Molecular Vision 2005;
11:603-608 <http://www.molvis.org/molvis/v11/a71/> Received 12 May 2005 | Accepted 5 August 2005 | Published 12 August 2005 |
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Versican splice variants in human trabecular meshwork and ciliary muscle
Section on Aging and Ocular Disease, National Eye Institute, National Institutes of Health, Bethesda, MD
Correspondence to: Paul Russell, 7 Memorial Drive, MSC 0703, Bethesda, MD, 20892; Phone: (301) 496-7471; FAX: (301) 496-1759; email: russellp@nei.nih.gov
Abstract
Purpose: Versican, chondroitin sulfate glycoprotein 2, is thought to play a role in regulating aqueous humor outflow and intraocular pressure via the human trabecular meshwork (HTM) of the eye. This protein was upregulated in HTM cells when they were treated with TGF-β. There are four splice variant forms of versican (V0-V3) with different numbers of glycoaminoglycan (GAG) attachment domains. In this study, we investigated the various isoforms of versican from ocular tissues and cultured cells.
Methods: HTM and human ciliary muscle (HCM) tissues were dissected from three pairs of donors eyes with no histories of eye disease. Cultures of HTM and HCM cells were established from five donor eyes, and HTM cells were treated for 72 h with 1 ng/ml of either with hrTGF-β1 or hrTGF-β2. Total RNA was isolated from cells and tissues from each of the samples. Relative quantitation of gene expression of each variant was detected by real-time PCR with SYBR Green dye.
Results: All four variants of versican existed in each sample. In cultured HTM cells, the two variants with the largest number of GAG domains predominate. The V1 form is about 20% greater than the V0 form. There is an upregulation, particularly in the V0 form, when the cells are cultured. In tissue, the V1 form is about five fold greater than the other three. In the ocular ciliary muscle, the V1 form is the most prominent, but with this tissue, the relative amount of the V0 form did not change when cells were cultured. There was an upregulation of all splice variants in HTM cells when they were cultured with either TGF-β1 or TGF-β2. The increase in expression in the HTM with TGF-β treatments were greatest with the V0 and V2 forms.
Conclusions: This is the first report about the presence of various forms of versican in the anterior segment of the eye and the alterations in the mRNA patterns of these forms when cells are placed in culture. The results indicate the variants with the largest numbers of GAG attachment domains are the most prominent in the HTM and HCM. The increases in these forms that have the most GAG domains would be consistent with the increases in chondroitin sulfate reported in glaucoma.
Introduction
Versican, one of a family of proteoglycans termed the hyalectans, has a tridomain structure [1,2]. The amino-terminal end, designated as G1, binds to hyaluronan. The carboxy-terminal domain, G3, has a lectin adjacent to two epidermal growth factor domains and a complement regulatory region. The central area of versican is encoded by two exons that specify chondroitin sulfate attachment regions. RNA splicing of these two exons results in four different forms of versican (Figure 1). The V0 form is encoded by the two exons, exon 7 and 8. The V1 form is encoded by exon 8 but not 7 and has the glycoaminoglycan (GAG)-β attachment domain. The V2 isoform is encoded by exon 7 and not 8 and has the GAG-α domain. V3 is not encoded by either exon 7 or 8 and has no GAG attachment domains. The potential numbers of glycoaminoglycan attachment sites for each form are: V0, 17-23; V1, 12-15; V2, 5-8; and V3, 0 [2]. The distribution of the forms in adult tissues varies [3] with some tissues and organs having all forms, such as stomach while others appear to have only one, such as the presence of V1 in uterus tissue.
In brain, the V0 and V1 forms double in concentration between E14 and birth and later decrease by more than 90% [4]. The V1 form has been shown to induce neural differentiation and promotes outgrowth of neurites [5]. The bovine V2 form increases during the postnatal period and the V2 form is the major extracellular matrix component of the mature bovine brain [4,6]. The V2 form has been reported to have neurite growth inhibitory activity [7]. In embryonic chick retina, the V0 form appears to be the predominant one [8].
The chondroitin sulfate chains of versican are highly negatively charged and contribute to the anti-adhesive properties associated with this proteoglycan. However, increased expression of the V3 isoform actually causes increased adhesion and inhibition of proliferation and migration of smooth muscle cells [9]. These effects are thought to be a result of either competition for hyaluronan binding with the larger forms or an alteration in the pericellular matrix. It has been reported that in arterial wall there is an increase in versican in the prelesion state that may be related to increased lipoprotein retention; but in the aneurysmal vessel wall, there is a decrease in versican perhaps by increased degradation and decreased synthesis [10,11]. Versican has been reported to be upregulated by transforming growth factor-β1 (TGF-β1) [12-14].
Versican has been reported in the HTM of the eye and this is the tissue that is thought to regulate aqueous humor outflow and intraocular pressure [15-17]. In primary open angle glaucoma, there is a large decrease in the hyaluronic acid content in the trabecular meshwork and an increase in chondroitin sulfate content [18]. A similar change was also reported in the ciliary body. In the aqueous humor of the glaucomatous eye, there is an increase in active TGF-β [19,20]. We reported an upregulation of versican in HTM cells when they were treated with TGF-β. In this report, we examined the various forms of versican from human trabecular meshwork and ciliary muscle tissues and cultured cells.
Methods
Tissue dissection, cell culture, and TGF-β treatment
Eight pairs of normal human eyes from donors with no history of eye diseases were obtained from the National Disease Research Interchange (Philadelphia, PA) around 30-36 h after death. Three pairs of eyes were dissected and used for HTM and human ciliary muscle (HCM) tissue samples, while the other five eyes were used for HTM and HCM cell culture. HTM tissues were dissected and the cells were cultured in a manner similar to that previously described [21]. The five HTM primary cell cultures were used for treatment with the TGF-βs. They were cultured in serum-free Dulbecco's Modified Eagle's Medium (GIBCO, Invitrogen, Carlsbad, CA). Three flasks of confluent cells from each individual, at passage levels three to five, were treated with either vehicle, 4 mM HCl containing 1% BSA (control), 1.0 ng/ml activated rhTGF-β1 (Roche, Mannheim, Germany), or 1.0 ng/ml activated rhTGF-β2 (R&D Systems, Minneapolis, MN) and incubated for 72 h. During this period, the medium was changed every 24 h and the same TGF-βs were added to the fresh medium. Six groups of samples were generated (HTM tissue, HCM tissue, HTM cells, HCM cells, and HTM cells with either TGF-β1 or TGF-β2).
RNA isolation and purification
Total RNA was isolated from the cells and tissues for each of the samples using TRIzol Reagent (GIBCO-BRL Life Technologies, Gaithersburg, MD). RNA was prepared following the protocol from the manufacturer. The RNA pellets were washed with 75% ethanol, centrifuged, and dried. The residual DNA was removed by treatment with DNase I. Pellets were resuspended in 30 μl of DEPC-treated water followed by the addition of 50 mM Tris, pH 7.5, 10 mM MgCl2, 20 U of RNase-free DNase I (Boehringer Mannheim, Indianapolis, IN), and 20 U of RNasin (Promega Corp., Madison, WI) in a total volume of 60 μl. Samples were incubated at 37 °C for 25 min. Then the RNA was cleaned using RNeasy Mini Kits (Qiagen, Valencia, CA) following the protocol by the manufacturer. RNA concentration and purity was determined by measuring optical density at 260 and 280 nm using a spectrophotometer.
Real-time RT-PCR
cDNA was generated from the total RNA samples by using the Taqman Reverse Transcription Reagents kit (Applied Biosystems, Foster City, CA). Primers were designed by using Primer Express Software version 2.0 (Applied Biosystems; Table 1). To make the cDNA, the total RNA from each sample was first incubated at 25 °C for 10 min, and then reverse transcribed at 48 °C for 30 min. Real-time RT-PCR was performed using a SYBR green dye (Applied Biosystems) with the ABI PRISM 7900HT equipment. The forward and reverse primers for each variant are found in Table 1. DNA polymerase was first activated at 95 °C for 10 min, denatured at 95 °C for 15 s, and annealed/extended at 60 °C for 1 min, for 40 cycles according to the manufacturer's protocol. The products were sequenced to insure that the correct gene sequence was being amplified. All PCR reactions were performed in triplicate. Relative quantitation of gene expression was carried out using the standard curve method (User Bulletin number 2, ABI PRISM 7700 Sequence Detection System; Applied Biosystems). For comparison of the transcript levels between samples, standard curves were prepared for both the target gene and the endogenous reference (18S ribosomal RNA). For each experimental sample, the amounts of target and endogenous reference were determined from the appropriate standard curves. Then, the target amount was divided by the endogenous reference amount to obtain a normalized target value. Each of the experimental normalized sample values was divided by the normalized control sample value to generate the relative expression levels.
Results
The levels of expression of the variant forms of versican were determined for both HTM dissected tissue and HTM cells grown in tissue culture. In both cases, the V1 form predominated (Figure 2). To get an estimate of the contribution of the various isoform to the total, the expression of the V1 form was given a value of 1.0 and all other isoforms were normalized to this form. In the cultured cells, the V0 form was present at a level of 80% of the V1 form. The V3 RNA was 7% or less of the V1 form while the V2 was less than 0.5% of V1. Thus, the V1 and V0 forms appeared to be the dominant forms expressed in the cultured cells. Trabecular meshwork from three donors was worked up separately. Some individual variations in the forms from human donor tissue were seen and an example is shown for the V0 form (Figure 3). Although mRNA from the V1 isoform was again the predominant RNA, the V0 and V3 isoforms were almost the same (each 14% of V1). The mRNA from the V2 isoform represented only about 4% of the V1 form. Comparison of the data from the cultured cells and the tissue showed that while V1 was the most prominent variant, when the cells are placed in culture the V0 form was upregulated by about 4.7 fold. In contrast, both the V3 and the V2 mRNA were downregulated by 2 and 10 fold, respectively. Clearly with both the cultured cells and the tissue, the V2 isoform contributed least to the total mRNA.
Cells from the ciliary muscle from the same five donors were placed in tissue culture in order to determine if a similar change in regulation occurred upon culturing. In the mRNA from the cultured human ciliary muscle cells and the ciliary muscle from the three donor eyes, there was good correlation of the amount mRNA encoded by the V1 and V0 isoforms. As with the meshwork, the V1 isoform was the predominate one with the V0 being about 25% of the V1 (Figure 4). There did not appear to be an upregulation for this isoform when the cells were placed in tissue culture as was observed with the meshwork cells. However, the V3 form appeared to be downregulated in the cultured cells. Upon culture, both the V3 and V2 forms were less than 3% of the V1 value. Similar to the meshwork, the mRNA for the V3 form represented a greater amount relative to the V2 in the ciliary muscle.
Previous microarray data indicated that there was a 4.4 fold increase in versican mRNA in HTM cells with TGF-β1 treatment and a 2.7 fold increase with TGF-β2 [22]. To explore more fully this upregulation, the mRNAs of the various forms of versican were examined in the same samples. For TGF-β1 treatment, V0 had a 9.2 fold increase (Table 2). The next largest increase was V2 with a 7.5 fold increase. The other two isoforms also had increases with V1 increasing 4.8 fold and V3 up by 2.7 fold. With TGF-β2 treatment, the increases in mRNA in the HTM cells were about a third the level observed the TGF-β1 treatment. The highest increase was a 2.5 fold increase with V0 followed by a 2 fold increase with V2. V1 and V3 had a 1.2 and 1.5 fold increase, respectively. If one determines the contribution of the increase in the mRNA from a particular form on the total versican mRNA, then the upregulation of the V3 and V2 mRNAs contributed little to the total since they had such a small amount of mRNA to begin with (Table 2). Since V0 had about 80% of the message of the V1 isoform in the HTM cells, the large upregulation of the V0 isoform constituted the largest increase to the mRNA in the treated cells. This was the case for both TGF-β1 and TGF-β2 treatments.
Discussion
Although the forms of versican have been identified in retina [23] and vitreous humor [24], this is the first report about the presence of the various forms of versican in the anterior segment of the eye and the alterations in the mRNA patterns of these forms when cells were placed in culture. In both the trabecular meshwork and the ciliary muscle, all forms of versican were present. In both tissues, the predominant form was V1.
Our findings clearly demonstrate that for human trabecular meshwork and ciliary muscle, placing cells in culture downregulated the mRNA of both the V2 and the V3 forms when compared to the V1 isoform. The reason for the downregulation of these two isoforms is unclear. The downregulation of the V3 form might indicate that the cells are suppressing this isoform, which might affect the adhesive properties when the cells are shifted to the plastic matrix, but the downregulation of the V2 forms is somewhat more difficult to explain. With the V0 isoform, a difference in the two types of cells was demonstrated with an upregulation of this isoform in the trabecular meshwork cells when they were cultured, but essentially no change relative to the V0 form in the ciliary muscle cells in similar culture conditions.
With TGF-β treatment, there were several changes in gene expression in the trabecular meshwork cells [22]. Both TGF-β1 and TGF-β2 upregulate versican gene expression with the TGF-β1 giving more robust changes than the TGF-β2. Because of the large amount of V0 in cultured cells, the increase in gene expression of this isoform had a much more profound effect on total versican expression in vitro after TGF-β treatment than it might have in vivo. If the results of increases in mRNA levels were similar in vivo then the largest contributor to the gene increase of versican would be V1. Clearly, the least influential isoform in the human trabecular meshwork cells and potentially in the meshwork itself would be the V2 form. The upregulation of total versican might explain the increase in chondroitin sulfate in glaucoma. The increase in active TGF-β2 in the aqueous humor of the glaucomatous eye could upregulate versican [19], particularly the V1 and V0 forms. Both of these forms have the largest number of GAG attachment domains. Since more domains for chondroitin sulfate might exist, this would be consistent with the increases in chondroitin sulfate reported with glaucoma. A possible way in which to increase outflow in glaucoma might be to downregulate the versican isoforms. Currently, the biochemical analyses of the forms of versican are complicated because all four splice variants are present in both trabecular meshwork and ciliary muscle. Antibodies that can differentiate all the forms when each of the four forms is expressed are not currently available.
Acknowledgements
The authors wish to acknowledge the National Disease Research Interchange for providing the donor material. This work was presented in part at the 2005 meeting of the Association for Research in Vision and Ophthalmology.
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