Molecular Vision 2004; 10:537-543 <http://www.molvis.org/molvis/v10/a66/> Received 16 April 2004 | Accepted 27 July 2004 | Published 9 August 2004

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Retinal selectivity of gene expression in rat retinal versus brain capillary endothelial cell lines by differential display analysis

Masatoshi Tomi,^1,2 Hayato Abukawa,¹ Yoko Nagai,³ Toshio Hata,³ Hitomi Takanaga,^2,3,4 Sumio Ohtsuki,^2,3,4 Tetsuya Terasaki,^2,3,4 Ken-ichi Hosoya^1,2
 
 

¹Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama, Japan; ²Core Research for Evolutional Science and Technology (CREST) and Solution Oriented Research for Science and Technology (SORST) of the Japan Science and Technology Agency, Kawaguchi, Japan; ³Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical Sciences and the ⁴New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan

Correspondence to: Ken-ichi Hosoya, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630, Sugitani, Toyama, 930-0194, Japan; Phone: +81-76-434-7505; FAX: +81-76-434-5172; email: hosoyak@ms.toyama-mpu.ac.jp

Abstract

Purpose: The retina is a neural tissue especially differentiated for vision and, thus, the inner blood-retinal barrier (inner BRB) specific molecules may play an essential role in maintaining neural functions in the retina. The purpose of the present study was to identify selectively expressed genes at the inner blood-retinal barrier compared with the blood-brain barrier (BBB).

Methods: A comparison of expressed genes between conditionally immortalized rat retinal (TR-iBRB) cell lines and brain capillary endothelial (TR-BBB) cell lines was performed using mRNA differential display analysis and quantitative real time PCR analysis. The rat M-cadherin gene was cloned by performing 5' RACE, and its protein expression was detected by immunoblot analysis.

Results: Eight clones were identified as highly expressed genes in TR-iBRB cells including GATA-binding protein-3 (GATA-3), cytosolic branched chain amino transferase (BCATc), and M-cadherin (cadherin-15). The rat M-cadherin gene was cloned from TR-iBRB cells, for the first time, and has >86% amino acid sequence identity to the previously cloned mammalian M-cadherins. Rat M-cadherin expression in TR-iBRB cells was much greater than that in TR-BBB cells as far as mRNA and protein levels were concerned.

Conclusions: M-cadherin, GATA-3, and BCATc are highly expressed in TR-iBRB cells compared with TR-BBB cells and may indeed be involved in unique functions at the inner BRB.

Introduction

In neural tissues like the retina and brain, the retinal and brain capillary endothelial cells form the inner blood-retinal (inner BRB) and blood-brain barriers (BBB), respectively. These barriers strictly regulate molecular transport between the circulating blood and neural tissues to maintain neural activities. It is believed that the molecules expressed at both barriers are almost identical and both barriers possess rigid tight-junctions to prevent the free diffusion of substances from the blood to neural tissues [1]. Several common transporters, such as D-glucose transporter 1 (GLUT1), creatine transporter (CRT), and P-glycoprotein, at both barriers are involved in the influx and efflux transport of essential substances and xenobiotics [2-7]. Nevertheless, the retina is especially differentiated for vision. This prompts the hypothesis that the inner BRB expresses different molecules from the BBB. We previously reported that the system x_c^- mediated L-cystine transport process is present at the inner BRB to protect the retina from light-induced oxidative stress, but it may not be at the BBB under normal conditions [8]. It is important to obtain more information about the inner BRB specific molecules, since they are intimately involved in retinal function.

We recently established conditionally immortalized rat retinal and brain capillary endothelial cell lines (TR-iBRB and TR-BBB cells, respectively) from transgenic rats harboring a temperature sensitive simian virus (SV) 40 large T-antigen gene [9,10]. TR-iBRB and TR-BBB cells possess endothelial markers and express GLUT1 and P-glycoprotein [9,10] which are expressed at the inner BRB and the BBB in vivo as shown by immunohistochemical analysis [2,3,6,7]. Thus, TR-iBRB and TR-BBB cells maintain certain in vivo functions and are a suitable in vitro model for the inner BRB and BBB, respectively [11].

The purpose of the present study was to identify selectively expressed genes at the inner BRB by mRNA differential display analysis of TR-iBRB and TR-BBB cells. The mRNA differential display technique works by systematic reverse transcription, polymerase chain reaction (PCR) amplification of the 3' termini of mRNAs, and resolution of those fragments on a polyacrylamide gel. This allows direct side-by-side comparison of the expressed genes under different conditions by using multiple primer combinations.

Methods

Cell culture

TR-iBRB2, TR-iBRB9, TR-BBB11 and TR-BBB13 cells were established and characterized as described previously [9,10]. Cells were seeded onto rat tail collagen type I-coat tissue culture dishes (Becton Dickinson, Bedford, MA). The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Moregate, Bulimbra, Australia) and 15 μg/L endothelial cell growth factor (Roche Diagnostics, Mannheim, Germany) at 33 °C in a humidified atmosphere of 5% CO₂/air. The permissive-temperature for TR-iBRB and TR-BBB cell culture is 33 °C due to the presence of the temperature sensitive SV 40 large T-antigen in both cells.

mRNA differential display analysis

Differential display was performed using the rhodamine version of a fluorescence differential display kit (Catalog number 6626; Takara, Shiga, Japan). Briefly, total RNA was prepared from TR-iBRB2, TR-iBRB9, TR-BBB11 and TR-BBB13 cells using Trizol reagent (Invitrogen, Carlsbad, CA). DNase I was added to RNA in the presence of RNase inhibitor and incubated at 37 °C for 20 min to get rid of any potentially contaminating genomic DNA that would interefere with subsequent differential display. RNA (300 ng) was reverse transcribed with a rhodamine labeled arbitrary anchored oligo dT primer (5'-rhodamine labeled-T_nVV-3', where n=13-15, and V represents any base except T). The resulting cDNA was PCR amplified using 48 combinations of the rhodamine labeled arbitrary anchored oligo dT primer and an arbitrary decamer (5'-NNNNNNNNNN-3') through 35 cycles at 94 °C for 30 s, 40 °C for 2 min, and 72 °C for 1 min. PCR products were mixed with formamide loading buffer and incubated for 3 min at 94 °C prior to loading on to 6% polyacrylamide gels. Gels were run at 30 W constant current, and DNA bands were visualized using a fluorescent image analyzer (FLA3000; Fujifilm, Tokyo, Japan). DNA bands differentially displayed between TR-iBRB and TR-BBB cells were excised from the gel and boiled in water for 30 min. The eluted DNA was reamplified using the same primer set and PCR conditions. Reamplified PCR products were run on 3% agarose gel containing H. A.-Yellow^TM (Catalog number HA002, Takara) and visualized using the fluorescent image analyzer. H. A.-Yellow selectively binds to AT base pairs, and thus loaded DNA fragments are separated according to their AT contents. Differentially displayed DNA bands were excised and the DNA was eluted using spin columns (Ultrafree-DA; Millipore, Billerica, MA). The eluted DNA was cloned into a plasmid (pBluescript SKII+; Stratagene, La Jolla, CA), and amplified in E. coli. Several clones were sequenced in both directions using a DNA sequencer (model 4200; Li-COR, Lincoln, NE). Similarities with other sequences in GenBank were examined using the BLAST program at the National Center for Biotechnology Information.

Quantitative real time PCR analysis

Quantitative real time PCR for specific genes was performed to confirm the differences in genes identified by differential display analysis. Single-strand cDNA was synthesized from TR-iBRB2, TR-iBRB9, TR-BBB11 and TR-BBB13 cell total RNA (1 μg) by reverse transcription using oligo dT as the primer. According to the manufacturer's protocol, quantitative real time PCR was performed using an ABI PRISM 7700 sequence detector system (PE-Applied Biosystems, Foster City, CA) with a 2X SYBR Green PCR master mix (PE-Applied Biosystems), reverse transcribed cDNA, and gene specific primers. To quantify the amount of target mRNA in the samples, a standard curve was prepared for each run using the plasmid containing the target gene. This enabled standardization of the initial mRNA content of cells relative to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequences of the specific primers were as follows: the sense sequence was 5'-GTT CAC CAA GGA TGA GTT CTT TAT GG-3' and the antisense sequence was 5'-AGG ATG GTG AAC CTG GCC ACC CAG TT-3' for rat M-cadherin, and the sense sequence was 5'-TGA TGA CAT CAA GAA GGT GGT GAA G-3' and the antisense sequence was 5'-TCC TTG GAG GCC ATG TAG GCC AT-3' for GAPDH. The data represent means±standard error of the mean. Statistical significance of differences among means of several groups was determined by one way analysis of variance (ANOVA) followed by the modified Fisher's least squares difference method.

5' Rapid amplification of cDNA ends (5' RACE)

Since differential display could only identify the 3' terminal of mRNAs, the 5' sequence was amplified using total RNA of TR-iBRB2 cells, a SMART RACE cDNA amplification kit (CLONTECH, Palo Alto, CA), and the clone 1 (Table 1) specific reverse primer (5'-AGA TGC CTG CTT TCA TAC AGA GGT GA-3'), according to the manufacturer's protocol. The purified 5' RACE PCR product was cloned into a plasmid (pBluescript SKII+) and then amplified in E. coli. Several clones were sequenced from both directions using a DNA sequencer.

Immunoblot analysis

Protein samples were obtained by dissolving cells in lysis buffer consisting of 1% sodium dodecyl sulfate (SDS), 10 mM Tris-HCl (pH 6.8), 10% glycerol, 1 mM EDTA, and 10 μL/mL protease inhibitor cocktail (Sigma, St. Louis, MO). Protein samples were boiled for 10 min and centrifuged at 8,000x g for 10 min at 4 °C. Supernatants were separated and used as a whole cell extract. The proteins (100 μg) were electrophoresed on an SDS-polyacrylamide gel and subsequently electrotransferred to a poly vinylidene difluoride membrane. The membranes were incubated with goat polyclonal anti-human M-cadherin antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) for 16 h at 4 °C as the primary antibody using blocking agent solution (Block Ace; Dainihon Pharmaceutical Co., Osaka, Japan). There was apparently sufficient antibody cross-reactivity between human and rat M-cadherin. The membranes were subsequently incubated with horseradish peroxidase conjugated anti-goat IgG as the secondary antibody. The bands were visualized with an enhanced chemiluminescence kit (Amersham, Buckinghamshire, UK).

Results

The differential display analysis of TR-iBRB2, TR-iBRB9, TR-BBB11, and TR-BBB13 cells showed 40 bands, which were selectively expressed more in TR-iBRB cells than in the TR-iBBB cells (Figure 1A). These DNA bands of 100 to about 450 bp were cloned and sequenced. Quantitative real time PCR using specific primers for each clone was performed to confirm the reproducibility. As a result of these analyses, eight clones were identified and found to be expressed to a greater extent in TR-iBRB cells than in TR-BBB cells (Table 1). The differential displayed clones included two sequences homologous to mouse genes, such as M-cadherin (cadherin-15; clone 1), GATA-binding protein 3 (GATA-3; clone 2), and one sequence identical to rat cytosolic branched chain amino transferase (BCATc; clone 4). The other five sequences (clones 3, 5, 6, 7, and 8) were only found in the expressed sequence tag (EST) or genomic sequences.

Clone 1, of 251 bp, which was expressed 213 fold more intensely in TR-iBRB cells than in TR-BBB cells, exhibited the largest difference in mRNA expression level of the eight clones. The sequence of clone 1 following nucleotide position 72 exhibited 77% nucleotide identity to the 3' terminal of the mouse M-cadherin gene (Figure 1B) [12]. By performing 5' RACE, a 2686 bp cDNA fragment was amplified as a rat M-cadherin. This sequence includes the clone 1 sequence and an open reading frame of 2355 nucleotides, which corresponds to 784 amino acid residues (Figure 2). The deduced amino acid sequence exhibits 97% and 86% identity to mouse and human M-cadherin, respectively [12,13], suggesting that the amplified DNA fragment represents the rat homolog of the M-cadherin gene.

The expression level of rat M-cadherin was compared between TR-iBRB and TR-BBB cells. Following quantitative real time PCR analysis using rat M-cadherin specific primers amplified at nucleotide position 790-916, the rat M-cadherin mRNA content relative to the amount of GAPDH mRNA (M-cadherin/GAPDH) in TR-iBRB2, TR-iBRB9, TR-BBB11, and TR-BBB13 cells was 2.38±0.41x10^-3, 6.93±0.67x10^-3, 7.32±4.80x10^-5, and 2.74±0.49x10^-5, respectively (Figure 3A). The mean value of the rat M-cadherin mRNA content in TR-iBRB cells was 92.9 fold greater than that in TR-BBB cells. Consistent with mRNA expression, immunoblot analysis revealed that the expression of rat M-cadherin protein at 130 kDa in TR-iBRB cells was greater than that in TR-BBB cells (Figure 3B). The molecular weight of rat M-cadherin at 130 kDa was identical to the reported value for rat L6 myoblasts [14].

Discussion

This study found eight genes expressed more in retina than in brain capillary endothelial cell lines using mRNA differential display analysis with 48 primer combinations (Table 1). Three hundred primer combinations are required to visualize all differentially displayed genes [15]. Therefore, 16% of mRNAs in both cell lines were screened in this study, and further 42 genes are expected to exist as a differentially displayed gene in TR-iBRB cells. For the analysis of differentially displayed genes between the inner BRB and BBB, it is important to use cells exhibiting normal physiological properties. In the case of isolated capillary endothelial cells from animals, it is difficult to avoid contamination from non-endothelial cells and obtain a sufficient number of cells due to very small dimensions of the retina. Using TR-iBRB and TR-BBB cells eliminates these concerns, since both cell lines were established from the same rat strain using the same procedure and cultured in the same conditions. Moreover, both cell lines possess the same endothelial markers and express several transporters [9-11], which have been reported to be expressed in vivo retinal and brain capillaries [2,3,6,7]. Therefore, the difference in expressed genes between these cell lines may indeed reflect the difference between the inner BRB and BBB in vivo.

Cadherin is an adherens junction protein and mediates calcium dependent cell-cell adhesion in a homophilic manner [16]. It has been reported that some cadherin-family proteins are expressed at the inner BRB and the BBB. VE-Cadherin (cadherin-5), an endothelial cell specific protein which is necessary for vessel formation in vivo [17], is localized to adhesion sites of endothelial cells in the retina and brain [18,19]. In embryonic chicken brain and retina, N-cadherin (cadherin-2) is expressed at contact zones between pericytes and endothelial cells and its concentration rapidly falls with the onset of barrier differentiation, suggesting that N-cadherin expression represents a signal for the expression of barrier properties [20]

The present study revealed that M-cadherin is highly expressed in TR-iBRB cells, but not in TR-BBB cells (Figure 3). TR-BBB cells originate from the cerebrum, and there is no report of M-cadherin being expressed in the cerebrum. On the other hand, when nls-lacZ reporter gene was introduced into the M-cadherin locus, strong β-gal activity was observed in the distinct cell layer of the retina [21], suggesting that M-cadherin is expressed in the retina. Ultrastructural comparison between the retinal and brain capillaries shows that retinal vessels have denser interendothelial junctions and a greater covering of pericytes than brain vessels [1]. In diabetes, the neovascularization following the loss of pericytes from vessels mostly occurs in the inner BRB, and may not take place in the BBB [22]. It is proposed that M-cadherin is intimately involved in these ultrastructural and pathological differences in cell-cell adhesion between the inner BRB and BBB.

Rat M-cadherin, which has been cloned for the first time, contains a hydrophobic signal sequence at amino acids 1-21 [23] and a postulated furin cleavage site of precursor polypeptides at amino acids 44-45 [23]. The deduced 740 amino acid sequence of mature M-cadherin protein consists of a long extracellular domain containing five cadherin extracellular subdomain repeats (EC1-EC5), a transmembrane domain, and a cytoplasmic domain. The extracellular domain of M-cadherin includes the characteristic cadherin consensus sequences DXD, LD(R/Y)E, and DXNDNXP which are involved in Ca²⁺ binding [24]. Furthermore, four cysteine residues conserved in cadherin are also present in the EC5 of M-cadherin. There are five N-glycosylation sites in the extracellular domain. The cytoplasmic domain of M-cadherin includes a membrane-proximal conserved domain (MPCD) [16], which is implicated in the basolateral sorting of cadherin molecules, and a catenin binding sequence (CBS) [16].

The 3' termini of GATA-3 and BCATc mRNAs were expressed 24 and 5.5 fold more in TR-iBRB than TR-BBB cells, respectively. A transcription factor, GATA-3, is predominantly expressed and required for optimal cytokine production in T helper type 2 cells [25]. Although the physiological role of GATA-3 at the inner BRB is not clear at the present time, GATA-3 may regulate the expression of some genes that are responsible for the unique functions of the inner BRB. Branched chain amino transferase (BCAT) is involved in de novo glutamate synthesis by transferring nitrogen from the branched-chain amino acids to α-ketoglutarate and vice versa primarily in the retina and the brain [26]. BCATc is proposed to play a more important role in de novo glutamate synthesis at the inner BRB than the BBB. This was supported by the finding that the percentages of glutamate input into the glutamate/glutamine cycle, which are provided by de novo glutamate synthesis, are about 30% and 20% in the retina and whole brain, respectively [26]. Further studies are needed to understand these functions in the retina in vivo. It is important to investigate whether other clones with an expression ratio between 2 and 5 (clones 5, 6, 7, and 8) reflect differences in the two tissues. This confirms that mRNA differential display analysis using both cell lines is a useful technique to determine differences in gene expression between the inner BRB and BBB.

In conclusion, eight clones were identified as highly expressed genes in TR-iBRB cells compared with TR-BBB cells using mRNA differential display analysis. Of these eight clones, M-cadherin, GATA-3, and BCATc were more abundantly expressed in TR-iBRB cells than TR-BBB cells and may indeed be involved in unique functions at the inner BRB. Moreover, rat M-cadherin gene, which has been cloned from TR-iBRB cells, for the first time, was expressed to a much greater extent in TR-iBRB than in TR-BBB and may be responsible for some cell-cell adhesion differences between the two tissues. The selective expression of genes at the inner BRB compared with the BBB may have important implications for the unique function of the inner BRB and the retina.

Acknowledgements

The authors thank Drs. Tadahiro Oshida, Hideaki Takeuchi, and Gozo Tsujimoto for valuable discussions. This study was supported, in part, by a Grant in Aid for Scientific Research from the Japan Society for the Promotion of Science.

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