Secreted protein acidic and rich in cysteine is a matrix scavenger chaperone

Abstract

Secreted Protein Acidic and Rich in Cysteine (SPARC) is one of the major non-structural proteins of the extracellular matrix (ECM) in remodeling tissues. The functional significance of SPARC is emphasized by its origin in the first multicellular organisms and its high degree of evolutionary conservation. Although SPARC has been shown to act as a critical modulator of ECM remodeling with profound effects on tissue physiology and architecture, no plausible molecular mechanism of its action has been proposed. In the present study, we demonstrate that SPARC mediates the disassembly and degradation of ECM networks by functioning as a matricellular chaperone. While it has low affinity to its targets inside the cells where the Ca(2+) concentrations are low, high extracellular concentrations of Ca(2+) activate binding to multiple ECM proteins, including collagens. We demonstrated that in vitro, this leads to the inhibition of collagen I fibrillogenesis and disassembly of pre-formed collagen I fibrils by SPARC at high Ca(2+) concentrations. In cell culture, exogenous SPARC was internalized by the fibroblast cells in a time- and concentration-dependent manner. Pulse-chase assay further revealed that internalized SPARC is quickly released outside the cell, demonstrating that SPARC shuttles between the cell and ECM. Fluorescently labeled collagen I, fibronectin, vitronectin, and laminin were co-internalized with SPARC by fibroblasts, and semi-quantitative Western blot showed that SPARC mediates internalization of collagen I. Using a novel 3-dimensional model of fluorescent ECM networks pre-deposited by live fibroblasts, we demonstrated that degradation of ECM depends on the chaperone activity of SPARC. These results indicate that SPARC may represent a new class of scavenger chaperones, which mediate ECM degradation, remodeling and repair by disassembling ECM networks and shuttling ECM proteins into the cell. Further understanding of this mechanism may provide insight into the pathogenesis of matrix-associated disorders and lead to the novel treatment strategies.

Figure 1. SPARC mediates disassembly of ECM networks.

(A) Collagen fibril formation at 37°C was almost completely prevented by SPARC. Density of collagen networks measured by opalescence at 2 h was decreased by 99.7±7.2% (F = 249) for 1∶1 and by 91.8±8.2% (F = 224) for 1∶2 molar ratio of SPARC to collagen monomer respectively, as compared with PBS control. At 1∶3 ratio, SPARC prevented formation of collagen fibrils by 44.9±14.2% (F = 46). BSA at the same ratios did not cause statistically significant change in fibril formation (F<3.92). (B) Degradation of pre-formed collagen fibrils at 47°C doubled when SPARC was addedat 1∶1 molar ratio to collagen monomer with the complete loss of opacityin 100 min. Collagen fibril degradation was only enhanced by SPARC in 1 mM Ca²⁺ (F = 60), and was completely abolished in 1 mM EDTA (F<3.90). The same amount of BSA or PBS without proteins did not affect degradation of collagen fibrils neither in Ca²⁺, nor EDTA (F<3.90).

Figure 2. Internalization of SPARC by fibroblastcells.

(A) After treatment with exogenous SPARC for indicated periods, bright intracellular vesicles containing internalized SPARC were detected by immunofluorescence in the cytoplasm of mouse NIH/3T3 fibroblasts. Exposure time was chosen to minimize interferenceof endogenously expressed SPARC, which can be detected in untreated cells. Lack of co-localization with the marker of caveolae and clathrin-coatedvesicles, dynamin, was apparent at 5 and 15 min time points. (B) Representative Western blot and (C) semi-quantitative analysisof SPARC in the cytoplasm of primary fibroblasts, isolated from SPARC-nullmice. After addition of 5 µg/ml of SPARC tothe culture media, it was detected in cell lysates in 1 min with increasing accumulation until 1–2 hours. Treatment with 1 µg/ml of SPARC resulted in weaker internalization which followed the same dynamic. (D) Representative Western blot and (E) semi-quantitative analysis of SPARC turnover in the pulse-chase assay. SPARC-null fibroblasts were treated with 5 µg/ml of SPARC added to themedia. The amount of SPARC in the media and internalized bythe fibroblasts at the end of pulse treatment is shown in the first lane.After SPARC was removed from the media, both media and cell lysates were collected at the indicated time points. Western blot analysis showed rapid, time-dependent release of SPARC from the fibroblasts, accompanied bya decrease in the amount of intracellular SPARC.

Figure 3. Co-internalization of SPARC and ECM proteins in fibroblasts.

(A) Primary mouseSPARC-null fibroblasts were attached to glass coverslips overnight and treated with red AF594-labeled collagen and green AF488-labeled SPARC. After 1 h of treatment, strong fibrillar collagennetworks were deposited at the trailing edge of a moving fibroblast (purple arrow). Fluorescent SPARC bound ECM tracks, deposited along the movement path (blue arrows) and was internalized at the leading edge of the fibroblast (white arrow). (B) SPARC-null fibroblasts were plated and treated with green AF488-labeled SPARC and red AF594-labeled ECM proteins as above. Allmatrix proteins were deposited into apparently normal extracellular networks and internalized by the cells. Strong co-localizationof all internalized ECM proteins with SPARC is evident by the yellow overlap of the two colors.

Figure 4. Effect of SPARC on collagen turnover.

(A) SPARC-null primary mouse fibroblasts were treated with green AF488-labeled SPARC (left panel), red AF594-labeled collagen (right panel) or their combination (middle panels). Formation of cytoplasmic vesicles containing internalized SPARC follows the same dynamic, as determined by Western blot analysis. Collagenwas deposited into fibrillar extracellular networks in 2–4 hafter the treatment. When significant uptake of collagen was observed, it was internalized in SPARC-positive vesicles. (B) Representative Western blot and (C) semi-quantitative analysis of exogenously added AF488-labeled collagen in the cytoplasm of SPARC-null fibroblasts. Treatment with SPARC led to a statistically significant (p<0.05) 2–3 times increase in collagen internalization at 30 min–48 h. Substantial degradation of collagen was also be observed in 24 and 48 h after the treatment.

Figure 5. Degradation of fluorescent ECM networks by SPARC.

(A) Deposition of fluorescently labeled ECM. NIH/3T3 fibroblasts were grown on Matrigel-covered coverslips in the presence of green AF488-labeled collagen. Fluorescent collagen was incorporated into the normal 3-dimentional ECM networks, deposited by live cells. After cells were removedby mild lysis, the fluorescently labeled matrix remained intact. (B) Networks were formed by NIH3T3 cells with green AF488-labeled collagenand red AF594-labeled fibronectin. After cell removal (NO cells), fluorescent networks remained intact in tissue culture conditions for at least 48 hours. SPARC knock-out fibroblasts (KO cells) grown on pre-formed fluorescent matrix re-arrangedthe networks by bending, merging and thickening fibrils around the cells. Wild type cells (WT cells) also remodeled the fibrils and caused matrix degradation, which was almost complete in 48 h. (C) Quantitative image analysis of the above experiment with separation of red (fibronectin) and green (collagen I) colors. Measured by the loss of fluorescence, matrix degradation was statistically significant (p<0.05) starting at 2 h after the attachment of the wild type cells (WT), compared with untreated matrices (NO). SPARC knock-out fibroblasts (KO) did not cause statistically significant decreasein fluorescence (p>0.05).