ARTICLE |
Correspondence to: E. Helene Sage, Dept. of Vascular Biology, Hope Heart Institute, 1124 Columbia Street, Suite 720, Seattle, WA 98104. E-mail: hsage@hopeheart.org
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Summary |
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SPARC (Secreted Protein, Acidic and Rich in Cysteine) is a matricellular glycoprotein that modulates cell proliferation, adhesion, migration, and extracellular matrix (ECM) production. Although SPARC is generally abundant in embryonic tissues and is diminished in adults, we have found that the expression of SPARC in murine lens persists throughout embryogenesis and adulthood. Our previous studies showed that targeted ablation of the SPARC gene in mice results in cataract formation, a pathology attributed partially to an abnormal lens capsule. Here we provide evidence that SPARC is not a structural component of the lens capsule. In contrast, SPARC is abundant in lens epithelial cells, and newly differentiated fiber cells, with stable expression in wild-type mice up to 2 years of age. Pertubation of the lens capsule in animals lacking SPARC appears to be a consequence of the invasion of the lens cells situated beneath the capsule. Immunoreactivity for SPARC in the lens cells was uneven, with minimal reactivity in the epithelial cells immediately anterior to the equator. These epithelial cells appeared essentially noninvasive in SPARC-null mice, in comparison to the centrally located anterior epithelial cells, in which strong labeling by anti-SPARC IgG was observed. The posterior lens fibers exhibited cytoplasmic extensions into the posterior lens capsule, which was severely damaged in SPARC-null lenses. The expression of SPARC in wild-type lens cells, together with the abnormal lens capsule in SPARC-null mice, indicated that the structural integrity of the lens capsule is dependent on the matricellular protein SPARC. The effects of SPARC in the lens appear to involve regulation of lens epithelial and fiber cell morphology and functions rather than deposition as a structural component of the lens capsule. (J Histochem Cytochem 51:503511, 2003)
Key Words: SPARC, basement membrane, lens capsule, lens epithelial cells, lens fiber cells, collagen type IV, ß1 integrin, matricellular protein
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Introduction |
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THREE rather unique components distinguish the mammalian ocular lens: (a) the lens capsule, a thick basement membrane and specialized extracellular matrix (ECM) that regulates lens cell growth and differentiation; (b) epithelial cells, which form a monolayer underneath the anterior capsule; and (c) differentiated transparent fiber cells that constitute the lens mass (Fig 1 A). During the development of the lens, epithelial cells exit from the cell cycle, migrate to the post-equatorial region, and undergo substantial morphological and biochemical changes to form concentric layers of transparent fiber cells (
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SPARC is a secreted, Ca2+-binding glycoprotein that interacts with a variety of ECM proteins and growth factors (reviewed in
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Materials and Methods |
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Animals
129SvEv x C57BL/6j wild-type (wt, +/+) and SPARC-null (-/-) mice were maintained in a pathogen-free facility. The treatment and use of the mice in this study conformed to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmology and Vision Research.
Immunohistochemistry
Eyeballs from mice (E12 to 18 months of age) were removed and were fixed by immersion in methyl Carnoy solution (60% methanol, 30% chloroform, and 10% glacial acidic acid) for 4 hr at room temperature (RT). The eyeballs were dehydrated in a series of ethanol solutions (70%, 90%, and 100%) and were embedded in paraffin.
Serial 5-µm sections were cut, deparaffinized, rehydrated, and washed in PBS. Auto/Zyme was used for unmasking of the tissue sections (Biomeda; Foster City, CA). Nonspecific binding sites were blocked by incubation in 20% Aqua serum (Biomeda) in PBS with 0.05% Tween-20 for 2 hr at RT. Sections were incubated for 2 hr with agitation at RT with the following antibodies: polyclonal goat anti-mouse SPARC IgG; polyclonal rabbit anti-mouse SPARC IgG (R & D Systems, Minneapolis, MN; 10 µg/ml); rabbit anti-mouse laminin-1 IgG (Sigma, St Louis, MO; 5 µg/ml); rabbit anti-mouse collagen type IV (Chemicon International, Temecula, CA; 5 µg/ml); rat anti-mouse ß1-integrin (Chemicon International; 5 µg/ml). Negative controls included replacement of primary antibodies by normal mouse IgG or PBS, or primary antibody applied to SPARC-null lens sections. Sections were washed three times for 10 min each in PBS, and were incubated in fluorescein isothiocyanate (FITC)- or rhodamine-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories; West Grove, PA).
Immunoblotting
Lens capsules were removed from the cell mass of the lens under a dissecting microscope. The lens epithelium was mechanically scraped off the capsule. The capsule was immersed in cold distilled water for 20 min to lyse any remaining epithelial cells and was washed thoroughly in PBS. The capsules were briefly immersed in Hoechst 33258 fluorochrome (0.1 µg/ml in distilled water; Flow Laboratories, McLean, VA) and were examined under fluorescence microscopy to ensure the complete removal of attached cells. Capsules were homogenized and were solubilized in 40 µl Laemmli's SDS-PAGE sample buffer containing 10 mM dithiothreitol, heated for 5 min at 100C, and analyzed by SDS-PAGE on 10% polyacrylamide minigels (2 capsules per lane). Standard immunoblotting procedure was followed (
RT-PCR
Total RNA was isolated from lens epithelial cells and lens fiber cells of wt and SPARC-null mice with a RNeasy Mini Kit (Qiagen; Valencia, CA). Lens fiber cells initially extracted in chloroform and isopropanol were homogenized in TRI reagent (Molecular Research Center; Cincinnati, OH). The quality and yield of recovered RNA were evaluated by absorption at 260 and 280 nm. Total RNA was reverse-transcribed into cDNA by the use of a reverse transcription kit (Omniscript RT kit; Qiagen). Equal amounts of RNA were transcribed from each preparation. cDNA was amplified using the following primer pairs: SPARC, ATGAGGGCCTGGATCTTCTTTC/GGAAGAGTCGAAGGTCTTGTTGTC; CX43, GTTCAGCCTGAGTGCGGTCTAC/TTCCCTTCACGCGATCCTT; and GAPDH, GACCCCTTCATTGACCTCAACT/ACCAAGTGTGGGGTAGTGT-TTG. The PCR program was 12 min at 94C, followed by 35 cycles of 45 sec at 95C, 59 sec at 65C, 2 min at 72C, followed by a final extension of 8 min at 72C. Products were separated on agarose gels and were visualized by staining with ethidium bromide.
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Results |
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Expression and Distribution of SPARC in Embryonic and Adult Lenses
The distribution of SPARC protein in the lens at three embryonic stages (E11.5, E13.5, and E17.5) and in the adult up to 18 months of age is shown in Fig 1. Reactivity with anti-SPARC IgG was first detected in the primary lens fiber cells at E11.5 (Fig 1B, arrow) and E13.5 (Fig 1D, arrow). At E17.5 (Fig 1F), SPARC was prominent in the anterior lens epithelium (single long arrow), and was diminished before the bow region (arrowhead). Behind the bow region, SPARC was evident at the basal surface of the lens fibers where they contact the posterior capsule (double long arrows). Secondary fibers (E17.5) appear essentially devoid of SPARC (short small arrows), although the lens nucleus (primary fibers) was weakly reactive with anti-SPARC IgG (*). Immunostaining in the anterior lens epithelium remained similar in both intensity and distribution pattern at postnatal day 5 (Fig 1H), day 13 (Fig 1I), 1 month (Fig 1J), 4 months (Fig 1K), and 18 months (Fig 1L), whereas the intensity of the stain in the primary fibers became insignificant (data not shown). The lens epithelial cells flatten, and the capsule thickens, after 1 month of age, but the intensity of the staining for SPARC was not decreased in the epithelium up to 18 months of age (Fig 1J1L). Importantly, the lens capsule was negative in all the ages examined (Fig 1J1L, arrowheads). SPARC-null lenses (Fig 1E and Fig 1G) exposed to the same conditions as wt lenses (Fig 1D and Fig 1F), and wt lens (E11.5) without primary antibody (Fig 1C), showed no immunoreactivity.
Two capsules (approximately 70 µg protein) were also examined for the presence of SPARC. By immunoblotting analysis, the levels of SPARC appeared similar in lenses from 1 month to 2 years of age (Fig 1M). Moreover, proteolytic cleavage of SPARC was not observed in the lenses, although the polyclonal antibody recognizes internal sequences of SPARC (
SPARC Is Not Detected in Murine Lens Basement Membrane
The murine lens capsule exhibited no immunoreactivity with anti-SPARC IgG (Fig 1), consistent with our observations in human (
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Cellular Localization of SPARC in Lens Epithelial Cells and Newly Differentiated Fiber Cells
The presence of SPARC in the lens was examined further in animals 1 month of age (Fig 3). The cytoplasm of the anterior lens epithelial cells was reactive with anti-SPARC IgG (Fig 3A, arrow, inset), whereas the cell nucleus was not (Fig 3A, arrowhead, inset). SPARC-null lens epithelial cells showed no reaction with the antibody (Fig 3B, arrow). The area showing the least amount of SPARC in the lens epithelial cells was in the region anterior to the equator (Fig 3C, between the arrows). Newly differentiated lens fiber cells exhibited immunoreactivity with anti-SPARC IgG, particularly at the basal surface near the contacts with the capsule (Fig 3D3F represent a continuous series of photographs toward the posterior pole). The lens capsule was not stained with anti-SPARC IgG in either the anterior (Fig 3A and Fig 3C) or posterior region (Fig 3D3F).
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SPARC mRNA was present in both lens epithelial cells and in fiber cells (Fig 3G). Connexin 43 (Cx43) was expressed specifically in the undifferentiated lens epithelial cells and not in fiber cells (
Absence of SPARC in the Lens Is Associated with a Disrupted Capsule
The absence of SPARC in the lens capsule supports the contention that SPARC does not contribute to the structure of this specialized ECM (
In the SPARC-null mouse there is substantial damage of the lens capsule, with many protrusions of lens cells into the capsule (
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Discussion |
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SPARC is essential for the maintenance of lens transparency and lens capsular integrity. Significant labeling of SPARC was detected in the epithelial cells and newly differentiated fiber cells in the murine lens up to 2 years of age (Fig 1). It is known that the expression of SPARC is both temporally and spatially regulated in normal tissues (
Lens basement membrane is a highly specialized ECM present at the epithelial and fiber interface of the lens. It is now evident that individual components of the basement membrane regulate specific biological activities, such as cell growth, migration, adhesion, and differentiation, all of which contribute to the development of tissues (
The distribution of collagen type IV was altered in the SPARC-null lens (
The depletion of SPARC appears to be associated with cellular protrusions into the lens capsule by epithelial cells and fiber cells (Fig 4). Fig 5 is a drawing summarizing our results. (a) SPARC is present in the lens epithelial cells and newly differentiated fiber cells, but not in the lens capsule. (b) wt lens epithelial cells showing minimal reactivity with anti-SPARC IgG were located anterior to the lens equator (Fig 5, gray area; see also Fig 3C). The corresponding cells in the SPARC-null lens showed no cellular invasion into the basement membrane (Fig 4E, Fig 4F, and Fig 5). It has been reported that expression of SPARC is significantly reduced or absent in ovarian cancer (
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Interestingly, the absence of SPARC causes no apparent pathology in the lens nucleus before mature cataract (
The macromolecular structure of the basement membrane has become more complex as additional components are discovered and characterized. For example, the laminin family, collagen type XVIII, and nidogen-2 were recently described in several basement membranes (
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Acknowledgments |
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Supported by National Institutes of Health grants EY13180 and GM 40711, and by a National Research Service Award F32-EY 06987 to QY.
We thank Rolf Brekken for the purification of polyclonal antibody against SPARC, David Graves for the SPARC primers, and Teri Seeberger for technical assistance. We thank our colleagues in the Sage lab for helpful discussions.
Received for publication September 30, 2002; accepted November 27, 2002.
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