Involvement of Hepatocyte Nuclear Factor-4 in the Expression of the Growth Hormone Receptor 1A Messenger Ribonucleic Acid in Bovine Liver

Honglin Jiang and Matthew C. Lucy

Department of Animal Sciences University of Missouri Columbia, Missouri 65211


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GH receptor 1A mRNA (GHR 1A mRNA) is one of the major GHR mRNA variants that differ in the 5'-untranslated region. The GHR 1A mRNA is unique because it is exclusively expressed in liver. The objective of the present study was to understand the mechanism for the liver-specific expression of the GHR 1A mRNA in the bovine. Twenty-six kilobases of 5'-flanking region of the bovine GHR gene was cloned and sequenced. The first exon (exon 1A) that corresponded to the 5'-untranslated region of the GHR 1A mRNA was 15,250 bp upstream from exon 2 in the GHR gene. The major transcription start site for the GHR 1A mRNA was 19 bp downstream from a putative TATA box. Transient transfection analyses of the 5'-flanking region of exon 1A in liver cell lines vs. nonliver cell lines did not reveal a positively regulatory region responsible for the liver-specific expression of the GHR 1A mRNA perhaps because the liver cell lines do not recapitulate the in vivo hepatic environment. A putative regulatory region was then found by deoxyribonuclease I footprinting analyses of the proximal 5'-flanking region of exon 1A with nuclear extracts from bovine liver tissue. This regulatory region contained a putative binding site for the liver-enriched transcription factor hepatocyte nuclear factor-4 (HNF-4). Binding of HNF-4 in bovine liver to this putative HNF-4 binding site was confirmed by electrophoretic mobility shift assays. Overexpression of HNF-4 enhanced the transcriptional activity of the 5'-proximal region of exon 1A in various cell lines. Mutation of the HNF-4 binding site abolished the transactivation. In addition, the HNF-4 mRNA was found to be primarily expressed in liver and absent in most nonhepatic tissues in the bovine. Collectively, these observations suggest that the liver-enriched transcription factor HNF-4 plays a role in the expression of GHR 1A mRNA in bovine liver.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GH receptor (GHR) is expressed in many tissues, with the highest level in liver (1). Binding of GH with the GHR in liver stimulates the synthesis and secretion of insulin-like growth factor-I (IGF-I) into blood and thereby regulates growth, metabolism, and many other physiological processes (2). The GHR mRNA transcripts in liver as well as other tissues are a heterogeneous pool consisting of variants that differ in the 5'-untranslated region (5'-UTR) (3, 4, 5, 6, 7, 8, 9, 10, 11). These GHR mRNA 5'-UTR variants are probably generated from transcription of different leader exons by different promoters or from alternative splicing (12). One of the GHR mRNA 5'-UTR variants, named 1A in sheep (5) and cattle (11), V1 in humans (3), V2 or GHR 1 in rats (4, 6), and L1 in mice (8), is exclusively expressed in liver. Studies in several species have indicated that the liver-specific GHR mRNA variant is transcribed from an alternative leader exon by a liver-specific GHR promoter (5, 13, 14). However, little is known about the regulatory mechanisms underlying the tissue-specific expression of this GHR mRNA variant.

In the bovine, the liver-specific GHR 1A mRNA appears to be the most important GHR mRNA variant because it is the most abundant GHR mRNA variant in liver, and its levels are highly correlated with the blood levels of IGF-I under various physiological conditions (15, 16). The objective of the present study was to understand how the liver-specific GHR 1A mRNA is generated in the bovine and to identify transcription factors that are involved in the liver-specific expression of this GHR mRNA variant. Our study indicates that, like its homologs in other species, the bovine GHR 1A mRNA is transcribed from an alternative leader exon (exon 1A) by a promoter (GHR P1) in the 5'-flanking region of exon 1A. In addition, our study indicates that the liver-enriched transcription factor hepatocyte nuclear factor-4 (HNF-4) plays a role in the expression of the GHR 1A mRNA in bovine liver.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Exon 1A Is Approximately 15.2 kb Upstream from Exon 2 in the Bovine GHR Gene
Approximately 26 kb of bovine GHR genomic DNA region were cloned and sequenced. This bovine GHR DNA region consisted of the first exon (exon 1A) of the GHR 1A mRNA, the entire intervening sequence between exon 1A and exon 2, and a 10.3-kb 5'-flanking region of exon 1A. An EcoRI restriction enzyme digestion map is presented in Fig. 1AGo. A comparison of the bovine GHR genomic DNA sequence with the GHR 1A mRNA sequence revealed that exon 1A was 15,250 bp upstream from exon 2 in the bovine GHR gene. The intervening sequence between exon 1A and exon 2 in the bovine GHR gene was flanked by a consensus splice donor site (GT) at its 5'- end and a consensus splice acceptor site (AG) at its 3'-end.



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Figure 1. Cloning and Sequencing of the 5'-Region of the Bovine GHR Gene

A, Schematic representation of a 25.8-kb 5'-region of the bovine GHR gene that contains exon 1A (open box on left), exon 2 (open box on right), a 10.3-kb 5'-flanking region of exon 1A (solid line), and a 15.2-kb intervening sequence (solid line) between exon 1A and exon 2. The sequence contains several restriction digestion sites for EcoRI (E). The 25.8-kb nucleotide sequence has been deposited in GenBank under accession no. U15731. B, Sequence comparison of the bovine proximal 5'-flanking region of exon 1A with its ovine (5 ), human (GenBank AJ131868), and mouse (GenBank U06224) homologs. Indicated in the alignment are the locations of transcription start sites for exon 1A (italic boldface), a putative TATA box (underlined), and a putative HNF-4 binding site (boldface). The two transcription start sites in the bovine sequence were identified by a 5'-RACE. The ovine and mouse transcription start sites are adopted from other studies (5 13 ). The human transcription start site is unknown. Also included in the alignment is the consensus sequence for HNF-4 binding site, NG/AGGNCAAAGG/TTCG/AN, where n = A, C, G, or T (34 ). Numbering refers only to the nucleotides in the bovine sequence and is relative to the major transcription start site, which is designated as +1.

 
An alignment of the nucleotide sequence of the approximately 240-bp 5'-proximal region of exon 1A in the GHR genes of various species indicated that the bovine sequence was approximately 97%, 87%, and 65%, similar to the ovine, human, and mouse sequences, respectively (Fig. 1BGo). A putative TATA box was conserved among species (Fig. 1BGo). A major difference between the sequences of different species was found in the length of a TG-repeat microsatellite, which contained 20 consecutive TG repeats in both the bovine and ovine sequences, and six and four consecutive TG repeats in the human and mouse sequences, respectively (Fig. 1BGo).

Identification of the Transcription Start Site for the GHR 1A mRNA
The transcription start site for the bovine GHR 1A mRNA was first determined by using a ribonuclease protection assay (RPA). Using an antisense riboprobe containing a 130-bp GHR DNA region that was composed of 105 bp of exon 1A and 25 bp of its 5'-flanking region, the RPA generated an abundant protected fragment and several less abundant fragments from bovine liver RNA (Fig. 2AGo). The size of the most abundant fragment was 105 nucleotides (nt) (Fig. 2AGo), and it placed a transcription start site at the nucleotide 19 bp downstream from a putative TATA box (Fig. 1BGo). This site was designated as +1 in Fig. 1BGo. The less abundant RPA bands may correspond to minor transcription start sites or may be artifacts of the RPA.



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Figure 2. Mapping of the Major Transcription Start Site for the Bovine GHR 1A mRNA by RPA

A, RPA of the bovine liver RNA using a probe that included the GHR DNA region -25 to +105 (see Fig. 1BGo). The RPA generated one major protected fragment that was 105 nt according to the sequencing ladders (lanes G, A, T, and C) of an unrelated DNA fragment. This fragment places a transcription start site at nucleotide +1 in the GHR DNA sequence presented in Fig. 1BGo. The less abundant bands are nonspecific RPA products. B, RPA of the bovine liver RNA using a probe that contained a 325-nt exon 1A region and a 52-nt exon 2 region. The most abundant RPA band was 262 nt long. This 262-nt RPA fragment also places a transcription start site at nucleotide +1 in the GHR DNA sequence presented in Fig. 1BGo. The less abundant bands are nonspecific RPA products.

 
To confirm the results of the RPA and to map other potential transcription start sites, a rapid amplification of cDNA 5'-ends (5'-RACE) was performed on total RNA of bovine liver. The 5'-RACE generated two cDNA fragments that were specific for the 5'-region of the GHR 1A mRNA. One 5'-RACE fragment consisted of a 52-bp exon 2 and a 210-bp exon 1A; the other 5'-RACE fragment consisted of an identical 52-bp exon 2 and a 325-bp exon 1A. The presence of a 52-bp exon 2 region in the 5'-RACE products was expected based on the location of the GHR-specific reverse primer used in the last round of PCR in the 5'-RACE. By aligning the sequences of these two 5'-RACE fragments with the GHR genomic sequence, two transcription start sites for the GHR 1A mRNA were located in the GHR gene. The short 5'-RACE product placed a transcription start site at nucleotide +1 in the GHR DNA sequence (Fig. 1BGo), the same transcription start site that was placed by the 105-nt RPA fragment (Fig. 2AGo). The long 5'-RACE product placed a second transcription start site at nucleotide -115 in the GHR DNA sequence (Fig. 1BGo). This upstream start site was not mapped by the RPA. The 5'-RACE did not generate products that matched with the less abundant bands on the RPA autoradiograph (Fig. 2AGo), suggesting that the less abundant bands on the RPA autoradiograph were artifacts.

Although the 5'-RACE identified a second transcription start site at nucleotide -115, the 5'-RACE did not indicate whether this start site was a major start site or a minor start site. For this reason, a second RPA using a probe corresponding to the long 5'-RACE product was performed to determine the relative abundance of the GHR 1A mRNA transcribed from this upstream transcription start site. This RPA generated one abundant and several less abundant protected fragments (Fig. 2BGo). The length of the most abundant RPA band was 262 nt, and it placed a transcription start site at nucleotide +1 (Fig. 1BGo), the same transcription start site that was mapped by both the short 5'-RACE product (262 bp) and the 105-nt product of the first RPA. A 377-nt RPA fragment corresponding to the long 5'-RACE product was not observed on the same autoradiograph, suggesting that the transcription start site corresponding to the long 5'-RACE product is a minor transcription start site. Like the first RPA, the second RPA also generated several less abundant bands (Fig. 2BGo), but none of these less abundant bands matched with those generated by the first RPA, further suggesting that the less abundant bands on both RPA autoradiographs were artifacts.

Promoter Analysis of the 5'-Flanking Region of Exon 1A in Cell Lines
To determine whether the 5'-flanking region of exon 1A serves as a liver-specific promoter (designated GHR P1) and to identify potential regulatory regions that control promoter activity, serially truncated 5'-flanking regions of exon 1A were tested in luciferase reporter gene constructs using human hepatoma-derived cell lines Hep G2 and PLC/PRF-5, baby hamster kidney-derived cell line BHK-21, and human cervix adenocarcinoma-derived cell line HeLa. The transcriptional activities of the various 5'-flanking regions of exon 1A were low and similar in both liver cell lines (Hep G2 and PLC/PRF-5) and nonliver cell lines (BHK-21 and HeLa) (Fig. 3Go). A relatively high transcriptional activity was observed in the approximately 500 bp 5'-proximal region (4 to 6 times the promoterless pGL2B activity) across all of the four cell lines (Fig. 3Go). The transfection analyses did not indicate a positively regulatory region for greater GHR P1 activity in liver cells than nonliver cells (Fig. 3Go). An RPA of the total RNA isolated from cultured Hep G2 and PLC/PRF-5 cells did not detect mRNA for the human liver-specific GHR mRNA variant (named V1 in Ref. 3) in either cell line (data not shown). Thus, Hep G2 and PLC/PRF-5 cells may not be optimal models for direct transcriptional analysis of the liver-specific bovine GHR P1.



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Figure 3. Transient Transfection Analysis of the GHR 5'-Flanking Region of Exon 1A in Hep G2, PLC/PRF-5, BHK-21, and HeLa Cells

The promoter-reporter plasmid constructs contained serial truncations of the GHR 5'-flanking region of exon 1A inserted upstream of the firefly luciferase reporter gene in pGL2B. The numbers in each construct refer to the locations of nucleotides in Fig. 1Go. Cotransfection of the GHR promoter-reporter pGL2 plasmid and pRL-SV40 (encoding the Renilla luciferase, used as a transfection efficiency control) were done by using the calcium phosphate method. Activities of the firefly luciferase and Renilla luciferase were assayed by using the Dual Luciferase Assay System (Promega Corp.). Activities of the promoter-reporter plasmids are expressed relative to that of the promoterless pGL2B plasmid, which is normalized to 1. Values are expressed as mean ± SE from three independent transfection analyses. Letters a, b, c, and d indicate statistical difference (P < 0.05) between groups within the same cell line.

 
The transient transfection analyses did indicate several negatively regulatory regions within the GHR P1. The GHR DNA region between -8,133 and -2,730 inhibited the activity of the GHR P1 in Hep G2, PLC/PRF-5, and HeLa cells; the GHR DNA region between -2,730 and -1,719 inhibited the activity of the GHR P1 in BHK-21 and HeLa cells; the GHR DNA region between -1,719 and -469 inhibited the GHR P1 activity in Hep G2 and BHK-21 cells (Fig. 3Go). Whether these regions that inhibited the GHR P1 activity in cell lines also inhibit the GHR P1 activity in bovine tissues is unknown.

Identification of Protein-Binding Sites within the Proximal GHR P1 by Deoxyribonuclease I (DNase I) Footprinting Analysis
The liver cell lines were not ideal models for transient transfection analysis of the bovine GHR P1. Therefore, DNase I footprinting analyses were used to analyze the bovine GHR P1 for transcription factor-binding sties using nuclear extracts from bovine liver and other tissues. Two DNA fragments corresponding to the -299/+21 and -469/-250 region of the proximal GHR P1 were incubated with nuclear extracts from bovine liver, kidney, and spleen tissues and then digested with DNase I. Incubation of the -299/+21 DNA fragment with liver nuclear extracts generated an extended footprint within the -218/-151 region on both strands of the DNA fragment (Fig. 4Go, A and B). Incubation of the -299/+21 DNA fragment with kidney nuclear extracts generated a weak footprint in the same region on the antisense strand but not on the sense strand (Fig. 4Go, A and B). Incubation of the same DNA fragment with spleen nuclear extracts did not generate a footprint on either strand (Fig. 4Go, A and B). These results together suggest that the -218/-151 region of the proximal GHR P1 contains binding sites for liver nuclear proteins. The liver nuclear proteins that bound to the -218/-151 region may not be expressed in spleen because the same region was not footprinted by the spleen nuclear extracts. The same proteins may be expressed in kidney because the same DNA region was footprinted by kidney nuclear extracts. The proteins in kidney are probably expressed at a lower level compared with liver because the footprint generated by kidney nuclear extracts was weaker and was only observed on one strand of the DNA fragment (Fig. 4Go, A and B). The footprinting analysis of the -469/-250 DNA fragment revealed a weak footprint in the -464/-446 region for the liver nuclear extracts and a weak footprint in the -423/-403 region for all of the three nuclear extracts (data not shown). The specificity of these two weak footprints was not further evaluated.



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Figure 4. DNase I Footprinting Analysis of the 500-bp Proximal GHR P1

A DNA fragment containing the GHR 5'-flanking region -299/+21 of exon 1A was single end-labeled on the antisense (panel A) or sense (panel B) strand using T4-polynucleotide kinase and 32P-{gamma}-ATP. Approximately 2 x 104 cpm probe was partially digested with DNase I in the absence (lane -) or presence of 40 µg of nuclear extracts from bovine tissues (liver, kidney, or spleen). The (G+A) lanes contain a Maxam-Gilbert sequencing ladder of the same DNA fragment. The vertical line beside each autoradiograph indicates the footprint. The numbers beside the autoradiograph indicate the location of the footprint relative to the major transcription start site (nucleotide +1 in Fig. 1BGo).

 
Confirmation of Binding of Nuclear Proteins to the -218/-151 Region by Electrophoretic Mobility Shift Assays (EMSAs)
The interaction of nuclear proteins from bovine tissues with the -218/-151 DNA region was further evaluated by EMSA. As shown in Fig. 5AGo, a double-stranded DNA fragment corresponding to the footprinted region -218/-151 formed a DNA-protein complex with liver nuclear extracts. The same DNA-protein complex was not formed when the DNA fragment was incubated with an equal amount (5 µg) of spleen nuclear extracts (Fig. 5AGo). The DNA fragment formed a weak DNA-protein complex with kidney nuclear extracts. The DNA-protein complex formed for kidney migrated faster than the DNA-protein complex formed for liver (Fig. 5AGo), suggesting that different proteins were involved in the two DNA-protein complexes.



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Figure 5. EMSA of the DNase I Protected GHR DNA Fragment -218/-151

A double-stranded oligonucleotide corresponding to the GHR DNA region nt -218 to -151 was labeled at both ends using T4-polynucleotide kinase and 32P-{gamma}-ATP. The 32P-labeled oligonucleotide was incubated with 5 µg nuclear extracts (NE) from the bovine liver, kidney, or spleen (panel A) or with increasing amounts of liver NE (panel B). In the competition assay (panel C) and supershift assay (panel D), the liver nuclear extracts were incubated with an increasing molar excess of unlabeled oligonucleotide (cold oligo) or anti-HNF-4, anti-C/EBP{alpha}, anti-C/EBPß, or nonspecific antiserum (NSA) before addition of the labeled oligonucleotide. Letters B, F, and SS indicate the positions of the DNA-protein binding complex, free probe, and supershifted DNA-protein complex, respectively.

 
The specificity of the DNA-protein complex formed between the -218/-151 DNA fragment and liver nuclear extracts was further evaluated. Greater amounts of DNA-protein complex were formed when greater amounts of liver nuclear extracts were added to the reactions in the EMSA (Fig. 5BGo). The formation of labeled DNA-protein complex was completely inhibited by the addition of an excess of unlabeled -218/-151 DNA fragment (Fig. 5CGo). These results indicated that the interaction between the -218/-151 DNA region and liver nuclear proteins was specific.

Identification of HNF-4 as the Bovine Liver Nuclear Protein Binding to the -218/-151 Region
The DNase I footprinting analysis and EMSA demonstrated that the -218/-151 DNA region contained binding sites for liver nuclear proteins. An analysis of the -218/-151 DNA sequence with the computer program MatInspector (17) revealed the presence of putative binding sites for Nkx-1, Sp1, C/EBPß, E4BP4, HNF-4, COUP-TF, VBP, and C/EBP{alpha} within this region. Among these putative binding proteins, HNF-4, C/EBP{alpha}, and C/EBPß were the most likely candidates because they are liver-enriched transcription factors that play a critical role in the regulation of many liver-specific gene promoters (18, 19). EMSAs using specific antibodies for C/EBP{alpha}, C/EBPß, and HNF-4 showed that the DNA-protein complex formed between the -218/-151 DNA fragment and the liver nuclear extracts could be supershifted by the anti-HNF-4 antibody (Fig. 5DGo). This result indicated that HNF-4 was present in the DNA-protein complex formed between the -218/-151 region and the liver nuclear extracts. The DNA-protein complex formed between the -218/-151 DNA fragment and the liver nuclear extracts was not supershifted by the anti-C/EBP{alpha} or anti-C/EBPß antiserum (Fig. 5DGo).

Confirmation of Binding of Liver HNF-4 Protein to the Putative HNF-4 Binding Site in the GHR P1
The putative HNF-4 binding site in the -218/-151 DNA fragment is located between nt -196 and -178 (Fig. 1BGo), as revealed by the computer program (17). To confirm the binding of this site by HNF-4 protein from bovine liver nuclear extracts, EMSAs were performed on a double-stranded oligonucleotide confined to the putative HNF-4 binding site (-196/-178). Incubation of the -196/-178 oligonucleotide with the bovine liver nuclear extracts resulted in the formation of two DNA-protein complexes (Fig. 6Go, A and B). Both complexes were efficiently competed away by an excess of an unlabeled oligonucleotide containing the consensus HNF-4 binding site (see Fig. 1BGo for the sequence of the consensus HNF-4 binding site) (Fig. 6AGo). Both complexes were supershifted to near completion by the anti-HNF-4 antibody (Fig. 6BGo). The observation of two DNA-protein complexes between the HNF-4 binding site and liver nuclear extracts is consistent with previous studies (20, 21). The formation of two DNA-protein complexes indicates that in addition to HNF-4, the bovine liver may express a second protein that contains an identical or similar DNA-binding region to the HNF-4 protein and an identical or similar region to the epitope against which the anti-HNF-4 antibody was raised (21). The possibility that this second protein is a splice variant of HNF-4 was not studied. Nevertheless, the results of the EMSA confirm that the -196/-178 region of the bovine GHR P1 is a HNF-4 binding site that can interact with the HNF-4 protein in bovine liver.



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Figure 6. EMSA of the Putative HNF-4 Binding Site -196/-178 within the GHR P1

The interaction between the -196/-178 DNA fragment and HNF-4 protein from bovine liver nuclear extracts was assayed in the presence of an increasing molar excess of unlabeled oligonucleotide (HNF-4 oligo) containing a consensus HNF-4 binding site (panel A) or in the presence of an antibody (panel B). In panel B, lane 2 contained 1 µl anti-HNF-4 antiserum and lane 3 contained 2 µl anti-HNF-4 antiserum. The EMSA was done as described in Fig. 5Go. Letters B, F, and SS indicate the positions of the DNA-protein binding complex, free probe, and supershifted DNA-protein complex, respectively.

 
Transactivation of the GHR P1 by HNF-4 in Cell Lines
To determine the functional importance of HNF-4 to the activity of GHR P1, a rat HNF-4 expression vector pMT7-rHNF-4{alpha}1 was cotransfected into Hep G2, PLC/PRF-5, BHK-21, and HeLa cell lines with the GHR P1-reporter construct -469/+21/pGL2. The cotransfection of 50, 200, and 800 ng HNF-4 expression vector all increased the activity of the GHR P1 in various cell lines (P < 0.05) (Fig. 7Go). Thus, HNF-4 protein can transactivate the GHR P1.



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Figure 7. Effects of Cotransfected HNF-4 on the Activity of GHR P1 in Cell Lines

Indicated amounts of a rat HNF-4 expression vector pMT7-rHNF-4{alpha}1 were cotransfected into Hep G2, BHK-21, PLC/PRF-5, and HeLa cells with 2 µg of GHR P1-reporter construct -469/+21/pGL2 that contains the HNF-4 binding site or with 2 µg of GHR P1-reporter construct -469/+21/delHNF-4/pGL2 that has a deletion in the HNF-4 binding site. Variation in transfection efficiency was controlled by cotransfecting pRL-SV40 plasmid that encodes the Renilla luciferase. The luciferase activity under each treatment is expressed relative to the luciferase activity of -469/+21/pGL2 in the absence of exogenous HNF-4, which is normalized to 1. Values are expressed as mean ± SE from four (Hep G2, HeLa) or three (BHK-21, PLC/PRF-5) independent transfection analyses. Letters a, b, c, and d indicate statistical difference (P < 0.05) between groups within the same cell line.

 
The extent to which the GHR P1 was activated by the cotransfected HNF-4 appeared to depend on the recipient cell line. An equal amount of HNF-4 expression vector had a greater effect on the GHR P1 activity in HeLa cells and BHK-21 cells when compared with Hep G2 or PLC/PRF-5 cells (Fig. 7Go). Similar cell type-dependent variability in the activation of target promoters by transfected HNF-4 was also shown in other studies (22, 23, 24, 25, 26, 27). The reason for the differential activation is unknown. The extent to which the GHR P1 was activated by the cotransfected HNF-4 also appeared to depend on the amount of cotransfected HNF-4 vector. In Hep G2 cells, the effect of cotransfected HNF-4 on the GHR P1 increased when the amount of cotransfected HNF-4 expression vector was increased from 50 ng to 800 ng. In other cell lines, the effect of HNF-4 on GHR P1 activity was saturated or nearly saturated by cotransfection of 50 ng of HNF-4 expression vector (Fig. 7Go). Saturation of the HNF-4 cotransfection in some cell lines, but not others, suggests that the differences in the efficiency of transfected HNF-4 expression may exist across the cell lines.

The HNF-4 Transactivates the GHR P1 by Binding to the HNF-4 Binding Site
To determine whether the HNF-4 binding site (-196/-178) within the GHR P1 was required for HNF-4 transactivation, HNF-4 expression vector was cotransfected into cells with the HNF-4 site-deleted GHR P1 construct (-469/+21/delHNF-4/pGL2). Deletion of the HNF-4 binding site completely blocked the effects of cotransfected HNF-4 on the GHR P1 in Hep G2, BHK-21, and PLC/PRF-5 cells (Fig. 7Go). Deletion of the HNF-4 binding site blocked approximately 90% of the effect of cotransfected HNF-4 on the GHR P1 in HeLa cells (Fig. 7Go). These results confirm that the cotransfected HNF-4 increased the activity of GHR P1 by binding to the HNF-4 binding site between nt -196 and -178.

When the HNF-4 expression vector was not cotransfected, the HNF-4 site-deleted GHR P1 and the HNF-4 site-intact GHR P1 had similar activities in each of the four cell lines (Fig. 7Go). This result suggests that either the cell lines do not contain endogenous HNF-4 or that the endogenous HNF-4 in these cell lines does not activate the bovine GHR P1. The Hep G2 cells do contain endogenous HNF-4, but the levels are 5 to 10 times less than in liver (Dr. F. M. Sladek, personal communication). Perhaps the levels of HNF-4 in Hep G2 cells are too low to fully or partially activate the transfected GHR P1.

Tissue Distribution of the HNF-4 mRNA and the GHR 1A mRNA in the Bovine
The above experiments demonstrated that the GHR P1 contained a HNF-4 binding site, and binding of HNF-4 to this site increased the activity of the GHR P1 in cell lines. These results suggest that HNF-4 may be involved in the expression of the GHR 1A mRNA in vivo. In rats, the HNF-4 mRNA was primarily expressed in liver (20). To determine whether HNF-4 was also primarily expressed in bovine liver, a 311-bp bovine HNF-4 cDNA fragment was cloned. The bovine HNF-4 cDNA sequence (deposited in GenBank under accession no. AF250028) was 90% identical to the human HNF-4 mRNA (Genbank accession no. NM_000457) and 87% identical to the rat HNF-4 mRNA (GenBank accession no. X57133). An RPA revealed that the HNF-4 mRNA in cattle was primarily expressed in liver. The HNF-4 mRNA was also detected in kidney, but the level in kidney was approximately 15% of that in liver (Fig. 8Go). The HNF-4 mRNA was not detected in other bovine tissues that were examined (Fig. 8Go). An RPA was also performed to determine the expression of the GHR 1A mRNA in the same samples in which the HNF-4 mRNA was measured. As shown in Fig. 8Go, the GHR 1A mRNA was detected only in liver (Fig. 8Go). The enrichment of HNF-4 in liver and absence of HNF-4 in most other bovine tissues support a role of HNF-4 in the liver-specific expression of the GHR 1A mRNA.



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Figure 8. Tissue Distribution of the HNF-4 mRNA and GHR 1A mRNA in Cattle

Total RNA from various bovine tissues (nonlactating cows) were analyzed for the expression of HNF-4 mRNA or GHR 1A mRNA by using RPA. In each assay, a probe specific for the bovine GAPDH mRNA was included as a loading control. The RPA-protected bands corresponding to HNF-4, GHR 1A, and GAPDH mRNA are indicated with arrows. The autoradiograph is representative of two assays. Sk, Skeletal.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GHR 1A mRNA is a liver-specific GHR mRNA 5'-UTR variant that has been identified in various species (3, 4, 5, 6). In the bovine, the GHR 1A mRNA appears to be the most important GHR mRNA 5'-UTR variant because it is the most abundant GHR mRNA variant in liver and its levels are highly correlated with blood IGF-I concentrations (15, 16). It is important, therefore, to understand how this liver-specific GHR mRNA variant is generated in the bovine. In the present study, we located the exon (exon 1A) that corresponds to the 5'-UTR of the GHR 1A mRNA at 15.2 kb upstream from exon 2 in the bovine GHR gene. This location of the bovine exon 1A is very similar to that of the ovine and mouse exon 1A, which were estimated to be 17 kb and 15 kb from exon 2, respectively (5, 28). In addition, we found that the intervening sequence between exon 1A and exon 2 in the bovine GHR gene is flanked by GT at the 5'-end and AG at the 3'-end, which are typical structural features for an intron. Thus, splicing of exon 1A onto exon 2 and generating the GHR 1A mRNA comply with the GT-AG rule of mRNA splicing. We also found that the major transcription start site for the bovine GHR 1A mRNA was located at a nucleotide 19 bp downstream from a putative TATA box in the bovine GHR gene. This relative location of a transcription start site to the TATA box is consistent with the role of the TATA box in determining the site of transcription initiation. In addition, the location of the major transcription start site for the GHR 1A mRNA in the bovine GHR gene was similar to that in the ovine and mouse GHR genes (Fig. 1BGo). Collectively, these observations indicate that the bovine GHR 1A mRNA variant, like its homologs in other species (5, 13, 14), is generated from transcription of a leader exon (exon 1A).

The mechanism for liver-specific expression of the GHR mRNA variants is unknown in any species (5, 13, 14). This mechanism, therefore, was the focus of the present study. An attractive hypothesis for this mechanism is that liver-specific transcription factors interact with respective DNA elements in the GHR P1 and thereby cause the transcription from exon 1A in liver. Such DNA elements are traditionally defined by transient transfection analysis in liver-derived cell lines vs. nonliver cell lines. However, the transient transfection analyses of the 4.5-kb ovine (5) or the 3.6-kb mouse (13) GHR 5'-flanking sequence in liver-derived cell lines vs. nonliver cell lines did not identify a positively regulatory region that defines the liver-specific activity of the GHR P1. The liver-specific regulatory regions may reside in more distal 5'-flanking regions. For this reason, the transfection analyses in the present study were extended to the 8-kb 5'-flanking DNA region of exon 1A in the bovine GHR gene. The analyses did not reveal a region within the 8-kb 5'-flanking region that conferred a significantly greater transcriptional activity in liver cell line Hep G2 or PLC/PRF-5 than in nonliver cell line BHK-21 or HeLa (Fig. 3Go). Subsequently, we found that Hep G2 and PLC/PRF-5 cells did not express the human liver-specific GHR mRNA variant V1, even though they were derived from human liver. The liver-specific GHR mRNA variant V1 was also not expressed in two other frequently used human liver-derived cell lines Hep3B and Huh7 (29, 30). Therefore, liver cell lines may not be optimal models for studying the transcriptional control of the liver-specific GHR mRNA variant. This may explain why the transient transfection analysis of the bovine GHR P1 in Hep G2 and PLC/PRF-5 cells in the present study, the transient transfection analysis of the ovine GHR P1 in Huh7 cells (5), and the transient transfection analysis of the mouse GHR P1 in Hep G2 cells (13) failed to identify a liver-specific regulatory region within the GHR P1.

Most, if not all, of the liver cell lines are derived from hepatomas. Because hepatomas lack the liver-specific GHR mRNA variant (30), we suspected that a liver cell line suitable for the transient transfection analysis of the liver-specific GHR promoter was probably not available. We therefore used DNase I footprinting analysis and EMSA to directly analyze the 500-bp proximal region of the GHR P1 for protein binding sites with nuclear extracts isolated from bovine liver. The 500-bp proximal GHR P1 was chosen to study because this region of the GHR gene shared a high degree of sequence similarity among species (Fig. 1BGo), and similarities in the 5'-flanking DNA sequence among different species may represent important regulatory regions. We found that the -218/-151 proximal GHR P1 region bound nuclear proteins from liver and that the protein binding to the putative HNF-4 binding site (-196/-178) was the liver-enriched transcription factor HNF-4. Overexpression of HNF-4 increased the activity of the cotransfected GHR P1 in various cell lines. In addition, we found that in the bovine, HNF-4 mRNA was enriched in liver and absent in many other tissues (Fig. 8Go). These observations together suggest that HNF-4 may play a role in the liver-specific expression of the GHR 1A mRNA in the bovine.

HNF-4 is a highly conserved member of the nuclear receptor/HNF-4 family (19). The HNF-4 binding site has been found in many genes (18, 19, 22, 31, 32, 33), and the reported HNF-4 binding sequences are heterogeneous (34). The consensus sequence binding to HNF-4 with high affinity is NG/AGGNCAAAGG/TTCA/GN, which consists of direct repeats of the hexamer AGGTCA (underlined) intervened by one or two nucleotides (34). The HNF-4 binding site in the bovine GHR P1, CTGGGCAAAGGTCGG, is only one nucleotide different from the consensus HNF-4 binding site, suggesting that the HNF-4 binding site in the bovine GHR P1 is a high-affinity binding site for HNF-4. A sequence comparison reveals that a similar HNF-4 binding site is also present in the ovine GHR P1 and human GHR P1 (Fig. 1BGo). Conservation of the HNF-4 binding site in the GHR P1 across species further supports a role for HNF-4 in the expression of the liver-specific GHR mRNA variant. Interestingly, the HNF-4 binding site is less conserved in the mouse GHR gene (Fig. 1BGo). Perhaps, the lack of a HNF-4 binding site is one of the reasons why the mouse GHR P1 is inactive except during pregnancy (8). A unique mechanism seems to be responsible for the activation of the mouse GHR P1 during pregnancy (35).

Given the fact that HNF-4 plays a critical role in the expression of several liver-specific genes (18, 19), we suspect that HNF-4 plays an important role in regulating the expression of the GHR 1A mRNA in bovine liver. However, in addition to liver, HNF-4 is also expressed in tissues such as kidney where the GHR 1A mRNA is not expressed (Fig. 8Go). Thus, HNF-4 alone is probably not sufficient for the expression of GHR 1A mRNA in liver. Because a strictly liver-specific transcription factor has never been identified for any liver-specific genes, we postulate that the liver-specific expression of GHR 1A mRNA is probably not regulated by one or two master liver-specific transcription factors but may be regulated by many transcription factors that are not necessarily liver specific. Thus, additional proteins remain to be identified in future studies to completely understand the mechanism underlying the liver-specific expression of the GHR 1A mRNA in the bovine.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Genomic Library Screening and Long-PCR Amplification
Genomic library screening and long-PCR amplification were used to clone the 5'-flanking region of the bovine GHR gene. A bovine genomic library in the vector EMBL3 SP6/T7 (CLONTECH Laboratories, Inc. Palo Alto, CA) was screened by using an exon 1A-specific probe according to the manufacturer’s instructions. Approximately 10 kb of 5'-flanking region and 5 kb of 3'-flanking region of exon 1A were obtained from the GHR genomic clones. The remaining sequence (~10 kb) between exon 1A and exon 2 that was not included in the clones screened from the bovine genomic library was amplified from the bovine genomic DNA by a long-PCR with the forward primer specific to the known most 3'-flanking sequence of exon 1A and the reverse primer specific to the known most 5'-flanking sequence of exon 2. The long PCR was carried out by using the TaqPlus Long PCR system (Stratagene, La Jolla, CA) as described previously (36). The long-PCR products were cloned in the PCR 2.1 vector (Invitrogen, Carlsbad, CA).

PCR and RT-PCR
A standard PCR was used to amplify the GHR DNA fragment from nt -25 to +105 (see Fig. 1Go for locations) with specific primers. The PCR product was cloned into the PCR 2.1 vector. This plasmid was then used to generate an antisense riboprobe for the RPA to map the transcription start site for the GHR 1A mRNA.

A standard RT-PCR was used to clone a bovine HNF-4 cDNA fragment. Briefly, the bovine liver total RNA was oligo-d(T) primed and reverse transcribed into first-strand cDNA. The first-strand cDNA was then amplified with a forward primer 5'-ATGAAGGAGCAGCTGCTGGT-3' and a reverse primer 5'-AAGAAGATGATGGCTTTGAGG-3' that were designed based on the human HNF-4 cDNA sequence (GenBank accession no. NM 000457). The PCR products were cloned into the pGEM-T vector (Promega Corp., Madison, WI). This HNF-4 cDNA plasmid was used to generate an antisense riboprobe for the RPA to analyze the expression of the HNF-4 mRNA in bovine tissues.

5'-RACE
The 5'-RACE was used to map the transcription start site in the GHR gene for the GHR 1A mRNA. The 5'-RACE was performed by using a commercially available 5'-RACE kit according to the manufacturer’s instructions (Life Technologies, Inc.). The starting RNA was total RNA isolated from bovine liver. The three GHR-specific reverse primers used in the reverse transcription and in the two sequential rounds of PCR reactions were 5'-ATTTAGGATTCCCAGA-3', 5'-CTGCAGACTCTGAGATGCTC-3', and 5'-CCTGCCACTGCCAAGGTCAA-3' that were each specific to the GHR exon 4, 3, and 2, respectively. The products from the second PCR were electrophoresed on agarose gels, and DNA bands of interest were isolated and cloned into the PCR2.1 vector.

Cloning and Sequencing
Cloning and subcloning were done according to standard procedures. The nucleotide sequence of cloned DNA/cDNA was determined by automated fluorescent sequencing (University of Missouri DNA Core Facility, Columbia, MO).

RNase Protection Assay
The RPA was used to map the transcription start site for the GHR 1A mRNA and to measure the expression of the GHR 1A mRNA and the HNF-4 mRNA in bovine tissues. Two different antisense probes were used in the RPAs for mapping the transcription start site for the GHR 1A mRNA. One probe corresponded to a GHR cDNA region that consisted of 52 bp exon 2 and 325 bp exon 1A. The other probe corresponded to the GHR DNA region nt -25 to +105 (Fig. 1Go). The former probe was also used in the RPA of the GHR 1A mRNA expression in bovine tissues. For the RPA that was used to determine the tissue distribution of the HNF-4 mRNA, the probe was generated from the HNF-4 cDNA clone that was cloned by the RT-PCR. The GHR 1A or HNF-4 mRNA- specific probe was generated by standard in vitro transcription and each probe had a specific activity greater than 5 x 108 cpm/µg. In each RPA, approximately 4 x 104 cpm of riboprobe were hybridized with 20 µg of total RNA. When used to analyze the tissue distribution of the GHR 1A or HNF-4 mRNA, the RPA also contained 4 x 104 cpm of bovine glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe. The bovine GAPDH probe was generated as described (15) with a specific activity of approximately 5 x 107 cpm/µg. The RPAs were performed essentially as described previously (10).

Construction of Promoter-Reporter Plasmids
The promoter-reporter plasmid constructs were made by inserting the GHR genomic DNA fragment into the polycloning sites upstream from the luciferase reporter gene in the promoterless vector pGL2B (Promega Corp.). The GHR DNA regions from nt -2,730 to +21, -1,719 to +21, and -469 to +21 (numbering relative to the transcription start site +1, Fig. 1Go) were each amplified by using standard PCR with specific primers linked with a SacI site and a NheI site. The PCR fragments were cloned in the SacI/NheI sites of pGL2B to form promoter-reporter plasmids -2,730/+21/pGL2, -1,719/+21/pGL2, and -469/+21/pGL2, respectively. A DNA fragment corresponding to the GHR 5'-flanking region nt -8,133 to -2,730 was amplified by using long-PCR with specific primers containing SacI linkers and cloned in the SacI site of plasmid –2,730/+21/pGL2 to form plasmid -8133/+21/pGL2.

Construct -469/+21/delHNF-4/pGL2 contained a deletion in the HNF-4 binding site (-196/-178) compared with construct -469/+21/pGL2. The deletion was made by using PCR-based site-directed mutagenesis as described previously (37). In all of the PCR reactions, proofreading polymerase Tli (Promega Corp.) was added to minimize PCR errors. The direction of the insert, insert-vector junction, and deletion were confirmed by DNA sequencing. The HNF-4 expression vector pMT7-rHNF4{alpha}1 that encodes rat HNF-4 was provided by Dr. Frances Sladek (University of California at Riverside, Riverside, CA) (20).

Transient Transfection Analysis
Hep G2, PLC/PRF-5, HeLa, and BHK-21 cells were each plated on 12-well plates and cultured according to the manufacturer’s instructions (ATCC, Manassas, VA). In the transient transfection analyses that were used to determine the transcriptional activity of the GHR 5'-flanking region of exon 1A, cells in each well were transfected with 0.5 pmol of GHR promoter-reporter plasmid, 0.002 µg of pRLSV40 plasmid, and an appropriate amount of pCR 2.1 plasmid (without insert) to bring the total amount of DNA to 5 µg/well. In the transient transfection analyses that were used to determine the effect of overexpressed HNF-4 on GHR P1, cells in each well were transfected with 2 µg of plasmid -469/+21/pGL2 or 2 µg of plasmid -469/+21/delHNF-4/pGL2, 0.002 µg of pRLSV40, the indicated amount of pMT7-rHNF-4{alpha}1, and an appropriate amount of pMT7 (without HNF-4 cDNA insert) to bring the total amount of DNA to 3 µg/well. The transfection was done by using the calcium phosphate method as described (10). Cotransfection of pRLSV40 plasmid was used as a control for transfection efficiency. The firefly luciferase activity encoded by the promoter-reporter plasmid and the Renilla luciferase activity encoded by the pRLSV40 plasmid were measured by using the Dual Luciferase Reporter Assay System (Promega Corp.), essentially as described (10). To correct for variance in transfection efficiency, the firefly luciferase activity of each GHR 1A promoter-reporter construct was divided by the Renilla luciferase activity of the cotransfected pRLSV40 plasmid. The transcriptional activity of a GHR promoter-reporter construct was expressed as relative to the activity of the promoterless vector pGL2B, which was arbitrarily designated as 1. Data from independent transfections were subjected to statistical analysis by using ANOVA and the Duncan’s multiple range test.

Isolation of Nuclear Extracts from Bovine Tissues
The nuclear protein extracts from bovine liver, kidney, and spleen were prepared as described (38) except that a protease inhibitor cocktail (1 ml/20 g tissue) (Sigma, St. Louis, MO) was included in the homogenization buffer (37). The bovine tissues were collected from nonlactating cows at slaughter.

DNase I Footprinting Analysis
The DNase I footprinting analysis was used to identify the 5'-proximal region of GHR exon 1A that bound to liver nuclear proteins. The footprinting analysis was done by using the Core Footprinting System (Promega Corp.) as described previously (37). Briefly, two DNA fragments corresponding to the 5'-flanking region of exon 1A nt -299 to +21 (-299/+21) and nt -469 to -250 (-469/-250) were released from their respective plasmids. The DNA fragments were labeled at the 5'-end of either the sense or the antisense strand with 32P by using 32P-{gamma}-ATP and T4 polynucleotide kinase (Promega Corp.). Approximately 2 x 104 cpm of the labeled DNA fragment were incubated with 40 µg of nuclear extracts in 50 µl of total volume on ice for 45 min in 1x binding buffer supplemented with 1 µg of poly d(A-T) and 1 µg of poly d(I-C) (Sigma). The probe-protein mixture was then digested with 0.01 U/µl DNase I in the presence of Ca2+/Mg2+ at room temperature for 1 min. The DNase I digestion was analyzed by electrophoresis on a 6% polyacrylamide sequencing gel. The (G+A) Maxam-Gilbert sequencing ladders of the same DNA fragment were generated essentially as described previously (39) and served as size markers.

EMSA
Complementary oligonucleotides were annealed in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 5 mM MgCl2 by heating to 90 C for 10 min and slowly cooling to 25 C over 1 h. Both 5'-ends of the double-stranded oligonucleotide were end-labeled with 32P as described above. The EMSA was performed as described previously (37). Briefly, 2 x 104 cpm (~0.02 ng) of probe were mixed with 5 µg or indicated amounts of nuclear extracts in a total volume of 20 µl that contained 1x binding buffer (Promega Corp.), 1 µg of poly d(A-T), 1 µg of poly d(I-C), and 2% Ficoll 400. For the competition shift assays, the nuclear extracts were incubated with a molar excess of the unlabeled oligonucleotide on ice for 45 min before the addition of the labeled oligonucleotide. For the supershift assay, the nuclear extracts were incubated with 1 µl or 2 µl of 1:5 diluted anti-HNF-4 antiserum (provided by Dr. Frances Sladek, University of California at Riverside) (20) or 1 µl of anti-C/EBP{alpha} or anti-C/EBPß antiserum (provided by Dr. Simon Williams, Texas Tech University) (40) before the addition of the labeled oligonucleotide. The binding reactions were further incubated on ice for 45 min and analyzed by electrophoresis on a native 5% polyacrylamide gel.


    ACKNOWLEDGMENTS
 
The authors thank Dr. Frances Sladek, University of California at Riverside and Dr. Simon Williams, Texas Tech University, for providing antibodies and plasmids. The authors also thank Cynthia K. Boyd and Charles W. Bolten for technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Dr. H. Jiang, 164 Animal Science Research Center, Department of Animal Sciences, University of Missouri, Columbia, Missouri 65211. E-mail: jiangh{at}missouri.edu

This work was supported in part by National Research Initiative Competitive Grants USDA CSREES 95–37205-2312 (M.C.L.) and 2001–35205-09963 (H.J.). This is contribution 13,127 from the Missouri Agricultural Experiment Station Journal Series.

Received for publication January 19, 2001. Revision received February 27, 2001. Accepted for publication March 7, 2001.


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