Clusterin gene transcription is activated by caudal-related homeobox genes in intestinal epithelium

Eunran Suh1, Zhengqi Wang2, Gary P. Swain1, Martin Tenniswood2, and Peter G. Traber1

1 Department of Internal Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; and 2 Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 45556


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Caudal-related homeobox (Cdx) proteins play an important role in development and differentiation of the intestinal epithelium. Using cDNA differential display, we identified clusterin as a prominently induced gene in a Cdx2-regulated cellular model of intestinal differentiation. Transfection experiments and DNA-protein interaction assays showed that clusterin is an immediate downstream target gene for Cdx proteins. The distribution of clusterin protein in the intestine was assessed during development and in the adult epithelium using immunohistochemistry. In the adult mouse epithelium, clusterin protein was localized in both crypt and villus compartments but not in interstitial cells of the intestinal mucosa. Together, these data suggest that clusterin is a direct target gene for Cdx homeobox proteins, and the pattern of clusterin protein expression suggests that it is associated with the differentiated state in the intestinal epithelium.

Cdx2; differential display; differentiation; transactivation; Caco-2


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MEMBERS OF THE CAUDAL-RELATED homeobox gene family, Cdx1 and Cdx2, appear to have important roles in the function of the intestinal epithelium. Cdx1 is expressed exclusively in the epithelial cells of the small intestine and colon of adult mice and humans (14) and is capable of transactivating intestinal genes (20). Mice null for Cdx1 demonstrate an anterior homeotic transformation of the axial skeleton caused by expression in the early embryo (16), but information on suggested abnormalities in the intestinal epithelium has not yet been published. Cdx2 is also expressed predominantly in the epithelial cells of the adult intestine and colon (3, 8, 9, 17). Cdx2 activates transcription of intestinal genes (17), transiently inhibits cell growth in intestinal cell lines, and induces a marked morphological differentiation and induction of intestinal genes in undifferentiated cell lines (18). Mice null for Cdx2 die early in gestation, between 3.5 and 5.5 days after coitus (4). Interestingly, heterozygous knockout mice exhibit an anterior homeotic transformation of the axial skeleton similar to the phenotype of the Cdx1 null mice and develop anterior metaplasia of the colonic epithelium by 3 mo of age (2, 4, 19).

Because of the marked effect of Cdx2 expression on induction of a differentiated phenotype in cell lines, we attempted to identify gene targets for Cdx2 that may be involved in the differentiation program. For these studies, we used the cDNA differential display method to identify genes that are differentially expressed during Cdx2-induced cellular differentiation. One mRNA that was markedly induced after expression of Cdx2 was clusterin, a secreted glycoprotein that has been associated with many cellular processes, including most prominently apoptosis. In transfection experiments we found that the rat clusterin promoter is a direct target for Cdx2 and that the ability of Cdx2 to induce transcription is embodied primarily in the immediate upstream promoter of the gene. To examine the functional role of clusterin in the differentiation process, we studied the cellular patterns of expression during intestinal development and overexpressed clusterin in intestinal epithelial cells. Immunohistochemistry experiments showed that clusterin protein was expressed in high levels in the mouse intestinal epithelium during the endoderm-intestinal transition. Several days later clusterin was expressed in the supranuclear region of enterocytes, and the pattern stayed the same in the adult. Over-expression of clusterin in intestinal epithelial cells did not result in the morphological changes that are found with expression of Cdx2. Therefore, clusterin is a downstream gene target of Cdx2, but it does not seem to play a primary role in directing epithelial morphogenesis previously seen in this system. Its developmental expression in the intestine suggests several potential functions.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell culture and maintenance. Small intestinal epithelial IEC-6 cells and IEC6-Cdx2L1 cells were maintained as previously described (18). Briefly, IEC-6 cells and stably transfected IEC-6 cells with a mouse Cdx2-inducible expression vector (LacSwitch system, Stratagene, La Jolla, CA) were cultured under an atmosphere of 5% CO2 in Dulbecco's modified Eagle's medium with 4.5 g/l glucose containing 5% fetal calf serum and 0.1 U/ml of insulin. For isolation of total RNA cells were plated at 20% confluence in 100-mm plates, and 24 h later the medium was replaced with medium with or without 4 mM isopropyl-beta -D-thiogalactopyranoside (IPTG).

RNA preparation and differential display analysis. Differential display was performed as previously described (11) with modifications. Total RNA was obtained from IEC-6 and IEC6-Cdx2L1 cells that were grown in the absence or presence of 4 mM IPTG for 3, 24, 48, 72, and 96 h and prepared using the guanidinium-CsCl procedure (15). In each case, cDNA was synthesized at 37°C for 1 h in the reverse transcription reaction containing 500 ng of total RNA, 2.5 µM reverse primer (T12CG), 20 µM dNTPs, 5 mM dithiothreitol, 40 units RNase inhibitor (Promega, Madison, WI), and 5× reaction buffer with 200 units of Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (GIBCO BRL, Gaithersburg, MD). At the end of this incubation, the temperature was increased to 95°C for 5 min. PCR amplification of a given reverse transcription reaction was prepared in 20 µl of PCR mixture containing 2 µl of the reverse transcription reaction, 2.5 µM reverse primer (T12CG), 0.5 µM arbitrary primer (5'-CTTGATTGCC 3'), all dNTPs, each at 100 µM, 10 mM Tris · HCl (pH 8.3), 2.5 mM MgCl2, 10 µCi of [32P]dCTP (3,000 Ci/mmol), and 2.5 units of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN). PCR was performed in a 9600 thermocycler (Perkin-Elmer, Foster City, CA) using the following parameters: 94°C for 2 min; 1 cycle of 94°C for 30 s, 42°C for 60 s, and 72°C for 60 s; and 35 cycles, the last of which was followed by a 5-min extension at 72°C. Three microliters of the PCR-amplified cDNA products were resolved on a 6% DNA sequencing gel. The gels were transferred to filter paper and dried on vacuum dryer. Glogos II autoradiographic markers (Stratagene, La Jolla, CA) were adhered to the dried gels to aid in subsequent alignment. The gels were then exposed for autoradiography.

Recovery and reamplification. The cDNA bands of interest were excised from the dried gel on the filter paper and transferred to Eppendorf tubes. Each gel slice was rehydrated for 30 min in 100 µl of diethyl pyrocarbonate-treated water at room temperature and incubated in boiling water for 20 min. After centrifugation for 10 min at 17,000 g to pellet any solid debris, the supernatant was removed and 10 µl of 3 M sodium acetate and 250 µl of 100% EtOH were added. The DNA was precipitated at -70°C for 1 h and collected by centrifugation. The pellet was resuspended in 10 µl of distilled water, and 5 µl of eluted DNA were used for PCR reamplification. PCR conditions were as described in RNA preparation and differential display analysis except that all dNTPs, each at 60 µM, were added without [32P]dCTP and the concentration of both primers was 2.5 µM in a final reaction volume of 40 µl. The reamplified PCR products were cloned using the TA cloning kit (InVitrogen, San Diego, CA) and sequenced. The sequence of cDNA was analyzed using the BLAST program.

Northern blot analysis. Total RNA (10 µg) was separated in a 1.4% agarose-formaldehyde gel and transferred to a Hybond-N membrane (Amersham, Arlington Heights, IL). Probes were synthesized from reamplified PCR products, followed by random priming in the presence of [alpha -32P]dCTP (3,000 Ci/mmol). Hybridization was performed as described previously (17).

Immunohistochemistry. Paraffin-embedded tissue sections were fixed with 4% paraformaldehyde as described previously (14) and treated by boiling for 6 min in a microwave oven to quench endogenous alkaline phosphatase activity. For immunostaining for clusterin, SGP-2 antibody (generous gift of Dr. Michael Griswold, Washington State University) was used in a 1:1,500 dilution. The primary antibody was visualized with goat anti-rabbit biotinylated antiserum and avidin/biotin system (ABC) according to the protocol provided by Vector Labs (Burlingame, CA). The slides were developed with 5-bromo-4-chloro-3-indolyl-phosphate-4-nitro blue tetrazolium chloride (Boehringer Mannheim). The tissue was counterstained with neutral red.

Stable transfection. The complete coding sequence of rat clusterin cDNA (testosterone-repressed prostate message-2; TRPM-2) was obtained from pG17H by excising with EcoR I. A 1.7-kb cDNA fragment was blunt ended and inserted with either orientation to construct sense (pMTtrpm) or antisense (pMTtrpmAS) into an EcoR V site in pMTCB6+, which contains the promoter of the sheep metallothionein I gene (6). IEC-6 and IEC6-Cdx2L1 cells were transfected as described previously (18) by electroporation with pMTtrpm or pMTtrpmAS, respectively.

Electrophoretic mobility shift assay. Electrophoretic mobility shift assay (EMSA) was performed with nuclear extract from COLO DM cells as described previously (25). Oligonucleotides for making probes were as follows: upstream Cdx2 binding element (UCB) top strand: 5'-GATCCATGTTTAGGTTTTATGCATCTCAA-3'; UCB bottom strand: 5'-GATCTTGAGATGCATAAAACCTAAACATG-3'; mutant UCB top strand: 5'-GATCCATGTTTAGGTGGGATGCATCTCAA-3'; mutant UCB bottom strand: 5'-GATCTTGAGATGCATCCCACCTAAACATG-3'; TATA top strand: 5'-GATCCAGAGCGCTATAAATAGGGCGCA-3'; TATA bottom strand: 5'-GATCTGCGCCCTATTTATAGCGCTCTG-3'; mutant TATA top strand: 5'-GATCCAGAGCGCAGCCAATAGGGCGCA-3'; and mutant TATA bottom strand: 5'-GATCTGCGCCCTATTGGCTGCGCTCTG-3'.

Plasmid reporter constructs and transfection analysis. The rat clusterin gene structure was reported previously (26). The Hind III-Pvu II fragment, representing 1,297 nucleotides of the rat clusterin promoter upstream of the transcription start site and 57 nucleotides downstream of the start site, was subcloned into the luciferase reporter pLuc-Link (5). A series of unidirectional deletional mutants were generated by exonuclease III digestion of the plasmid using the Erase-a-Base system (Promega, Madison, WI). Plasmids for transfection were purified by two rounds of CsCl gradient centrifugation, and cells were transfected and analyzed as previously described (23, 27, 28).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Clusterin mRNA is induced in intestinal cell lines that express Cdx2. We previously showed (18) that induction of Cdx2 expression in IEC-6 cells, an undifferentiated rat intestinal epithelial cell line, results in dramatic changes in proliferation, morphology, and expression of intestinal genes. To identify other genes that are induced as part of the differentiation program, we used the cDNA differential display method to compare patterns of mRNA expression between IEC-6 cells that express Cdx2 and those that do not (see EXPERIMENTAL PROCEDURES). For these experiments, we used the IEC6-Cdx2L1 cell line that we engineered to conditionally express Cdx2 when IPTG is included in the culture medium (18). In one condition of differential display, cDNA was generated by reverse transcription using T12CG, which anneals to a subset of polyadenylation tails. PCR amplification was then performed with T12CG and a second primer containing a sequence of 10 nucleotides that were chosen at random. A 160-bp cDNA was detected in RNA isolated from IEC6-Cdx2L1 cells that had been cultured in medium containing IPTG to induce expression of Cdx2 but not from RNA isolated from either uninduced IEC6-Cdx2L1 cells or the parental IEC-6 cell line (Fig. 1A). This cDNA fragment was recovered from the gel, reamplified, subcloned, sequenced, and identified as the 3'-untranslated region of the rat clusterin mRNA (26).


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Fig. 1.   Differential display of IEC6-Cdx2L1 mRNA. A: IEC-6 and IEC6-Cdx2L1 cells were grown in the presence and absence of induction medium containing 4 mM isopropyl-beta -D-thiogalactopyranoside (IPTG) and RNA isolated after 1 day. RNA was reverse transcribed and PCR amplified as described in EXPERIMENTAL PROCEDURES. Arrow indicates the differentially expressed cDNA, which were isolated for reamplification. The photograph is a negative image of the radiographic film. B: Northern blot analysis using the reamplified 160-bp cDNA (top panels) and caudal-related homedeox (Cdx) 2 (middle panels) as a probe. Total RNA (10 µg) was isolated from IEC6-Cdx2L1 cells at the indicated time and from IEC-6 cells at 120 h after culture with (right panels) or without (left panels) 4 mM IPTG. RNA loading was assessed by ethidium bromide staining of 28S and 18S rRNA (bottom panels), and the fold induction of the clusterin mRNA was quantified and normalized with ribosomal RNA.

The clusterin cDNA was used to examine the expression of clusterin mRNA using Northern blot analysis (Fig. 1B). Parental IEC-6 cells express low levels of clusterin mRNA, as do IEC6-Cdx2L1 cells cultured in medium that did not contain IPTG. In contrast to the parietal cells, IEC6-Cdx2L1 cells were stimulated with IPTG to express Cdx2 as early as 2 h after induction. By 8 h after induction with IPTG there was already a small increase in clusterin mRNA, and by 20 h there was a threefold increase in clusterin mRNA that was sustained through 120 h of culture. There was a slight increase in clusterin mRNA at 120 h in the uninduced IEC6-Cdx2L1 cells caused by leaky expression of Cdx2 (Fig. 1B; Ref. 18). These results show that the expression of the clusterin gene is induced in IEC-6 cells as a result of expression of Cdx2. Thus clusterin may be a downstream gene in the regulatory network leading to the differentiated phenotype in IEC-6 cells.

Expression of clusterin in mouse intestinal tract. Clusterin is a heterodimeric glycoprotein encoded from a single mRNA that is expressed widely in tissues and is found circulating in the serum (13). Clusterin has been isolated and cloned from multiple species resulting in multiple synonyms for clusterin, including SGP-2, apolipoprotein J, and TRPM-2. Clusterin is widely expressed and is relatively abundant in testes, brain, and liver under normal conditions. It has been implicated in several biological processes such as cell-cell interactions, apoptosis, sperm maturation, membrane remodeling, lipid transport, and regulation of complement-induced cell lysis. In a number of tissues, clusterin is expressed during cellular differentiation and development. Clusterin mRNA is induced in the intestinal epithelium at embryonic day 16.5, just after the start of a marked change in the morphology of the epithelium from a stratified endoderm to a columnar intestinal epithelium (7).

To further explore the potential role of clusterin in the intestinal tract, we examined the expression of clusterin protein in the developing mouse intestinal tract and in the adult small intestine and colon. At postcoital day 13.5, a time when the endoderm of the developing intestine has a pseudostratified structure, there was very light staining of the endodermal cells (Fig. 2A). Intense immunostaining for clusterin was seen in the developing intestine immediately after the transition from endoderm to the columnar intestinal epithelium (Fig. 2, B and C). At postcoital day 14.5, when the stratified endoderm begins to form villi, much more intense cytoplasmic staining is noted in the nascent villi and intervillus epithelial cells (Fig. 2B). At postcoital day 15.5, when the simple columnar epithelium has been fully established, clusterin immunostaining was concentrated in the supranuclear area of epithelial cells (Fig. 2C). This pattern of protein expression at the time of the endoderm-intestinal transition is similar to the previously described pattern of expression of clusterin mRNA (7).


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Fig. 2.   Localization of clusterin protein in embryo and adult mouse intestine. Mouse embryos and proximal jejunum from adult mice were fixed, paraffin embedded, and immunostained with anti-SGP-2 (1:1,500), as described in EXPERIMENTAL PROCEDURES. A: clusterin protein was only weakly detectable in the endodermal cells (En) and not in the mesodermal cells (Me) of the postcoital day 13.5 embryo. B: At embryonic day 14.5, the cytoplasm of epithelial cells (Ep) on nascent villi and in the intervillus regions was positive for clusterin protein. Mesoderm-derived cells (Md) were unstained. C: a high level of clusterin expression was sublocalized in the cytoplasm of all epithelial cells by postcoital day 15.5. Arrowheads in A, B, and C indicate examples of positively stained cells. D: apical regions of the nuclei were stained in the epithelium of the proximal jejunum at postnatal day 9. Submucosa layer (SM) was not stained. E: in the adult, epithelial cells in both villus (V) and crypt (C) regions were positive for clusterin. F: high-magnification view of inset in E shows more detail of supranuclear staining (arrowheads) with clusterin antibody. Arrows indicate nuclei of epithelial cells. A and C: ×200; B: ×160; D: ×125; E: ×100; F: ×600.

In the small intestinal epithelium of a 9-day-old (Fig. 2D) and an adult (Fig. 2E) mouse, clusterin protein was exclusively expressed in epithelial cells. Both villus and crypt cells were positive for clusterin staining, but there was a gradient of activity with higher expression in the villus compartment than in the crypts. A high-power view shows more detail of supranuclear staining (Fig. 2F).

Overexpression of clusterin is insufficient to initiate a differentiation program in IEC-6 cells. Early expression of clusterin in IEC-6 cells expressing Cdx2, as well as the developmental timing of clusterin gene expression in the intestinal epithelium, suggested that the clusterin protein may have a regulatory role in the differentiation process. Therefore, we made stable IEC-6 cell lines that expressed the rat clusterin cDNA under the direction of a sheep metallothionein promoter (see EXPERIMENTAL PROCEDURES). Three cell lines were characterized that expressed clusterin when treated with medium containing zinc sulfate. These cell lines did not show changes in proliferation or morphology on induction of clusterin protein expression (data not shown). Therefore, although clusterin is induced as a result of Cdx2 expression, expression of clusterin by itself is incapable of initiating the same differentiation program. This experiment showed that clusterin alone is insufficient for induction of differentiation, but it does not address the issue of whether clusterin is necessary for differentiation in this cell model. To answer this question, we attempted to inhibit the expression of clusterin in IEC6-Cdx2L1 cells by introducing an antisense clusterin cDNA under the control of the zinc-inducible promoter. Although we were able to isolate cell lines, we were not able to identify a cell line that showed decreased clusterin mRNA expression. Therefore, the issue of whether clusterin gene expression is required for induction of differentiation in this model remains unresolved.

Cdx2 binding elements in the rat clusterin gene 5'-flanking region. Cdx2 might induce clusterin mRNA levels either by stimulation of gene transcription or by prolongation of the mRNA half-life. Moreover, activation of gene transcription might be a direct effect of Cdx2 on the gene promoter or an effect on other genes that subsequently regulate clusterin gene transcription. We inspected the promoter of the rat clusterin gene and identified two elements located at ~740 and 25 nucleotides upstream of the transcriptional start site that had a consensus sequence for Cdx2 binding elements (Fig. 3A). The UCB (nucleotide -740) contains two adjacent binding elements, as in the sucrase-isomaltase promoter where the Cdx2 binding elements were originally described (17). The element located at nucleotide -25 contains a TATA box consensus sequence and is located appropriately to be the binding site for TATA box binding protein (TBP) in the process of transcriptional initiation. To examine direct binding of Cdx2 protein to the two binding elements, EMSA was performed using nuclear extract from COLO DM cells, which express a high level of Cdx2 proteins (17, 18). Supershift experiments with specific Cdx2 antibodies and competition experiments with mutant oligonucleotides showed that both elements interacted specifically with Cdx2 (Fig. 3B). Previously, we showed (12, 20) that Cdx1 could bind to DNA elements similarly to Cdx2 and could activate transcription. Therefore, we performed EMSA using nuclear extracts from IEC-6Cdx1MT1 cells (12). Supershift experiments with Cdx1 antibodies demonstrated that Cdx1 was also able to bind to the UCB and the TATA element (data not shown). Thus Cdx1 and Cdx2 proteins can bind to both of these potential regulatory elements in the rat clusterin gene.


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Fig. 3.   Specific binding of Cdx2 protein to DNA elements in the clusterin promoter. A: comparison of Cdx2 binding sites in the clusterin promoter. Cdx binding sites, upstream Cdx2 binding element (UCB), within TATA sequences, and the SIF1 element in the sucrose-isomaltase gene promoter are shown by underlines. B: nuclear proteins isolated from the COLO DM cells were used for EMSA to assess for specific binding of Cdx2 protein (arrow) to 2 elements (UCB and TATA) of the clusterin promoter. For competition experiments, a 100-fold excess of the unlabeled SIF1, UCB, or TATA double-stranded oligonucleotides was used. Anti-Cdx2 antibody (alpha Cdx2) was used for supershift experiments demonstrating the presence of the Cdx2 protein in the complex. Arrowheads indicate the complex that supershifted with addition of anti-Cdx2 antibody.

Activation of the clusterin promoter in cell lines. The ability of the rat clusterin promoter to drive transcription in intestinal and nonintestinal cell lines was tested using various lengths of the 5'-flanking region of the rat clusterin gene linked to the luciferase reporter gene (Fig. 4). The construct -771-luc contained the UCB Cdx binding site, and the -732-luc construct had this element deleted. In the mouse fibroblast cell line NIH/3T3, the clusterin promoter was able to activate transcription with very little difference between the three different constructs tested. Caco-2 cells were used as a model intestinal cell line; they have a small amount of endogenous Cdx2 protein and can support low-level expression of Cdx-responsive genes such as sucrase-isomaltase (17). The shortest construct (-421-luc) had only a slightly greater level of activation in Caco-2 cells than in NIH/3T3 cells. In contrast, the -771-luc construct was expressed at fourfold greater levels in Caco-2 cells than in the fibroblast line, with -732-luc in between the -421-luc and -771-luc constructs. As a control, when the clusterin promoter was inserted into the luciferase vector in the inverse orientation, there was minimal constitutive expression in either NIH/3T3 or Caco-2 cells (Fig. 4). These results suggest that there are factors in Caco-2 cells that are able to activate DNA regulatory elements located between nucleotides -421 and -771. Although not responsible for all the additional activation, these results also suggest that the 39-base element located between -771 and -732, which contains two Cdx binding sites, is able to augment clusterin gene transcription in an intestinal cell line.


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Fig. 4.   Functional analysis of deletional mutants of the rat clusterin gene promoter. Mutants in which the deleted clusterin gene was linked to luciferase were transfected into Caco-2 and NIH/3T3 cells. Testosterone-repressed prostate message-2 (TRPM) contains -1297 to +57 fragment of clusterin gene in inverted orientation in the luciferase plasmid vector pLL. CMV-beta gal was used as a control for transfection efficiency. Results are expressed as the absolute values of light units generated per unit of beta -galactosidase activity (means ± SD, n = 3).

Cdx proteins transactivate the rat clusterin promoter. We next examined the ability of Cdx proteins to directly activate transcription of the rat clusterin promoter. The minimal clusterin promoter, containing nucleotides -48 to +57, was not able to activate transcription in NIH/3T3 cells. As shown in Fig. 4, 771 nucleotides of the 5'-flanking sequence resulted in moderate constitutive activation of the promoter-reporter construct. Removal of the Cdx binding element between -771 and -732 did not change the constitutive activation of the promoter in NIH/3T3 cells (Figs. 4 and 5). Cotransfection of an expression vector for either Cdx1 or Cdx2 resulted in marked activation of each of the clusterin promoter constructs (Fig. 5). The level of induction over constitutive expression was greatest for the -48 to +57 construct, with the same level of induced expression for the -732 to +57 construct. Addition of the UCB Cdx binding element (-771 to -732) resulted in a further induction by both Cdx1 and Cdx2 expression vectors with no change in constitutive activation. Similar results on induction by either Cdx1 or Cdx2 on the -48, -732, and -771 clusterin promoter constructs were obtained from five independent transfection experiments (data not shown). Removal of the NH2-terminal domain of Cdx2 (HD2CD), which embodies the activation domain of the protein, eliminated the transcriptional activation of the clusterin promoter constructs (Fig. 5). This deletion construct of Cdx2 has been shown in other studies to effectively bind to DNA but is not capable of activating the sucrase-isomaltase promoter (21). As a final negative control, transfection with an expression construct for hepatocyte nuclear factor-1beta (HNF-1beta ) failed to activate transcription of the clusterin constructs.


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Fig. 5.   Transactivation of the clusterin promoter by murine Cdx1 and Cdx2. NIH/3T3 cells were cotransfected with deletional luciferase constructs of clusterin gene and expression vectors pRcCMV (control), pRc-Cdx1, pRc-Cdx2, HD2CD, or HNF-1beta . HD2CD contains amino acids 165-247 from Cdx2 coding region, which includes the DNA binding homeodomain but lacks the transcriptional activation domain. The localization of the Cdx binding elements in the clusterin promoter, UCB and the TATA box, are shown. Values are means ± SD, n = 3.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our results identify the clusterin gene as a direct target for Cdx transcription factors in intestinal epithelial cells. Moreover, we found that clusterin protein is expressed in a complex pattern during intestinal epithelial cells development. These findings have several implications for the mechanism of Cdx proteins in transcriptional regulation as well as the potential functions of the clusterin protein in intestinal development and differentiation.

The rapid induction of clusterin mRNA on expression of Cdx2 in IEC-6 cells provides evidence that regulation of the clusterin gene is part of a cascade of events initiated by Cdx2 expression. Transfection experiments using the rat clusterin gene promoter show that Cdx proteins can be direct regulators of transcriptional initiation of the clusterin gene. The 5'-flanking region of the clusterin gene revealed two areas with specific binding sites for the Cdx2 protein, one located between -771 and -732 nucleotides upstream of the transcriptional start site (UCB) and one that overlaps the putative TATA box. Both of these sites appear to play a role in clusterin gene regulation by Cdx proteins.

The most robust activation of the rat clusterin gene promoter by both Cdx1 and Cdx2 appears to be mediated primarily via a short promoter region encompassing nucleotides -48 to +57. The only Cdx binding site within this region is the putative TATA box, which is positioned properly with respect to the transcriptional start site to be the site of binding for TBP, an important component of the basal transcriptional apparatus. A number of controls show that transcriptional induction from this promoter is due to Cdx2 expression. First, when the promoter region is inverted in the luciferase reporter plasmid, there is no activation of transcription by Cdx proteins. Second, activation of the promoter is dependent on the presence of the activation domain of Cdx2. Finally, cotransfection of HNF-1beta , another transcription factor that contains a homeodomain, failed to activate transcription of the clusterin promoter. These results are even more interesting given the fact that another gene with a Cdx2 binding site overlying the TATA box has been shown to be inhibited by Cdx2 (10). However, the experiments described herein do not definitively prove that Cdx proteins activate transcription by interacting with the TATA region of the clusterin promoter. Experiments to show the dependence of transcriptional activation upon DNA binding of Cdx proteins to this promoter sequence are problematic because of the potential function of the TATA box in basal transcription. Simple transfection experiments with mutations in the TATA region are not sufficient to address this question. To examine this issue further, targeted mutations in the TATA box and initiator regions of the promoter would need to be tested in assays that use in vitro transcription. In this way, the effect of purified proteins, such as Cdx1, Cdx2, TFIID, and others, could be tested individually. The interrelationship of transcriptional proteins such as Cdx that can interact with the TATA box may provide additional insight into the complexities of transcriptional initiation of tissue-specific genes.

The UCB site is positioned at a distance from the transcription start site and only has a small effect on transcription of the clusterin promoter. However, the effect on enhancement of transcription of the clusterin promoter by this element is consistent in both Caco-2 transfections and cotransfection experiments with Cdx expression vectors in NIH/3T3 cells. The experiments in Caco-2 cells also suggest that there may be other elements that enhance transcription of the gene between nucleotides -421 and -771 including the UCB. These results suggest that the UCB element is capable of binding Cdx protein and that it likely plays a role in Cdx-mediated transcriptional induction of the clusterin gene.

Expression of clusterin appears to be associated with the process of differentiation in both the IEC-6 cell differentiation model and in the developing intestinal epithelium. Clusterin is first expressed at high levels during the endoderm-intestinal epithelium transition in the late fetal period in mouse. This corresponds to the time when the columnar epithelium of the intestine is first established (24). There are many morphological changes at this time with the development of cellular junctions, polarization, and the expression of many intestinal genes. In the adult intestine, clusterin is expressed in both the crypt and villus. In the IEC cell culture model of intestinal differentiation, clusterin expression in IEC-6 cells occurs after induction of Cdx2, which leads to the development of a differentiated columnar epithelium. Therefore, the patterns of clusterin expression suggest that the protein may have a role in a variety of intestinal cells during development and in the adult.

Although these studies of clusterin expression suggest an association with intestinal differentiation, the data provide little additional insight on the function of clusterin in intestinal epithelial cells. Many functions have been ascribed to clusterin, including a prominent association with apoptosis in many tissues and cells (13). Our data suggest that in the normal intestinal epithelium clusterin is not associated with apoptosis. Apoptotic cells are normally seen in the developing epithelium at the endoderm-intestinal epithelium transition, particularly associated with false lumens in the endoderm. Apoptotic cells are also seen occasionally in normal intestinal crypts and at the villus tips in adult intestine, but this is relatively uncommon. We did not see a concentration of clusterin protein in these locations. Furthermore, a previous study in the intestine showed that clusterin mRNA is not concentrated in apoptotic intestinal epithelial cells after irradiation (1).

In other cell systems, clusterin has also been found in association with differentiation (22). Although the function of clusterin in differentiation of the intestinal epithelium is not elucidated by the current studies, it appears that expression of clusterin is associated with epithelial differentiation. Clusterin is first expressed in the intestinal epithelium during the endoderm-intestinal transition, the most important developmental transition in the morphogenesis of the intestinal epithelium. Once the mature architecture of the epithelium is attained, clusterin expression is distributed in a very specific cellular and spatial pattern. It is expressed in highest levels in villus-associated enterocytes that reside in the nonproliferating, differentiated compartment of the intestinal epithelium. Because many genes are expressed in this pattern in the epithelium, this does not suggest a specific function but only that expression is associated with the differentiated phenotype, as suggested by the expression in IEC-6 cells expressing Cdx2. In enterocytes, clusterin protein appears to be distributed throughout the cytoplasm and not only in a supranuclear Golgi distribution as might be found for an exclusively secreted protein product. In addition to enterocytes, clusterin is expressed in higher levels in goblet cells. Immunohistochemistry performed on both paraffin-fixed and frozen sections indicates that clusterin protein is present both in the apical goblet cell and in the mucous globule and luminal mucus (data not shown). Therefore, it appears that clusterin may be secreted by goblet cells into the intestinal lumen. There is no evidence from our data that clusterin is expressed in either Paneth cells or enteroendocrine cells.

Together, our results show that clusterin expression is associated with the differentiation process in the intestinal epithelium. Moreover, clusterin might be a direct target gene for Cdx1 and Cdx2, which are transcription factors that participate in directing differentiation of intestinal epithelial cells. Thus clusterin is likely to be one of many genes regulated by Cdx genes that are important either for the differentiated process itself or for defining differentiated cellular function.


    ACKNOWLEDGEMENTS

Technical assistance in the performance of these studies was provided by Nadine Blanchard.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-46704 (to P. G. Traber) and the Morphology Core of the Penn Center for Molecular Studies in Digestive and Liver Disease (P30-DK-50306).

Address for reprint requests and other correspondence: E. R. Suh, GI Division, School of Medicine, Univ. of Pennsylvania, Suite 600 CRB, 415 Curie Blvd., Philadelphia, PA 19104 (E-mail: suher{at}mail.med.upenn.edu).

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.

Received 23 August 1999; accepted in final form 17 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 280(1):G149-G156
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