|Molecular Vision 2006;
Received 12 December 2005 | Accepted 4 July 2006 | Published 6 July 2006
Characterization of the integrin receptor subunit profile in the mammalian sclera
Ravikanth Metlapally, Andrew I. Jobling, Alex Gentle,
Neville A. McBrien
Department of Optometry and Vision Sciences, University of Melbourne, Carlton, Victoria, Australia
Correspondence to: Neville A. McBrien, Department of Optometry and Vision Sciences, University of Melbourne, Parkville, Victoria, Australia, 3010. Phone: 0011 613 8344 7401; FAX: 0011 613 93497474; email: email@example.com
Purpose: During the increased eye growth that results in myopia, the sclera undergoes biochemical and biomechanical remodeling. The cell surface integrin receptor family has important roles during tissue remodeling, regulating the extracellular matrix environment and cellular biomechanical properties. As integrin receptors may have a role in remodeling during myopia, this study detailed subunit gene expression in the mammalian sclera.
Methods: Several tissues, including sclera, were isolated from the tree shrew, a mammalian model used in eye growth studies. Total RNA was purified, reverse transcribed and primers for the α- and β-integrin subunits were designed to the published human sequence in areas of high inter-species homology. PCR was used to amplify products of predetermined size and all tree shrew integrin subunits were sequenced to confirm their identity. Multiple PCR conditions were used to identify the scleral integrin subunits, and positive control tissues were included to reduce the possibility of false negative results.
Results: Integrin PCR products corresponding to the β1-, β4-, β5-, and β8-integrin subunits and the α-integrin subunits, α1-6-, α9-11- and αv-integrin were identified in the sclera and in scleral fibroblast cultures. The respective sequences showed a high identity (>81%) to their human counterparts. The β2-, β3-, β6-, β7-, α7-, and α8-integrin subunits were not detected in tree shrew scleral samples, despite being present in the respective positive controls. Association of the 4 β-integrin subunits with the 10 α-integrin subunits suggests that the mammalian sclera is capable of expressing 13 of the 24 identified integrin receptors.
Conclusions: This is the first systematic description of the integrin subunit expression profile in the sclera. Due to the multiple roles of integrin receptors during tissue remodeling, the identification of these scleral integrins is an important preliminary step in determining the role of these receptors during normal eye growth and myopia development.
The manner in which individual cells communicate with their immediate surroundings, including neighboring cells, is crucial to the proper functioning of a tissue/organ. The integrin family of cell surface receptors is known to form an integral part of this "cellular awareness". Integrin receptors are heterodimers comprised of an α- and β-integrin subunit. At present 18 α- and 8 β-integrin subunits have been described, which combine to form 24 functional receptors, each with characteristic extracellular matrix ligand-binding properties [1,2]. Integrins are involved in cell attachment, signaling, growth factor interaction, and the conversion of mechanical stresses into chemical signals (mechanotransduction) [1,3-5]. Functionally, they regulate diverse processes such as survival, proliferation, migration, and differentiation [6-9].
Due to their extensive functional interactions, it is not surprising that multiple integrin receptors have been described in several ocular tissues, such as cornea, lens, retina, choroid, and trabecular meshwork [10-13]. These ocular receptors play important roles during development, with the α4β1-integrin receptor controlling retinal cell survival, α6-integrin being involved in lens epithelial cell differentiation, and αvβ3- and β1-integrin containing receptors implicated in corneal cell migration during development [14-16]. Integrin receptors are also important for maintaining ocular homeostasis, being involved in the phagocytosis of shed photoreceptor outer segments (αvβ5), activation of TGFβ1 in lens capsule (αvβ6) and formation of corneal epithelial cell hemidesmosomes (α6β4) [17-19].
Alterations in integrin receptor expression have also been associated with several ocular diseases. A decreased expression of β4-integrin has been reported in the corneal basement membranes of patients suffering keratoconus, while β6-integrin showed a twofold increase in mRNA expression in corneas with bullous keratopathy [20,21]. Integrins have also been implicated in retinal neovascularization, with the αvβ3- and αvβ5-integrin receptors expressed on retinal vasculature from patients with proliferative diabetic retinopathy (PDR) . The importance of these receptors is further evidenced by the fact that addition of an antagonist to αvβ3- and αvβ5-integrin inhibited new blood vessel formation in a murine model of retinal angiogenesis. Blockade of the α4-integrin subunit has also been suggested as a useful therapeutic strategy for the treatment of autoimmune ocular diseases .
By far the best characterized role of ocular integrin receptors is in corneal wound healing. In response to corneal wounding, α9-integrin protein and mRNA are enhanced during migration and restratification of the epithelium, while α6- and β4-integrins are possibly involved in the disassembly of hemidesmosomes, allowing epithelial cell migration [24,25]. In addition to the epithelial changes during corneal healing, the stroma undergoes significant extracellular matrix (ECM) remodeling and during this time keratocyte integrins are upregulated [24,26]. Integrins, possibly the α5β1-integrin receptor, are also involved in the differentiation of the quiescent keratocytes into α-smooth muscle actin expressing myofibroblasts. This formation of myofibroblasts is critical to matrix deposition and corneal wound repair . Like the corneal stroma, the sclera is a tissue rich in ECM which undergoes remodeling during normal ocular growth, as well as during the excessive eye growth that occurs in myopia. Significant scleral ECM alterations have been documented during myopia development, with changes in collagen and glycosaminoglycan content reported and alterations in matrix metalloproteinase activity also observed . In addition to the ECM changes during myopia development, alterations in cell biomechanical properties may also occur, with the sclera known to contain a myofibroblast cell population . As integrins play a key role in ECM remodeling and myofibroblast differentiation, these receptors are likely to play critical roles in regulating scleral architecture. A recent study has highlighted the role of integrins in the control of ocular growth, reporting decreased expression of the α1-, α2-, and β1-integrins during myopia development . Despite this, it is not known which integrin receptors are expressed in the sclera, with only a limited number of subunits identified [31-34].
This study used RT-PCR and automated sequencing to determine which of the 18 α- and 8 β-integrin subunits are expressed in the sclera of the tree shrew, a commonly used mammalian model for eye growth studies. By designing individual primers to areas of high inter-species sequence identity and including positive and negative controls, the experimental design limited the false negative and false positives which can occur using other techniques. The identification of scleral integrin profiles will allow future experiments to investigate which of these receptors are involved in normal and myopic ocular growth.
Animal model and tissue collection
The tree shrew (Tupaia belangeri) was used in this study since it is close to the primate line, has a similar scleral structure to that of humans and is a well-established model for eye growth and myopia . Maternally reared tree shrews were used 15 days after eye opening, a time when they are most susceptible to eye growth changes . The light/dark cycle was maintained at 14/10 h, respectively, with the light level at approximately 250 lux and food and water were available ad libitum. All animals used in this study were treated in accordance with the Association for Research in Vision and Ophthalmology resolution on the use of animals in research.
During tissue isolation, animals were deeply anesthetized with a mixture of ketamine hydrochloride (90 mg/kg) and xylazine (10 mg/kg) and a terminal dose of pentobarbitone sodium (120 mg/kg) was administered. The eyes were enucleated, an incision made posterior to the limbus and the anterior portion of the eye removed. A seven mm punch was taken from the posterior eye cup using a surgical trephine. The retina and choroid were dissected away and the optic nerve head was removed. Other tissues, including skin, lung, liver, heart, and kidney were also collected to act as positive controls for the various integrin subunits. All tissues were immediately placed in liquid nitrogen after dissection and were subsequently stored at -80 °C until use.
For primary scleral fibroblast cell culture, tissues were isolated as described above, however, whole scleral samples were taken and placed in Dulbecco's Modification of Eagles Media (DMEM; Invitrogen, Carlsbad, CA) on ice. Scleral samples were immediately cultured as described below.
Scleral fibroblast cell culture
Scleral explants were placed in culture dishes (Nunc, Roskilde, Denmark) together with DMEM (Invitrogen) supplemented with 25 mM HEPES, 10% fetal calf serum (FCS), 50 U/ml penicillin, and 50 μg/ml streptomycin (JRH, Melbourne, Australia). The explants were maintained in a humidified incubator at 37 °C with 5% CO2 and confluence was reached after 3 weeks. Cell monolayers were passaged using a 0.25% (w/v) trypsin-EDTA solution (Invitrogen) and cell numbers were estimated. Scleral fibroblasts between passage numbers two and six were used for this study since they displayed stable growth characteristics.
Total RNA isolation and reverse transcription
Total RNA was isolated from the posterior scleral tissues using phenol-chloroform extraction . Samples were digested with DNase I (Promega Corp., Madison, WI) to eliminate genomic contamination, and were subsequently re-extracted. Cultured fibroblast cell total RNA was isolated (0.5-1x107 cells) using commercial spin columns (RNeasy; Qiagen, Chatsworth, CA) according to the manufacturer's instructions. RNA quantity and purity was estimated spectrophotometrically (UV 2501 PC; Shimadzu, Kyoto, Japan) and 0.5 μg RNA was reverse transcribed (M-MLV reverse transciptase; Promega) using an oligo-(dT)15 primer (1 μM). The reaction was incubated for one h at 42 °C and was terminated by increasing the temperature to 94 °C for two min. All the samples were diluted 5 fold with dH2O and stored at -20 °C.
Integrin subunit PCR
Due to lack of information on tree shrew integrin receptor sequences in genomic databases, oligonucleotide primers were designed to human integrin subunit sequences in regions that showed high inter-species identity (Table 1, Table 2). The expression of all β-integrin subunits was initially investigated. Primers were subsequently designed only to those α-integrin subunits that are known to be associated with the β-integrin subunits identified in the sclera.
Amplification of the integrin products was performed using a commercial hot-start polymerase (HotStarTaq master mix, Qiagen) on a thermal cycler (Techne; Progene, Cambridge, UK). A general amplification protocol consisted of denaturation at 95 °C for 15 min followed by 35 to 45 cycles of 95 °C for 45 s, 55 °C for 45 s, and 72 °C for 1 min. Specifically, magnesium chloride titrations from 1.5 mM to 5 mM were performed for all the genes of interest at an annealing temperature of 55 °C. Failure to detect the specific integrin subunits with these conditions resulted in a stepwise increase in annealing temperature (58 °C, 60 °C, and 62 °C). For those subunits which failed to amplify given the above conditions, a touchdown protocol (72 to 55-62 °C, 1 °C/cycle) was used with magnesium chloride titrations (1.5 mM to 5 mM) performed at annealing temperatures of 55 °C, 58 °C, 60 °C, and 62 °C. Positive controls were included in all amplifications and the use of total RNA as a template for the reaction controlled for genomic contamination.
In order to confirm scleral product identity, fragments were separated on agarose gels (1.5% w/v), purified using commercial spin columns (QIAquick, Qiagen) and sequenced (CEQ 8000 Genetic Analysis System; Beckman Coulter, Fullerton, CA). All integrin receptor subunit sequences were compared with their human counterparts using Clustal W.
As there are very limited data on integrin receptor expression in the sclera, RT-PCR was used to identify which of the 18 α- and 8 β-integrin subunits are expressed in the mammalian sclera. As observed in Figure 1, single products of correct sizes were obtained for β1- (Figure 1A; 184 bp), β4- (Figure 1B; 383 bp), β5- (Figure 1C; 276 bp), and β8-integrin (Figure 1D; 179 bp) subunits in scleral cDNA samples. Similar products were observed in scleral fibroblast cell samples. These specific products were not due to genomic contamination, since amplifications using the corresponding RNA samples did not produce specific products (Figure 1; lanes 2, 4, and 6). Skin cDNA was used as a positive control for these integrin subunits, since their expression has been previously documented in this tissue [37-39]. Sequencing of the β-integrin subunit products showed them to have a high identity to their human counterparts (β1-; 81%, β4-; 90%, β5-; 88%, and β8-integrin; 91%; GenBank DQ453506, DQ453507, DQ453508, and DQ453509).
Despite the use of the various PCR conditions outlined in the methods section, the β2-, β3-, β6-, and β7-integrin subunits were not detected in scleral or scleral fibroblast cell samples (see Figure 2). Amplification products of the correct size (Figure 2A; β2-, 347 bp, Figure 2B; β3-, 391 bp, Figure 2C; β6-, 306 bp, and Figure 2D; β7-integrin, 392 bp) were, however, detected in the respective positive controls (lung, liver, kidney, and leukocyte). These tissues have been previously shown to express the above subunits in human, mouse, and rhesus monkey [40-43]. The faint band observed for β7-integrin in the scleral fibroblast sample (Figure 2D, lane 3) was re-amplified, sequenced and found to be a nonspecific amplification product. The use of total RNA during amplification (Figure 2; lanes 2, 4, and 6) again shows no genomic contamination.
As integrins are composed of α- and β-integrin heterodimers and it is known which subunits associate to form functional receptors, the identification of β1-, β4-, β5-, and β8-integrin subunits in the sclera, allowed a more targeted approach to be used for the identification of the α-integrin subunits. Primers were designed to the 12 α-integrin subunits that could associate with the above identified β-integrin subunits and RT-PCR was performed. As observed in Figure 3, products were amplified for α1- (Figure 3A; 198 bp), α2- (Figure 3B; 179 bp), α3- (Figure 3C; 657 bp), α4- (Figure 3D; 383 bp), α5- (Figure 3E; 388 bp), α6- (Figure 3F; 375 bp), α9- (Figure 3G; 436 bp), α10- (Figure 3H; 439 bp), α11- (Figure 3I; 459 bp), and αv-integrin (Figure 3J; 420 bp) in the sclera and scleral fibroblast cell line. Again, the use of the corresponding RNA samples ruled out genomic contamination. Skin cDNA was used as a positive control since the expression of these subunits had been previously determined in this tissue [22,37-39,44,45]. Isolation and sequencing of the amplified products showed the tree shrew subunits to again have a high identity to the respective human mRNA (α1-; 85%, α2-; 93%, α3-; 91%, α4-; 88%, α5-; 96%, α6-; 93%, α9-; 90%, α10-; 93%, α11-; 91%, and αv-integrin; 94%; GenBank DQ457076, DQ457077, DQ457078, DQ457079, DQ457080, DQ457081, DQ457082, DQ457083, DQ457084, and DQ457085).
Of the 12 possible α-integrin subunits that could associate with β1-, β4-, β5-, and β8-integrins, the α7- and α8-integrin subunits were not detected in the sclera, nor scleral fibroblasts (Figure 4A,B), despite using several different PCR conditions. Products of the correct size (α7-; 410 bp and α8-integrin; 530 bp) were, however, detected in heart and lung cDNA samples, which has been previously reported in human and rat [46,47]. As with all the previous amplifications, no products were obtained using the corresponding RNA samples.
It is known that the association of one of the α-integrin subunits with one of the β-integrin subunits produces a functional integrin receptor. The 14 integrin subunits that the mammalian sclera expresses have the potential to form 13 functional integrin receptors. Table 3 lists these proposed receptors together with their preferred extracellular matrix ligands. As can be observed, all of the ligands have already been identified in the sclera.
Remodeling of the scleral ECM is critical to normal eye growth and also occurs during the excessive growth that results in myopia. As integrin receptors have roles in ECM remodeling within the eye and in other tissues, they are likely to play an important role in ocular growth. This is the first study to report the expression profile of integrin receptor subunits in the sclera. In total, 10 α- and 4 β-integrin subunits were detected in scleral samples as well as in a primary culture of scleral fibroblast cells. These subunits are able to combine to form 13 possible integrin receptors, α1β1-, α2β1-, α3β1-, α4β1-, α5β1-, α6β1-, α6β4-, α9β1-, α10β1-, α11β1-, αVβ1-, αVβ5-, and αVβ8-integrin, each of which has specific ECM ligand interactions and signaling events.
While several other studies have utilized gene array technology and reported α6-, αv-, β5-, and β1-integrin expression [33,34], which have been confirmed in this study, this is the first report to provide a detailed profile of integrin expression in the sclera. As about 90% of the scleral exracellular matrix is comprised of caollagen , it is not surprising that the subunits which combine to produce the collagen-binding α1β1-, α2β1-, α10β1-, and α11β1-integrins are expressed in the sclera and its fibroblasts. While they are all able to bind collagen, each of these receptors has preferred subtypes, with α1β1-integrin preferentially binding basement membrane collagens such as collagen type IV and type XIII, while α2β1-integrin associates with fibrillar collagens such as types I, III, and V. α10β1-Integrin is similar to α1β1-integrin, while α11β1-integrin binds more efficiently to collagen type I rather than type IV. As the majority of scleral collagen is type I, and types III and V are thought to be critical in scleral collagen fibril association , the α2β1-integrin receptor may be a key scleral fibroblast receptor.
In addition to linking the cell to the surrounding ECM, integrin receptors are also able to regulate ECM gene expression and matrix contraction. The α1β1- and α2β1-integrin receptors are able to regulate collagen and matrix metalloproteinase synthesis in various tissues [50,51]. In addition, specific function blocking antibodies to the α2β1-integrin receptor have highlighted its importance in fibroblast-mediated matrix contraction . As ECM constituents, such as collagen and glycosaminoglycans, are altered during scleral remodeling, and fibroblast-mediated contraction may contribute to the biomechanical properties of the sclera [28,29], these receptors are likely to be important in the remodeling that occurs during normal and myopic eye growth. This role of the collagen-binding integrin receptors in eye growth is further substantiated by a recent report indicating time-dependent decreases in α1-, α2-, and β1-integrin subunit mRNA expression during myopia induction .
The present study also identified the subunits which can form the fibronectin-binding integrin receptors, α4β1-, α5β1-, αvβ1-, αvβ5-, and αvβ8-integrin in the sclera. Fibronectin and its receptors trigger a range of signaling cascades regulating multiple processes during morphogenesis . Within the eye, fibronectin is potentially important during ocular growth, with patients suffering from nanophthalmos exhibiting higher scleral fibronectin expression than that found in normal sclera . In addition, fibronectin integrin receptors, such as α5β1-integrin, have been implicated in the differentiation of the highly contractile myofibroblast cell , which may suggest a role for these receptors in maintaining/regulating the recently identified scleral myofibroblast population .
The α9β1-integrin receptor, whose subunits were also identified in this study, binds the tenascin family of ECM proteins. In the cornea, α9β1-integrin and tenascin-C were shown to be differentially regulated in extracellular matrix remodeling during corneal erosion syndrome and in response to corneal epithelial injury [24,55]. Furthermore, one of the members of the tenascin family, tenascin-X, has been associated with Ehlers-Danlos syndrome, characterized by hyperextensibility of skin caused by defective collagen metabolism . Interestingly, a thin sclera and myopia are common ophthalmological complications of Ehlers-Danlos syndrome , suggesting that tenascin may play an important role in maintaining scleral architecture.
Based on evidence in other tissue systems, regulation of these scleral integrin subunits would be critical for proper cell/tissue function. Of the numerous proteins which are reported to control integrin expression, several are also thought to play an important role during myopia development. Transforming growth factor beta (TGF-β) is a positive regulator of numerous integrin subunits such as α2-, αv-, α8-, and β1-integrin, is decreased during myopia and may play an important role in scleral remodeling [58-61]. Other molecules such as retinoic acid and collagen also regulate integrin gene expression and are also reported to be involved in signalling and scleral remodeling during myopia development [28,62,63]. Obviously, further work is required to assess the effect of these regulatory proteins on the scleral integrin subunit gene expression.
Despite being present in their respective positive controls, several integrin subunits were not detected in the sclera, or its fibroblasts. While the inclusion of the positive control tissue indicates the appropriateness of the amplification conditions, it is difficult to definitively prove these subunits are not expressed in the sclera. However, previous reports of subunit distribution generally provide support for the data obtained by this study. The β6-integrin subunit is restricted to cells of epithelial origin , while receptors containing the β2-, β3-, and β7-integrins are predominantly expressed on cells associated with the vasculature [64-66] and thus might not be found in the largely avascular sclera. The α8-integrin subunit, unlike the restricted expression pattern of the above subunits, has been reported in a wide range of cell types and the α8β1-integrin receptor typically binds to fibronectin which is found in the sclera [47,67]. The apparent lack of the α8-integrin subunit in the sclera may reflect the redundancy observed in integrin receptors, since its ligand can be bound by other integrin receptors whose subunits were detected in the sclera (for example, α5β1- and α9β1-integrin).
Although the α7-integrin subunit was not detected in the tree shrew sclera in this study, despite being present in tree shrew heart tissue, Young et al.  detected it in human scleral samples using gene array technology. While species differences may explain this discrepancy, so may differences in the technique used. One potential drawback of the gene array analysis is the use of "global" hybridization protocols. Such conditions can lead to the presence of false negative and false positive results which is evidenced by the fact that the major collagen binding subunits, α1- and α2-integrin, have not been reported in these scleral array studies.
The experimental design used in this study was chosen specifically to maximize the likelihood of detecting the various integrin subunits. RT-PCR is highly sensitive which is particularly important for low copy number genes. This technique also allows false positive and false negative results to be minimized through the use of total RNA/sequencing and specific positive control tissues. This, together with the fact that amplification can be optimized for each integrin subunit, results in an increased specificity.
This is the first study to report a comprehensive gene profile of integrin subunits in the mammalian sclera and serves as a basis to determine which receptors are critical to scleral architecture. Based on their roles in other tissues including those in the eye, integrins are likely to significantly contribute to the scleral ECM remodelling that occurs during normal and myopic eye growth.
This work was supported by The National Health and Medical Research Council of Australia, grant number 251557.
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