Title: SPARC-Null Mice Display Abnormalities in the Dermis Characterized by Decreased Collagen Fibril Diameter and Reduced Tensile Strength
Abstract: Although collagen and elastic fibers are among the major structural constituents responsible for the mechanical properties of skin, proteins that associate with these components are also important for directing formation and maintaining the stability of these fibers. We present evidence that SPARC (secreted protein acidic and rich in cysteine) contributes to collagen fibril formation in the dermis. The skin of SPARC-null adult mice had approximately half the tensile strength as that of wild-type skin. Moreover, the collagen content of SPARC-null skin, as measured by hydroxyproline analysis, was substantially reduced in adult mice. At 2 weeks of age, no differences in collagen content were observed; within 2 months, however, the dermis of SPARC-null mice displayed a reduced collagen content that persisted through adulthood until ≈20 months, when collagen levels of SPARC-null skin approximated those of wild-type controls. The collagen fibrils present in SPARC-null skin were smaller and more uniform in diameter, in comparison with those of wild-type skin. At 5 months of age, the average fibril diameter in SPARC-null versus wild-type skin was 60.2 nm versus 87.9 nm, respectively. Extraction of soluble dermal collagen revealed a relative increase in collagen VI, accompanied by a decrease in collagen I, in SPARC-null mice. A reduction in the relative amounts of higher-molecular weight collagen complexes was also observed in extracts of dermis from SPARC-null animals. Thus the absence of SPARC compromises the mechanical properties of the dermis, an effect that we attribute, at least in part, to the changes in the structure and composition of its collagenous extracellular matrix. Although collagen and elastic fibers are among the major structural constituents responsible for the mechanical properties of skin, proteins that associate with these components are also important for directing formation and maintaining the stability of these fibers. We present evidence that SPARC (secreted protein acidic and rich in cysteine) contributes to collagen fibril formation in the dermis. The skin of SPARC-null adult mice had approximately half the tensile strength as that of wild-type skin. Moreover, the collagen content of SPARC-null skin, as measured by hydroxyproline analysis, was substantially reduced in adult mice. At 2 weeks of age, no differences in collagen content were observed; within 2 months, however, the dermis of SPARC-null mice displayed a reduced collagen content that persisted through adulthood until ≈20 months, when collagen levels of SPARC-null skin approximated those of wild-type controls. The collagen fibrils present in SPARC-null skin were smaller and more uniform in diameter, in comparison with those of wild-type skin. At 5 months of age, the average fibril diameter in SPARC-null versus wild-type skin was 60.2 nm versus 87.9 nm, respectively. Extraction of soluble dermal collagen revealed a relative increase in collagen VI, accompanied by a decrease in collagen I, in SPARC-null mice. A reduction in the relative amounts of higher-molecular weight collagen complexes was also observed in extracts of dermis from SPARC-null animals. Thus the absence of SPARC compromises the mechanical properties of the dermis, an effect that we attribute, at least in part, to the changes in the structure and composition of its collagenous extracellular matrix. SPARC (secreted protein acidic and rich in cysteine/BM-40/osteonectin) is a secreted glycoprotein that belongs to the matricellular class of proteins. Matricellular proteins associate with the extracellular matrix (ECM) but are thought not to serve a structural function (as established for classical ECM proteins such as collagen), but to act as modulators of cell–ECM interaction (Bornstein and Sage, 2002Bornstein P. Sage E.H. Matricellular proteins. Extracellular modulators of cell function.Curr Opin Cell Biol. 2002; 14: 608-616Crossref PubMed Scopus (708) Google Scholar). Thrombospondin 1 and 2, tenascin C and X, osteopontin, and SPARC are examples of matricellular proteins, the absence of which leads to changes in ECM assembly and composition (Bornstein and Sage, 2002Bornstein P. Sage E.H. Matricellular proteins. Extracellular modulators of cell function.Curr Opin Cell Biol. 2002; 14: 608-616Crossref PubMed Scopus (708) Google Scholar). For example, thrombospondin-2 null mice display altered collagen fibril morphology in the skin (Kyriakides et al., 1998Kyriakides T.R. Zhu Y.-H. Smith L.T. et al.Mice that lack thrombospondin 2 display connective tissue abnormalities that are associated with disordered collagen fibrillogenesis, an increased vascular density, and a bleeding diathesis.J Cell Biol. 1998; 140: 419-430Crossref PubMed Scopus (386) Google Scholar). Moreover, mutations in tenascin X have been implicated in a form of Ehlers–Danlos syndrome (Burch et al., 1997Burch G.H. Gong Y. Liu W. et al.Tenascin X deficiency is associated with Ehlets-Danlos syndrome.Nature Gen. 1997; 17: 104-108Crossref PubMed Scopus (259) Google Scholar), and result in diminished deposition of collagen in the skin (Mao et al., 2002Mao J.R. Taylor G. Dean W.B. et al.Tenascin-X deficiency mimics Ehlers–Danlos syndrome in mice through alteration of collagen deposition.Nat Genet. 2002; 30: 421-425Crossref PubMed Scopus (185) Google Scholar). Apparently, cells express matricellular proteins in a tissue-specific manner to modify and construct the appropriate ECM required for tissue function. SPARC is expressed at high levels during development in many areas of the embryo, and SPARC mRNA and protein decrease concomitantly with the differentiation of specific cell types and organization of tissues (Lane and Sage, 1994Lane T.F. Sage E.H. The biology of SPARC, a protein that modulates cell–matrix interactions.FASEB J. 1994; 8: 163-173Crossref PubMed Scopus (466) Google Scholar). Expression of SPARC often recurs in response to injury and during ECM turnover in adult animals. For example, increased amounts of SPARC are observed during dermal wound repair and in association with angiogenesis (Reed et al., 1993Reed M.J. Puolakkainen P. Lane T.F. Dickerson D. Bornstein P. Sage E.H. Differential expression of SPARC and thrombospondin 1 in wound repair: Immunolocalization and in situ hybridization.J Histochem Cytochem. 1993; 41: 1467-1477Crossref PubMed Scopus (185) Google Scholar; Brekken and Sage, 2001Brekken R.A. Sage E.H. SPARC, a matricellular protein: at the crossroads of cell-matrix communication.Matrix Biol. 2001; 19: 815-827Crossref Scopus (36) Google Scholar). In many cases, the production of SPARC accompanies induction of collagen I (Brekken and Sage, 2001Brekken R.A. Sage E.H. SPARC, a matricellular protein: at the crossroads of cell-matrix communication.Matrix Biol. 2001; 19: 815-827Crossref Scopus (36) Google Scholar). SPARC binds to collagens I–V, and cleavage of SPARC by certain matrix metallo-proteinases results in an increased affinity of SPARC for these ECM proteins (Sage et al., 1989Sage H. Vernon R.B. Funk S.E. Everitt E.A. Angello J. SPARC, a secreted protein associated with cellular proliferation, inhibits cell spreading in vitro and exhibits Ca2+-dependent binding to the extracellular matrix.J Cell Biol. 1989; 109: 341-356Crossref PubMed Scopus (310) Google Scholar; Sasaki et al., 1997Sasaki T. Göhring W. Mann K. et al.Limited cleavage of extracellular matrix protein BM-40 by matrix metalloproteinases increases its affinity for collagens.J Biol Chem. 1997; 272: 9237-9243Crossref PubMed Scopus (134) Google Scholar).Iruela-Arispe et al., 1996Iruela-Arispe M.-L. Vernon R.B. Wu H. Jaenisch R. Sage E.H. Type I collagen-deficient mov-13 mice do not retain SPARC in the extracellular matrix: Implications for fibroblast function.Dev Dyn. 1996; 207: 171-183Crossref PubMed Scopus (51) Google Scholar reported that mice that do not express collagen I in mesenchymal tissues fail to deposit SPARC into ECM in vivo and in vitro. Hence, production of collagen I appears to be requisite for the association of SPARC with embryonic ECM in the mouse. SPARC-null mice display a number of different abnormalities that appear to reflect, at least in part, changes in the structure and composition of tissue-specific ECM (Bradshaw and Sage, 2001Bradshaw A.D. Sage E.H. SPARC, a matricellular protein that functions in cellular differentiation and tissue response to injury.J Clin Invest. 2001; 107: 1049-1054Crossref PubMed Scopus (490) Google Scholar). Early onset cataractogenesis was observed in three separate SPARC-null transgenic backgrounds (Gilmour et al., 1998Gilmour D.T. Lyon G.J. Carlton M.B.L. et al.Mice deficient for the secreted glycoprotein SPARC/osteonectin/BM40 develop normally but show severe age-onset cataract formation and disruption of the lens.EMBO J. 1998; 17: 1860-1870Crossref PubMed Scopus (204) Google Scholar; Norose et al., 1998Norose K. Clark J.I. Syed N.A. Basu A. Heberkatz E.S. Sage E.H. Howe C.C. SPARC deficiency leads to early onset cataractogenesis.Invest Ophthalmol Vis Sci. 1998; 39: 2674-2680PubMed Google Scholar).Yan et al., 2002Yan Q. Clark J.I. Wight T.N. Sage E.H. Alterations in the lens capsule contribute to cataractogenesis in SPARC-null mice.J Cell Sci. 2002; 115: 2747-2756PubMed Google Scholar reported substantial alterations in the distribution of collagen IV and laminin in the SPARC-null lens capsule basement membrane, which was more permeable to water and small molecules than the capsule synthesized in wild-type mice. Presumably, a lack of SPARC contributed to early onset cataractogenesis by disruption of the ion and fluid balance across the basement membrane of the lens. SPARC-null mice also developed severe osteopenia by 6 mo of age, due in part to a decrease in the synthesis of new bone by osteoblasts (Delany et al., 2000Delany A.M. Amling M. Priemel M. Howe C. Baron R. Canalis E. Osteopenia and decreased bone formation in osteonectin-deficient mice.J Clin Invest. 2000; 105: 915-923Crossref PubMed Scopus (218) Google Scholar). In addition, mice that do not produce SPARC exhibited enhanced closure of dermal wounds (Bradshaw et al., 2002Bradshaw A.D. Reed M.J. Sage E.H. SPARC-Null mice exhibit accelerated cutaneous wound closure.J Histochem Cytochem. 2002; 50: 1-10Crossref PubMed Scopus (137) Google Scholar). The authors proposed that the connective tissue of the dermis made in the absence of SPARC was intrinsically more contractile than that of wild-type mice, a property due, at least in part, to the decrease in collagen content of the skin in SPARC-null mice (Bradshaw et al., 2002Bradshaw A.D. Reed M.J. Sage E.H. SPARC-Null mice exhibit accelerated cutaneous wound closure.J Histochem Cytochem. 2002; 50: 1-10Crossref PubMed Scopus (137) Google Scholar). We sought to investigate further the function of SPARC in the design and composition of the ECM in skin. We observed that the decrease in collagen content of SPARC-null skin began at ≈–2 mo of age and became more substantial with age. We found that the dermal collagen fibrils assembled in the absence of SPARC were smaller and more uniform in diameter than those of wild-type dermis. We also showed that the altered SPARC-null dermal ECM resulted in a decreased tensile strength of SPARC-null skin in comparison with wild-type skin. We conclude that expression of SPARC is required for maturation of collagen in the dermal ECM, and that the absence of this glycoprotein leads to aberrant ECM in murine skin that affects its mechanical strength. C57BL/6J X 129SVJ mice with a targeted deletion of the SPARC gene as described byNorose et al., 1998Norose K. Clark J.I. Syed N.A. Basu A. Heberkatz E.S. Sage E.H. Howe C.C. SPARC deficiency leads to early onset cataractogenesis.Invest Ophthalmol Vis Sci. 1998; 39: 2674-2680PubMed Google Scholar were used in these studies. Wild-type control mice generated from periodic heterozygous crosses were maintained under identical conditions as SPARC-null mice to ensure minimal variability in genetic background and environment between transgenic and wild-type animals. Experiments were conducted under a protocol approved by the Institutional Animal Care and use Committee of the Hopeheart Institute and Fred Hutchinson Cancer Research Center, Seattle, WA, USA. Skin from age-matched wild-type and SPARC-null animals was fixed in either formalin (histologic stains), methyl Carnoy's (immunohistochemistry for SPARC), or Karnovsky's fixative (EM). Masson's trichrome and picro-sirius red stains were carried out as described (Bradshaw et al., 2002Bradshaw A.D. Reed M.J. Sage E.H. SPARC-Null mice exhibit accelerated cutaneous wound closure.J Histochem Cytochem. 2002; 50: 1-10Crossref PubMed Scopus (137) Google Scholar). Immunohistochemistry was performed with an affinity-purified polyclonal antibody that was generated in goat against murine SPARC purified from PYS-2 cells (Sage et al., 1989Sage H. Vernon R.B. Funk S.E. Everitt E.A. Angello J. SPARC, a secreted protein associated with cellular proliferation, inhibits cell spreading in vitro and exhibits Ca2+-dependent binding to the extracellular matrix.J Cell Biol. 1989; 109: 341-356Crossref PubMed Scopus (310) Google Scholar). Secondary antibodies against goat IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA) were used to detect and amplify primary immunoreactivity in sections of skin. Subsequent incubation of sections with 3,3′-diaminobenzidine substrate (Sigma, St Louis, MO) was used to identify immunoreactivity for SPARC. Slides were counterstained with toluidine blue. Skin from wild-type and SPARC-null mice was immersed in Karnovsky's fixative for EM and processed for routine transmission EM (Karnovsky, 1965Karnovsky M.J. A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy.J Cell Biol. 1965; 272: 137aGoogle Scholar). EM taken at ×20,000 magnification were scanned into Adobe Photoshop (Fremont, WA) on an Epson flat bed scanner. Three wild-type and six SPARC-null animals were used for quantification, with a minimum contribution of 200 fibrils from each mouse. Measurement of fibril diameter was performed with the NIH image software program, and data were transferred to the Microsoft Excel program (Redmond, WA) to generate average lengths and distributions of diameters. Full thickness (2 cm in length×1 cm in width) strips of 5 mo wild-type (n=9) and SPARC-null skin (n=12) were used to determine the tensile strength by an Instron tensiometer. The tissue was allowed to stretch and break under a given load (kilogram-force) in the tensiometer. The unit of measurement is force per cross-sectional area=kg per mm2. The results are reported as ratios between the mean tensile strength of SPARC-null and wild-type mice. The significance of the difference between the two genotypes was determined by Student's t test for unpaired values. A minimum of three mice of each genotype contributed to each time point. Sections of ≈1 cm2 were collected from the dorsum of each animal. Skin pieces uniform in color were chosen for analysis to ensure similar stages of hair follicle morphogenesis between samples (Yamamoto and Yamauchi, 1999Yamamoto K. Yamauchi M. Characterization of dermal type I collagen of C3H mouse at different stages of the hair cycle.Br J Dermatol. 1999; 141: 667-675Crossref PubMed Scopus (12) Google Scholar; Muller-Rover et al., 2001Muller-Rover S. Handjiski B. van der Veen C. et al.A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages.J Invest Dermatol. 2001; 117: 3-15Abstract Full Text Full Text PDF PubMed Google Scholar). Hydroxyproline analysis was carried out according toWoessner, 1961Woessner J.F. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid.Arch Biochem Biophys. 1961; 93: 440-447Crossref PubMed Scopus (3258) Google Scholar, as described inBradshaw et al., 2002Bradshaw A.D. Reed M.J. Sage E.H. SPARC-Null mice exhibit accelerated cutaneous wound closure.J Histochem Cytochem. 2002; 50: 1-10Crossref PubMed Scopus (137) Google Scholar. Equal weights of skin taken from the dorsa of wild-type and SPARC-null mice were minced and placed in cold 0.5 M acetic acid. The skin pieces were stirred overnight at 4°C. Soluble collagen was separated from insoluble collagen by centrifugation at 12,000×g for 30 min, and the supernate was dialyzed against 0.1 M acetic acid at 4°C and was lyophilized. Equal amounts of lyophilized protein (by weight) were resuspended in cold 0.1 M acetic acid and were tumbled from 4 to 20 h. Equal volumes of wild-type and SPARC-null protein were neutralized with 1 M Tris-base (pH 11), boiled in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer with and without 0.1 M dithiothreitol, and resolved by SDS–PAGE on 8% polyacrylamide gels (Laemmli, 1970Laemmli U.K. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4.Nature. 1970; 277: 680-685Crossref Scopus (202752) Google Scholar). For detection of collagen VI, proteins were transferred to PVDF membranes. Three separate collagen VI polyclonal antibodies (provided by Dr Eva Engvall, Burnham Institute, La Jolla, CA) were immunoreactive with a ≈140 kDa band (Engvall et al., 1986Engvall E. Hessle H. Klier G. Molecular assembly, secretion, and matrix deposition of type VI collagen.J Cell Biol. 1986; 102: 703-710Crossref PubMed Scopus (183) Google Scholar) that was sensitive to pepsin digestion, characteristic of type VI collagen (Timpl and Engel, 1987Timpl R. Engel J. Type VI collagen.in: Mayne R. Burgeson R.E. Structure and Function of Collagen Types. Academic Press, San Diego1987: 105-143Crossref Google Scholar). Through the course of routine handling of the SPARC-null mice in our colony, we observed that the skin of mice lacking SPARC tended to be more pliable and sensitive to tearing than that of wild-type mice. In addition, the tails of SPARC-null mice always curled at the tips by ≈3 mo of age. We reasoned that collagen-rich ECM present in the skin and tendon of the tail might be altered in the absence of SPARC. Shown in Figure 1 are sections of skin from adult wild-type (Figure 1a,c) and SPARC-null mice (Figure 1b,d). Collagen fibers stained by Masson's trichrome reagent (Figure 1a,b) are blue. The SPARC-null dermis appeared to be less dense than that of wild-type animals. Picrosirius red (Figure 1c,d) stains mature collagen fibers red, and less cross-linked fibers as yellow and green, when viewed under polarized light (Sweat et al., 1964Sweat F. Puchtler H. Rosenthal S.I. Sirius red F3BA as stain for connective tissue.Arch Pathol. 1964; 78: 69-72PubMed Google Scholar). A preponderance of smaller, yellow and green fibers was present in the SPARC-null dermis, a result indicating that the majority of collagen fibers formed in the absence of SPARC were more immature and less cross-linked than those of wild-type dermis. EM of dermis from SPARC-null (Figure 2a,c) and wild-type mice (Figure 2b,d) revealed further differences in collagen organization (Figure 2). Collagen fibrils in SPARC-null dermis were smaller and more uniform in diameter than the corresponding fibrils in wild-type skin. The difference in collagen fibrils was especially pronounced when deeper regions of wild-type and SPARC-null reticular dermis were compared. Typical of normal murine skin, a broad range of fibril diameters was noted in this region (Craig et al., 1987Craig A.S. Eikenberry E.F. Parry D.A. Ultrastructural organization of skin: classification on the basis of mechanical role.Connect Tissue Res. 1987; 16: 213-223Crossref PubMed Scopus (57) Google Scholar). In contrast, SPARC-null collagen fibrils displayed similar diameters throughout the dermis. Collagen fibril diameters were quantified from EM taken predominantly from the reticular dermis of three wild-type and six SPARC-null animals. The size distribution profiles are shown in Figure 3. Average diameters of 87.9 nm for wild-type and 60.2 nm for SPARC-null collagen fibrils were found. Hence, the absence of SPARC resulted in significant alterations in the ultrastructure of the dermal ECM.Figure 3Quantification and distribution of collagen fibril diameters from wild-type and SPARC-null skin. The frequency of collagen fibrils with a given diameter from wild-type (white bars) and SPARC-null skin (black bars) is shown in the histogram. At least 200 fibrils from six SPARC-null and three wild-type animals at 5 mo of age were used for the analysis. The average fibril diameter in SPARC-null skin was 60.2 nm, whereas wild-type fibrils averaged 87.9 nm. Collagen fibrils larger than 110 nm in diameter are not present in SPARC-null dermis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We hypothesized that the changes in the structure and composition of the ECM in SPARC-null skin might adversely affect the tensile strength of the tissue. Pieces of skin from the dorsa of 5 mo wild-type and SPARC-null animals were placed on an Instron tensiometer to measure their tensile strength (Eming et al., 1999Eming S.A. Whitsitt J.S. He L. Krieg T. Morgan J.R. Davidson J.M. Particle-mediated gene transfer of PDGF isoforms promotes wound repair.J Invest Dermatol. 1999; 112: 297-302Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). As shown in Figure 4, the skin of adult SPARC-null mice (black bar) exhibited a substantially lower tensile strength than the skin of adult wild-type animals (white bar). We reported previously that the collagen content of SPARC-null skin is approximately half that of wild-type skin in adult animals (Bradshaw et al., 2002Bradshaw A.D. Reed M.J. Sage E.H. SPARC-Null mice exhibit accelerated cutaneous wound closure.J Histochem Cytochem. 2002; 50: 1-10Crossref PubMed Scopus (137) Google Scholar). We asked whether the decrease in collagen accumulation in the skin was affected by age. Hydroxyproline analysis was carried out on skin samples from age-matched wild-type and SPARC-null animals at seven separate ages (Figure 5). Interestingly, animals that were 2 wk of age did not display significant differences in the content of dermal collagen. A substantial decrease in the amount of collagen in SPARC-null vs wild-type skin was noted at 3 mo of age, when the period of rapid growth ends. The decrease in collagen content of SPARC-null skin was maintained over the first year. Although the differences in hydroxyproline content at 6 mo were not statistically significant between wild-type and SPARC-null skin, the trend toward diminished levels of collagen in the absence of SPARC was apparent. At ages >20 mo, the levels of collagen in SPARC-null skin appro-ximate that of wild-type skin (Figure 5). To begin to address the function of SPARC in ECM organization in the skin, we performed immunohistochemistry with an anti-SPARC polyclonal antibody on sections of skin from wild-type animals of different ages (Figure 6). The expression of SPARC in adult skin has been described previously for rat and human species (Reed et al., 1993Reed M.J. Puolakkainen P. Lane T.F. Dickerson D. Bornstein P. Sage E.H. Differential expression of SPARC and thrombospondin 1 in wound repair: Immunolocalization and in situ hybridization.J Histochem Cytochem. 1993; 41: 1467-1477Crossref PubMed Scopus (185) Google Scholar; Hunzelman et al., 1998Hunzelman N. Hafner M. Anders S. Krieg T. Nischt R. BM-40 (osteonectin, SPARC) is expressed both in the epidermal and in the dermal compartment of adult human skin.J Invest Dermatol. 1998; 110: 122-126Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In accordance with these studies, we found SPARC immunoreactivity in adult dermis to be primarily associated with hair follicles, blood vessels, and occasional fibroblasts (Figure 6e,f). The incidence of SPARC was substantially reduced in adult skin in comparison with that observed in sections from postnatal day 1 (Figure 6, compares Figure 6e,f with Figure 6a,c). In fact, immunoreactivity for SPARC was sharply reduced in skin from animals 2 wk of age (Figure 6b,d) in comparison with newborn mice (Figure 6a,c). Immunoreactivity in the skin of newborn animals appeared within fibroblasts and was associated with extracellular material. Although resolution at the level of the light microscope does not provide definitive evidence for the localization of SPARC to the ECM of skin in newborn animals, the intensity and prevalence of staining throughout the sections were consistent with an association of SPARC with dermal ECM at postnatal day 1. By 2 wk of age, immunoreactivity for SPARC appeared to be largely intracellular. Levels of SPARC in the skin appear to diminish with increasing age, consistent with the decrease in SPARC observed in other tissues that coincides with differentiation and the cessation of growth (Bradshaw and Sage, 2001Bradshaw A.D. Sage E.H. SPARC, a matricellular protein that functions in cellular differentiation and tissue response to injury.J Clin Invest. 2001; 107: 1049-1054Crossref PubMed Scopus (490) Google Scholar). To assess potential biochemical alterations in the expression and/or modification of collagens in the dermis, we extracted soluble collagen from skin with acetic acid (Sage and Bornstein, 1982Sage E.H. Bornstein P. Preparation and characterization of procollagens and procollagen-collagen intermediates.Methods Enzymol. 1982; 82: 96-127Crossref PubMed Scopus (40) Google Scholar). We also treated the insoluble collagen fraction with pepsin to release collagen that was highly cross-linked from the matrix. No differences were observed between SPARC-null and wild-type collagen released by pepsin from the insoluble matrix (data not shown). Shown in Figure 7 is an SDS–polyacrylamide gel of soluble collagens extracted from wild-type and SPARC-null skin from animals of increasing age. Equal amounts of lyophilized collagen soluble in acetic acid were resolved by SDS–PAGE. The major collagen in skin is collagen type I (Figure 7, small arrows) (Klein and ChandraRajan, 1977Klein L. ChandraRajan J. Collagen degradation in rat skin but not in intestine during rapid growth: Effect on collagen types I and III from skin.Proc Natl Acad Sci USA. 1977; 74: 1436-1439Crossref PubMed Scopus (36) Google Scholar). The band ≈140 kDa present in extracts from mice 2 wk of age (Figure 7, lanes 1 and 2, dashed arrow) was shown to be collagen α 1(III) by interrupted electrophoresis following pepsin digestion (data not shown). Collagen type III has been shown to be present in higher amounts in the skin of younger animals relative to older animals (Shuttleworth and Forrest, 1975Shuttleworth C.A. Forrest L. Changes in guinea-pig dermal collagen during development.Eur J Biochem. 1975; 55: 391-395Crossref PubMed Scopus (28) Google Scholar; Ramshaw, 1986Ramshaw J.A. Distribution of type III collagen in bovine skin of various ages.Connect Tissue Res. 1986; 14: 307-314Crossref PubMed Scopus (36) Google Scholar). We did not observe significant differences between wild-type and SPARC-null samples, with respect to the profile and amount of soluble collagen extracted from mice 2 wk of age; however, there were differences in the amount of collagen extracted from mice at 2 mo of age [wild-type: 70 mg per g tissue (dry protein per wet weight of tissue); SPARC-null: 50 mg per g tissue], as well as a decrease in the amount of higher molecular weight collagen complexes (Figure 7, lanes 3 and 4, large arrows). The collagenous nature of the higher molecular weight bands was confirmed by sensitivity to collagenase (data not shown). At 4–5 mo of age, the amount of soluble collagen extracted from SPARC-null skin continued to be less than that extracted from wild-type skin (wild-type: 50 mg per g tissue; SPARC-null: 25 mg per g tissue). In addition, we noted the appearance of a ≈140 kDa band with a slightly lower mobility than the α1(I) collagen chain in extracts from mice 4 mo of age and older (Figure 7, lanes 5 and 6, upper arrowheads). The ≈140 kDa chain was consistently over-represented in collagen extracted from SPARC-null mice, in comparison with wild-type samples, in animals between 4 and 6 mo of age (see below). As reported byShuster et al., 1975Shuster S. Black M. McVitie E. The influence of age and sex on skin thickness, skin collagen and density.Br J Dermatol. 1975; 93: 639-643Crossref PubMed Scopus (388) Google Scholar, in aged animals (>20 mo) the amount of soluble collagen was greatly decreased compared with that from younger animals (wild-type and SPARC-null=20 mg per g tissue) (Figure 7, lanes 7 and 8). We did not observe differences in the amount of soluble collagen extracted from wild-type and SPARC-null skin at this age, consistent with the result shown in Figure 5, that the overall amount of collagen in the skin of both genotypes of older ages was not significantly different. Hence, the influence of SPARC on collagen content in the skin appeared to be greatest in young adult to middle-aged animals. Collagen type VI is expressed in the dermis and is composed of three subunits: α1(VI)/α2(VI)/α3(VI) (1 : 1 : 1). The α1(VI) and the α2(VI) subunits migrate at ≈140 kDa, whereas the larger α3(VI) subunit migrates at ≈230 kDa (Engvall et al., 1986Engvall E. Hessle H. Klier G. Molecular assembly, secretion, and matrix deposition of type VI collagen.J Cell Biol. 1986; 102: 703-710Crossref PubMed Scopus (183) Google Scholar). Whereas the processed forms of collagen I and III soluble in acetic acid are resistant to pepsin digestion, soluble collagen VI is cleaved by pepsin. The ≈140 kDa collagenous protein over-represented in SPARC-null acetic-acid extracts from the skin of mice 4 mo