~ ) Pergamon Progress in Growth Factor Research, Vol. 6. Nos. 2-4, pp. 131-140, 1995

Published by Elsevier Science Ltd Printed in Great Britain

0955-2235/95 $29.00 + 00

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DEXAMETHASONE STIMULATION OF RAT INSULIN-LIKE GROWTH FACTOR BINDING

PROTEIN-1 (IGFBP-1) PROMOTER ACTIVITY INVOLVES THE INTERACTION OF MULTIPLE

TRANSCRIPTION FACTORS

Dae-Shik Suh,* Yuehua Zhou, Guck T. Ooi 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, MD 20892-1758, U.S.A.

Using an improved procedure for transient transfection of H4-1I-E rat hepatoma cells, we characterized the cis elements in the proximal promoter of the rat insulin-like growth factor binding protein-1 (rat IGFBP-1) gene that are required for basal (unstimulated) and dexamethasone-stimulated promoter activity. Three sites are required for optimal basal promoter activity: an AP-2 site (nt -286 to -293), the M4 region of the insulin response element (nt -108 to -99), and a hepatocyte nuclear factor-1 (HNF-1) site (nt 4 2 to -50). In addition to the glucocorticoid response element (nt -91 to -77), participation of two of three accessory sites is required for optimal stimulation by dexamethasone: the M4 and HNF-I sites, and a third site located between nt -252 and -236. Further study will focus on how the interactions o f tissue-specific and hormonally-responsive transcription factors are integrated.

Keywords: Dexamethasone, glucocorticoid response element (GRE), insulin response element (IRE), hepatocyte nuclear factor (HNF), accessory factor.

INTRODUCTION

Dexamethasone stimulates transcription of the rat IGFBP-1 gene [1] and rat IGFBP-1 promoter activity [2, 3] in H4-II-E rat hepatoma cells. The stimulation of promoter activity required that both half-sites of a glucocorticoid response element (GRE) located between nt -91 and -77 (with respect to the tran- scription start site, +1) be intact (Fig. 1; [2,3]). Deletion of the promoter region

*Correspondence to: D.-S. Suh, Tel 301-496-2483, Fax 301-402-4136.

131

132 D.-S. Suh et al.

upstream from the GRE (to nt -92), however, did not diminish dexamethasone stimulation [2].

Unexpectedly, the stimulation of rat IGFBP-1 promoter activity by dexametha- sone was greatly reduced when a mutation in the M4 region, corresponding to nt -108 to -99, was introduced into a rat IGFBP-1 promoter fragment whose 5' end was at nt -135 (Fig. 1; [2]). The M4 region corresponds to the left half of a palin- dromic sequence that is identical in the human and rat IGFBP-1 promoter, and mediates the inhibition of IGFBP-1 promoter activity by insulin, leading to its designation as an insulin response element (IRE) [4]. These results suggested that transcription factors which bound to sites in the proximal promoter region other than the GRE interacted with the glucocorticoid receptor to regulate dexametha- sone stimulation of promoter activity.

-327 I

d e x a m e t h a s o n e

-92 +79 II +

-91 -77

GRE

.sJtmu.latltn_ d e x a m e t h a s o n e

- 135 -92 +79 p135 [ I ~ ~ +

p92 ~ + -108 -99

p135M4 ~ PI P " " - " ' - -

M4 GRE (IRE)

FIGURE 1. (Top) Both half-sites of the glucocorticoid response element (GRE, nt -91 to -77) are required for dexamethasone stimulation of rat IGFBP-I promoter activity. Mutants were constructed in plasmid p327 containing a nt -327 to nt +79 promoter fragment coupled to a luciferase reporter gene. The hexanu- cleotide half-sites of the wild-type GRE are shown by open boxes, mutated half-sites by an "X". Mutation of either the right or the left half-site abolished dexamethasone stimulation of promoter activity. (Bottom) Dexamethasone stimulation of the rat IGFBP-I promoter is abolished in constructs containing a mutation in the M4 region of the insulin response element (IRE, nt -108 to -99) despite the presence of an intact GRE. The M4 mutation (denoted by an "X") was introduced in plasmid p135 (containing a promoter frag- ment whose 5' end was at nt -135). The M4 site is not essential for dexamethasone stimulation in plasmid p92 which contains an intact GRE, but must be intact for dexamethasone stimulation of promoter activity to occur in plasmid p135.

Dexamethasone Regulation of Rat IGFBP-1 Promoter Activity 133

ATTEMPTS TO IDENTIFY THE DNA BINDING SITE FOR A PUTATIVE INHIBITOR OF DEXAMETHASONE STIMULATED PROMOTER

ACTIVITY

The previous results could be explained by a model which postulated that, although the nt -92 to +79 promoter fragment containing the GRE was sufficient to allow dexamethasone-stimulation of rat IGFBP-1 promoter activity, an inhibitor of dexamethasone stimulation bound somewhere within the nt-135/-93 region, and that this inhibitory factor was neutralized by another factor which bound to the M4 region [2, 5]. According to this model, inhibition would occur if the M4 site were mutated so that the putative neutralizing factor could not bind, allowing the puta- tive inhibitor to act unopposed. However, if the site to which the inhibitor bound were deleted or mutated, the rat IGFBP-1 promoter would be stimulated by dexa- methasone even in the presence of an M4 mutation. As seen in Fig. 2, dexametha- sone stimulation was still inhibited in constructs containing the M4 mutation and a deletion of nt -135 to -110 (p109), and in constructs containing the M1, M2, M3, or M5 regions (spanning nt -135 to -93) in addition to the M4 mutation. These results indicate that the putative inhibitory factor, if it exists, does not bind to the region between nt -135 and-93.

E 120 I .O

100 E

O~ ==[ 8o o.>,

e- = 6 0

x~ ~ ~ 40

~ 20

o

p109 120 - p135

100 -

80 •

60 -

40 •

20 -

0 - - W T M4 W T M I M 4 M2M4 M3M4 M4 M5M4

F I G U R E 2. Attempts to identify the DNA binding site of a postulated inhibitor of dexamethasone stimula- tion of rat IGFBP-1 promoter activity. Plasmids were transfected into H4-1I-E cells as previously described [2]. The cells were incubated 8-12 h in serum-containing medium, followed by 16 h in serum-free medium. I)examethasone (1 ~M) was added for 24 h, after which the cells were lyscd and luciferase activity assayed in cell lysates. (Left) The effect of the M4 mutation (nt -108 to -99) on dexamethasone stimulation of promoter activity in plasmid p109 containing a ut -109 to +79 promoter fragment, p109 plasmids contain- ing the wild-type (WT) or mutant (M4) sequences of the M4 region were transfected into H4-11-E ceils. Half of the cultures were incubated with dexamethasone. Stimulation by dexamethasone (+Dex/-Dex) is expressed relative to that in cells transfeeted with wild-type plasmid p109. Dexamethasone stimulation in wild-type p109 transfectants was 20 and 85-fold in two experiments. (Right) The effect of the M4 mutation on dexamethasone stimulation in constructs containing a second substitution mutation in the ut -135 to nt -93 region. Transfections were performed with a series of p135 plasmids containing the wild-type sequence (WT), a substitution mutation in the M4 region alone, or the M4 mutation together with a second substitution mutation in region M1 (ut -135 to -126), M2 (nt -125 to -116), M3 (nt -119 to -110), or M5 (nt -96 to -93). The stimulation of luciferase activity by dexamethasone (+Dex/-Dex) was determined for each construct, and is expressed relative to the stimulation in wild-type p135 (5 to 60-fold in three experiments).

134 D.-S. Suh et al.

SITES REQUIRED FOR OPTIMAL BASAL PROMOTER ACTIVITY

Although the M4 mutation appeared to inhibit dexamethasone stimulation selec- tively, the possibility remained that its predominant effect was on basal (unstimu- lated) promoter activity. To evaluate this possibility, it was necessary to modify the transient transfection procedure to increase its sensitivity because otherwise basal promoter activity was too low to detect a further decrease.

After optimization of the transfection procedure, we examined the effect of several 5' deletions (p327, p278, p235, p135 and p92) on rat IGFBP-1 promoter activity in the absence of dexamethasone treatment. Promoter activity was >50 times background in the p327 construct, decreased 80% when the region between nt-327 and-278 was deleted (p278), and remained low in constructs p235, p135 and p92 that contained more extended 5' deletions. The region between nt -327 and -278 cotained sequences that might bind transcription factors AP-1 (nt -308 to -302) or AP-2 (nt -286 to -293). Substitution mutations were introduced in these sites in plasmid p327 to determine whether either of these sites was essential for optimal basal promoter activity. Mutation of the AP-1 site did not affect basal promoter activity, whereas mutation of the AP-2 site reduced promoter activity by 70%. We conclude that AP-2 or a related transcription factor that binds to the AP-2 site is required for optimal basal promoter activity. Deletion of the AP-2 site could account for the decreased basal promoter activity observed in p278.

Next, we evaluated whether a potential binding site for hepatocyte nuclear factor- 1 (HNF-1) located at nt -62 to -50 in the rat IGFBP-1 promoter was important for basal promoter activity, as has been reported for the corresponding site in the human IGFBP-1 promoter [6-8]. The consensus HNF-1 site is a 13 bp palindrome that has been identified in many genes that are specifically or preferentially expressed in liver [9, 10]. It binds homodimers or heterodimers of a homeodomain protein, HNF-la, which is predominantly expressed in differentiated liver. The HNF-1 site in the rat IGFBP-1 promoter is identical to the consensus sequence at 11 of 13 positions, and to an HNF-1 site in the human IGFBP-1 promoter at 12 of 13 position [2]. When the left half-site of the HNF-1 site in the rat IGFBP-1 promoter was mutated in plasmid p327 to a sequence previously shown to interfere with the binding of recombinant HNF-1 binding domain [6], basal promoter activ- ity was reduced by 90%. This indicates that an intact HNF-1 site is required for optimal basal activity of the rat IGFBP-1 promoter.

We then asked whether mutation in the M4 region of the rat IGFBP-1 promoter affected basal promoter activity. This region is identical in the human IGFBP-1 promoter [2, 4], and constitutes half of a palindromic sequence that appears to be involved in both dexamethasone stimulation (see below) and insulin inhibition of the rat [2, 3, 11] and human [4, 12] IGFBP-1 promoters. It binds HNF-3fl [13, 14] and other transcription factors [13, 15]. Introduction of an M4 mutation in plasmid p327 decreased basal rat IGFBP-1 promoter activity ~80%. Unterman et al. [13] showed that mutation of nt -105 to -99 in the M4 region decreased the stimulation of basal promoter activity in NIH/3T3 fibroblasts following cotransfection with an HNF-3fl expression plasmid. However, in another publication from the same labo- ratory, Goswami et al. [3] reported that internal deletion of nt -113 to -92 in plasmid p320 increased basal activity in H4-II-E cells by 3-fold. The reason for these differences is unknown.

Dexamethasone Regulation of Rat IGFBP-1 Promoter Activity 135

Thus, as summarized in Fig. 3, we have identified three sites in the proximal rat IGFBP-1 promoter that are required for optimal basal promoter activity: an AP-2 site (nt -286 to -293), the M4 region of the IRE (nt -108 to -99), and an HNF-1 site (nt --62 to -50).

SITES PARTICIPATING IN DEXAMETHASONE STIMULATION

Using the same deletion and substitution mutations, we next determined whether the three sites that are important for basal promoter activity also are important in regulating dexamethasone stimulation of rat IGFBP-1 promoter activity. As pre- viously reported [2, 3], the promoter region could be deleted up to nt -92, one nucleotide upstream from the GRE, without reducing dexamethasone-stimulated promoter activity. In view of the fact that deletion of the region between nt -327 and -278 removed an AP-2 site that was essential for optimal basal promoter activ- ity, we specifically examined the effect of this deletion on dexamethasone stimula- tion. In contrast to its effect on basal promoter activity, deletion of nt -327 to -278 did not decrease dexamethasone-stimulated promoter activity.

The effect on dexamethasone-stimulated promoter activity of the same substitu- tion mutation in the HNF-1 site that decreased basal activity of the rat IGFBP-1 promoter was examined in different 5' deletion constructs. In plasmids p92, p135, and p235, the HNF-1 mutation decreased stimulation by dexamethasone to less than 10%o of the level seen in the corresponding deletion containing a wild-type HNF-1 region. In contrast, no decrease was observed when the HNF-1 mutation was introduced into plasmids p278 or p327. This suggested that the loss of a func- tional HNF-1 site could be compensated by a site located between nt -278 and -235, but not by an intact M4 region (which was present in the inactive p135 and p235 constructs that contained an HNF-1 mutation).

Similar results were observed when the M4 region of the IRE was altered by site- mutagenesis in the same deletion constructs. Introduction of the M4 mutation in plasmids p235 and p135 decreased dexamethasone stimulation of promoter activity to 25% of the level in the corresponding deletion containing a wild-type M4 region. This confirms the previously reported inhibition of dexamethasone stimulation in p135M4 [2]. The inactive p135M4 and p235M4 constructs contain an intact HNF- 1 site, indicating that an intact HNF-1 site could not compensate for a mutant M4

I , ; O O

I

-327 -278 -235 - 135 -92 H

FIGURE 3. Schematic diagram showing the three sites that are important for optimal basal activity of the rat IGFBP-1 promoter in H4-11-E cells. They are an HNF-1 site (nt --62 to nt -50), an AP-2 site (nt -286 to -293), and site M4 in the IRE (nt -108 to -99). Mutation in any one of these sites decreased unstimu- lated promoter activity by 70-90%.

136 D.-S. Suh et al.

region in these constructs. In contrast, but similar to the results observed with mutant HNF-1 sites, the M4 mutation did not decrease dexamethasone stimulation in p327 or p278 plasmids. Goswami et aL [3] reported similar results, namely, that an internal deletion of nt -107 to -102 in the M4 region of plasmid p320 did not significantly decrease dexamethasone stimulation. Thus, as with the HNF-1 site, the loss of a functional M4 region of the IRE could be compensated by the presence of a site between nt -278 and -235.

As summarized in Fig. 4, the preceding results indicate that three sites partici- pate, together with the GRE, in dexamethasone-stimulated promoter activity: the region between nt -278 and -235, the M4 region of the IRE, and the HNF-1 site. The results suggest that the presence of any two of these three sites is sufficient

-252/-236 M4 GRE HNF-1 + 1

F - m

I , I I i I -327 -278 -235 -135 -92 +79

X I

X I

m X (-) X

FIGURE 4. Schematic diagram summarizing the evidence that three accessory sites interact with the gluco- corticoid receptor/GRE to enable dexamethasone stimulation of rat IGFBP-1 promoter activity. These are HNF-1, M4, and a site between nt-252 and-236. The relative locations of these sites are indicated on the nt -327 to +79 promoter fragment. Wild-type (hatched ovals) and mutant ("X") sequences in the HNF-1 and M4 sites are indicated. The upstream site between nt -252 and -236 (hatched rectangles) is present in p278 (and longer) constructs, but is absent from p235 (and shorter) constructs. Constructs that gave dexa- methasone stimulation are indicated by an upward arrow; constructs in which dexamethasone stimulation was not observed are designated (-). As discussed in the text, two of the three accessory sites (HNF-1, M4, -252/-236) must be present and intact for dexamethasone stimulation to be observed.

Dexamethasone Regulation of Rat IGFBP-1 Promoter Activity

-278 I

-265 -252

137

-236 I

Dexamethasone Stimulation -236 in p278HNF1m

(+)

(-)

-258 -236 Footprint

-252 -236 Gel Shift

FIGURE 5. Schematic diagram summarizing the evidence that the region from nt -252 to -236 of the rat IGFBP-1 promoter is important for dexamethasone stimulation. Substitution mutations were introduced in plasmid p278 containing a mutation in the HNF-1 site (p278HNF-lm) in nt -265/-253 or nt -252/-236 (hatched bars). Dexamethasone stimulation was retained in the construct containing the nt -265/-253 muta- tion, but was lost in the construct containing the nt -252/-236 mutation. Proteins in H4-1I-E nuclear extract that bind to the nt -252/-236 region have been observed in DNase I footprinting assays, and in electro- phoretic mobility shift assays.

for dexamethasone stimulation. However, the alternative possibility remained that an intact nt -278/-235 site by itself was sufficient to allow dexamethasone stimula- tion in the presence of either an HNF-1 or an M4 mutation. This possibility was excluded by the demonstration that dexamethasone was unable to stimulate rat IGFBP-1 promoter activity in p278 constructs that contained mutations in both the M4 and HNF-1 sites.

ATTEMPT TO IDENTIFY THE SITE IN THE NT - 2 7 8 T O - 2 3 5 REGION THAT PARTICIPATES IN DEXAMETHASONE STIMULATION

Preliminary experiments have been performed to identify the site in the nt -278 to -235 region that can compensate for the loss of a functional HNF-1 or M4 site and allow stimulation of promoter activity by dexamethasone. DNase I footprint- ing assays were performed using H4-II-E cell nuclear extract and a radiolabeled DNA fragment corresponding to nt -327 to +79. The region between nt -258 and -235 was protected against nuclease degradation by proteins in the extract. This region contains a sequence (-258 CAAAAACA -251) that is similar to the so- called D-site of the albumin promoter, one of the sites that determines its liver- specific expression [16]. A transcription factor that binds to this site, the D-site binding protein or DBP, has been cloned from rat liver [16].

In electrophoretic mobility shift assays using a radiolabeled nt -278/-236 oligo- nucleotide fragment, four complexes were observed with H4-II-E nuclear extract. They were designated A, B, C and D in decreasing order of mobility. Formation of all four bands was inhibited by excess unlabeled -278/-236 oligonucleotide. None of the four bands was decreased by an oligonucleotide containing the high affinity binding site for HNF-3 [17], making it unlikely that HNF-3 was one of the proteins

138 D.-S. Suh et al.

in H4-II-E extract that bound to this DNA fragment. In contrast, band D, but not bands A, B and C, was inhibited by a 50-fold excess of unlabeled oligonucleotide containing the albumin D-site. This suggested that DBP might be present in H4-II- E extract and be responsible for forming band D in the gel shift assays. Consistent with this possibility, DBP expressed in bacteria (kindly provided by Christopher Muller) formed a complex with the nt -278 to -236 oligonucleotide probe.

We then asked whether mutation of the putative DBP binding site (nt -258 to -251) affected dexamethasone stimulation in plasmid p327, or plasmid p327 containing a mutation in the HNF-1 site (as the nt -278 to -235 region could restore dexamethasone stimulation to constructs that contained a mutant HNF-1 site). Dexamethasone stimulation was not inhibited in either of the constructs containing the DBP-site mutation. Thus, the putative DBP-site does not appear to be the element in the nt -278/-235 region that is involved in dexamethasone stimulation.

In contrast, substitution of nt -252/-236 in the p278 plasmid containing an HNF- 1 mutation virtually abolished the ability of dexamethasone to stimulate promoter activity. Dexamethasone stimulation was retained in a construct containing a substitution mutation in nt -265/-253, the region that contains the putative DBP- site. These results localize to nt -252 to -236 the site in the nt -278/-235 region that participates with the M4 region of the IRE, the HNF-1 site, and the GRE to give optimal dexamethasone stimulation Fig. 5.

Consistent with this result, the nt -252/-236 oligonucleotide, but not the nt -265/-253 oligonucleotide, competitively inhibited the formation of the A, B and C protein complexes with radiolabeled nt -278/-236 oligonucleotide probe in gel shift assays using H4-II-E nuclear extract. Further studies are required to identify the factors in H4 extracts that bind to this region.

CONCLUSIONS

Unstimulated and hormonally-stimulated regulation of rat IGFBP-1 promoter activity requires the participation of multiple c/s-elements and presumably multiple transcription factors. We have shown that an intact HNF-1 site and M4 region of the IRE (which also is involved in inhibition by insulin) are required for unstimulated promoter activity, and participate in dexamethasone-stimulated promoter activity. Whether the same factors binding to these sites participate in both processes remains to be determined. Basal promoter activity also requires an AP-2 site, which is not involve in dexamethasone stimulation. Conversely, a site localized to nt -252 to -235 participates in dexamethasone stimulation but not in basal promoter activity.

The enhancer activity of a GRE may be augmented by a second GRE or by sites that bind other transcription factors (such as the CAAT-box factor, Spl, octamer transcription factor, CACCC-box factor, or nuclear factor 1 [18]). The human IGFBP-1 promoter appears to use both mechanisms [12]. The rat IGFBP-1 promoter, containing a single GRE, appears to use a complex series of interactions of transcription factors binding to three distinct sites to achieve maximal stimula- tion by dexamethasone. The specific factors binding to these sites are not presently known. Some of them, such as HNF-1, are enriched in liver. HNF-3fl binds to the M4 region of the IRE in both the human and rat IGFBP-1 promoters, and muta- tions that reduce HNF-3fl binding decrease dexamethasone stimulation [13, 14].

Dexamethasone Regulation o f Rat IGFBP-1 Promoter Act iv i ty 139

Despite these associations, it remains to be demonstrated that HNF-3fl is the factor responsible for dexamethasone stimulation. Further delineation of the transcription factors that effect glucocorticoid receptor-mediated stimulation of rat IGFBP-1 transcription by dexamethasone, promises to provide important new insights into the mechanisms by which the integrated regulation of transcription by tissue- specific and hormonally-responsive transcription factors is achieved.

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17. Overdier DG, Porcella A, Costa RH. The DNA-binding specificity of the hepatocyte nuclear factor 3/forkhead domain is influenced by amino acid residues adjacent to the recognition helix. Mol Cell Biol. 1994; 14: 2755-2766.

18. Schiile R, Muller M, Kaltschmidt C, Renkawitz R. Many transcription factors interact syner- gistically with steroid receptors. Science. 1988; 242: 1418-1420.