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First published online 26 January 2006
doi: 10.1242/dev.02262

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Caspase-dependent secondary lens fiber cell disintegration in A-/B-crystallin double-knockout mice

Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Building 7, 7 Memorial Drive, MSC 0704 Bethesda, MD 20892, USA.

^* Author for correspondence (e-mail: morozovv{at}nei.nih.gov)

Accepted 21 December 2005

	SUMMARY

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
B-crystallin has been demonstrated, in tissue culture experiments, to be a caspase 3 inhibitor; however, no animal model studies have yet been described. Here, we show that morphological abnormalities in lens secondary fiber cells of A-/B-crystallin gene double knockout (DKO) mice are consistent with, and probably result from, elevated DEVDase and VEIDase activities, corresponding to caspase 3 and caspase 6, respectively. Immunofluorescence microscopy revealed an increased amount of caspase 6, and the active form of caspase 3, in specific regions of the DKO lens, coincident with the site of cell disintegration. TUNEL labeling illustrated a higher level of DNA fragmentation in the secondary fiber lens cells of DKO mice, compared with wild-type mice. Using a pull-down assay, we show interaction between caspase 6 and A- but not B-crystallin. These studies suggest that -crystallin plays a role in suppressing caspase activity, resulting in retention of lens fiber cell integrity following degradation of mitochondria and other organelles, which occurs during the apoptosis-like pathway of lens cell terminal differentiation.

Key words: Caspase 3, Caspase 6, A-crystallin, B-crystallin, Double knockout mice

	INTRODUCTION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The vertebrate lens offers a unique model system for studying mechanisms of cell death. The mature ocular lens consists of three cell types: epithelial cells, primary lens fiber cells and secondary lens fiber cells. Primary fiber cells are generated in early embryogenesis from elongated epithelial cells at the posterior region of the lens vesicle. Removal of nuclei and other organelles from these cells occurs through accumulation of osmiophillic bodies that are later digested in vesicles with proteolytic enzymes, followed by removal of the breakdown products from the cell (Vrensen et al., 1991). Following this process, these cells remain unchanged in the central part of the lens, known as the lens nucleus. However, the lens constantly grows throughout the life of the organism, by formation of new secondary lens fiber cells from the epithelial cells at the lens equator, which differentiate and elongate. These newly formed fiber cells form an onion-like array of concentric layers. Maturation of the secondary lens fiber cells parallels many classical apoptosis events: chromatin condensation, DNA fragmentation, nucleopore clustering with subsequent nuclear breakdown, and disappearance of mitochondria and other organelles. However, lens fiber cell differentiation shows signs of attenuated or arrested apoptosis: breakdown of nuclei occurs in days, not hours; the cytoskeleton remains intact throughout the remaining life of the organism; and the lens fiber cells show increased resistance to apoptotic agents such as staurosporine and etoposide (Dahm, 1999; Wride, 2000).

Differentiation of the secondary fiber cells is accompanied by lens-specific expression of several families of crystallin genes (Piatigorsky, 1981). Crystallins are the predominant soluble proteins (20-60% of the lens wet weight) in the mammalian lens, reaching a physiological concentration of 300 mg/ml (Harding, 1991). One crystallin family found in all vertebrate species examined (Wistow and Piatigorsky, 1988), is -crystallin, which consists of two subunits, A (Cryaa, Crya1, Hspb4) and B (Cryab, Crya2, Hspb5) in an approximate 3:1 ratio, with 55% amino acid sequence identity. The -crystallins are also members of the small heat-shock protein family, and B-crystallin is a bona fide heat shock protein.

Earlier studies described a potential role for -crystallin as an anti-apoptotic agent because of its ability to protect the cell from hypertonic (Dasgupta et al., 1992), heat (Aoyama et al., 1993), oxidative and TNF-induced (Mehlen et al., 1995) stress. This list of stress factors is continually expanding. -Crystallin has been shown to prevent apoptosis induced by a variety of agents including UV radiation (Harding, 1991), TNF (Andley et al., 2000; Mehlen et al., 1996a), staurosporine (Andley et al., 2000; Mehlen et al., 1996a; Mehlen et al., 1996b; Mao et al., 2004), hydrogen peroxide (Mao et al., 2001), sorbitol (Mao et al., 2004), etoposide (Mao et al., 2004) and okadaic acid (Li et al., 2001). Recent reports have begun to elucidate the molecular mechanisms by which -crystallin inhibits apoptosis. Experiments with cell extracts and in tissue culture suggest that B-crystallin increases resistance to the apoptotic inducers etoposide and TNF by decreasing caspase 3 activity via inhibition of autocatalytic maturation of procaspase 3 (Kamradt et al., 2001; Kamradt et al., 2002). Coimmunoprecipitation of B-crystallin with caspase 3 and its uncleaved precursor form suggests a direct interaction between these proteins (Kamradt et al., 2001). Binding of the molecular chaperones A- and B-crystallin to the proapoptotic factors Bax and Bcl-Xs prevents their translocation into mitochondria, and could serve as an additional mechanism of apoptosis inhibition (Mao et al., 2004).

So far, all evidence that the small heat shock proteins A- and B-crystallin inhibit apoptosis are based on in vitro or tissue culture experiments. No animal models have yet been analyzed to elucidate the roles of these proteins in protecting cells from programmed cell death. In the present report, we describe a physiological role of -crystallin in mammalian lens development, employing a knockout mouse model. We chose to eliminate both A- and B-crystallin because they both might affect apoptosis, and absence of only one subunit could be partially or fully compensated by the remaining subunit. Our data suggest that the absence of -crystallin causes elevated caspase activity in lens secondary fiber cells. As a result, lens secondary fiber cells fail to halt their apoptosis-like maturation program after degradation of the cell nuclei, mitochondria and all other organelles, and progress to the stage of cell disintegration.

	MATERIALS AND METHODS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Generation of A-/B-crystallin DKO mouse
The A-/B-crystallin DKO mouse line was produced by breeding A-crystallin KO mice (Brady et al., 1997) to B-crystallin KO mice (Brady et al., 2001). Both KO mice were generated and maintained in the 129Sv background. These mice also lack the HSPB2 gene product (Brady et al., 2001).

Preparation of the lens extract
The eyes were removed from mice immediately after euthanasia, and then further dissected with microsurgical scissors. Lenses were removed and cleaned from bound tissues. Dissected lenses, typically 8 to 10, were placed immediately into 200 µl of ice-cold caspase 3 assay buffer (20 mM HEPES, pH 7.4, 2 mM EDTA, 0.1% CHAPS, 5 mM DTT). Lenses were disrupted on ice in a 1.5 ml microtube, with a disposable polypropylene pestle rotated by a cordless drive unit. Homogenized extracts were centrifuged at 20,000 g for 30 minutes at 0°C. After centrifugation, the supernatant was collected, aliquoted and stored at -80°C for future use. Protein concentration was measured with a Coomassie protein assay reagent kit (Pierce), which is based on the Bradford method (Bradford, 1976). A commercial solution of bovine serum albumin (BioRad) was used to prepare protein concentration standards from which standard curves were plotted.

Caspase 3 and caspase 6 activity assays
The colorimetric caspase 3 assay kit and fluorimetric caspase 6 assay kit (both from Sigma) were used in 96-well plate format, according to manufacturer's instructions. For each reaction, 160-230 µg of lens extract protein were used. Reactions usually were incubated 16-20 hours at room temperature, and the amount of cleaved product was measured on the microplate reader (Bio-Tek Instruments), or on a fluorometer (Perkin Elmer). Specific activity for caspase 3 was calculated in nmol of pNA/hr/ng of total protein, and for caspase 6 in nmol of AMC/hr/mg of total protein. Caspase 3 and caspase 6 activity data were analyzed using linear models in R (Version 2.1.1). In all cases, P0.007 and was highly significant.

Histology and immunofluorescence
For light microscopy, paraffin sections of mouse lenses at the different ages were stained with Hematoxylin and Eosin. Images were collected on a Zeiss Axioplan 2 photomicroscope with an attached CCD camera (OPELCO). For immunohistochemistry, frozen sections were used. A rabbit anti-active caspase 3 monoclonal antibody (BD PharMingen) was used undiluted, and a rabbit anti-caspase 6 polyclonal antibody (Sigma) was diluted 1:100 in 1xPBS. Freshly dissected eyes were embedded in OCT compound (Tissue-Tek, Miles Scientific) and 10-15 µm slices were cryosectioned at -20°C. Cryosections were fixed in acetone at -20°C for 5 minutes, washed three times in room temperature PBS, and blocked for 30 minutes with 10% normal goat serum (Vector Laboratories) in PBS at room temperature. Sections were incubated with primary antibody overnight at 4°C, then washed three times for 10 minutes with PBS. They were then incubated with Alexa Fluor 488-conjugated anti-rabbit IgG secondary antibodies (Molecular Probes), diluted 1:100 in 1x PBS, for 1 hour at room temperature, followed by a thorough washing (three times) with PBS; 0.1% Tween 20. For negative controls, primary antibodies were omitted. Distilled water was used for the final wash. Samples were mounted with aqueous mounting medium containing anti-fading agents (Biomeda) and examined with a Zeiss Axioplan 2 photomicroscope equipped with epifluorescence and a CCD camera (OPELCO). Fluorescence intensity was analyzed and quantified with Image-Pro Plus Scientific Software, version 5.1 (Media Cybernetics). Fluorescence intensity value for anti-active caspase 3 and anti-caspase 6 staining was calculated as a difference between staining with and without primary antibodies.

TUNEL reaction
Paraffin-embedded eye sections were deparaffinized and rehydrated to PBS. Samples were treated with 10 µg/ml Proteinase K (ICN) in 10 mM Tris-HCl (pH 7.4) for 15 minutes at room temperature. Sections were washed three times in PBS solution after proteinase treatment. TUNEL labeling was performed with the In Situ Cell Death Detection Kit, Fluorescein (Roche) according to manufacturer recommendations (1 hour at 37°C). Fluorescein-labeled nucleotides, supplied with the kit, were used in the TUNEL reaction. For negative controls, terminal transferase was omitted. Sections were washed with PBS/0.1% Tween 20. If DAPI labeling was performed, sections were finally incubated with DAPI reagent (Molecular Probes) diluted 1:2500 in PBS for 15 minutes at room temperature and then washed with PBS/0.1% Tween 20. Distilled water was used for the final wash. Samples were mounted with aqueous mounting medium containing anti-fading agents (Biomeda) and examined with a Zeiss Axioplan 2 photomicroscope equipped with epifluorescence and a CCD camera (OPELCO).

Caspase 6 and -crystallin pull-down interaction assay
We used the commercially available ProFound Pull-Down PolyHis Protein:Protein Interaction Kit (Pierce Biotechnology) to study possible direct protein-protein interactions. The assay was performed according to manufacturer's instructions. Purified recombinant C-terminal histidine tagged caspase 6 (Sigma) (7.2 µg in 400 µl of wash solution) was immobilized on the cobalt chelate gel as bait. In control experiments, caspase 6 was omitted. Lens extracts (68 µg), prepared as described above from 4.5-week-old wild-type mice, were used as the source of prey proteins, and were incubated in 400 µl of wash solution for 1 hour at room temperature with immobilized caspase 6. All wash and elution steps were performed as recommended in the assay manual.

Gel electrophoresis and western blot analysis
Proteins isolated from lens extract by the pull-down assay were analyzed by western blot. Proteins eluted from the cobalt chelate gel, 30 µl per lane, were electrophoresed on pre-cast 10% NuPAGE Bis-Tris gels with MES running buffer (Invitrogen). Proteins in the gel were then electroblotted onto 0.45 µm pore size nitrocellulose membranes (Schleicher & Schuell) for 120 minutes at 30 V in an XCell II Blot Module (Invitrogen). After transfer, membranes were blocked for 1 hour at room temperature with 0.2% I-Block reagent (Tropix) in PBS with 0.1% Tween 20, then probed with specific primary antibodies. The following primary antibodies were used in western blot analyses: rabbit anti-rhA-crystallin polyclonal antibody (from Dr Joseph Horwitz, UCLA) diluted 1:5,000; rabbit anti-rhB-crystallin polyclonal antibody (from Dr Joseph Horwitz, UCLA) diluted 1:5,000; and rabbit anti-caspase 6 polyclonal antibody (Sigma) diluted 1:2,500. Nitrocellulose membranes were incubated for 1 hour with primary antibodies, then washed three times for 5 minutes in PBS/0.1% Tween 20. Caspase 6 and A/B-crystallin were then visualized with the Western-Star (Tropix) chemiluminescent immunoblot detection system, according to the manufacturer's instructions.

	RESULTS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Morphological changes in DKO mouse secondary lens fiber cells
Lenses from DKO mice (Fig. 1B-F) are appreciably smaller and softer than those from wild-type mice (Fig. 1A). The DKO lenses are opaque at birth, and exhibit a series of abnormal histological changes with age. By contrast, all lenses from wild-type animals were completely transparent.

Histological sections of a DKO mouse lens at 6 weeks of age shows major disruption of normal architecture only at the equator (Fig. 1B). This has been seen in other genetically altered mice, and may be due to physical forces exerted on a weakened structure by zonule fibers, which attach the lens to the musculature involved in visual focusing. No other morphological abnormalities are apparent. However, with age, the lenses of A-/B-crystallin DKO mice show cellular disintegration in the region of the secondary fiber cells (Fig. 1C-F). Lenses from wild-type (Fig. 1A) and B-crystallin single KO mice (data not shown) never displayed this type of cell disintegration. However, A-crystallin single KO mouse lenses display a similar pattern of cell death in much older animals (data not shown). This could be additional evidence for the compensatory activity of the two -crystallin subunits, particularly in the lens, where -crystallin concentration is exceptionally high.

View larger version (85K): [in this window] [in a new window]   Fig. 1. Lens fiber cells disintegrate in A-/B-crystallin double knockout mice. Fixed sections of the lens from a wild-type mouse at the age of 3 months (A), and from DKO mice at the ages of 6 weeks (B), 8 weeks (C), 3 months (D), 10 months (E) and 13 months (F) were stained with Hematoxylin and Eosin. eq, equatorial/bow region of lens; ln, lens nucleus; df, disintegrated fiber cells. Scale bar: 250 µm.

Without a firm foundation to build on, newly differentiating cells at the lens equator of older mice fail to form neatly arrayed fiber cells that elongate symmetrically to the anterior and posterior lens, but instead they form amorphous globular cells that migrate to the anterior part of the lens (Fig. 1C-F). Unlike normal lens fiber cells (Fig. 1A), these globular cells do not appear to follow the normal apoptosis-like differentiation pathway (Boyle et al., 2003). Eventually, the accumulated cell mass in the anterior lens pushes the hard lens core through the posterior lens capsule (Fig. 1F), obliterating any semblance of a lens.

Immunofluorescent examination of the lens section
The process of secondary lens fiber formation has been described as a case of an `attenuated form of cell death' (Dahm, 1999). Our observation of morphological abnormalities in DKO lenses raised two important questions: (1) do these abnormalities in the lenses of A-/B-crystallin DKO mice result from failure to arrest the apoptosis-like maturation process?; and (2) does secondary lens fiber cell disintegration result from increased caspase activity in A-/B-crystallin DKO mice? The answer to the second question could clarify the first and suggest a possible caspase-dependent apoptotic pathway for lens secondary fiber cell differentiation. Data from other laboratories suggested direct interaction with, and inhibition of, caspase 3 by B-crystallin (Mao et al., 2001; Kamradt et al., 2001; Kamradt et al., 2002). Detection of the active form of caspase 3 in lenses of DKO mice was chosen as a starting point for this study. We used immunohistochemical labeling of the active form of caspase 3 in frozen eye sections from the 7-week-old DKO mice (Fig. 2). Almost no signal was detected when primary anti-active caspase 3 antibody was omitted and only secondary antibody was used (Fig. 2C). However, anti-active caspase 3 antibody gives a strong signal in the same area where secondary lens fiber cell disintegration is evident in older mice (Fig. 2A). Merging the bright-field image of the same section (Fig. 2B) with the immunofluorescence image (Fig. 2A) allowed us to see that the majority of active caspase 3 localized in the oldest secondary lens fiber cells (Fig. 2D). The presence of active caspase 3 in the area of imminent cellular disintegration, before cell disintegration becomes histologically apparent, suggests a caspase-dependent cell death pathway. In other words, the elevated level of caspase activity is probably causing the observed cell disintegration, which encouraged us to continue this line of research. The next logical step was to compare caspase activity in the lens from wild-type and A-/B-crystallin DKO mice.

Caspase activity in wild-type and A/B-crystallin DKO mouse lens extracts
For the initial screening of caspase activities in lens extracts, we used a wide spectrum of specific caspase inhibitors from R&D Systems: Z-VAD-FMK; Z-WEHD-FMK; Z-VDVAD-FMK; Z-DEVD-FMK; Z-YVAD-FMK; Z-VEID-FMK; Z-IETD-FMK; Z-LEHD-FMK; Z-AEVD-FMK; Z-LEED-FMK (data not shown). The most promising candidates for further detailed study, chosen from the preliminary screening, were caspase 3 and caspase 6. Both caspase 3 and caspase 6 belong to the subclass of executioner caspases. Interaction of -crystallin and caspase 3 has already been described in tissue culture systems. However, almost nothing is known about the possible role of -crystallin in regulation of caspase 6 activity or maturation.

Commercially available kits were used for the determination of caspase activity. Protease activity was assayed using chromogenic or fluorogenic sequence-specific substrates, DEVD-pNA for caspase 3 and VEID-AMC for caspase 6, in the presence or absence of specific inhibitors. This type of caspase activity determination has obvious flaws. Any protease present in the lens extract that can digest caspase 3 or caspase 6 substrate and can be blocked with the specific caspase inhibitor will be classified as caspase 3 or caspase 6. However, for easier reading, this paper will refer to DEVDase activity as caspase 3 activity and VEIDase activity as caspase 6 activity. Wild-type and DKO mouse lenses from three age groups [4-, 8- (or 9-) and 21- (or 23-) week-old] were chosen for extract preparation and protease assay. Caspase activities were assayed in wild-type and DKO lens extracts for each age group, in the presence or absence of a specific caspase inhibitor (Fig. 3). It is notable that, for all ages tested, DKO mouse lens extracts showed elevated activities of both caspase 3 (Fig. 3A) and caspase 6 (Fig. 3B) compared with wild type, with the DKO mice exhibiting two- to fourfold higher caspase 3 activity than wild type (Fig. 3A,C). Caspase 6 activity in DKO mouse lens extract was 1.3- to 3-fold higher than in wild-type depending on the age of the animal. Interestingly, both caspase 3 and caspase 6 activity levels from DKO mouse lenses demonstrate age dependency.

View larger version (70K): [in this window] [in a new window]   Fig. 2. Immunostaining of DKO mouse lens with anti-active caspase 3 antibodies. Both primary anti-active caspase 3 antibody and secondary anti-rabbit IgG antibodies, conjugated with Alexa Fluor 488, were used for detection (A). The bright-field image was captured (B). As a negative control, the primary antibody was omitted, and only the secondary antibody was used (C). The bright field image (B) and active caspase 3 fluorescent image (A) were merged for morphological comparison (D). Scale bar: 250 µm; A-C are at the same magnification.

Age dependent caspase activity in wild-type and A/B-crystallin DKO mouse lens extracts

Immunostaining of DKO and wild-type mouse lenses with anti-active caspase 3 and anti-caspase 6 antibodies
To strengthen the caspase activity data from wild-type and DKO mouse lens extracts, we employed immunofluorescence microscopy of frozen sections stained with anti-active caspase 3 or anti-caspase 6 antibodies (Fig. 4). Frozen sections from 7-week-old mouse eyes were labeled with antibody (described in detail in the Materials and methods section), and all images were captured with identical parameters. For fluorescent microscopy analysis, we selected an anterior portion of the lens, consisting of secondary fiber cells, and approximately in the middle of secondary fiber cell region, as illustrated on the lens diagram (Fig. 4A). The negative controls (Fig. 4D,E,H,I), in which primary antibodies were omitted, were carried out simultaneously with the experimental samples. All controls showed low background signal compared with sections where both primary and secondary antibodies were used (Fig. 4B,C,F,G). These immunofluorescence staining experiments for active caspase 3 and caspase 6, in a specific region of the lens, support our biochemical data demonstrating the presence of higher levels of caspase 3 and caspase 6 activities in DKO lenses. Secondary lens fiber cells from DKO mice showed a much more intense signal for both the active form of caspase 3 (Fig. 4C) and caspase 6 (Fig. 4G), than cells from the similar lens regions in wild-type mice (Fig. 4B,F).

View larger version (16K): [in this window] [in a new window]   Fig. 3. Caspase 3 and caspase 6 activities. Caspase 3 (A,C) and caspase 6 (B,D) activities were measured in the presence (+) or absence (-) of a specific inhibitor (A,B) in lens extracts from wild-type and DKO mice from three age groups. The activities plotted in C and D as a function of animal age represents the difference in caspase activity measurements in the presence or absence of a specific inhibitor. Specific activities are in nmol of pNA/hr/ng of total extract protein for caspase 3 or in nmol of AMC/hr/mg of total extract protein for caspase 6. The error bars represent data error analyzed using linear models in R (Ver. 2.1.1). *P<0.001; **P=0.007; ***P=0.003.

View larger version (30K): [in this window] [in a new window]   Fig. 4 . Caspase 3 and caspase 6 immunostaining. Immunostaining of the anterior region of secondary lens fiber cells (A) in wild-type (B,D,F,H) and DKO (C,E,G,I) lenses with anti-active caspase 3 (B,C) and anti-caspase 6 (F,G) antibodies. In negative controls (D,E,H,I), primary antibodies were omitted and only the secondary anti-rabbit IgG antibody, conjugated with Alexa Fluor 488, was used. (J) Image-Pro Plus v.5.1 software was used to quantitate fluorescence intensity in sections of lens from wild-type and DKO mice. Scale bar: 25 µm.

TUNEL reaction
Fragmentation of nuclear DNA, which is often measured in situ by TUNEL reaction, is one of the hallmark characteristics of apoptotic cells. We employed TUNEL techniques to assess apoptosis in the bow regions of lenses from 7-week-old wild-type and 7.5-week-old DKO mice (Fig. 5). Most cells in the lens loose there nuclei, except for cuboidal epithelial cells at the lens anterior, which actively divide and form new layers of secondary lens fiber cells in equatorial/bow region. Compared with wild-type (Fig. 5A), lenses from DKO mice suffered severe disintegration in the bow region (Fig. 5B). We therefore selected for TUNEL analysis the area posterior to the bow region, which had remnants of the degenerated cells and their nuclei (Fig. 5B, boxed). Higher magnification of the selected regions of the bright field images (Fig. 5A,B, boxed) are shown in Fig. 5C,D. Contrary to the morphologically well organized fiber cells in lenses of wild-type mice (Fig. 5C), cells in different stages of disintegration are evident in lenses from DKO mice (Fig. 5D).

TUNEL analysis of lens sections from wild-type and DKO mice revealed a dramatic difference in DNA fragmentation. TUNEL-positive cells in lenses of wild-type mice (indicated by arrows in Fig. 5E) coincide with the narrow area of differentiating secondary fiber cells (merged images of bright field and TUNEL fluorescent signal are presented in Fig. 5G). Normally, these cells undergo an apoptosis-like process of maturation that includes DNA fragmentation and removal of nuclei. Compared with wild-type mice, significantly stronger TUNEL signal was observed in lenses from DKO mice (Fig. 5F). The large number of the TUNEL-positive cells in lenses from DKO mice coincides with the region of cell disintegration (indicated by arrows in Fig. 5H). Labeling conditions and imaging parameters were identical for wild-type and DKO samples. In both cases, wild-type (Fig. 5I) and DKO (Fig. 5J) lens sections demonstrate only background fluorescence when terminal transferase was omitted from the TUNEL reaction.

In order to determine the number of cells undergoing apoptosis in the lenses of wild-type and DKO mice, we combined TUNEL staining with DAPI, which labels nuclear DNA, on the same sections (Fig. 6). Similar areas of lens bow regions from wild-type and DKO mice were identically labeled and imaged. Cells in the peripheral zone of the lens bow region of wild-type mice have well stained nuclei (Fig. 6A) that gradually disappear in the area of secondary fiber cells maturation (Fig. 6G and marked with arrowheads in Fig. 6E). A majority of nuclei in lenses from wild-type mice was TUNEL negative (Fig. 6C). DNA fragmentation was detected in the narrow area of secondary fiber cell denucleation (Fig. 6C,E,G). Only one cell in this field from the thin area of secondary fiber cell maturation was double labeled with DAPI and TUNEL (arrow in the Fig. 6E). Other cells in this region, which exhibit punctuate TUNEL staining, have probably progressed to a late stage of DNA degradation with the nuclei completely disintegrated. In contrast to wild-type mice, the majority of nucleated fiber cells in lenses from DKO mice was TUNEL positive (Fig. 6B,D, see arrow in Fig. 6F), suggesting a high level of DNA fragmentation, which is one of the key characteristics of apoptosis. TUNEL-positive cells in lenses from DKO mice are coincident with the area of progressive cell disintegration and cell loss (Fig. 5H, Fig. 6H), suggesting the apoptotic character of this process. In negative control samples, no signal was detected above background fluorescence in wild-type (Fig. 6I) or DKO (Fig. 6J) lenses when terminal transferase was omitted in TUNEL reaction.

View larger version (61K): [in this window] [in a new window]   Fig. 5. TUNEL labeling of lens secondary fiber cells. Bright-field image (A-D), TUNEL reaction fluorescence (E,F) and merge of both (G,H) sections from 7-week-old wild-type (A,C,E,G,I) and 7.5-week-old DKO (B,D,F,H,J) mouse lenses. In negative controls (I,J), calf thymus terminal deoxynucleotidyl transferase was omitted from the TUNEL reaction. C,D are enlargements of boxed areas in A,B. Arrows in E,F,H indicate TUNEL-positive nuclei. Scale bar: 125 µm in A,B; 25 µm in C-J.

View larger version (41K): [in this window] [in a new window]   Fig. 6. Secondary lens fiber cell apoptosis. DAPI labeling fluorescence (A,B), TUNEL reaction fluorescence (C,D), merge of both (E,F) and merge of DAPI and TUNEL with bright field (G,H) of lens sections from 7-week-old wild-type (A,C,E,G,I) and 7.5-week-old DKO (B,D,F,H,J) mice. Lenses on all images are oriented such that the upper right corner points towards the bow region and the lower left corner points towards the lens nucleus. In negative controls (I,J), terminal transferase was omitted from the TUNEL reaction. Arrows in E,F indicate nuclei double labeled with DAPI and TUNEL. Arrowheads in E indicate the area of secondary fiber cell maturation. Scale bar: 25 µm.

-Crystallin interaction with caspase 6

View larger version (21K): [in this window] [in a new window]   Fig. 7. Western blots of cobalt chelate gel column elution fractions. Recombinant caspase 6 (0.6 µg, left lane), 30 µl of the elution fraction from the control column, where caspase 6 was omitted (middle lane), and 30 µl of the elution fraction from the caspase 6-Co2+ column (right lane) were separated on a 10% NuPage gel and detected by western blot with anti-caspase 6, anti-B-crystallin or anti-A-crystallin antibodies.

	DISCUSSION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A caspase-dependent pathway of cell death was supported in our caspase activity experiments. Caspase 3 and caspase 6 activities are higher in the lenses of DKO mice than of wild-type mice under all conditions tested. Increased activities of apoptotic executioner caspases in these DKO lenses contribute to cell disintegration, which is consistent with -crystallin inhibition of caspase activity in normal lenses, where -crystallin is highly abundant. Interestingly, caspase 3 and 6 activity levels in wild-type mouse lenses remained relatively constant at all ages tested, and the lenses remained transparent throughout the life of the animal. However, activity levels of caspase 3 and caspase 6 in DKO mouse lenses fluctuated with the age of the animal, and changes in caspase activities were consistent with changes in lens morphology. Increased activities of apoptotic executioner caspases in the lenses of DKO mice at earlier ages contributed to the cell disintegration that was readily apparent at the age of 8 weeks. Partially disintegrated lenses have a decreased number of intact fiber cells capable of generating new caspase activity, which would lead to a decrease in the overall level of caspase activity in the lens. Increases in caspase 6 and caspase 3 activities at ages 23 and 21 weeks could reflect the impact of the other, non-fiber lens cells and result in further cell disintegration.

Comparing immunofluorescence signal intensities of similar regions in wild-type and DKO mouse lenses revealed an increase in caspase 6 and the active form of caspase 3 in lens secondary fiber regions in DKO mice. Quantitation of the fluorescent images show increased levels of both caspase 6 and the active form of caspase 3 in lens of DKO mice, compared with wild-type mice. Immunostaining of the active form of caspase 3 is consistent with the caspase 3 activity assay. The greater difference in staining intensity for caspase 6 in lenses from wild-type and DKO mice, compared with the data from caspase 6 activity analysis, can be explained by presence of caspase 6 degradation products in the lens. For both caspase 3 and caspase 6, elevated activities in this area are consistent with the site of future cell disintegration. Further characterization of caspases involved in terminal differentiation of lens secondary fiber cells is a crucial part of future studies.

As the majority of literature regarding the inhibition of apoptosis by -crystallin has been focused on caspase 3, we chose this as our primary target. However, according to our preliminary caspase screening experiments with a wide spectrum of caspase inhibitors, caspase 6 plays a similar, or even more important, role than caspase 3 in secondary lens fiber cells maturation. A role for caspase 6 in lens secondary fiber cell maturation was suggested earlier (Wride et al., 1999; Foley et al., 2004), however, some data suggest that VEIDase activity in the lens is not attributed to caspase 6 (Zandy et al., 2005). Nevertheless, possible inhibition of caspase 6 by -crystallin had not been previously described. Employing a pull-down assay we were able to demonstrate direct interaction between caspase 6 and A-crystallin, which could provide a possible mechanism for modulation of caspase 6 activity. Interestingly, using the same assay, we did not find any interaction between caspase 6 and B-crystallin. These data are consistent with our observation that lenses of A-, but not B-crystallin, single KO mice display secondary lens fiber cell disintegration, but only in much older animals. Perhaps A-crystallin controls activity of caspase 6, which appears to be more important for lens fiber cell maturation, and B-crystallin controls activity of caspase 3. Although interactions between A-crystallin and caspase 3 have not yet been reported, we cannot rule out modulation of caspase 3 activity by A-crystallin. This is an important goal of our ongoing study.

Employing a TUNEL technique, we visualized the precise narrow region in lenses from wild-type animals, where secondary fiber cells undergo terminal maturation and where denucleation occurs. In contrast to DKO animals, TUNEL-positive lens cells in wild-type mice were identified only in this area. TUNEL signal in lenses from DKO mice was more intense, suggesting a higher level of DNA fragmentation compared with samples from wild-type mice. In addition, almost every nucleated fiber cell in the lens of DKO mice, regardless of location in the lens, was TUNEL positive. Regions of morphological change, or cell loss, in lenses from DKO mice coincide with intense TUNEL signal, suggesting an apoptotic character of cell disintegration. A high level of endonucleolysis, and complete cleavage of nuclear DNA, is considered the key event of apoptosis. Elevated TUNEL signal in lenses from DKO mice suggests an inhibitory role of -crystallin in apoptosis. This is consistent with the increased level of ischemia-induced apoptosis observed in the hearts of B-crystallin/HSPB2 gene knockout mice compared with wild type (Morrison et al., 2004).

The mechanism by which A- and B-crystallin inhibit caspase activity is unknown. Data presented in this report and in publications from other laboratories suggest possible pathways of inhibition: B-crystallin could interact with factors promoting apoptosis, e.g. Bax and Bcl-Xs (Mao et al., 2004), or the interaction could be directly between caspase 6 and A-crystallin and between caspase 3 and B-crystallin (Kamradt et al., 2001; Kamradt et al., 2002). If a direct protein-protein interaction is involved in regulation of caspase activity by -crystallin, it could be at the stage of maturation of the executioner procaspase(s) to their fully processed, active form, as suggested earlier (Kamradt et al., 2001; Kamradt et al., 2002), or direct inhibition of the fully processed caspase, or both. Further elucidation of the mechanisms regulating caspase activities is essential.

	ACKNOWLEDGMENTS

 
This research was supported by the Intramural Research Program of the NIH, NEI. We thank Dr Terry A. Cox from Statistical Methods and Analysis Section, Biometry Branch, NEI, NIH for performing statistical analysis of caspase activity data; Dr Robert Fariss and Ms Amanda Bundek, both from NEI, NIH, for the confocal microscopy study not included in this report, which supports our immunofluorescent data and suggests elevated level of active caspase 3 in the lens of DKO mouse. We thank Dr Joseph Horwitz for providing antibodies to A- and B-crystallin; and Dr Steven Bassnett for helpful discussions and sharing his unpublished findings.

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TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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