|Molecular Vision 2006;
Received 13 February 2006 | Accepted 9 October 2006 | Published 26 October 2006
Vitronectin is present in epithelial cells of the intact lens and promotes epithelial mesenchymal transition in lens epithelial explants
Lavinia Taliana,1 Margaret D.M. Evans,2 Sharyn
Ang,1 John W. McAvoy1
1Save Sight Institute and Department of Anatomy and Histology, University of Sydney and 2Commonwealth Scientific and Industrial Research Organisation, Molecular and Health Technologies, Sydney, NSW, Australia
Correspondence to: Professor John McAvoy, Save Sight Institute/Department of Anatomy and Histology, GPO Box 4337 Sydney 2001, NSW, Australia; email: firstname.lastname@example.org
Purpose: Extracellular matrix (ECM) accumulates during the development of posterior capsule opacification (PCO). Vitronectin, an ECM component that is generally prominent in wound healing, has been detected in PCO specimens. Here we set out to investigate the distribution of vitronectin in the lens and determine how it, and other ECM components, influence the lens epithelial phenotype.
Methods: Rat lens epithelial explants were cultured on vitronectin, fibronectin, and laminin substrata. Explants were monitored for cell migration and the appearance of markers for epithelial mesenchymal transition (EMT), using phase contrast microscopy and immunohistochemistry, respectively. Explants were also monitored for evidence of Smad signaling. Vitronectin expression was analyzed in embryonic and postnatal rodent lens development by immunohistochemistry, western blotting, and in situ hybridization.
Results: Vitronectin, like fibronectin and laminin, provided a good substratum for cellular attachment and migration. However, in the case of vitronectin and fibronectin, this was accompanied by a major phenotypic change. On either vitronectin or fibronectin, but not laminin, most of the cells became elongated, spindle-shaped and were strongly reactive for filamentous α-smooth muscle actin. In these respects this transition was typical of the well known TGFβ-induced EMT. In explants cultured on vitronectin and fibronectin, but not laminin, cell nuclei showed prominent reactivity for Smad 2/3. Vitronectin was also shown to be expressed during embryonic and postnatal development. Initially mRNA and protein were detected in all lens cells, however as development progressed, expression became restricted to cells of the epithelium and transition zone.
Conclusions: The results clearly show that lens cell engagement with a vitronectin or a fibronectin, but not laminin, substratum has a potent EMT promoting effect and that Smad 2/3 signaling is involved. Thus when considering strategies to slow or prevent PCO, these results highlight the need to take into account ECM molecules such as vitronectin that have the capacity to promote EMT.
In the embryo the lens arises from ectoderm situated next to the optic vesicle. By processes of thickening and invaginating, the ectoderm forms the lens vesicle. Cells in the posterior and anterior hemispheres of the lens vesicle form the primary fibers and epithelial cells, respectively. These lens cells are contained within a thick layer of extracellular matrix, the lens capsule, which originates from their basal lamina. Initially, as with the remainder of the ectoderm, the presumptive lens cells have a typical basal lamina but as the lens placode/pit and optic vesicle/cup become closely associated, accumulation of basal laminar material becomes evident. As the lens grows, progressive multilayering of basal lamina material results in the formation of a thick lens capsule [1-3].
The major components of the lens capsule are type IV collagen, heparan sulphate proteoglycans, laminin, and entactin . The presence of lesser amounts of other extracellular matrix components, including fibronectin and type I and III collagen, has also been reported [3-6]. Clearly one important role for capsular ECM components is to maintain the structural integrity and functional properties of the capsule. Other important roles include providing a substratum for lens cell attachment and migration . Despite the importance of these functions, little is known about the capsule components that the lens cells adhere to and migrate on in vivo. The need to identify key substrata for lens cell attachment and migration has taken on some urgency due to the recognition that a common complication of cataract removal is caused by aberrant differentiation and growth of cells left in the lens capsular bag after surgery. Cataract surgery involves removal of the fiber mass and insertion of an intra-ocular lens into the capsular bag. However, many lens epithelial cells remain firmly attached to the capsule and commonly undergo an epithelial mesenchymal transition (EMT). These cells proliferate and migrate along the exposed capsule surface and form plaques of fibroblastic/myofibroblastic cells that cause capsule wrinkling and eventually distort the passage of light along the visual axis (for reviews see [8,9]). This results in a condition, most commonly referred to as posterior capsule opacification (PCO). Further treatment is often given to try to restore some visual acuity; however, it is not without complications and adds a further cost to what is the most common surgery carried out in Western countries [9-11].
Clearly it is important to understand the molecular basis of PCO in order to devise molecular strategies to slow or prevent its progression. In recent years, progress has been made in identifying factors that regulate some of the cellular processes involved in PCO. Work on various rodent models has shown that TGFβ induces lens epithelial cells to undergo EMT and acquire features and markers of PCO [8,9,12-14]. In addition, studies on human lens cells in vitro, as well as analysis of post-operative human cataract material, also indicate that TGFβ is a key regulator of cellular processes in PCO [8,9,15,16]. During the process of posterior migration the cells produce new ECM components that contribute to the formation of fibrotic plaques. As it is well known that ECM, on its own and in concert with growth factors, can profoundly influence cell phenotypes , it is important to determine how such novel ECM components that accumulate during the formation of PCO, influence lens cell behavior. Components deposited include fibronectin and collagen types I and III . These are all commonly involved with wound healing in many systems, and in the lens some of these components have been shown to be associated with extensive cell migration and EMT . Vitronectin is another ECM constituent that has been identified in PCO [19,20]. Vitronectin binds collagens and heparan sulphate proteoglycans and is known to act as a cell adhesion molecule. It has been reported to be a major protein present in the event of wound repair, tumor progression, and development of a variety of tissues . However, so far there is no information on a role for vitronectin in the lens, although lens epithelial cells are known to be sensitive to vitronectin for adhesion to plasticware during primary culture .
In this study we investigated the effects of explanting lens epithelial cells on vitronectin and several other substrata. We found that vitronectin, like fibronectin but unlike laminin, promoted an EMT that is similar to that induced by TGFβ. The similarity to TGFβ-induced EMT extended to the presence of key components of the TGFβ signaling pathway, Smad 2/3, in the nuclei of vitronectin- and fibronectin-treated cells. We also show that vitronectin is expressed in lens epithelial cells during embryonic and postnatal development. These results raise questions about the function of vitronectin in the lens and its role in normal lens biology.
Lens explant studies
Petri dishes (35 mm) were pre-coated with 250 μl vitronectin (1 μg/ml, CSIRO, North Ryde, Sydney, Australia), laminin (10 μg/ml, Calbiochem, San Diego, CA) and fibronectin (10 μg/ml, CSIRO) for 30 min at 37 °C prior to use. Control dishes were left uncoated. Excess matrix solution was removed from each dish and filled with 1 ml of M199 medium supplemented with 0.1% bovine serum albumin (BSA, Sigma, St Louis, MO), fungizone and antibiotic additives. 12 day old rat lens capsules with adherent epithelial cells were removed with fine forceps as previously described  and pinned down with the cells facing the vitronectin/laminin/fibronectin substratum (Figure 1). For further testing, 200 ng of TGFβ was added to some dishes as a positive control to investigate Smad signalling. As another control, a Pan-specific TGFβ antibody (R&D Systems, Inc., Minneapolis, MN) was used to block any endogenous TGFβ that may have influenced Smad activity in the explants on laminin, fibronectin and vitronectin.
Explants were cultured at 37 °C in a humidified 5% carbon dioxide incubator. After 3 days of culture, explants were rapidly fixed in methanol for a maximum of 60 s. Immunohistochemistry was conducted using antibodies to α-smooth muscle actin, ZO1, and phospho-Smad 2/3 (see immunohistochemical techniques below). Samples were viewed by confocal microscopy.
Immunohistochemistry of lens tissue
Mouse embryos at embryonic day 12.5 (E12.5) and E17.5 and rat eyes at post-natal day 7 (P7) and P21 were fixed in 10% neutral buffered formalin, embedded in paraffin and cut into 5 μm sections onto superfrost glass slides. Samples were dried overnight at 37 °C. The following day, samples were dewaxed and rehydrated in a xylene and ethanol series. Specimens were then treated with citrate buffer pH 6.0 for 20 min at 70 °C, cooled to 40 °C, and then placed in PBS prior to immunohistochemistry. All samples were then blocked at RT for 30 min in 3% bovine serum albumin. Following this, samples were incubated in 1:100 dilution of a mouse monoclonal antibody against vitronectin (Ab62; CSIRO) in PBS for 60 min at RT. Samples were then washed three times in PBS and incubated in a 1:20 dilution of FITC-conjugated rabbit anti-mouse secondary antibody (DAKO, Glostrup, Denmark) for 60 min at RT in the dark. Sections were then washed three times in PBS and mounted in 50% glycerol/PBS before viewing by laser confocal microscopy.
Immunohistochemistry of lens explant outgrowth
All dishes were washed three times in PBS plus 1% bovine serum albumin (BSA) post fixation and blocked at RT for 30 min in 3% normal goat serum or 3% rabbit serum. Explants were then incubated with a mouse monoclonal antibody against α-smooth muscle actin (1:100; DAKO) or with a rabbit polyclonal antibody against ZO1 (1:100; Zymed, San Francisco, CA) or a goat polyclonal antibody against phospho-Smad 2/3 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) for 60 min at RT. Samples were then washed three times in PBS and incubated in the dark with goat-anti-mouse Alexa 488, or goat anti-rabbit Alexa 546 or rabbit-anti-goat Alexa 546 secondary antibody, respectively, for 60 min at RT (Molecular Probes, Carlsbad, CA). Explants were then washed three times in PBS and mounted with Universal Mountant (Invitrogen Life Technologies, Carlsbad, CA). In some experiments, explant nuclei were also stained with Hoechst dye.
SDS-PAGE and western analysis
Rat lens capsules (containing lens epithelial cells) were treated and prepared for western blot analysis as described previously . Rat tissue was chosen for western blot analysis to ensure a sufficient quantity of protein lysate. Approximately 20 μg of protein per lane was separated by electrophoresis under reducing conditions using 1 mm 10% gels (BioRad Laboratories, Hercules, CA) using SDS-PAGE and transferred to nitrocellulose membranes (0.2 m Protran; Schleicher & Schuell, Keene, NH). Membranes were then blocked for non-specific binding with 5% skim milk powder in Tris-buffered saline plus 0.05% Tween-20®. Vitronectin was detected by western blot analysis using a mouse monoclonal antibody to vitronectin (Ab62; CSIRO) for 60 min at RT. The nitrocellulose membrane was washed three times in PBS and treated with goat anti-mouse HRP for ECL detection. A sample of vitronectin purified from bovine serum (CSIRO) was used as a positive control.
Adult rat lenses were treated with TRIzol reagent (Life Technologies, Sydney Australia) to isolate total RNA. Following the synthesis of cDNA, this was then amplified using specific primers for vitronectin (5'-GCT GAC CAA GAG TCA TGC AA-3', 3'-GGT TTC CTC CGG GTA GTC AT-5'). These primers amplified a 201 base pair segment of vitronectin fragment.
In situ hydbridization
In situ hybridization was performed as described previously . A probe against vitronectin (previously described) was applied to paraffin sections (5 μm) from embryonic mice (FVB/N) and postnatal rats. After hybridization, sections were washed twice in 4X SSC at 65 °C for 30 min followed by two washes in STE (4X SSC, 20 mM Tris-HCl, pH 7.4, 1 mM EDTA) at 37 °C for 20 min each wash. Sections were then washed in 2X SSC at 65 °C for 10 min, 10 min in 0.5X SSC, twice in 0.1X SSC for 20 min each, and finally 0.1X SSC at RT. Digoxigenin reactivity was detected using an anti-digoxigenin, alkaline phosphatase-linked antibody (Roche, Nutley, NJ) accordingly at 1:100 for 90 min at RT. Samples were then reacted with NBT/BCIP at RT until a color change was observed. Sections were then be washed three times in PBS and mounted in 50% glycerol before viewing by light microscopy.
Vitronectin and fibronectin, but not laminin, promote epithelial mesenchymal transition
Previous studies showed that explanted lens epithelial cells attached and migrated on a laminin or fibronectin substratum in the absence of any serum or growth factor additives . To determine if lens cells could attach and migrate on a vitronectin substratum, we set up explants so that the capsule was uppermost and epithelial cells were face down and in contact with the vitronectin substratum. In this setting cells attached and within several days had migrated out from under the capsule (Figure 2A). On vitronectin the cells took on irregular, migratory morphologies and lost their characteristic polarity and cobblestone-like packing arrangement. Loss of lens epithelial phenotype was also indicated by reduced localization of ZO-1 at the cell margins and strong filamentous reactivity for α-smooth muscle actin throughout the cells (Figure 2B,C).
We also assessed the behavior of cells explanted on fibronectin or laminin substrata. Explants set up on fibronectin behaved in a very similar fashion to explants set up on vitronectin, both in terms of morphologic and molecular changes (Figure 2D-F). On laminin, as on the other substrata, the cells migrated out from under the capsule. However, in contrast to the cells on vitronectin and fibronectin, most of the cells retained a polarized cobblestone-like packing arrangement. The only exceptions were the cells at the leading edge of the migratory sheet (Figure 2G). Localization of ZO-1 showed reactivity at the cell margins and defined a cobblestone-like packing arrangement (Figure 2H). Even though the cells maintained their epithelial morphology, reactivity for α-smooth muscle actin was evident in many cells. However, in contrast to the filamentous arrangement of α-smooth muscle actin in explants on vitronectin and fibronectin, this reactivity in explants on laminin tended to be diffuse, the exception being a few cells at the leading edge of the migrating cell mass (Figure 2I). Cells that migrated on laminin had similar characteristics to cells in standard explants, i.e., explants set up with the capsule in contact with the culture dish and the cells facing uppermost. In standard explants, the cells remained attached to the capsule, did not migrate onto the culture dish, and exhibited a polarized cobblestone-like packing arrangement (Figure 2J) that was highlighted by localization of ZO-1 at the cell margins (Figure 2J,K). Non-filamentous α-smooth muscle reactivity was present in some cells scattered throughout the explant (Figure 2L).
Vitronectin and fibronectin, but not laminin, promote features of TGFβ/Smad signaling
The morphologic and molecular changes induced by explanting lens epithelial cells onto vitronectin and fibronectin were characteristic of the effects that TGFβ has on lens epithelial explants [12,13]. To investigate if the vitronectin and/or fibronectin substrata influenced TGFβ signaling through the Smad pathway, we investigated the subcellular distribution of phospho-Smad 2/3. Using an antibody that recognized both phospho-Smads, we conducted an immunohistochemical analysis of explants cultured on the different extracellular matrix substrata. Some reactivity for phospho-Smad 2/3 was detected in the cytoplasm of cells cultured on laminin but little or no Smad 2/3 reactivity was detected in their nuclei (Figure 3B,C). In contrast, there was distinct localization of phospho-Smad 2/3 in the nuclei of cells that migrated on vitronectin and fibronectin (Figure 3E,F and Figure 3H,I). In additional experiments, when TGFβ was added to the cultures, the cells on laminin underwent the characteristic TFGβ-induced morphologic and molecular changes and phospho-Smad 2/3 was localized in their nuclei (Figure 3K,L). The addition of TGFβ had no effect on the explants cultured on fibronectin or vitronectin (data not shown).
Supplementary experiments were conducted to determine if a pan-specific TGFβ antibody could block the TGFβ-like effects induced by the vitronectin or fibronectin substrata. The addition of 50 μg/ml of TGFβ antibody had no effect on cells migrating on vitronectin or fibronectin. The cells still showed the irregular, migratory morphologies and lost their characteristic polarity and cobblestone-like packing arrangement whether the TGFβ antibody was present or not (compare Figure 4B with Figure 4A and Figure 4D with Figure 4C). Confirmation that this particular antibody is capable of blocking the effects of TGFβ is shown by adding it to standard explants treated with 250 pg/ml of TGFβ2. The characteristic TGFβ-induced phenotypic changes (Figure 4E) are blocked by the presence of 50 μg/ml the TGFβ antibody (Figure 4F). As expected, the addition of the TGFβ antibody had no effect on cells migrating on laminin. These cells maintained their polarity and cobblestone-like packing arrangement (Figure 4G,H).
Expression of vitronectin in the lens during embryonic and postnatal development
In other systems, vitronectin has been shown to be expressed in normal as well as pathological development. To determine if vitronectin is expressed in the rodent lens during development, we carried out immunohistochemical and in situ hybridization analyses for proteins and mRNA, respectively.
Immunohistochemistry was carried out using specific vitronectin antibodies to detect protein. This showed that most lens cells at embryonic days 12.5 (E12.5) and E17.5 had a diffuse cytoplasmic reactivity for vitronectin (Figure 5A,D and Figure 5B,E). At later embryonic stages, reactivity for vitronectin became weaker in the primary fibers but was stronger in the differentiating epithelium (not shown). This pattern continued postnatally and by postnatal day 21 (P21), reactivity was absent from the fibers but remained strong throughout the epithelium and transitional zone (Figure 5C,F). Vitronectin was not evident in the lens capsule at any time point studied.
Western blot analysis confirmed the presence of vitronectin in the rodent lens. Lysates of capsules (with adherent epithelial cells) from P21 mouse lenses probed for vitronectin showed the presence of bands that matched the purified vitronectin bands at 70 kDa and 65 kDa. The vitronectin molecule typically denatures readily, as with boiling in this case, to produce these distinctive bands (Figure 5G, arrows).
In situ hybridization analysis of rodent lenses showed that vitronectin mRNA was expressed in most lens cells at E12.5 (Figure 5H). At later embryonic stages expression progressively became weaker in the fibers but remained strong in the epithelium and transitional zone. Reduced expression in fibers was detected at E17.5 (Figure 5I). Postnatally at P7 and P21, a strong signal for vitronectin mRNA was present in the epithelium and transitional zone but was undetectable in the fibers (Figure 5J,K).
This study has shown that vitronectin, like fibronectin and laminin, provides a good substratum for cellular attachment and migration. However, in the case of vitronectin and fibronectin, this was accompanied by major phenotypic changes. On either vitronectin or fibronectin, but not laminin, most of the cells became elongated, spindle-shaped, and were strongly reactive for filamentous α-SMA. In these respects this transition was typical of the well known TGFβ-induced EMT that has been described in several cataract models [12,14,16,27-29]. Our study also showed that attachment and migration on vitronectin and fibronectin, but not laminin, coincided with the translocation of Smad 2/3 into lens cell nuclei. As lens epithelial cells are known to express all three TGFβ isoforms it is possible that this substratum-induced EMT is mediated by endogenous TGFβ . Evidence against this possibility came from the experiment that showed inclusion of the pan specific TGFβ blocking antibody had no effect on the vitronectin or fibronectin-induced EMT. While at this stage we cannot rule out involvement of some residual TGFβ, the results clearly show that lens cell engagement with a vitronectin or a fibronectin, but not laminin, substratum has a potent EMT promoting effect.
Interestingly, this study has shown that vitronectin- or fibronectin-induced EMT is accompanied by increased Smad 2/3 reactivity in cell nuclei. An important role for Smad signaling in EMT has previously been shown in studies on Smad 3 null mice . Although it is well known that nuclear translocation of Smad proteins is a predominant feature of TGFβ signaling , evidence is now emerging that the Smad activation that is central to EMT can also occur independently of TGFβ . In one example that is particularly relevant to this study, it has been shown that in the presence of vitronectin (with no added TGFβ), dermal fibroblasts increased promoter activity of Smad 3 which caused the transformation of these cells into scleroderma fibroblasts that are associated with fibrosis . In addition, earlier studies of lens epithelial cells suspended in collagen gels indicated that newly expressed integrins were involved in promoting EMT . Taken together, this indicates that pathways, other than TGFβ-activated Smad signaling, may have an important role in the induction/promotion of EMT in the lens.
Given the potency of vitronectin and fibronectin to bring about EMT, it may appear surprising that these ECM components have been detected in the lens. Fibronectin has previously been detected, although in much smaller amounts than laminin, in bovine lens capsule . Now, for the first time, the current study has shown that lens cells express vitronectin during embryonic and postnatal development. During embryonic development, both mRNA and protein were detected in lens epithelial cells and primary fibers. As development progressed, both protein and mRNA expression were reduced in the maturing fiber cells in the lens cortex. Surprisingly, and although vitronectin is often present as an ECM component in other tissues, no clear localization was evident in the lens capsule. This does not necessarily mean that it is restricted to the intracellular compartment, as key epitopes may be masked by interactions with other components (see for example, ). Further biochemical analysis of the capsule will be needed to rule out this possibility. Nevertheless, strong reactivity was present in lens cells, notably the epithelial cells and early elongating fibers in the transitional zone at the lens equator. The role of vitronectin in this location is not clear. In other systems it has a well recognized role in wound healing and can be released from the cells after trauma (such as cataract surgery) to participate in wound healing events . Alternatively, it may have some other function. Recent studies on other molecules that are well known for their structural roles have identified nuclear translocation and signaling functions. For example, SPARC is an ECM molecule that is generally found in basement membranes and has a role in the wound healing response. However, in the lens, SPARC has been shown to be restricted to the intracellular compartment and to enter the nucleus [36,37]. Its role in this setting is not known, although knockout studies have shown that in its absence postnatal lens development is defective and cataract develops . Similar studies may help establish a role for vitronectin in the lens.
While much needs to be learned about vitronectin (and fibronectin) and related integrin signaling, the current study shows that lens cell attachment to these ECM components promotes EMT. Since vitronectin and fibronectin have been detected in PCO specimens, it is quite likely that in vivo they have similar EMT promoting effects as shown in our explant cultures [9,19,20]. With this in mind it would appear that in any approaches aimed at preventing PCO, consideration needs to be given to including strategies that block lens cell attachment to ECM molecules such as vitronectin that have the capacity to promote EMT.
The authors would like to thank Barbara Bojarski of CSIRO Molecular and Health Technologies for assistance with the histology. Supported by grants from Sydney Foundation for Medical Research, NIH (R01 EY0-3177), and NHMRC Australia. Portions of this study were presented at the 2005 ARVO meeting in Fort Lauderdale, FL (JW McAvoy, L Taliana, MDM Evans. SL Ang, B Bojarski. A role for vitronectin in the lens. 2005. 2875/B428.)
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