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Endocrinology Vol. 140, No. 1 187-196
Copyright © 1999 by The Endocrine Society

ARTICLES

Expression of Hepatocyte Growth Factor-Like Protein Is Repressed by Retinoic Acid and Enhanced by Cyclic Adenosine 3',5'-Monophosphate Response Element-Binding Protein (CREB)-Binding Protein (CBP)1

Rebecca S. Muraoka2, Susan E. Waltz and Sandra J. Friezner Degen

Graduate Program in Developmental Biology, University of Cincinnati College of Medicine (R.S.M.), and the Division of Developmental Biology, Children’s Hospital Research Foundation (S.E.W., S.J.F.D.), Cincinnati, Ohio 45229-3039

Address all correspondence and requests for reprints to: Dr. Sandra J. F. Degen, Children’s Hospital Research Foundation, Division of Developmental Biology, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039. E-mail: sandra.degen{at}chmcc.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In an effort to understand the molecular mechanisms involved in the regulation of expression of the gene encoding hepatocyte growth factor-like protein (HGFL), it was found that all-trans-retinoic acid dramatically represses expression of the endogenous HGFL gene in HepG2 cells, a human hepatocyte-derived cell line. This repression requires the sequence between nucleotides -135 and -105 in the 5'-flanking sequence of the HGFL gene, a site that has previously been shown to bind the transcription factor hepatocyte nuclear factor-4 (HNF-4). Electrophoretic mobility shift analysis suggests that the retinoic acid receptor does not bind to this site, and that retinoic acid does not alter binding of HNF-4 to this DNA site. However, the transcriptional coactivator, CREB-binding protein (CBP) coactivates expression of this gene through an indirect interaction with the HNF-4-binding site, and overexpression of CBP in HepG2 cells eliminates retinoic acid repression of reporter gene expression driven by the HGFL promoter. Overexpression of CBP also protects the endogenous HGFL gene from down-regulation by retinoic acid. These results suggest that HGFL gene expression requires CBP, and competition for limiting amounts of CBP by retinoic acid receptor may be a means of modifying the activity of HNF-4 at the HGFL gene promoter.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GROWTH factor known as hepatocyte growth factor (HGF)-like protein/macrophage-stimulating protein (HGFL/MSP) (1, 2, 3, 4, 5) is a heterodimeric serum glycoprotein that is synthesized primarily in hepatocytes (6, 7) and is thought to be an inflammatory mediator (4, 5, 6, 8, 9). HGFL is a member of a family of growth factors that is characterized by common structural motifs, including kringle domains and a nonproteolytic serine protease-like domain (2, 3, 10). This family of growth factors also includes hepatocyte growth factor (HGF), a well characterized growth factor that is involved in stimulating migration, proliferation, invasion, and polarization of many epithelial cell types and is clearly required for liver development and embryogenesis (11, 12, 13, 14). The biological functions and physiological significance of HGFL are not fully understood, but based on its similarity to HGF, it is thought that HGFL may invoke similar cellular responses.

The cell surface receptor for HGFL is Ron (15, 16, 17), a receptor tyrosine kinase that is homologous to the HGF receptor, c-Met (12). These receptors are composed of an extracellular ligand-binding domain and a highly conserved intracellular region that contains the tyrosine kinase domain. Binding of Ron by HGFL results in an increase in tyrosine kinase activity and signal transduction into the intracellular environment (18, 19). In doing so, HGFL activates macrophages (4, 5, 10, 20), stimulates bone resorption and DNA synthesis in osteoclasts (21, 22), induces megakaryocyte maturation (23), and increases proliferation and migration of keratinocytes (24).

The liver-specific regulation of HGFL requires DNA sequences within the first 135 bp immediately upstream of the proposed initiator methionine (25). Specifically, the sequences contained between -135 to -105 are vital for high levels of liver-specific HGFL promoter activity in HepG2 (human hepatocellular carcinoma) cells. The transcription factor hepatocyte nuclear factor-4 (HNF-4) was shown to bind to the -135 to -105 region of the HGFL 5'-flanking sequence, resulting in liver-specific activation of the HGFL gene.

HNF-4 is a liver-enriched transcription factor that binds to DNA exclusively as a homodimer and has been shown to play a positive role in the regulation of many liver-specific genes (26, 27, 28, 29, 30). HNF-4 is a member of the nuclear receptor superfamily, which also includes the steroid hormone receptors and the retinoid/thyroid hormone receptors (31). Recently, HNF-4 has been shown to interact with the transcriptional coactivator CREB-binding protein (CBP) in a ligand-free manner and uses CBP to enhance its ability to trans-activate transcription of HNF-4-dependent reporter genes (32). However, the physiological significance of this interaction has not been demonstrated. CBP is a 265-kDa protein that is for the most part functionally interchangeable with the 300-kDa adenovirus E1A-associated protein, p300 (33). CBP has been shown to interact with other nuclear receptors, such as retinoic acid receptor (RAR), estrogen receptor, thyroid hormone receptor, glucocorticoid receptor, and progesterone receptor (34, 35). It is thought that CBP enhances transcriptional activation by recruiting components of the basal transcription machinery to gene promoters (33) and altering the framework of transcriptionally repressive chromatin by means of the intrinsic histone acetyltransferase activity of CBP (36, 37) or its associated factors (38, 39).

There is growing evidence that HGFL may be a critical regulatory component in mediating inflammatory responses, and it is important to understand the regulation of the HGFL gene in response to various biological stimuli. For example, it was found that HGFL expression increases in response to injury of the liver and lung (6, 9). In an effort to understand the molecular mechanisms underlying the regulation of HGFL, experiments were conducted examining expression of the human HGFL gene in response to various factors. Our results provide evidence that all-trans-retinoic acid (RA) dramatically decreases HGFL gene expression. We show for the first time that CBP and HNF-4 are vital components in the transcriptional regulation of an endogenous gene in response to RA. The transcriptional coactivator CBP enhances the expression of HGFL through its interaction with HNF-4. Competition with HNF-4 for limiting amounts of CBP by ligand-activated RAR represses the ability of HNF-4 to activate transcription at the HGFL gene promoter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
HepG2 (human hepatocellular carcinoma) cells and 293 (transformed human primary embryonal kidney) cells were grown in DMEM supplemented with 10% charcoal-treated FBS, 2 mM L-glutamine, and 50 µg/ml gentamicin in 5% CO2 at 37 C. Cells were treated with or without one of the following factors for 0–48 h, at which point total RNA was extracted: RA (2 µg/ml; Sigma Chemical Co., St. Louis, MO), estrogen (as 17ß-estradiol; 1 µg/ml; Sigma Chemical Co.), dexamethasone (2 µg/ml; Sigma Chemical Co.), progesterone (2 µg/ml; Sigma Chemical Co.), or T3 (thyroid hormone; 2 µg/ml; Sigma Chemical Co.).

RNA preparation and Northern analysis
Total RNA from HepG2 cells was isolated using Trizol (Life Technologies, Grand Island, NY). RNA (10 µg) was resolved on a 1% denaturing agarose gel and transferred to a nylon membrane (GeneScreen Plus, DuPont NEN, Boston, MA). A 2.2-kb EcoRI fragment coding for nucleotides 17–2235 of the human HGFL complementary DNA (3) was labeled with [{alpha}-32P]deoxy-CTP and used as a probe for the detection of HGFL transcripts. Hybridization was carried out as previously described (1).

Construction of plasmids
Chimeric human HGFL promoter-chloramphenicol acetyltransferase (CAT) plasmids were described by Waltz and co-workers (25). The plasmids pMT2.HNF4 (26) and pRc/RSV-mCBP.HA.RK (40) were gifts from Dr. Francis M. Sladek (University of California-Riverside) and Dr. Mark Montminy (Harvard University, Boston, MA), respectively. The plasmid pW1-RAR{alpha} was provided by Dr. Melissa Colbert (Children’s Hospital Research Foundation, Cincinnati, OH).

Transfection of cultured cells
Equimolar amounts of DNA (10-12 mol) were introduced into HepG2 or 293 cells by lipid-mediated transfection (pFx-2, Invitrogen, San Diego, CA) according to the manufacturer’s protocol, then incubated in DMEM plus 10% charcoal-treated FBS for 48 h, at which point cells were harvested for analysis. The transfected cells were treated with or without RA (2 µg/ml) for the final 16 h of the 48-h incubation. To overexpress the transcription factors HNF-4, CBP, or RAR{alpha} in transient transfections, cells were transfected with 1 µg pMT2-HNF4, pRc/RSV-mCBP.HA.RK, or pW1.RAR{alpha}, respectively, or were transfected with increasing amounts of pRc/RSV.mCBP.HA.RK, ranging from 0.2–2 µg, where indicated. To obtain stable transformants with pRc/RSV-mCBP.HA.RK, HepG2 cells were transfected with 2 µg of the expression plasmid as described above. After transfection (36 h), transfected cells were selected with 800 µg/ml G418 (Geneticin). Selection resistant clones were isolated and maintained in 400 µg/ml G418.

Analysis of transfected cells
Transfected HepG2 or 293 cells were collected and resuspended in 0.25 M Tris-HCl, pH 7.5, and analyzed as previously described (25). The supernatant containing the cell extract was assayed for protein concentration by Bradford analysis (Bio-Rad Laboratories, Inc., Richmond, CA), then snap-frozen and stored at -80 C. Cell extracts (40 µg) were assayed for the amount of CAT protein produced using a CAT ELISA kit (5 Prime 3 Prime, Boulder, CO) according to the manufacturer’s protocol. All assays were performed in duplicate and repeated at least three times. The amount of CAT protein produced was determined by comparison to a standard curve, followed by normalization for ß-galactosidase activity.

Preparation of nuclear extracts and electrophoretic mobility shift assay
HepG2 cell nuclear extracts were harvested as described previously (25). The protein concentrations of the nuclear extracts were determined by Bradford analysis (Bio-Rad Laboratoris, Inc.). Complementary oligonucleotides were synthesized encompassing the (-135/-105) region of the human HGFL promoter (25) and the natural retinoic acid response element of the human RARß promoter (coding strand 5'-CGGGGTAGGGTTCACCGAAAGTTCACTCGACA-3') (41). Complementary oligonucleotides were annealed for use as probes for electrophorectic mobility shift assay, then end labeled with [{gamma}-32P]deoxy-ATP using T4 polynucleotide kinase and purified on 15% polyacrylamide gels. Nuclear extracts (20 µg) were incubated in binding buffer [20 mM HEPES (pH 7.9), 15% glycerol, 5 mM KCl, 0.1 mM EDTA, and 0.2 µg/ml poly(dI-dC)], and probe (3000 cpm) was added with or without unlabeled competitors, and the incubation was continued on ice for 30 min. Reactions were electrophoresed, and gels were dried and exposed to x-ray film overnight at -80 C (25).

Immunoprecipitation and Western analysis
Nuclear extracts were prepared from HepG2 cells transiently transfected with pRc/RSV-mCBP.HA.RK as described above. Proteins from nuclear extracts (500 µg) were subjected to immunoprecipitation using a control rabbit polyclonal antibody against human HGFL or a rabbit polyclonal antibody against the amino-terminal region of human CBP (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA). Immunoprecipitation using the CBP antibody was performed in the presence or absence of a CBP-blocking peptide composed of residues 2–22 of human CBP (Santa Cruz Biotechnologies, Inc.). For immunoprecipitation, the nuclear extract was incubated in the presence of the indicated antibodies for 16 h at 4 C on a rocking platform. Protein A-agarose (50%, vol/vol) in dilution buffer [100 µl; 10 mM Tris-HCl (pH 8.0), 140 mM NaCl, 0.025% NaN3, 0.1% Triton X-100, and 0.1% BSA] was added, and incubation was performed for 3 h at 4 C on a rocking platform. The mixture was centrifuged at 15,000 x g at 4 C for 30 min. The pellet was washed twice in RIPA buffer [50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 0.1% Triton X-100, and 0.1% protease inhibitor cocktail; Sigma Chemical Co.], resuspended in SDS loading buffer (20 µl), heated to 90 C for 2 min, then resolved on a 10% reducing SDS-polyacrylamide gel. The resolved proteins were transferred to polyvinylidine fluoride membranes in 25 mM Tris-HCl, 192 mM glycine, and 20% methanol at 4 C for 3 h at 250 V. The antibody used for immunodetection was a rabbit polyclonal antibody directed against a carboxyl-terminal peptide of rat HNF-4, which has been shown to cross-react with human HNF-4 (30). The antibody ({alpha}455) was a gift from Dr. Francis M. Sladek. The membrane was incubated with antibody against HNF-4, then incubated with a biotinylated goat antirabbit IgG antibody, followed by incubation with an avidin-biotinylated horseradish peroxidase complex (Vector Laboratories, Inc., Burlingame, CA). A chemiluminescent visualization system (ECL, Amersham, Arlington Heights, IL) was applied according to the manufacturer’s protocol, then the membranes were exposed to x-ray film.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HGFL expression is down-regulated by retinoic acid
Sequence analysis of the human HGFL 5'-flanking region revealed a number of potential regulatory sequences that conform to the consensus sequence reported for response elements for RA (RAREs) that lie within regions of the promoter that are conserved between the human and mouse HGFL genes (1, 25), suggesting that HGFL gene expression may be responsive to RA. The region between -141 and -100 contains two potential RAREs, a single 9-cis-retinoic acid response element, a thyroid hormone response element, and the previously identified binding site for HNF-4 (Fig. 1AGo) (25). To determine whether HGFL is regulated by retinoic acid, HepG2 cells, a human hepatocyte-derived cell line that synthesizes HGFL (25), were treated with RA for times ranging from 0–48 h, and the expression of endogenous HGFL messenger RNA (mRNA) was determined by Northern analysis (Fig. 1BGo). In response to RA, there was a dramatic decrease in the level of HGFL mRNA, which appeared as early as 1 h after treatment with RA. Among several other putative transcription factor-binding sites found further upstream in the HGFL gene promoter region, there was a single response element for granulocyte-macrophage colony-stimulating factor, two interleukin-6 response elements, and response elements for glucocorticoid, estrogen, and progesterone (25). Treatment of HepG2 cells with 9-cis-retinoic acid, granulocyte-macrophage colony-stimulating factor, or interleukin-6 did not confer any change in the level of HGFL expression (data not shown). However, treatment of HepG2 cells with thyroid hormone, dexamethasone, progesterone, or estrogen caused a decrease in the expression of HGFL mRNA, similar to what was observed for retinoic acid (Fig. 1Go, B and C). The initial repression of expression of HGFL lasted at least 4 h in the presence of retinoic acid and thyroid hormone and for at least 24 h with dexamethasone. There appears to be a later biphasic response to either retinoic acid or thyroid hormone where expression found to be present at 8 h is again repressed at 24 h.



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  		Figure 1. Retinoic acid decreases expression of HGFL. A, The sequence of both strands of the 5'-flanking region of the human HGFL gene from -141 to -100 relative to the initiator methionine (25 ) contains repeats of the half-site sequence recognized by nuclear receptors (AGGTCA and derivatives thereof) (31 ). These are present as direct repeats separated by one (DR1) or two (DR2) nucleotides and as an inverted repeat (IR0). The positions of these sites are indicated within brackets and are labeled as follows: RARE, retinoic acid response element; TRE, thyroid hormone response element; HNF-4, HNF-4 response element; and RXRE, retinoid-X receptor response element. Other putative transcription factor-binding sites within the HGFL 5'-flanking region are reported by Waltz and co-workers (25 ). B, Total RNA from HepG2 cells treated for the indicated times (0–48 h) with RA (2 µg/ml), thyroid hormone (2 µg/ml), or dexamethasone (2 µg/ml) was analyzed for expression of HGFL mRNA by Northern analysis (top). Blots were reprobed with a labeled human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complementary DNA fragment to control for RNA loading (bottom). C, Quantitation of HGFL mRNA expression in HepG2 cells treated for 4 h with the indicated factors using PhosphorImager analysis (ImageQuant 1.1, Becton Dickinson, Mountain View, CA; black bars). Levels of HGFL mRNA were normalized for expression of GAPDH and calculated in reference to the amount of HGFL mRNA in untreated HepG2 cells, which was given a value of 1 (gray bars). Values represent the average from three independent experiments. The results of Northern analyses of RNA isolated from cells treated with estrogen (2 µg/ml) and progesterone (2 µg/ml) are not shown in B.

 
The region between -135 and -105 in the HGFL gene is required for responsiveness to retinoic acid
To delineate the specific regions of the HGFL 5'-flanking region that may be required for retinoic acid repression, transient transfection analyses were performed using a series of HGFL promoter-CAT constructs in which the 5'-end of the HGFL promoter sequence was progressively removed (Fig. 2AGo) (25). In response to retinoic acid, there was an approximately 30% decrease in CAT expression in the cells transfected with the pL5(-4154/+1) construct compared with that in cells receiving no retinoic acid. A continued decrease in relative CAT expression was observed in response to retinoic acid despite the progressive 5'-deletions of the HGFL promoter from -4154 through -135 (Fig. 2AGo). Deletion of the region between -135 and -105 of the HGFL promoter resulted in a coincident loss in retinoic acid-mediated repression, suggesting that the -135/-105 region of the HGFL promoter is required for the repression induced by retinoic acid.



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  		Figure 2. The -135/-105 region of the HGFL gene is required for repression by retinoic acid. A, HepG2 cells were transfected with the chimeric plasmids containing serial deletions of the human HGFL 5'-flanking sequence ligated to a CAT reporter gene (top), then treated with (black bars) or without (white bars) retinoic acid (2 µg/ml) and analyzed for CAT expression (bottom), using a CAT sandwich ELISA. Nucleotides included from the HGFL gene are identified in parentheses next to the corresponding construct. The amount of CAT expression was determined by comparison with a standard curve, and the level of CAT expression was calculated in reference to the amount of CAT expression obtained from cells transfected with the promoterless CAT vector pBLCAT6, which was given a value of 1. All experiments were repeated at least three times, and all samples were assayed in duplicate for CAT expression. Error bars represent the SE. B, Chimeric plasmids (top) containing the minimal herpes simplex virus-thymidine kinase (HSV-tk) promoter (black bars) and the HGFL (-135/-105) sequence (striped bar) ligated to the CAT reporter gene were transfected into 293 cells alone or in conjunction with the HNF-4 expression vector pMT2.HNF4 (HNF4). The amount of CAT expression was determined (bottom) in transfected cells that were treated with (black bars) or without (white bars) retinoic acid (2 µg/ml) for 6 h. The amount of CAT expression was calculated in reference to the amount of CAT expression obtained from cells transfected with pBLCAT5 (top; pBLCAT5 contains the minimal HSV-tk promoter and the CAT reporter gene), which was given a value of 1. Error bars represent the SE. The transfected plasmids are indicated on the x-axis.

 
To determine whether the -135/-105 region of the HGFL promoter, which contains the HNF-4-binding site (25), is independently responsive to retinoic acid, this region was examined in the context of a heterologous promoter in the vector pL5CAT5(-135/-105) (Fig. 2BGo). Human embryonic kidney-derived 293 cells were used in this experiment, because they do not normally express HGFL or HNF-4, allowing us to dissect the potential contribution of HNF-4 in the retinoic acid-mediated repression of HGFL. Cotransfection of pL5CAT5(-135/-105) with the HNF-4 expression vector pMT2.HNF4 (26), produced a 31-fold increase in CAT expression over that obtained by the parental plasmid pBLCAT5, consistent with previous reports (25), and with the fact that HNF-4 activates expression of HGFL through this element. When these cells were subsequently treated with retinoic acid, there was a striking decrease in the levels of CAT expression, suggesting that the sequences contained in the HGFL -135/-105 promoter region are necessary and sufficient to mediate repression of the HGFL promoter by retinoic acid. The fact that retinoic acid had no effect on CAT expression in the absence of HNF-4 in 293 cells is evidence that HNF-4 is somehow involved in the repression of HGFL gene expression by retinoic acid.

Based on the facts that HNF-4 has been shown to bind the -135/-105 region of the HGFL promoter (25), and that HNF-4 and retinoid X receptors (RXRs) have been shown to bind to identical DNA sequences (30, 31, 42), it was proposed that repression of the HGFL promoter may occur through direct competition for DNA-binding sites between HNF-4 and RXR. However, the ligand for RXR, 9-cis-retinoic acid, had no effect on the expression of the endogenous HGFL mRNA, nor did it influence CAT expression from the HGFL promoter-CAT constructs in HepG2 cells that overexpress RXR (data not shown), suggesting that RXR is not involved in the repression of HGFL.

Binding of HNF-4 to the -135 to -105 region of the HGFL gene is not perturbed by RAR or retinoic acid
To explore the possibility that RAR may compete with HNF-4 for access to DNA sequences within the HGFL -135 to -105 region, an electrophoretic mobility shift assay was used to determine whether this sequence is recognized by RAR or if binding of this region by HNF-4 is affected by retinoic acid (Fig. 3Go). Proteins in nuclear extracts prepared from HepG2 cells were able to produce a single band shift of a labeled oligonucleotide comprising the HGFL -135/-105 sequence (Fig. 3AGo, lane 2). The protein binding to this region has previously been identified as HNF-4 (25). In the presence of an unlabeled RARE from the human RARß promoter or an unlabeled random oligonucleotide, binding of HNF-4 was not affected (Fig. 3AGo, lanes 4 and 5). Similar results were observed using nuclear extracts from HepG2 cells that overexpress RAR{alpha} (data not shown). The converse experiment was performed with similar results. When the oligonucleotide comprising the natural RARE from the RARß promoter was labeled, nuclear extracts prepared from HepG2 cells produced a single band shift (Fig. 3AGo, lane 7), consistent with the mobility shift produced by RAR/RXR heterodimers (42). A 500-fold excess of unlabeled HGFL -135/-105 was unable to compete against RAR/RXR for binding to the RARE (Fig. 3AGo, lane 9), suggesting that RAR does not bind to the HGFL -135/-105 sequence. Furthermore, in vitro binding of HNF-4 to HGFL -135/-105 was maintained even in the presence of retinoic acid (Fig. 3BGo). Therefore, retinoic acid represses expression of HGFL without directly interfering with the ability of HNF-4 to bind the HGFL -135/-105 sequence.



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  		Figure 3. Electrophoretic mobility shift analysis comparing binding of HNF-4 to HGFL (-135/-105) vs. RARE. A, Oligonucleotides (31 bp) comprising sequences of HGFL (-135/-105; lanes 1–5) or the RARE from the human RARß promoter (lanes 6–10) were labeled and used as probes. HepG2 nuclear extracts were added (lanes 2–5 and 7–10). Competition experiments against the HGFL (-135/-105) probe were performed using a 100-fold excess of unlabeled HGFL (-135/-105; lane 3), a 500-fold excess of unlabeled RARE (lane 4), or a 500-fold excess of a 31-bp unlabeled random oligonucleotide sequence (nonsp.; lane 5). Competition experiments against the RARE probe were performed using a 100-fold excess of unlabeled RARE (lane 8), a 500-fold excess of unlabeled HGFL (-135/-105; lane 9), or a 500-fold excess of unlabeled random oligonucleotide (lane 10). All experiments were conducted in the presence of 10-6 M RA. B, The 31-bp oligonucleotide comprising the -135 to -105 sequence of the HGFL gene was labeled and used as a probe. HepG2 nuclear extracts were added in the presence or absence of RA (10-6 M) and an unlabeled RARE, as indicated.

 
CBP increases HGFL promoter activity
Based on the fact that HNF-4 and RAR both interact with CBP (32, 34, 35), we explored the possibility that retinoic acid interferes with HNF-4-mediated trans-activation by activating RAR, which squelches CBP. HepG2 cells were transiently transfected with pL5(-1554/+1), a construct that contains the region of the HGFL 5'-flanking sequence required for repression by retinoic acid (Fig. 2AGo). Increasing amounts of the CBP expression vector pRc/RSV.CBP.HA.RK were cotransfected into these cells (Fig. 4AGo). This expression vector encodes a human full-length CBP linked to a hemagglutinin (HA) epitope (CBP-HA). Cotransfection of pL5(-1554/+1) with CBP-HA resulted in an increase in CAT expression over levels achieved with pL5(-1554/+1) alone (Fig. 4AGo). This increase in HGFL promoter activity was dose dependent on the amount of pRc/RSV.CBP.HA.RK that was cotransfected, demonstrating that CBP enhances the activity of the HGFL promoter and is consistent with the hypothesis that CBP is present in the cell at limiting concentrations.



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  		Figure 4. CBP enhances HGFL promoter activity and protects against repression by retinoic acid. A, Increasing amounts of the CBP-HA expression plasmid pRc/RSV.CBP.HA.RK were transfected into HepG2 cells in conjunction with the promoterless CAT vector pBLCAT6 (white bars) or with pL5(-1554/+1 (black bars). The amount of CAT expression is presented relative to the amount of CAT expression obtained from cells transfected with the pBLCAT6 alone, which was set at a value of 1. All experiments were repeated three times. Each sample was analyzed in duplicate for CAT expression. Error bars represent the SE. B, HepG2 cells were transfected with the plasmid pL5(-1554/+1) alone or in conjunction with 0.2 µg of the CBP-HA expression vector pRc/RSV.CBP.HA.RK (CBP) and treated with (black bars) or without (white bars) retinoic acid (2 µg/ml) for 6 h, and the amount of CAT expression was determined relative to the amount of CAT expression that was obtained from HepG2 cells transfected with the plasmid pL5(-1554/+1), which was given a value of 1. Each experiment was repeated three times. Each sample was analyzed in duplicate for CAT expression. Error bars represent the SE. The transfected plasmids are indicated on the x-axis. C, 293 cells were transfected with the pL5CAT5(-135/-105) plasmid containing the heterologous promoter and were cotransfected with the HNF-4 expression vector pMT2.HNF4 (HNF4) alone or in conjunction with the CBP-HA expression vector pRc/RSV.CBP.HA.RK (CBP) and treated with (black bars) or without (white bars) retinoic acid (2 µg/ml) for 6 h, and the amount of CAT expression was determined relative to the amount of CAT expression obtained from cells transfected with the vector pBLCAT5, which was given a value of 1 (data not shown). Each experiment was repeated four times, and the samples were analyzed in duplicate for CAT expression. Error bars represent the SE. The transfected plasmids are indicated on the x-axis. D, HGFL expression in HepG2 cells that overexpress CBP-HA, obtained by stable transfection of HepG2 cells with the CBP-HA expression vector pRc/RSV.CBP.HA.RK. CBP.17 and CBP.18 refer to two independently derived stable transformants. Expression of CBP-HA was analyzed by Western analysis (data not shown). CBP.17, CBP.18, or HepG2 cells were treated with retinoic acid (2 µg/ml) for 6 h. Total RNA was isolated and analyzed for expression of HGFL mRNA by Northern analysis (top). The positions of the 18S and 28S ribosomal RNAs are indicated. The ethidium-stained agarose gel is shown (bottom).

 
Overexpression of CBP-HA reduces repression of HGFL expression by retinoic acid
The effects of CBP overexpression on retinoic acid-mediated repression were examined in transient transfections using pL5(-1554/+1) in HepG2 cells (Fig. 4BGo). Cells were cotransfected with a minimal amount of the pRc/RSV.CBP.HA.RK expression vector (0.2 µg) to avoid a large increase in CAT expression in untreated cells. CBP-HA overexpression eliminated repression of CAT expression in response to retinoic acid.

To examine the consequence of CBP overexpression on the activity of the -135 to -105 region of the HGFL promoter in the presence or absence of HNF-4, 293 cells were transiently transfected with the pL5CAT5(-135/-105) plasmid containing the heterologous promoter (Fig. 4CGo). As previously shown, CAT expression from pL5CAT5(-135/-105) required coexpression of HNF-4, and this expression could be repressed by retinoic acid. Coexpression of CBP-HA with HNF-4 increased CAT expression from pL5CAT5(-135/-105), suggesting that there is an interaction between CBP and HNF-4 at the HGFL -135 to -105 region, and it appears that CBP is involved in activating expression from this region through its interaction with HNF-4. Overexpression of CBP-HA prevented the repression of CAT expression by retinoic acid, suggesting that CBP and HNF-4 are both involved in the retinoic acid-mediated repression.

To investigate the effect of CBP-HA overexpression on endogenous HGFL gene expression, stable transformant HepG2 cells were created using the pRc/RSV.CBP.HA.RK expression vector. Overexpression of CBP-HA was confirmed by Western analysis using an antibody against the HA epitope (data not shown). Untransfected HepG2 cells underwent the characteristic decrease in HGFL mRNA in response to retinoic acid (Fig. 4DGo). In striking contrast, two independently derived CBP-HA-overexpressing cell lines were less affected by the treatment with retinoic acid. This suggests that CBP is critically involved in the expression of HGFL in the context of the endogenous HGFL promoter, and that ligand-activated RAR prevents HNF-4 and CBP from activating the HGFL gene.

HNF-4 interacts with CBP
To determine whether an interaction between CBP and HNF-4 exists, coimmunoprecipitation experiments were performed in nuclear extracts prepared from HepG2 cells that were transiently transfected with pRc/RSV.CBP.HA.RK to ensure that the amount of CBP would not limit the potential interaction between HNF-4 and CBP. Complexes immunoprecipitated with antibody to CBP (in the presence or absence of a CBP blocking peptide), or an anti-HGFL antibody were denatured, and the components were identified by Western analysis (Fig. 5Go). It was found that an antibody against CBP was able to precipitate CBP, but not in the presence of a CBP-blocking peptide (data not shown). The presence of HNF-4 in the immunoprecipitated complexes was determined using the antibody {alpha}455, directed against a carboxyl-terminal peptide of rat HNF-4, which has also been shown to cross-react with human HNF-4 (30). The HNF-4 antibody interacts with a band of the appropriate size (~54 kDa) that is not precipitated in the presence of a CBP-blocking peptide or HGFL antibody. These results demonstrate a direct interaction between CBP and HNF-4.



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  		Figure 5. HNF-4 coimmunoprecipitates with CBP. Immunoprecipitation of nuclear extract prepared from HepG2 cells that were transiently transfected with 2 µg of the CBP-HA expression plasmid pRc/RSV.CBP.HA.RK. Immunoprecipitation (IP) was carried out using a nonspecific anti-HGFL antibody (NS; lane 2), an anti-CBP antibody (CBP; lane 3), or an anti-CBP antibody in the presence of a CBP-blocking peptide (lane 4). Nuclear extracts (30 µg; lane 1) or immunoprecipitated complexes (lanes 2–4) were resolved on a 10% denaturing SDS-polyacrylamide gel and analyzed by Western analysis. The immunoblot was incubated with antibody against HNF4.

 
HNF-4 overexpression reduces HGFL promoter repression by retinoic acid
To more directly examine the competitive nature of the repression by retinoic acid, HNF-4 was overexpressed in HepG2 cells that were cotransfected with pL5(-1554/+1). The level of CAT expression observed in the presence of HNF-4 overexpression is comparable to previous observations (25). Whereas retinoic acid was able to decrease CAT expression under normal HNF-4 levels, when HNF-4 was overexpressed, the HGFL promoter was unaffected by retinoic acid (Fig. 6Go). The converse experiment was carried out, in which the RAR{alpha} expression vector pW1-RAR{alpha} was cotransfected with the pL5(-1554/+1) construct. Overexpression of RAR{alpha} further decreased CAT expression from pL5(-1554/+1) in response to retinoic acid. These results suggest that there is competition occurring between HNF-4 and retinoic acid-activated RARs.



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  		Figure 6. HNF-4 overexpression prevents repression by retinoic acid. HepG2 cells were transfected with the chimeric plasmid pL5(-1554/+1) alone or in combination with 2 µg of either the HNF-4 expression vector pMT2.HNF4 (HNF4) or the RAR{alpha} expression vector pW1-RAR{alpha} (RAR). The cells were treated with (black bars) or without (white bars) retinoic acid (2 µg/ml) for 6 h. The amount of CAT expression is calculated relative to the amount of CAT expression obtained from untreated HepG2 cells transfected with only pL5(-1554/+1), which is given a value of 1. Each experiment was performed twice with pMT2.HNF4 and four times with pW1-RAR{alpha}. Each sample was analyzed in duplicate for CAT expression. Error bars represent the SE. The transfected plasmids are indicated on the x-axis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This report demonstrates that CBP enhances HGFL gene expression through its interaction with HNF-4. The association of CBP with HNF-4 has been previously implied (32), using the yeast two-hybrid and in vitro translation systems. We provide evidence for a mechanism by which HNF-4 activity is modulated by an integrated set of signals that converge upon CBP in the context of both the endogenous and exogenous HGFL promoters. This is intriguing in light of the fact that HNF-4 is an orphan receptor and suggests an indirect approach to modifying HNF-4 activity in the absence of a direct ligand for HNF-4. Because HNF-4 binds to sequences within the HGFL gene promoter, the association of HNF-4 with CBP may recruit members of the basal transcription machinery to the HGFL promoter (Fig. 7Go). Interactions between CBP and certain components of the basal transcription machinery have been demonstrated, such as with TATA-binding protein, TFIIB, or RNA polymerase II (43, 44). Another mechanism by which CBP may facilitate HGFL gene expression is by perturbing the local chromatin structure surrounding the HGFL gene promoter. CBP could perform this by means of its intrinsic histone acetyltransferase activity (36, 37) or by its association with other histone acetyltransferases, such as p/CAF, p/CIP, or ACTR (38, 39). Activation of RAR by retinoic acid may interfere with transcription of HGFL, as we have observed, by titrating out CBP, preventing an interaction between HNF-4 and CBP (Fig. 7Go). This type of indirect competitive transcriptional repression could also be performed by other ligand-activated nuclear receptors. This hypothesis is supported by the fact that thyroid hormone, dexamethasone, estrogen, and progesterone all cause similar decreases in HGFL gene expression (Fig. 1Go). It is important to note that direct interactions with CBP have been demonstrated for RAR, thyroid hormone receptor, glucocorticoid receptor, estrogen receptor, and progesterone receptor, but only in the presence of their unique ligands (33, 34, 35). A similar scenario of transcriptional interference has been observed to occur between the glucocorticoid receptor and AP-1, the result of competition between the two transcription factors for association with CBP, which causes the apparent cross-talk and mutual inhibition of their target genes. Because many transcription factors outside of the nuclear receptor family also associate with CBP, such as CREB (40), activating protein-1 (34), p53 (45), c-Myb (46), sterol regulatory element-binding protein (47), and MyoD (48), it will be important to understand the extent of hormonal cross-talk and transcriptional coordination that occurs via CBP. Although we have shown that nuclear hormone receptors can interfere with HNF-4-mediated trans-activation, it would be interesting to determine whether HNF-4 competes with members of other signaling systems for association with CBP. It is of further interest to determine how the chromatin might be altered by CBP at HNF-4 target genes, and how this influences gene expression.



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  		Figure 7. Model for the interaction of HNF-4 and CBP on the HGFL promoter. Sequence-specific binding of HNF-4 to the -135/-105 region of the HGFL promoter recruits the transcriptional coactivator CBP (top). Recruitment of components of the basal transcription machinery and other transcriptional coactivators, such as the histone acetyltransferases pCAF and p/CIP, onto the HGFL promoter is enhanced by CBP, allowing for efficient activation of HGFL gene expression. Competition for association with CBP by ligand-activated nuclear receptors, such as RAR, estrogen receptor, or glucocorticoid receptor, can prevent the interaction between CBP and HNF-4 at the HGFL promoter, resulting in a decrease in HGFL gene expression (bottom). This model predicts that competition for CBP is one mechanism used by the cell to integrate information from various signals to organize the transcriptional output of the cell.

 
For the most part, transcription factors recruit CBP to specific promoters in a signal- or ligand-dependent manner. An association between HNF-4 and CBP is intriguing, in that HNF-4 is the first example of a transcription factor that interacts with CBP in the absence of a known signal, as it is considered to be an orphan receptor (30). Based on the fact that HNF-4 has been shown to interact with CBP in a ligand-independent manner, HNF-4 appears to have an intrinsic affinity for CBP (32). This affinity may be sufficient under normal circumstances to associate with CBP and trans-activate gene expression, but may not be able to compete for association with CBP in the presence of other ligand-activated nuclear receptors.

The involvement of CBP in the transcription of immunologically relevant genes has previously been implied, based on the fact that p65, a component of the transcription factor NF-{kappa}B, interacts with CBP to stimulate transcription from p65-dependent promoters (49). NF-{kappa}B encompasses a family of signal-dependent transcription factors that activate transcription of many genes in response to injury or inflammation, such as interleukin-1, tumor necrosis factor-{alpha}, inducible nitric oxide synthase, and granulocyte macrophage colony-stimulating factor. CBP also cooperates with the immunologically relevant transcription factor STAT2 (signal transducer and activator of transcription) in response to signaling by interferon-{alpha} (50). HGFL is thought to serve a critical role in the inflammatory process, and evidence presented here shows that CBP plays a pivotal role in the expression of HGFL. HGFL has been shown to be involved in macrophage activation, a phenomenon characterized by the secretion of proteases, morphological changes, chemotaxis, and an increase in phagocytosis (4, 5, 10). HGFL also inhibits the synthesis of nitric oxide by macrophage in response to bacterial endotoxins (20), suggesting that HGFL regulates the inflammatory response of macrophage. Accordingly, HGFL-deficient mice are subject to a delay in the onset of macrophage activation and are mildly impaired in particular inflammatory responses, such as in response to challenge with acute colitis (8), confirming that HGFL is involved in mediating specific inflammatory responses. Consistent with its proposed role in mediating inflammation, HGFL expression increases upon injury to the lung or liver (6, 9). Coordination of HGFL gene expression may be required to establish an appropriate inflammatory response to infection or injury. The positive effect of CBP on HGFL gene expression may be a reflection of the larger role of CBP in the expression of many inflammatory factors.

These results have demonstrated that CBP and HNF-4 cooperate to activate HGFL gene transcription, and that repression of HGFL gene transcription may be due to competition between HNF-4 and RAR as well as other nuclear receptors. These results manifest the central role played by CBP in the integration of many signals to generate an orchestrated transcriptional response and provide a model in which HNF-4 directs transcription on the HGFL promoter.


   Acknowledgments
 
The authors acknowledge Drs. Bruce Aronow and Michelle Barton for insightful discussions, suggestions, and critical reading of the manuscript. We thank Drs. Mark Montminy, Francis Sladek, and Melissa Colbert for generously providing critical reagents and the members of the laboratory for technical support.


   Footnotes
 
1 This work was supported in part by USPHS Grant DK-47003 from the NIDDK, NIH (to S.J.F.D.), NIH Training Grant HL-07527 (to R.S.M.), National Research Scientist Award Postdoctoral Fellowship (to S.E.W.), and a Board of Trustees Fellowship from the Children’s Hospital Research Foundation (to S.E.W.). Back

2 Current address: Department of Immunology and Microbiology, Vanderbilt University School of Medicine, Nashville, Tennessee 37235. Back

Received August 4, 1998.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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