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Am J Physiol Gastrointest Liver Physiol 290: G737-G746, 2006. First published December 29, 2005; doi:10.1152/ajpgi.00394.2005
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MUCOSAL BIOLOGY

Intestinal alkaline phosphatase gene expression is activated by ZBP-89

Madhu S. Malo,1 Moushumi Mozumder,1 Xiao Bo Zhang,1 Shaluk Biswas,1 Alexander Chen,1 Long-Chuan Bai,2 Juanita L. Merchant,2 and Richard A. Hodin1

1Gastrointestinal Unit and Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts; and 2Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan

Submitted 24 August 2005 ; accepted in final form 26 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Intestinal alkaline phosphatase (IAP) is an enterocyte differentiation marker that functions to limit fat absorption. Zinc finger binding protein-89 (ZBP-89) is a Kruppel-type transcription factor that appears to promote a differentiated phenotype in the intestinal epithelium. The purpose of this study was to investigate the regulation of IAP gene expression by ZBP-89. RT-PCR, quantitative real-time RT-PCR, Western blot analyses, and reporter assays were used to determine the regulation of IAP by ZBP-89 in HT-29 and Caco-2 colon cancer cells. ZBP-89 knockdown was achieved by specific short interfering (si)RNA. EMSA and chromatin immunoprecipitation (ChIP) were performed to examine the binding of ZBP-89 to the IAP promoter. The results of RT-PCR, quantitative real-time PCR, and Western blot analyses showed that ZBP-89 was expressed at low levels in Caco-2 and HT-29 cells, whereas IAP was minimally expressed and absent in these cells, respectively. Transfection with ZBP-89 expression plamid increased IAP mRNA and protein levels in both cell lines, whereas knockdown of endogenous ZBP-89 by siRNA reduced basal levels of IAP gene expression in Caco-2 cells. IAP-luciferase reporter assays, EMSA, and ChIP established that ZBP-89 activated the IAP gene through a response element (ZBP-89 response element: 5'-CCTCCTCCC-3') located between –1018 and –1010 bp upstream of the AUG start codon. We conclude that ZBP-89 is a direct transcriptional activator of the enterocyte differentiation marker IAP. These findings are consistent with the role that this transcription factor is thought to play as a tumor suppressor and suggests its possible function in the physiology of fat absorption.

carcinogenesis; development; enterocyte differentiation; eukaryotic promoter; transient transfection

INTESTINAL ALKALINE PHOSPHATASE (IAP) is a membrane-bound glycoprotein that hydrolyzes monophosphate esters at high pH optima and plays an important role in fat and phosphate metabolism (12, 13, 27). In rats, the IAP gene is only expressed in villus-associated enterocytes, and hence it has been well recognized as an enterocyte differentiation marker (8, 16). IAP protein has been identified as a component of the surfactant-like particle (SLP), a unilamellar secreted membrane associated with the process of fat absorption and isolated from the apical surface of enterocytes (20). Recent work by Narisawa et al. (27) on IAP knockout mice showed that IAP limits fat absorption by enterocytes. Compared with pair-fed littermates, IAP knockout mice gained more weight and became obese under conditions of a high-fat diet (27). We (16, 17) have previously reported that IAP gene expression is silenced by starvation, suggesting the existence of a fundamental survival mechanism designed to maximize gut fat absorption once feeding is reinitiated.

Given the apparent physiological importance of IAP levels in regard to fat absorption and the development of obesity, we were interested in understanding the molecular mechanisms of IAP gene regulation. The present study was focused on the Krüppel-type transcription factor zinc finger binding protein-89 (ZBP-89), a transcriptional regulator that appears to play an important role in intestinal cell proliferation, differentiation, and oncogenesis (6, 7, 26, 30). Similar to Sp1/Sp3 factors, ZBP-89 is ubiquitously expressed and interacts with GC-rich DNA sequences (26). ZBP-89 functions as either a transcriptional activator or repressor depending on the target promoter, e.g., ZBP-89 represses gastrin, ENA-78, vimentin, and beta-integrin CD11b gene transcription (18, 26, 29, 42), whereas it activates STAT1, lck, and stromelysin genes (4, 39, 41). ZBP-89 induces growth arrest and apoptosis in human gastrointestinal cell lines (2, 5, 6), primarily through stabilization of p53 protein (7), and it also potentiates sodium butyrate-mediated activation of the p21 gene. Heterozygosity of ZBP-89 is embryonically lethal, and two functional alleles of ZBP-89 are required for normal development of fetal germ cells (36). Taken together, the data on ZBP-89 suggest that this transcription factor functions to promote a more differentiated phenotype in the intestinal epithelium and that it may play a role as a tumor suppressor.

In this study, we showed that ZBP-89 positively regulates endogenous IAP gene expression in the human colorectal adenocarcinoma HT-29 and Caco-2 cell lines. This IAP gene transactivation appears to be mediated through an interaction between ZBP-89 and its response element (ZBPRE) located between –1018 and –1010 bp upstream from the AUG start codon. These results provide a molecular mechanism by which ZBP-89 activates an enterocyte differentiation marker that plays a functional role in limiting dietary fat absorption.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. DNA restriction enzymes and DNA modifying enzymes were purchased from New England Biolaboratory (Beverly, MA) and Fisher Scientific (Pittsburgh, PA). TRIzol reagent for RNA preparation, the SuperScript III Platinum Two-Step qRT-PCR kit, and the Oligofectamine kit for short interfering (si)RNA transfection were obtained from Invitrogen (Carlsbad, CA). Taq DNA polymerase and the TNT T7 Quick Coupled Transcription/Translation System were obtained from Promega (Madison, WI). Anti-IAP antibody was obtained from GeneTex (San Antonio, TX). Anti-beta-actin antibody, the SYBER Green JumpStart Taq ReadyMix kit, and poly(dI-dC).poly(dI-dC) were purchased from Sigma (St. Louis, MO). SuperFect transfection reagent, the kit for DNA extraction from agarose gel, and also the kit for large-scale DNA preparation were obtained from Qiagen (Valencia, CA). Radionucleotides were obtained from Perkin-Elmer Life Sciences (Boston, MA), and oligonucleotides were synthesized by Sigma Genosys (The Woodlands, TX). The generation of polyclonal antibody against human ZBP-89 (htbeta) has been previously described (26). The chromatin immunoprecipitation (ChIP) kit (ChIP-IT) was purchased from Active Motif (Carlsbad, CA).

Plasmids. The ZBP-89 expression plasmid pCMV/Myc-ZBP-89-FLAG was constructed by cloning full-length rat ZBP-89 cDNA (amino acids 1–794) into the eukaryotic expression vector pcDNA3 (Invitrogen), which carries the cytomegalovirus (CMV) and T7 promoter upstream of ZBP-89 cDNA (3). To construct the full-length IAP-luciferase reporter plasmid pFRL7-IAP-2574, we cloned the 2.6-kb KpnI-NarI fragment from the plasmid pIAP–2574/–49 (23), which carries the human IAP promoter region (–2574 to –49, relative to translation initiation codon AUG), into the mammalian promoter-detection vector pFRL7, a derivative of pFRL2 (22) that lacks the CMV promoter. pFRL7 derivatives carrying various 5' deletions of the IAP promoter were constructed by transferring the KpnI-NarI fragments from the pGL3 derivatives (23) into pFRL7 digested with KpnI-NarI. The plasmid pFRL7-IAP-2574mZBPRE was generated by PCR-mediated mutagenesis following the protocol previously described (23). Mutagenic PCR primers were synthesized with specific substitution mutations followed by PCR amplification and restriction digestion and ligation of PCR products into the appropriate plasmid. The nature of the novel joint(s) as well as targeted mutations in respective plasmids were verified by DNA sequencing (32, 35), which was performed at the Sequencing Core Facility at the Department of Molecular Biology, Massachusetts General Hospital (Boston, MA), using dye-labeled dideoxynucleotide chain terminators.

Cell culture. HT-29 and Caco-2 human colorectal adenocarcinoma cell lines were purchased from the American Type Culture Collection (ATCC; Rockville, MD). The Cos-7 fibroblast cell line (derived from African green monkey kidney) was also purchased from the ATCC. Cells were maintained in DMEM (Invitrogen) supplemented with 10% FBS (Sigma), 2 mM L-glutamine, and 100 U/ml each of penicillin and streptomycin (Invitrogen). Cells were grown at 37°C in the presence of 5% CO2 and were split by trypsinization when they reached about 80–90% confluence.

RNA preparation and RT-PCR. HT-29 and Caco-2 cells (80% confluence) were transfected with the ZBP-89 expression plasmid (15 µg DNA/10-cm dish), and total RNA was prepared after 48 h using TRIzol reagent from Invitrogen following the manufacturer’s instructions. RNA was also prepared from control untransfected cells. First-strand cDNA was synthesized from individual samples of RNA isolated from untransfected and transfected cells using the SuperScript III Platinum Two-Step qRT-PCR kit (Invitrogen). Briefly, the RNA sample (1 µg) was incubated with oligo(dT)20 (2.5 µM) and SuperScript III reverse transcriptase in the presence of dNTPs (400 µM) at room temperature for 10 min, followed by incubation at 42°C for 1 h. The enzyme activity was terminated by treating the sample at 85°C for 5 min. PCR was then performed on the synthesized cDNA (20 ng) with gene-specific primers (0.2 µM) using Taq DNA polymerase (2.5 units) from Promega. PCR conditions were as follows: first denaturation step at 94°C for 2 min and then 32 cycles of 94°C for 1 min (denaturing), 55°C for 1 min (annealing), and 72°C for 1 min (extension), followed by 5 min of a final extension step at 72°C. As a control, we also performed PCR on RNA samples alone to confirm that the amplified RT-PCR products were not derived from any contaminated DNA in the RNA samples. PCR products were electrophoresed in a 2% agarose gel containing 0.025 µg/ml ethidium bromide. Gels were photographed under UV light using the Gel Doc 2000 Gel Documentation System from Bio-Rad (Hercules, CA). The RT-PCR primers were 1) hIAP2146F: 5'-GCAACCCTGCAACCCACCCAAGGAG-3'; 2) hIAP2423R: 5'-CCAGCATCCAGATGTCCCGGGAG-3'; 3) hbAct601F: 5'-GGGTCTGGACCTGGCTGGCCGGGACCTG-3'; 4) hbAct1100R: 5'-GGGCCGCCGATCCACACGGAGTACTTGC-3'; 5) hZBP.414F: 5'-CGCTGTGATGAATGTGGTGATGAGAC-3'; and 6) hZBP.698R: 5'-CCCAGCTCTATTATCATTTACATTC-3'.

Quantitative real-time PCR. The SYBER Green JumpStart Taq ReadyMix kit for quantitative real-time PCR was obtained from Sigma and used following the manufacturer’s protocol. Primers and conditions for real-time PCR were the same as described in RNA preparation and RT-PCR for RT-PCR. Real-time PCR was performed in the DNA Engine Opticon 2 System (MJ Research; Waltham, MA).

siRNA-mediated gene silencing. For siRNA-mediated knockdown of ZBP-89 gene expression, we synthesized ZBP-89-specific wild-type siRNA as well as a mutant derivative of this siRNA. The 5'-end of the oligonucleotide starts at +144 in relation to the AUG start codon. The respective complementary oligonucleotide was also synthesized. The sequences of wild-type and mutant siRNA oligonucleotides (antisense) were 1) ZBP-89 siRNA-wt: 5'-AAGAUCGAAGUAUGCCUCACCUU-3' (wild type) and 2) ZBP-89 siRNA-mut: 5'-AAGAUCGAACGUGUCCUCACCUU-3' (mutant); mutant bases are underlined.

Complementary oligonucleotides were annealed to form double-stranded siRNA and were then used in transfection at a final concentration of 50 nM. Caco-2 cells were transfected with siRNA using the Oligofectamine kit from Invitrogen following the manufacturer’s protocol. After 24 h, cells were lysed, RNA was extracted, and RT-PCR was performed (see above).

Western blot analysis. Western blot analysis was performed following the protocol described in Malo et al. (23). Whole cell lysates were prepared from untransfected HT-29 and Caco-2 cells as well as from these cells transfected with ZBP-89 expression plasmid. Approximately 15 µg of plasmid DNA were used to transfect 50% confluent cells in a 10-cm culture dish. Forty-eight hours after transfection, cells were lysed by incubating the cells for 15 min on ice in the lysis buffer [50 mM Tris·HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 1% Triton X-100], which also contained a complete range of protease inhibitors (Roche Applied Science; Indianapolis, IN), whole cell lysates were prepared, and protein concentrations were determined. IAP protein was detected using anti-IAP antibody (GeneTex; San Antonio, TX), and control beta-actin protein was detected with anti-beta-actin antibody (Sigma). Equal amounts (60 µg) of each lysate were individually mixed with 6x loading dye [10.28% SDS, 0.6 M DTT, 36% glycerol, 0.35 M Tris·HCl (pH 6.8), and 0.006% bromophenol blue] boiled for 10 min, and the lysates were then electrophoresed through a Tris·HCl Ready Gel (10% polyacrylamide resolving gel and 4% stacking gel, Bio-Rad) in Tris-glycine-SDS running buffer (Boston Bioproducts; Ashland, MA). Proteins were electrotransferred onto a nitrocellulose membrane (0.2 µm, Bio-Rad), which was then blocked with 5% (wt/vol) nonfat milk (Bio-Rad) overnight at 4°C. The blot was then incubated overnight at 4°C in the presence of the anti-IAP antibody (1:1,000 dilution). The membrane was washed to remove excess anti-IAP antibody, and horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution, Bio-Rad) was then added to the blot and incubated for 2 h at room temperature. The IAP protein band was identified by developing the blot with an Immune-Star horseradish peroxidase chemiluminescent kit from Bio-Rad. The blot was then "stripped" by incubating it in "strip" solution [62.5 mM Tris·HCl (pH 6.7), 2% SDS, and 0.71% beta-mercaptoethanol] for 10 min at 50°C and reprobed with the beta-actin antibody.

Transient transfection and luciferase reporter assays. Transient transfection and luciferase reporter assays were performed following the protocols as described previously (23). HT-29 cells were plated at a density of 300,000 cells/well of a six-well plate. Cells were grown overnight in DMEM containing 10% FBS, and transient transfection was performed using SuperFect reagent and IAP-luciferase reporter plasmid DNA (1.5 µg/well). Whenever indicated, ZBP-89 expression plasmid DNA was used in cotransfection, and the total amount of DNA was kept the same for each transfection by adding nonspecific plasmid TF12 DNA. After transfection, cells were grown for a further 48 h in DMEM containing 10% FBS. Firefly and Renilla luciferase assays were then performed on cell lysates using the Dual-Luciferase Reporter Assay System (Promega) as per the manufacturer’s instructions. Control Renilla luciferase activity was used to determine transfection efficiency as well as to calculate the relative firefly luciferase activity (normalization) as a percentage of the Renilla luciferase activity. The activation ratio (fold activation) was determined by dividing the normalized firefly luciferase counts in the presence and absence of ZBP-89 (ZBP89+/ZBP89–). Results were obtained from >3 independent experiments, and values are expressed as means ± SD (P < 0.05).

In vitro protein synthesis. The TNT T7 Quick Coupled Transcription/Translation System (Promega) was used for in vitro synthesis of the rat ZBP-89 protein from pCMV/Myc-ZBP-89-FLAG (3), the derivative of pcDNA3 (Invitrogen) carrying the relevant coding sequence under the control of the T7 promoter.

Preparation of nuclear extract. Nuclear extract was prepared from 2 x 106 HT-29 cells using NE-PER Nuclear and Cytoplasmic Extraction Reagents kit from Pierce (Rockford, IL) following the protocols from the manufacturer. Halt Protease Inhibitor Cocktail (Pierce) was added to the extract and stored at –80°C in small aliquots.

EMSA. EMSAs were performed according to the protocol previously described (23). Complementary oligonucleotides were annealed and radiolabeled by the kinasing reaction with T4 polynucleotide kinase in the presence of [{gamma}-33P]ATP. The radiolabeled probe was purified twice by passage through Micro Bio-Spin 6 chromatography columns (Bio-Rad), followed by determination of the specific activity, which usually measured about 108 counts·min–1·µg DNA–1. Approximately 10 ng of radiolabeled probe were incubated at room temperature for 20 min with 2 µl of HT-29 nuclear extract or in vitro-synthesized ZBP-89 protein in 10 µl of binding buffer containing 20 mM HEPES (pH 7.7), 50 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 5 µM nonspecific oligonucleotide, 10% glycerol, and 2 µg of poly(dI-dC).poly(dI-dC). Samples were electrophoresed in a 5% polyacrylamide gel in a cold room (4°C), followed by drying of the gel and autoradiography.

ChIP. The ChIP-IT kit from Active Motif (Carlsbad, CA) was used to perform ChIP assays on untransfected HT-29 cells as well as on HT-29 cells transfected with the ZBP-89 expression plasmid. Cells were grown on a 150-mm plate (Becton Dickinson; Franklin Lakes, NJ) to ~80% confluence and then transfected with 25 µg of ZBP-89 expression plasmid. After 48 h, cells were treated with 1% (vol/vol) formaldehyde for 10 min to cross-link DNA with the associated proteins. Cross-linking was stopped with glycine stop-fix solution; cells were harvested, lysed, and then subjected to sonication using a Fisher model Sonic Dismembrator with a 2-mm microtip (Fisher Scientific; Pittsburgh, PA). Samples were sonicated for twelve 15-s pulses at a setting of 25%, with cooling of 30 s between pulses so that chromatin was converted mostly into mono-, di-, or trinucleosomes (<500-bp DNA fragments). Each chromatin sample (1 ml) was precleared with the provided preblocked protein G agarose beads, and an aliquot was collected to use as "input" DNA. The precleared chromatin samples, 350 µl each, were then individually incubated with the anti-ZBP-89 antibody (5 µg) overnight at 4°C. The antibody-chromatin complex was mixed with protein G beads for 1 h and then centrifuged. The precipitated immune complex was then washed with the provided buffers, nucleosomes were eluted, and cross-links were reversed by an overnight incubation at 65°C. After samples were treated with proteinase K for 2 h, DNA was column purified, eluted in 50 µl of elution buffer, and then subjected to PCR. The concentration of template DNA in untransfected and transfected input DNA samples was determined, and, accordingly, ZBP-89-targeted immunoprecipitated template DNA from the proportionate amount of the respective nucleosome sample was used in PCR that ensured an equal amount of starting nucleosomes in each ChIP reaction sample. With the use of the DNA Engine PTC-200 thermal cycler (MJ Research), PCR was performed in 1x Taq DNA polymerase buffer containing 2.5 units Taq DNA polymerase (Promega), 0.2 mM dNTPs, and 0.2 µM forward and reverse primers. PCR samples were denatured for 2 min at 94°C first and then subjected to amplification for 32 cycles at 94°C for 1 min (denaturing), 60°C for 1 min (annealing), and 72°C for 1 min (extension), followed by 5 min of a final extension step at 72°C. PCR products were electrophoresed in a 2% agarose gel, and DNA bands were documented using the Bio-Rad gel documentation system. The following primers were used in PCR for amplification of the IAP sequence around ZBPRE as well as the sequence located 8 kb upstream of the ZBPRE: IAP-1140F, 5'-GCAGTGTTGAGTACACGCACAGTGTTG-3'; 1AP-987R, 5'-GAATGGAGGTTGCCTGAGGCTGAG-3'; IAP-8326F, 5'-CAGTTCCAGCACGATTCAGAGTCGGC-3'; and IAP-8028R, 5'-GCTTCTGTCCCCAGAGCACAGGATTG-3'.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ZBP-89 activates IAP gene expression. We first determined the endogenous levels of ZBP-89 expression in Caco-2 and HT-29 colon cancer cell lines. Total RNA samples were subjected to RT-PCR using the primer sets described in MATERIALS AND METHODS. The expected PCR product of 285 bp was obtained for ZBP-89 cDNA using primers hZBP.414F and hZBP.698R, indicating expression of endogenous ZBP-89 in these cell lines (Fig. 1A, top, lanes 2 and 6, respectively). Cells were also transfected with the ZBP-89 expression plasmid to verify the specificity of the RT-PCR product as well as to determine the effects of exogenous ZBP-89 on IAP gene expression. The results confirmed that ZBP-89 is overexpressed in the transfected cells (Fig. 1A, top, lanes 4 and 8, respectively).


Figure 1
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  		Fig. 1. Expression levels of zinc finger binding protein-89 (ZBP-89) and intestinal alkaline phosphatase (IAP) in HT-29 and Caco-2 human colon cancer cell lines. A: RT-PCR showing expression levels of ZBP-89 and IAP mRNAs before (endogenous) and after (exogenous) transfection with the ZBP-89 expression plasmid. mRNA levels were determined by RT-PCR (primers are shown in MATERIALS AND METHODS). Cells untransfected (–) or transfected (+) with the ZBP-89 expression plasmid (pZBP-89) are indicated. PCR products obtained without (–) and with (+) the addition of reverse transcriptase (RT) are shown. RT-PCR on human beta-actin was used as a control to verify the equal input of RNA. PCR was repeated at least 3 times in each case, and the photograph of a representative gel is shown. B: quantitative real-time PCR analyses of IAP expression. Real-time PCR was performed using the same cDNAs and primers as used in the case of RT-PCR (see MATERIALS AND METHODS). Data shown were generated from 3 independent experiments. C: Western blots showing IAP expression in HT-29 and Caco-2 cells. Whole cell lysates were prepared from untransfected cells as well as cells transfected with the ZBP-89 expression plasmid. Anti-IAP antibody was used to detect the IAP protein band, whereas anti-beta-actin antibody was used as the control to detect equal loading of lysates in each lane (see MATERIALS AND METHODS). Western blotting was performed multiple times, and a photograph of a representative autoradiograph is shown. D: expression levels of ZBP-89 and IAP mRNAs in Caco-2 cells after transfection with wild-type and mutant short interfering (si)RNAs. RT-PCR was performed on RNAs obtained from Caco-2 cells transfected with wild-type (WT) or mutant (Mut) ZBP-89 siRNA. The primers were the same as used for RT-PCR in A and B. The experiment was repeated 3 times, and photographs of representative gels are shown. E: quantitative real-time PCR showing the effects of ZBP-89 silencing on IAP activation. The same cDNAs and primers as used in RT-PCR (D) were used in real-time PCR. Data were obtained from at least 3 independent experiments.

 
We next used RT-PCR to determine the effects of ZBP-89 on IAP gene regulation. We designed primers hIAP2146F and hIAP2423R (see MATERIALS AND METHODS) to amplify a 278-bp IAP cDNA fragment. We performed RT-PCR on the same total RNA (see above) obtained from the untransfected cell line (endogenous) as well as on RNA obtained from the same cell line transfected with the ZBP-89 expression plasmid (exogenous). The results showed the expected 278-bp IAP band from cDNAs of untransfected Caco-2 cells, whereas virtually no PCR product was seen in the case of untransfected HT-29 cells (Fig. 1A, middle, lanes 2 and 6, respectively). With ZBP-89 transfection, there was a marked increase in the levels of IAP expression in both cell lines (Fig. 1A, middle, lanes 4 and 8, respectively), indicating that the IAP gene was activated by ZBP-89 in both HT-29 and Caco-2 cells. To verify an equal template concentration in each sample, RT-PCR was also performed on equal amounts of cDNAs using hbAct601F and hbAct1100R human beta-actin primers (see MATERIALS AND METHODS). Amplification of the expected 500-bp beta-actin fragments with approximately equal band intensity in all samples suggested that there was an equal template concentration in each RT-PCR sample (Fig. 1A, bottom). The absence of any target DNA amplification from PCR on RNA alone (–reverse transcriptase) confirmed that the RT-PCR products were not derived from contaminated DNA in the RNA samples.

We then performed quantitative real-time RT-PCR on all isolated RNA samples described above using gene-specific primers. The results are shown in Fig. 1B. Real-time PCR data also showed that levels of IAP expression are dramatically increased in Caco-2 and HT-29 cells transfected with the ZBP-89 expression plasmid, thus corroborating the RT-PCR data described above.

We used Western blot analyses to determine IAP protein levels in untransfected HT-29 and Caco-2 cells as well as cells transfected with a plasmid overexpressing ZBP-89 protein. The results show that both cell lines produce small amounts of IAP (Fig. 1C, lanes 1 and 3, respectively). IAP protein levels were markedly increased in Caco-2 and HT-29 cells when the cell lines were transfected with the ZBP-89 expression plasmid (Fig. 1C, lanes 2 and 4, respectively), indicating that both IAP mRNA and protein are induced by ZBP-89.

IAP gene expression is reduced by siRNA silencing of ZBP-89. We determined the effects of siRNA-mediated ZBP-89 knockdown (silencing) on the regulation of the IAP gene in Caco-2 cells. A 23-bp double-stranded wild-type siRNA (ZBP-89 siRNA-wt) was transfected into Caco-2 cells, and, for control purposes, a mutant siRNA (ZBP-89 siRNA-mut) was also used (see MATERIALS AND METHODS). The RT-PCR results showed that the wild-type siRNA dramatically decreased the levels of ZBP-89 (Fig. 1D, top, lane 4), whereas the mutant siRNA did not silence ZBP-89 expression (Fig. 1D, top, lane 6), confirming the specificity of wild-type siRNA. As a consequence of ZBP-89 silencing, the levels of IAP expression were also significantly reduced, whereas mutant siRNA had no effect on IAP levels (Fig. 1D, middle, lanes 4 and 6, respectively). The actin control was used to quantify the amount of template cDNA in each PCR, and the results suggested that there was an approximately equal amount of template in each reaction (Fig. 1D, bottom). As shown above, the absence of any target DNA amplification from PCR on RNA alone (–RT) confirmed that the RT-PCR products were not derived from contaminated DNA in the RNA samples. These results confirm that ZBP-89 is a positive regulator of IAP gene expression.

We also performed quantitative real-time PCR to verify the RT-PCR data described above. The results (Fig. 1E) showed that wild-type ZBP-89 siRNA dramatically knocked down ZBP-89 expression, whereas, as expected, mutant ZBP-89 siRNA had no effect on ZBP-89 mRNA levels. As a consequence of ZBP-89 silencing, IAP levels were also dramatically reduced, whereas mutant ZBP-89 siRNA had no effect on IAP expression, thus confirming ZBP-89-mediated activation of the IAP gene. Real-time PCR data confirmed the RT-PCR data shown above.

ZBP-89 activates IAP-luciferase reporter gene expression. To further explore the mechanism of IAP gene activation by ZBP-89, we determined the effects of this transcription factor on the expression levels of an IAP-luciferase reporter gene. We constructed the IAP-luciferase reporter plasmid pFRL7-IAP-2574, which carries the 2.5-kb 5' flanking region of the human IAP gene proximal to its AUG start codon. We transiently transfected HT-29 cells with pFRL7-IAP-2574, cotransfected them with either the ZBP-89 expressing plasmid or a control nonspecific plasmid, and then determined luciferase activities after 48 h. The results (Fig. 2A) showed that ZBP-89 activates the IAP gene by approximately fivefold in HT-29 cells. We observed similar activation of the IAP-luciferase reporter gene when Caco-2 and Cos-7 cells were transfected with the ZBP-89 expression plasmid (data not shown). To further confirm the effects of ZBP-89 on IAP activation, we examined the dose-dependent effects of ZBP-89 (Fig. 2B). HT-29 cells were transiently cotransfected with a fixed amount of pFRL7-IAP-2574 and increasing amounts of ZBP-89 expression plasmid; the results demonstrated that ZBP-89 activates the IAP gene in a dose-dependent manner. We also determined the temporal effects of exogenous ZBP-89 (Fig. 2C). Minimal effects of exogenous ZBP-89 were observed after 6 h, and maximal effects on IAP transcription were seen at 48 h, a time course that is similar to that observed by RT-PCR analyses.


Figure 2
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  		Fig. 2. Regulation of the IAP-luciferase reporter gene by ZBP-89. The IAP-luciferase reporter plasmid pFRL7-IAP-2574 is the derivative of the eukaryotic promoter-detection vector pFRL7 (see MATERIALS AND METHODS) and carries the 2.5-kb proximal promoter region of the human IAP gene (from –49 to –2574 bp upstream of the AUG start codon). HT-29 cells (~80% confluence) were transfected with the vector pFRL7 or its derivative pFRL7-IAP-2574. When indicated, cells were also cotransfected with the plasmid expressing ZBP-89. Cells were lysed 48 h after transfection, the relative firefly luciferase activity was determined, and the fold activation (ZBP89+/ZBP89–) was calculated. Results were obtained from 6 independent experiments, and values are expressed as means ± SD (P < 0.05). A: activation of the IAP-luciferase gene by ZBP-89 in HT-29 cells. Equal amounts (1.5 µg) of pFLR7 or pFRL7-IAP-2574 were used in transfection. When indicated, cells were also transfected with a fixed amount (2 µg) of the ZBP-89 expression plasmid. B: effects of increasing doses (dose response) of ZBP-89 on the regulation of the IAP-luciferase reporter gene. Different samples of HT-29 cells were transfected with a fixed amount of pFRL7-IAP-2574, and samples were also cotransfected with increasing amounts of ZBP-89 expression plasmid. C: temporal effects of exogenous ZBP-89 on IAP activation. Cells were transfected with fixed amounts of pFRL7-IAP-2574 and ZBP-89 expression plasmid, and relative luciferase activities were determined after specified periods of time.

 
Localization of ZBPRE within the IAP gene. We constructed various IAP-luciferase reporter plasmids carrying sequential 5' deletions of the IAP promoter region. HT-29 cells were transiently transfected with an individual IAP-luciferase construct along with the ZBP-89 expression plasmid. The results (Fig. 3) showed that ZBP-89 activates the full-length IAP plasmid (pFRL7-IAP-2574) by ~10-fold; however, there was a major decrease in reporter gene activation in the case of pFRL7-IAP-750, in which the levels were equal to that seen in the case of the pFRL7 vector alone (~2-fold). Plasmids carrying shorter IAP promoter fragments also showed the low basal level of activity, thereby localizing the ZBPRE between –1107 and –750, a 357-bp region (Fig. 3). Detailed sequence analyses of this 357-bp region suggested that the sequence between –1018 and –1010 (5'-CCTCCTCCC-3') could be the ZBPRE (IAP-ZBPRE; see Table 1) given its close homology to a previously characterized ZBPRE from the p21 gene (3).


Figure 3
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  		Fig. 3. Localization of the ZBP-89 response element (ZBPRE) in the 5' flanking region of the human IAP gene. Various 5' deletion mutants of pFRL7-IAP-2574 were constructed by deleting specific restriction fragments from the 5'-end of the 2.5-kb proximal IAP promoter, and the resultant IAP-luciferase reporter plasmids were used to transfect HT-29 cells (~80% confluence) (see MATERIALS AND METHODS). When indicated, cells were also cotransfected with 2 µg of the plasmid expressing ZBP-89. Cells were lysed 48 h after transfection, the relative firefly luciferase activity was determined, and fold activation (ZBP89+/ZBP89–) was calculated. Results were obtained from 6 independent experiments, and values are expressed as means ± SD (P < 0.05). Be, BstEII; Bt, BtrI; Bu, Bsu36I; Bx, BstXI; Ml, MluI; Pf, PflMI; Ps, PstI; Pv, PvuII; Sc, SacI; Sm, SmaI; Sp, SphI; Xh, XhoI.

 

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  		Table 1. Properties of the oligonucleotides used in mapping of the IAP-ZBPRE

 
ZBP-89 protein binds to putative IAP-ZBPRE in vitro. We used EMSAs to determine the binding of the in vitro-synthesized ZBP-89 protein to the putative IAP-ZBPRE described above. We synthesized a double-stranded oligonucleotide carrying the putative IAP-ZBPRE sequence along with 13 and 11 extra nucleotides at its 5'- and 3'-ends, respectively (Table 1). For control purposes, we also synthesized the p21-ZBPRE (3) and a mutant version of the IAP-ZBPRE (mZBPRE) (Table 1). The EMSA result showed that in vitro-synthesized ZBP-89 protein efficiently binds to the IAP-ZBPRE, thus generating a shifted band (Fig. 4A, lane 2). Fifty-fold excess of cold competitor IAP-ZBPRE (Fig. 4A, lane 3) or p21-ZBPRE (Fig. 4A, lane 4) could compete with the IAP-ZBPRE probe, whereas mZBPRE could not compete (Fig. 4A, lane 5), thus confirming the specificity of binding. This result indicates the presence of a genuine ZBPRE in this IAP sequence (IAP-ZBPRE oligonucleotide; Table 1).


Figure 4
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  		Fig. 4. Binding of ZBP-89 to its response element in the IAP gene analyzed by EMSA. Sequences of the oligonucleotides used in EMSA are shown in Table 1. Double-stranded oligonucleotide ZBPRE in the IAP promoter (IAP-ZBPRE) was 5'-end labeled with 33P and used as a probe in EMSA with either ZBP-89 protein synthesized in vitro using reticulocyte lysate or nuclear extract from HT-29 cells. A: binding of the in vitro-synthesized ZBP-89 protein to the putative IAP-ZBPRE. B: binding of nuclear protein(s) from HT-29 cells to the putative IAP-ZBPRE.

 
We next used EMSA to determine whether the endogenous ZBP-89 protein in HT-29 nuclear extract binds to the IAP-ZBPRE. EMSA was performed using a 33P-labeled IAP-ZBPRE probe (Table 1) and HT-29 nuclear extract along with the anti-ZBP-89 antibody for supershift analyses. The results showed a clear shifted complex with nuclear extract (Fig. 4B, lane 2), which could be supershifted by anti-ZBP-89 antibody (Fig. 4B, lane 6). When 50-fold excess of cold competitor oligonucleotides was tested, IAP-ZBPRE and p21-ZBPRE were able to compete, whereas mutant mZBPRE could not (Fig. 4B, lanes 3–5, respectively), thus demonstrating the specificity of binding. These results show that HT-29 cells express ZBP-89 protein that is capable of specifically binding to the IAP-ZBPRE in vitro.

ZBP-89 protein binds to putative IAP-ZBPRE in vivo. We performed ChIP to determine whether ZBP-89 protein binds tothe IAP promoter region, specifically in the region surrounding the identified ZBPRE located between –1018 and –1010 bp upstream of the AUG start codon (see above). We expect that, like most transcription factors, ZBP-89 protein initiates transcription by binding to the ZBPRE, and hence it would not bind further upstream of the ZBPRE. Accordingly, as a nonspecific control, we also investigated whether ZBP-89 binds to the IAP promoter region about 8 kb upstream of the start codon. We investigated ZBP-89 binding in chromatin from untransfected HT-29 cells as well as from cells transfected with the ZBP-89 expression plasmid. We designed the target primers IAP-1140F and 1AP-987R to determine the specific binding of ZBP-89 around the IAP-ZBPRE and also used the control primers IAP-8326F and IAP-8028R to examine any binding of ZBP-89 in the 8 kb upstream region of the IAP gene (see MATERIALS AND METHODS). The results showed that both sets of primers were able to amplify the expected DNA fragments from input DNA that were extracted from untransfected and transfected cells and were not subjected to treatment with any antibody, thus validating the authenticity of the primers (Fig. 5, lanes 1 and 2, respectively). The target fragment containing the ZBPRE was amplified from anti-ZBP-89 antibody-targeted immunoprecipitated DNA samples, thus indicating in vivo binding of ZBP-89 to the IAP chromatin of untransfected as well as transfected HT-29 cells (Fig. 5, top, lanes 3 and 4, respectively). No PCR product was obtained when control primers (IAP-8326F and IAP-8028R) were used to PCR amplify the immunoprecipitated DNA from untransfected and transfected cells (Fig. 5, bottom, lanes 3 and 4, respectively). This result confirms that ZBP-89 does not bind to the chromatin 8 kb upstream of the AUG start codon. Lane 5 in Fig. 5, top and bottom, shows PCR products amplified with primers only (no template DNA), and, as expected, no DNA band was visible in this lane, indicating that the bands in the other lanes were specific products and were not from any contaminated template DNA.


Figure 5
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  		Fig. 5. Chromatin immunoprecipitation (ChIP) assay showing binding of ZBP-89 to the IAP-ZBPRE. Nucleosomes were isolated from HT-29 cells transfected with the ZBP-89 expression plasmid as well as from untransfected cells. PCR was performed on input DNA (In) as well as on immunoprecipitated (IP) DNA using specific (targeted to the IAP promoter region containing ZBPRE) and nonspecific (targeted to the IAP promoter region 8 kb upstream from the start codon) primers (see MATERIALS AND METHODS). PCR was repeated at least 3 times in each case, and photographs of representative gels are shown. Pmr, primer only (no template DNA); Un, untransfected; TF, transfected.

 
IAP-ZBPRE is functionally active. To assess the biological importance of the identified ZBPRE in the IAP promoter (IAP-ZBPRE), we performed site-directed mutagenesis to construct an IAP-luciferase reporter plasmid carrying substitution mutations of the ZBPRE (pFRL7-IAP-mZBPRE; Table 2). HT-29 cells were transfected with the mutant plasmid along with the ZBP-89 expression plasmid, and relative luciferase activity was determined. The results (Fig. 6) showed that the mutations rendered the IAP-ZBPRE biologically inactive, confirming that the identified IAP-ZBPRE is the major biologically active ZBPRE in the IAP promoter. Figure 7 depicts the 2.5-kb nucleotide sequence of the proximal 5' flanking region of the IAP gene, illustrating the ZBPRE along with the other previously identified cis-elements that respond to Kruppel-like factor 4 (KLF4) (15), thyroid hormone receptor (23), caudal-related homeobox transcription factor (CDX) (1), and hepatocyte nuclear factor-4 (HNF-4) (28).


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  		Table 2. Site-directed mutagenesis of the IAP-ZBPRE

 

Figure 6
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  		Fig. 6. Functional role of the IAP-ZBPRE in regard to ZBP-89-mediated regulation of the IAP gene. The mutant IAP-luciferase reporter plasmid pFRL7-IAP-mZBPRE carrying nucleotide-specific substitution mutations in the putative IAP-ZBPRE was constructed by in vitro site-directed mutagenesis (the sequence of the mutations is shown in Table 2; see also MATERIALS AND METHODS). Wild-type and mutant IAP-luciferase reporter plasmids were used to separately transfect HT-29 cells, and, where indicated, cells were also cotransfected with the plasmid expressing ZBP-89. Cells were lysed 48 h after transfection, the relative firefly luciferase activity was determined, and fold activation (ZBP89+/ZBP89–) was calculated. Results were obtained from 6 independent experiments, and values are expressed as means ± SD (P < 0.05).

 

Figure 7
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  		Fig. 7. Nucleotide sequence of the 5' flanking region of the IAP gene showing the ZBPRE and other cis-elements. The sequence is numbered relative to the ATG translation initiation site (marked as +1). The ZBPRE is underlined and marked in bold. The putative TATA box (pTATA) is also marked in bold. The Sp1/KLF4 binding site (IF-III/Sp1/GKLFRE), thyroid hormone response element (TRE), CDX response element (CDXRE), and hepatic nuclear factor-4 response element (HNFRE) are underlined and marked in bold. Restriction sites relevant to construction of the 5' deletion plasmids are marked and underlined. Transcription start sites [major (>>>) and minor (>)] are also marked. The translation initiation site ATG is marked in bold.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Alkaline phosphatases are membrane-bound glycoproteins that hydrolyze a wide variety of monophosphate esters at high pH optima (11–13, 24) and are widely distributed in different tissues. Considering their tissue distribution and biochemical nature, alkaline phosphatases have been classified as placental, placental-like, intestinal (fetal and adult), and liver/bone/kidney (L/K/B) forms. The human IAP gene produces a 528-amino acid protein, and it maps to chromosome 2q34–37 along with the genes for placental and placental-like alkaline phosphatase (13, 31). IAP is exclusively expressed in villus-associated enterocytes and hence serves as an excellent marker for crypt-villus differentiation (8, 16). IAP is a component of the unilamellar membranes that surround fat droplets in enterocytes to form SLPs (9, 10). SLPs are formed inside enterocytes and are then secreted into either the intestinal lumen or bloodstream through lymphatic vessels in the intestinal lamina propria (21, 34, 40). IAP functions as the receptor for Aeromonas sobria hemolysin, indicating some role in the pathogenesis of diarrhea caused by this bacterial toxin (38). On the other hand, IAP also may function as a host defense factor by reducing the toxicity of bacterial lipopolysaccharides (19). A recent report (27) on IAP knockout mice indicates a major role for this protein in regard to the regulation of dietary fat absorption, because the knockout mice became obese when fed a high-fat diet. We (16, 17) have previously shown that IAP expression is silenced during starvation both in vitro and in vivo. Taken together, IAP gene knockout data along with IAP regulation by starvation have led us to hypothesize that IAP gene silencing may be an important adaptive response of the gut epithelium, designed to prepare the animal for maximal absorption of fat once food is available.

Given the importance of IAP in fat absorption, delineation of its regulatory mechanisms might identify one or more therapeutic targets that could be exploited in the clinical setting to either enhance or inhibit weight gain. In addition, the mechanisms that govern IAP gene expression will almost certainly be applicable to other enterocyte-specific genes and thus provide a broader understanding of the crypt-villus differentiation program. In previous work, we have described regulation of the IAP gene by homeobox transcription factors, CDX1 and CDX2 (1), thyroid hormone (23), and gut-enriched KLF4 (15). Furthermore, synergistic activation of the IAP gene was seen in regard to thyroid hormone and KLF4 (33). Recently, Olsen et al. (28) have shown differentiation-dependent activation of the IAP gene by HNF-4 in the Caco-2 cell line. The present work adds the Kruppel transcription factor ZBP-89 to the network of transcription factors that regulate IAP gene expression (see Fig. 7).

ZBP-89 is a Kruppel-type transcription factor that requires zinc for its function and is ubiquitously expressed (26). Using RT-PCR, quantitative real-time PCR, and Western blot analyses, we determined the levels of endogenous ZBP-89 and/or IAP in the two well-characterized enterocyte-like cell lines HT-29 and Caco-2 (Fig. 1, A–C). RT-PCR showed that the two cell lines had similar levels of endogenous ZBP-89, but, compared with Caco-2 cells, the endogenous levels of IAP were extremely low in HT-29 cells, suggesting that one or more cell line-specific factors exist in Caco-2 cells and activate the IAP gene. When the cells were transfected with the ZBP-89 expression plasmid, IAP mRNA and protein levels increased in both cell lines (Fig. 1, A–C). These results indicate that the IAP gene can be activated by exogenous ZBP-89. In addition, we have also documented an increase in overall alkaline phosphatase enzyme activity in response to ZBP-89 (data not shown).

To assess the effects of endogenous ZBP-89 on IAP expression, we employed siRNA to knock down (silence) endogenous ZBP-89 expression (Fig. 1, D and E). ZBP-89 silencing resulted in a marked decrease in IAP levels, confirming that IAP is a ZBP-89 target gene. Transient transfections were employed to examine the molecular mechanisms by which ZBP-89 transcriptionally activates the IAP gene. We found that the approximate fivefold activation of the IAP gene was mediated largely by a biologically functional ZBPRE (IAP-ZBPRE: 5'-CCTCCTCCC-3') located between –1018 and –1010 (Figs. 2–4). Remarkably, this response element is identical to the antisense strand of the previously characterized p21-ZBPRE (3). Because the ZBPRE is an enhancer element, it is not surprising that it can function in either orientation.

EMSA demonstrated that the purified ZBP-89 protein was able to bind to the IAP-ZBPRE, i.e., no binding partner was required. Furthermore, endogenous ZBP-89 protein from HT-29 cells could also bind to the IAP-ZBPRE. The ChIP assay confirmed the binding of ZBP-89 to the ZBPRE region. It will be of interest in the future to examine the status ofhistone acetylation and the involvement of other chromatin-modulating enzymes in relation to ZBP-89-mediated regulation of the IAP gene.

Given that the ZBPRE is a GC-rich sequence, it is possible that other transcription factors, such as KLF4, Sp1, and Sp3 might also bind to the IAP-ZBPRE. Previously, we (15) have shown that KLF4 binds to a GC-rich cis-element (IF-III) located in the proximal IAP gene promoter between –153 and –146 (see Fig. 7). Interestingly, we have observed that in vitro-synthesized ZBP-89 protein is also able to bind to this GC-rich cis-element (data not shown). Although the present study suggests that the identified ZBPRE is the major functional response element in the IAP gene, it will be worthwhile in future studies to determine whether ZBP-89 also interacts with the proximal promoter region to regulate IAP gene expression.

The role that ZBP-89 plays in gut epithelial biology remains somewhat unclear. For example, ZBP-89 appears to play a role in the growth arrest of the gastric adenocarcinoma AGS cell line (30), and yet it has been shown to be overexpressed in gastric cancers (37). The fact that ZBP-89 binds to the p21 promoter and interacts with p53 strongly suggests that its major function relates to an inhibition of cellular proliferation (2, 3, 5). ZBP-89-mediated activation of a differentiation marker, like IAP, further supports its role in the linked processes of growth arrest and enterocyte differentiation. Our previous results have shown that among the short-chain fatty acids, sodium butyrate maximally activates the IAP gene, inducing a differentiated phenotype in HT-29 cells (14, 17, 25). We (3) have also shown that sodium butyrate activates ZBP-89 expression and that its antiproliferative function is probably mediated via its activation of the p21 gene. It will be of interest in the future to investigate a possible role for p21 in the activation of the IAP gene.

The precise role that ZBP-89 plays in IAP gene regulation cannot be determined from the present results. It is clear that many transcription factors are involved in the regulation of the IAP gene, and some of these factors likely interact with each other to govern IAP activation or repression. Studies using animal model systems will be needed to further delineate the physiological role for ZBP-89 in regard to the IAP gene and in particular to determine whether this factor plays a role in the IAP gene silencing seen with starvation.

In conclusion, we have shown that IAP is a target gene for the ubiquitously expressed Kruppel-like zinc finger transcription factor ZBP-89. As such, IAP is the only enterocyte-specific gene identified as a target of ZBP-89. Given the multitude of functional roles reported for ZBP-89 and the recently identified role of IAP in fat absorption, these results will have important implications in regard to numerous cellular processes, including gut development, differentiation, and homeostasis.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Research Grants RO1-DK-47186 and RO1-DK-50623 (to R. A. Hodin) and R01-DK-55732 (to J. L. Merchant).


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. A. Hodin, Dept. of Surgery, Massachusetts General Hospital, Gray 504, 55 Fruit St., Boston, MA 02114 (e-mail: rhodin{at}partners.org)

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.


   REFERENCES
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 MATERIALS AND METHODS
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 DISCUSSION
 GRANTS
 REFERENCES
 

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