Expression of retinoic acid receptor and retinoid X receptor subtypes in rat liver cells: Implications for retinoid signalling in parenchymal, endothelial, Kupffer and stellate cells

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European Journal of Cell Biology 77, 111-116 (1998, October) . © Gustav Fischer Verlag· Jena

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Expression of retinoic acid receptor and retinoid X receptor subtypes in rat liver cells: Implications for retinoid signalling in parenchymal, endothelial, Kupffer and stellate cells Stine M. Diven a, Vasanti Natarajanh, Kirsten B. Holvena, Torunn Ll2lvdaIe, Trond Berge, Rune Blomhoff1)a a b C

Institute for Nutrition Research, School of Medicine, University of Oslo, Oslo/Norway Institute of Medical Biochemistry, School of Medicine, University of Oslo, Oslo/Norway Institute for Molecular and Cell Biology, University of Oslo, Oslo/Norway

Received February 12, 1998 Received in revised version June 15, 1998 Accepted July 9, 1998

Retinoic acid receptors - retinoid X receptors - liver cells mRNA expression - reverse transcriptase polymerase chain reaction In the present study, a systematic examination of the relative expression pattern of the nuclear retinoid receptors (RAR and RXR) in various liver ceUs was performed. Our data demonstrate that RXRa is the dominant receptor in aU liver ceUs, and that RARIl is also expressed at a high level in most cells. More specifically, RARIl and RARa were the most predominant of the RAR subtypes in parenchymal cells, while all three RAR subtypes were equally expressed in endothelial and Kupffer cells. The total level of expression of all the RXR subtypes were larger than the total level of expression of all the RAR subtypes in all liver cells. This is in agreement with the observation that RXR is a heterodimer partner not only for RAR, but also for other members in the steroid/thyroid receptor superfamily of ligand. dependent transcription factors.

Abbreviations: PBS Phosphate-buffered saline. - RAR Retinoic acid receptor. - RT-PCR Reverse tronscriptase polymerase chain reaction. - RXR Retinoid X receptor.

J) Prof. Dr. Rune Blomhoff, Institute for Nutrition Research, School of Medicine, University of Oslo, P. O. Box 1046, Blindern, N-0316 OslolNorway, e-mail: [email protected], Fax: +4722851396.

Introduction Vitamin A is involved in the regulation of proliferation and differentiation of many cell types during fetal development and throughout life [22]. The main players that orchestrate the metabolism and function of vitamin A have been elucidated. This includes the mechanisms of absorption and transport of vitamin A in chylomicrons and by retinol-binding protein, the cellular uptake of these plasma carriers, the important role of liver stellate cells in storage of vitamin A, and the central role of cellular retinoid-binding proteins in regulating the intracellular metabolism [8]. The discovery of several nuclear receptors for retinol metabolites has provided an understanding of one of the underlying mechanisms of actions of vitamin A. The three retinoic acid receptors (RARa, ~ and y) [6, 12, 17, 24, 33] and the three retinoid X receptors (RXRa, ~ and y) [28] belong to the steroid/thyroid receptor superfamily of ligand-dependent transcription factors [29]. The physiological ligands for aU the RARs appear to be all-trans retinoic acid [2], 9-cis retinoic acid [2], 3,4 didehydro-retinoic acid [2] as well as 4-oxo-retinoic acid and 4-oxo-retinol [1, 11]. The RXRs are more selective and are only activated by 9-cis retinoic acid [19]. It has been shown that RARa and RXR~ mRNA are widely distributed in most tissues and cells, while the expression of RAR~, RARy, RXRa and RXRy mRNA is more specific [21, 26,28,44,45]. The nuclear retinoid receptors bind to retinoic acid response elements as RXR-RAR heterodimers [25,31] or as RXR-RXR homodimers [27,46]. The RXR subtypes also bind to various other response elements as heterodimer partners for a number of other nuclear receptors such as the vitamin D receptor and the thyroid hormone receptor [30]. The liver consists of several cell types including the parenchymal cells (hepatocytes), endothelial cells, Kupffer cells (macrophages) and stellate cells. Vitamin A has been shown to

112 S. M. Ulven, V. Notorojon, K. B. Halven et 01. regulate various functions of these cells including regulation of proliferation [14], expression of growth factors and hormones [20,35,37], extracellular matrix components [32,36], retinoid binding proteins [34] as well as some of the nuclear retinoid receptors [23,40]. These effects of vitamin A appear to be mediated via the RAR and the RXR subtypes. The parenchymal cells (hepatocytes) account for 85-90 % of the total liver mass [43]. The nonparenchymal cells (i.e. endothelial cells, Kupffer cells and stellate cells) are considerably smaller than the parenchymal cells and account for about 6 % of the total liver mass. The endothelial cells form the continuous lining of the liver sinusoids and represent a barrier between blood and the parenchymal cells [43]. The liver endothelial cells represent about 72 % of the total nonparenchymal cells [4]. These cells have an important function in the clearance and degradation of various plasma components by receptor-mediated endocytosis. The Kupffer cells are the largest group of tissue macrophages in most mammals. A large part of the cell surface is exposed to the blood flow through the liver and to the space of Disse. Their main function is to phagocytose foreign particles, such as bacteria or colloids [43]. They comprise about 17 % of the number of nonparenchymal cells [4]. The perisinusoidal stellate cells (also called fatstoring cells, lipocytes or Ito cells) are localised within the space of Disse. The hepatic stellate cells store a major portion (50-80 %) of the body's vitamin A content [10]. However, the stellate cells represent about 8 % of the cells in liver [4]. We have measured the expression of the RAR subtypes and the RXR subtypes in isolated liver cells to characterise the physiological roles of each receptor subtype. This was performed by semi-quantitative RT-PCR expression analysis using total RNA isolated from pure suspensions of liver cells. Our data demonstrate that there is a differential expression pattern of the RAR and RXR subtypes in each cell type.

Materials and methods Isolation of liver cells

The study protocol was in accordance with the official governmental guidelines on the care and use of laboratory animals. Male Wistar rats (250 g) were fed an ordinary pelleted diet (AREX, Ml1lllesentralen, Oslo, Norway). Total liver cell suspensions were prepared by a collagenase liver perfusion technique [9]. Collagenase (type IV, 3.4.24.3) was obtained from Sigma (Poole, Dorset/United Kingdom). Parenchymal cells were isolated from the total liver cell suspension by differential centrifugation [9] and centrifugal elutriation. Beckman JE-5 elutriator rotor (Beckman Instruments, Palo Alto, CAlUSA) equipped with a standard chamber was used in a J-6MIE type Beckman centrifuge. Parenchymal cells were introduced into the elutriation system in a 0.2M phosphate buffer, pH 7.2, containing 0.15 M NaCi (PBS) and 1 % bovine serum albumin at a flow rate of 25 ml/min. The flow rate was increased stepwise to 40 ml/min at 1400 rpm. About 95 % of the isolated parenchymal cells were viable as determined by the trypan blue exclusion test. The parenchymal cells were contaminated with less than 4 % Kupffer cells. Nonparenchymal cells were prepared from total liver cell suspensions by differential centrifugation [9]. The endothelial cells and Kupffer cells were further purified by centrifugal e1utriation. These cells were introduced into the elutriation system in PBS buffer containing 1 % bovine serum albumin at a flow rate of 22 ml/min at 2500 rpm. The endothelial cells were washed out of the elutriation chamber at this flow rate. Kupffer cells were separated by increasing the flow stepwise to 55 ml/min. Kupffer cells were identified cytochemically by positive peroxidase reaction and endothelial cells by negative peroxidase reaction [42]. The endothelial cells were contaminated

with less than 2 % Kupffer cells, and Kupffer cells were contaminated with less than 17 % endothelial cells. Pure fractions of stellate cells were isolated from total liver cell suspensions by differential centrifugation followed by density gradient centrifugation using Percoll [9]. Percoll was purchased from Pharmacia Fine Chemicals AB (Sweden). Percoll gradients were prepared by mixing 25 ml Percoll solution (9 parts (v/v) of Percoll to 1 part (v/v) of 9 % NaCI with 15 ml nonparenchymal cell suspension, and centrifuged at 15000 rpm for 90 min at 4°C. Stellate cells were identified in a Leitz fluorescence microscope equipped with an A filter block (no. 513596) by positive autofluorescence when excited by 326 nm ultraviolet light [7]. The yield of endothelial, Kupffer and stellate cells were 109 x 106 cells, 15 x 106 cells and 2 x 106 cells.

Isolation of total RNA

Total RNA was extracted from liver and liver cells by the acid guanidinium thiocyanate/phenol/chloroform method [13]. Liver tissue was removed from the rat and frozen immediately in liquid nitrogen and stored at -80°C until use.

Reverse trancriptase-polymerase chain readion (RT-PCR)

Total RNA was reverse transcribed using PCR buffer (20 mM TrisHCI, 50mM KCI), 5mM MgCI2, ImM of each dNTP, 2.5U reverse trancriptase (M-MLV), 1 U RNase inhibitor, 2.5!tM oligo d(T)16 primer at 42°C for 15 minutes using a Perkin Elmer 9600 Thermal cycler. All solutions and enzymes used for reverse-transcription were obtained from the GeneAmp RNA PCR kit (Perkin-Elmer, Roche Molecular Systems, Inc., Branchburg, NJIUSA). PCR was carried out using a mixture including 60 pmol of primers specific for RARa, ~, y and RXRa, ~,y and ~-actin (Tab. I), PCR buffer, optimal MgCh concentration for each primer pair (1-2.5 mM) and 2.5 U Ampli Taq DNA polymerase was used. The reaction mixture (loo!tl) was heated to 95°C for 2 minutes in order to denature the cDNA. Ampli Taq DNA polymerase was then added during a hot-start at 70°C. The reaction consisted of 30 cycles of denaturation at 95 °Cfor 30 s, annealing at 55°C for 30 s, extension at 72 °C for 45 s and an additional extension at 72 °C for 4 min after the last cycle. After PCR, onefifth of the amplified products were analysed by electrophoresis on an 1.5 % agarose gel (SeaKem LE, FMC Bioproducts, Rockville, MD/ USA) in 0.5 x Tris-Borate EDTA buffer (TBE), stained in 0.5 !tglml ethidium bromide for 30min, and visualised using a F1uorlmager screen (Molecular Dynamics, Sunnyvale, CAlUSA). The amount of DNA was then quantified using the software ImageQuaNT™ (Molecular Dynamics) and corrected for background. Water was used to replace cDNA and served as a negative control in each run of PCR. The expression levels of RAR and RXR subtypes are normalised to the expression of the housekeeping gene ~-actin. Data represent the means of four independent experiments ± S.D. The sizes of amplified RARa, ~ and y and RXRa, ~ and y cDNA fragments were 195 bp, 156 bp, 156 bp, 165 bp, 175 bp, and 191 bp, respectively. The detected size of amplified product was 165 bp for ~-actin.

Primer design Common for all members of the steroid/thyroid receptor superfamily of ligand-dependent transcription factors is the high homology in the DNA-binding domain. The retinoic acid receptors also have a high degree of similarity in the ligand-binding domain. In order to make specific primers, these regions were avoided at least for one of the primers in a primer pair. The primers also span two exons to avoid amplification of genomic DNA. Since the different sets of primers were to be used in the same PCR profile, the primers were designed such that the GC content and the length of products was comparable (Tab. I). The specificity of each primer pair was confirmed by crossreaction experiments, i.e. when the primer pair specific for RARa was used in a reaction where the template was RAR~ or RARy, no PCR product was detectable under the experimental condition (data not shown). The identity of the PCR products were also confirmed by sequencing (MediGenomix, GmbH, PlanegglMartinsried, Germany).

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Expression of RAR and RXR subtypes in rat liver cells

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Results and discussion The RT-PCR conditions Several aspects of the experimental setup of the RT-PCR reaction were standardised in order to semi-quantitate the levels of RARa, RARB, RARy and RXRa, RXRB, RXRy mRNA in different liver cells. Since the same PCR profile was to be used (i.e. time and temperature protocol) for all the receptors, primers with a similar percentage of GC content were chosen and the sizes of the amplified fragments were also similar (156--195 bp) (Tab. I). In addition, the MgClz concentration was optimised for each primer pair. To test the linearity of the RT-PCR reaction, 0-2 flg total RNA isolated from total liver were used. Figure 1 shows the results from the experiments with the RARa primers. Similar results were also observed with the other primers (data not shown). To obtain semi-quantitative conditions in the following experiments, 1 flg total RNA was used for all receptors except RXRa where 0.3 flg total RNA was used. To analyse the expression pattern in stellate cells, one flg total RNA was used for all receptors. Thus, only qualitative data is presented from the stellate cells. J..Lg total R

RT-PCR analysis of RAR and RXR subtypes in

liver cells

The expression patterns of RAR and RXR subtypes in liver cells are shown in Figure 2. Of the RAR subtypes, RARB was the most predominant receptor in parenchymal cells and stellate cells. This was also observed in total liver (data not shown). These cells did also express RARa and RARy although at a lower level. In endothelial and Kupffer cells, all RAR subtypes were equally expressed. Among the RXR subtypes (and all of the receptors), RXRa showed the highest expression in all cells. RXRB receptors were also expressed at a significant level in all cells. Only small amounts of RXRy were expressed in parenchymal, Kupffer and endothelial cells. In stellate cells, no RXRy was detected. Tab. I.

Fig. 1. Titration of retinoic acid receptor RARa mRNAin total rat liver. Total RNA was extracted from rat liver as described in Materials and methods and total RNA (0, 0.25, 0.5, 1.0, and 2.0 ~g, lanes 1-5 respectively) was reverse transcribed using 2.5 ~M oligo d(T)16 primer. The synthesised cDNA was amplified by using sequence-specific primers (Tab. I) in the PCR. The reaction included 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72 °c for 45 s. The expected size for RARa PCR product was 195 bp. The gels were analysed by using FluorImager, and quantitated using the software ImageQuant and corrected for background. The amount of DNA loaded on the gel was onefifth of the amplified products. Size marker is a 123 bp DNA ladder from Gibco BRL. The values from ImageQuant were calculated in excel microsoft and R 2 = 0.99 was the regression value for RARa mRNA.

The oligonucleotides used for reverse transcriptase-polymerase chain reaction (RT-PCR).

Gene/species* Primer sequence

Position in sequence (nt) 1domain

GC content (%)

Expected product size (bpj

Ref.

RARalh Sense Antisense

5'-CCCAGCCACCATTGAGAC-3' 5'-TACACCATGTTCTTCTGGATGC-3'

81-98/A 275-254/C

53

195

[33]

5'-TCGAGACACAGAGTACCAGC-3' 5'-GAAAAAGCCCTTGCACCCCT-3'

161-180/B 316-297/C

55

156

[18J

5'-GCCTCCTCGGGTCTACAAG-3' 5'-ATGATACAGTTTTTGTCGCGG-3'

246-264/B 401-381/C

54

156

[24J

5'-ATGAAGCGGGAAGCTGTG-3' 5'-CATGTTTGCCTCCACGTATG-3'

613-630/C+0 777-758/0

53

165

[25]

5'-TCAACTCCACAGTGTCGCTC-3' 5'-TAAACCCCATAGTGCTTGCC-3'

23Q-249/A+B 404-385/C

53

175

[25]

54

191

[25J

50

165

[3]

RAR~/m

Sense Antisense RARy/h Sense Antisense RXRalm Sense Antisense RXRWm Sense Antisense RXRy/m Sense Antisense ~-actin/m

Sense Antisense

5'-CTTCTTCAAAAGGACCATCAGG-3' 483-504/C 5'-CTGCCTCACTCTCTGCTCG-3' 673-655/0 5'-TGTTACCAACTGGGACGACA-3' 5'-GGGGTGTTGAAGGTCTCAAA-3'

147-166 311-292

* m: mouse, h: human. The rat sequence that corresponds to the primer sequence used is not available or published for the receptors with the exception of RARu. The homology between the human and rat sequence are 95 and 91 %, respectively, for the two RARa primers.

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Fig. 2. Fig. 2 A~ shows the semi-quantitative mRNA levels of the RAR and RXR subtypes in parenchymal cells, endothelial cells and Kupffer cells by using RT-PCR. Total RNA (1 f.lg for all primers, except for RXRa, where 0.3 f.lg total RNA was used) extracted from pure fractions of liver cells was reverse transcribed using 2.5 f.lM oligo d(T)16 primer. Primers and conditions used for the RT-PCR assay are given in Table I and Materials and methods. The synthesised cDNA was used for PCR as described in the legend for Figure 1. Each experiment was performed four times and the error bars show the range of values. The gels were analysed by using a F1uorImager, and quantitated using the software ImageQuant and corrected for background. The values obtained were expressed as relative expression compared to ~-actin mRNA. Data represent mean ± SD (n = 4). Insert is a typical gel from

one experiment. The expected sizes of PCR products are RARa 195bp (lanel), RARy 156bp (lane2), RXRa 165bp (lane3), RXR~ 175 bp (lane 4), RXRy 191 bp (lane 5), ~-actin 165 (lane 6), RARj3 156 bp (lane 7) and ~-actin 165 bp (lane 8). Fig. 2D shows the qualitative level of the RAR and RXR subtypes in Percoll-isolated hepatic stellate cells (n = 4). Total RNA (1 f.lg) extracted from stellate cells was reverse transcribed using 2.5 f.lM oligo d(T)16 primer. The expected sizes of PCR products are RARa 195bp (lane1) , RAR~ 156bp (lane2) RARy 156bp (lane3), RXRa 165bp (lane4), RXR~ 175bp (lane 5), RXRy 191 bp (lane 6), ~-actin 165 (lane 7). The amount of DNA loaded on the gel was one-fifth of the amplified products. Size marker is a 123 bp DNA ladder from Gibco-BRL.

In parenchymal cells, the relative abundance of the nuclear retinoic acid receptors are in the order of RXRa > RAR~ > RXR~ > RARa = RARy > RXRy. This expression pattern is almost similar to that observed for total liver (data not shown). This is not surprising, as parenchymal cells comprise more than 90 % of the liver mass. In a previous report, Weiner et a1. [40] observed no detectable mRNA for RARa, RAR~ and RARy in rat liver parenchymal cells using the less sensitive Northern blot analysis. By using RT-PCR and Northern blot analysis, however, Wan and co-workers detected RAR~ in one out of three hepatoma cell lines [39]. RARa and RARy were observed in all cell lines. The relative expression of all RAR subtypes and RXR subtypes was 1.8 and 3.7, respectively. This observation is consistent with the assumption that the RXR subtypes not only form heterodimers with RAR subtypes, but also with other nuclear receptors in addition to RXR homodirners [30]. The relative abundance of the receptors is the same for both Kupffer and endothelial cells: RXRa > RARa = RAR~ = RARy = RXR~ > RXRy which is different from that of total liver and parenchymal cells. The relative expression of all RAR subtypes and RXR subtypes was 2.1 and 3.6 for the endothelial cells, and 2.4 and 5.9 for the Kupffer cells. There are no previous reports on nuclear receptor expression in pure

cultures of liver endothelial cells. In a mixed Kupffer/endothelial cell preparation, Weiner et al. [40] studied the expression of RAR subtypes by Northern blotting. They observed a similar expression of RARa and RARy. However, they did not detect RAR~ mRNA in pure cultures of Kupffer cells. Stellate cells play a major role in vitamin A storage and metabolism [7, 10], and the distribution of the retinoic acid receptors are of particular interest since some of the proteins involved in these processes are regulated by the nuclear receptors in these cells [15,38]. Stellate cells treated with retinoids show suppressed proliferation, and reduced collagen and TGF-f3 expression. All these findings support a link between the cellular status of vitamin A metabolism and activation of stellate cells. The exact function the retinoid receptors have in these phenomena remains to be elucidated. Our analysis demonstrates that all three RAR subtypes are expressed in stellate cells, in agreement with a previous report from Ohata et a1. [32] where they used RT-PCR to detect the different RAR and RXR subtypes. Also in agreement with Ohata et aI., we demonstrate that RXRa and RXRf3 are expressed, but no or not detectable RXRy is expressed in this cell type. Friedman et a1. [16] detected by Northern blot analysis RARa and RAR~ mRNA in stellate cells. However, no expression of RARy was detected. This disagreement might be because of different sen-

EJCB sitivity between the different methods used. Weiner et al. [40] have reported that all RAR subtypes are expressed in stellate cells, but stellate cells cultured for 7 days or more contained very little vitamin A and contained no detectable RAR~ mRNA. Weiner and co-workers also reported that stellate cells isolated from rats with CCl 4-induced hepatic fibrosis had a drastic decrease in their RAR~ mRNA levels compared to normal stellate cells [40]. These results are extended by Ohata et al. [32] who showed that stellate cells isolated from rats with cholestatic liver fibrosis had a decreased level of vitamin A and a decreased level of both RARj3 and RXRa mRNA. It is interesting that the expression of both components of the RARj3-RXRa heterodimer seem to be regulated by the vitamin A level in stellate cells. In summary, a systematic examination of the relative expression pattern of RAR and RXR subtypes in various liver cells has been performed. Our data demonstrate that RXRa is the receptor with highest expression in all liver cells, and that RARj3 is also expressed at a high level in most cells. Interestingly, the expression of both of these receptors has been observed to be sensitive to the vitamin A status. Kato et al. [23] reported that vitamin A deficiency caused the levels of RARj3 transcripts in liver to decrease drastically, while no significant changes in the levels of RARa and RARy mRNA were observed. In stellate cells it has been observed that both RARj3 and RXRa can be regulated during cholestatic liver fibrosis where the vitamin A content is reduced [32,40]. In vitro, Leid et al. [25], have demonstrated that all three RXR subtypes stimulate binding of all three RAR subtypes to a retinoic acid-responsive element (~RARE) without any preferential binding of different sets of RAR-RXR heterodimers. The contribution of RAR-RXR heterodimers to activation of transcription from a particular responsive promotor within a given cell may depend on the actual concentration of RAR, RXR, the ligands all-trans-retinoic acid and 9-cisretinoic acid. The thyroid hormone receptor (TR) , the peroxisomal proliferator-activated receptor alpha (PPARa) and LXRa are all members of the steroidlthyroidlretinoic acid receptor family which bind to RXR and are expressed in liver [5,41]. The total level of expression of all RAR subtypes is lower compared to all the RXR subtypes. If protein levels reflect mRNA expression, these data will be in agreement with the observation that RXR subtypes in liver not only form heterodimers with RAR subtypes, but also with other members of the nuclear receptor superfamily.

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