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Molecular Endocrinology 18 (1): 63-69
Copyright © 2004 by The Endocrine Society

A Silencer Element in the First Intron of the Glutamine Synthetase Gene Represses Induction by Glucocorticoids

Frank Gaunitz, Kerstin Heise and Rolf Gebhardt

Institut für Biochemie, Medizinische Fakultät, Universität Leipzig, 04103 Leipzig, Germany

Address all correspondence and requests for reprints to: Frank Gaunitz, Institut für Biochemie, Medizinische Fakultät, Universität Leipzig, Liebigstrasse 16, 04103 Leipzig, Germany. E-mail: frank.gaunitz{at}medizin.uni-leipzig.de.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The enzyme glutamine synthetase (GS) ranks as one of the most remarkable glucocorticoid-inducible mammalian genes. In many tissues and cell lines, the synthetic glucocorticoid dexamethasone alone increases GS expression several fold. The direct response is mainly mediated by a cellular glucocorticoid receptor that, upon binding of the hormone, interacts with glucocorticoid responsive elements (GREs) of the gene. In cells of hepatocellular origin the response is mediated by a GRE located in the first intron of the gene. Surprisingly, hepatocytes do not respond to glucocorticoids with enhanced GS expression, despite the presence of an intact glucocorticoid receptor, which, in the same cells, stimulates expression of other genes such as tyrosine amino transferase. Reporter gene assays identified a sequence element downstream from the intronic GRE that inhibits the enhancement of expression by glucocorticoids. This silencer was designated GS silencer element of the rat. Gel mobility shift assays demonstrate the binding of a factor in hepatocyte nuclear extract. This yet unknown factor was designated GS silencer-binding protein. It is absent in FAO cells that respond to glucocorticoids with enhanced expression of GS and present in HepG2 cells that do not respond.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ENZYME GLUTAMINE synthetase (GS) is well known to respond to the presence of glucocorticoids with enhanced activity in many tissues (1). Thus, GS is inducible by glucocorticoids in fully differentiated 3T3-L1 adipocytes (2, 3), in glia cells from the retina of the chicken (4), and in muscle (5, 6), but only to a small extent in liver (7). In fact, hepatocytes respond to glucocorticoids only with a small permissive effect in the presence of GH (8). Of course, this lack of inducibility cannot be attributed to the missing of a functional glucocorticoid receptor, because liver cells are well known to have a glucocorticoid receptor, and many genes in liver are under the control of glucocorticoids. Among them are genes encoding enzymes such as tyrosine amino transferase or enzymes from the urea cycle (9, 10, 11). This raises the question of how GS is prevented from being induced by glucocorticoids, although its gene carries appropriate glucocorticoid response elements (GREs) that function in other tissues. Recently, it was demonstrated that a GRE within the first intron of the GS gene can be activated in hepatocytes after their transformation into hepatoma cells, such as the immortal rat hepatoma FAO cell line (12). From these data, the question arose whether the acquisition of GS inducibility in the course of transformation may be caused by the loss of a factor that silences GS activity in hepatocytes. In the work presented, an intensive investigation was initiated to identify mechanisms responsible for the prevention of glucocorticoid stimulation of GS activity in hepatocytes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoid Control of Tyrosine Amino Transferase (TAT) Activity and Glutamine Synthetase Activity in Primary Cultured Hepatocytes and FAO Cells
In the first experiment it was asked whether hepatocytes isolated from rat liver by collagenase perfusion respond to the synthetic glucocorticoid dexamethasone (DEX) with regard to GS activity. Hepatocytes were isolated from the liver and 24 h after isolation, cells were treated with DEX at a concentration of 10-6 M or with vehicle alone. Another 24 h later the activities of TAT and GS were measured. As can be seen in Fig. 1Go there is in fact a strong response of TAT to DEX (Fig. 1BGo) and almost no effect with regard to the endogenous GS activity (Fig. 1AGo). For comparison, the response of FAO cells to the hormone with regard to GS activity (Fig. 1CGo) and TAT activity (Fig. 1DGo) are shown. Because GS activity is already high in total hepatocyte preparations that only contain approximately 7% of GS-positive hepatocytes derived from the region around the central veins (13), we wondered whether the remaining 93% of GS-negative hepatocytes might respond to the presence of the hormone what may be undetectable in total hepatocyte preparations. Therefore, cells from a preparation that only contains GS-negative hepatocytes were cultured in the absence and presence of DEX. For this experiment GS activities from six independently treated wells were used for each condition. GS activity in the absence of DEX was measured to be 19.8 ± 0.7 mU/mg. In the presence of DEX it was determined to be 20.3 ± 0.7 mU/mg. Obviously, there is no induction of GS by glucocorticoids in GS-negative hepatocytes. However, the observed induction of TAT (Fig. 1BGo) indicates that a functional glucocorticoid receptor must be present in the cells, since the induction of TAT by glucocorticoids is well known to be dependent on a functional glucocorticoid receptor.



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  		Fig. 1. GS Activity and TAT Activity in Primary Cultured Hepatocytes and FAO Cells Under the Influence of DEX

A number of 0.5 x 106 hepatocytes (A and B) and of 0.25 x 106 FAO cells (C and D) were cultivated per well of a six-well plate. Cells were cultivated for 24 h after seeding the cells, and before they received either 10-6 M dexamethasone or vehicle alone. Incubation in the presence of hormone was done for 24 h, and then GS (A and C) and TAT activity (B and D) were determined. Each bar represents the mean and SD from six independently treated wells.

 
Reporter Gene Assays with Sequences from the First Intron of the GS Gene Identify a Silencer Element Suppressing the Function of the GS GRE
It was previously shown that FAO cells strongly respond to the presence of the glucocorticoid DEX with an enhanced expression of GS (12). This response is mainly mediated by a GRE, located within the first intron of the gene. Therefore, it was asked whether this element is also functional in primary cultured hepatocytes. In the experiment presented in Fig. 2Go different reporter genes with different sequences from the first intron of the GS gene, cloned into the vector pT81, were transfected into primary hepatocytes. As expected, the GRE was in fact able to confer sensitivity to DEX to the corresponding reporter genes as observed with FAO cells (12). Interestingly, two of the three reporter genes containing the response element exhibited an increase of luciferase expression of almost 6- to 7-fold. However, the longest construct that included DNA 3'-downstream from the GRE, namely the region from position 1676 to position 1938, shows only a 2-fold induction. This induction is higher than the induction of the endogenous GS gene, which is only 1.2-fold (Fig. 1Go), but one should keep in mind that two different enzymatic activities are compared in Figs. 1Go and 2Go. Although this does not rule out the possibility that other silencers are active in regulating GS activity, the data from Fig. 2Go indicate that a silencer element must be located within the first intron that is able to repress induction of GS in primary cultured hepatocytes. To determine the position of the element within this region in greater detail, additional reporter genes were constructed. These reporter genes contained progressive deletions from position 1938 down to position 1690. As can be seen in Fig. 3Go, the removal of the DNA sequences located between position 1818 and position 1764 makes the reporter genes responsive to the stimulation by glucocorticoids, thus strongly indicating that the silencing activity is mediated by an element located within this region.



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  		Fig. 2. Enhancement of Reporter Gene Activity from Different Constructs with Intronic DNA from the GS Gene, Controlling a Luciferase Gene Under the Influence of DEX, in Primary Hepatocytes

The left part of the figure indicates the intronic DNA sequences used for the construction of the reporter genes. A part of the intron with restriction enzyme recognition sites used for the construction is shown. Numbers indicate base pair position with regard to the first base of the intron. The known glucocorticoid response element at position 1537 is indicated by a small black bar. The right panel shows the enhancement of luciferase expression in the presence of DEX. Each bar is the result of six independent transfection experiments in the presence and absence of glucocorticoid.

 


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  		Fig. 3. Enhancement of Luciferase Activity Obtained from Reporter Genes with Different DNA Sequences from the First Intron Under the Influence of DEX

For details, see legend to Fig. 2Go.

 
DNA-Protein Interactions within the Silencer Region Detected by Gel Retardation Assays
The next question asked was whether a protein that is able to specifically bind DNA from the silencer region can be detected in nuclear extracts from hepatocytes. Therefore, gel retardation assays were performed with a labeled DNA probe, encompassing the DNA from position 1677 to position 1818. To probe the specificity of binding and to further delimit the silencer, different competitors were used. In Fig. 4AGo, the result of an experiment is shown, in which two different competitors and a self-competition were used. The retardation assay shown in Fig. 4AGo demonstrates the binding of a factor in the hepatocyte nuclear extract to DNA (+ in Fig. 4AGo). In addition, the competitors used demonstrate that binding must occur above position 1764 and below position 1818, which is in agreement with the data from the reporter gene assays shown in Fig. 3Go. Experiments with nuclear protein extracts from the line HepG2, which does not respond to glucocorticoids with enhanced GS activity (29.32 ± 0.99 mU/mg in the absence of DEX and 29.76 ± 0.51 mU/mg in its presence), also detected a retardation signal that is even stronger than the one observed with extracts from primary hepatocytes (Fig. 4BGo). Using protein extracts from the line FAO, no binding activity was observed (Fig. 4BGo). This correlation between the presence of a DNA binding protein in HepG2 cells and hepatocytes and its absence in FAO cells, leads to the attractive hypothesis that this protein prevents induction of GS by DEX in primary hepatocytes and HepG2 cells.



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  		Fig. 4. Retardation Assay with Sequence 1677/1818 from the First Intron

A, A gel retardation assay was performed with a labeled DNA sequence from the first intron, encompassing the DNA from position 1677–1818 (position with regard to the first base of the intron). As competitors, DNA from position 1764–1818 and 1677–1764 were used, as well as the sequence from 1677–1818 for a self-competition. Numbers on top indicate the molar excess used for competition. B, Gel retardation assay with the sequence 1677/1818 and nuclear extracts from different cells. In each lane 3 µg of nuclear protein were used from HepG2 cells (HG2), FAO cells (F), and primary hepatocytes (Hep). As unspecific competitor, 4 µg of poly(dI-dC)·poly(dI-dC) were used in each binding reaction.

 
Reporter Gene Assays with the Silencer Element in Different Cells
It was then asked whether the presence of the intronic silencer is able to suppress the induction of reporter genes with the GRE of the GS gene in correlation with the absence or presence of the intron binding protein. Therefore, transfection experiments were performed using reporter genes with the GS-GRE with (1818 in Fig. 5Go) or without the silencer (1764 in Fig. 5Go). The reporter genes were transfected into FAO cells that do not show a binding activity (Fig. 4BGo) and into hepatocytes and HepG2 cells that were shown to have a binding factor (Fig. 4BGo). In addition, the reporter gene pT81 was transfected, which was used for the construction of these reporter genes. As can be seen in Fig. 5Go (top panel), a strong induction of the reporter genes is observed in FAO cells from both reporter genes. On the other hand, hepatocytes (middle panel) and HepG2 cells (lower panel) respond significantly to the presence of the silencer. Although the glucocorticoid induction of expression from the shorter reporter gene is smaller in hepatocytes and HepG2 cells, both cells show a loss (hepatocytes) or a reduced specific inducibility (HepG2) in the presence of the silencer. In both cases, the response in the presence of the silencer is close to the unspecific effect shown by the control plasmid pT81. Whether the lower inducibility of the reporter gene 1764 in HepG2 cells and hepatocytes is dependent on the presence of other silencer elements must be addressed by further experiments. However, the result presented is in agreement with the hypothesis that cells expressing the silencer binding factor are prevented from induction by DEX if the silencer element between position 1764 and position 1818 is present.



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  		Fig. 5. Enhancement of Luciferase Activity Obtained from Reporter Genes under the Influence of DEX in Different Cells

FAO cells, hepatocytes, and HepG2 cells were transfected with reporter genes containing sequences from the first intron in the absence and presence of DEX, and the fold enhancement of expression was determined. For further details, see legend to Fig. 2Go.

 
Transfection of the Silencer Element into GS-Negative Hepatocytes
With regard to the phenomenon of restriction of GS expression to hepatocytes surrounding the central veins of the liver (14), it is tempting to speculate whether the silencer may be involved in the phenomenon of zonation. Therefore, it was asked whether it is possible to titrate the negative factor in GS-negative primary hepatocytes, inducing them to become GS positive. To answer this question, transfection experiments were performed with the silencer element using a PCR product obtained by using the primers I1_1818_down and I1_1677_up. One microgram of the PCR product was transfected into 0.5 x 106 cells from a subpopulation of hepatocytes that is completely negative for GS. In this experiment, the GS-negative cells from the periportal zone of the liver acinus did not respond with an enhanced GS activity to the presence of the high amounts of the binding sequence (-DEX, untransfected: 19.8 ± 0.7 mU/mg; -DEX, transfected: 19.7 ± 1.3 mU/mg; +DEX, untransfected: 20.3 ± 0.7 mU/mg; +DEX, transfected: 20.7 ± 0.9 mU/mg). The observation that the overrepresentation of the silencer does not make GS-negative hepatocytes responsive to the hormone does not, of course, definitely imply that the factor does not silence the gene. In fact, many experiments done in recent years demonstrate that the GS-negative phenotype of hepatocytes is controlled at several levels of regulation (14, 15, 16). However, it is an attractive hypothesis that the presence of the silencer binding factor, designated GSS-BP (GSSEr-binding protein), may contribute to zonation by preventing induction of GS by glucocorticoids under physiological conditions.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the adult liver of rat and other mammalian species, the expression of GS [E.C. 6.3.1.2] is restricted to a small population (~7%) of hepatocytes, forming a continuous layer of one to three cells around the central veins (13, 17, 18). In contrast to other enzymes, distributed heterogeneously in the liver, the expression pattern of GS has proven stable even under extreme experimental conditions (19, 20, 21, 22, 23). Two experimental examples demonstrating an induction of GS in originally GS-negative hepatocytes are the cocultivation of GS-negative hepatocytes with a liver epithelial cell line (24) and the induction after transplantation into interscapular fat bodies (25). Interestingly, GS expression is frequently observed in tumors of hepatocellular origin (26) and has been discussed as a possible marker for tracing cell lineage relationships during hepatocarcinogenesis (14, 27). At this point, it should be noted that there is now compelling evidence that GS-producing tumors may have a growth advantage (28), and hepatocellular carcinomas expressing GS appear to have a higher metastatic potential (29). Whether inducibility of GS by DEX in hepatoma cells may contribute to their growth advantage (30) remains to be demonstrated.

Although the inducibility of GS by glucocorticoids in hepatoma cells was already described 30 yr ago (31), up to now nothing was known about the mechanisms or regulatory elements involved. With the experiments presented, it is now obvious that a silencer, downstream from the GRE in the first intron of the GS gene, mediates a negative effect on the induction by glucocorticoids in primary hepatocytes. This silencer was termed GSSEr (GS silencer element of the rat) in accordance with a silencer element found in the GS gene of the chicken (4), although it is highly speculative whether both elements share a similar binding factor. As demonstrated by gel retardation assays, the GSSEr binds a nuclear protein of unknown nature that is detectable in primary hepatocytes and cells from the line HepG2, but is absent in FAO cells. This factor was termed GSS-BP. Of course, the absence of GSS-BP binding to the GSSEr in FAO cells, as judged by the retardation experiments, must not necessarily imply that it was lost in the course of dedifferentiation. Another possibility may be that the induction of another protein factor that interacts with GSS-BP may prevent it from binding to its recognition sequence, but, so far, this cannot be judged. Although the glucocorticoid receptor is known to cross-talk to other factors such as activator protein 1 (32, 33), the silencing activity of GSS-BP does not appear to be the result of a direct interaction with the glucocorticoid receptor itself, because other glucocorticoid-responsive genes, as shown for TAT, are still inducible by DEX in primary hepatocytes. It is tempting to speculate that loss of GSS-BP function may well be involved in the up-regulation of other genes that must be discovered in further experiments.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents and Kits
If not stated otherwise, all reagents were purchased from Sigma-Aldrich Corp. (Taufkirchen, Germany). DNA-modifying enzymes were obtained from Promega Corp. (Heidelberg, Germany) and from Roche Diagnostics (Mannheim, Germany). Kits for DNA isolation and purification were purchased from QIAGEN (Hilden, Germany).

Cells and Cell Lines
For the preparation of hepatocytes, male Sprague Dawley rats weighting 220–280 g were used. Total hepatocytes were isolated by in vitro perfusion technique with collagenase (34). Subpopulations of hepatocytes from the periportal zone of the liver containing only GS-negative hepatocytes were obtained using the method of Gebhardt et al. (34). All hepatocytes were plated at a density of 0.5 x 106 cells per 35-mm dish of a six-well plate [Macroplate TC (TPP), Trasadingen, Switzerland], precoated with collagen as described previously (35) and cultured in William’s medium E (BioWhittaker, Verviers, Belgium), supplemented with 10% fetal calf serum (PAA Laboratories, Cölbe, Germany). The cells were incubated in 1.0 ml medium per well, at 37 C and 90% humidity, in an atmosphere containing 5% CO2 and 95% air. Two hours after seeding, medium was removed and fresh medium was added. Cells from the lines HepG2 and FAO were removed from confluent culture flasks by use of accutase (PAA Laboratories) and plated at the densities indicated. Cells from the line HepG2 were cultured in William’s medium E, and cells from the line FAO received a medium consisting of 50% Ham’s F12 and 50% NCTC109, both supplemented with 5% fetal calf serum, pretreated with active coal to remove trace amounts of glucocorticoids. All other conditions were like that used for primary hepatocytes.

Vectors, Reporter Genes, and DNA Probes for Gel Retardation Assays
Reporter genes were constructed using standard procedures (36). The reporter genes with the heterologous thymidine kinase promoter of Herpes simplex were constructed by use of the plasmid pT81 (37). Some of the reporter gene constructs were previously described (12, 16). The reporter gene (735/1938)_pT81 was constructed by cloning an EcoRI/BamHI fragment (after blunting) from the first intron into the SmaI site of the vector pT81. The reporter genes with deletions in the intron region between the EcoRI position (1938) and the SmaI position (1676) were constructed by use of PCR products. These products were generated by use of the reporter gene (735/1938)_pT81 as template and a forward primer designated I1_735_up (5'-CGGGGATCCTCCCCCAACG-3'). The corresponding backward primers for the individual constructs were designated I1_1875_down (5'-AAGATTTCCTGAGATCTGAC-3'), I1_1818_down (5'-CTACACCAGATCTCAATGGC-3'), I1_1765_down (5'-TCCAGAGACAGATCTCAATG-3') and I1_1690_down (5'-CCACCAGATCTTATTTTCTG-3'). Each backward primer introduced a BglII site into the PCR products that was used for the detection of the desired orientation of the sequences after ligation into the BamHI site of the vector pT81. The sequence 1677/1818 that was used in retardation assays and for titrating the GSSEr was obtained by a PCR, using the primers I1_1677_up: 5'-GGGTCTTTATTAGAGGTGGG-3' and I1_1818_down.

All constructs were verified by restriction analysis and nucleotide sequencing.

Transfection and Enzyme Assays
Cells were transiently transfected using Effectene (QIAGEN), according to the manufacturer’s suggestions. Firefly luciferase activity of reporter genes was determined as described previously (38). Light emission was measured with a Microlumat Luminescence Reader (Berthold Technologies, Bad Wildbad, Germany). Enzyme activities were determined at least in duplicate and corrected for background activity in nontransfected cells. GS activity (50 mM Tris buffer, pH 7.4) was measured by the glutamyltransferase assay (39). The activity of TAT was determined as described previously (8). Protein was determined by the method of Bradford.

Gel Retardation Assays
Nuclear protein isolation was performed as described previously (40). For the preparation of probes, DNA restriction fragments were dephosphorylated and gel purified. PCR products were used directly after gel purification. The labeling reactions were performed according to standard protocols (36) using T4 polynucleotide kinase (Roche Clinical Laboratories) and [{gamma}-32P]ATP (5000 Ci/mmol, Amersham Pharmacia Biotech, Freiburg, Germany). The labeled fragments were purified by adsorption to silica gel particles (QIAEX desalting protocol, QIAGEN) using a modified adsorption buffer (5 M NaCl, 40 mM sodium acetate, pH 5.0). Labeled fragments (2000–5000 cpm/fmol) were stored in 10 mM Tris (pH 7.5) at -20 C. For mobility shift assays, nuclear protein was preincubated for 10 min at 4 C in a final volume of 20 µl binding buffer [5 mM MgCl2, 60 mM KCl, 1 mM dithiothreitol, 10% glycerol, 250 µg/ml BSA, 15 mM HEPES (pH 7.4) including poly(dI-dC)·poly(dI-dC), additional competitor DNA and further components, as indicated in the figure legends]. After adding the probe (20,000 cpm), incubation proceeded at 16 C for 35 min. Electrophoresis was performed in a 4% polyacrylamide gel (40:1) in a low ionic strength buffer (6.8 mM Tris/HCl, 3.3 mM sodium acetate, pH 7.9) at 10 C and 10 V/cm.


   FOOTNOTES
 
This work was supported by Grant GA 428/1-1 from the Deutsche Forschungsgemeinschaft.

Abbreviations: DEX, Dexamethasone; GRE, glucocorticoid response element; GS, glutamine synthetase; GSS-BP, GSSEr, GS silencer element of the rat; TAT, tyrosine amino transferase.

Received for publication February 26, 2003. Accepted for publication October 10, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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