Collagen accumulation is decreased in SPARC-null mice with bleomycin-induced pulmonary fibrosis

Thomas P. Strandjord1, David K. Madtes2, Daniel J. Weiss2, and E. Helene Sage3

1 Department of Pediatrics, University of Washington, Seattle 98195-6320; 2 Fred Hutchinson Cancer Research Center, Seattle 98109; and 3 Hope Heart Institute, Seattle, Washington 98122


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Secreted protein acidic and rich in cysteine (SPARC) has been shown to be coexpressed with type I collagen in tissues undergoing remodeling and wound repair. We speculated that SPARC is required for the accumulation of collagen in lung injury and that its absence would attenuate collagen accumulation. Accordingly, we have assessed levels of collagen in SPARC-null mice in an intratracheal bleomycin-injury model of pulmonary fibrosis. Eight- to ten-week-old SPARC-null and wild-type (WT) mice received bleomycin (0.0035 U/g) or saline intratracheally and were subsequently killed after 14 days. Relative levels of SPARC mRNA were increased 2.7-fold (P < 0.001) in bleomycin-treated WT lungs in comparison with saline-treated lungs. Protein from bleomycin-treated WT lung contained significantly more hydroxyproline (191.9 µg/lung) than protein from either bleomycin-treated SPARC-null lungs or saline-treated WT and SPARC-null lungs (147.4 µg/lung, 125.4 µg/lung, and 113.0 µg/lung, respectively; P < 0.03). These results indicate that SPARC is increased in response to lung injury and that accumulation of collagen, as indicated by hydroxyproline content, is attenuated in the absence of SPARC. The properties of SPARC as a matricellular protein associated with cell proliferation and matrix turnover are consistent with its participation in the development of pulmonary fibrosis.

secreted protein acidic and rich in cysteine; fibroblast; osteonectin; BM-40; lung injury; immunohistochemistry; extracellular matrix


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DEVELOPMENT OF PULMONARY FIBROSIS requires a complex interplay between lung epithelial and interstitial cells, inflammatory cells, inflammatory mediators, cytokines, and extracellular matrix (3). One class of proteins with a potential for modulating fibrosis is the "matricellular" proteins that bind to matrix components as well as to cell surface receptors (4). They include tenascin, the thrombospondins, and secreted protein acidic and rich in cysteine (SPARC), structurally distinct secreted proteins with counteradhesive properties (38). Tenascin expression has been shown to be increased in rat lung in the bleomycin-injury model of pulmonary fibrosis during early stages of inflammation (57).

SPARC, also known as osteonectin and BM-40, is an ~34-kDa glycoprotein that has a number of properties indicative of potential importance in tissue repair after injury (30, 40). SPARC has been shown to be associated with tissues undergoing morphogenesis and remodeling. Immunohistochemical staining of adult mouse tissues demonstrated its association with epithelia exhibiting high rates of turnover such as gut, skin, and glandular tissue (41). Expression of SPARC is also widespread in fetal tissues, including fetal lung (41, 50). It has been localized in fibroblasts at the margins of experimental wounds in rats (37) and in fibroblasts in the lungs of patients with idiopathic pulmonary fibrosis (28). In these fibrotic lungs, SPARC was in the cytoplasm of fibroblasts in areas of active connective tissue synthesis.

Experiments from several laboratories indicate that SPARC might participate in the regulation of matrix turnover. Treatment of cultured fibroblasts with SPARC stimulates production of collagenase [matrix metalloproteinase (MMP)-1], stromelysin (MMP-3), and 92-kDa gelatinase (MMP-9) (53). The expression of SPARC is closely associated with that of other matrix molecules. It is coexpressed with type I collagen (41) and can bind collagen and thrombospondin-1 (11, 42). Finally, SPARC has counteradhesive properties in vitro and inhibits the spreading of cultured endothelial cells and fibroblasts. These observations suggest that SPARC may participate in the regulation of tissue remodeling and repair after injury.

Intratracheal instillation of bleomycin is a well-described model of pulmonary fibrosis (5). Administration of bleomycin leads to a patchy fibrotic process in many species of animals, including several strains of mice (44, 45, 47, 51). Animals injured with bleomycin undergo a characteristic sequence of inflammatory cell migration, edema, cellular proliferation, and accumulation of collagen, analogous to many fibrosing pathological states in human lungs (8, 31). Extensive remodeling of the extracellular matrix follows bleomycin injury of the lung. Changes include increases in steady-state levels of type I and III procollagen mRNAs, macrophage metalloelastase (MMP-12), tissue inhibitor of metalloproteinase-1, fibronectin, and gelatinase A (MMP-2) (24, 51). Whether SPARC is involved in the regulation of any of these responses to injury has not been examined.

Recently, a transgenic mouse model of SPARC deficiency has been produced that allows for the investigation of the role of SPARC in pulmonary fibrosis. SPARC-null mice have been reported to develop cataracts (18, 33). Studies of mesenchymal cells cultured from SPARC-null mice have demonstrated increased rates of cellular proliferation (6) and decreased synthesis of type I collagen (17). No other phenotypic abnormalities in the homozygous mutants in vivo have yet been reported. Pulmonary morphology and function have not been studied in these mutant mice.

Our objective in this study was to determine whether the absence of SPARC attenuated collagen accumulation in the bleomycin model of pulmonary fibrosis. Transgenic SPARC-null mice and wild-type (WT) controls were given bleomycin or saline by intratracheal instillation. We found increased expression of SPARC in bleomycin-injured WT lungs in comparison with saline-treated control WT lungs and decreased accumulation of collagen in injured SPARC-null lungs relative to injured WT lungs. These results were consistent with the participation of SPARC in the regulation of pulmonary fibrosis.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Bleomycin-induced lung injury. Specific pathogen-free female and male homozygous SPARC-null mice (33) and WT mice (both produced by crossing SPARC-null 129 and C57BL/6 mice maintained on site in a specific pathogen-free facility) 8- to 10-wk old, weighing 18-25 g, were given 0.0035 U/g of bleomycin sulfate (Pharmacia, Kalamazoo, MI) in 2.33 µl/g of sterile saline via transtracheal puncture under intraperitoneal avertin anesthesia (44). Control mice received saline alone. This bleomycin dose has been demonstrated to produce pulmonary fibrosis consistently, with a mortality rate of <10% in preliminary experiments with mice of similar genetic background. Fourteen days after injection, the mice were killed by exsanguination under deep anesthesia with intraperitoneal avertin. The lungs were exposed by a mid-thoracotomy incision, and the pulmonary arteries were perfused with RNase-free PBS (0.01 M NaH2PO4 and 0.15 M NaCl, pH 7.6) via right ventricular puncture. The right lung was isolated with a ligature at the right hilum, resected, rinsed in RNase-free PBS, and finely minced. Minces were divided into two aliquots, one each for tissue hydroxyproline and RNA isolation. Each aliquot of the minced right lung was weighed, frozen in liquid nitrogen, and stored at -70°C for further analysis. The left lung was inflated with 4% neutral buffered paraformaldehyde or methyl Carnoy's solution (60% methanol-30% chloroform-10% glacial acetic acid) instilled at 30 cmH2O of pressure through the trachea for 120 min. The trachea was tied, and the lung was immersed in 4% buffered paraformaldehyde for 24 h or, in the case of methyl Carnoy's fixation, transferred to 70% ethanol before being embedded in paraffin. Five-micrometer-thick sections of paraformaldehyde-fixed lung were exposed to Masson's trichrome stain to visualize fibrillar collagen in the lung.

Hydroxyproline quantification. Total right lung collagen content was determined by assay of lung hydroxyproline content after hydrolysis in 6 N HCl as previously described (54). A minced aliquot of right lung was added to 800 µl of 6 N HCl and hydrolyzed overnight at 110°C. To 200 µl of hydrolysate were added 100 µl of 0.02% methyl red and 20 µl of 0.04% bromthymol blue. The sample volume was adjusted to 2 ml with 0.5× assay buffer (0.024 M citric acid, 0.02 M acetic acid, 0.088 M sodium acetate, and 0.085 M NaOH), and the pH was adjusted to 6.5-7.0. The colorimetric assay was performed by addition of 1 ml of chloramine T solution to the sample, incubation at room temperature for 20 min, and subsequent addition of 1 ml of dimethyl benzaldehyde solution with incubation at 60°C for 15 min. The absorbance at 550 nm was measured for each lung sample. Whole lung hydroxyproline values were determined by normalization of the hydroxyproline values obtained from colorimetric assay of the minced lung aliquots to the wet weight of the whole right lung and are expressed as micrograms of hydroxyproline per lung.

RNA isolation. Total cellular RNA was isolated from the aliquot of frozen minced right lung by a modification of the method of Chirgwin et al. (10) with CsCl density gradient centrifugation (48).

Probes. Mouse SPARC cDNA (32) was labeled with a random-prime labeling kit (Promega, Madison, WI) with [alpha -32P]deoxycytidine triphosphate (NEN Life Science Products, Boston, MA) (14, 15). An 18S rRNA was used as an internal control for RNA loading (49). The 5'-end of the 24-base 18S sequence was labeled with [gamma -32P]ATP (NEN Life Science Products) in the presence of T4 polynucleotide kinase (Promega) (43).

Northern analysis. Northern analysis for SPARC mRNA was performed as previously described (49). Briefly, total cytoplasmic RNA (15 µg/lane) from each animal was resolved by electrophoresis through a 1% agarose-formaldehyde gel (43) and transferred to a nylon membrane (Nytran 0.45-mm pore size; Schleicher and Schuell, Keene, NH). The membrane was hybridized with the SPARC cDNA probe (1.1 × 106 dpm/µg DNA, 1 × 106 counts/min of probe in each milliliter of hybridization solution) at 42°C for 18 h. The membrane was washed in 0.1× SSC (1× SSC = 150 mM NaCl and 15 mM sodium citrate, pH 7.0)-0.1% (wt/vol) SDS at 65°C for 15 min. An autoradiograph of the hybridized membrane was made by exposure of PhosphorImager storage plates at room temperature for 3 days. The plates were scanned with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The membrane was stripped of SPARC cDNA probe in 0.01× SSC-0.01% (wt/vol) SDS at 100°C for 20 min (repeated twice). The stripped membrane was hybridized with an 18S oligonucleotide probe as an internal control for RNA loading of the gel as described previously (49). Quantification of bands was performed by densitometry in conjunction with ImageQuant software (Molecular Dynamics). The densitometry value for each SPARC mRNA was normalized for variations in total RNA loading to the densitometry value of 18S rRNA in the same lane.

Immunohistochemistry. Polyclonal rabbit anti-SPARC antibodies were affinity purified as previously described (2). Rabbit antiserum 5944 was produced against mouse parietal yolk sac cell SPARC (42). Immunoglobulins were precipitated with 50% ammonium sulfate and dialyzed against PBS. Approximately 10 µg of murine SPARC were subjected to SDS-PAGE through a 12% polyacrylamide gel, transferred to a 0.2-µm nitrocellulose membrane, and visualized by staining with amido black. The bands containing SPARC were excised and incubated in a solution containing 1% nonfat powdered milk dissolved in buffer A (PBS and 0.05% Tween 20) and ~250 µg/ml of anti-SPARC immunoglobulins. The membrane was washed with buffer A, and affinity-purified immunoglobulins were eluted with 0.2 M glycine-HCl (pH 2.2), neutralized with 1 M Tris · HCl (pH 10), and dialyzed against PBS. Five-micrometer-thick sections of lung fixed with methyl Carnoy's solution were stained with anti-SPARC antibodies according to the avidin-biotinylated horseradish peroxidase complex (ABC) method, as previously described (48), with the Vectastain Elite ABC Peroxidase kit (Vector Laboratories, Burlingame, CA). The sections were counterstained with methyl green. Photomicrographs were made with a Nikon Eclipse E600 photomicroscope (Melville, NY) and Ektachrome 64T film (Kodak, Rochester, NY). Slides were scanned digitally with a Nikon LS-1000 slide scanner and were edited with Adobe Photoshop 5.0 software (Adobe Systems, Mountain View, CA) under identical conditions.

Statistical analysis. All statistical analyses were performed with SPSS 8.0 for Windows (SPSS, Chicago, IL). Mean values were compared by one-way ANOVA with Scheffé's test of multiple comparisons (1). Results are presented as mean values ± SE if they represent mean values of multiple measures for each case or as mean values ± SD if they represent mean values of single measures for each case.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nine WT and ten SPARC-null mice were given bleomycin (5 WT and 5 SPARC-null mice) or saline by intratracheal instillation. Fourteen days after instillation, the mice were killed, and their lungs were removed. Examination by histology showed no gross differences in morphology between WT and SPARC-null lungs in the saline-treated control mice (Fig. 1, A-D). Airway morphology appeared normal in the SPARC-null lungs, and alveolar architecture was not disordered. Bleomycin-treated WT and SPARC-null lungs both showed the typical patchy pattern of fibrosis (Fig. 1, E-H). The overall distribution of lung injury appeared similar in the WT and SPARC-null lungs.


View larger version (85K):
[in this window]
[in a new window]
 
Fig. 1.   Wild-type (WT) and secreted protein acidic and rich in cysteine (SPARC)-null saline-treated control lungs have similar histology in saline controls and in response to bleomycin injury. WT (A, B, E, and F) and SPARC-null mice (C, D, G, and H) were treated with intratracheal saline (A-D) or bleomycin (E-H) and stained with Masson's trichrome stain to accentuate distribution of fibrillar collagen. Collagen-rich areas stain dark blue. Locations of higher-power frames (B, D, F, and H) are marked by squares in lower-power frames (A, C, E, and G). A, C, E, and G: bars = 500 µm. B, D, F, and H: bars = 50 µm. Arrows point to examples of fibrotic areas.

Immunohistochemistry was performed to determine the extent of SPARC expression in normal, uninjured lung as well as any possible changes in distribution in bleomycin-injured lung. Five-micrometer-thick sections from WT and SPARC-null lungs treated with saline or bleomycin were stained with affinity-purified antibodies against SPARC (Fig. 2). No immunoreactive protein was identified in SPARC-null lungs (Fig. 2, B and D). SPARC was rarely seen in isolated cells in saline-treated WT lung (Fig. 2A). However, in bleomycin-treated WT lung, immunoreactive SPARC was prominent in the cytoplasm of intra-alveolar cells, the morphology of which appeared consistent with their identification as fibroblasts, as well as in alveolar septal cells (Fig. 2C). The distribution and increased number of cells associated with immunoreactive SPARC indicated that its accumulation was increased in regions of lung injury.


View larger version (117K):
[in this window]
[in a new window]
 
Fig. 2.   Augmented expression of SPARC in bleomycin-injured WT lung. WT (A and C) and SPARC-null mice (B and D) were treated with saline (A and B) or bleomycin (C and D). Paraformaldehyde-fixed lung sections were stained with affinity-purified polyclonal anti-SPARC antibodies in conjunction with an avidin-biotinylated horseradish peroxidase complex technique and were counterstained with nuclear stain methyl green. Areas containing immunoreactive SPARC stained brown. Arrow, an example of a collection of intra-alveolar cells staining for immunoreactive SPARC; arrowheads, immunoreactive cells in alveolar septa; bars = 50 µm.

Northern analysis for SPARC mRNA was performed to further study changes in the production of SPARC in response to injury. Fifteen micrograms of total cellular RNA were analyzed from each animal (Fig. 3A). As expected, no detectable SPARC mRNA was seen in any of the RNA samples from SPARC-null animals. In contrast, SPARC mRNA was present in the lungs of both saline- and bleomycin-treated WT animals. Steady-state levels of SPARC mRNA, corrected for 18S rRNA, were increased significantly in bleomycin-treated lungs in comparison with saline-treated lungs (2.7-fold, P < 0.001; Fig. 3B). The increased levels of SPARC mRNA, in addition to the detection of immunoreactive SPARC in areas of lung injury, provided strong evidence that synthesis of SPARC is increased in response to bleomycin-induced lung injury.


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3.   SPARC mRNA levels were increased in WT bleomycin-injured lung relative to saline controls. A: total cellular RNA was isolated from homogenates of right lungs 14 days after intratracheal instillation of saline or bleomycin from WT and SPARC-null mice. Each lane represents 15 µg of RNA from each animal, which were resolved by electrophoresis, transferred to a nylon membrane, and hybridized with 32P-labeled mouse SPARC cDNA. Nylon membrane was stripped and was rehybridized with 32P end-labeled 18S oligonucleotide as a control for loading of RNA. Mouse 3T3 cell and mouse liver RNAs (15 µg) were also used as positive and negative controls, respectively. B: SPARC mRNA levels were determined by densitometry and were adjusted for variations in loading by normalization to 18S rRNA bands in same lanes. Mean normalized values ± SD from each treatment/genotype group [saline-treated SPARC-null (n = 5 mice), saline-treated WT (n = 4), bleomycin-treated SPARC-null (n = 5), and bleomycin-treated WT (n = 5)] are shown. * Bleomycin-treated WT group had significantly more SPARC mRNA than all other groups by one-way ANOVA and Scheffé's multiple comparison test (P < 0.0001). ** Saline-treated WT group had significantly more SPARC mRNA than either SPARC-null group (P < 0.001).

We next sought to determine whether SPARC was important in the regulation of collagen metabolism in the bleomycin model of pulmonary fibrosis. Total collagen content was determined by measurement of the content of hydroxyproline in the right lung of each animal (Fig. 4). Bleomycin-treated WT lung had significantly more hydroxyproline (191.9 µg/lung) than either the bleomycin-treated SPARC-null or saline-treated WT and SPARC-null lungs (147.4 µg/lung, 125.4 µg/lung, and 113.0 µg/lung, respectively; P < 0.03). The hydroxyproline content of bleomycin-treated SPARC-null lungs was not significantly greater than that of the saline-treated lungs by one-way ANOVA. Thus, despite the patchy distribution of injury (as seen in Fig. 1, E and G), which might be expected to mask or result in underestimation of the alterations in collagen content, there was significant attenuation in the accumulation of collagen in SPARC-null mouse lungs in response to injury.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   Accumulation of collagen is increased in bleomycin-treated WT lungs in comparison with SPARC-null and saline-treated lungs. Collagen content in right lungs of WT and SPARC-null mice treated with saline or bleomycin was determined by hydroxyproline levels in duplicate measurements for each animal. Mean hydroxyproline values ± SE for each treatment/genotype group [saline-treated SPARC-null (n = 5), saline-treated WT (n = 4), bleomycin-treated SPARC-null (n = 5), and bleomycin-treated WT (n = 5)] are shown. * Bleomycin-treated WT group had significantly more collagen than any other group (including bleomycin-treated SPARC-null group) by one-way ANOVA and Scheffé's multiple comparison test (P < 0.03).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have demonstrated that SPARC is induced in the bleomycin model of pulmonary fibrosis. The lungs of bleomycin-treated WT mice have increased SPARC mRNA levels and increased numbers of cells with immunoreactive protein. Immunostaining for SPARC was particularly prominent in areas of bleomycin injury. That the collagen content of the bleomycin-injured WT lungs was increased over that of bleomycin-injured SPARC-null lungs indicates that the absence of SPARC may lead to a blunted fibrotic response to lung injury.

SPARC has been associated with pulmonary development in several studies. It has been localized in embryonic mouse lung by immunohistochemistry (41) and in fetal rat lung by immunohistochemistry and in situ hybridization (50). Branching morphogenesis was arrested after neutralizing antibodies against SPARC were added to explant cultures of fetal rat lungs (50). However, the morphology of lungs from adult SPARC-null mice is grossly normal. Morphometric studies comparing WT and SPARC-null lungs are required to determine whether more subtle differences in lung morphology exist. The relatively normal appearance of SPARC-null lung indicates that some other protein(s) is able to compensate for the absence of SPARC in the SPARC-null mouse lung morphogenesis. Several proteins with a high degree of homology to SPARC have been isolated. These include QR-1, which is expressed by quail retinal cells (9, 21), and SC-1 (known as hevin in humans) (19, 46). Hevin has been shown to have properties similar to those of SPARC in vitro in that treatment of endothelial cells with hevin inhibited attachment and spreading (20). Abundant SC-1 mRNA has been noted in fetal mouse lung; thus it is possible that this protein may compensate for the absence of SPARC in the developing SPARC-null lung (46).

Our results demonstrate that expression of SPARC is increased after bleomycin injury in WT lung. Steady-state levels of SPARC mRNA were increased, and the extent of immunostaining was augmented. The kinetics of SPARC induction after bleomycin-induced lung injury, however, are not known and will require the study of multiple time points after injury. In uninjured saline-treated lungs, immunoreactivity for SPARC was detected only rarely. That immunoreactive SPARC was seen so seldom in the lungs of saline-treated WT mice is somewhat surprising given the presence of SPARC mRNA by Northern analysis of whole homogenates from these animals. It is possible that our immunohistochemical approach to detection of SPARC may be less sensitive than Northern analysis and would therefore miss low levels of SPARC expression. Alternatively, SPARC production may, in part, be posttranscriptionally regulated so that levels of SPARC mRNA would not be correlated directly with levels of immunoreactive SPARC. In contrast, 14 days after the instillation of bleomycin, immunoreactive SPARC was prominent, particularly in the cytoplasm of intra-alveolar cells that resembled fibroblasts. This result is consistent with the observation that SPARC immunoreactivity was most evident in the cytoplasm of intra-alveolar fibroblasts in lung biopsies from patients with idiopathic pulmonary fibrosis (28). These foci are formed by the invasion of fibroblasts into the damaged alveoli, where they produce extracellular matrix (29). The counteradhesive properties of SPARC observed in vitro (42) might promote the invasion of fibroblasts into the air spaces. Decreased adhesion of cells to substrate could facilitate the mobility of cells and allow migration into injured areas. For example, treatment of endothelial cells with the integrin-binding competitor echistatin increased or decreased their mobility on fibronectin as a function of the concentration of echistatin and the density of fibronectin (56). Migration of fibroblasts could be facilitated by the decreased adhesion to the extracellular matrix induced by SPARC in areas of injury.

The mechanism of induction of SPARC expression in the injured lung is not known. SPARC could be produced by fibroblasts as part of their proliferative response. SPARC has been associated with proliferating and migrating cells in a "culture-shock" model in vitro (39, 40). It could also be increased in injured lungs secondarily to expression of transforming growth factor (TGF)-beta 1. Several studies have demonstrated a prominent role for TGF-beta in the development of pulmonary fibrosis (7, 12, 52). TGF-beta expression was increased in the lung after bleomycin injury in rats (27) and mice (34). Treatment of human fibroblasts with TGF-beta 1 can stimulate synthesis of SPARC through a posttranscriptional mechanism (55). It is thus possible that the expression of SPARC is increased in lung fibroblasts in the bleomycin-injured lung through stimulation by TGF-beta 1.

Previous studies have shown that total lung collagen content is increased up to twofold after bleomycin-induced lung injury (35, 36). As expected, total lung collagen as estimated by hydroxyproline content was increased in the WT animals treated with intratracheal bleomycin in comparison with both WT and SPARC-null animals treated with saline. It is possible that some of the increase in hydroxyproline content in the bleomycin-treated lungs could be due to an increased content of elastin in the lungs. Elastin is also increased in the lung as much as twofold after injury with bleomycin (47). Although elastin contains low levels of hydroxyproline (16), most, if not all, of the hydroxyproline that we measured was derived from collagen. The SPARC-null animals treated with bleomycin accumulated significantly less collagen than the WT bleomycin-treated animals. This attenuation of the fibrotic response could be due to several activities of SPARC. 1) SPARC has been shown to induce the expression of type I plasminogen-activator inhibitor (PAI)-1 in bovine aortic endothelial cells (23). Increased expression of PAI-1 has been shown to decrease fibrinolytic activity and enhance the fibrotic response to bleomycin-induced lung injury (13). Conversely, PAI-1-deficient mice demonstrated enhanced fibrinolytic activity and attenuated lung collagen accumulation in response to bleomycin injury (13). Expression of PAI-1 may be decreased in the SPARC-null mice, resulting in increased fibrinolytic activity and decreased fibrosis. 2) Recently, we noted that mesangial cells from SPARC-null mice exhibit reduced production of type I collagen relative to WT cells (17). Expression of SPARC has been closely linked to collagen remodeling, and SPARC has been associated with fibroblasts at the edges of healing skin wounds in rats (37). The Mov-13 strain of mice lacks type I collagen protein in the extracellular matrix (22, 26). Treatment of fibroblasts cultured from Mov-13 homozygous mice with exogenous SPARC enhanced the ability of these cells to remodel type I collagen gels (25). These studies indicate that SPARC has significant effects on cellular remodeling of collagen. The attenuated accumulation of collagen in SPARC-null animals might therefore be due to decreased production of collagens, increased degradation of collagens, or both.

In summary, we have demonstrated that SPARC expression is augmented in response to bleomycin injury of the lung and that SPARC-null mice accumulate less collagen in response to bleomycin injury. The factors regulating production of SPARC in lung have not yet been defined, and the role of SPARC in the regulation of collagen accumulation is also not presently understood. Lung injury brings into play a complex balance of factors that regulate matrix remodeling, which could lead to fibrosis (matrix production) or emphysema (matrix degradation). SPARC is likely to be important in the regulation of this balance given its increased expression after lung injury and its multiple effects on matrix turnover.


    ACKNOWLEDGEMENTS

We acknowledge Andréa Snyder for assistance with immunochemistry, Sandra Guidotti for assistance with mouse surgery, Andrew Elston for technical assistance, and James Bassuk for assistance and advice.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-03017 (to T. P. Strandjord) and HL-49401 (to D. K. Madtes) and by National Institute of General Medical Sciences Grant GM-40711 (to E. H. Sage).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: T. P. Strandjord, Univ. of Washington, Dept. of Pediatrics, Box 356320, Seattle, WA 98195-6320 (E-mail: tps{at}u.washington.edu).

Received 5 March 1999; accepted in final form 13 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Armitage, P., and G. Berry. Statistical Methods in Medical Research. Oxford, UK: Blackwell Scientific, 1987.

2.   Bassuk, J. A., T. Birkebak, J. D. Rothmier, J. M. Clark, A. Bradshaw, P. J. Muchowski, C. C. Howe, J. I. Clark, and E. H. Sage. Disruption of the SPARC locus in mice alters the differentiation of lenticular epithelial cells and leads to cataract formation. Exp. Eye Res. 68: 321-331, 1999[Medline].

3.   Bienkowski, R. S., and M. G. Gotkin. Control of collagen deposition in mammalian lung. Proc. Soc. Exp. Biol. Med. 209: 118-140, 1995[Abstract].

4.   Bornstein, P. Diversity of function is inherent in matricellular proteins: an appraisal of thrombospondin 1. J. Cell Biol. 130: 503-506, 1995[Medline].

5.   Bowden, D. H. Unraveling pulmonary fibrosis: the bleomycin model. Lab. Invest. 50: 487-488, 1984[Medline].

6.   Bradshaw, A. D., A. Francki, K. Motamed, C. Howe, and E. H. Sage. Primary mesenchymal cells isolated from SPARC-null mice exhibit altered morphology and rates of proliferation. Mol. Biol. Cell 10: 1569-1579, 1999[Abstract/Free Full Text].

7.   Broekelmann, T. J., A. H. Limper, T. V. Colby, and J. A. McDonald. Transforming growth factor beta 1 is present at sites of extracellular matrix gene expression in human pulmonary fibrosis. Proc. Natl. Acad. Sci. USA 88: 6642-6646, 1991[Abstract].

8.   Brown, R. F. R., R. B. Drawbaugh, and T. C. Marrs. An investigation of possible models for the production of progressive pulmonary fibrosis in the rat. The effects of repeated intratracheal instillation of bleomycin. Toxicology 51: 101-110, 1988[Medline].

9.   Casado, F. J., C. Pouponnot, J. C. Jeanny, O. Lecoq, G. Calothy, and A. Pierani. QR1, a retina-specific gene, encodes an extracellular matrix protein exclusively expressed during retina differentiation. Mech. Dev. 54: 237-250, 1996[Medline].

10.   Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, and W. J. Rutter. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294-5299, 1979[Medline].

11.   Clezardin, P., L. Malaval, A. S. Ehrensperger, P. D. Delmas, M. Dechavanne, and J. L. McGregor. Complex formation of human thrombospondin with osteonectin. Eur. J. Biochem. 175: 275-284, 1988[Abstract].

12.   Denis, M. Neutralization of transforming growth factor-beta 1 in a mouse model of immune-induced lung fibrosis. Immunology 82: 584-590, 1994[Medline].

13.   Eitzman, D. T., R. D. McCoy, X. Zheng, W. P. Fay, T. Shen, D. Ginsburg, and R. H. Simon. Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene. J. Clin. Invest. 97: 232-237, 1996[Abstract/Free Full Text].

14.   Feinberg, A. P., and B. Vogelstein. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13, 1983[Medline].

15.   Feinberg, A. P., and B. Vogelstein. Addendum: a technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137: 266-267, 1984[Medline].

16.   Foster, J. A. Elastin structure and biosynthesis: an overview. Methods Enzymol. 82: 559-570, 1982[Medline].

17.   Francki, A., A. D. Bradshaw, J. Bassuk, J. Carbon, C. Howe, and E. H. Sage. SPARC regulates collagen type I and TGF-beta1 expression in mouse mesangial cells (Abstract). Mol. Biol. Cell 9: 167a, 1998.

18.   Gilmour, D. T., G. J. Lyon, M. B. L. Carlton, J. R. Sanes, J. M. Cunningham, J. R. Anderson, B. L. M. Hogan, M. J. Evans, and W. H. Colledge. 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. 17: 1860-1870, 1998[Free Full Text].

19.   Girard, J. P., and T. A. Springer. Cloning from purified high endothelial venule cells of hevin, a close relative of the antiadhesive extracellular matrix protein SPARC. Immunity 2: 113-123, 1995[Medline].

20.   Girard, J. P., and T. A. Springer. Modulation of endothelial cell adhesion by hevin, an acidic protein associated with high endothelial venules. J. Biol. Chem. 271: 4511-4517, 1996[Abstract/Free Full Text].

21.   Guermah, M., P. Cristani, D. Laugier, P. Dezelee, L. Bidou, B. Pessac, and G. Calothy. Transcription of a quail gene expressed in embryonic cells is shut off sharply at hatching. Proc. Natl. Acad. Sci. USA 88: 4503-4507, 1991[Abstract].

22.   Harbers, K., M. Kuehn, H. Delius, and R. Jaenisch. Insertion of retrovirus into the first intron of alpha 1(I) collagen gene to embryonic lethal mutation in mice. Proc. Natl. Acad. Sci. USA 81: 1504-1508, 1984[Abstract].

23.   Hasselaar, P., D. J. Loskutoff, M. Sawdey, and E. H. Sage. SPARC induces the expression of type 1 plasminogen activator inhibitor in cultured bovine aortic endothelial cells. J. Biol. Chem. 266: 13178-13184, 1991[Abstract/Free Full Text].

24.   Hoyt, D. G., and J. S. Lazo. Alterations in pulmonary mRNA encoding procollagens, fibronectin and transforming growth factor-beta precede bleomycin-induced pulmonary fibrosis in mice. J. Pharmacol. Exp. Ther. 246: 765-771, 1988[Abstract].

25.   Iruela-Arispe, M. L., R. B. Vernon, H. Wu, R. Jaenisch, and E. H. Sage. Type I collagen-deficient Mov-13 mice do not retain SPARC in the extracellular matrix: implications for fibroblast function. Dev. Dyn. 207: 171-183, 1996[Medline].

26.   Jaenisch, R., K. Harbers, A. Schnieke, J. Löhler, I. Chumakov, D. Jähner, D. Grotkopp, and E. Hoffmann. Germline integration of Moloney murine leukemia virus at the Mov13 locus leads to recessive lethal mutation and early embryonic death. Cell 32: 209-216, 1983[Medline].

27.   Khalil, N., O. Bereznay, M. Sporn, and A. H. Greenberg. Macrophage production of transforming growth factor beta and fibroblast collagen synthesis in chronic pulmonary inflammation. J. Exp. Med. 170: 727-737, 1989[Abstract].

28.   Kuhn, C., and R. J. Mason. Immunolocalization of SPARC, tenascin, and thrombospondin in pulmonary fibrosis. Am. J. Pathol. 147: 1759-1769, 1995[Abstract].

29.   Kuhn, C., III, J. Boldt, T. E. King, Jr., E. Crouch, T. Vartio, and J. A. McDonald. An immunohistochemical study of architectural remodeling and connective tissue synthesis in pulmonary fibrosis. Am. Rev. Respir. Dis. 140: 1693-1703, 1989[Medline].

30.   Lane, T. F., and E. H. Sage. The biology of SPARC, a protein that modulates cell-matrix interactions. FASEB J. 8: 163-173, 1994[Abstract/Free Full Text].

31.   Lazenby, A. J., E. C. Crouch, J. A. McDonald, and C. Kuhn III. Remodeling of the lung in bleomycin-induced pulmonary fibrosis in the rat. An immunohistochemical study of laminin, type IV collagen, and fibronectin. Am. Rev. Respir. Dis. 142: 206-214, 1990[Medline].

32.   Mason, I. J., A. Taylor, J. G. Williams, H. Sage, and B. L. M. Hogan. Evidence from molecular cloning that SPARC, a major product of mouse embryo parietal endoderm, is related to an endothelial cell "culture shock" glycoprotein of Mr 43,000. EMBO J. 5: 1465-1472, 1986[Abstract].

33.   Norose, K., J. I. Clark, N. A. Syed, A. Basu, E. Heber-Katz, E. H. Sage, and C. C. Howe. SPARC deficiency leads to early-onset cataractogenesis. Invest. Ophthalmol. Vis. Sci. 39: 2674-2680, 1998[Abstract].

34.   Phan, S. H., and S. L. Kunkel. Lung cytokine production in bleomycin-induced pulmonary fibrosis. Exp. Lung Res. 18: 29-43, 1992[Medline].

35.   Phan, S. H., R. S. Thrall, and C. Williams. Bleomycin-induced pulmonary fibrosis. Effects of steroid on lung collagen metabolism. Am. Rev. Respir. Dis. 124: 428-434, 1981[Medline].

36.   Quinones, F., and E. Crouch. Biosynthesis of interstitial and basement membrane collagens in pulmonary fibrosis. Am. Rev. Respir. Dis. 134: 1163-1171, 1986[Medline].

37.   Reed, M. J., P. Puolakkainen, T. F. Lane, D. Dickerson, P. Bornstein, and E. H. Sage. Differential expression of SPARC and thrombospondin 1 in wound repair: immunolocalization and in situ hybridization. J. Histochem. Cytochem. 41: 1467-1477, 1993[Abstract/Free Full Text].

38.   Sage, E. H., and P. Bornstein. Extracellular proteins that modulate cell-matrix interactions. SPARC, tenascin, and thrombospondin. J. Biol. Chem. 266: 14831-14834, 1991[Free Full Text].

39.   Sage, H., J. Decker, S. Funk, and M. Chow. SPARC: a Ca2+-binding extracellular protein associated with endothelial cell injury and proliferation. J. Mol. Cell. Cardiol. 21, Suppl. 1: 13-22, 1989[Medline].

40.   Sage, H., C. Johnson, and P. Bornstein. Characterization of a novel serum albumin-binding glycoprotein secreted by endothelial cells in culture. J. Biol. Chem. 259: 3993-4007, 1984[Abstract/Free Full Text].

41.   Sage, H., R. B. Vernon, J. Decker, S. Funk, and M. L. Iruela-Arispe. Distribution of the calcium-binding protein SPARC in tissues of embryonic and adult mice. J. Histochem. Cytochem. 37: 819-829, 1989[Abstract].

42.   Sage, H., R. B. Vernon, S. E. Funk, E. A. Everitt, and J. Angello. 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. 109: 341-356, 1989[Abstract].

43.   Sambrook, J., E. F. Fritsch, and T. Maniatis. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.

44.   Schrier, D. J., R. G. Kunkel, and S. H. Phan. The role of strain variation in murine bleomycin-induced pulmonary fibrosis. Am. Rev. Respir. Dis. 127: 63-66, 1983[Medline].

45.   Snider, G. L., B. R. Celli, R. H. Goldstein, J. J. O'Brien, and E. C. Lucey. Chronic interstitial pulmonary fibrosis produced in hamsters by endotracheal bleomycin. Lung volumes, volume-pressure relations, carbon monoxide uptake, and arterial blood gas studied. Am. Rev. Respir. Dis. 117: 289-297, 1978[Medline].

46.   Soderling, J. A., M. J. Reed, A. Corsa, and E. H. Sage. Cloning and expression of murine SC1, a gene product homologous to SPARC. J. Histochem. Cytochem. 45: 823-835, 1997[Abstract/Free Full Text].

47.   Starcher, B. C., C. Kuhn, and J. E. Overton. Increased elastin and collagen content in the lungs of hamsters receiving an intratracheal injection of bleomycin. Am. Rev. Respir. Dis. 117: 299-305, 1978[Medline].

48.   Strandjord, T. P., J. G. Clark, W. A. Hodson, R. A. Schmidt, and D. K. Madtes. Expression of transforming growth factor-alpha in mid-gestation human fetal lung. Am. J. Respir. Cell Mol. Biol. 8: 266-272, 1993[Medline].

49.   Strandjord, T. P., J. G. Clark, and D. K. Madtes. Expression of TGF-alpha , EGF, and EGF receptor in fetal rat lung. Am. J. Physiol. 267 (Lung Cell. Mol. Physiol. 11): L364-L389, 1994.

50.   Strandjord, T. P., E. H. Sage, and J. G. Clark. SPARC participates in the branching morphogenesis of developing fetal rat lung. Am. J. Respir. Cell Mol. Biol. 13: 279-287, 1995[Abstract].

51.   Swiderski, R. E., J. E. Dencoff, C. S. Floerchinger, S. D. Shapiro, and G. W. Hunninghake. Differential expression of extracellular matrix remodeling genes in a murine model of bleomycin-induced pulmonary fibrosis. Am. J. Pathol. 152: 821-828, 1998[Abstract].

52.   Toti, P., G. Buonocore, P. Tanganelli, A. M. Catella, M. L. D. Palmieri, R. Vatti, and T. A. Seemayer. Bronchopulmonary dysplasia of the premature baby: an immunohistochemical study. Pediatr. Pulmonol. 24: 22-28, 1997[Medline].

53.   Tremble, P. M., T. F. Lane, E. H. Sage, and Z. Werb. SPARC, a secreted protein associated with morphogenesis and tissue remodeling, induces expression of metalloproteinases in fibroblasts through a novel extracellular matrix-dependent pathway. J. Cell Biol. 121: 1433-1444, 1993[Abstract].

54.   Woessner, J. F., Jr. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch. Biochem. Biophys. 93: 440-447, 1961.

55.   Wrana, J. L., C. M. Overall, and J. Sodek. Regulation of the expression of a secreted acidic protein rich in cysteine (SPARC) in human fibroblasts by transforming growth factor beta . Comparison of transcriptional and post-transcriptional control with fibronectin and type I collagen. Eur. J. Biochem. 197: 519-528, 1991[Abstract].

56.   Wu, P., J. B. Hoying, S. K. Williams, B. A. Kozikowski, and D. A. Lauffenburger. Integrin-binding peptide in solution inhibits or enhances endothelial cell migration, predictably from cell adhesion. Ann. Biomed. Eng. 22: 144-152, 1994[Medline].

57.   Zhao, Y., S. L. Young, and J. C. McIntosh. Induction of tenascin in rat lungs undergoing bleomycin-induced pulmonary fibrosis. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L1049-L1057, 1998[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 277(3):L628-L635
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society