Targeted Deletion of the SPARC Gene Accelerates Disc Degeneration in the Aging Mouse
Department of Orthopaedic Surgery (HEG,JI,ENH) and Department of Biostatistics (HJN), Carolinas Medical Center, Charlotte, North Carolina, and Hope Heart Program, The Benaroya Institute at Virginia Mason, Seattle, Washington (EHS,SF)
Correspondence to: Helen E. Gruber, PhD, Director, Orthopaedic Research Biology, Cannon Bldg., 3rd Floor, Carolinas Medical Center, PO Box 32861, Charlotte, NC 28232. E-mail: helen.gruber{at}carolinashealthcare.org
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Summary |
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(J Histochem Cytochem 53:11311138, 2005)
Key Words: intervertebral disc disc degeneration matricellular proteins SPARC
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Introduction |
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In other tissues, SPARC is known to play dynamic roles in modulating interactions between cells and their ECM; in collagen fibrillogenesis, deposition, and remodeling; and in growth factor efficacy (Bradshaw and Sage 2001). Because SPARC has been shown to bind to protein constituents of the ECM (collagen types IV and VIII, vitronectin, and thrombospondin-1) (Brekken and Sage 2001
), it has been proposed that SPARC modulates cell-ECM interactions and influences cell behavior during remodeling of the ECM (Sweetwyne et al. 2004
). Cleavage of SPARC by certain matrix metalloproteinases increases the affinity of SPARC for collagens I, IV, and V (Sasaki et al. 1997
), and such activity may serve to direct SPARC to sites of ECM remodeling.
Examination of SPARC-null mice has revealed that these animals have osteopenia and decreased bone formation (Delany et al. 2000), as well as defects in collagen fibril formation that influence responses to insults such as dermal wounding (Bradshaw et al. 2001
,2002
), implants (Puolakkainen et al. 2003
), and tumor cells (Brekken et al. 2003
). Such studies support the hypothesis that SPARC has specific functions in specific sites and that regulated expression of SPARC is important during development and in the response to alterations in tissue homeostasis.
To explore the function and activity of SPARC in the homeostasis of intervertebral disc ECM, we have studied discs in SPARC-null mice. Specifically, we have asked whether SPARC affects the morphology of the disc and whether the absence of SPARC predisposes the disc to accelerated aging.
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Materials and Methods |
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Lateral radiographs of the lumbar spine were obtained with a MicroFocus Imaging system with high-resolution Kodak X-OMAT TL mammography film (Kodak; Rochester, NY). Specimens were fixed in either 10% neutral buffered formalin or 70% ethanol and were decalcified in a solution of 22.5% formic acid (Allegiance; McGaw Park, IL) and 10% sodium citrate (Sigma; St Louis, MO). Complete decalcification was determined by radiography. The spine was cut in sagittal section, embedded in paraffin, and sectioned at 4 µm. Sections were stained with Masson-trichrome for evaluation of general disc features and with picrosirius red-Alcian blue for evaluation of proteoglycan content (Gruber et al. 2002).
Adjacent sections were processed for immunohistochemical localization of collagens I, II, III, and VI. Paraffin sections were cut at 4 µm, collected on PLUS slides (Allegiance), dried at 60C, and deparaffinized in xylene (Allegiance). Sections were rehydrated through graded alcohol changes (AAPER; Shelbyville, KY) to distilled water. The immunohistochemistry procedure was performed with the Dako Autostainer Plus (DakoCytomation; Carpinteria, CA) automated stainer. Endogenous enzyme was blocked using 3% H2O2 (Sigma) in methanol (Allegiance) for 5 min. Slides were rinsed with Tris-buffered saline containing 0.05% Tween 20 (TBST) (DakoCytomation) and were treated with 10% normal goat serum (Sigma). Normal sera were diluted in a solution of 5% BSA (Sigma) and 4% nonfat dried milk (Carnation; Young America, MN) in PBS, pH 7.4 (Roche, Indianapolis, IN). The blocking solution was blown off the slides without rinsing and the primary antibody was applied. All antibodies were purchased from Biodesign International (Saco, ME). Anticollagen I and II IgG were used at a 1:100 dilution, anticollagen III was used at a 1:200 dilution, and anticollagen VI IgG was used at a 1:400 dilution. All antibodies were diluted in 10% normal goat serum. Ten percent normal goat serum was used as a negative control. Slides were incubated in primary antibody for 90 min, rinsed in TBST, and treated with biotinylated goat anti-rabbit IgG (Vector Laboratories; Burlingame, CA) diluted 1:50 in 10% normal goat serum for 1 hr. Slides were rinsed in TBST and were treated with streptavidin conjugated horseradish peroxidase (DakoCytomation) for 20 min. Slides were rinsed in TBST and treated with the Chromagen Vector NovaRed (Vector Laboratories) for 5 min. Slides were rinsed in water, removed from the Autostainer, counterstained with Light Green (Polysciences; Warrington, PA), dehydrated, cleared, and mounted with Cytoseal XYL (Allegiance).
Light microscopy was performed on the lumbar spines of the following WT animals: 11 mice aged 0.32.9 months, 15 3-month-old mice, 8 mice aged 4.55 months, and 6 mice aged 620 months. The following SPARC-null mice were studied: 16 mice aged 0.32.9 months, 12 3-month-old mice, and 15 mice aged 621 months.
Histomorphometry was performed on the annulus to determine cell densities in WT and SPARC-null mice. Quantitative histomorphometry was performed on tissue sections stained with Masson-trichrome dye using the OsteoMeasure system (OsteoMetrics; Atlanta, GA).
For ultrastructural studies, discs from two WT and two SPARC-null mice were minced, immersed in Karnovsky's fixative, postfixed with osmium tetroxide, embedded in Spurr resin, thin-sectioned, and grid-stained with uranyl acetate and lead citrate. Sections were viewed on a Philips CM10 transmission electron microscope. Analysis of collagen fibril diameters was performed with the OsteoMeasure system (OsteoMetrics).
Standard statistical methods were performed with SAS version 8.2 (SAS; Carey, NC). Data are presented as mean ± SD (n).
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Results |
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As shown in Figure 1A, in a 2-month-old WT animal, disc spaces were regular and showed no wedging or endplate calcification. In contrast, radiologic images of 2-month-old SPARC-null mice showed wedging, endplate calcification, and sclerosis. Figure 1B shows loss of disc space and spontaneous fusion (arrow). As the SPARC-null mouse ages, radiological signs of degeneration appeared earlier compared with age-matched WT spines. Note that the lumbar spine of the 6-month-old SPARC-null mouse in Figure 1D showed substantially more prominent wedging and endplate calcification than observed in the spine of an 11-month-old WT mouse (Figure 1C).
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Characterization of Intervertebral Discs
No herniations have been identified in any of the WT discs. In SPARC-null specimens, however, histological evidence of herniated discs has been found in mice aged 14, 19, and 20 months (Figure 4). These herniations projected dorsally from the disc and often appeared to impinge on the spinal cord. Multiple herniated discs have been observed in the same spine (as shown in Figures 4B and 4C, which depict adjacent discs).
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Discussion |
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Mice with targeted deletions of the matricellular proteins are valuable research models. Deletions of most of the matricellular proteins studied to date result in either grossly normal or subtle phenotypes that are exacerbated during injury. Framson and Sage (2004) have recently reviewed the major phenotypic characteristics of SPARC-null mice. These mice exhibit defects in the lens capsule associated with early cataract formation (Yan et al. 2002
), osteopenia (Delany et al. 2000
), reduction of the dermis, with small collagen fibrils and decreased tensile strength of the skin (Bradshaw et al. 2003b
), and increased adipose tissue (Bradshaw et al. 2003a
). Our data contribute to the number and type of tissues adversely affected by the absence of SPARC to include alterations of the ECM of the intervertebral disc associated with accelerated disc degeneration during aging.
The SPARC-null mouse also exhibits an altered response to injury, reflected in accelerated closure of cutaneous wounds (Bradshaw et al. 2002), diminished encapsulation of implanted materials (Puolakkainen et al. 2003
), reduced tumor growth of mammary carcinoma (Sangaletti et al. 2003
), and enhanced growth and metastasis of Lewis lung carcinoma and B-cell lymphoma (Brekken et al. 2003
). Such changes in the SPARC-null phenotype reflect the significant role played by SPARC in the design, maintenance, and repair of a diversity of tissues types. As pointed out by Framson and Sage (2004)
, the common theme revealed by these defects is that SPARC plays a critical role in the production or assembly of the ECM. The results presented here indicate that discs from young SPARC-null mice can meet the structural and remodeling demands made on them, but with aging and loss of disc cells, the ECM fails, with resultant herniations.
From what is known of the role of SPARC in other tissues, our data are consistent with several possible mechanisms that could account for the disc ECM failure in SPARC-null mice. One candidate is the substantial downregulation of type I collagen that is seen in vitro and is reflected in alterations such as the thin dermis of mice lacking SPARC (Bradshaw et al. 2003b). TGF-ß is a recognized mitogen for disc cells (Gruber et al. 1997
), and it is known that SPARC affects cellular levels of TGF-ß, its receptor activation, and components of its signal transduction (Francki et al. 1999
; Schiemann and Schiemann 2003
).
The decreased proteoglycan content seen in herniated discs was an important finding, consistent with observations in human disc aging and degeneration wherein glycosaminoglycan content decreases with age (Antoniou et al. 1996). Because proteoglycans bind water and contribute to the "shock absorber" function of the disc, the changes in biochemical disc composition observed here in herniated discs of SPARC-null mice probably have significant functional consequences, and further work is needed to investigate proteoglycan loss in discs of the SPARC-null mouse.
In summary, the accelerated degeneration in discs from SPARC-null mice indicates that SPARC has an essential role in normal disc ECM remodeling and matrix homeostasis. The absence of SPARC adversely affects: (a) the number of disc cells in older animals, (b) the ultrastructure of collagen fibrils (evidenced by alterations in fibril diameter and shape in both young and old mice), (c) type I collagen content (apparent reduction in the annulus of both young and old mice), (d) spinal structure (apparent from radiologic wedging and endplate changes in young and old mice), and (e) the structural integrity of the disc (seen in the development of disc herniations in older mice).
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Footnotes |
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Literature Cited |
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