Ubiquitin–Proteasome Pathway Function Is Required for Lens Cell Proliferation and Differentiation

Abstract

purpose. The ubiquitin proteasome pathway is involved in the regulation of many cellular processes, such as cell cycle control, signal transduction, transcription, and removal of obsolete proteins. The objective of this work was to investigate roles for this proteolytic pathway in controlling the differentiation of lens epithelial cells into lens fibers.

methods. bFGF-induced cell proliferation was monitored in rat lens epithelial explants by bromodeoxyuridine (BrdU) incorporation. Indicators of lens differentiation included expression of crystallins, lens major intrinsic protein 26 (MIP26), CP49, and filensin and morphologic changes such as cell multilayering and elongation or loss of nuclei. Clasto-lactacystin-β-lactone, the proteasome-specific inhibitor, was used to study the role of the proteasome in controlling the proliferation and differentiation processes.

results. Explants treated with bFGF initially underwent enhanced proliferation, as indicated by BrdU incorporation and multilayering of the epithelial cells. By 4 days of bFGF treatment, most cells withdrew from the cell cycle, as indicated by diminished BrdU incorporation. After 7 days of treatment with bFGF, lens epithelial explants displayed characteristics of lens fibers, including higher ratios of crystallins to other cytoplasmic proteins and expression of large quantities of MIP26, CP49, and filensin. Adding the proteasome inhibitor to the medium simultaneously with bFGF (day 0) or at day 4 prohibited or delayed bFGF-induced cell proliferation and differentiation. This was indicated by reduced BrdU incorporation and decreased expression of β- and γ-crystallins, MIP26, CP49, and filensin. Proteasome inhibition also significantly decreased the number of layers and the sizes of differentiating fibers.

conclusions. These data show that proteasome activity is required not only for lens cell proliferation but also required for the transition from the epithelial phenotype to the fiber phenotype.

The lens is composed of epithelial cells and fiber cells. A single layer of epithelial cells covers the anterior surface of the lens, and fiber cells occupy the rest of the volume of the lens. Fiber cells are derived from epithelial cells. Undifferentiated cells in the central epithelium are nonmitotic and cuboidal. ^{1 2} Proliferation is restricted to the epithelial cells. Epithelial cells adjacent to the equator show the highest proliferative index, whereas cells in the central region do not normally proliferate. ³ During differentiation into fiber cells, lens epithelial cells exit the cell cycle and undergo morphologic and biochemical changes that result in an elongated cell devoid of nuclei and other organelles. ⁴ Fibers with increasingly advanced stages of differentiation accumulate concentrically at the interior of the lens. ⁵ Proper execution of the differentiation program and the formation of mature fibers is required for lens transparency, and abnormalities that result in incomplete degradation of intracellular organelles are associated with various forms of cataract. ^{6 7}  

In mammals, the fibroblast growth factor (FGF) family contains 23 members. FGFs have been implicated in the regulation of many key cellular responses associated with and involved in developmental and physiologic processes. These include proliferation, differentiation, migration, apoptosis, angiogenesis, and wound healing. ⁸ Basic FGF (bFGF), also called FGF-2, is a dominant member of the FGF family. This growth factor induces rat and human lens epithelial cells to proliferate, migrate, spatially reorganize such that cells make a 180° rotation, and differentiate into fiber cells. ^{1 2 9 10} Whereas low concentrations of FGF, such as 5 ng/mL, stimulate cell proliferation, higher concentrations (40–100 ng/mL) of FGF are required to induce fiber differentiation. ^{9 11 12} In early models of differentiation in cells maintained in culture, the inhibition of actin microfilaments by treatment with cytochalasin was shown to disrupt the early stages of differentiation. ^{2 13}  

The ubiquitin–proteasome pathway (UPP) is a major cytosolic proteolytic pathway in most eukaryotic cells. There are two stages in the UPP: substrate recognition by covalent ligation of ubiquitin to substrate proteins to form polyubiquitinated protein conjugates, and subsequent degradation of the ubiquitin conjugates by the 26S proteasome in an adenosine triphosphate (ATP)–dependent reaction. ¹⁴ As documented in several types of cells, the UPP is involved in the regulation of multiple processes, including cell cycle regulation, transcription, protein quality control, DNA repair, and immune responses, and defects in this proteolytic pathway have been shown to play important roles in several human diseases, such as Parkinson disease, Alzheimer disease, and certain types of cancer. ^{15 16 17 18 19 20 21}  

In earlier studies, we demonstrated that lens epithelial cells have a fully functional UPP ^{22 23 24 25 26 27 28 29} and that ubiquitin conjugation activity increases during early stages of lens fiber cell differentiation. ²⁸ Using a rat lens explant model, we showed that several ubiquitin-conjugating enzymes were activated in differentiating lens fibers, whereas the cullin subunit of a specific ubiquitin ligase was downregulated. ³⁰ Based on these data and information gleaned from other types of cells, we hypothesized that the UPP plays a role in controlling lens cell proliferation, establishing the differentiation phenotype, and executing the morphologic remodeling required during lens cell proliferation, differentiation, and lens organogenesis. In this study, we tested this hypothesis by determining the effects of proteasome inhibition on bFGF-induced lens cell proliferation and differentiation in rat lens explants. We found that treatment with proteasome inhibitor delays bFGF-induced proliferation and differentiation, including biochemical changes, such as expression of fiber-specific proteins, β- and γ-crystallins, MIP26, CP49, and filensin, and morphologic changes including elongation and enlargement of lens fibers. These data indicate that proteasome activity is required for lens cell proliferation and differentiation. 

Materials and Methods

Materials

Rabbit polyclonal anti–β- and –γ-crystallin antibodies were kind gifts from Nicolette Lubsen (University of Nijmegen, Netherlands). Rabbit polyclonal anti-MIP26 antibody was from Joseph Horwitz (Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA), and rabbit polyclonal antibodies to CP49 and filensin were from Paul FitzGerald (University of California, Davis, CA). bFGF was purchased from PeproTech (Rocky Hill, NJ). Medium 199 was purchased from Invitrogen (Carlsbad, CA), and clasto-lactacystin β-lactone, hereafter called lactacystin, was purchased from Boston Biochem (Cambridge, MA). The mouse monoclonal antibody to β-actin and all chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or Bio-Rad (Hercules, CA). 

Preparation of Lens Epithelial Explants

All procedures involving the use of animals were in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care and Use Committee (ACUC) of this Center. 

Five-day-old Wistar rats were killed, and the lenses were removed and dissected. The whole lens capsule, including the anterior region and the germinative zone, was peeled from the lens and pinned to tissue culture dishes, as previously described, ^{30 31 32 33} such that the capsule was in contact with the bottom of the tissue culture dish. To determine the morphologic features of cell proliferation and differentiation, only the anterior region of the explant was examined. Proteins from the whole explant were used in Western blotting. Lens epithelial explants were cultured in medium 199 supplemented with 1.0 μg/mL insulin, 0.1% bovine serum albumin (BSA), 100 IU/mL penicillin, 100 μg/mL streptomycin, 2.5 μg/mL amphotericin B, and 25 mM HEPES at 37°C in 5% CO₂. To induce differentiation, bFGF was added to the culture medium at a final concentration of 100 ng/mL. Lower levels of bFGF were also tried (data not shown). To investigate the effect of proteasome inhibition on the proliferation and differentiation of rat lens epithelial cells, lactacystin was added to the culture medium at a final concentration of 10 μM. This is the minimal level of lactacystin that inhibits more than 90% of the chymotrypsin-like activity of the proteasome in cultured lens epithelial cells (Shang et al., unpublished data, January 2004). Explants were incubated in this medium at 37°C for 2 to 21 days, and the medium was changed every 3 days. To determine DNA synthesis, 100 μM bromodeoxyuridine (BrdU) was added to the medium for 2 hours, and the explants were fixed in 10% neutral-buffered formalin for 1 hour and stored at 4°C in 70% ethanol. The explants were rinsed with PBS, pre-embedded in 3% agar, dehydrated through a series of ethanol gradients, and washed with xylene before they were embedded in paraffin. The embedded explants were sectioned at 5 μm, dewaxed, hydrated, and stained with hematoxylin and eosin. To detect BrdU incorporation, the sections were incubated in 50% formamide and 2× SSC (0.3 M NaCl, 0.03 M sodium citrate) at 65°C for 2 hours. After the sections were washed with 2× SSC and subsequently incubated in 2 M HCl for 30 minutes at 37°C, they were washed once with 0.1 M borate buffer (pH 8.5) for 10 minutes. The sections were blocked with 10% horse serum for 1 hour and incubated with anti-BrdU mouse monoclonal antibody in Tris-buffered saline (TBS; 50 mM Tris, 150 mM NaCl, pH 7.4) at 4°C overnight. After rinses in TBS, the sections were incubated with biotinylated horse antimouse immunoglobulin G (IgG; Jackson ImmunoResearch, West Grove, PA) for 4 hours at room temperature. Avidin–biotin complex reagent (Vector Laboratories, Burlingame, CA) in TBS was applied to the sections, and the sections were incubated for 1 hour. Next, 0.25 mg/mL diaminobenzidine, along with 0.01% H₂O₂ and 0.04% nickel chloride, was applied as a substrate for the peroxidase reaction for 5 minutes. The sections were then thoroughly washed and mounted with coverslips for examination. 

SDS-PAGE and Western Blot Analysis

Explants were rinsed once with cold PBS and homogenized in Laemmli buffer, and the proteins were resolved by SDS-PAGE. The gels were stained by Coomassie brilliant blue. Levels of β- and γ-crystallins, CP49, filensin, and MIP26 were also determined by Western blot analysis using the antibodies mentioned. In brief, the samples were resolved on 12% SDS-PAGE gels and then transferred to a nitrocellulose membrane. The membrane was blocked with TST (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 0.02% Tween-20) containing 2.5% milk proteins for 1 hour and incubated with the primary antibodies overnight at 4°C. The membrane was then washed four times with TST and incubated with a horseradish peroxidase (HRP)–conjugated secondary antibody for 1 hour at room temperature. Immunocomplexes were visualized by incubating the membrane with enhanced chemiluminescence reagents (Super Signal; Pierce, Rockford, IL) and exposed to x-ray film. For quantification of the expression of specific proteins, the density of the corresponding bands was quantified with an imaging densitometer (Amersham Biosciences, Sunnyvale, CA) and evaluated by an image analyzer (Image Quant software, ver. 3.3; Molecular Dynamics, Sunnyvale, CA). 

Morphologic Analysis

Lens epithelial explants were sectioned as described. To accurately define the boundaries of differentiating fibers on the explants, cell membranes were immunostained with MIP26 antibody. Cross-sectional cell areas were calculated with commercial software (Scion Corporation, Frederick, MD). Denucleation and nuclear elongation were determined by counting and measuring nuclei within predefined fields on the slides of explants. 

Statistical Analysis

Data were expressed as the mean ± SD and were analyzed (Excel 2002; Microsoft, Redmond, WA). Statistical analysis was performed by paired Student t test. Statistical significance was defined as P < 0.05. 

Results

Proteasome Activity Is Required for Proliferation and Differentiation, as Indicated by Morphologic Features of Multilayering, Elongation, and Enlargement

Cell proliferation, as indicated by BrdU incorporation, started within 1 day of bFGF treatment and reached a maximum rate by 2 days of bFGF treatment. ^{4 9 30} At this time, approximately 25% of the nuclei were BrdU positive, and BrdU incorporation occurred in the upper layers of cells and in the original basal epithelial monolayer of cells (Fig. 1B) . Thereafter, the cells started to withdraw from cell cycle. By day 4 of bFGF treatment, most of the cells completed the proliferation program, and only approximately 5% of nuclei showed BrdU incorporation (Fig. 1E) . This was limited to the original epithelial monolayer. Explants not treated with bFGF showed basal levels of proliferation and did not show multilayering (Figs. 1A 1D) . In these explants, the mitotic index remained at approximately 6% throughout the period of the experiments (Figs. 1A 1D) . When the proteasome inhibitor was included, bFGF-stimulated proliferation was markedly attenuated (Fig. 1 , compare panels C versus B), and multilayering of the cells was markedly inhibited (Fig. 1 , compare panels C versus B and panels F versus E). Mitotic indices of the differently treated lens epithelial explants are summarized in Figure 1G . Taken together, these data corroborate previous reports that bFGF stimulates cell proliferation. Specifically, they indicate that, under these conditions, cell proliferation continues for 2 days and that cells in all layers of the explant remain in the cell cycle. However, by 4 days or in the presence of the proteasome inhibitor, most cells withdraw from the cell cycle and proliferation is limited, continuing only in the original epithelial cell layer. 

Differentiation in the fibers is characterized by a sequence of morphologic changes that occur as they mature. These include cell elongation, multilayering, increased cross-sectional area, fiber denucleation, and elongation of nuclei. ^{30 34 35 36} As shown in Figure 2A , extending the time in culture from 4 to 15 days in the presence of bFGF increases the average number of cell layers from 5.4 to 11.2. Inhibition of the proteasome attenuated the multilayering by approximately 70%. bFGF treatment also induced cell elongation (Fig. 2B)and increased the cross-sectional cell area (Fig. 2C) . After 15 days of culture in the presence of bFGF, cell length doubled compared with explants not exposed to bFGF. Incubation with the proteasome inhibitor attenuated this by approximately 50% (Fig. 2B) . Treatment with bFGF for 15 days also resulted in a fivefold increase in the cross-sectional area of the cells (Fig. 2C) . The cross-sectional area increased only by approximately half as much in the presence of lactacystin. Thus, adding lactacystin to the bFGF-treated lens explants thwarted each of these differentiation processes, indicating that proteasome activity is required for these differentiation-related processes to occur. Loss of nuclei and lengthening of nuclei (data not shown) also indicated bFGF-induced differentiation. 

It appeared possible that some of the effects noted resulted from lactacystin-induced limitation in proliferation because the inhibitor was added at day 0. To determine whether the proteasome controls differentiation independent of proliferation, we further tested the effects of inhibition of the proteasome on differentiation by adding lactacystin at day 4, when most cell proliferation was complete. As shown in Figure 3 , treatment with bFGF for 15 days increased the cross-sectional cell area by sixfold (Fig. 3) . Adding lactacystin at day 0 reduced cell size, as noted. Adding lactacystin at day 4 was also associated with a similar decrease in the cross-sectional area of cells. If proteasome activity was not required for postmitotic differentiation-related events, then including the proteasome inhibitor at day 4 would have had only limited effects. Thus, proteasome activity appears to be required not only for proliferation but also for the differentiation-related enlargement of epithelial cells as they differentiate into fibers. 

Proteasome Activity is Required for Differentiation, as Evaluated by Expression of Crystallins, MIP26, CP49, Filensin, and Actin Relocalization

In the mammalian lens, epithelial and fiber cells contain α-crystallin, but the expression of β- and γ-crystallin is thought to be limited to fiber cells. Therefore, higher levels of total crystallins and specific enhancements in expression of β- and γ-crystallins serve as biochemical markers for fiber differentiation. ^{37 38 39 40} We defined cytoskeletal proteins, including actin, vimentin, and α-tubulin ^{13 41} (Fig. 4A) , as proteins expressed in cultured lens cells and within these explants. These protein levels were held constant and were used to normalize for the expression of crystallins (Fig. 4B) . That bFGF significantly promoted lens fiber differentiation is indicated by the twofold to threefold higher ratio of crystallins to cytoskeletal proteins in explants exposed to bFGF. After 7 days of bFGF treatment, crystallins were the most abundant proteins in the lens explants (Fig. 4A , compare lanes 2 and 1 in 4A; 4B). When the proteasome inhibitor was added simultaneously with bFGF (day 0), crystallin levels were significantly reduced compared with levels of cytoskeletal proteins (Fig. 4A , lane 3). Because the initial proliferation of epithelial cells is a prerequisite for subsequent fiber differentiation, the effect of the proteasome inhibitor on expression of crystallins might have resulted from the inhibition of cell proliferation. To determine whether an effect of proteasome inhibition on crystallin expression was independent of cell proliferation, as with the morphologic investigation noted, we added the proteasome inhibitor after 4 days of bFGF treatment, when most of the cells had completed the proliferation program. Adding the proteasome inhibitor at day 4 also reduced the expression of crystallins, as indicated by a decreased ratio of crystallins to cytoskeletal proteins (Fig. 4A , compare lanes 4 and 2). These results imply that the proteasome is required for cell proliferation and lens cell differentiation. 

Mousa and Trevithic ¹³ and Sussman et al. ⁴¹ noted in cells in culture and in lens tissue explants, respectively, that as differentiation progresses, actin relocalizes and that in differentiated cells in tissues, the highest levels appear at the cell margins. Comparison of actin staining in Figure 4Cshows that actin was clearly observed at the cell margins when lens tissues were maintained and allowed to differentiate in culture in the presence of bFGF. In comparison, when differentiation was stalled because of the absence of growth factor in the medium, actin staining at the intercellular margins, between the cuboidal epithelial cells, was less pronounced. 

The requirement for a functional proteasome for differentiation was further investigated using β- and γ-crystallins as differentiation markers. bFGF treatment for 7 days enhanced the expression of β- and γ-crystallin in the explants by approximately 3.5- and 18-fold, respectively. However, adding lactacystin at day 0 prevented these increases in β- and γ-crystallins, as indicated by lower ratios of the levels of these crystallins to the level of β-actin (Fig. 5A and 5B ; compare lanes 3 and 2). Inhibition of the proteasome had a stronger effect on the expression of γ-crystallins than on the expression of β-crystallins. Adding lactacystin at day 4 also decreased the expression of β- and γ-crystallin, though the effect was less than that observed when proteasome inhibitor was added at day 0 (Fig. 5A and 5B ; compare lanes 4 and 2). 

The major intrinsic protein 26 (MIP26), also called aquaporin 0, is the most abundant membrane protein and is specifically expressed in lens fiber cells. ³⁸ MIP26 plays an important role in maintaining lens transparency by reducing the interfiber space, as suggested by its ability to function as a weak water channel and possibly as an adhesion molecule. ⁴² Mutations in the MIP gene have been linked to genetic cataracts in mice. ⁴³ In this study, we tracked the expression of MIP26 as a specific marker of lens cell differentiation. We found that MIP26 levels were 8.5-fold higher in explants treated with bFGF for 7 days (Fig. 5C ; compare lanes 2 and 1). As with γ-crystallins, adding lactacystin to the bFGF-treated lens explants at day 0 prevented FGF-induced expression of MIP26 (Fig. 5C , lane 3). Adding lactacystin at day 4 also decreased the expression of this differentiation marker (Fig. 5C , lane 4). 

CP49 and filensin, components of beaded filament proteins, are also only expressed in differentiated lens fiber cells. ^{44 45 46} Accordingly, these proteins provide additional markers of lens fiber differentiation. At day 7, levels of CP49 and filensin (Figs. 5D 5E ; compare lanes 2 and 1) were 5.4-fold and 1.9-fold higher in explants treated with bFGF, respectively. Adding lactacystin to the bFGF-treated lens explants at day 0 prevented CP49 expression, and adding the inhibitor at day 4 also decreased the expression of CP49. Results with filensin were similar to those obtained for CP49 and for the other differentiation markers noted (Figs. 5A 5B 5C 5D 5E ; compare lanes 3 and 4 with lane 2). 

Discussion

Temporally and spatially controlled proliferation and differentiation are required for lens formation and the maintenance of lens function. ^{47 48 49 50 51} The UPP regulates cell proliferation and differentiation in many eukaryotic cells but has not been explored systematically in eye tissues. Based on our previous observation that ubiquitin conjugation activity increases during the early stages of lens cell differentiation ^{28 30} and that the UPP is essential for controlling the cell cycle in cultured lens epithelial cells and other types of cells, ^{52 53} we hypothesized that the UPP plays a role in regulating lens cell proliferation and differentiation. Consistent with our hypothesis, we found that inhibition of the proteasome diminished the initial proliferation and subsequent differentiation of lens cells in the in vitro lens differentiation model. 

A boost of proliferation preceding terminal differentiation is a common phenomenon in many types of stem or progenitor cells. This expansion of available cells provides enough mass of cells for tissue formation upon terminal differentiation. Thus, this initial proliferative burst can be considered the first stage of the differentiation process. ¹¹ Consistent with previous reports, we found that the proteasome is required for the initial proliferation of lens epithelial cells during bFGF-induced lens cell differentiation. ^{30 32} In addition, we demonstrated that proteasome activity is required for differentiation-related morphologic changes, such as multilayering, cell lengthening, and enlargement of cellular volume. Inhibition of proteasome activity reduced the bFGF-induced multilayering and enlargement of the cross-sectional area of the differentiating fibers by as much as 50%. Our results regarding the role of the proteasome in lens cell differentiation are consistent with data obtained from other differentiation systems. For example, a requirement for proteasome activity is noted for the cAMP-induced differentiation of neuroblastoma cells. ^{54 55} Furthermore, it has been reported that proteasomal degradation of ubiquitinated proteins plays an important role in the enucleation of mammalian erythroblasts. ⁵⁶ Proteasome activity is also required for adipocyte differentiation ⁵⁷ and for the regulation of myogenic differentiation through the activation of NF-κB. ⁵⁸ Some reports also indicate that the inhibition of proteasome activity is positively associated with withdrawal from the cell cycle and with induction of differentiation processes in oligodendroglial cells and neuroblastoma cells. ^{59 60 61}  

In addition to morphologic changes, we demonstrated that proteasome activity is also required for the expression of several fiber-specific proteins in postmitotic fiber cells. These include β- and γ-crystallins, MIP26, CP49, and filensin. However, it is not clear at present whether the effects of proteasome inhibition on the expression of fiber-specific proteins result from blockage of the transcription processes or from blockage of the signal transduction process that triggers the expression of these fiber-specific proteins. It was reported that the UPP is involved in proliferation-independent transcription processes, such as chromatin remodeling and degradation of transcription factors. ⁶² It is plausible that proteasome-dependent degradation of repressors, which block the transcription of fiber-specific proteins in epithelial cells, is required for lens differentiation. Future identification of such repressors will allow us to elucidate molecular mechanisms by which the UPP executes its role in controlling lens cell differentiation. 

These data also indicate that cells at the anterior surface of the lens, which in vivo are quiescent, can reenter the cell cycle on stimulation with bFGF (Lavker RM, et al. IOVS 2005;46:ARVO E-Abstract 2410). They open the possibility of using such proliferative potential for lens remodeling and repair. ^{63 64} Because some proliferation occurs even in the presence of lactacystin, it appears either that some cells can escape the restriction of proliferation imposed by the inhibition of the proteasome or that the inhibition is incomplete. We have observed the latter (data not shown). 

Taken together, the data presented here demonstrate that the UPP is not only required for the proliferation of lens cells, but it is also required for the subsequent differentiation and maturation of lens fibers. This work sets the stage for our continuing studies regarding the molecular mechanisms by which the UPP controls biochemical and morphologic phenomena associated with transitions from the epithelial to the differentiated fiber phenotype. 

 Supported in part by National Institutes of Health Grants EY13250 (AT) and EY11717 (FS); National Eye Institute Core Grants EY13078 and EY14083–01A2 (AT); the Johnson & Johnson Focused Giving Program Award (AT); the US Department of Agriculture, under agreements No. 58-1950-9-001 and 1950-51000-060-01A.

 Submitted for publication February 25, 2005; revised August 4, 2005, and January 4, 2006; accepted April 17, 2006.

 Disclosure: W. Guo, None; F. Shang, None; Q. Liu, None; L. Urim, None; M. Zhang, None; A. Taylor, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Cell proliferation and subsequent differentiation are delayed by inhibition of the proteasome. Lens capsules were peeled from 5-day-old rat lenses and cultured in the absence (A, D) or presence of 100 ng/mL bFGF alone (B, E) or 100 ng/mL bFGF together with 10 μM lactacystin (C, F) for 2 (A–C) and 4 days (D–F). Explants were labeled with 100 μM BrdU for 2 hours at day 2 (A–C) or day 4 (D–F). After fixing and sectioning, BrdU incorporation was detected with antibodies to BrdU. The yellow/brown nuclei (arrows) indicate BrdU incorporation and cell proliferation. The percentage of BrdU-positive cells is summarized (G). Ld0, lactacystin added at day 0.

Figure 2.

 

Proteasome inhibitor decreases or delays differentiation-related multilayering and enlargement of the lens cells. Lens explants were cultured as indicated in Figure 1for up to 15 days. Where indicated, 10 μM lactacystin was added at day 0. The number of cell layers (A), the cell length (B), and a cross-sectional cell area (C) were determined as indicated in Materials and Methods. Results shown are from four explants examined at the same time. The experiment was repeated three additional times with similar results. *P < 0.05 compared with control; #P < 0.05 compared with bFGF; †P < 0.05 compared with 4-day bFGF. Scale bars: (A) 50 μm; (B) 30 μm.

Figure 3.

 

Proteasome activity is required for the differentiation of lens fiber cells. Lens explants were cultured for 15 days in the absence or presence of 100 ng/mL bFGF alone or of 100 ng/mL bFGF with 10 μM lactacystin added at day 0 (bFGF+Ld0) or day 4 (bFGF+Ld4). Preparation of the sections is described in Materials and Methods. (A) Changes in cross-sectional cell area. (B) Typical MIP26 staining pattern indicates cellular boundaries of differentiating fibers. *P < 0.05 compared with control; #P < 0.05 compared with bFGF.

Figure 4.

 

bFGF promotes, and proteasome inhibitor limits or delays, differentiation. (A) Lens capsules were cultured as described in the legend to Figure 3 . Explants were collected at 7 days and lysed in 1× Laemmli buffer. Proteins in the lysate were resolved by 12% SDS-PAGE. The region labeled “Cyto” contains actin, tubulin, vimentin (as indicated by Western blotting), and other unknown proteins. The region labeled “Crys” contains all three classes of crystallins and some unknown proteins. (B) The gel was scanned, and the ratio of levels of crystallins (Crys.) to levels of cytoskeletal proteins (Cyto.) was calculated as indicated in the text. *P < 0.05 compared with control; #P < 0.05 compared with bFGF. (C). Lens explants were cultured for 7 days, as described in the legend to Figure 1 , in the absence (top) or presence (bottom) of 100 ng/mL bFGF, which also contains 10 μM lactacystin. Arrowhead: intercellular juncture between nondividing cuboidal epithelial cells in the lens explant. This is not as clearly decorated with actin as is observed at the cell juncture (full arrow) between dividing and differentiating fiber cells in the lens explant. Explants treated with nonimmune antiserum did not reveal anything (data not shown).

Figure 5.

 

Proteasome inhibitor decreases or delays bFGF-induced expression of β- and γ-crystallin, MIP26, CP49, and filensin. Lysates prepared from explants cultured for 7 days in the absence or presence of 100 ng/mL bFGF alone or 100 ng/mL bFGF with 10 μM lactacystin, added at day 0 or day 4, were resolved on SDS-PAGE gels. Lysates with equivalent amounts of cytoskeletal proteins were loaded. Relative levels of (A) β-crystallin, (B) γ-crystallin, (C) MIP26, (D) CP49, and (E) filensin were determined using Western blotting and were quantified by densitometry. *P < 0.05 compared with control; #P < 0.05 compared with bFGF.

The authors thank Peggy Zelenka and John McAvoy for their technical guidance in establishing the lens explant system and Madeleine Zetterberg, Elizabeth Whitcomb, and Ed Dudek for critical review of the manuscript.