Retinal Pigment Epithelial Cells Synthesize Laminins, Including Laminin 5, and Adhere to Them...

Retinal Pigment Epithelial Cells Synthesize Laminins, IncludingLaminin 5, and Adhere to Them through α3- and α6-ContainingIntegrins

Sabine Aisenbrey1,2,3, Minlei Zhang1,3, Daniel Bacher1,3, Jason Yee1,3, William J.Brunken1,3,4, and Dale D. Hunter1,3,41Department of Neuroscience, Tufts University, Boston, Massachusetts3Tufts Center for Vision Research, Tufts University, Boston, Massachusetts4Department of Anatomy and Cellular Biology, Tufts University, Boston, Massachusetts

AbstractPurpose—Retinal diseases are often accompanied by changes in the structure of the multilayeredextracellular matrix underlying the retina, Bruch's membrane (BrM). These structural revisionspotentially lead to alterations in retinal pigment epithelium (RPE) adhesion, likely via modificationof interactions with extracellular matrix (ECM) proteins including laminins in BrM. The purpose ofthis study was to identify specific laminins in BrM and their receptors in RPE cells.

Methods—The laminin composition of BrM was determined using biochemical, molecularbiological, and immunohistochemical techniques of rat, bovine, and human tissue and cell lines. Anadhesion assay was used to test RPE attachment to laminins and the receptors used for this attachment.

Results—BrM contained laminin chains that could form laminin heterotrimers including laminins1, 5, 10, and 11. RPE cells synthesized these laminin chains in vitro. Therefore, RPE cells maysynthesize BrM laminins. The RPE cells preferentially adhered to potential BrM laminins. Althoughthe cells adhered to the BrM component collagen IV, these cells preferentially adhered to laminins.Of the laminins tested, the RPE cells adhered preferentially to laminin 5. The cells interacted withthese laminins via specific integrins and attained a different morphology on each laminin. Inparticular, the RPE cells rapidly attached and flattened on laminin 5.

Conclusions—BrM contains specific laminins, and RPE cells express integrin receptors for thoselaminins. The interaction of these specific laminins and integrins most likely leads to differentialbehavior of RPE cells.

The interface between the neural retina and retinal pigment epithelium (RPE) is formed duringthe unusual juxtaposition of two epithelial apical surfaces as the optic cup folds in from theneural tube. The inner limit of the retina is formed by the epithelial basal surface of the neuralretina, the structured basement membrane known as the inner limiting membrane. Because theapical surface of the neural retina is juxtaposed to the apical surface of the RPE, the outer limitof the retina is also formed by an epithelial basal surface: that of the RPE, Bruch's membrane(BrM). BrM serves functions analogous to basement membranes in other tissues, including

Corresponding author: Dale D. Hunter, Tufts Center for Vision Research, Tufts University, 136 Harrison Avenue, Boston, MA 02111;[email protected] affiliation: Department of Ophthalmology, University of Tübingen, Tübingen, Germany.Disclosure: S. Aisenbrey, None; M. Zhang, None; D. Bacher, None; J. Yee, None; W.J. Brunken, P; D.D. Hunter, P

NIH Public AccessAuthor ManuscriptInvest Ophthalmol Vis Sci. Author manuscript; available in PMC 2010 September 8.

Published in final edited form as:Invest Ophthalmol Vis Sci. 2006 December ; 47(12): 5537–5544. doi:10.1167/iovs.05-1590.

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anchoring subjacent cells, acting as a barrier and a filter, and stabilizing the structure of thetissue.1

Epithelial basement membranes are minimally composed of a structural framework built froma combination of members of several families of glycoproteins.2,3 These families includefibronectins, the polymer-forming collagens (collagens type IV), and the laminins. Together,this complex forms the electrondense structure visible in electron micrographs apposed to thebasal surface of epithelial cells.

BrM is a five-layered structure that consists of two basement membranes bordering an innercore of two collagenous layers (composed largely of collagen type I) and an elastic layer(composed largely of elastin). The choroidal border of BrM is formed by the basementmembrane of the choriocapillaris. Subjacent to the RPE itself is another classic basementmembrane, the RPE basement membrane, which, like other basement membranes, containsfibronectin, collagen type IV, and laminins.4–6

One of the many changes associated with the RPE basement membrane during disease ispotentially critical: a splitting of Bruch's membrane between the basement membrane of RPEcells and the inner collagenous layer of BrM. This splitting occurs in retinas of patients withprotein and lipid deposits known as drusen, during the development of choroidalneovascularization and, most obviously, in pigment epithelium detachment (either serous orfibrovascular). Changes in the composition and distribution of BrM extracellular matrix (ECM)components, including proteins promoting adhesion such as collagen IV, fibronectin, andlaminins, can promote this splitting of BrM. Changes in these BrM ECM components also leadto changes in BrM hydroconductivity, thereby promoting pigment epithelium detachment.7,8However, it is not known which of these ECM components—in particular, which laminins—are present and functional in the RPE basement membrane.

Laminins are large heterotrimeric glycoproteins consisting of an α, a β, and a γ chain.2,9Vertebrates produce five α chains, three β chains, and three γ chains; these chains combine toform at least 15 different laminins.10,11 The distinct biological activity of each of these lamininsis the result of combined properties of the individual chains. Of the 15 reported laminins, severalhave quite restricted tissue distribution and elicit distinct biological responses in cells withwhich they interact. One of the most distinctive is laminin 5, a critical component of skinstability.12

Although it is possible to purify heterotrimeric laminins from some tissues, the presence of agiven laminin heterotrimer is usually inferred by the presence of its component chains. Mostchains are components of several heterotrimers; however, the notable exceptions are the β3and γ2 chains, which are thought only to exist as components of laminin 5. Thus, thepresence of the β3 or γ2 chains infers the presence of the laminin 5 heterotrimer.

Epithelial cells adhere to their basement membranes via myriad membrane-associatedreceptors including dystroglycan13 and, critically for many laminins, a large family oftransmembrane receptors, the integrins. Individual integrin heterodimers show some ligandspecificity but also can be somewhat promiscuous in their binding.14

Integrins are composed of one each of the 18 α and 8 β subunits.14 Integrin subunits that arepresent on the basal side of the RPE include α3, α5, α6, and β1.15–17 Of these, potentialheterodimeric laminin receptors are α3β1 and α6β1. Antibodies against the β1 subunit blockattachment of RPE cells on isolated BrM18 and inhibit migration of RPE cells on isolated BrM,19 suggesting that β1-containing integrins can function in RPE interactions with laminins ofBruch's membrane.

Aisenbrey et al. Page 2

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Genetic disruptions demonstrate that interactions between integrins on epithelial cells andlaminins in their subjacent basement membranes are critical for tissue function (see thereferences in the Discussion section). We propose that similar interactions between integrinsin RPE and specific laminins in the RPE-basement membrane are vital for RPE stability andRPE-BrM interface integrity.

MethodsAnimals and Tissue Preparation

All procedures involving animals were approved by the Tufts University animal carecommittee and were in accordance with the National Institutes of Health Guide for the Careand Use of Animals and the ARVO Statement for the Use of Animals in Ophthalmic and VisionResearch. Rats and mice were killed by exposure to CO2. Bovine eyes were obtained from alocal abattoir. Human retinal sections, unfixed and fixed, were provided by Ann Milam (ScheieEye Institute, University of Pennsylvania, Philadelphia, PA) and from tissue sharing at Tufts-NEMC. All human tissue was obtained in accordance with the Declaration of Helsinki.

ImmunohistochemistryImmunohistochemistry was performed as previously described,20,21 with the exception thatautofluorescence of human sections was reduced with a proprietary commercialautofluorescence reduction reagent (Autofluorescence Eliminator Reagent, catalog no. 2160;Chemicon, Temecula. CA), according to the manufacturer's instructions. Adult rat or bovineeye cups were embedded in optimal cutting temperature (OCT) compound (Miles, Elkhart, IN)and frozen by immersion in liquid nitrogen-cooled isopentane. Transverse, 10-μm-thicksections were cut with a cryostat (Leica, Bannockburn, IL) and placed on slides (SuperfrostPlus; Fisher Scientific, Pittsburgh, PA). Slides were stored at −20°C (or, for long-term storage,at −80°C) until use. For use, slides were returned to room temperature, immersed briefly inacetone (or, interchangeably, for all but the α5, β3, and γ2 chains, MeOH) at −20°C, washedin phosphate-buffered saline (PBS; 137 mM NaCl, 2.68 mM KC1, 10 mM Na2HPO4, and 1.76mM KH2PO4, pH 7.4), and then incubated in primary antibodies for 2 hours at roomtemperature or overnight at 4°C. Primary antibodies (Table 1) were diluted in PBS containing2% goat serum, 2% bovine serum albumin, or both. Sections were washed in PBS and incubatedin species-appropriate, affinity-purified, fluorescently labeled secondary antibodies diluted in2% goat serum in PBS for 1 hour at room temperature. Some sections were counterstained with1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI). After washes in PBS, slides were mountedin 90% glycerol and 10% water, containing para-phenylenediamine (1 mg/mL; Sigma-Aldrich,St. Louis, MO), to reduce photobleaching, or in an antifade reagent (Prolong; Invitrogen-Molecular Probes, Eugene, OR). Sections were examined with epifluorescence on an uprightmicroscope (Carl Zeiss Meditec, Inc. Oberkochen, Germany) and photographed with a cooledcharge-coupled device (CCD) camera (Apogee Instruments, Auburn, CA) driven bymicroscopy imaging and processing software (MicroCCD; Diffraction Limited, Ottowa, ON,Canada), or with a scanning confocal system on an upright microscope (TCS SP2 AOBS;Leica) driven by the manufacturer's software. Images were adjusted for contrast and cropped(Photoshop; Adobe Systems, San Jose, CA).

Cell CultureTwo widely used and extensively described RPE cell lines, rat RPE-J30 and humanARPE-19,31 and the rat Müller cell line RMC,32 were grown as described in the originalpublications. Cells were split every week or, for adhesion assays, the day before the assay.



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Reverse Transcription–Polymerase Chain ReactionRNA was isolated from mouse retina and skin and from the rat RPE-J and human ARPE-19RPE cell lines (Trizol; Invitrogen, Carlsbad, CA), according to the manufacturer's procedures.RNA was reverse-transcribed (Superscript II; Invitrogen) with the reverse transcriptase primedwith random hexamer or oligonucleotide primers. The resultant cDNAs were amplified inreactions with PCR master mix (High Fidelity; Roche, Indianapolis, IN) and the use of primerschosen in the laboratory for sequences of high similarity among species for each laminin chain.These sequences were designed by identifying regions of amino acid identity, comparingsequences manually and sequentially among the species for which sequences have beenreported, and choosing regions of the highest sequence identity. Degenerate primers were usedwhen necessary (see Table 2). The reaction product was analyzed on a 1% agarose gel, stainedwith ethidium bromide, and photographed.

Protein-Transfer (Western) Blot AnalysisProtein-transfer blot analyses were performed as previously described.20,21 Briefly, RPE wasextracted from rat or bovine eyes or RPE cells were scraped from dishes. Extracts of ECMprotein were made in 25 mM Tris-HCl (pH 7.4) containing 0.2 mg/mL α2-macroglobulin and1% phenylmethylsulfonyl fluoride. Proteins were analyzed by protein-transfer (Western) blotwith anti-laminin antibodies (Table 1).

Adhesion AssaysFour spots of substrates (2 μL each) were allowed to adhere to 12-well or 24-well tissue cultureplates for 60 minutes at ambient temperature. The wells were blocked with 10 mg/mL bovineserum albumin (BSA; Sigma-Aldrich) in PBS for 30 minutes and washed twice with PBS.ARPE-19 cells were dissociated from monolayer cultures using an enzyme-free dissociationsolution (Specialty Media, Phillipsburg, NJ) to preserve cell surface receptors and resuspendedin Hanks' balanced salt solution (Cambrex, East Rutherford, NJ). The cells (105, 12-well; and5 × 104, 24-well in DMEM/F12; Cambrex) were added to each well and were allowed to adherefor 60 to 90 minutes. Wells were washed with PBS and adherent cells were fixed with 2%paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS. Cells were stainedwith 10 μg/mL acridine orange in PBS, washed in PBS, photographed and counted (ImageJsoftware; available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/ij; developed byWayne Rasband, National Institutes of Health, Bethesda, MD). Nonspecific binding (to theBSA surrounding the adhesive substrate) was zero (e.g., Fig. 7). For assessment of the blockingof adhesion with anti-integrin antibodies, cells were preincubated with antibody for 20 minutesbefore addition to wells, and adhesion was continued in the presence of antibody for 60 minutesat ambient temperature. Laminin 1 from mouse EHS tumor was obtained from Invitrogen.Laminin 5 was a generous gift of Manuel Koch (University of Cologne, Germany); laminin10/11 from human placenta was from Chemicon. Adhesion assays were performed in at leasttwo independent experiments, each of which contained eight independent cell counts per assaycondition.

AntibodiesAnti-laminin antibodies used are listed in Table 1. Rabbit anti-nidogen-1 was a gift of RupertTimpl (Max-Planck-Institut für Biochemie, Martinsried, Germany). Function-blocking anti-integrin antibodies were obtained from Chemicon.

ResultsTo detect the presence of particular laminins in BrM, we analyzed the presence ofheterotrimeric laminins and their component chains, using several antibodies (Table 1) on



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http://zippy.nimh.nih.gov/

http://rsb.info.nih.gov/ij

cryostat sections of human, rat, and bovine retinas. Laminin immunoreactivity nearly alwaysdoes not survive aldehyde fixation; therefore, these immunohistochemical studies wereperformed on fresh-frozen cryostat sections.

Several examples of laminin immunoreactivity in bovine BrM are shown in Figure 1. Lamininimmunoreactivity present at the basal surface included the previously demonstrated laminin 1(α1β1γ1) as well as the individual chains of laminin 1: α1, β1, and γ1 (Fig. 1). The additionalcomponent chains (α5, β2) of laminin 10 (α5β1γ1) and 11 (α5β21) were also expressed in BrM(Fig. 1). Laminin chains including laminin β2 colocalized with the basement membranecomponent nidogen-1, demonstrating the presence of laminins in basement membranes (Fig.1). Additional laminins are potentially present in BrM: laminin 5 and its component chains,including α3, and laminins, including laminins 13 (α3β2γ3) and 15 (α5β2γ3), that contain thelaminin γ3 chain (Fig. 1). Similar results were obtained in human and rat BrM (not shown).

To probe biochemically the presence of laminin chains in BrM, we performed protein transferblots of ECM extracts from purified RPE/BrM. BrM was stripped from bovine neural retina,matrix fractions were isolated, run on denaturing gels, and transferred to blots. Blots were thenprobed with laminin chain-specific antibodies.

Several laminin chains, including α3, α5, β2, β3, γ1, γ2, and γ3, were present in matrix fractionsof bovine RPE/BrM extracts (Fig. 2). Similar results were obtained in rat RPE/BrM extracts(not shown). These biochemical data, together with the immunohistochemical data presentedearlier, are consistent with the presence of the heterotrimeric laminins 5 (α3β3γ2), 11(α5β2γ1), 13 (α3β2γ3), and 15 (α5β2γ3) in BrM.

Laminins present in the RPE basement membrane are probably secreted into the basementmembrane from RPE cells themselves. To determine whether RPE cells produce laminins, weperformed RT-PCR for laminin chains on RNA isolated from the human RPE cell lineARPE-19.31 As positive controls for laminin chains that did not amplify from some sources,we used tissues in which we knew those chains were expressed: for the chains of laminin 5,skin, and for other chains, Müller cells.18 As a source of purified Müller cell RNA, we usedthe rat Müller cell line RMC.32

Primers used were either already in our laboratory or were designed for regions of extensivehomology among mouse, human, and rat (when available) sequences for individual chains.Degenerate primers were synthesized when necessary. Positive control experiments for lamininchains for which we had no product (e.g., α4 in ARPE-19) were run in parallel. Products wereobtained for 8 (α1,3,5; β1,2,3; γ1,2) of the 11 laminin chains in ARPE-19 cells (Table 2). RNAsencoding the laminin α2, α4, and γ3 chains were not detectable in ARPE-19 cells, althoughthey were present in the control tissues (Table 2): muscle and skin (α2), RMC cells (α4), andwhole retina (γ3).

These data are consistent with the synthesis of at least laminins 1 (α1β1γ1), 5 (α3β3γ2), 10(α5β1γ1), and 11 (α5β2γ1) by RPE cells. Therefore, RPE cells are competent to synthesizespecific laminins potentially present in native BrM.

To determine whether ARPE-19 cells translate laminin RNAs into laminin proteins, weperformed protein transfer blots on extracts of ARPE-19 cells. Cells were grown on uncoatedplastic dishes for 3 to 6 days, during which they synthesized their own matrix, and then werescraped from the dishes. Extracts were made and run on denaturing gels, and transferred toblots. Blots were then probed with laminin chain-specific antibodies. Several laminin chainswere present in ARPE-19 extracts, including α1, α3, α5, β1, β2, γ1, and γ2 (Fig. 3). ARPE-19cells appeared to make both the α3A and α3B chains, and both chains were processed at boththeir N and C termini, at least by molecular weight (Fig. 3). ARPE-19 cells did not produce



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α2 or α4 chain protein (by protein transfer blot, not shown), consistent with the lack of α2 andα4 chain RNA. These biochemical data are consistent with the synthesis of laminins 1(α1β1γ1), 5 (α3β3γ2), 10 (α5β1γ1), and 11 (α5β2γ1) by human RPE (ARPE-19) cells.

To determine whether ARPE-19 cells assemble laminin heterotrimers and secrete them, wegrew ARPE-19 cells on uncoated plastic and allowed them to synthesize their own ECM. After3 to 6 days, cells grew to confluence and began to attain their differentiated hexagonal shape(Fig. 4). Cells were then examined for laminin chain expression. The presence of individualchains in the ECM necessarily reflects the presence of laminin heterotrimers, as secretionrequires assembly of the trimer.

Flocculent laminin immunoreactivity was present surrounding the ARPE-19 cells, consistentwith secretion of laminins into ARPE-19 ECM. The laminin chains α3, α5, β1, β3, and γ2 andheterotrimeric laminin 5 are shown in Figure 4. We also tested the rat cell line RPE-J, and thesecells also produced laminin RNAs and laminin proteins (not shown). These data (and data notshown) are consistent with the synthesis and secretion of several laminins, potentially includinglaminins 1 (α1β1γ1), 5 (α3β3γ2), 10 (α5β1γ1), and 11 (α5β2γ1) by RPE cells regardless ofspecies.

Together, our immunohistochemical and biochemical data suggest that RPE cells make andsecrete laminins of differing functional activities—in particular, laminins 1, 5, 10, and 11 —into BrM (Figs. 1-4). To determine whether RPE express receptors for particular laminins, weused a short-term in vitro binding assay to ascertain whether RPE cells bind to specific lamininisoforms: laminin 1 from mouse EHS tumor, affinity-purified laminin 5 from a squamouscarcinoma cell line, and laminin 10/11 (contains a mixture of laminins 10 and 1133,34) fromhuman placenta. ARPE-19 cells bound to laminins 1, 5, and 10/11 in a dose-dependent manner(Fig. 4). Under these conditions, RPE cells bound to laminins rapidly and avidly—preferentially to laminins rather than another component of BrM, collagen type IV (not shown).In addition, a dramatically higher number of cells bound to laminin 5 than to laminin-1 or-10/11 (Fig. 5). These data suggest that RPE cells preferentially bind to laminin 5.

Binding of cells to laminins is often mediated by integrins. For some cell types, the particularintegrins used as receptors for specific laminins has been elucidated. We began to investigatethe interactions of RPE cells with laminins by asking whether the binding of ARPE-19 cellsto laminins could be inhibited by antibodies to integrins expressed by RPE cells. RPE cellsexpress the integrin subunits α3, α6, and β1 on their basal surface, apposed to the laminin-containing RPE basement membrane.15–17

ARPE-19 cell binding to laminin 1 was markedly inhibited by antibodies against the α6 andβ1 subunits, whereas anti-α3 did not inhibit binding (Fig. 6). These data demonstrate that RPEcells, like other cells, bind to laminin 1 via α6β1 and not α3β1.

ARPE-19 cell-binding to laminin 5 was partially inhibited by antibodies against the α6 subunitand markedly by anti-β4, whereas anti-α3 did not inhibit binding (Fig. 6). These datademonstrate that RPE cells, like other cells, bind to laminin 5 via the laminin 5 receptor,α6β4.

Finally, ARPE-19 cell binding to laminin 10/11 was partially inhibited by antibodies againstα3 and α6 subunits and markedly by anti-β1 (Fig. 6). These data demonstrate that RPE cells,like other cells, bind to laminin 10/11 via α3β1 and α6β1.

RPE cells express other integrins in vivo: α5β1 (fibronectin receptors) on the basal surface,16,17 and αvβ5 (vitronectin receptors) on the apical surface.16 As expected, given the apicallocation of αvβ5 in vivo, where there are no basement membrane laminins, antibodies against



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αv did not inhibit binding to the basement membrane components laminins 1, 5, or 10/11 (notshown).

Epithelial cells respond to different laminins in two seemingly opposite ways: adhesion andmigration. As a first step in determining the functional activity of individual laminins for RPEcells, we examined the morphology of RPE cells on different laminin isoforms. Laminins 1,5, and 10/11—all potential components of BrM—were adhesive for ARPE-19 cells (Figs. 5,6).However, the morphology of the cells was remarkably different on the three substrates, evenat short times of adherence (60 minutes; Fig. 7). Laminin 5 promoted the most epithelialphenotype; that is, cells were flattest on laminin 5, whereas they were rounded and had theappearance of migrating cells on laminin 10/11 (Fig. 7). Thus, we propose that laminin 5preserves the stability of the mature RPE by promoting an adherent state.

DiscussionThe data presented herein demonstrate that, in our study, RPE cells produced specific lamininsincluding laminins 1, 5, and 10/11. RPE cells bound to these laminins using an integrin-mediated mechanism. Adhesion of RPE cells to laminins 1 and 10/11 is robust and mediated,in part, by integrin receptors for these laminins used by other epithelial cells. In addition, RPEcells synthesize and preferentially bind to laminin 5, a laminin previously thought to be largelyassociated with the dermis. In the skin, laminin 5 is a critical mediator of attachment. Wepropose that interactions between integrins in the RPE and laminins including laminin 5 in theRPE-BM are vital for RPE stability.

In other cell types, the highest affinity integrin receptor for laminin 1 is α6β1, and α3β1 is nota receptor for laminin 1.35 The best-characterized receptor for laminin 5 is α6β4,36 and laminin10/11 binds to many integrins, including α3β1 and α6β1.34 Our data suggest that RPE cells useall these mechanisms as well.

Some of the retinal degenerative diseases (RDDs) are produced by mutations in proteinsintrinsic to the photoreceptors and RPE cells. For example, several genetic RDDs are the resultof mutations in the signal transduction, structural, or metabolic components of thephotoreceptor, including those that cause autosomal dominant retinitis pigmentosa (e.g.,mutations in rhodopsin37 and in peripherin38), Stargardt disease (mutations in ABCA439), andsome forms of Leber congenital amaurosis (e.g., mutations in CRX40,41). Other RDDs resultfrom mutations in genes of the RPE, including Best disease (mutations in VMD2 [bestrophin]42) and some forms of Leber congenital amaurosis (e.g., mutations in RPE6543). In addition,some RDDs are produced by mutations that affect the structure and function of BrM. Theseinclude Doyne's honeycomb retinal dystrophy (mutation in the ECM component, fibulin-3,also known as EFEMP144,45) and Sorsby fundus dystrophy (mutation in the matrixmetalloproteinase inhibitor, TIMP-346). Thus, disruptions in the structure and function of BrMcan lead to RDD.

Other mutations correlate with predisposition for age-related macular degeneration (AMD).The most recently characterized of these are in complement factor H and point to aninflammatory component as a resolute (and perhaps necessary) feature in the multifactorialAMD.47–49 However, mutations in several ECM components also correlate with apredisposition to AMD; these include mutations in the ECM components fibulin-550 and -6,also known as hemicentin.51,52

In addition, there are ocular defects associated with mutations in several other ECM-relatedcomponents, including a gross ocular defect in Marfan syndrome (mutation in the ECMcomponent, fibrillin-153) and muscle-eye-brain disease, in which there is defectiveglycosylation in the ECM receptor, β-dystroglycan.54 Deficiencies in several laminin chains



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also lead to ocular defects, including a recently characterized mutation in the laminin β2 chain.55

Together, these observations point to an intimate relationship between changes in BrM andRPE function. Diminishing of this function can lead to RDD, and changes in BrM are linkedto predisposition to AMD. Alterations in the basic biological interplay between the RPE andBrM can affect the health of the retina; in humans, these changes can be particularly disturbingin the foveal and parafoveal areas because of our reliance on these regions for high-acuityvision.

Defining the function of each integrin and laminin present in RPE cells and BrM is critical indeveloping a model for stability of the RPE based on interactions between integrins andlaminins at the RPE-BrM interface. The composition must maintain RPE quiescence,adherence, polarity, and health, yet be capable of supporting proliferation after damage andmigration into a wound. Our data predict that laminins including 1, 5, and 10/11 and integrinsincluding α3β1, α6β1, and α6β4 are differential players in these phenomena.

AcknowledgmentsSupported by the Köln Fortune Program (SA), National Institute of Neurological Disorders and Stroke Grant R01-NS39502 (WJB, DDH), and National Eye Institute Grants R01-EY12676 (WJB, DDH) and P30-EY13078.

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55. Zenker M, Aigner T, Wendler O, et al. Human laminin beta-2 deficiency causes congenital nephrosiswith mesangial sclerosis and distinct eye abnormalities. Hum Mol Genet 2004;13:2625–2632.[PubMed: 15367484]



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Figure 1.BrM contained several potential laminins. Fluorescent images of bovine sections reacted withantibodies to laminins and laminin chains—in one case, doubly with anti-laminin β2 and anantibody to the basement membrane component, nidogen-1. Bottom right: nuclei of RPE cellscounterstained with DAPI. BrM contained several laminin chains, consistent with the presenceof multiple laminin trimers. bv, blood vessel; no 1°, no primary antibody.



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Figure 2.Laminins were present in the RPE matrix. ECM extracts from bovine BrM were denatured,electrophoresed, transferred, and reacted with antibodies to laminin chains. Bruch's membranecontains several laminin chains, consistent with the presence of multiple laminin trimers.



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Figure 3.The human RPE cells produced laminin proteins. ECM extracts from ARPE-19 cells weredenatured, electrophoresed, transferred, and reacted with antibodies to laminin chains.ARPE-19 matrix contained several laminin chains, consistent with the expression of multiplelaminin trimers by RPE cells. The sizes of the α3A and α3B chains, and their processed forms,are shown (left). The truncated form of the γ2 chain (γ2t) is also noted.



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Figure 4.The human RPE cells secreted laminins. ARPE-19 cells grown on uncoated glass coverslipswere reacted with antibodies against individual laminin chains and an antiserum againstlaminin 5 The presence of flocculent extracellular immunoreactivity for laminin chainsdemonstrates the secretion of laminins by RPE cells. Bar, 20 μm.



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Figure 5.The RPE cells bound to laminins. Laminins 1, 5, and 10/11 were spotted onto plastic atincreasing concentrations. ARPE-19 cells were allowed to bind, and adherent cells were fixedand counted. RPE cells bound saturably to all three substrates, but more cells bound to laminin5 than to laminin 1 or 10/11. Data are from results ± SD of two independent experiments.



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Figure 6.RPE binding to laminins 1, 5, and 10/11 was blocked by specific anti-integrins. ARPE-19 cellsbound to laminins (10 μg/mL); integrin antibodies modulate binding. Cells were allowed toadhere for 60 minutes in the presence of increasing concentrations of anti-integrin antibodiesand then were rinsed, fixed, and counted. Data shown are results ± SEM of at least threeindependent experiments.



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Figure 7.The morphology of the RPE cells was influenced by laminin type. Cells were plated in wellson spots of substrates. (A–C) Outlined edges of spots; (D–F) central areas of the spots. (G–L) Different cell morphology on various laminins, shown at a higher power. The cells attaineda much flatter morphology on laminin 5 than on laminin 1 or 10/11, and they elongated onlaminin 10/11. The cells were allowed to adhere for 60 minutes and then were rinsed and fixed.Bar: (A–F) 20 μm; (G–L) 10 μm.



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Tabl

e 1

Ant

ibod

ies U

sed

for L

amin

in C

hain

s

Lam

inin

Cha

in

Ant

ibod

ySp

ecie

s Rea

ctiv

ity

Nam

eSo

urce

Ref

eren

ceB

ovin

eH

uman

Rat

α1A

L-4

Che

mic

on−

++

NT

α25H

2C

hem

icon

−−

+N

T

α3B

M2

Hun

ter*

22+

++

α4JS

4Sa

nes

23N

T+

+

α54C

7En

gval

l24

, 25

++

+

α540

5So

roki

n26

++

+

β1LT

3C

hem

icon

−+

+N

T

β1C

21Sa

nes

27+

−+

β2C

4H

unte

r†27

++

+

β2D

5H

unte

r28

+−

+

β36F

12H

unte

r*22

++

+

γ1D

18H

unte

r29

++

+

γ2G

B3

Har

lan

−+

++

γ3R

96H

unte

r*‡

++

+

α1β1γ1

(lam

inin

1)

Ant

i-lam

Invi

troge

n−

++

+

α3β3γ2

(lam

inin

5)

9LN

5H

unte

r*11

++

+

All

antib

odie

s fun

ctio

n fo

r im

mun

ohis

toch

emis

try; a

ll bu

t C21

func

tion

for p

rote

in tr

ansf

er (W

este

rn) b

lots

ana

lysi

s. N

T, n

ot te

sted

.

* From

col

labo

ratio

n w

ith th

e B

urge

son

and

Bru

nken

labo

rato

ries.

† From

cel

ls o

btai

ned

from

ATC

C.

‡ Li, B

runk

en, K

och,

and

Hun

ter,

man

uscr

ipt I

n pr

epar

atio

n.


NIH


NIH


NIH



Tabl

e 2

AR

PE-1

9 C

ells

Pro

duce

Lam

inin

RN

As

Lam

inin

Cha

in

Prim

er (5′–

3′)

AR

PE-1

9 C

ells

Con

trol

s

Forw

ard

Rev

erse

Skin

Ret

RM

C

α1TGTAGATGGCAAGGTCTTATTTCA

CTCAGGCAGTTCTGTTTGATGT

+N

D+

+

α2CAAAGAYATTGAAATTTCAAG

GAGTTAATAACAAGRTTCCA

−+

+−

α3CTCATGGTGAGATACAAACT

TTGAAAGGTCTGAAACCCAAA

++

ND

ND

α4GGATGCGCCTTCATGGG

GTCAGGCTGTGGGACAGGA

−N

D+

+

α5GAAGCTGGCTCTTGTCATCC

GCATAATCCAGGCCAAAGAA

++

++

β1TAACGAGGTGGAGTCCGGTTA

AAGGCCCGTCTGGTGAATCAA

+N

DN

D+

β2CTTCGCTTGGGCCTACTTCT

GGATGGTGACCATCGGAACA

+N

D+

+

β3AATGTAGTGGGCCCCAAATG

CTCCTTCTTGCTTCGGTAC

++

ND

+

γ1ACGGCTACTTTGGAGACCCT

GTCCAAACCCAAAGTGGTTG

+N

DN

DN

D

γ2ACCCCCGCAGCTGCAAGCCRTGT

GCAAGCTCGACACTTGTCTGCT

++

++

γ3TACGGTAACGCCTTCTCAGG

CAAGTGGGTGACATTTGCAG

−N

D+

ND

γ3*

TATGGCAACCCTTTCGCGGG

CCAGTGGGTGACACTTGCAG

−N

DN

DN

D

The

hum

an R

PE c

ell l

ine

AR

PE-1

9 an

d se

vera

l oth

er c

ell t

ypes

wer

e an

alyz

ed fo

r exp

ress

ion

of R

NA

enc

odin

g al

l 11

lam

inin

cha

ins.

ND

, not

det

erm

ined

.

* “Hum

aniz

ed”

prim

ers.


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Retinal Pigment Epithelial Cells Synthesize Laminins, Including Laminin 5, and Adhere to Them...

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