promoter region

Journal of Hepatology 43 (2005) 126–131 www.elsevier.com/locate/jhep

The RL-ET-14 cell line mediates expression of glutamine synthetase through the upstream enhancer/promoter region Marianna Kruithof-de Julio1, Wilhelmina T. Labruye`re1, Jan M. Ruijter2, Jacqueline L.M. Vermeulen1, Vesna Stanulovic´1, Jan M.P. Stallen1, Alicja Baldysiak-Figiel3, Rolf Gebhardt3, Wouter H. Lamers1, Theodorus B.M. Hakvoort1,* 1 AMC Liver Center, Academic Medical Center, Amsterdam, The Netherlands Department of Anatomy & Embryology, Academic Medical Center, Amsterdam, The Netherlands 3 Institut fu¨r Biochemie, Universita¨t Leipzig, Leipzig, Germany

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Background/Aims: The expression of glutamine synthetase (GS) in the mammalian liver is confined to the hepatocytes surrounding the central vein and can be induced in cultures of periportal hepatocytes by co-cultivation with the ratliver epithelial cell line RL-ET-14. We exploited these observations to identify the regulatory regions of the GS gene and the responsible signal-transduction pathway that mediates this effect. Methods: Fetal hepatocytes of wild-type or GS-transgenic mice were co-cultured with RL-ET-14 cells to induce GS expression. Small-interfering RNA was employed to silence b-catenin expression in the fetal hepatocytes prior to co-culture. Results: Co-cultivation of RL-ET-14 cells with fetal mouse hepatocytes induced GS expression 4.2-fold. The expression of another pericentral enzyme, ornithine aminotransferase and a periportal enzyme, carbamoylphosphate synthetase, were not affected. Co-culture of RL-ET-14 cells with transgenic fetal mouse hepatocytes demonstrated that GS expression was induced via its upstream enhancer located at K2.5 kb and that the signal mediator required a functional b-catenin pathway. Conclusions: The ‘RL-ET-14’ factor specifically induces GS expression, working via its upstream enhancer in a b-catenin-dependent fashion. q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Glutamine synthetase; Upstream enhancer; b-Catenin; Ornithine aminotransferase; Carbamoylphosphate synthetase; Fetal hepatocytes

1. Introduction Glutamine synthetase (GS; EC 6.3.1.2) catalyses the ATP-dependent conversion of ammonia and glutamate to Received 25 August 2004; received in revised form 6 January 2005; accepted 26 January 2005; available online 11 April 2005 * Corresponding author. Address: AMC Liver Center, Academic Medical Center, Meibergdreef 71, 1105 BK Amsterdam, The Netherlands. Tel.: C 31 20 5665412; fax: C31 20 5669190. E-mail address: [email protected] (T.B.M. Hakvoort). Abbreviations: GS, glutamine synthetase; OAT, ornithine aminotransferase; CPS, carbamoylphosphate synthetase; GSK-3b, glycogen synthase kinase-3b.

glutamine. In the liver, the expression of GS is confined to a rim of hepatocytes surrounding the central vein [1]. In rodents, this expression pattern develops in the late fetal period and is maintained after birth [2,3]. GS gene expression is detected from ED12 onward with a transient peak at ED13-15 and a second sharp rise to adult levels at birth [4]. The pattern of expression of GS protein is identical to that of its mRNA [5–7] and is complementary to the periportal expression pattern of another ammoniadetoxifying enzyme, carbamoylphosphate synthetase (CPS). Mechanistically, GS expression seems to be controlled by intrahepatic factors [8,9] and may depend on the direction of the sinusoidal blood flow [10].

0168-8278/$30.00 q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2005.01.036

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In earlier studies with transgenic mice, we showed that the developmental appearance and pericentral expression of GS in the liver is determined at the transcriptional level by elements present in the upstream region of the GS gene [4]. In vitro experiments have since shown that interactions between the upstream enhancer at K2.5 kb and several intronic regulatory elements determine the degree of activation of the GS promoter [11–14]. GS transcriptional activity in the liver is upregulated by the b-catenin pathway [15,16]. The epithelial cell line RL-ET-14, which was established from a 10-day-old Sprague–Dawley rat liver, induces, upon co-cultivation, GS expression in GS-negative periportal hepatocytes [17]. We found that RL-ET-14 cells also induce GS in fetal mouse hepatocytes upon co-culture. We further determined the GS regulatory region that mediates the effect of RL-ET-14 cells by co-cultivating these cells with fetal hepatocytes from two different transgenic mouse lines, containing different parts of the upstream promoter/enhancer region of the GS gene. Our results show that a 244 bp region in the upstream enhancer is sufficient for upregulating the expression of the transgene and that a functional b-catenin pathway is required.

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2.3. Culture of embryonic mouse hepatocytes The RL-ET-14 co-cultures were established as described [17] with the addition of 0.1 mM dexamethasone. Fetal hepatocytes were suspended in culture medium and seeded at two livers per well for ED11 and ED12 preparations and one liver per well thereafter. The non-adhering hematopoietic cells were washed away with pre-warmed medium after 2 h of culture. The remaining cells were harvested after 48 h. To inhibit GSK-3b activity, LiCl (20 mM) was added during the first 24 h of culture, while an equivalent amount of NaCl was added to control cultures [20].

2.4. Transfection of fetal hepatocytes Hepatocytes were seeded in 24-well plates at one liver per two wells. After 2 h, the cultures were washed with pre-warmed medium and transfected with 0.1 mg pRL-CMV (Promega, Leiden, The Netherlands) and the luciferase reporter construct containing GS-UE2 (Fig. 1) or the same construct lacking the 244 bp upstream enhancer element (GScontrol), using Lipofectin (Invitrogen). After 4 h, the medium was changed to normal culture medium. The cells were harvested after 48 h and activity was measured using dual-luciferase reporter assay system (Promega) in an Autolumat Plus (Berthold, Vilvoorde, Belgium).

2.5. Total RNA extraction and first strand cDNA synthesis After detaching the cells with 0.2% trypsin, total RNA was extracted with the RNeasy mini RNA-isolation kit (Qiagen, Leusden, The Netherlands). Contaminating genomic DNA was eliminated with RQ1 RNase-free DNaseI digestion (Promega). First strand cDNA was transcribed from total RNA as in [21].

2. Material and methods 2.6. Real-time polymerase-chain reaction 2.1. Animal care FVB transgenic mice were maintained on a 12 h light/12 h dark cycle with free access to water and food. Pregnant females were sacrificed 11–17 days after the detection of a vaginal plug (ED11–ED17). Fetal hepatocytes were isolated as described [18]. The study was performed in accordance with the Dutch guidelines for the use of experimental animals.

PCR amplification and analysis were carried out using the LightCyclere with software version 3.0 (Roche, Almere, The Netherlands). Reaction mix preparation and amplification were done as described [21]. Primers, annealing temperature and Mg concentrations are reported in Table 1. Calculations were performed with our real-time PCR data analysis program [22]. When reverse transcriptase was omitted, a product never formed before cycle 30.

2.2. Transgenic animals

2.7. Calculation of mRNA content

The GS-UR transgenic line (Fig. 1) contains the 5 0 -flanking region of the rat GS gene from position K3150 to C59 with respect to the transcription-start site [19]. The GS-UE2 transgenic line contains the 244 bp AflIII-AflIII fragment of the upstream enhancer of the GS gene (located at K2500 bp relative to the transcription-start site) fused to the promoter at position K965, the first and the second exon up to the translation-start site, and a minimized first intron [11].

The fold induction was calculated as the ratio of the specific mRNA concentration in co-cultures over plain embryonic liver-cell cultures, both normalized for their 18S RNA content. This ratio underestimates the fold induction of gene expression, since the co-cultures contain both RL-ET-14 and liver cells. Because the total RNA content of RL-ET-14 cells was approximately 8-fold lower than that of embryonic liver cells, this effect was limited and not adjusted for.

Fig. 1. Schematic representation of the constructs present in the respective transgenic mice. The GS-UR mouse contains the entire 3.1 kb GS-upstream region (PstI-Ksp632I fragment) coupled to the chloramphenicol-acetyltransferase gene (CAT) and the SV40 small-t intron and polyadenylation sequence. The GS-UE2 mouse contains the 244 bp AflIII-AflIII fragment of the upstream enhancer region and the BglII-NcoI fragment containing the promoter, the 1st exon, minimized 1st intron and 2nd exon up to the translation-start site, which is coupled to the firefly-luciferase gene (luc) and the bovine growth-hormone polyadenylation sequence. The arrow indicates the translation-start site in exon II. Abbreviations: A, AflIII; K, Ksp632I; N, NcoI; P, PstI. The representation is not drawn to scale.

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Table 1 Experimental conditions and nucleotide sequence of primers used for quantitative real-time RT-PCR Gene

Primer sequence (5 0 /3 0 )

Temperature (8C)

MgCl2 (mM)

Glutamine synthetase

F: G G C T T C C G G T T A T A C T T G R: T G G C C A C C T C A G C A A G T T F: C C G A A T T G C C A T T G C G R: A G C C C G T T A T C T C G A A G F: C T A C A T C A G A C T G G C T C A A R: T T G G T G A G G A G A G C A A T F: G A G G C A T T T C A G T C A G T T G C R: T A G A A A C T G C C G G A A A T C G T C F: C A G T C A A G T A A C A A C C G C G A R: C T C T G A T T T T T C T T G C G T C G A F: T T C G G A A C T G A G G C C A T G A T R: C G A A C C T C C G A C T T T C G T T C T F: A C A C A T G A A C A T C T C C T T C C A A G G T R: G A G A C T G C A G A T C T T G G A C T G G A C A

55

3

58

4

58

4

60

4

58

3

58

3

65

1.5

Ornithine aminotransferase Carbamoylphosphate synthetase Chloramphenicol-acetyltransferase Luciferase 18S ribosomal RNA b-Catenin

2.8. Transfection with RNA oligonucleotides The double-stranded b-catenin small-interfering (si)RNA oligonucleotide (HPP grade) (sense strand: 5 0 -GUAGCUGAUAUUGACGGGCdTdT3 0 ) was purchased from Qiagen. The oligonucleotide was resuspended in 100 mM potassium acetate, 30 mM HEPES-KOH (pH 7.4) and 2 mM magnesium acetate. To disrupt aggregates, the oligonucleotide was heated for 1 min at 90 8C and incubated at 37 8C for 1 h prior to use. A scrambled siRNA oligonucleotide (sense strand: 5 0 -UGGACUGAUACUGUCGGCGdTdT-3 0 ) was used as a control. siRNA oligonucleotides were validated both at the protein level by immunohistochemistry and Western blot and at the DNA level by PCR (see Table 1 for primer sequence, MgCl2 and annealing temperature). Newly isolated hepatocyte cultures were transfected with b-catenin siRNA oligonucleotide (final concentration 50 nM), using Oligofectamine (Invitrogen, Breda, The Netherlands). After 4 h the medium was changed to normal culture medium and RL-ET-14 cells were added at a density of 1! 104 cells per well in a 6-well plate. The medium was refreshed after 24 h. Total RNA was isolated and reverse transcribed as described except that the first strand was synthesized by b-catenin-specific priming.

3. Results 3.1. RL-ET-14-mediated induction is specific for GS Upon co-cultivation with RL-ET-14 cells, fetal mouse hepatocytes started to accumulate GS. A typical co-culture of ED15 hepatocytes (Fig. 2B) shows the presence of GS

2.9. Immunohistochemistry After rinsing with Hank’s salt solution, the cultures were fixed with an ice-cold mixture of methanol/acetone/water (2/2/1), followed by immunohistochemical staining as described [18]. Monoclonal GS antibody (1:1000) was from Transduction Laboratories, Lexington, KY and monoclonal b-catenin antibody (1:500) from Santa Cruz Biotechnology, Inc., California, USA. For immunofluorescence microscopy, cells were grown on collagen-coated glass-cover slips in the presence of 20 mM LiCl or NaCl for 0, 1, 2, 4, 8, 24, and 48 h. After washing with PBS and fixing in 3.5% formaldehyde at 4 8C for 30 min, the cells were permeabilized with PBS containing 0.1% Tween-20 (PBST), blocked in blocking solution (PBS, 0.1% Tween-20, 10% fetal bovine serum) for 30 min, and incubated for 2 h at room temperature with mouse monoclonal b-catenin antibody (1:500 in blocking solution; BD Transduction Laboratories). After washing with PBST, cells were incubated with Cy2-conjugated anti-mouse IgG (1:200 in blocking solution; Dianova, Hamburg, Germany) for 1 h at room temperature. Finally, cells were washed in PBST and in 500 nM propidium iodide (Molecular Probes).

2.10. Statistical analysis Differences in the GS induction were tested with a one-way analysis of variance (ANOVA; SPSS version 11.5) per transgene between time points and per time point between transgenes. To avoid false positives, which could arise from the large number of comparisons, we applied a Bonferroni correction and used aZ0.01 as the significance level.

Fig. 2. Effect of co-culturing RL-ET-14 cells with primary mouse hepatocytes. Panels A and B: primary fetal mouse hepatocytes (ED15) do not accumulate the glutamine-synthetase protein when cultured alone (A), but show a positive staining for GS, when co-cultured with RL-ET-14 cells (B). Panels C and D: stimulation of endogenous GS, CPS, OAT (C) and reporter-gene expression (D) upon co-cultivation of fetal hepatocytes with RL-ET-14 cells. Primary hepatocytes from GSUR and GS-UE2 transgenic mice (cf. Fig. 1) were (co-) cultured in the presence of 0.1 mM dexamethasone and in the presence or absence of the RL-ET-14 cell line. Data were normalized for 18S rRNA and show mean (GSEM) fold induction for 42 (endogenous genes) and 4–15 (reporter genes) independent experiments. Induction was calculated as the ratio of mRNA content in co-cultured over non-co-cultured hepatocytes.

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after 48 h of culture. As observed earlier [17], many islands of GS-expressing hepatocytes surrounded by RLET-14 cells were found. GS staining was absent in the nuclei, as expected for a cytosolic enzyme. Fig. 2C shows that the GS mRNA content of embryonic hepatocytes increased 4.2-fold when co-cultured with RL-ET-14 cells and that the effect was specific for GS, as the mRNAs for CPS and OAT were not induced. These effects did not differ between fetal hepatocytes of ED11 to ED17 (ANOVA PZ0.975). For this reason, time points were pooled for the analysis of treatment effects. The RL-ET-14 cells contained less than 0.01% of the GS mRNA that primary hepatocytes in co-culture can accumulate, and expressed no CPS or OAT mRNA as determined from real-time PCR (data not shown). 3.2. RL-ET-14-mediated induction of transgenic constructs Fig. 2D shows a significant 2.8-fold induction in expression of the reporter gene of the GS-UR transgene upon co-cultivation with RL-ET-14 cells. To delineate the sequence in the upstream region that mediated the ‘RL-ET14’ effect, RL-ET-14 cells were co-cultured with transgenic GS-UE2 fetal hepatocytes. In transfected primary hepatocytes cultures, the sequence present in this transgene (Fig. 1) mediated a 3-fold increase in reporter-gene activity compared to the promoter alone (Fig. 3) and a narrow pericentral expression upon the luciferase reporter gene in adult liver in vivo (not shown). Upon co-culture of RL-ET14 cells with GS-UE2 fetal hepatocytes, the transgene was induced 2.5-fold, which is not significantly different from the effect of the entire upstream region (ANOVA PO0.5). 3.3. b-Catenin is required for GS induction In cultures of primary ED15 hepatocytes that were treated with 20 mM LiCl, GS mRNA levels were 2.1-fold higher than in NaCl-treated controls (nZ2). The expression of the reporter genes in the GS-UR and GS-UE2 lines increased 6.2- and 4.8-fold, respectively (nZ1 each;

Fig. 3. Luciferase activity in transfected fetal hepatocytes. Luciferase activity in fetal hepatocytes transfected with plasmid GS-UE2 (nZ4), containing the GS upstream enhancer element and the corresponding control plasmid, only containing the 965 bp promoter (GS-control). Reporter gene activity is shown as activityGSEM.

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Fig. 4A). To establish whether this inducing effect of LiCl and RL-ET-14 was mediated by b-catenin, we transfected fetal hepatocytes with double-stranded siRNA oligonucleotide directed against the b-catenin sequence prior to co-cultivation. The siRNA oligonucleotides (50 nM) effectively reduced b-catenin expression, as demonstrated at the mRNA (Fig. 4E) and protein level (compare Fig. 4B1 and B2, and Fig. 4D lanes 1 and 2; nZ2). Transfection reagent only or scrambled siRNA (50 nM) were without effect (Fig. 4D, compare lanes 1 and 3). The transfection of hepatocytes with b-catenin siRNA also blocked the induction of GS and the GS-UE2 reporter gene in hepatocytes co-cultivated with RL-ET-14 cells effectively (Fig. 4C). The mobilization of b-catenin to the nucleus is shown in Fig. 4F. In the presence of LiCl, an increase of cellular b-catenin and a shift towards a cytoplasmic/nuclear localization was detected as early as 8 h (data not shown), and was most clear at 24 h (Fig. 4F) and 48 h.

4. Discussion The aim of this study was to identify the region of the GS gene that is activated by the ‘factor’ secreted by RL-ET-14 cells. Fetal hepatocytes are a good vehicle because they are easy to culture and respond to RL-ET-14 cells with a strong GS induction, irrespective of the age of the embryo from which they were taken. 4.1. The ‘RL-ET-14’ factor targets the GS upstream enhancer/promoter region The GS upstream region is necessary for normal expression of the gene in the liver in vivo [15,19]. The 244 bp AflIII-AflIII fragment of the upstream enhancer (present in GS-UE2) behaves, when fused to the promoter at position K367, as a bona-fide enhancer element in transient transfection experiments [11,13,14], but the promoter has to be extended to position K965 (GS-UE2) to obtain pericentral reporter-gene expression in the liver in vivo (our unpublished observations). In fetal mouse hepatocytes co-cultured with RL-ET-14 cells, the 244 bp AflIII-AflIII fragment conferred a similar increase in reporter gene expression upon transient transfection (Fig. 3) and when stably integrated as a transgene in the genome (Fig. 2B). The AflIII-AflIII fragment, therefore, suffices for the response of the GS gene to the factor secreted by RL-ET-14 cells. However, the RL-ET-14mediated induction of the expression of the GS-UR and GS-UE2 transgenes amounted to only 60–70% of that measured in the same extracts for the endogenous GS gene (Fig. 2C and D). Some enhancing sequences that are quantitatively important for GS induction by RL-ET-14 cells may, therefore, be absent from our transgenic constructs. In this respect, the positive interaction of

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Fig. 4. Inhibition of GSK-3b stimulates GS gene expression in fetal hepatocytes. Panel A: fold induction by LiCl treatment of primary hepatocyte cultures only. Primary transgenic hepatocytes from GS-UR and GS-UE2 were cultured in the presence of 0.1 mM dexamethasone and in the presence or absence of 20 mM LiCl. Panel B: ED15 primary hepatocytes only cultured in the absence (B1) or presence (B2) of the b-catenin siRNA oligonucleotide, and stained for b-catenin. Panel C: induction of GS (black bars) and GS-UE2 reporter-gene (grey bars) mRNA (GSEM) in primary hepatocytes co-cultured with the RL-ET-14 cell line in the absence (control) or presence of transfection reagent and 0 or 50 nM b-catenin-specific or scrambled siRNA oligonucleotide. Panels D and E: cellular concentration of b-catenin is reduced at the mRNA level as demonstrated by PCR products loaded on a 1.5% MS-8 agarose gel (panel E; nZ2) and at the protein level as shown by the Western blot (panel D; nZ2). Lanes shown are hepatocyte cultures treated with the transfection reagent (1), with 50 nM b-catenin siRNA (2) or a scrambled siRNA oligonucleotide (3). Panel F shows ED15 hepatocytes cultured in the presence of NaCl (F2) or LiCl (F1), and stained for b-catenin. Cultures were fixed after 24 h and processed for visualization of b-catenin (green) and propidium-iodide (red). Fetal hepatocytes show a pronounced cytoplasmic (green) and nuclear (yellow) accumulation of b-catenin, while cells surrounding the hepatocyte islands only weakly stain for b-catenin. (For interpretation of the reference to color in this legend, the reader is referred to the web version of this article.)

the upstream enhancer with the 5 0 -end of intron 1 of the GS gene may prove to be important [11,23,24]. 4.2. The ‘RL-ET-14’ factor induces GS expression via b-catenin Cell–cell interactions are not necessary for RL-ET-14 cells to induce GS expression in periportal hepatocytes [17], suggesting that a secreted factor is involved [8]. Okadaic acid at a concentration that selectively inhibits protein phosphatase A2 completely inhibits induction of GS in adult pig hepatocytes upon co-culture with RL-ET14 cells [8]. This finding suggests that phosphorylation of a signal-transduction component can prevent the upregulation of GS expression by RL-ET-14 cells. Recently, we showed that an adenovirus expressing b-catenin lacking the N-terminal phosphorylation site causes induction of GS expression in all infected hepatocytes [15]. The link

between b-catenin activation and GS expression was further strengthened when it was shown that tumors carrying b-catenin mutations were GS-positive, whereas no GS expression was seen in those that did not [15]. b-Catenin is phosphorylated by GSK-3b. When we tested the effect of the non-competitive GSK-3b inhibitor LiCl [20] on hepatocyte cultures, GS expression was indeed enhanced (Fig. 4A). Furthermore, a small-interfering RNA oligonucleotide directed against b-catenin effectively suppressed GS and GS-UE2 expression (Fig. 4C). These findings show that b-catenin is essential for GS activation by RL-ET-14 cells and that its effect is largely mediated via the AflIII-AflIII enhancer fragment. We found no differences between embryonic and fetal hepatocytes in GS inducibility, even though embryonic hepatocytes are reported to contain much higher cellular levels of b-catenin than fetal hepatocytes [25]. We interpret these data to mean that it is the activation of b-catenin rather

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than its total cellular concentration that regulates GS expression and that cellular b-catenin levels in fetal hepatocytes still suffice to induce GS expression. It remains, of course, possible that the co-culture conditions modify hepatic b-catenin expression. Our data show the ‘RL-ET-14 factor’ that induces GS expression, acts via activation of b-catenin. Previously, we showed that adenoviral expression of constitutively active b-catenin induces expression of the pericentral proteins GS, OAT, and the glutamate transporter GLT-1 in all hepatocytes, suggesting that b-catenin could be a key factor in determining the pericentral phenotype of hepatocytes. Our present data show that this model must be too simple, as the ‘RL-ET-14 factor’ activates b-catenin, but does not induce the pericentrally expressed OAT enzyme. RL-ET-14 must therefore also secrete a factor that inhibits the b-catenindependent expression of OAT. The involvement of b-catenin in RL-ET-14-mediated GS expression in hepatocytes makes the family of Wnt proteins likely candidates for the ‘RL-ET-14 factor’. In agreement, we have observed the presence of several Wnt mRNA species in RL-ET-14, Wnt 5A, 7A, and 7B being the most prominent (unpublished results).

Acknowledgements Part of this study was supported by grant Ge 465/8-1 to RG and NWO 902-23-196 to WHL.

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