Regulation of alpha 1-antitrypsin gene expression in human intestinal epithelial cell line Caco-2 by HNF-1alpha and HNF-4

Chaobin Hu and David H. Perlmutter

Departments of Pediatrics, Cell Biology, and Physiology, Washington University School of Medicine, Division of Gastroenterology and Nutrition, St. Louis Children's Hospital, St. Louis, Missouri 63110


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

There is still relatively limited information about mechanisms of gene expression in enterocytes and mechanisms by which gene expression is regulated during enterocyte differentiation. Using the human intestinal epithelial cell line Caco-2, which spontaneously differentiates from a cryptlike to a villouslike enterocyte, we have previously shown that there is a marked increase in transcription of the well-characterized alpha 1-antitrypsin (alpha 1-AT) gene during enterocyte differentiation. In this study we examined the possibility of identifying the cis-acting elements and trans-acting DNA-binding proteins responsible for expression of the alpha 1-AT gene in Caco-2 cells during differentiation. Footprint analysis and electrophoretic mobility shift assays showed that hepatocyte nuclear factor-1alpha (HNF-1alpha ), HNF-1beta , and HNF-4 from nuclear extracts of Caco-2 cells specifically bound to two regions in the proximal promoter of the alpha 1-AT gene. Cotransfection studies showed that HNF-1alpha and HNF-4 had a synergistic effect on alpha 1-AT gene expression. RNA blot analysis showed that HNF-1alpha and HNF-4 mRNA levels and electrophoretic mobility shift assays showed that HNF-1alpha binding activity increase coordinately with alpha 1-AT mRNA levels during differentiation of Caco-2 cells. Finally, overexpression of antisense ribozymes for HNF-1alpha in Caco-2 cells resulted in a selective decrease in endogenous alpha 1-AT gene expression. Together, these results provide evidence that HNF-1alpha and HNF-4 play a role in the mechanism by which the alpha 1-AT gene is upregulated during enterocyte differentiation in the model Caco-2 cell system.

enterocyte differentiation; hepatocyte nuclear factor-1alpha ; hepatocyte nuclear factor-4


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

THERE IS STILL RELATIVELY little information about specific cis-acting elements and trans-acting DNA binding proteins that are responsible for gene expression in enterocytes. A 20-nucleotide element in the gene for the intestinal fatty acid binding protein acts as a suppressor of gene activation in crypt enterocytes and Paneth cells (44). A 22-nucleotide element in the sucrase-isomaltase (SI) gene, which binds caudal-related gene Cdx2, appears to be involved in the expression of SI in enterocytes (47, 52). Two elements further upstream in the 5'-flanking region of the SI gene, which bind hepatocyte nuclear factor-1alpha (HNF-1alpha ) and HNF-1beta , are also involved in expression of SI in enterocytes (52, 56). The HOXC11 homeodomain protein interacts with a 15-nucleotide element in the promoter of the lactase gene to direct its transcription in enterocytes (31). Another recent study has shown that a 9-nucleotide element in the gene for intestinal trefoil factor, which binds a distinct nuclear transcription factor, is involved in expression in intestinal goblet cells (36).

There is also some limited information about mechanisms of expression for genes that are expressed in both enterocytes and hepatocytes. A specific element in the alpha -fetoprotein gene binds HNF-1 in both hepatocytes and enterocytes (55). A cis-acting element in the apolipoprotein B gene is involved in its expression in both hepatocytes and enterocytes (38). Expression of apolipoprotein CIII in hepatocytes and enterocytes involves a cis-acting element, which binds apolipoprotein regulatory protein 1, Ear3/COUP-TF, and HNF-4 (30). There is even less information in the literature about cis-acting elements and trans-acting DNA-binding proteins involved in the regulation of gene expression that accompanies differentiation of enterocytes. One transcription factor, Cdx2, originally identified as a regulator of SI gene expression, appears to play a defined role in the differentiation program of enterocytes (47).

In previous studies we have shown that the alpha 1-antitrypsin (alpha 1-AT) gene, which is well known as a product of hepatocytes, is also expressed in human enterocytes in vivo (32) and in human enterocyte-like cell lines (20, 32, 33, 41). Moreover, in the human enterocyte-like cell line Caco-2 there is a marked increase in alpha 1-AT gene expression during spontaneous differentiation from the cryptlike to the villouslike enterocyte (41). Because the structure and function of the alpha 1-AT gene have been well characterized, we have now used it as a model gene for studies designed to identify specific mechanisms for gene expression in undifferentiated enterocytes as well as during the differentiation program of the enterocytes. The expression and regulation of the alpha 1-AT gene in the enterocyte are also of interest because previous data have raised the possibility that alpha 1-AT is part of a local response to injury and/or inflammation at the epithelial surface of the intestine (20, 33).

alpha 1-AT is an ~55-kDa serum glycoprotein that inhibits the destructive neutrophil proteases elastase, cathepsin G, and proteinase 3 (reviewed in Ref. 39). It is the archetype of a family of serum proteins, which are called serine protease inhibitors or SERPINS (reviewed in Ref. 24). It is also a classical positive acute phase protein in that plasma concentrations increase three- to fivefold during the host response to tissue injury and/or inflammation. Inherited deficiency of alpha 1-AT is associated with destructive lung disease and emphysema, and a subgroup of deficient individuals develops liver disease (reviewed in Ref. 49).

Plasma alpha 1-AT is predominantly derived from the liver as shown by studies of changes in alpha 1-AT allotypes after orthotopic liver transplantation (1, 23). There is abundant synthesis of alpha 1-AT in hepatoma cell lines and hepatocytes in primary culture, and alpha 1-AT mRNA is extremely abundant in hepatocytes in human liver as determined by in situ hybridization analysis (32). There is also evidence for extrahepatic sites of synthesis, notably that in blood monocytes and tissue macrophages (40) as well as in enterocytes.

Several things are known about the mechanism by which alpha 1-AT gene expression is regulated in enterocytes. Our previous studies have shown that the increase in alpha 1-AT gene expression during differentiation of Caco-2 cells can be in large part accounted for by an increase in rate of transcription (20). Second, expression of the alpha 1-AT gene in undifferentiated and differentiated Caco-2 cells is regulated by cytokines in a manner that recapitulates the expression of the alpha 1-AT gene in the intestinal epithelium during inflammation in vivo and is characteristic of the physiological acute phase response (20, 33). Transcription is initiated at the downstream "hepatocyte" specific transcription initiation site during basal and differentiated expression of alpha 1-AT in Caco-2 cells but is initiated at both the downstream and the upstream "macrophage" specific transcription initiation sites during modulated [interleukin-6 (IL-6)-activated] expression in Caco-2 cells (20) and in human intestinal epithelial cells during inflammation in vivo (32).

In the current study we examined the possibility of identifying the cis-acting element(s) and trans-acting DNA-binding protein(s) responsible for alpha 1-AT gene expression in Caco-2 cells, whether these were different from those responsible for alpha 1-AT gene expression in hepatoma Hep G2 cells and whether we could identify a mechanism or mechanisms responsible for increased transcription during differentiation.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmid constructs and site-directed mutagenesis. Progressive deletions of the human alpha 1-AT promoter region from -1951 to -2 were generated by PCR amplification using a human genomic alpha 1-AT clone hAAT7zf (kindly provided by Dr. K. Ponder, St. Louis, MO) as template. These PCR fragments were subcloned into the Kpn I/Hind III site of the pGL3 basic luciferase reporter vector (Promega) to generate alpha 1-AT promoter-luciferase fusion plasmids. The internal site-directed mutations within the potential HNF-1 and HNF-4 binding sequences of this fragment were prepared by overlap extension PCR (22) using gene-specific oligonucleotide primers that flank the region to be altered. All PCR fragments were subjected to DNA sequence analysis (48) to show that the designated mutations had been made, that the remaining sequence was identical to the original template, and that the proper orientation had been maintained.

Two different HNF-1alpha constructs were used. For transfection studies we used a rat HNF-1alpha cDNA kindly provided by Dr. R. J. Gonzalez (Bethesda, MD). A fragment spanning nucleotide +65 to +2483 was inserted into the cloning site of plasmid pCMV4 (28). For RNA blot analysis, a 248-base pair fragment corresponding to the homeodomain (nucleotides 595-843) was generated by PCR using the rat HNF-1alpha cDNA as template.

The HNF-1beta construct pBJ5-HNF-1beta (11) was kindly provided by Dr. G. R. Crabtree (Palo Alto, CA). Full length HNF-4 cDNA subcloned into the pMT2 expression vector (42) was provided by Dr. J. Rottman (St. Louis, MO). The beta -galactosidase expression vector pSV-beta -gal was purchased from Promega.

Cell culture, DNA transfections, and luciferase assay. The Hep G2, HeLa, and Caco-2 cells were grown as previously described (32). The promoter-luciferase plasmids were cotransfected with the indicated amount of HNF-1alpha , HNF-1beta , and/or HNF-4 expression vectors. A pGL3-basic and pGL3-control vector containing SV40 promoter and enhancer sequences were used as controls in all transfection experiments, and the pSV-beta -gal vector was included as an internal control to monitor and normalize for transfection efficiency. Cells (1-1.5 × 106) were plated on 60-mm tissue culture dishes and incubated for 24 h before transfection. Duplicate dishes were transfected using the calcium phosphate method. Cells were shocked with 15% glycerol 24 h after transfection and harvested 48 h after transfection. For transfection of HeLa cells the Tfx-20 reagent from Promega was used. Luciferase activity was detected on the Turner Designs luminometer (model TD-120/20, Promega).

Preparation of double-strand oligonucleotides and labeling. The wild-type and mutant oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer in the Nucleic Acid Chemistry Laboratory, Biotechnology Center, Washington University School of Medicine (St. Louis, MO). The potential HNF-1-binding sequence corresponds to the alpha 1-AT promoter sequence from -86 to -50 (5'-ATAACTGGGGTGA-CCTTGGTTAATATTCACCAGCAG-3'). The potential HNF-4-binding sequence corresponds to the sequence from -125 to -96 (5'-ATCCAGCCAGTGGACTTAGCCCCGTTTG-3') with respect to the downstream transcriptional start site. The oligonucleotides were purified by denaturing PAGE, annealed at 65°C for 1 h, allowed to cool slowly at room temperature, and then labeled using [alpha -32P]dCTP and the Klenow fragment of DNA polymerase I. Radiolabeled oligonucleotides were separated from free nucleotide by nondenaturing PAGE.

Nuclear extracts and DNase I footprint analysis. Nuclear extracts of Caco-2 and Hep G2 cells were prepared according to the protocol described by Dignam et al. (15). Nuclear extracts from HeLa cells (HeLa Scribe nuclear extract, in vitro transcription grade) were purchased from Promega. A fragment of the human alpha 1-AT gene that spans nucleotides -157 to -2 was generated by PCR and subcloned into Hind III/Pst I site of the pL-TG vector (kindly provided by Dr. G. U. Ryffel, Karlsruhe, Germany). This fragment was labeled with 32P by a 3'-end filling reaction. For DNase I footprint analysis several different concentrations of nuclear extracts were resuspended in buffer so that a 20-µl reaction volume contained a final concentration of 60 mM KCl, 25 mM HEPES, pH 7.6, 1 mM dithiothreitol, 0.1 mM EDTA, 7.5% glycerol, 1 µg poly(dI-dC), and 2% polyvinyl alcohol. Nuclear extract dialysis buffer and BSA were used as negative controls. After a preincubation at room temperature for 15 min, labeled probe [~8 × 104 counts/min (cpm)] was added. The reaction was mixed well and then incubated for another 20 min at room temperature. DNase I (Worthington Biochemical, Freehold, NJ) was added to a final concentration 1-10 ng of enzyme per reaction. After a 1- or 2-min incubation at room temperature, the reaction was stopped by adding 1 volume of DNase I stop solution containing 20 mM EDTA, 200 mM NaCl, 1% SDS, 10 µg of yeast tRNA, and 40 µg of proteinase K followed by extraction with phenol-chloroform and ethanol precipitation. The samples were analyzed by electrophoresis on 6% polyacrylamide-8 M urea gels, and autoradiography was performed for 12 h at -70°C.

Electrophoretic mobility shift assays. Complementary oligonucleotide probes for the HNF-1 and HNF-4 sequences in the alpha 1-AT promoter and mutant oligonucleotide probes were labeled with [32P]dCTP by the 3'-end filling reaction and 2 × 104 cpm were incubated for 20-40 min at room temperature with nuclear extracts from Hep G2, Caco-2, and HeLa cells. The nuclear extracts were prepared exactly as previously described. The binding reaction was performed using the same protocol that was used for DNase I footprint analysis except that lower concentrations of nuclear proteins were used and a 10-µl reaction volume was used. Unlabeled oligonucleotide in 10-, 50- and 100-fold molar excess was used in designated experiments. Rabbit polyclonal anti-HNF-1alpha TC 284 (kindly provided by M. Yaniv and M. Pontoglio, Paris, France) and anti-HNF-4 (kindly provided by I. Talianidis, Crete) were used in designated experiments by incubation with nuclear extracts for 5 min at 22°C before addition of radiolabeled oligonucleotide probe. The products were analyzed on 5% polyacrylamide-2.5% glycerol gels cast in 0.5 × Tris-borate-EDTA.

RNA blot analysis. Total cellular RNA was isolated from cell lines by guanidine isothiocyanate extraction and ethanol precipitation (8). Poly(A)+ RNA was isolated using oligo(dT) cellulose column chromatography (43). RNA was then subjected to agarose-formaldehyde electrophoresis and transferred to nylon filters (50) for hybridization with 32P-labeled DNA probes.

Construction of antisense ribozymes for HNF-1alpha . Overlap PCR methods of Zillman and Robinson (60) were used to generate two hammerhead antisense ribozymes for HNF-1alpha , the F1(1) and F1(2) PCR products. In each case a GUC codon was targeted [nucleotides 378-380 for F1(1) and 455-457 for F1(2)] and 35-flanking nucleotides were included on each side to optimize ribozyme activity according to previous studies (12). The HNF-1alpha plasmid HA (2), kindly provided by M. Yaniv (Paris, France), was used as template. Results of transient transfections indicated that the F1(1) antisense ribozyme had the highest inhibitory activity so it was used for the stable transfection studies. For F1(1) the first primer set was CGCTGTGGGATGTTGT and added a Xho I restriction site at the 5' terminus. The second primer set was TTCTGCAGGAGGACCCG and added a BamH I restriction site at the 5' terminus. The target was reacted separately with these two primer sets. The PCR products were then gel purified and reacted with the two primer sets to give the final ribozyme-encoding cassette. This cassette was ligated into the Xho I and BamH I sites in the multiple cloning site of plasmid pcDNA3 (Invitrogen, San Diego, CA). This plasmid was expressed in Caco-2 cells by transient and stable transfection protocols. For transient transfection, Caco-2 cells at 80% confluence were cotransfected with the antisense ribozyme HNF-1alpha expression plasmid and the alpha 1-AT promoter (-137 to -2)-luciferase reporter plasmid using lipofectamine (GIBCO BRL, Gaithersburg, MD) and the conditions described by the manufacturer's instructions. After 64 h, luciferase activity was measured. For stable transfection, Caco-2 cells at 80% confluence were transfected with the antisense ribozyme HNF-1alpha expression plasmid alone and the lipofectamine reagent. After 48 h, transfected cells were subjected to selection in G418.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Localization of cis-acting elements within upstream flanking region responsible for expression of the alpha 1-AT gene in Caco-2 cells and Hep G2 cells. To determine whether there were cis-acting regulatory elements within the upstream flanking region for expression of the alpha 1-AT gene in Caco-2 cells and whether these differed from those responsible for expression in Hep G2 cells, we constructed seven alpha 1-AT-luciferase fusion plasmids differing in the length of the alpha 1-AT 5'-flanking sequence upstream of the luciferase coding sequence (Fig. 1). We examined the expression of these plasmids in undifferentiated Caco-2 cells as well as Hep G2 cells. The results show that deleting 451 nucleotides from -1951 to -1500 results in a small drop in expression in Caco-2 cells. Deletion of another 509 nucleotides from -1500 to -991 results in an increase in expression in Caco-2 cells. There is a substantial drop in expression in these cells on deletion of 330 nucleotides from -991 to -661. There is no effect on expression in Caco-2 cells of deleting 391 nucleotides from -661 to -270 but significant drops occur on deletion of 133 nucleotides from -270 to -137 and deletion of 135 nucleotides from -137 to -2. The results therefore indicate that there are cis-acting elements for expression in Caco-2 cells between -1951 and -1500, -1500 and -991, -991 and -661, -270 and -137, and -137 and -2. The results also indicate that there are cis-acting elements for expression in Hep G2 cells between -1500 and -991, between -490 and -270, between -270 and -137, and between -137 and -2. Analysis of the percent change in luciferase activity (Fig. 1C) suggests that there are at least three major differences between Caco-2 and Hep G2 cells: negative element(s) between -1500 and -991 and positive elements in regions from -991 to -661 and -137 to -2. We decided to first examine the more proximal region, -137 to -2, in more detail.


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Fig. 1.   Expression in Caco-2 and Hep G2 cells of alpha 1-antitrypsin (alpha 1-AT)-luciferase fusion plasmids carrying progressive deletion of 5'-flanking sequences. A: schematic structure of 5'-flanking deletions. Continuous lines indicate alpha 1-AT gene sequences. Boxes represent luciferase coding sequences. B: relative luciferase activity in fold increase over basic vector activity. Undifferentiated Caco-2 cells (day 1) and Hep G2 cells were cotransfected with alpha 1-AT promoter-luciferase fusion plasmids (7.5 µg) and the pSV-beta -galactosidase control vector (1 µg). After 48 h luciferase and beta -galactosidase activities were measured, and ratio was plotted on horizontal axis as normalized luciferase activity in fold increase over activity of basic vector alone. Results represent means ± SD for 4 separate determinations. Open bars, Caco-2 cells; solid bars, Hep G2 cells. In this form of data display, basic vector gives value of 1.0 by definition. C: relative luciferase activity in percent maximal activity. Results are expressed as percent maximal activity compared with activity of plasmid carrying longest 5'-flanking sequence, which was assumed to give 100% luciferase activity. Open bars, Caco-2 cells; solid bars, Hep G2 cells. In this form of data display, basic vector gives a value of 0.16%.

Binding sites for nuclear proteins from Caco-2 cells within proximal upstream region of the alpha 1-AT gene. We first used DNase I footprint analysis to examine the possibility that nuclear proteins from Caco-2 bound to the region of the alpha 1-AT promoter spanning nucleotides -137 to -2 (Fig. 2). The results showed that nuclear proteins from Caco-2 protected a region within the promoter that corresponded to nucleotides -47 to -80. The protection was concentration dependent and greater for nuclear proteins from "differentiated" Caco-2 cells (day 9) than from undifferentiated Caco-2 cells (day 1). This region contains a sequence highly homologous to the consensus sequence for binding HNF-1 and is also protected by nuclear extracts from liver cells (3, 7, 9, 12, 14, 34). The increase in protection in differentiated Caco-2 cells corresponds to the increase in expression of alpha 1-AT in differentiated Caco-2 cells (41). There is also protection, although to a significantly lesser extent, in a region that corresponds to nucleotides -96 to -102. This region contains part of a potential HNF-4 binding sequence within the alpha 1-AT promoter.


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Fig. 2.   Binding sites for nuclear protein(s) from Caco-2 cells within proximal upstream region of alpha 1-AT gene as assessed by DNase I footprint analysis. A fragment of the human alpha 1-AT gene corresponding to nucleotides -157 to -2 was end-labeled on noncoding strand and then incubated with nuclear extracts from undifferentiated (day 1) and differentiated (day 9) Caco-2 cells in absence or presence of DNase I. Nuclear extract dialysis buffer (Naked) and BSA were used as negative controls. Sequencing reactions (G+A, C+T) shown in first 2 lanes on left were used to map protected regions. Amount of nuclear extract in microgram protein and duration of DNase I digestion in minutes are indicated at top. Brackets at right correspond to 2 protected regions. Results were identical when coding strand of alpha 1-AT fragment was used in footprint analysis (data not shown). The sequence of the alpha 1-AT promoter is shown at bottom with protected areas bracketed.

We used this information to generate two oligonucleotide probes for electrophoretic mobility shift assay. The alpha 1-AT HNF-1 oligo corresponds to nucleotides -86 to -50 within the alpha 1-AT promoter and the alpha 1-AT HNF-4 oligo corresponds to the HNF-4 binding site from -125 to -96. These oligonucleotides were labeled, reacted with nuclear extracts from undifferentiated Caco-2 cells (day 1) as well as from differentiated Caco-2 cells (day 4 and day 9), and analyzed by electrophoretic mobility shift assay (Fig. 3A). The results show complexes formed with each oligonucleotide. The complexes formed with the alpha 1-AT HNF-1 oligo are competed away with unlabeled alpha 1-AT HNF-1 oligo in concentration-dependent fashion. Similarly the complexes formed with the alpha 1-AT HNF-4 oligo are competed with unlabeled alpha 1-AT HNF-4 oligo in concentration-dependent fashion. For the alpha 1-AT HNF-1 oligo there are two complexes that migrate very close to each other. This is typical for HNF-1alpha and HNF-1beta . HNF-1alpha usually migrates slightly slower than HNF-1beta (7). It was of some interest to us to see that the relative amounts of these two bands change progressively during differentiation of Caco-2 cells. On day 1 there is more of the faster migrating band. On day 4 there are approximately equivalent levels of the two bands. On day 9 there is substantially more of the slower migrating band, suggesting that there has been an increase in HNF-1alpha binding. To ensure that the slower migrating complex with the alpha 1-AT HNF-1 oligo was truly HNF-1alpha , we preincubated the nuclear extract with anti-HNF-1alpha antiserum (Fig. 3B). This antiserum eliminated the slower migrating complex, leaving only the more rapidly migrating complex unaffected. This result confirms the prediction that the slower migrating complex is HNF-1alpha . The more rapidly migrating complex is identical to HNF-1beta as reported in previous studies (7). In Fig. 3B, right, antiserum to HNF-4 supershifted the complex of Caco-2 nuclear extracts with the alpha 1-AT HNF-4 oligo, providing further evidence that this complex truly represents HNF-4. It is not yet known why antibody to HNF-4 generates two supershift complexes. The complexes generated with both HNF-1 and HNF-4 oligos were also shown to be specific by competition with unlabeled oligos in Fig. 3C. The complexes with the alpha 1-AT HNF-1 oligo were completely blocked by unlabeled alpha 1-AT HNF-1 oligo but not by unlabeled irrelevant GRE oligo. The complexes formed with the alpha 1-AT HNF-4 oligo were completely blocked by unlabeled alpha 1-AT HNF-4 oligo but not by unlabeled irrelevant GRE oligo.



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Fig. 3.   Trans-acting proteins from Caco-2 cells that bind to proximal upstream region of alpha 1-AT gene as assessed by electrophoretic mobility shift assay. A: nuclear extracts from undifferentiated (day 1) and differentiated (day 4 and day 9) Caco-2 cells were reacted with radiolabeled alpha 1-AT hepatocyte nuclear factor-1 (HNF-1) and alpha 1-AT HNF-4 oligonucleotides in absence (-) or presence of unlabeled oligonucleotides (competitor) in 10-fold (10×) or 100-fold (100×) molar excess. Reaction products were then subjected to PAGE. Relative migration of specific DNA-binding proteins is indicated by arrows at left for HNF-1 (solid arrow, presumed HNF-1alpha ; open arrow, presumed HNF-1beta ) and at right for HNF-4. B: nuclear extracts from undifferentiated (day 1) and differentiated (day 8) Caco-2 cells were reacted with radiolabeled alpha 1-AT HNF-1 or alpha 1-AT HNF-4 oligonucleotides in absence or presence of antiserum specific for HNF-1alpha or HNF-4. Nuclear extracts were preincubated for 5 min at 22°C with antisera before labeled probe was added to reaction mixture. Relative migration of HNF-1alpha (solid arrow) and HNF-1beta (open arrow) is indicated at left and that of HNF-4 (solid arrow) is indicated at right. C: nuclear extracts from differentiated (day 4) Caco-2 cells were reacted with radiolabeled alpha 1-AT HNF-1 in absence (-) or presence of unlabeled alpha 1-AT HNF-1 oligo or unlabeled irrelevant GRE oligo in 100-fold molar excess (left). These extracts were also reacted with radiolabeled alpha 1-AT HNF-4 in absence (-) or presence of unlabeled alpha 1-AT HNF-4 oligo or unlabeled irrelevant GRE oligo in 100-fold molar excess (right). Relative migration of HNF-4 (solid arrow) is indicated at far right.

Taken together, these results indicate that there are nuclear proteins in Caco-2 cells that bind to the HNF-1 binding and HNF-4 binding sequences in the upstream proximal promoter of the alpha 1-AT gene, and that these proteins resemble HNF-1alpha , HNF-1beta , and HNF-4. The results also suggest that there is increased HNF-1alpha -binding activity in Caco-2 cells correlating temporally with the increase in alpha 1-AT transcription that accompanies the spontaneous differentiation of these cells. There is no change in HNF-4 binding activity during differentiation of Caco-2 cells.

Expression of the HNF-1alpha and HNF-4 genes during differentiation of Caco-2 cells. Next we used RNA blot analysis to examine steady-state levels of HNF-1alpha , HNF-4, and alpha 1-AT mRNA in undifferentiated and differentiated Caco-2 cells compared with Hep G2 and HeLa cells (Fig. 4). The results show that HNF-1alpha and HNF-4 are not expressed in HeLa cells, but there is a similar amount of HNF-1alpha and HNF-4 mRNA in Hep G2 and undifferentiated Caco-2 cells. There is a marked increase in HNF-1alpha and HNF-4 mRNA levels in differentiated Caco-2 cells that corresponds temporally and in magnitude to that for alpha 1-AT mRNA levels. The increase cannot be attributed to RNA loading as shown by the absence of any change in steady-state levels of glyceraldehyde-3-phosphate dehydrogenase (GADPH) mRNA levels. Taken together with the other results of this study, these data provide evidence that the increase in alpha 1-AT gene expression during differentiation of Caco-2 cells correlates with an increase in HNF-1alpha and HNF-4 mRNA levels and an increase in the binding of HNF-1alpha to the alpha 1-AT promoter.


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Fig. 4.   Steady-state levels of HNF-1alpha , HNF-4, alpha 1-AT, and glyceraldehyde-3-phosphate dehydrogenase (GADPH) mRNA in HeLa cells, Hep G2 cells, and undifferentiated and differentiated Caco-2 cells. Relative migration of RNA markers is indicated at left and that of each specific mRNA is indicated by arrows at right.

Evidence for a synergistic effect of HNF-1alpha and HNF-4 on alpha 1-AT gene expression in Caco-2 cells. Next we cotransfected undifferentiated Caco-2 and Hep G2 cells with the alpha 1-AT promoter (-137 to -2)-luciferase reporter plasmid and HNF-1alpha , HNF-1beta , and HNF-4 expression plasmids. The luciferase activity was normalized to beta -galactosidase activity conferred by a cotransfected pSV-beta -gal plasmid (Fig. 5). The results show that HNF-1alpha and HNF-4 mediate concentration-dependent increases in luciferase activity, but the effect of HNF-1beta on Caco-2 cells is negligible (Fig. 5A). In each case the effect of HNF-1alpha and HNF-4 reaches a plateau when 2 µg are transfected. At this concentration HNF-1alpha mediates an ~15.9-fold and HNF-4 an ~5.7-fold increase in luciferase activity (Fig. 5A). HNF-1alpha and HNF-4 also mediate increases in luciferase activity in Hep G2 cells (Fig. 5B). HNF-1alpha mediates an ~3.5-fold and HNF-4 an ~1.5-fold increase in luciferase activity, but HNF-1beta has no significant effect. Thus there is a lower magnitude in the effect of HNF-1alpha and HNF-4 on this promoter in Hep G2 cells. This difference is unlikely to be due to differences in efficiency of transfection because the data were normalized to beta -galactosidase activity conferred by the cotransfected pSV-beta -gal plasmid and the galactosidase activity of transfected Caco-2 cells was very similar to that in Hep G2 cells in these experiments (data not shown). It is also unlikely to be due to different relative levels of endogenous HNF-1alpha or HNF-4 expression because RNA blot analysis indicates that the steady-state level of HNF-1alpha and HNF-4 mRNA are similar in undifferentiated Caco-2 and Hep G2 cells (see Fig. 4). Interestingly, the basal luciferase activity of the promoter was over 20-fold higher in Hep G2 cells than Caco-2 cells. These data suggest the possibility that there are differences in endogenous cofactors or other transcription factors in the two cell lines. There was no significant basal luciferase activity for the promoter in transfected HeLa cells (data not shown).


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Fig. 5.   Effect of HNF-1alpha , HNF-1beta , and HNF-4 on expression of alpha 1-AT reporter constructs in undifferentiated Caco-2 cells (A) and Hep G2 cells (B). Cells were cotransfected with alpha 1-AT promoter (-137 to -2)-luciferase reporter plasmid (7.5 µg) and pSV-beta -galactosidase control vector (1 µg) in absence or presence of HNF-1alpha , HNF-1beta , or HNF-4 expression plasmids in several different concentrations as shown on horizontal axis. After 48 h luciferase and beta -galactosidase activities were measured and the ratio was plotted on vertical axis as normalized luciferase activity. Results are reported as means ± SD for duplicate samples from 3 separate experiments.

Because previous studies have suggested that HNF-1 and HNF-4 may have synergistic effects on some genes we examined the effect of cotransfecting both HNF-1alpha and HNF-4 or HNF-1beta and HNF-4 together with the alpha 1-AT promoter-luciferase reporter plasmid in undifferentiated Caco-2 cells. The results were normalized for beta -galactosidase activity and are reported in Fig. 6A as relative activation compared with the alpha 1-AT promoter in the absence of HNF-1alpha , HNF-1beta , and HNF-4. The results show that HNF-1alpha and HNF-4 have a profound synergistic effect, but HNF-1beta has no effect on luciferase activity by itself or together with HNF-4. When 2 µg of the HNF-1alpha plasmid and 1 µg of the HNF-4 plasmid are cotransfected there is an ~66-fold increase in luciferase activity. HNF-1alpha and HNF-4 also had a synergistic effect on the reporter in Hep G2 cells, but it was much lower in magnitude (Fig. 6B). At its maximal, the synergistic effect in Hep G2 cells resulted in an ~9.5-fold increase in luciferase activity. HNF-1alpha and HNF-4 also have a synergistic effect on the alpha 1-AT promoter in cotransfected HeLa cells (Fig. 6C).


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Fig. 6.   Effect of HNF-1alpha or HNF-1beta together with HNF-4 on expression of alpha 1-AT reporter constructs in undifferentiated Caco-2 cells (A), Hep G2 cells (B), and HeLa cells (C). Cells were cotransfected with alpha 1-AT promoter (-137 to -2)-luciferase reporter plasmid, pSV-beta -gal plasmid, in absence (-) or presence of HNF-1alpha alone, HNF-1beta alone, HNF-4 alone, HNF-1alpha together with HNF-4, or HNF-1beta together with HNF-4. Concentrations of HNF-1alpha , HNF-1beta , and HNF-4 are shown at bottom. After 48 h luciferase and beta -galactosidase activities were measured. Luciferase activity was normalized for transfection efficiency by comparing it with galactosidase activity. Relative activation of promoter shown on vertical axis represents normalized luciferase activity of each condition compared with normalized luciferase activity in cells transfected with alpha 1-AT promoter alone. Results are reported as means ± SD for duplicate samples from 3 separate experiments.

To determine whether the synergistic effect of HNF-1alpha and HNF-4 on expression of the reporter construct could be attributed to binding to their respective binding sites in the alpha 1-AT promoter, we generated several mutants of the alpha 1-AT promoter (Fig. 7A). For Mut-2, we altered three nucleotides within the HNF-1 binding region that would be predicted to disrupt HNF-1alpha binding (TGG at nucleotides -70 to -68 were substituted by ATT). As a positive control, Mut-1, we introduced one new nucleotide and substituted another nucleotide that would be predicted to make the region a more "perfect" HNF-1alpha binding site (G between nucleotides -63 and -62 and A for C at nucleotide -59). A naturally occurring mutation in the HNF-1-binding region of the alpha -fetoprotein gene is associated with increased HNF-1 binding affinity and hereditary persistence of alpha -fetoprotein (29). For Mut-4 we altered three nucleotides within the HNF-4 binding. Before testing the effects of these mutations on luciferase activity after transfection, we tested their effect on binding activity by generating the corresponding oligonucleotides for gel retardation assays. The results showed that there was a significant decrease, but not elimination of, binding of HNF-1alpha and HNF-1beta to the Mut-2 oligo and an increase in binding of HNF-1alpha to the Mut-1 oligo. There was absolutely no binding of HNF-4 to the Mut-4 oligo (data not shown).


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Fig. 7.   Effect of HNF-1alpha , HNF-4, or HNF-1alpha together with HNF-4 on expression of wild-type and mutant reporter constructs in undifferentiated Caco-2 cells. A: map of human alpha 1-AT proximal promoter and mutations used to generate the Mut-1, Mut-2, and Mut-4 plasmid constructs. B: cells were cotransfected with alpha 1-AT promoter (-137 to -2)-luciferase reporter plasmid, mutant promoter-luciferase reporter plasmid (as indicated at bottom), and the pSV-beta -gal promoter in absence (-) or presence of HNF-1alpha alone, HNF-4 alone, HNF-1alpha together with HNF-4. Concentrations of HNF-1alpha and HNF-4 are shown at bottom. After 48 h luciferase and beta -galactosidase activities were measured. Luciferase activity was normalized for transfection efficiency by comparing it with galactosidase activity. Relative activation of promoter shown on vertical axis represents normalized luciferase activity of each condition compared with normalized luciferase activity in cells transfected with basic vector alone. Results are reported as means ± SD for duplicate samples from 3 separate experiments.

Now that we had established that Mut-2 decreased HNF-1alpha binding, Mut-1 increased HNF-1alpha binding and Mut-4 decreased HNF-4 binding in electrophoretic mobility shift assay, we examined the function of these mutations in the proximal upstream region of the alpha 1-AT promoter-reporter plasmid by cotransfection of Caco-2 cells. These mutants were cotransfected with HNF-1alpha expression plasmid alone, HNF-4 plasmid, or both HNF-1alpha and HNF-4 plasmids. The results are reported in Fig. 7B as fold activation compared with the basic vector to see the effects on basal activity as well as on activity in the presence of HNF-1alpha and HNF-1beta . The results show the wild-type promoter construct induces luciferase activity by ~25-fold in the absence and ~400-fold in the presence of HNF-1alpha and HNF-4. There is a significant decrease in luciferase activity for the Mut-2 construct. The induction of Mut-2 is ~6-fold in the absence and ~100-fold in the presence of HNF-1alpha and HNF-4. Thus there is a fourfold reduction in trans-activation when HFN-1alpha binding is reduced by using the Mut-2 construct. This suggests that the collaborative action of HNF-4 and HNF-1alpha at least in part involves binding to the HNF-1-binding regulatory element. The residual trans-activation of Mut-2 is probably due to the fact that the mutation of the HNF-1alpha binding site in this construct did not completely eliminate HNF-1 binding (Fig. 6A). The effect on the Mut-2 construct is specific as shown by using the Mut-1 construct. This mutation of the HNF-1 binding sites is associated with ~100-fold activation in the absence and 700-fold in presence of HNF-1alpha and HNF-4. The increase in activity of the Mut-1 construct in the absence of HNF-1alpha and HNF-4 suggests the presence of endogenous HNF-1alpha and that endogenous HNF-1alpha binds more effectively to the mutant consensus sequence. The Mut-4 construct is associated with ~20-fold activation in the absence and 300-fold activation in the presence of HNF-1alpha and HNF-4. These activities are not significantly different from those of the wild type. The Mut-4 construct is activated by HNF-1alpha alone ~70-fold, indicating that the effect of HNF-1alpha on this construct is comparable to that on the wild-type construct. In contrast the Mut-4 construct is not activated at all by HNF-4. Taken together, these data suggest that the role of HNF-4 in the synergistic activation mechanism does not depend on binding to the cis-acting HNF-4 binding site.

Effect of antisense hammerhead ribozyme for HNF-1alpha on alpha 1-AT gene expression in Caco-2 cells. To provide further evidence for the role of HNF-1alpha and for the synergistic effects of HNF-4 and HNF-1alpha on the increase in alpha 1-AT gene expression during differentiation of Caco-2 cells, we examined the effect of antisense ribozymes for HNF-1alpha . First, we generated two hammerhead antisense ribozymes (Fig. 8A). Next, we examined the effect of the two constructs on expression from the alpha 1-AT promoter (-137 to -2) reporter plasmid in Caco-2 cells after transient transfection. The F1(1) anti-sense ribozyme had a more potent inhibitory activity (Fig. 8B). If mediated a concentration-dependent decrease in luciferase activity. At the highest concentration used there was a reduction to -40% levels present in cells transfected with the reporter alone. The F1(1) antisense ribozyme in the pcDNA3 plasmid was then used to establish stable transfected Caco-2 cell lines. Total cellular RNA was harvested from untransfected Caco-2 cells, Caco-2 cells transfected with the pcDNA3 plasmid (Caco-2-pcDNA3) alone and Caco-2 cells transfected with the ribozyme (Caco-2-ribozyme) 1 and 7 days after reaching confluence. RNA blot analysis (Fig. 8C) shows that there is a marked decrease in steady-state levels of alpha 1-AT mRNA on day 1 and day 7 in the Caco-2-ribozyme cell line, but no change in alpha 1-AT mRNA levels in the control Caco-2-pcDNA3 cell line. The decrease is not due to a general effect as shown by the results of RNA blot analysis with the GADPH cDNA probe (Fig. 8C, right). If anything there is slightly more GADPH mRNA in the Caco-2-ribozyme cell line. These data therefore provide further evidence for the role of HNF-1alpha in alpha 1-AT gene expression in Caco-2 cells and for the increase in alpha 1-AT gene expression during differentiation of Caco-2 cells. However, because there is still a small increase in alpha 1-AT mRNA levels in the Caco-2-ribozyme cell line on day 7 compared with day 1, the effect of HNF-1alpha may not completely explain the increase in alpha 1-AT gene expression during differentiation of Caco-2 cells.




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Fig. 8.   Effect of antisense hammerhead ribozyme for HNF-1alpha on alpha 1-AT gene expression in Caco-2 cells. A: alignment of HNF-1alpha mRNA sequence with that of F1(1) antisense ribozyme that was designed. Ribozyme was targeted to GUC at nucleotides 378-380 and included flanking sequences corresponding to nucleotide 343 at 5' end and nucleotide 415 at 3' end. Helices and loop of ribozyme and putative cleavage site of HNF-1alpha mRNA are indicated. B: effect of ribozyme on expression of alpha 1-AT reporter construct in transiently transfected Caco-2 cells. Caco-2 cells at 80% confluence were transfected with alpha 1-AT promoter (-137 to -2) reporter plasmid alone or cotransfected with reporter plasmid and ribozymes in several different concentrations as indicated at bottom. pcDNA3 plasmid was added to ensure that same amount of DNA was present in each set of samples; 64 h later, cells were harvested and cell extracts subjected to luciferase assays. Results are reported as means ± SD for 3 samples as percent maximal luciferase activity relative to luciferase activity of reporter plasmid alone. Results are representative of 2 separate experiments. C: effect of ribozyme on expression of endogenous alpha 1-AT mRNA levels in stable transfected Caco-2 cells. Total cellular RNA was collected from Caco-2 cells, Caco-2 cells after stable transfection of pcDNA3 plasmid alone (Caco-2-pcDNA3) and Caco-2 cells after stable transfection of the pcDNA3 plasmid with antisense hammerhead ribozyme for HNF-1alpha (Caco-2-ribozyme) 1 and 7 days postconfluence. RNA samples were subjected to RNA blot analysis with radiolabeled alpha 1-AT and GADPH cDNA. Relative migration of ribosomal RNA is indicated at right.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

alpha 1-AT is the major circulating inhibitor of destructive neutrophil proteases such as elastase, cathepsin G, and proteinase 3. Because it is also an acute-phase reactant it is thought to provide proteinase inhibitor activity during the host response to inflammation and tissue injury. Liver is the predominant site of synthesis of plasma alpha 1-AT, and alpha 1-AT has been studied as a "liver-specific" gene. These studies have shown that the cis-acting elements required for expression of the human alpha 1-AT gene in liver are localized to the 5'-flanking region of the gene (14). In fact, there appears to be three regions, distal elements at -488 to -356 and -261 and -208 and a proximal element at -137 to -37 (14). The minimal proximal element has been shown to be sufficient for liver-specific transcription in vivo in cultured hepatoma cells compared with HeLa cells and in vitro with rat liver nuclear extracts compared with nuclear extracts from spleen (9, 12, 34). Five different trans-acting factors from nuclear extracts of liver have been shown to bind to this proximal element: HNF-1alpha /LFB1, HNF-4/LFA1, HNF-1beta /vHNF1/LFB3, LFB2 and LFC (34). HNF-1alpha /LFB1 and HNF-4/LFA1 appear to be particularly important in expression of the human alpha 1-AT gene. Two distinct regions within the proximal element bind these two transcription factors (53). In fact, substitution of five nucleotides at -77 to -72 disrupts binding of HNF-1alpha and dramatically reduces expression of the human alpha 1-AT gene in the liver of transgenic mice (53). Substitution of four nucleotides at -118 to 115 disrupts binding of HNF-4 but does not alter expression of the human alpha 1-AT gene in liver of adult transgenic mice. The latter mutation does result in a reduction in expression of human alpha 1-AT in the liver during embryonic development.

The human alpha 1-AT gene is also expressed in extrahepatic tissues and cell types. Expression of this gene in small intestinal epithelial cells has been studied by in situ hybridization analysis of normal and inflamed human intestine and by metabolic labeling studies in human intestinal epithelial cell lines. The results of these studies indicate that alpha 1-AT is synthesized by enterocytes, that it is secreted at both poles of the enterocytes, that its expression increases progressively during differentiation from crypt to villous tip, and that its expression is increased by inflammation in vivo and by inflammatory cytokines in cell culture. Thus the results raise the possibility that alpha 1-AT expression in intestinal epithelial cells plays a role in the local response to inflammation/injury at the mucosal surface and is a target of the enterocyte differentiation program.

In this study we examined the possibility of identifying the cis-acting elements and trans-acting proteins that direct expression of alpha 1-AT in enterocytes, whether they are different from those involved in expression in hepatocytes and how they mediate the increase in transcription during differentiation. We first examined the proximal upstream flanking region of the alpha 1-AT gene, which appears to be involved in liver alpha 1-AT gene expression. The results show that HNF-1alpha and HNF-4 have a synergistic effect on alpha 1-AT gene in Caco-2-derived enterocytes. HNF-1alpha and HNF-4 also have a synergistic effect on alpha 1-AT gene expression in Hep G2-derived hepatocytes, but it is considerably lower in magnitude.

The results of the current study also suggest that HNF-1alpha and HNF-4 play an important role in the increase in transcription of the alpha 1-AT gene during enterocyte differentiation. First, there is an increase in protection of the HNF-1 and HNF-4 regulatory elements from DNase I digestion during differentiation of Caco-2 cells (Fig. 2). Second, there is an increase in HNF-1alpha binding activity in nuclear extracts from differentiated Caco-2 cells (Fig. 3). Third, there is a marked increase in HNF-1alpha and HNF-4 mRNA levels that correlates temporally with the increase in alpha 1-AT mRNA levels during differentiation of Caco-2 cells (Fig. 4). Interestingly, Tripodi et al. (53) have reported that expression of human alpha 1-AT gene in the gut is abolished by mutation of the HNF-1alpha or HNF-4 binding site in transgenic mice. This result indicates that expression of the alpha 1-AT gene in the model cell culture system Caco-2 in general recapitulates the expression of this gene in intestine in vivo. Fourth, overexpression of antisense ribozymes for HNF-1alpha in stable transfected Caco-2 cell lines resulted in a marked decrease in endogenous alpha 1-AT gene expression (Fig. 8).

HNF-1alpha was one of the first "liver-specific" transcription factors that was characterized (5, 6, 10, 18, 21, 26). As with many other transcription factors, which were originally believed to be tissue- and or cell-specific, subsequent studies of HNF-1alpha have shown that its expression is not restricted to liver cells. Its expression has been demonstrated in intestine, stomach, pancreas, kidneys, and lung (reviewed in Refs. 14, 18, and 54). Indeed, it has been shown to regulate transcription of a number of genes in intestine.

HNF-4 is a member of the steroid hormone receptor superfamily (46). Binding sites for this molecule are found in many genes expressed in liver cells, and these binding sites are required for expression after transfection in hepatoma cells (45). However, HNF-4 mRNA and protein can be detected in intestine, kidney, and pancreas in addition to liver. Expression in pancreas may be particularly important because mutations in the HNF-1alpha and HNF-4 genes appear to be associated with maturity-onset diabetes of the young (27, 57, 58). Recent studies have suggested that HNF-4 also plays a role in early development and in differentiation (16, 25, 51). Results of the current study indicate that HNF-4 regulates alpha 1-AT gene expression in intestinal epithelial cells during differentiation, but it does not appear that the contribution of HNF-4 to this synergistic effect is due to binding to its own regulatory element in the alpha 1-AT promoter (Fig. 7B). Instead, the results raise the possibility that there is an interaction between HNF-4 and HNF-1alpha , which enhances HNF-1alpha binding to its own regulatory element and/or that the collaborative action of HNF-4 and HNF-1alpha induces trans-activation of the proximal promoter of alpha 1-AT by another endogenous transcription factor. Indeed HNF-4 has recently been shown to directly bind to HNF-1alpha , and this interaction has been implicated in the mechanism by which HNF-1alpha negatively regulates HNF-4-dependent genes in hepatoma cells (26). Although the results indicate that the HNF-1 binding sequence in the proximal promoter is crucial for the synergistic effect of HNF-4 and HNF-1alpha , it is not entirely clear why there is no increase in HNF-4 binding activity during differentiation of Caco-2 cells (Fig. 3A) despite the increase in HNF-4 mRNA levels over the same interval. This may reflect competition for the HNF-4 binding site by members of the HNF-3 family of transcription factors expressed endogenously. Recent studies by Duncan et al. (17) suggest that HNF-3alpha and HNF-3beta compete in opposite ways for the regulatory activity of HNF-4 and HNF-1alpha (17). In preliminary studies we have also found that HNF-3alpha and HNF-3beta have opposite and marked effects on regulatory activity of the proximal promoter of the alpha 1-AT gene (unpublished data).

These studies also indicate that other cis-acting elements in the alpha 1-AT gene and trans-acting factors are involved in its expression in enterocytes and in its upregulation during enterocyte differentiation. There is still a small increase in alpha 1-AT gene expression during differentiation of the Caco-2 cell line which overexpresses the antisense ribozyme for HNF-1alpha (Fig. 8). Moreover, there appears to be a positive element, or elements, between nucleotides -991 to -661 in the 5'-flanking region of the alpha 1-AT gene, which has a much greater effect on expression in Caco-2 cells than in Hep G2 cells (Fig. 1). Based on the known sequence in this region and comparison to consensus sequences for known cis-acting regulatory elements, there are many candidates for this particular regulatory effect on the alpha 1-AT gene, and thus further detailed studies will be necessary for its elucidation.

Several genes that are expressed in both enterocytes and hepatocytes have been studied for mechanism of transcriptional activation. A proximal promoter with an HNF-1 binding sequence and HNF-1 itself appears to be involved in alpha -fetoprotein gene expression in enterocytes and hepatocytes (55). Moreover, a substitution in an HNF-1-binding site upstream of the alpha -fetoprotein gene which results in tighter binding of HNF-1 is associated with hereditary persistence of alpha -fetoprotein expression (29). Expression of apolipoprotein B in Caco-2 and Hep G2 cells appears to involve a similar cis-acting element and three nuclear proteins, but there is a different amount of the three proteins in Caco-2 compared with Hep G2 cells (38). Distal regulatory elements may also have different effects on apolipoprotein B expression in Caco-2 compared with Hep G2 (37), and elements as far upstream as 70 kb may be necessary for expression in the intestine in vivo (35). Expression of apolipoprotein CIII also involves a similar cis-acting regulatory element in Caco-2 and Hep G2 cells (30). This element binds apolipoprotein AI regulatory protein 1, Ear3/COUP-TF, and HNF-4 in both Caco-2 and Hep G2 cells.

There are also a number of similarities in the mechanism by which the alpha 1-AT gene is activated in Caco-2 cells and that of the enterocyte-specific gene SI. HNF-1alpha activates transcription of SI in Caco-2 cells (56). HNF-1beta binds to the SI promoter but does not affect its transcription (56). The expression of both alpha 1-AT and SI increases during differentiation of Caco-2 cells in culture and enterocytes in vivo, implicating HNF-1alpha in a more general role in the differentiation program of enterocytes. However, there is a dramatic increase in SI gene expression late in Caco-2 cell differentiation that does not occur for alpha 1-AT, suggesting that additional SI-specific mechanisms determine its activation during the most terminal phases of enterocyte differentiation. Expression of both alpha 1-AT and SI in enterocytes is modulated by inflammation and specifically IL-6, with alpha 1-AT gene expression increasing and SI gene expression decreasing in the presence of IL-6 in cell culture or in the presence of inflammation in vivo (20, 33, 41, 59). There are also some similarities in mechanisms of expression of the alpha 1-AT gene and the enterocyte-specific gene lactase in Caco-2 cells. Lactase gene expression in Caco-2 cells is activated by the synergistic effect of HNF-1alpha and the HOXC11 homeodomain protein (31).

It was also interesting to note that HNF-1alpha did not bind to the endogenous alpha 1-AT promoter in HeLa cells, which do not express the alpha 1-AT gene. HNF-1beta did bind to the alpha 1-AT promoter but only weakly. There was, however, induction of the alpha 1-AT-reporter when HeLa cells were cotransfected with HNF-1alpha , or with both HNF-1alpha and HNF-4, but not with HNF-4 alone. Thus HeLa cells have all the machinery necessary to activate a naked alpha 1-AT reporter but lack the critical role played by expression of the HNF-1alpha gene. Previous studies have shown that extinction of both HNF-4 and HNF-1alpha gene expression is also associated with the absence of alpha 1-AT gene expression in fibroblasts (4, 19).


    ACKNOWLEDGEMENTS

We are indebted to Joyce Williams and Barbara Hermann for preparation of the manuscript.


    FOOTNOTES

The studies were supported by the National Institutes of Health Grants DK-45085 and HL-37784, as well as a Burroughs Wellcome Experimental Therapeutics Scholar Award.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: D. H. Perlmutter, Dept. of Pediatrics, Washington Univ. School of Medicine, One Children's Place, St. Louis, MO 63110.

Received 25 August 1998; accepted in final form 4 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 276(5):G1181-G1194
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