS cells. The transfected cells were seeded in complete medium and incubated for 24 h before further treatment with either 1% O2 or 300 μm CoCl2. VEGF was measured with an ELISA kit (DuoSet VEGF ELISA; R&D Systems, Minneapolis, MN).

Statistical analysis

Data are presented as means ± sem. Statistical significance for single comparisons of normally distributed data were determined by Student's t test or, for data that was not normally distributed, determined by Mann-Whitney rank sum test. For multiple comparisons, one-way ANOVA followed by the Bonferroni correction were performed. All statistics were analyzed with the program SigmaStat (Jandel Scientific, San Rafael, CA).

Results

AGS gastric carcinoma cells were chosen for the following experiments because previous studies have established that gastrin gene expression can be induced in AGS cells by multiple factors, including epidermal growth factor and cAMP (22). Because AGS cells do not normally express functional CCK2R (23), AGS cells stably transfected with the CCK2R (AGS-CCK2R cells) were also included to determine the role of the CCK2R (21).

Hypoxia and CoCl2 induce HIF-1α protein and gastrin mRNA

HIF-1α concentrations were measured in AGS and AGS-CCK2R cells after exposure to 1% O2 (referred to hereafter as hypoxia) or the hypoxia mimetic, 300 μm CoCl2, for 16 h in the presence or absence of the CCK2R antagonist RP73870A (50 nm). Hypoxia increased HIF-1α concentrations in both AGS and AGS-CCK2R cells by 3.2 ± 1.2- and 3.5 ± 0.8-fold, respectively, compared with the untreated controls (Fig. 1, A and B). CoCl2 induced a greater increase in HIF-1α concentrations than hypoxia with an 11.5 ± 3-fold increase in AGS cells and a 12.2 ± 1.5-fold increase in AGS-CCK2R cells. Treatment of AGS and AGS-CCK2R cells with the CCK2R antagonist had no effect on HIF-1α induction.

Fig. 1.

Fig. 1.

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Hypoxia or CoCl2 increases HIF-1α protein and gastrin mRNA expression. Western blots (A) and densitometric analysis (B) of HIF-1α protein expression in AGS (black bars) and AGS-CCK2R cells (gray bars) after treatment with 1% O2 or 300 μm CoCl2 for 16 h in the presence (hatched bars) or absence (plain bars) of CCK2R antagonist (50 nm). Glyceraldeyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. C, Gastrin mRNA expression in AGS and AGS-CCK2R cells after treatment with 1% O2 or 300 μm CoCl2 for 16 h in the presence (hatched bars) or absence (plain bars) of CCK2R antagonist. Gastrin mRNA concentrations were measured using real-time PCR and normalized to the amount of 18S rRNA. Values are expressed as the fold increase compared with untreated cells and are the mean ± sem of at least three separate treatments. *, P < 0.05 vs. untreated control; #, P < 0.05 vs. treated AGS cells. D, In experiment 1 proliferation of AGS cells was assessed by counting cell numbers after exposure to 1% O2 (gray bars) or 300 μm CoCl2 (black bars) for 24 h in a serum-free medium (white bars). In experiment 2 proliferation of quiescent AGS cells was assessed under normoxic conditions after exposure to the three conditioned media from experiment 1. Values are expressed as a percentage of untreated cells (experiment 1) or as a percentage of cells treated with control conditioned medium (experiment 2) and are the mean ± sem of three separate treatments. *, P < 0.05 vs. control.

Treatment of AGS or AGS-CCK2R cells with hypoxia or 300 μm CoCl2 also induced a parallel increase in gastrin mRNA concentrations (Fig. 1C). The 4.9 ± 0.8-fold increase in gastrin mRNA expression induced by CoCl2 was greater than the 2.1 ± 0.4-fold increase stimulated by 1% O2 in AGS cells. A similar trend was also observed in AGS-CCK2R cells, in which the increase in gastrin mRNA transcription stimulated by CoCl2 was 10.1 ± 1.5-fold in contrast to the 4.1 ± 0.3-increase stimulated by 1% O2. Further the greater fold increase in gastrin mRNA stimulated by CoCl2 and 1% O2 in AGS-CCK2R compared with AGS cells can be attributed to the previously identified positive feedback loop, whereby any secreted gastrin will bind to the CCK2R and stimulate a further increase in gastrin expression (21). To test this hypothesis the stimulation of gastrin mRNA by 1% O2 or CoCl2 in AGS-CCK2R and AGS cells was measured in the presence of the CCK2R antagonist. Gastrin expression in response to 1% O2 or CoCl2 in antagonist-treated AGS-CCK2R cells was significantly lower (1.8 ± 0.1- or 4.6 ± 1.1-fold, respectively) than in untreated cells (4.1 ± 0.3-fold or 10.1 ± 1.5-fold, respectively) (Fig. 1C).

Effect of hypoxia on cell proliferation

To determine whether exposure of AGS cells to hypoxia had any effect on proliferation, cell numbers were counted after the incubation of AGS cells under hypoxic conditions for 24 h. Neither 1% O2 nor 300 μm CoCl2 had any effect on proliferation of AGS cells (Fig. 1D). To investigate whether hypoxia induced the secretion of growth factor(s) into the medium, AGS cells were treated with 1% O2 or 300 μm CoCl2 for 24 h, and conditioned medium was collected. Medium conditioned by AGS cells treated with 1% O2, but not 300 μm CoCl2, significantly increased proliferation of quiescent AGS cells under normoxic conditions by 166 ± 28% compared with medium conditioned by untreated AGS cells (Fig. 1D).

Hypoxia and CoCl2 stimulate expression of amidated and nonamidated gastrin

To confirm that the increased transcription leads to an increase in cellular expression as well as secretion of gastrin peptides, the concentrations of Gamide, Gly, and progastrin in cell extracts and in conditioned media from AGS and AGS-CCK2R cells were measured after a 16-h incubation in 1% O2 or in the presence of 300 μm CoCl2 (Fig. 2). In agreement with the observed increase in gastrin mRNA, after CoCl2 treatment the Gamide concentration in the conditioned media and in the cell extracts from AGS cells increased by 26.2 ± 14.3 and 12.2 ± 7.3-fold, respectively (Fig. 2, A and B). Similarly, treatment of AGS cells with 1% O2 led to a 1.9 ± 0.2-fold increase in the Gamide concentration in the conditioned medium. However, the greater increase in gastrin mRNA in AGS-CCK2R cells after hypoxia or CoCl2 treatment did not result in a correspondingly greater increase in secreted or stored Gamide. Treatment of AGS and AGS-CCK2R cells with CoCl2 led to nearly 2-fold increases in Ggly expression in conditioned medium and cell extract, but there was no significant increase in Ggly expression after 1% O2 treatment in either cell type (Fig. 2, C and D). As with Gamide, treatment of AGS cells with 1% O2 led to a greater increase (6.3 ± 3.5-fold) in the progastrin concentration in the conditioned medium than with AGS-CCK2R cells (1.2 ± 0.5-fold). There was no significant increase in cellular progastrin in either cell type (Fig. 2F).

Fig. 2.

Fig. 2.

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Hypoxia or CoCl2 increases gastrin peptide expression. Concentrations of Gamide (A and B), Ggly (C and D), and progastrin (E and F) in conditioned medium (A, C, and E) and cell extracts (B, D, and F) after treatment of AGS (black bars) or AGS-CCK2R (gray bars) cells with 1% O2 or 300 μm CoCl2 for 16 h were measured by RIA. Values are expressed as the mean ± sem of at least three separate experiments. Because there was large variation in the peptide concentration in the untreated control between each separate experiment, the data are expressed as the fold increase in peptide concentration. *, P < 0.05 vs. untreated control. Gamide concentrations in the conditioned medium of untreated AGS and AGS-CCK2R cells were 1.9 ± 0.9 and 2.9 ± 1.1 fmol/ml/million cells, respectively, and in cell extracts were 4.4 ± 1.1 and 3.6 ± 1.8 fmol/million cells, respectively. Ggly concentrations in the conditioned medium of untreated AGS and AGS-CCK2R cells were 12.1 ± 3.5 and 14.3 ± 4.9 fmol/ml/million cells, respectively, and in cell extracts were 19.4 ± 5.6 and 22.7 ± 6.5 fmol/million cells, respectively. Progastrin concentrations in the conditioned medium of untreated AGS and AGS-CCK2R cells were 0.52 ± 0.35 and 0.5 ± 0.4 fmol/ml/million cells, respectively, and in cell extracts were 9.5 ± 1.9 and 4.7 ± 1.2 fmol/million cells, respectively. #, P < 0.05 vs. treated AGS cells.

HIF expression is stimulated, and the gastrin promoter activated, in AGS cells by CoCl2 in a dose-dependent manner

Treatment with CoCl2 induced HIF-1α expression in a dose-dependent manner in both AGS and AGS-CCK2R cell lines (Fig. 3, A and B). However, there was no significant difference in the level of HIF-1α induction in AGS cells compared with AGS-CCK2R cells.

Fig. 3.

Fig. 3.

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Gastrin promoter activity is increased in a dose-dependent fashion by CoCl2. Western blots (A) and densitometric analysis (B) of HIF-1α protein expression in AGS (black bars) or AGS-CCK2R (gray bars) cells after treatment with the indicated concentrations of CoCl2 for 16 h. Actin was used as a loading control. C, AGS or AGS-CCK2R cells were cotransfected with the gastrin promoter construct 1300pGASLuc and were treated with increasing concentrations of CoCl2 in serum-free medium for 16 h. The promoter activity was calculated as the fold increase of relative luciferase activity compared with untreated control for each cell line. Values are expressed as the mean ± sem of at least three separate treatments. *, P < 0.05 vs. untreated control; #, P < 0.05 vs. treated AGS cells.

To examine the stimulatory effects of CoCl2 on the gastrin gene, luciferase reporter assays were performed using a gastrin promoter construct carrying 1300 bp of the 5′-flanking region of the human gastrin gene. Treatment of transfected AGS and AGS-CCK2R cells with CoCl2 significantly stimulated the gastrin promoter activity in a dose-dependent manner (Fig. 3C). Treatment of AGS-CCK2R cells with 300 μm CoCl2 caused a significantly greater increase (4.0 ± 0.3-fold) in gastrin promoter activity in contrast to AGS cells where the maximal stimulation was 2.4 ± 0.3-fold. A similar difference in the increase in gastrin mRNA was attributed to a gastrin-CCK2R positive feedback loop.

Putative HRE sequences are not responsible for gastrin gene activation by 1% O2 or CoCl2

Because gene activation by hypoxia often involves the binding of HIF-1 to a hypoxia response element (HRE: 5′-CGTG-3′) within the regulatory sequence of the gene, the 1300-bp gastrin promoter region was screened to identify HRE sequences. Seven potential HIF binding sites (HBS) were identified in this region either in the forward (5′-CGTG-3′) or reverse (5′-CACG-3′) orientation (Fig. 4A). To determine whether hypoxia or CoCl2 responsiveness was mediated via putative HBS in the gastrin promoter, short fragments of the gastrin promoter containing progressively fewer putative HBS were cloned in the pGL3 reporter vector (Fig. 4A). The deletion constructs were transiently transfected into AGS cells, which were then treated with 1% O2 or 300 μm CoCl2 (Fig. 4B). The promoter activity of the deletion constructs was not statistically different from that of the longest gastrin promoter construct 1147pGASLuc. In particular, the observation that there was no reduction in activity in the 365pGASLuc construct, which contains no putative HBS, indicated that activation of the gastrin gene by hypoxia was independent of the binding of HIF-1α to the proximal gastrin promoter.

Fig. 4.

Fig. 4.

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Deletion of putative HBS in the gastrin promoter does not affect induction of the gastrin promoter by hypoxia. A, 5′-deletions of the gastrin promoter (1147pGASLuc, 948pGASLuc, 666pGASLuc, 365pGASLuc) were constructed, in which the putative HIF-1 binding sites (HBS, black vertical lines) were progressively deleted. Numbering is from the transcription start site in the gastrin promoter. B, Promoter activities of the deletion constructs were measured after transient transfection in AGS cells, and treatment with either 1% O2 (gray bars) or 300 μm CoCl2 (black bars) for 16 h. The firefly luciferase activity of each construct was normalized to Renilla luciferase activity and expressed as the fold increase relative to the corresponding untreated construct. The basal luciferase activity in the cells transfected with the pGL3 empty vector was 0.8 ± 0.2% of the value for 365pGASLuc transfectants. Although the luciferase activity of the empty vector was increased by hypoxia or CoCl2, the magnitude of the change (1.7% and 2.1%, respectively) was much less than with the 365pGASLuc construct. *, P < 0.05 vs. untreated control.

HIF is not responsible for hypoxic activation of the gastrin gene

To establish whether HIF-1α has any indirect role in stimulating the gastrin promoter, HIF-1α expression in AGS cells was reduced by stable transfection with shRNA vectors. Knockdown of HIF-1α expression was confirmed by Western blot (Fig. 5, A and B). The AGS cells that had been stably transfected with either control shRNA (AGS HIF WT) or HIF knockdown shRNA (AGS HIF KD) were then transiently transfected with the 365pGASLuc construct and treated with 1% O2 or 300 μm CoCl2. The observation that the gastrin promoter activity after either treatment in AGS HIF KD cells was not statistically different from the activity in AGS HIF WT cells that had undergone the same treatment (Fig. 5C) indicated that hypoxic activation of the gastrin gene was not dependent on any HIF-1α involvement either direct or indirect.

Fig. 5.

Fig. 5.

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Activation of the gastrin promoter is not dependent on HIF. Western blots (A) and densitometric analysis (B) of HIF-1α protein expression in control vector-transfected (AGS HIF WT, black bars) and HIF-1α shRNA vector-transfected (AGS HIF KD, gray bars) cells after treatment with 1% O2 or 300 μm CoCl2 for 16 h. HIF-1α protein expression was normalized to Glyceraldeyde-3-phosphate dehydrogenase (GAPDH), and expressed as the fold increase relative to untreated cells. C, AGS HIF WT or AGS HIF KD cells were transfected with the 365pGASLuc construct and treated with either 1% O2 or 300 μm CoCl2 for 16 h. The relative luciferase activity was normalized to the total protein concentration and expressed as the fold increase relative to untreated controls. D, AGS cells were transiently transfected with the 365pGASLuc construct in combination with either 200 nm HIF-1β siRNA or lamin A/C siRNA and treated with 1% O2 or 300 μm CoCl2 for 16 h. The relative luciferase activity was normalized to the total protein concentration and expressed as the fold increase relative to untreated controls. E, VEGF was measured in the medium conditioned by AGS cells transfected with either 200 nm HIF-1β siRNA or lamin A/C siRNA. Values are expressed as the mean ± sem of at least three separate treatments. *, P < 0.05 vs. untreated control; #, P < 0.05 vs. treated AGS HIF WT cells or AGS lamin A/C siRNA cells. WT, Wild type; KD, knockdown.

To eliminate the possible involvement of all three isoforms of HIF (HIF-1α, HIF-2α, and HIF-3α) in the stimulation of gastrin expression by hypoxia, gastrin promoter activity was measured after transient transfection of AGS cells with HIF-1β siRNA. Knockdown of HIF-1β, which is a component of all forms of HIF, had no effect on the stimulation of gastrin promoter activity by either 1% O2 or 300 μm CoCl2 (Fig. 5E). HIF-1β knockdown was confirmed by the observation that HIF-1β siRNA completely blocked the increase in VEGF in response to either 1% O2 or 300 μm CoCl2 (Fig. 5E).

GC-rich regions are not involved in hypoxic activation of the gastrin gene

The 365-bp gastrin promoter region encompasses two GC-rich regions that are responsible for basal gastrin gene expression (24). In addition, the binding of the transcription factor Sp1 to the more 3′-GC-rich region is responsible for epidermal growth factor-stimulated gastrin promoter activity in AGS cells (25, 26). To investigate the role of Sp1 in hypoxia-induced gastrin gene expression, the GC boxes were therefore mutated singly or in combination (Fig. 6A). However, mutation of either one or both of the GC boxes in the minimal promoter region did not affect gastrin promoter activity upon stimulation with 1% O2 or CoCl2 (Fig. 6B). These observations suggest that Sp1 is not involved in the hypoxic activation of the gastrin gene in AGS cells.

Fig. 6.

Fig. 6.

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Activation of the gastrin promoter is not dependent on Sp1. A, Site-directed mutagenesis of the GC boxes in the promoter construct 365pGASLuc. The wild-type construct contains two perfect GC sequences (GGGCGGG), and the corresponding mutated sequences are shown above each GC box. B, AGS cells were transfected with the wild-type 365pGASLuc construct, the GC 1 mutation construct, the GC 2 mutation construct, or the double-GC mutation construct. Transfected cells were treated with either 1% O2 (gray bars) or 300 μm CoCl2 (black bars) for 16 h. Values (mean ± sem of at least three separate treatments) are expressed relative to the untreated wild-type 365pGASLuc construct. *, P < 0.05 vs. untreated control.

Hypoxic activation of the gastrin gene in colorectal cancer cell lines

To investigate whether the hypoxic activation of gastrin mRNA expression was tissue or cell specific, gastrin mRNA was measured in the colorectal carcinoma cell lines Lovo, DLD-1, and Colo320, and in Colo320 cells transfected with a plasmid encoding the CCK2R, after incubation in a 1% O2 environment. The maximal stimulation of 6.9 ± 0.5-fold in gastrin mRNA expression was observed with LoVo cells (Fig. 7). Although LoVo cells have been reported to express endogenous CCK2R (27), no receptors were detected with radiolabeled CCK8 ligand (data not shown). Gastrin mRNA expression was stimulated 2.0 ± 0.5-fold by 1% O2 in Colo320 cells transfected with CCK2R cells; however, 1% O2 failed to stimulate any significant gastrin expression in wild-type Colo320 cells. As with AGS-CCK2R cells, the higher increase in Colo320-CCK2R cells may be attributed to a positive gastrin feedback loop.

Fig. 7.

Fig. 7.

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Effect of hypoxia on gastrin mRNA expression in CRC cell lines. Cells were treated with 1% O2 for 16 h, and gastrin mRNA was measured by real time PCR and expressed relative to the amount of 18S rRNA. Values (mean ± sem of at least three separate treatments) are expressed as the fold increase relative to the corresponding untreated cells. *, P < 0.05 vs. untreated control.

Discussion

Up-regulation of the gastrin gene in tumor cells can be a direct consequence of intrinsic factors such as tumor suppressor or oncogene mutations. For example, mutations in the APC and K-ras genes are associated with gastrin gene overexpression in colon adenocarcinoma (28, 29). However, the effects of hypoxia on gastrin gene regulation in tumors remain largely unexplored. In this study, we have shown, for the first time, that hypoxia induces gastrin expression at the promoter, mRNA, and protein levels, and that this hypoxic induction of the gastrin gene is observed in both gastric (AGS) and some colorectal cancer cell lines (LoVo). This hypoxic induction of gastrin expression has biological significance because gastrin is a stimulant of angiogenesis and proliferation in human gastrointestinal tumors (6).

Although hypoxia itself had a modest effect (2.1 ± 0.4-fold increase) on gastrin expression in wild-type AGS cells, the presence of CCK2R on these cells led to a more pronounced stimulation of promoter activity and subsequent gastrin mRNA transcription (Fig. 1C). The difference in the magnitude of stimulation of gastrin expression in CCK2R-positive and -negative cells is likely due to the presence of a previously described positive feedback loop involving the CCK2R (21). The greater increase in gastrin expression in CCK2R-positive cells may be due to the fact that Gamide secreted as a result of hypoxic stimulation can then activate CCK2R and further stimulate gastrin mRNA transcription. Furthermore the inhibition of this amplified gastrin increase by a CCK2R antagonist in AGS-CCK2R cells confirmed that the increase in gastrin expression in AGS-CCK2R cells is mediated by a positive feedback loop involving the CCK2R. The fact that the concentration of Gamide in medium conditioned by AGS-CCK2R cells is lower than in medium conditioned by AGS cells (Fig. 2A) may be attributed to binding of Gamide to the CCK2R, rapid endocytosis of the Gamide-CCK2R complex, and lysosomal degradation of Gamide, as previously reported in this cell line (30). The observation that there was no significant difference in the concentration of Ggly in media conditioned by AGS-CCK2R or AGS cells is consistent with the fact that Ggly does not bind to CCK2R and cannot be endocytosed by the same mechanism (Fig. 2B).

Expression of CCK2R in gastrointestinal tumors has been controversial. The consensus that has emerged is that, although most gastric and colorectal carcinomas do not express CCK2R, the CCK2R is found on polymorphs within the tumor stroma (5, 31, 32). In contrast the CCK2R is often overexpressed by neuroendocrine and gastrointestinal stromal tumors (31). Gastric, colorectal, and pancreatic cancers also express gastrin transcripts and produce significant amounts of progastrin and its processing intermediates (Ggly) (31). The observations that Gamide can induce the expression of gastrin mRNA (33) and of CCK2R mRNA (34) highlights the possibility that even a small increase in Gamide concentration in response to an external signal such as hypoxia could lead to a significant increase in gastrin mRNA transcription via the Gamide/CCK2R-positive feedback loop in cells expressing the CCK2R. The possible involvement of the Gamide/CCK2R-positive feedback loop in the overexpression of nonamidated forms of gastrin and their role in cancer progression clearly warrants further investigation.

Using reporter gene assays, previous studies have demonstrated that a functional HBS in a HIF-1α-inducible gene is essential for promoter responsiveness to hypoxia (35–37). Although both chemical hypoxia and true hypoxia increase HIF-1α protein concentrations in AGS cells, the gastrin promoter activity is independent of direct HIF-1α binding because hypoxic stimulation of the gastrin gene was not affected by deletion of all the putative HBS in the gastrin promoter (Fig. 4). Furthermore, the observation that induction of the gastrin promoter by either chemical hypoxia or true hypoxia is not affected by HIF-1α knockdown in AGS cells (Fig. 5C) confirms that hypoxic regulation of the gastrin gene is independent of HIF-1α. This finding is consistent with a recent report that the stimulatory effect of Ggly on the expression of the proangiogenic factor VEGF is HIF independent (15). The fact that deletion of all the HBS did not affect induction of the gastrin promoter by hypoxia further suggests that other members of the family of hypoxia-inducible transcription factors (i.e. HIF-2α and HIF-3α) are not involved in direct regulation of gastrin promoter because all HIFs bind to the same core recognition sequence (5′-CGTG-3′) (38). Furthermore, the observation that HIF-1β knockdown did not affect the inducibility of the gastrin promoter in AGS cells (Fig. 5D) suggests that stimulation of gastrin expression by hypoxia is independent of any indirect involvement of HIF-2α and HIF-3α. We conclude that the inducibility of gastrin by hypoxia in AGS cells is independent of any direct or indirect involvement of HIF.

In addition to HIF, several other transcription factors have been shown to participate in gene activation by hypoxia (39–41), and several genes are activated by HIF-1α-independent pathways (42–44). In the gastrin promoter, two GC-rich elements, which are essential for constitutive expression of the gastrin gene (24) and are involved in activation of the gastrin gene by epidermal growth factor, have been previously identified (45). Both of the GC boxes bind the Sp1 transcription factor, a regulator that has been suggested in recent years to be frequently involved in the cellular hypoxic response (46). However, our results suggest that GC elements in the gastrin promoter are not responsible for hypoxic activation of the gastrin gene because mutation of one or both of these GC elements did not change the induction of the gastrin promoter by either chemical hypoxia or true hypoxia.

Previously it has been shown that gastrin expression is increased after hypoxia in rats (17) and in newborn calves (18). Because oral administration of HCl inhibited the rise of gastrin concentration induced by hypoxia in rats, the authors concluded that gastrin release was secondary to the rise in gastric pH due to hypoxia (17). However, hypoxia may be affecting many other pathways, and in fact the in vitro data presented here indicate that there is also a direct effect of hypoxia on gastrin promoter activity and subsequent transcription in vitro. Because the culture medium used in our experiments was supplemented with 25 mm HEPES buffer (pH 7.4), the possibility that the increase in gastrin expression is due to a change in the pH of culture medium can be eliminated. Further work will be required to establish the relative contributions of the direct and gastric pH-dependent hypoxic effects in vivo.

Iron is an integral part of the porphyrin ring of the heme in hemoglobin and hence is essential for supplying cells and tissues with oxygen. Several studies have established that there are tight links between iron availability and oxygen delivery and that iron deprivation and oxygen deprivation (i.e. hypoxia) have very similar consequences at the molecular level (47). Under hypoxia, the expression of the major genes responsible for iron homeostasis, including hepcidin, transferrin, transferrin receptor, ceruloplasmin, and heme oxygenase-1, provides increased iron availability for erythropoiesis in an attempt to enhance the uptake of oxygen and its delivery to the hypoxic cell. The possibility that in normal physiology gastrins may play an important role in increasing the availability of iron under hypoxia is supported by the fact that there is a clear relationship between gastrin expression and iron homeostasis (33, 48).

Chemical hypoxia appears to differ from true hypoxia (49). Thus the magnitude of gastrin induction at all three levels of regulation [i.e. promoter activity (Fig. 4), mRNA (Fig. 1C), and protein (Fig. 2)] was much higher in the case of cobalt chloride compared with hypoxia. Transition metal ions such as cobalt, nickel, and manganese can substitute for ferrous ions in many iron-containing proteins, and this substitution also forms the molecular basis of HIF-1α induction by Co2+ ions (50). The greater magnitude of gastrin stimulation by the hypoxia mimetic CoCl2 compared with true hypoxia in AGS cells may be due to multiple additional pathways activated as a result of the interference by Co+2 ions with various iron-containing enzymes and proteins. Our study is in agreement with the previous conclusion that chemical hypoxia is quite different from true hypoxia (49), and further studies are warranted to understand the different mechanisms involved.

In conclusion, our study has demonstrated, for the first time, that hypoxia up-regulates the promoter activity of the gastrin gene in AGS cells and that this regulation is HIF independent. The hypoxic effects are amplified in the presence of the gastrin-CCK2R via positive feedback mechanisms. Further studies are required to identify the regulatory elements in the promoter region responsible for increased gastrin transcription. The fact that most tumors have regions of hypoxia and that hypoxia stimulated gastrin production may provide potential targets for novel treatments of gastrointestinal cancers.

Acknowledgments

We thank Mildred Yim (University of Melbourne, Heidelberg, Australia) for performing the gastrin RIA.

This work was supported by grant 5 RO1 GM065926 from the National Institutes of Health (to G.S.B., A.S.), and grants 454322 (to G.S.B.) and 566555 (to G.S.B., A.S.) and 628390 (to A.S., O.P., G.S.B.) from the National Health and Medical Research Council of Australia.

Author Contributions: A.S., G.S.B., O.P.: study concept and design; L.X., S.K., M.C.: acquisition of data; L.X., S.K., M.C., O.P.: analysis of data; L.X., O.P.: drafting of the manuscript; All authors: critical revision of the manuscript.

Disclosure Summary: The authors declare no competing interests.

Footnotes

Abbreviations:
CCK2R
Cholecystokinin receptor 2
Gamide
amidated gastrin
Ggly
glycine-extended gastrin
HBS
HIF binding site
HIF-1
hypoxia-inducible factor 1
HRE
hypoxia-responsive element
shRNA
short hairpin RNA
siRNA
small interfering RNA
VEGF
vascular endothelial growth factor.

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