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Am J Physiol Cell Physiol 290: C592-C600, 2006. First published October 5, 2005; doi:10.1152/ajpcell.00278.2005
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RECEPTORS AND SIGNAL TRANSDUCTION

Hypoxia induces epithelial amphiregulin gene expression in a CREB-dependent manner

Susan M. O’Reilly,1 Martin O. Leonard,1 Niamh Kieran,1 Katrina M. Comerford,1 Eoin Cummins,1 Marc Pouliot,2 Sean B. Lee,3 and Cormac T. Taylor1

1UCD Conway Institute, University College Dublin, Dublin, Ireland; 2Centre de Recherche en Rhumatologie-Immunologie du Centre Hospitalier de l’Université Laval, Quebec City, Quebec, Canada; and 3Genetics of Development and Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland

Submitted 21 June 2005 ; accepted in final form 29 September 2005


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hypoxia occurs during a number of conditions in which altered epithelial proliferation is critical, including tumor development. Microarray analysis of colon-derived epithelial cells revealed a hypoxia-dependent increase in the expression of amphiregulin, an EGF receptor (EGFR) ligand that activates epithelial proliferation and has been associated with the development of colonic tumors. Amphiregulin expression was also induced in tissues from mice exposed to whole animal hypoxia. The hypoxic upregulation of amphiregulin was independent of the classic transcriptional response mediated via hypoxia-inducible factor (HIF)-1{alpha}. Transfection of HeLa cells with truncated amphiregulin promoter reporter constructs revealed that a 37-bp segment upstream from the TATA box retained hypoxic sensitivity. This sequence contains an evolutionarily conserved cAMP response element (CRE) that constitutively binds the CRE binding protein (CREB). Deletion of the CRE abolished sensitivity to hypoxia. Thus hypoxia promotes intestinal epithelial amphiregulin expression in a CRE-dependent manner, an event that may contribute to increased proliferation. These data also further support a role for CREB as an HIF-independent hypoxia-responsive transcription factor in the regulation of intestinal epithelial gene expression.

adenosine 3',5'-cyclic monophosphate response element binding protein; transcription; cancer

CELLULAR HYPOXIA, which occurs when metabolic oxygen demand exceeds supply, is associated with a diverse array of pathophysiological processes, including inflammation, ischemia, and cancer (16, 50). Mammalian cells adapt to hypoxia through regulation of gene expression (16). Classically, hypoxia activates hypoxia-inducible factor (HIF)-1 to increase expression of adaptive genes such as VEGF, adrenomedullin, and erythropoietin (12, 29, 40, 41, 54). The activation of these and other genes leads to the induction of an adaptive phenotype with the promotion of angiogenesis, vasodilatation, erythropoiesis, and glycolysis, all of which aid tissue survival (30, 42). Furthermore, tissue-specific adaptive genes such as intestinal trefoil factor (ITF) and CD73 that protect barrier function are upregulated in an HIF-1-dependent manner in intestinal epithelial cells (13, 47). A primary component of the adaptive response to hypoxia is the induction of soluble factors that alter cellular proliferative responses in physiological processes such as angiogenesis and wound healing; however, such responses may also support tumor development through increased proliferation.

While investigating global gene expression in intestinal epithelial cells in response to hypoxia, we (13) identified increased expression levels of known HIF-1-dependent genes such as adrenomedullin and ITF (14, 33). However, this work also revealed increased expression of a number of genes not previously associated with HIF-1-dependent regulation. These included amphiregulin, a glycoprotein that binds to and activates the EGF receptor (EGFR) to regulate growth and proliferation of epithelial, smooth muscle, and neural stem cells (10, 19). A role for amphiregulin has been proposed in wound healing and tumor development, two events in which hypoxia is a vital component (35, 39, 44). Increased amphiregulin expression has been associated with a number of tumors (8, 28, 34); however, the mechanism of transcriptional upregulation is less well understood. Recent work has demonstrated the upregulation of amphiregulin in bronchial epithelial cells in response to fine particulate matter in a manner inhibited by antioxidant treatment (3). Lee et al. (24) also demonstrated a role for the Wilms tumor suppressor transcription factor (WT1) in the control of amphiregulin expression in renal development.

An evolutionarily conserved cAMP response element (CRE) sequence exists close to the transcriptional start site of the amphiregulin gene. We recently (6, 49, 50) demonstrated a role for the CRE binding protein (CREB) in controlling hypoxia-regulated gene expression. CREB was initially described as a signal-dependent activator of genes such as somatostatin, via PKA-dependent phosphorylation of Ser133 (1, 31, 32). However, we (6, 49, 51) and others (56) have also reported that CREB may act as a repressor for genes including TNF-{alpha}. Thus CREB plays an ambiguous role in the regulation of transcription that appears to be gene specific. In each case, however, CREB binds to an 8-bp palindrome (5'-TGACGTCA-3') known as the CRE that regulates transcription.

Hypoxic sensitivity of the amphiregulin promoter is retained in a short fragment directly upstream from the TATA box that contains a sole CRE motif. Interestingly, this motif was recently demonstrated to mediate amphiregulin expression in response to prostaglandin E2 (43). We previously demonstrated (49, 51) that hypoxia alters CREB expression and subsequently transcriptional activity in intestinal epithelial cells, an event dependent on decreased activity of protein phosphatase-1{gamma}.

Thus hypoxia activates intestinal epithelial amphiregulin expression in transformed cells in a manner dependent on functional activity of a CRE motif situated 37 bp upstream of the transcriptional start site. We believe that hypoxia-elicited amphiregulin expression may contribute to continued cell proliferation in focal areas of hypoxia found in a range of epithelial tumors. Furthermore, this work highlights the role of CREB as a hypoxia-responsive transcriptional regulator that can function independently of the classic HIF-1-dependent pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and hypoxia. T84 intestinal epithelial cells were grown in a 1:1 mixture of Dulbecco’s/Vogt’s modified Eagle’s medium and Ham’s F-12 medium (Sigma, St. Louis, MO) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5% fetal calf serum (GIBCO). HeLa cells were grown in Dulbecco’s minimum essential medium (Sigma) containing 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml nonessential amino acids, and 5% fetal calf serum. Monolayers were subcultured from flasks every 7 days using trypsinization (0.1% trypsin and 0.9 mM EDTA in Ca2+- and Mg2+-free PBS). Cells were exposed to hypoxia on 60-mm tissue culture dishes under standard hypoxic conditions [PO2 < 10 Torr (1 Torr = 133 Pa), PCO2 35 Torr, with a balance of N2 and water vapor (48)] in a hypoxia chamber (Coy Labs). Normoxic controls were exposed to atmospheric O2 concentrations (PO2 147 Torr and PCO2 35 Torr within a tissue culture incubator).

Animal model of hypoxia. Tissues were obtained from Dr. Sean Colgan, Brigham and Women’s Hospital, Boston, MA. Mice were exposed to hypoxia (8% atmospheric oxygen) for 0–4 h as described previously (51). This protocol was in accordance with National Institutes of Health guidelines for the use of live animals and was approved by the Institutional Animal Care and Use Committee at Brigham and Women’s Hospital, Boston, MA. The receipt of these tissues was approved by the Animal Ethics Committee of The Conway Institute. After being exposed, colonic mucosal tissue was removed, dissected along the mesenteric border, washed, and snap frozen for further analysis.

Gene array analysis. Microarray analysis was carried out as described previously (27, 51) using T84 cells exposed to hypoxia (0, 6, and 18 h) with the balance of time in normoxia. The mRNA profile was assessed using quantitative gene chip expression arrays (Affymetrix, Santa Clara, CA; Ref. 27). This array experiment was carried out once, and any genes identified as putatively hypoxia inducible were confirmed by PCR and Western blot analysis as described below.

Western blotting. Western blotting was carried out with standard techniques as described previously (49). CREB was detected using immunoblotting with an anti-CREB antibody that recognizes total CREB levels (Cell Signaling). Phospho-EGFR was detected using a panel of antibodies that recognize different sites of phosphorylation (Biosource).

Transient transfection of cells. HeLa cells were grown to 90% confluence and transfected with 2 µg of amphiregulin promoter-luciferase reporter construct pGL2[C] (24). For HIF-1 and mutant HIF-1 overexpression, cells were transfected as described previously (25). In cotransfection studies, HeLa cells were transfected with 2 µg of the reporter pGL2[C] with or without 100, 200, or 400 ng of pCMV-CREB wild-type expression vector or pCMV-KCREB mutant expression vector (Clontech). The total amount of DNA in each transfection was 2.4 µg (pGL2Basic was used to supplement the total DNA to this level). All transfections were carried out with Effectene transfection reagent (Qiagen) according to the manufacturer’s guidelines. After transfection, the cells were incubated overnight at 37°C in a humidified atmosphere of 5% CO2. Fresh medium was then added to the cells. Forty-eight hours after transfection the cells were treated with normoxic or preequilibrated hypoxic (1% O2) medium and returned to the growth conditions described above. PMA (100 nM) treatment was for 8 h. After incubation of the cells in normoxic or hypoxic conditions for 22 h, whole cell extracts were harvested. Medium was aspirated, and cells were washed with 2 ml of 1x PBS (ice cold). Two hundred microliters of 1x lysis buffer (Stratagene) were added for 2–3 min at room temperature. The cells were then scraped, transferred into a microfuge tube, and placed on ice. Each sample was sonicated for 2–3 s and centrifuged at 20,000 g for 6 min at 4°C. The cell lysate was transferred into a fresh microfuge tube and allowed to warm to room temperature before being assayed for luciferase activity. One hundred microliters of room temperature luciferase substrate (Stratagene) were added to twenty microliters of lysate and mixed thoroughly by pipetting in a glass luminometer tube. Luminescence was measured using a luminometer (Berthold Technologies Junior LB 9509). All readings were normalized to protein (Bradford method; Bio-Rad).

In control experiments to determine equal transfection efficiency and demonstrate that the effects of CREB overexpression were specific for amphiregulin and not due to a general downregulation in transcriptional activity, cells were cotransfected with 1.5 µg of pGL2[C], 0.25 µg of pCMV-Renilla luciferase, and 100–400 ng of pCMV-CREB or pCMV-KCREB. In the assay for Renilla luciferase activity, 50 µl of whole cell extract were added to 50 µl of Renilla luciferase substrate coelenterazine (Sigma).

Analysis of mRNA levels by RT-PCR. Confluent monolayers of T84 cells were exposed to normoxia or hypoxia for 0–18 h. Total cellular RNA was obtained with the RNeasy system (Qiagen) according to the manufacturer’s recommendations. One microgram of each sample was used for conversion into cDNA. Total RNA was reverse transcribed to cDNA using a first-strand cDNA synthesis kit for RT-PCR (Roche). Each RNA sample was denatured by being heated to 65°C for 15 min followed by 5-min incubation on ice. One microgram (5 µl) of total RNA was then added to 10 mM Tris, 50 mM KCl (pH 8.3), 5 mM MgCl2 containing 1 mM deoxynucleotide mix (dNTP), 1.6 µg of oligo-p(dT) primer, 50 U of RNase inhibitor, and 20 U of avian myeloblastosis virus reverse transcriptase. Each sample reaction volume was adjusted to 20 µl with sterile water. Conversion of each RNA sample to cDNA was achieved by being incubated at 25°C for 10 min followed by 1 h at 40°C and then 99°C for 5 min and subsequently being cooled to 4°C for 5 min. The cDNA was then stored at –20°C.

Each cDNA sample (5 µl) subsequently acted as a template for amplification of amphiregulin by PCR (50 µl). The components of each PCR contained 5 µl of cDNA in reaction buffer containing 50 mM KCl, 10 mM Tris·HCl (pH 9), and 1% Triton X-100 along with 1.5 mM MgCl2, 0.2 mM dNTPs (Promega), 50 pmol of the upstream primer (5'-ACCTACACTCTGGGAAGCTGA-3'), 50 pmol of the downstream primer (5'-AGCCAGGTATTTGTGGTTCG-3') (Genoysis), 1.25 U of Taq DNA polymerase (Invitrogen), and 34.2 µl of sterile water, for a total reaction volume of 50 µl. The amplification reaction included a 2-min denaturation at 94°C, followed by 22 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 1 min, with a final extension of 72°C for 5 min. {beta}-Actin amplification of each sample cDNA was used as a control in the RT-PCR. The target gene sequence was amplified using 50 pmol of a primer mix (Continental Laboratory Products). The remaining components of each reaction were identical to those used to amplify amphiregulin mRNA. Each reaction was denatured at 95°C for 2 min; this was followed by 25 cycles of 94°C for 30 s, 55°C for 1 min, 72°C for 1 min, and a final extension at 72°C for 10 min. Each PCR product was visualized using a 1% agarose gel containing 0.5 µg/ml ethidium bromide (EtBr; Sigma).

For TNF-{alpha} and somatostatin RT-PCR, first-strand cDNA synthesis was performed with Superscript II (Invitrogen Life Technologies, Carlsbad, CA) under recommended conditions. Amplification of cDNA was performed using 1 µg of cDNA, 1.5 mM MgCl2, 0.2 mM dNTP, 100 nM primers, and 1 U of Taq polymerase (Amersham Biosciences, Piscataway, NJ) in a reaction volume of 50 µl and carried out in a PTC-200 thermal cycler (MJ Research, Waltham, MA). After an initial denaturation step at 94°C for 2 min, cycling was carried out with the following conditions: 94°C for 30 s, 55°C for 45 s, and 72°C for 30 s for up to 40 cycles, with a final elongation step at 72°C for 10 min. At the indicated cycle numbers, a 10-µl aliquot was withdrawn from the reaction vial and loaded onto a 2% agarose gel in 0.5x Tris-acetic acid-EDTA, stained with EtBr, and visualized under UV illumination. The up- and downstream primers for somatostatin were 5'-cccagactccgtcagtttc-3' and 5'-ccgggtttgagttagcagat-3', respectively. The up- and downstream primers for TNF-{alpha} were 5'-cgtctcctaccagaccaagg-3' and 5'-ccaaagtagacctgcccaga-3', respectively.

ELISA. Confluent monolayers of T84 cells were exposed to 0- to 18-h hypoxia. Conditioned medium from the cells was collected, and amphiregulin levels were quantified by capture ELISA. A 96-well plate was coated with 100 µl of mouse anti-human amphiregulin MAb (2 µg/ml; R & D Systems) and incubated overnight at room temperature. Wells were washed three times with 400 µl of wash buffer (0.05% Tween 20 in PBS; pH 7.4). Three hundred microliters of blocking buffer (PBS containing 1% bovine serum albumin, 5% sucrose, and 0.05% NaN3) were added at room temperature for 1 h. The blocking buffer was aspirated, and wells were washed three times. One hundred microliters of each sample or known standards (0–2,000 pg/ml) were added to the wells, covered with an adhesive strip, and incubated for 2 h at room temperature. Plates were washed three times before addition of 100 µl of 1:2,500 biotinylated human amphiregulin affinity-purified polyclonal detection antibody (R&D Systems) and incubation for 2 h at room temperature. Wells were washed three times. One hundred microliters of streptavidin-horseradish peroxidase (HRP) were then added to each well, and the plate was covered and incubated at room temperature for 20 min. After three washes, 100 µl of substrate solution containing a 1:1 mixture of H2O2 and tetramethylbenzidine (Sigma) were added. The plate was allowed to develop in the dark for 30 min. The reaction was stopped by the addition of 50 µl of 1 M H2SO4 (Sigma) to each well. Optical density was determined using spectrophotometry at 450 nm with 570 nm as reference filter. The amphiregulin levels in each sample were determined using elucidation from the standard curve.

DNA binding assays. To investigate HIF-1 and CREB binding to consensus hypoxia response element (HRE) and CRE sequences, we used an ELISA-based approach according to the manufacturer’s instructions (TransAm Active Motif). Briefly, T84 cells were exposed to increasing periods of hypoxia, and nuclear extracts were prepared. Extracts were incubated in 96-well plates that contained bound oligonucleotides containing either the CRE or the HRE consensus motif. After the capture of the relevant transcription factor by the oligonucleotide, a primary antibody to either HIF-1 or CREB was added. An HRP-conjugated secondary antibody was used to quantify the DNA binding activity of the transcription factor in question. In separate studies, protein-DNA interaction analysis was carried out with EMSA as described previously (25).

Statistical analysis. All data are presented as means ± SE for n independent experiments. Statistical significance (P < 0.05) was evaluated using Student’s t-test for paired data.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hypoxia induces amphiregulin expression in vitro and in vivo. We initially investigated global gene expression patterns in an in vitro model of intestinal epithelial hypoxia. Microarray analysis was used to screen differential gene expression in T84 colonic epithelial cells after 0, 6, and 18 h of hypoxia. Although the majority (96%) of genes remained unaltered in hypoxia, a significant number demonstrated time-dependent upregulation (30). The expression of epithelial tight junctional protein ZO-1 mRNA remained unaltered. In contrast, mRNA levels for adrenomedullin and ITF were increased in hypoxia (Fig. 1A). ITF is released apically and is known to protect the epithelium from injury and promote repair (36, 52). Adrenomedullin is a potent vasodilator (23, 46) that has been implicated in the pathogenesis of both inflammation and cancer (7, 23, 46). Transcriptional regulation of both ITF and adrenomedullin in hypoxia was previously characterized as HIF-1 dependent (13, 33). The expression of a number of genes not previously associated with upregulation through the classic HIF-1-dependent pathway was also observed. This group of genes included amphiregulin, the expression of which increased in a time-dependent manner (Fig. 1A).



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  		Fig. 1. Hypoxia regulates amphiregulin expression in vitro. T84 cells were incubated in hypoxia (1% O2) for 0, 6, and 18 h. A: global gene expression patterns were investigated with microarray analysis. Although ZO-1 gene expression remained stable, mRNA levels for adrenomedullin (Adr), intestinal trefoil factor (ITF), and amphiregulin (Amphi) were increased in hypoxia in a time-dependent manner. B: increased amphiregulin gene expression was confirmed by RT-PCR of total RNA isolated from T84 cells. N, normoxia; H, hypoxia. C: amphiregulin protein levels ([amphiregulin]) in soluble supernatants from T84 cells were also increased in hypoxia compared with normoxic controls. D: amphiregulin protein levels ([AREG]) in soluble supernatants from T84 cells grown on permeable supports demonstrate a predominantly basolateral release profile. E: amphiregulin expression is transiently increased in colonic mucosal tissues from mice exposed to whole animal hypoxia. F: Western blot analysis of whole T84 cell lysates exposed to 24-h hypoxia shows selective phosphorylation of sites on the EGF receptor (EGFR). The results in B are representative of 3 individual experiments, whereas the data in C represent means ± SE of 3 independent experiments. *P < 0.05.

 
RT-PCR analysis of mRNA from hypoxic T84 cells confirmed an increase in amphiregulin gene expression after 6 h (58% increase) and 18 h (110% increase) of hypoxia compared with normoxic controls (Fig. 1B). {beta}-Actin mRNA levels remained stable. In a parallel study, cell supernatants were harvested from T84 cells exposed to hypoxia and assayed for amphiregulin protein levels using an in-house ELISA that we developed for the purpose of this experiment. The levels of amphiregulin secreted into the cell supernatant increased significantly in hypoxia (6 and 18 h; P < 0.05) compared with normoxic controls (Fig. 1C). Furthermore, hypoxia-induced amphiregulin release is in a basolaterally polarized direction (Fig. 1D). To confirm the physiological relevance of our in vitro observations, we investigated the impact of hypoxia on amphiregulin expression in mucosal tissue from mice exposed to increasing periods of hypoxia (0–4 h). Hypoxia caused an increase in mucosal amphiregulin levels at 2 h that was resolved by 4 h (Fig. 1E). The transient nature of this response is indicative of the induction of a physiological adaptation to tissue hypoxia. mRNA levels of the classically hypoxia-responsive gene VEGF as determined using real-time PCR analysis demonstrate a 3.13-fold increase at 2 h of hypoxia, confirming the perception of hypoxia by the tissue (data not shown). As a readout of amphiregulin activity, T84 cells treated overnight in hypoxia were examined for the phosphorylation of the EGF receptor (Fig. 1F). Hypoxia caused detectable phosphorylation at Tyr1173 and Tyr845 but not at Tyr1068 or Tyr1448.

Transcriptional mechanisms underlying hypoxia-induced amphiregulin. We next investigated the transcriptional regulatory elements in the amphiregulin promoter that control expression in hypoxia. Initially, we examined the classic hypoxia-dependent activation of HIF-1{alpha}. HIF-1{alpha} was increased in hypoxia in HeLa cells in a time-dependent manner as demonstrated by transcription factor ELISA, which measures HIF-1-DNA binding (Fig. 2A). This response was consistent in a number of cell types including T84, HeLa, and HK-2 human kidney cells (data not shown). However, overexpression of wild-type HIF-1{alpha} or a constitutively active stable mutant, which was sufficient to induce the expression of known HIF-1 targets such as adrenomedullin, did not induce amphiregulin mRNA (Fig. 2B). This indicates an alternative mechanism for hypoxia-elicited amphiregulin expression.



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  		Fig. 2. Hypoxia-inducible factor (HIF)-1 accumulation does not account for hypoxia-induced amphiregulin. HeLa cells were exposed to preconditioned hypoxic medium for 0–60 min. A: transcription factor ELISA demonstrates that HIF-1 binding to the hypoxia response element (HRE) increased in a time-dependent manner in hypoxia. This binding was inhibited with specific wild-type oligonucleotides (WTO) directed against the HRE but not with mutant oligonucleotides (MTO), which do not inhibit HRE binding. B: overexpression of wild-type HIF-1{alpha} (WT) or a stable mutant HIF-1{alpha} (MT) was sufficient to drive adrenomedullin (AM) expression; however, amphiregulin mRNA was not upregulated. Transcription factor ELISA results are expressed as optical density readings at 450 nm and normalized to protein content. These data represent means ± SE of at least 3 independent experiments.

 
Initial characterization of the amphiregulin promoter has revealed a consensus CRE close to the TATA box (24, 35). Bioinformatic analysis of this motif by promoter alignment using the Matinspector software tool (Genomatix, Munich, Germany) demonstrated that it is evolutionarily conserved among mouse, rat, and human sequences (data not shown). This conservation likely implicates a functional role for this element in transcriptional regulation.

We investigated the molecular mechanisms underlying increased amphiregulin expression in hypoxia. Addition of the cAMP analog 8-bromo-cAMP, which activates the phosphorylation of CREB at Ser133, had only mild effects on amphiregulin expression (Fig. 3A), indicating that the phosphorylation of CREB at Ser133 is not the primary mechanism of hypoxia-induced activity. Because of difficulties in T84 cell transfection, we used HeLa cells for these studies. Amphiregulin promoter luciferase reporters containing fragments of the promoter 612 bp (pGL2[A]) and 37 bp (pGL2[C]) upstream from the TATA box were transiently transfected into HeLa cells (21). PMA (100 nM) was used as a positive control to activate the amphiregulin promoter after transfection (45). We demonstrated previously (26) that both hypoxia and PMA (100 nM) activated pGL2[A] to stimulate luciferase transcription (3- to 4-fold), indicating that this larger fragment [which contains both the CRE and WT1 response element (WRE) motifs] contains a hypoxia-sensitive sequence. Absolute luciferase activity was generally higher in cells expressing the pGL2[A] construct compared with those expressing the pGL2[C] construct (data not shown). This implicates a role for the WRE in the regulation of basal promoter activity. However, qualitatively similar responsiveness to hypoxia is retained in the pGL2[C] promoter fragment that contains only the CRE 37 bp upstream from the TATA box (2.5- to 3-fold; Fig. 3B). Notably, although hypoxia sensitive, this sequence contains no HRE motifs. To determine the importance of the CRE sequence in conferring hypoxic sensitivity in the amphiregulin promoter, we used a pGL2[C] construct in which the CRE was deleted (pGL2[{Delta}C]). This deletion within the CRE significantly decreased, but did not abolish, basal promoter activity by 77.3 ± 2.9% (P < 0.05) and abolished the responsiveness to both hypoxia and 100 nM PMA (Fig. 3C). Thus the CRE 37 bp upstream from the TATA box of the amphiregulin gene is critical in the hypoxic sensitivity of the promoter.



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  		Fig. 3. The cAMP response element (CRE) site of the amphiregulin promoter confers hypoxic sensitivity. A: T84 cells were exposed to hypoxia after 1 h pretreatment with 8-bromo-cAMP (8-Br; 100 µM) or H89 (30 µM). After 24-h exposure, basolateral amphiregulin release was assayed by performing ELISA. B: HeLa cells were transiently transfected with the truncated amphiregulin promoter luciferase (Luc) reporter construct pGL2[C]. Transfected cells were placed in hypoxia (1% O2) or normoxia (21% O2) for 22 h or treated with 100 nM PMA as a positive control. Hypoxia induced reporter gene activity in pGL2[C]-transfected cells. C: in cells transfected with PGL[C]-{Delta}CRE with a deleted CRE, hypoxic inducibility was lost. Results are expressed as the induction of luciferase activity relative to normoxia and represent means ± SE of 3 independent experiments. *P < 0.05.

 
CREB overexpression inhibits hypoxia-induced activation of amphiregulin promoter. CREB was originally identified as an activator of transcription for the somatostatin gene (32, 55). However, we (6, 49, 51) have also identified CREB as a repressor of transcription for genes such as TNF-{alpha}. Thus the role of CREB as an activator or repressor of gene transcription appears to be gene specific. Semiquantitative PCR analysis of T84 cells exposed to increasing periods of hypoxia demonstrated downregulation in somatostatin mRNA expression and a coincidental upregulation in TNF-{alpha} expression (Fig. 4A). These data would indirectly support a role for decreased CREB activity in hypoxia. Because of this ambiguous role for CREB, we investigated the mechanism by which it controls amphiregulin gene expression in hypoxia.



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  		Fig. 4. Overexpression of wild-type CREB inhibits hypoxia-induced activation of the amphiregulin promoter. A: T84 cells were exposed to increasing periods of hypoxia (0–72 h), and specific gene expression was measured using semiquantitative PCR. Although the expression of TNF-{alpha} mRNA was increased in a time-dependent manner in hypoxia, somatostatin expression was significantly decreased. B: HeLa cells were transiently cotransfected with pGL2[C] and increasing amounts of pCMV-CREB to overexpress wild-type CREB (100–400 ng). CREB overexpression inhibited hypoxia-dependent increases in pGL2[C] activity in a concentration-dependent manner. CTL, control. C: HeLa cells were transiently cotransfected with pGL2[C] and increasing amounts of pCMV-KCREB to overexpress non-DNA-binding CREB (100–400 ng). Mutant CREB was without effect on hypoxia-elicited pGL2[C] activity. The effects of CREB overexpression were specific for amphiregulin promoter constructs, because CREB or KCREB overexpression was without effect on CMV-controlled Renilla luciferase activity in the same cell type (inset). Results are expressed as induction relative to normoxia and represent means ± SE of 3 independent experiments. *P < 0.05. RLU, relative luminescence units.

 
The pGL2[C] amphiregulin promoter construct was transiently cotransfected with increasing amounts of wild-type CREB (pCMV-CREB) or a mutant form of CREB (pCMV-KCREB) that is mutated in the DNA binding domain and thus cannot bind to the CRE. Overexpression of wild-type CREB caused a concentration-dependent inhibition of hypoxia-induced PGL2[C]-driven luciferase expression (Fig. 4B), whereas overexpression pCMV-KCREB was without effect (Fig. 4C). The inactivity of KCREB ensures that the effects of CREB overexpression are not simply due to a "mopping up" of the CREB binding protein (CBP) coactivator and a general repressive effect. Furthermore, the effects of CREB overexpression were specific for the amphiregulin promoter, because CREB or KCREB overexpression did not effect reporter activity in cells transfected with a Renilla luciferase under the control of the constitutively active cytomegalovirus promoter (CMV) (Fig. 4, inset). These findings are consistent with a role for CREB as a repressor of transcription at the amphiregulin promoter, which is removed in hypoxia, allowing transcription to take place. Such a model is consistent with our previous studies (49) of the role of CREB in the regulation of TNF-{alpha} gene expression in intestinal epithelial cells in hypoxia.

HIF-1 does not bind to unmasked CRE. HIF-1 normally binds to the HRE, and CREB normally binds to the CRE. However, previous studies demonstrated cross-specificity for CREB and HIF-1 in binding to CRE and HRE motifs (11, 21, 22). Furthermore, within the CRE sequence exists a core motif that is 80% similar to the HRE. Because of this, we investigated the possibility that HIF-1, which is increased in hypoxia, might bind to a sequence within the unmasked CRE in the amphiregulin promoter to induce transcription. To investigate this possibility, we used specific transcription factor ELISAs for both HIF-1 and CREB binding to consensus HRE and CRE sequences, respectively. In increasing periods of hypoxia, quantifiable HIF-1-HRE binding occurred (Fig. 2A). As we have previously described (49), CREB DNA binding activity to the consensus CRE (100% similar to that in amphiregulin) was decreased in a time-dependent manner (data not shown). In cross-binding experiments, increasing periods of hypoxia did not increase HIF-1 binding to the amphiregulin CRE motif (Fig. 5A), indicating that HIF-1 binding is not an initiating factor in the control of CRE-dependent amphiregulin gene expression in hypoxia. Furthermore, using gel shift analysis, we demonstrated a hypoxia-dependent decrease in CREB-CRE binding with a concomitant increase in the binding of a protein of lower molecular mass (Fig. 5B). Future studies will identify this protein; however, the fact that it is smaller than CREB (42 kDa) rules out the possibility that it is HIF-1{alpha} (which has a molecular mass of 125 kDa).



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  		Fig. 5. HIF-1 does not bind to an unmasked CRE. A: with a specific transcription factor ELISA assay, HIF-1 binding to the consensus CRE was investigated in hypoxia. Cells were exposed to hypoxia (0–24 h), and nuclear extracts were made. Nuclear proteins were allowed to bind to an immobilized CRE, and HIF-1-CRE binding was detected with a specific anti-HIF-1 secondary antibody. HIF-1 binding to the CRE was significantly lower than CREB and did not alter over the time course of hypoxia, indicating that HIF-1 does not bind to an unmasked CRE in the amphiregulin promoter in hypoxia. Results are expressed as induction relative to normoxia and represent means ± SE of 3 independent experiments. *P < 0.05. B: T84 cells were grown to confluence and exposed to increasing periods of hypoxia (0–48 h). Nuclear extracts were generated and coincubated with double-stranded, 32P-labeled DNA primers containing the amphiregulin CRE motif. DNA-protein complexes were resolved on a nondenaturing polyacrylamide gel and transblotted onto nitrocellulose membrane. With increasing periods of hypoxia (left to right), there was a time-dependent decrease in levels of a protein-DNA complex consistent with CREB-CRE. Furthermore, we observed a concomitant increase in binding of another unidentified protein (X).

 

   DISCUSSION
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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Hypoxia-elicited pathways play a critical role in tumor development through the promotion of processes including angiogenesis, glycolysis, and vasodilatation via HIF-1-dependent mechanisms (9). Separate hypoxia-elicited pathways may also contribute to epithelial tumor progression by increasing cell proliferation. In this study, we have demonstrated that hypoxia positively regulates the expression of amphiregulin, an epithelial mitogen, in intestinal epithelial cells. Amphiregulin expression is elevated in a range of tumors, including those of breast, ovarian, and colonic origin (8, 28, 34). Furthermore, amphiregulin contributes to cell proliferation and tumor growth. However, the mechanism governing the expression of amphiregulin in tumors remain poorly defined. Thus a greater understanding of the molecular mechanisms by which tumor hypoxia increases amphiregulin expression will identify novel therapeutic windows of opportunity.

The impact of hypoxia on cellular proliferation differs depending on cell type. Fibroblast and B lymphocyte proliferation is decreased in a manner dependent on HIF-1 activation (15). In contrast, endothelial and smooth muscle cell proliferation is increased in hypoxia (17, 38). Furthermore, the induction of angiogenic events in hypoxia mediated through the HIF-1-dependent expression of VEGF has been documented extensively (42). These contrasting effects on cell proliferation may be accounted for by differing transcriptional profiles activated by hypoxia in different cell types.

Activation of the EGFR is a potent stimulus for epithelial cell proliferation (34). Microarray analysis confirmed the basal expression of the EGFR in T84 cells as previously described (20). In injury, epithelial cell proliferation was recently described to be due to the association of EGF ligand and receptor, which are normally separated by the tight junction in the polarized epithelium (53). This process aids reestablishment of the epithelial barrier through a rapidly activated proliferative response that is then silenced upon reformation of a polarized monolayer. In this study, we have demonstrated that hypoxia increases the expression of amphiregulin in transformed intestinal epithelial cells in vitro and in colonic mucosal tissue from mice exposed to whole body hypoxia. Amphiregulin is an EGFR ligand that was shown previously to increase proliferation in a range of cell types (10, 19). Furthermore, amphiregulin expression is increased in tumors and in wound healing (8, 28, 39). Because hypoxia is also a feature of tumor development and wound healing, we became interested in the mechanism by which hypoxia upregulates intestinal epithelial amphiregulin expression.

To date, the regulation of amphiregulin gene transcription is not fully understood; however, the transcription factor WT1 has been demonstrated to regulate expression in the developing kidney (24). Truncations of the amphiregulin promoter lacking the WT1 binding site but containing a conserved CRE sequence had a lower basal of activity but maintained sensitivity to upregulation by hypoxia. These data indicate that although WRE activity is important in the maintenance of basal amphiregulin expression, hypoxic sensitivity is conferred by the CRE sequence.

Amphiregulin and TNF-{alpha} are both upregulated in hypoxia in a CRE-dependent manner. Interestingly, both the amphiregulin CRE (–274 to –267) and the TNF-{alpha} CRE (–106 to –109) lie within 100 bp of the TATA box, the suggested region of functional relevance. Both amphiregulin and TNF-{alpha} CRE sequences are the same consensus 8-bp palindrome 5'-TGACGTCA-3'. The inducibility of both of these genes by hypoxia is dependent on the removal of CREB, and each gene is induced to approximately the same level.

Although CREB-CRE binding is constitutive, cAMP-dependent phosphorylation of Ser133 by PKA results in transactivation and subsequent transcription of specific genes such as somatostatin (31, 55). This promotes transcription through interaction with coactivators such as CBP and ultimately with the basal transcription complex to drive gene expression (31). Interestingly, in hypoxia, cAMP levels are significantly diminished, indicating that PKA-mediated Ser133 phosphorylation and subsequent transactivation are unlikely to account for the effects of hypoxia on amphiregulin expression (49). Furthermore, we demonstrate that the addition of the cAMP analog 8-bromo-cAMP has only mild effects on amphiregulin expression. Previous studies in neurons demonstrated a phosphorylation of Ser133 of CREB in acute hypoxia in neurons (2); however, we have found that longer periods of hypoxia do not alter Ser133 phosphorylation in our model (49).

CREB was previously reported to act as a repressor of transcription for some genes (5). Members of the CREB family of proteins have also been shown to bind activator protein (AP-1) target DNA sequences, thus inhibiting AP-1-mediated transcription in differentiating keratinocytes (18, 37). We (49, 51) and others (56) have found evidence to support a role for diminished CREB repressor activity in hypoxia-induced expression of proinflammatory genes such as TNF-{alpha} in intestinal epithelial cells. We have demonstrated (51) that CREB is targeted to degradation in a phosphorylation-dependent manner, leading to the induction of gene transcription.

Because of the ambiguous nature of CREB-dependent transcriptional control, we investigated whether CREB acts as a stimulated activator or diminished repressor of amphiregulin gene expression in hypoxia. CREB-CRE binding is decreased in hypoxia, and overexpression of wild-type (CREB) but not mutant (KCREB) overcame the hypoxic induction of amphiregulin promoter activation. These findings support the idea that CREB acts as a tonic repressor of amphiregulin expression, which is removed in hypoxia, allowing transcription to occur.

Because hypoxia-elicited decrease of CREB binding to the sole CRE in the amphiregulin promoter is sufficient to drive transcriptional upregulation, we hypothesized that some other as yet unidentified transcription factor might bind to the unmasked CRE to initiate the formation of the basal transcriptional complex. A number of studies have demonstrated cross-specificity between HIF-1 and CREB at the level of binding to HRE and CRE sequences (4, 11, 21, 22). Specifically, CREB has been shown to have binding affinity for the HRE sequence and may be of importance in the hypoxic upregulation of genes such as lactate dehydrogenase. Furthermore, the CRE consensus motif (5'-TGACGTCA-3') contains within it a sequence 80% similar to the HRE consensus motif (5'-RCGTG-3'). For these reasons, we investigated whether HIF-1 that accumulated in these cells in hypoxia bound to the unmasked CRE. Using a recently developed DNA-binding ELISA approach, we demonstrated that although HIF-1 activity is indeed increased in hypoxia, HIF-1 does not bind to the amphiregulin CRE in hypoxia. Furthermore, overexpression of HIF-1{alpha}, which induces HIF-1-dependent genes, does not alter amphiregulin expression. Initial gel shift analysis demonstrates the possibility that another protein might bind the CRE in a hypoxia-dependent manner. Future studies will identify such potential hypoxia-sensitive transcriptional regulators, which might bind to the unmasked CRE in the induction of the transcriptional complex.

In summary, this study demonstrates for the first time that hypoxia upregulates intestinal epithelial amphiregulin expression in a basolaterally polarized manner, an event that may be of significance in the regulation of epithelial proliferation in wound healing and/or tumor development where hypoxia occurs. Furthermore, hypoxia-induced amphiregulin is dependent on the liberation of CREB repressor activity and independent of the classic HIF-1-dependent transcriptional pathway.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from The Wellcome Trust, The Science Foundation of Ireland, and The Health Research Board of Ireland (to C. T. Taylor and K. M. Comerford).


   ACKNOWLEDGMENTS
 
We acknowledge technical assistance from Annemarie Griffin. The Renilla luciferase construct was a kind gift of Dr. Thilo Hagen, University College London, London, UK. Hypoxic mouse tissues were obtained from Dr. Sean Colgan, Brigham and Women’s Hospital (Boston, MA).


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. T. Taylor, Dept. of Medicine and Therapeutics, The Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland (e-mail: cormac.taylor{at}ucd.ie)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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