Hepatocyte Nuclear Factor 1 and the Glucocorticoid Receptor Synergistically Activate Transcription of the Rat Insulin-like Growth Factor Binding Protein-1 Gene

Dae-Shik Suh and Matthew M. Rechler

Growth and Development Section Molecular and Cellular Endocrinology Branch National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The insulin-like growth factor (IGF) binding proteins (IGFBPs) are a family of proteins that bind IGF-I and IGF-II and modulate their biological activities. IGFBP-1 is distinctive among the IGFBPs in its rapid regulation in response to metabolic and hormonal changes. The synthetic glucocorticoid, dexamethasone, increases IGFBP-1 mRNA abundance and gene transcription in rat liver and in H4-II-E rat hepatoma cells. A glucocorticoid response element (GRE) located at nucleotide (nt) -91/-77 is required for dexamethasone to stimulate rat IGFBP-1 promoter activity in transient transfection assays in H4-II-E cells. In addition to the GRE, three accessory regulatory sites [a putative hepatocyte nuclear factor-1 (HNF-1) site (nt -62/-50), an insulin-response element (nt -108/-99), and an upstream site (nt -252/-236)] are involved in dexamethasone stimulation under some, but not all, circumstances. The present study begins to address the mechanism by which transcription factors bound to the putative HNF-1 site act synergistically with the glucocorticoid receptor (GR) bound to the GRE. In gel shift assays, HNF-1{alpha} and HNF-1ß in H4-II-E extracts bind to the palindromic HNF-1 site. Both half-sites are required. Overexpression of HNF-1ß enhances dexamethasone-stimulated promoter activity. Both the HNF-1 site and the GRE must be intact for stimulation to occur. By contrast, overexpression of HNF-1{alpha} does not enhance dexamethasone-stimulated promoter activity, although, as also observed with overexpression of HNF-1ß, it inhibits basal promoter activity. Thus, the synergistic effects of HNF-1ß and the GR on dexamethasone-stimulated promoter activity require that they are bound to the HNF-1 site and the GRE, respectively, and may involve protein-protein interactions between the transcription factors, or between them and the basal transcription machinery or a steroid receptor coactivator. Synergy between the ubiquitously expressed GR and HNF-1, which is developmentally regulated and expressed in a limited number of tissues, provides a possible mechanism for tissue- and development-specific regulation of glucocorticoid action.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The insulin-like growth factor (IGF) binding proteins (IGFBPs) are a family of proteins that bind IGF-I and IGF-II and modulate their insulin-like and mitogenic actions (1, 2, 3). IGFBP-1 is distinctive among the IGFBPs in its rapid regulation in response to metabolic and hormonal changes (4). IGFBP-1 and IGFBP-1 mRNA are increased in insulin-deficient diabetes (5, 6) and by glucocorticoids (7), glucagon (8), and cytokines that are increased in a variety of inflammatory and catabolic conditions (9). Primary regulation of rat (r) IGFBP-1 gene transcription (6, 10, 11, 12, 13, 14) results in corresponding changes in IGFBP-1 mRNA and protein due to their rapid turnover (15, 16). The functional consequences of these transcriptional changes are determined, in part, by posttranscriptional modifications. Phosphorylated IGFBP-1 binds IGF-I with high affinity, forming inactive complexes that inhibit IGF action; less highly phosphorylated or nonphosphorylated forms bind IGF-I with lower affinity and may potentiate IGF action (17, 18, 19). Neutralization of the insulin-like effects of IGF-I have been observed in vivo after administration of IGFBP-1 to rats (20) and in some IGFBP-1 transgenic mice (21), resulting in hyperglycemia under fasting conditions and in response to a glucose challenge. Inhibition of the mitogenic effects of the IGFs in IGFBP-1 transgenic mice causes a striking inhibition of brain growth (16, 21) and, in some studies, modest decreases in birth weight and postnatal weight gain (21). IGFBP-1 potentiates wound healing by IGF-I or IGF-II in rat, rabbit, and human models (22, 23). IGF-independent stimulation of cell migration by IGFBP-1 due to the interaction of its RGD motif with the fibronectin receptor ({alpha}5ß1 integrin), as described in Chinese hamster ovary (CHO) (24) and vascular smooth muscle (25) cells, may contribute to this potentiation.

Glucocorticoids are important physiological and pathological regulators of IGFBP-1. Plasma IGFBP-1 is increased after cortisol infusion in human volunteers (7). Glucocorticoid deficiency after adrenalectomy reduces the dramatic increase in hepatic IGFBP-1 mRNA seen in insulin-deficient diabetic rats, suggesting that glucocorticoids play a permissive role in the regulation of the IGFBP-1 gene by insulin (26). Administration of glucocorticoids to 4-week-old rats increased hepatic IGFBP-1 mRNA (13), and dexamethasone treatment of pregnant rats increased IGFBP-1 mRNA in fetal liver in association with severe fetal growth retardation (27). Glucocorticoids also increased IGFBP-1 in cultured human bone cells (28).

We have studied the mechanism of glucocorticoid regulation of rIGFBP-1 promoter activity in the H4-II-E rat hepatoma cell line in which the synthetic glucocorticoid, dexamethasone, stimulates rIGFBP-1 gene transcription (10) and promoter activity (14, 29). Of the three sites in the proximal rIGFBP-1 promoter that bind recombinant human glucocorticoid receptor (GR) (29), the site at nucleotides (nt) -91/-77 with respect to the transcription initiation site corresponds to a consensus glucocorticoid response element (GRE) that must be intact for dexamethasone stimulation to occur (14, 29). Although this single GRE is necessary for dexamethasone stimulation of rIGFBP-1 promoter activity, maximal stimulation also requires the participation of one or more additional cis-elements (30). These include: nt -62/-50, which closely resembles the consensus binding sequence for hepatocyte nuclear factor-1 (HNF-1) (31); nt -108/-99, an insulin response element (IRE) that is necessary for the inhibition of promoter activity by insulin; and the region between nt -252/-236. Under most circumstances, maximal stimulation by glucocorticoids can occur with different combinations of these accessory sites, suggesting that the rIGFBP-1 promoter uses these accessory sites to compensate for the low affinity of its single functional GRE as proposed by Schüle et al. (32). The transcription factors binding to the three accessory sites in the rIGFBP-1 promoter have not been identified.

To begin to elucidate the mechanism by which transcription factors binding to these accessory sites might act synergistically with the glucocorticoid receptor (GR) bound to the GRE, we have focused, in this study, on the putative HNF-1 site. The HNF-1 consensus binding sequence is found in many genes that are preferentially expressed in liver (31) and binds the related homeodomain-containing transcription factors, HNF-1{alpha} and HNF-1ß, as homodimers or heterodimers (33, 34, 35). In the present study, we demonstrate that HNF-1{alpha} and -ß are present in H4-II-E extracts, and that they bind to the HNF-1 site. Overexpression of HNF-1ß enhances dexamethasone-stimulated promoter activity in plasmids in which the HNF-1 site and the GRE are intact, whereas overexpression of HNF-1{alpha} does not affect dexamethasone-stimulated promoter activity.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Gel Shift Assays Indicate That Both Half-Sites of the Putative HNF-1 Site (nt -62/-50) of the rIGFBP-1 Promoter Are Required to Bind Proteins in H4-II-E Nuclear Extracts
We previously demonstrated that mutation of nt -62/-50 of the rIGFBP-1 promoter decreased dexamethasone-stimulated promoter activity in H4-II-E cells (30). Eleven of 13 nucleotides in this region are identical to the HNF-1 consensus binding sequence (Fig. 1AGo) that has been shown to bind two related transcription factors, HNF-1{alpha} and HNF-1ß, both of which occur in at least three isoforms (36). HNF-1{alpha} and HNF-1ß have similar DNA-binding domains and bind to the canonical HNF-1 sequence with comparable affinity (31, 33, 34, 35).



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Figure 1. Gel Shift Assay Showing the Competition of Oligonucleotide Probes Containing Wild Type and Mutant HNF-1 Sequences for Binding of the Wild Type Probe to Proteins in H4-II-E Nuclear Extracts

A, Sequences of oligonucleotides containing wild type or mutant HNF-1 sites. The nucleotide sequences of the four oligonucleotides spanning nt -72/-40 of the rIGFBP-1 promoter that were used in gel shift assays are shown. The sequence of the nt -62/-50 region of the rIGFBP-1 promoter is shown in bold and compared with the consensus HNF-1 site. WT is the wild type oligonucleotide; ML-3 and ML-6 are oligonucleotides containing mutations in three or six bases in the 5'-half of the putative HNF-1 site, respectively; MR-6 contains a six-base mutation in the 3'-half of the HNF-1 site. The two bases in the WT sequence that differ from the HNF-1 consensus sequence are underlined. The nucleotides in the mutated sequences that differ from the WT sequence are shown in lower case. B, Electrophoretic mobility shift assay. The wild type oligonucleotide probe (WT) was incubated with (lanes 2–12) or without (lane 1) 6.7 µg of H4-II-E nuclear extract. The indicated unlabeled oligonucleotides were incubated with the probe in 20- or 100-fold molar excess as competitors (lanes 3 to 10): WT (lanes 3 and 4), ML-6 (lanes 5 and 6), ML-3 (lanes 7 and 8), MR-6 (lanes 9 and 10), or a nonspecific competitor (NS, lanes 11 and 12, nt -135/-92 of the rIGFBP-1 promoter). Electrophoresis and autoradiography were performed as described in Materials and Methods.

 
Electrophoretic mobility shift assays were performed to identify the protein(s) that bind to the nt -62/-50 region of the rIGFBP-1 promoter (Fig. 1BGo). As seen in lane 2, a predominant DNA-protein complex was observed when untreated H4-II-E cell nuclear extract was incubated with an oligonucleotide probe corresponding to nt -72/-40 of the wild type rIGFBP-1 promoter.1 [The pattern was unchanged when nuclear extracts prepared from dexamethasone-treated H4-II-E cells were used (results not shown).] Complex formation was inhibited competitively by coincubation with unlabeled wild type probe added at 20- or 100-fold molar excess (lanes 2 and 3), but not by a nonspecific oligonucleotide (nt -135/-92 of the rIGFBP-1 promoter, lanes 11 and 12).

Both half-sites of the putative HNF-1 site (nt -62/-50) were required for DNA-protein complex formation (Fig. 1BGo). Oligonucleotides, in which all six nucleotides in either the 5'-half-site (ML-6, lanes 4 and 5) or the 3'-half-site (MR-6, lanes 8 and 9) of the putative HNF-1 site were substituted, failed to inhibit the formation of complexes with the wild type oligonucleotide probe even when added at 100-fold excess. Oligonucleotide ML-3, used in earlier studies by us (30) and by Powell and colleagues (37, 38), in which only three of the nucleotides in the 5'-half-site were mutated, retained some affinity for the H4-II-E nuclear proteins (lanes 6 and 7). The presence of an MR-6 substitution mutation in a nt -327/+79 probe fragment also abolished protection of the nt -62/-50 region by H4-II-E nuclear extract in deoxyribonuclease I (DNase I) protection assays (results not shown). Thus, both half-sites of the HNF-1 consensus sequence must be intact for the observed complex to form.

HNF-1{alpha} and HNF-1ß in H4-II-E Nuclear Extracts Bind to the Putative HNF-1 Site
To determine whether the protein(s) in H4-II-E extracts that bound to the wild type nt -72/-40 oligonucleotide probe were related to HNF-1{alpha} or HNF-1ß, gel shift assays were performed after preincubation of H4-II-E nuclear extract with nonimmune rabbit serum, or antiserum to HNF-1{alpha} or HNF-1ß (Fig. 2Go). Incubation with nonimmune serum did not affect complex formation (lanes 2 and 3). By contrast, incubation with either antiserum to HNF-1{alpha} (lane 4) or antiserum to HNF-1ß (lane 5) decreased the abundance of these DNA-protein complexes, in part due to the formation of multiple supershifted complexes. Thus, the observed DNA-protein complexes contain both HNF-1{alpha} and HNF-1ß.



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Figure 2. HNF-1{alpha} and HNF-1ß in H4-II-E Cell Nuclear Extract Bind to the Putative HNF-1 Site of the rIGFBP-1 Promoter

The wild type oligonucleotide probe was incubated with (lanes 2–5) or without (lane 1) 6.7 µg of H4-II-E nuclear extract. Where indicated, the nuclear extract was incubated with normal rabbit serum (NRS, lane 3), anti-HNF-1{alpha} antiserum (lane 4), or anti-HNF-1ß antiserum (lane 5) before addition of the probe. Gel shift analysis was performed.

 
Overexpression of HNF-1ß but Not HNF-1{alpha} Enhances Dexamethasone Stimulation of rIGFBP-1 Promoter Activity
To determine the functional effect of HNF-1{alpha} or -ß on dexamethasone-stimulated rIGFBP-1 promoter activity, H4-II-E cells were cotransfected with reporter plasmid p327 containing the nt -327/+79 fragment of the rIGFBP-1 promoter, together with an HNF-1{alpha} expression plasmid, an HNF-1ß expression plasmid, or a control plasmid. When cotransfected with control plasmid, the promoter activity of the p327 construct was stimulated about 17-fold by dexamethasone (shown as 100% in Fig. 3Go) relative to the promoter activity in cells not treated with hormone. Overexpression of HNF-1{alpha} by cotransfection of the HNF-1{alpha} expression plasmid did not affect dexamethasone stimulation, whereas overexpression of HNF-1ß by cotransfection of the HNF-1ß expression plasmid enhanced dexamethasone stimulation of p327 by 230% compared with cotransfection of the control plasmid (Fig. 3Go, left panel).



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Figure 3. Effect of Overexpression of HNF-1{alpha} or HNF-1ß on Dexamethasone-Stimulated (left panel) or Basal (right panel) rIGFBP-1 Promoter Activity (left panel)

Dexamethasone stimulation (left panel). The plasmid p327 was transiently cotransfected into H4-II-E cells together with control vector (Control), or plasmids expressing HNF-1{alpha} or HNF-1ß. Duplicates of each transfection were incubated with or without dexamethasone, and luciferase activity was determined in cell lysates. Luciferase activity was normalized for the GH content of the media to control for transfection efficiency. The fold stimulation by dexamethasone was determined in dexamethasone-treated cells compared with untreated cells [(+Dex/-Dex) x100] and is expressed relative to the fold stimulation when control vector was cotransfected [16.8 ± 6.6 SD (n = 6), plotted as 100%]. The results are the mean ± SD from six experiments. P = 0.82 for the HNF-1{alpha} transfection and 0.0002 for the HNF-1ß transfection (Mann-Whitney Rank Sum Test). Basal (right panel): The luciferase activity of untreated cells from the same experiments shown in the left panel was compared after cotransfection with control vector (Control) or plasmids expressing HNF-1{alpha} or HNF-1ß. Results are expressed relative to the control vector (taken as 100%) and are plotted as the mean ± SD. Control basal activity is approximately 20 times greater than the background activity.

 
Since cotransfection of HNF-1{alpha} had no effect on dexamethasone stimulation, we wanted to verify that functional HNF-1{alpha} was expressed. Overexpression of HNF-1{alpha} (37) or HNF-1ß (39) had been reported to enhance basal activity of the human (h)IGFBP-1 promoter in several human cell lines, irrespective of whether the cells expressed HNF-1. We examined the effect of HNF-1{alpha} overexpression on basal promoter activity of the rIGFBP-1 gene in H4-II-E cells (Fig. 3Go, right panel). In H4-II-E cells, overexpression of HNF-1{alpha} or HNF-1ß decreased basal promoter activity of p327 by 73%, compared with the basal promoter activity of p327 cotransfected with the control plasmid.2 These results indicate that: 1) the HNF-1{alpha} expression vector is functional in H4-II-E cells; 2) the effects of HNF-1{alpha} and HNF-1ß on dexamethasone-stimulated and basal promoter activity are different; and 3) the functional interaction of overexpressed HNF-1ß with the GR that results in enhanced dexamethasone stimulation of rIGFBP-1 promoter activity is not seen with overexpression of HNF-1{alpha}.

Both the HNF-1 site and the GRE must be intact for overexpression of HNF-1ß to enhance dexamethasone stimulation of rIGFBP-1 promoter activity (see Figs. 4Go and 5Go). When H4-II-E cells were cotransfected with p327 and an HNF-1ß expression plasmid, dexamethasone stimulation was increased by 220%, compared with contransfection with a control vector (Fig. 4Go). By contrast, when HNF-1ß was overexpressed in H4-II-E cells transfected with an HNF-1 construct containing an MR-6 mutation in the 3'-half site (p327HNF-1 m), dexamethasone stimulation was not enhanced. Thus, the HNF-1 site must be intact for HNF-1ß to enhance dexamethasone-stimulated promoter activity.



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Figure 4. Enhancement of Dexamethasone Stimulation by Overexpression of HNF-1ß Requires an Intact HNF-1 Site in the rIGFBP-1 Promoter

Wild type plasmid p327, or p327 plasmid containing an MR-6 mutation in the HNF-1 site (p327HNF-1 m), was transiently cotransfected into H4-II-E cells with control vector (Control) or HNF-1ß expression vector (HNF-1ß). Half of the transfected cultures were incubated with dexamethasone. The fold stimulation by dexamethasone was determined in dexamethasone-treated cells compared with untreated cells (+Dex/-Dex) after normalization for the GH content of the media. The stimulation when control vector was cotransfected was 16.8 ± 6.6 SD for p327, and 9.1 ± 2.9 SD for p327HNF-1m. The results are the mean ± SD from six experiments. P values for both the p327 and p327HNF-1m transfections were 0.002 (Mann-Whitney Rank Sum Test). The reduction observed in dexamethasone-stimulated luciferase activity in p327HNF-1m after cotransfection of HNF-1ß (57 ± 15% of control) is unexplained.

 


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Figure 5. Enhancement of Dexamethasone Stimulation by Overexpression of HNF-1ß Requires an Intact GRE in the rIGFBP-1 Promoter

Wild type p327 plasmid, or a p327 plasmid containing a mutation in the GRE (p327GREm), was transiently cotransfected into H4-II-E cells together with control vector (Control) or HNF-1ß expression vector (HNF-1ß). Half of the transfected cultures were incubated with dexamethasone. The fold stimulation by dexamethasone was determined in dexamethasone-treated cells compared with untreated cells (+Dex/-Dex) after normalization for the GH content of the media. The stimulation when control vector was cotransfected was 15.4 ± 8.0 SD for p327, and 4.0 ± 3.0 SD for p327GREm (mean ± SD, n = 6). P values for p327 and p327GREm are 0.002 and 0.94, respectively (Mann-Whitney Rank Sum Test).

 
The enhancement of dexamethasone-stimulated activity of the rIGFBP-1 promoter by overexpression of HNF-1ß also requires that the GRE in the rIGFBP-1 promoter be intact (Fig. 5Go). H4-II-E cells were cotransfected with the HNF-1ß expression vector and a reporter plasmid containing either a wild type IGFBP-1 promoter fragment (p327) or a fragment containing a mutation in the 3'-half-site of the GRE (p327GREm). Overexpression of HNF-1ß increased dexamethasone-stimulated promoter activity of p327 by 230%, but did not enhance promoter activity in p327GREm. Binding of the activated GR to the functional GRE of the rIGFBP-1 promoter was required for HNF-1ß to enhance dexamethasone stimulation of rIGFBP-1 promoter activity.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Maximal induction of rIGFBP-1 promoter activity by glucocorticoids requires not only binding of the GR to the single low-affinity GRE, but the participation of three accessory sites: an HNF-1 site, the IRE, and a site located between nt -252 and -236 (30). The best defined of the three accessory sites is the potential binding site for HNF-1, a transcription factor that is expressed in a limited number of tissues including liver and kidney (31, 35), and appears to be required for the tissue-specific developmental program in these tissues (40, 41). Although HNF-1 is required for the basal expression of promoter activity of many genes that are preferentially expressed in liver (31), including human and rat IGFBP-1, HNF-1 had not been shown to participate in the regulation of gene transcription by glucocorticoids at the time our studies were initiated. For example, mutation of the HNF-1 site in the Xenopus laevis Bß-fibrinogen subunit gene decreased basal promoter activity by 90% without affecting dexamethasone stimulation (42). The present studies were undertaken to determine whether HNF-1 in H4-II-E nuclei bound to the putative HNF-1 site in the rIGFBP-1 promoter, and the effects of overexpression of HNF-1 on dexamethasone-stimulated promoter activity, to better define the mechanisms responsible for the synergistic interactions between the HNF-1 site and the GRE in the stimulation of rIGFBP-1 promoter activity by dexamethasone.

The HNF-1 site is required for optimal dexamethasone stimulation of rIGFBP-1 promoter activity (30). Dexamethasone-stimulated promoter activity was reduced to 54% of wild type levels when a stringent HNF-1 mutation (MR-6) was introduced into plasmid p327 containing the nt -327/+79 promoter fragment (Fig. 4Go). This promoter fragment contains the other two accessory sites, indicating that they cannot fully substitute for the requirement of an intact HNF-1 site.3

Two related transcription factors, HNF-1{alpha} and HNF-1ß, bind to the HNF-1 site as homodimers or heterodimers (31, 33, 34). HNF-1{alpha} and HNF-1ß have similar NH2-terminal dimerization and central DNA-binding domains (43), but differ in their COOH-terminal transactivation domains (34). Consistent with the binding of HNF-1{alpha} and HNF-1ß as homodimers or heterodimers, both half-sites of the HNF-1 element in the rIGFBP-1 promoter are required for dexamethasone stimulation. Electrophoretic mobility shift assays using antisera to HNF-1{alpha} and HNF-1ß demonstrated that both factors are present in H4-II-E nuclear extracts, consistent with previous studies using rat hepatoma cell lines related to H4-II-E; these earlier studies showed that both HNF-1{alpha} and HNF-1ß are expressed in differentiated liver cell lines such as H4-II-E, whereas only HNF-1ß is expressed in undifferentiated cells (35). Both transcription factors form DNA-protein complexes with an oligonucleotide corresponding to nt -72/-40 of the rIGFBP-1 promoter that contains the HNF-1 site.

To study the effect of HNF-1 on glucocorticoid stimulation of the rIGFBP-1 promoter, H4-II-E cells were cotransfected with plasmid p327 and with expression plasmids for HNF-1{alpha} or HNF-1ß. Overexpression of HNF-1ß enhanced dexamethasone stimulation of rIGFBP-1 promoter activity. This increased stimulation required that both the GRE and HNF-1 sites on the rIGFBP-1 promoter were intact, indicating that the synergistic effects of HNF-1ß on dexamethasone-stimulated promoter activity involved the interaction of transcription factors binding to the two nearby sites.

By contrast, overexpression of HNF-1{alpha} did not enhance dexamethasone-stimulated rIGFBP-1 promoter activity. This was not due to a failure to express the HNF-1{alpha} plasmid, since overexpression of HNF-1{alpha} inhibited basal promoter activity.

Differences in transcriptional activity between HNF-1{alpha} and HNF-1ß probably reflect differences in their transactivation domains (34). For the human C-reactive protein promoter (35), HNF-1ß was more active than HNF-1{alpha} in HeLa cells, whereas HNF-1{alpha} was more active than HNF-1ß in Fr3T3 cells; neither cell expresses HNF-1{alpha} or ß. Although overexpression of HNF-1{alpha} and HNF-1ß previously have been reported to increase the basal activity of many promoters (33, 35, 37, 39, 44), little information has been available suggesting possible participation of HNF-1 in gene regulation by glucocorticoids. Despite intensive study of sites involved in glucocorticoid stimulation of phosphoenolpyruvate carboxykinase promoter activity in liver-derived cells (45, 46), and although HNF-1{alpha} synergistically stimulated basal activity of the phosphoenolpyruvate carboxykinase promoter in a mouse hepatoma cell line cotransfected with a CAAT/enhancer binding protein-{alpha} expression plasmid (47), HNF-1 involvement in glucocorticoid stimulation has not been reported. While the present manuscript was in preparation, Faust et al. (48) reported that HNF-1 and the GR participated in dexamethasone stimulation of the mouse phenylalanine hydroxylase (PAH) promoter. The presence of a 3.8-kb enhancer region from the PAH gene in reporter plasmids containing either the PAH promoter or a heterologous thymidine kinase promoter enabled dexamethasone to induce promoter activity in a cell line closely related to H4-II-E. Full inducibility was localized to a 360-bp fragment that contained three GREs and two HNF-1 sites. Dexamethasone stimulation of the thymidine kinase promoter was greatly decreased by mutation of one of the HNF-1 sites.

The mechanism by which HNF-1 bound to the HNF-1 site and the GR bound to the GRE act synergistically to increase dexamethasone-stimulated rIGFBP-1 promoter activity is unknown. Possibilities include 1) protein-protein interactions between HNF-1 and the GR while both factors are bound to their respective cis-regulatory elements, or 2) interaction of one or both factors with the basal transcription machinery or with a coactivator for steroid hormone receptors. Direct protein-protein interactions with the GR have been described for c-jun (49), CREB (50), and other transcription regulatory proteins. The only protein other than HNF-1 that is known to interact with HNF-1 is an 11-kDa dimerization cofactor for HNF-1 (DCoH) (43). DCoH binds to the N-terminal regions of HNF-1{alpha} and HNF-1ß, stabilizing HNF-1 dimers and increasing their transactivation potential.

Direct protein-protein interactions between HNF-1ß and the GR may occur despite the fact that the HNF-1 site and the GRE must be intact for optimal dexa-methasone-stimulated promoter activity. In the ICAM-1 (intercellular adhesion molecule-1) gene, which is synergistically activated by Stat1 (signal transducer and activator of transcription) and the transcription factor Sp1, the cis-elements that bind both factors are required for functional activity despite the fact that Stat1 and Sp1 can be coimmunoprecipitated from solution (51). The binding of either factor to its respective cis-element may induce a conformational change that facilitates its binding to the other protein.

Activated steroid receptors may contact the basal transcription machinery directly or indirectly by forming complexes with coregulatory proteins (52). Synergistic interactions of HNF-1 may arise by regulating the interaction between the GR and one of its coactivators. As precedent for this model, the retinoblastoma protein directly interacts with the steroid receptor coactivator hBrm to up-regulate GR-mediated transcriptional activation (53).

In contrast to the stimulatory effect of HNF-1ß overexpression on dexamethasone-stimulated rIGFBP-1 promoter activity, overexpression of HNF-1{alpha} or HNF-1ß inhibited basal rIGFBP-1 promoter activity, as previously described for the ApoCIII, ApoAI, and HNF-1{alpha} genes (54). The reason for this inhibition is unclear. It appears to be, at least in part, an indirect effect on the rIGFBP-1 promoter since some partial inhibition is observed even when the HNF-1 site is mutated (results not shown).4

Synergistic interactions between HNF-1{alpha} and HNF-1ß, transcription factors that are developmentally regulated and expressed in only a limited number of tissues (31, 35, 40, 41), and the GR, a nuclear receptor that is capable of acting at many tissues, provide a potential mechanism for a ubiquitously expressed hormone to exert tissue-selective actions.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Chemicals and reagents were obtained from the sources previously specified (30). Acrylamide (30% solution) was purchased from National Diagnostics (Atlanta, GA). Rabbit polyclonal antisera specific for HNF-1{alpha} or HNF-1ß were raised to amino acids 541–561 in the COOH terminus of HNF-1{alpha} (33) and to amino acids 66–94 in the amino terminus of HNF-1ß (34) and were kindly provided by Drs. Cereghini (Institut Pasteur, Paris, France) and Crabtree (Stanford University, Palo Alto, CA), respectively. Expression plasmids for HNF-1{alpha} (55) and HNF-1ß (34) were graciously provided by Dr. Crabtree. A control plasmid (pcDL-SR{alpha}296) for cotransfection experiments containing the same promoter and vector sequence, but lacking a cDNA insert encoding HNF-1{alpha} or HNF-1ß, was kindly provided by Drs. S. Najjar and S. Taylor (NIDDK).

Plasmid Construction
Plasmid p327LUC (abbreviated p327) contains a nt -327/+79 (with respect to the transcription initiation site, +1) fragment of the rIGFBP-1 promoter ligated into plasmid pA3LUC (kindly provided by Dr. William M. Wood, University of Colorado, Denver, CO) in the sense orientation relative to a promoterless firefly luciferase reporter gene (14). Plasmid p327GREm, previously designated p327M6 (14), contains a substitution mutation in the 3'-half of the GRE.

The 3'-half of the HNF-1 site (-62 GACAATCATTAAC -50) was mutated in a nt -327/+79 promoter fragment by PCR amplification of two fragments overlapping at the BamHI site (nt -82/-77) using the conditions previously reported (30). The 3'-fragment was amplified using a 5'-primer that began at the BamHI site and included the mutated HNF-1 site (-62 GACAATCcggcca -50). The two PCR fragments were annealed and ligated into pA3LUC. The resulting construct contains the MR-6 mutation and was designated p327HNF-1 m.

All plasmids used in transfection studies were prepared using a Qiagen plasmid extraction kit (Qiagen, Inc, Chatsworth, CA), and were quantitated by absorbance at 260 nm. The sequence of the IGFBP-1 promoter insert was confirmed using PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit and 370A DNA Sequencer (Applied Biosystems, Foster City, CA).

Transfection and Luciferase Assay
Stock cultures of the H4-II-E cell line, established from the well differentiated Reuber H35 rat hepatoma (56), were grown as monolayer cultures (14) and were transfected using diethylaminoethyl-dextran as previously described (30). Each 60-mm culture dish of H4-II-E cells received 5 µg plasmid DNA containing rIGFBP-1 fragments and 1.4 µg of plasmid pXGH5 expressing human GH to control for transfection efficiency (14). [For cotransfection experiments, 2 µg of HNF-1{alpha} or HNF-1ß expression plasmid or 2 µg of the control plasmid were added at the same time]. The medium was replaced with serum-free DMEM containing 0.1% BSA and the cells were incubated overnight. Some of the media were collected before dexamethasone addition for quantification of GH using a solid phase RIA kit (Allegro HGH, Nichols Institute Diagnostics, San Juan Capistrano, CA), after which the medium was replaced with serum-free DMEM containing 0.1% BSA with or without dexamethasone (1 µM) as indicated. After 24 h incubation, the cells were lysed and luciferase activity was assayed in cell lysates as previously described (30).

Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared from confluent H4-II-E cells as described by Hennighausen and Lubon (57) with minor modification of the buffers (30). Oligonucleotides corresponding to nt -72/-40 and nt -135/-92 of the rIGFBP-1 promoter were synthesized. Equimolar quantities of complementary strands were annealed by heating at 100 C for 5 min and cooled to room temperature. The annealed fragments were labeled with [{gamma}-32P]ATP (>7000 Ci/mmol) using T4 polynucleotide kinase. H4-II-E cell nuclear extract (6.7 µg) was incubated with the oligonucleotide probe (2–4 fmol, 20,000 cpm) for 30 min at room temperature in a reaction mixture (20 µl, final volume) consisting of 2 µg of poly(dI-dC)·poly(dI-dC) acid buffer [1 mM Tris, pH 7.4, 5% glycerol, 1 mM MgCl2, 1 mM EDTA, and 1 mM dithiothreitol]. In competition studies, unlabeled double-stranded oligonucleotides were added immediately before the probe. The reaction mixtures were then loaded onto a 4% nondenaturing polyacrylamide gel that had been preelectrophoresed at 150 V for 1 h at 4 C. The gel and electrophoresis buffer contained 1 x and 0.5 x TBE (1 x TBE = 44.5 mM Tris-borate, 1 mM EDTA, pH 8.3), respectively. Electrophoresis was carried out at 10 mA for 2 h. Gels were dried and autoradiographed at -70 C using intensifying screens. In studies designed to immunologically identify the protein component of the protein-DNA complexes, 1 µl of antiserum against HNF-1{alpha} or HNF-1ß was incubated with the nuclear extract for 30 min at room temperature, after which the probe was added for an additional 30 min (final volume 20 µl).


    ACKNOWLEDGMENTS
 
We thank Peter Nissley, Guangren Zhang, and Guck T. Ooi for critical reading of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Matthew M. Rechler, NIH, Building 10, Room 8D08, 10 Center Drive, MSC 1758, Bethesda, Maryland 20892-1758.

1 Although only a single complex is evident in Fig. 1bGo, multiple DNA-protein complexes could be distinguished when gels with better resolving power were used (results not shown). These may arise from different combinations of the three isoforms of HNF-1{alpha} and HNF-1ß generated by alternative splicing (33 36 ). Back

2 Inhibition of basal rIGFBP-1 promoter activity by overexpression of HNF-1{alpha} or HNF-1ß was observed when transfections were performed with various amounts (0.01 µg to 3 µg) of HNF-1{alpha} or HNF-1ß expression plasmids (results not shown). No significant inhibition of basal promoter activity was observed after overexpression of HNF-1{alpha} or HNF-1ß in constructs in which the HNF-1 site was mutated (results not shown). Back

3 We previously reported that an intact HNF-1 site only was required for optimal stimulation of rIGFBP-1 promoter activity by dexamethasone under some, but not all, experimental conditions (30 ). In plasmid p92, in which the HNF-1 site was the only accessory site present in addition to the GRE, the HNF-1 site was necessary. By contrast, in plasmid p327 containing the nt -327/+79 promoter fragment, which includes all three accessory sites (the HNF-1 site, the IRE, and the nt -252/-236 site), the presence of any two sites was sufficient for optimal dexamethasone-stimulated promoter activity, suggesting that the sites were interchangeable. The HNF-1 mutation (ML-3) used in these experiments (30 ), however, retained some affinity for the HNF-1 site (Fig. 1BGo). When the more stringent MR-6 mutation was introduced into plasmid p327 and used in transfection assays, dexamethasone-stimulated promoter activity now was reduced to 54% of wild type levels despite the presence of the other two accessory sites (Fig. 4Go). Back

4 The effect of overexpressing a particular isoform of HNF-1{alpha} or HNF-1ß might differ from the activity of endogenous HNF-1. Inhibition might occur if endogenous HNF-1 dimers (or particular isoforms) stimulate basal promoter activity, whereas overexpression of a particular isoform of either protein favors the formation of inactive dimers. Back

Received for publication June 20, 1997. Revision received August 14, 1997. Accepted for publication August 19, 1997.


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