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* Department of Dermatology, University Medical Centre Mannheim, Ruprecht-Karls University of Heidelberg, Mannheim, Germany;
Hope Heart Program, Benaroya Research Institute at Virginia Mason, Seattle, WA 98101; and
M. D. Anderson Cancer Center, University of Texas, Houston, TX 77030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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SPARC binds to a number of ECM components, including vitronectin, entactin/nidogen, and several collagens (3), affects cell-ECM interactions, and negatively regulates cell adhesion (4). During development, SPARC is expressed abundantly at sites of organogenesis and cell migration/differentiation, but in the adult it is restricted to sites of active ECM turnover (1, 5). Mice with targeted disruptions of SPARC exhibit an overall deficiency of connective tissue and collagenous ECM, as well as early onset cataractogenesis, due in part to a defective lens capsule basement membrane (6). The induction of SPARC in response to injury, as well as its susceptibility to tissue transglutaminase, indicates a role in the assembly of ECM permissive for cell migration, proliferation, and differentiation (7, 8).
SPARC-mediated ECM assembly is an essential component of the wound healing process (9, 10, 11). However, the role of SPARC in tissue remodeling and response to injury is not restricted to modifications of the ECM, but includes an antiproliferative effect achieved by the modulation of growth factor activity. Direct interaction of SPARC with platelet-derived growth factor and vascular endothelial growth factor interferes with their binding to cognate receptors and decreases their mitogenic potency. SPARC also counteracts the proliferative capacity of fibroblast growth factor-2 on smooth muscle cells (4, 12) and negatively regulates the production of TGF-
(1).
SPARC modulates angiogenesis, i.e., the formation of new vessels from extant vasculature, by multiple mechanisms including the promotion of endothelial cell deadhesion, modulation of vascular ECM, and enhancement of the permeability of endothelial monolayers (3). Purified SPARC was shown to be a potent inhibitor of angiogenesis and, consequently, the growth of neuroblastoma in vivo (13). In vitro, peptides produced by the cleavage of SPARC by stromelysin-1 have differential effects on angiogenesis (14). Moreover, SPARC synergizes with its ortholog hevin to produce antiangiogenic effects in the foreign body response (15).
Tissue-specific effects of SPARC on ECM assembly and angiogenesis are consistent with a context-dependent role in tumor biology. Examination of mammary carcinoma in SPARCnull mice revealed reduced tumor growth and vascularization, decreased collagen IV deposition, and massive parenchymal infiltration of leukocytes (16). On a different genetic background, the ectopic growth of Lewis lung carcinoma in SPARCnull mice was characterized by enhanced tumor growth and metastasis, decreased tumor encapsulation, and diminished recruitment of macrophages (17). Although these models demonstrated different outcomes with respect to tumor size and dissemination, they indicate that SPARC modulates tumor-stromal cell interactions that are dependent on vascularization, immune reactions, and the type of malignancy (18).
Despite the identification of multiple effects of SPARC in development and pathology, the cellular receptor(s) transducing responses to SPARC, as well as regulating its activity and extracellular concentration, has not been identified. In this study, we performed phage display and identified stabilin-1 as a cellular receptor for SPARC. Stabilin-1 interacts with SPARC through the extracellular epidermal growth factor (EGF)-like domain containing the sequence FHGTAC, and it mediates the internalization and delivery of SPARC to the endocytic pathway in stably transfected CHO cells. Alternatively activated macrophages stimulated by IL-4 in combination with dexamethasone endocytose SPARC efficiently, in contrast to stabilin-1-negative macrophages stimulated with only IL-4 or IFN-
. Stabilin-1 appears to be the major macrophage receptor mediating the uptake of SPARC and its targeting through an endosomal pathway to lysosomal degradation. We propose that clearance of SPARC from the extracellular space by alternatively activated macrophages regulates tissue remodeling and ECM synthesis by cells that normally respond to this matricellular protein in the context of its location and concentration.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Generation of F4 rabbit polyclonal Abs directed against the stabilin-1 cytoplasmic tail has been described (19). Commercial primary mouse mAbs against early endosome Ag 1 (EEA1) and p62lck were purchased from BD Biosciences. Secondary Abs used for immunofluorescence were: Cy2-labeled donkey anti-mouse IgG, Cy3-labeled donkey anti-rabbit IgG, and Cy5-labeled sheep anti-mouse IgG (Dianova). Transferrin-Alexa 546 and acetylated low density lipoprotein (acLDL)-Alexa 488 were purchased from Molecular Probes.
Recombinant human SPARC was produced in Sf9 cells and was purified by anion exchange chromatography (14, 20). An FITC-coupling kit (Sigma-Aldrich) was used for the fluorescent labeling of purified SPARC according to the manufacturer’s protocol. After each coupling reaction, proteins were resolved by SDS-PAGE and stained with Gel Code (Pierce Biotechnology). No degradation of the FITC-coupled SPARC was detected.
Fragments of stabilin-1 corresponding to amino acids 2302–2570 (P9) and 1332–1385 (SPARC-binding domain) were cloned into EcoRI/XhoI sites of the pGEX4T1 vector (Invitrogen Life Technologies). GST-fusion proteins and GST were expressed in Escherichia coli strain BL21-CodonPlus-RIL (Stratagene) and purified under nondenaturing conditions as described (21).
Phage display screening
A phage display random peptide library was used that displayed the insert CX7C (where X is any amino acid and C is a cysteine residue). The phage input was 3 x 109 transducing units. Recombinant human SPARC was coated onto microtiter wells (22, 23), and phage-binding assays on purified proteins were performed as previously described (24). SPARC, hevin (25), bovine type I collagen (Vitrogen; Collaborative Biomedical Products), and BSA (Pierce Biotechnology) at 1 µg in 50 µl of PBS/0.1 mM Ca2+ were coated onto microtiter wells overnight at 4°C. Wells were washed twice with PBS, blocked with PBS/3% BSA for 1 h at room temperature, and incubated with 2 x 109 transducing units of phage (CIRFHGTAC) or fd-tet phage (control) in 50 µl of PBS/1.5% BSA, for 2 h at room temperature. Subsequently, wells were washed with PBS 10 times, and phage were recovered by bacterial infection. Three rounds of panning were performed.
In vitro-binding assay
Stabilin fragments were immobilized in wells of Nunc Maxisorp 96-well ELISA plates (Fisher Scientific) overnight at 4°C, blocked with 5% casein acid hydrolysate/0.1% Tween 20 in HBSS for 2 h at room temperature, and incubated with recombinant human SPARC (2 µg/well) overnight at 4°C. SPARC standards ranged from 0 to 4 µg/well. Wells were washed, incubated with anti-human SPARC Ab no. 08063-14 (U.S. Biological) for 2 h at room temperature, washed again, and incubated with peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. After a final wash, 1-Step Ultra TMB-ELISA substrate (Pierce Biotechnology) was added and the reaction was subsequently stopped with 10% H3PO4. Color development was measured by absorbance at 450 nm.
Primary macrophages and cell lines
Human monocytes/macrophages were isolated and cultivated as described (26). The cells were purified from individual buffy coats by density gradient centrifugations. Monocyte-enriched fractions were subjected to positive CD14+ magnetic cell sorting using CD14 magnetic beads (Miltenyi Biotec) resulting in 90–98% monocyte purity as confirmed by flow cytometry. Macrophages were cultivated in X-Vivo 10 serum-free medium (Cambrex) at a concentration of 1 x 106 cells/ml with the following stimulants: IFN- at 1000 U/ml; IL-4 and IL-10 at 10 ng/ml (Tebu Bio); and dexamethasone at 1 x 10–7 M (Sigma-Aldrich). The cells were incubated in the presence of 7.5% CO2 for 6 days before further analysis.
Chinese hamster ovary (CHO) cells stably transfected with the pLP-IRESneo vector (BD Clontech) and with the expression construct pLP-IRESneo-hstabilin-1 have been described (27). Stable expression of stabilin-1 in cell lines was confirmed by immunofluorescence as previously described (27).
Endocytosis
An endocytosis assay, immunofluorescence and FACS with CHO-K1 stably transfected cells was performed as described (27). For macrophages, fluorescent ligands were added to the conditioned X-Vivo medium. Incubation with fluorescent ligands was performed at 37°C. Durations of exposure to ligands and ligand concentrations are indicated in Results and in the figure legends. Cessation of endocytosis was achieved by placing cells on ice or by immediate fixation in paraformaldehyde (PFA) as described previously (26). For immunofluorescence, macrophages were subjected to cytospin preparation as described (26).
FACS analysis
Quantification of bound/internalized fluorescent ligands was performed with FACSCalibur (BD Biosciences) according to standard protocols. The data were visualized and analyzed with WinMDI 2.8 software.
Immunofluorescence and confocal microscopy
Cells on coverslips or cytospins were fixed for 10 min in 2% PFA in PBS, permeabilized for 15 min in 0.5% Triton X-100 in PBS, and fixed for 10 min with 4% PFA in PBS (26). Immunofluorescent staining was performed as described (26, 27). Confocal laser scanning microscopy was performed with a Leica TCS SP2 laser scanning spectral confocal microscope, equipped with a 63 x 1.32 objective. Excitation was with an argon laser emitting at 488 nm, a krypton laser emitting at 568 nm, and a helium/neon laser emitting at 633 nm. Data were acquired and analyzed with Leica confocal software. All two- or three-color images were acquired using a sequential scan mode.
Statistical analysis
A paired two-tailed t test was performed using the TTEST function of Excel program (Microsoft Office 2000 Pro).
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Results |
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SPARC-binding peptides were initially isolated by the screening of a phage library displaying random CX7C (where X is any amino acid and C is a cysteine residue) peptide inserts on purified native recombinant human SPARC immobilized on 96-well plates. Negative controls were BSA, collagen I, and the SPARC ortholog hevin. After enrichment as a result of successive rounds of panning, phage were sequenced from randomly selected clones after three rounds of selection (data not shown). Phage clone no. 54, displaying the sequence CIRFHGTAC, bound to SPARC but not to hevin, collagen I, or BSA (Fig. 1A). A basic local alignment search tool search of the sequence in phage no. 54 revealed significant identity to a short sequence in the scavenger receptor stabilin-1 (Fig. 1B). The large extracellular portion of stabilin-1 is composed of seven fasciclin domains and multiple EGF-like domains that are potentially involved in specific protein-protein interactions (19). The FHGTAC motif is a part of the extracellular EGF-like domain of stabilin-1, located between fasciclin domains 4 and 5 (Fig. 1).
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The binding of SPARC to the extracellular EGF-like domain of stabilin-1 indicated that the interaction between these two proteins could occur on the cell surface and/or in association with the ECM. Therefore, we next asked whether stabilin-1 functions as a receptor for SPARC.
SPARC is specifically internalized by stabilin-1 in stably transfected CHO cells
We have produced CHO cell lines stably transfected with stabilin-1 and have demonstrated that stabilin-1 functions as a specific endocytic receptor for acLDL in this model system (27). Here, we used these cell lines to analyze stabilin-1-mediated endocytosis of SPARC. The endocytosis assay was performed with FITC-labeled SPARC. After a 30-min incubation with the ligand, bound/internalized ligand was quantified by FACS. A strong signal was observed in CHO-stabilin-1 cells, but not in control CHO cells stably transfected with empty vector (Fig. 2). Similar results were obtained with SPARC labeled with Alexa Fluor 488 (data not shown). The increased FACS signal in CHO-stabilin-1 cells was specific for SPARC, as both CHO-stabilin-1 and CHO-vector control cells exhibited similar constitutive endocytic activity, measured previously as well as in concomitant experiments by transferrin-Alexa 546 endocytosis (27) (data not shown). We demonstrated recently that acLDL was internalized efficiently by stabilin-1 in CHO cells. Additional confirmation of stabilin-1-mediated binding of FITC-SPARC by CHO-stabilin-1 cells was obtained by competition experiments using increasing concentrations of nonlabeled acLDL, as shown in Fig. 2A. acLDL was added to serum-free F12 medium directly before addition of labeled SPARC. A reduction of SPARC-FITC signal in CHO-stabilin-1 cells was observed in the presence of 1 µg/ml acLDL, an effect that was concentration dependent (Fig. 2A). In contrast, CHO-vector control cells showed a slight increase in FACS signal in the presence of higher concentrations of acLDL, data indicating that acLDL can increase nonspecific binding of labeled SPARC to CHO cells.
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Stabilin-1 mediates endocytosis of SPARC in alternatively activated macrophages
Expression of stabilin-1 by macrophages requires stimulation with IL-4 and dexamethasone (26). This combination of factors results in generation of alternatively activated macrophages in vitro (28). We measured the endocytosis of SPARC by stabilin-1 by immunofluorescence and confocal microscopy. IL-4/dexamethasone-stimulated human macrophages derived from the PBMC of three healthy donors were used for this assay. After 15 and 45 min of continuous endocytosis, FITC-labeled SPARC was efficiently internalized by stabilin-1-positive macrophages. Single macrophages differentiated under stimulation with IL-4 and dexamethasone express various levels of stabilin-1 (26). Immunofluorescence analysis revealed that the amounts of internalized SPARC corresponded to the expression levels of stabilin-1 in these macrophages, but not to the expression levels of EEA1 (Fig. 3). After 15 min of endocytosis, SPARC was colocalized with stabilin-1, and this coincidence occurred preferentially in EEA1-positive early/sorting endosomes (Fig. 3). These data indicate that expression of stabilin-1 was required for the receptor-mediated endocytosis of SPARC. After 45 min of continuous endocytosis, a portion of labeled SPARC was observed in stabilin-1/EEA1-negative vesicles, a result indicating that stabilin-1 does not accompany SPARC in later steps of the endocytic pathway.
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or IL-4 (prototypic Th1 or Th2 cytokine) in the absence of glucocorticoids do not express stabilin-1 (Ref. 19 , and our unpublished data). Monocyte-derived macrophages from four healthy individuals, cultured for 6 days under different conditions, were incubated with FITC-labeled SPARC for 30 min, and SPARC binding/internalization was subsequently quantified by FACS. IFN-- and IL-4-stimulated macrophages exhibited low signals, whereas IL-4- and dexamethasone-stimulated macrophages demonstrated a strong, positive signal by FACS (Fig. 4). Immunofluorescence analysis confirmed that SPARC-FITC was efficiently internalized and delivered to early endosomes in macrophages stimulated with only IL-4 and dexamethasone. Barely detectable levels of FITC-SPARC were detected in EEA-1-negative small vesicles in macrophages stimulated with IFN-. IL-4-stimulated macrophages internalized more SPARC-FITC, in comparison to IFN--stimulated macrophages; however, SPARC-FITC was rarely detected in EEA1-positive endosomes (Fig. 4). By confocal microscopy, we confirmed that SPARC was internalized via a receptor-mediated pathway in stabilin-1-positive macrophages. In the absence of stabilin-1, macrophages failed to internalize and deliver SPARC-FITC efficiently to the endocytic pathway. The minimal presence of SPARC-FITC in EEA1-positive endosomes in macrophages stimulated with IL-4 is indicative of an alternative, stabilin-1-independent, receptor-mediated mechanism for the endocytosis of SPARC. However, stabilin-1 appears to be the major endocytic receptor for SPARC on macrophages.
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Upon receptor-mediated internalization, many of the ligands are subsequently sorted into late endosomes and lysosomes for degradation (29). To define the trafficking pathway of internalized SPARC in macrophages, we incubated labeled SPARC with macrophages for different time periods and assessed its intracellular localization by immunofluorescence and confocal microscopy. After 15 and 30 min of continuous endocytosis, SPARC was preferentially localized in stabilin-1-positive early endosomes (Fig. 5). For examination of later steps of SPARC endocytic trafficking (1–3 h), SPARC was added to the macrophage culture medium for 35 min to allow its efficient intracellular accumulation, after which the medium was changed to fresh X-Vivo medium, with continued incubation. One and 2 h after the beginning of endocytosis, the amount of intracellular SPARC-FITC was significantly decreased, and SPARC-FITC was only rarely detected in stabilin-1-positive vesicles. Three hours after initiation of endocytosis, the majority of SPARC was degraded. Because acLDL, the only stabilin-1 ligand established to date, is a classical scavenger receptor ligand that is transported through early and late endosomes to lysosomes for degradation, the parallel endocytic experiment with acLDL-Alexa 488 was performed to compare trafficking of acLDL-Alexa 488 and SPARC-FITC in IL-4- and dexamethasone-stimulated macrophages (compare Figs. 5 and 6). Both SPARC-FITC and acLDL-Alexa 488 were partially colocalized with p62lck and Rab7 in late endosomes between 30 min and 2 h after the inception of endocytosis; the ratio of colocalization for both fluorescent ligands with late endosome markers was similar (data not shown). The timing of SPARC endocytic trafficking and degradation was similar to that of acLDL, additionally confirming that stabilin-1 targets SPARC through the classical endocytic route for lysosomal degradation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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-ear-containing, Arf-binding monomeric clathrin adaptors (19, 26). Several lines of evidence indicate that stabilin-1 is a scavenger receptor: 1) the high structural similarity of stabilin-1 with the classical scavenger receptor of liver sinusoidal endothelial cells, stabilin-2 (19); 2) the localization of stabilin-1 in lysosome-targeted endocytic pathways in both macrophages (26) and sinusoidal endothelial cells (30); 3) identification of acLDL as a stabilin-1 endocytic ligand (27, 31). However, we have recently shown that stabilin-1 is not restricted to a scavenging function. Interaction of stabilin-1 with Golgi-localized, -ear-containing, Arf-binding monomeric clathrin adaptors, its shuttling between endosomes and the trans-Golgi network, and its potential involvement in secretion of a novel chitinase-like protein, SI-CLP (32), indicate that stabilin-1 functions as a sorting receptor in alternatively activated macrophages. Thus, in addition to its scavenging function, stabilin-1 is involved in the delivery of newly synthesized proteins from biosynthetic to secretory pathways. In this study, we demonstrated that stabilin-1 mediates SPARC internalization and endosomal delivery in stably transfected CHO cells, and that acLDL competes with SPARC for efficient stabilin-1-mediated internalization. In primary human macrophages, the expression of stabilin-1 is a prerequisite for the internalization. Endocytic experiments with acLDL and SPARC in alternatively activated macrophages demonstrated that both are transported along the endocytic pathway to lysosomes. These results indicate that stabilin-1 acts as a highly specific and efficient receptor in macrophages that mediates internalization of extracellular SPARC and its targeting to lysosomes.
Both SPARC and stabilin-1 are expressed in normal adult tissues and under pathological conditions characterized by exuberant ECM production and/or cellular turnover. Thus, high amounts of SPARC are found in normal adult bone marrow, gut epithelium, steroidogenic cells, hair follicles, and bone (3). SPARC is also produced by endothelial cells, fibroblasts, and macrophages in vivo during wound healing, the foreign body response, and angiogenesis (9, 15, 33). Stabilin-1 is expressed by cells specialized in the clearance of "unwanted self" and in the maintenance of tissue homeostasis (19, 34, 35). These cells include differential subsets of tissue macrophages, as well as noncontinuous endothelial cells lining sinusoidal structures in lymph nodes, liver, spleen, and bone marrow. Stabilin-1 is also induced on continuous endothelial cells undergoing angiogenesis in wound-healing tissue, in cutaneous T cell lymphoma, in psoriasis, and in melanoma metastases (36). Given the multiple activities and biochemical interactions in which SPARC participates, it becomes apparent that a mechanism for the temporal regulation of SPARC concentration in the extracellular space is needed. Stabilin-1 might indeed fulfill that regulatory function.
Macrophages regulate tissue homeostasis and orchestrate inflammatory and anti-inflammatory reactions by secretion and clearance of extracellular mediators. Selectivity of uptake of particular protein ligands is achieved by a highly specific pattern of scavenger receptors expressed by resident tissue macrophages as well as by newly recruited monocytes undergoing differentiation. Our data describing stabilin-1-mediated uptake and degradation of SPARC lead to the proposal that macrophages resolve inflammation and promote angiogenesis in part by their removal of excess SPARC from the extracellular milieu. Thus, the concentration of SPARC is adjusted to the actual physiological needs of the tissue. That only alternatively activated macrophages (stimulated with IL-4 and dexamethasone), but not inflammatory Th1- and Th2-polarized macrophages, mediate SPARC uptake indicates a specialized role of alternatively activated macrophages in wound healing and in the establishment and maintenance of tissue homeostasis. However, potential consequences of stabilin-1-mediated endocytosis vis-à-vis signaling cascades should also be considered. For example, the uptake of specific ligand by stabilin-1 may induce or block its function as an intracellular sorting receptor, and change the secretory repertoire of macrophages independently of transcription.
Stabilin-1 is expressed only by cell types specialized for clearance, tolerance induction, and modulation of tissue remodeling, and SPARC is known to affect stabilin-1-negative cells. These data support the hypothesis that SPARC may engage other extracellular partners with low affinity and act as an antagonist for specific ligand-receptor interactions (37). In turn, macrophages might use stabilin-1-mediated endocytosis to adjust extracellular concentrations of SPARC in a temporal- and tissue-specific manner. In this way, alternatively activated macrophages could regulate the context of SPARC-mediated extracellular protein complexes. Certain structural properties of ECM were suggested to be regulated by tissue transglutaminase-mediated cross-linking of SPARC (1, 7). Cleavage of SPARC by stromelysin-1 (matrix metalloprotease-3) results in the production of bioactive peptides that affect different aspects of angiogenesis (14). In light of our previous finding that dexamethasone suppresses the production of matrix metalloproteases and tissue transglutaminase in IL-4-stimulated macrophages (38), it is possible that alternatively activated macrophages could counteract SPARC-induced modifications of the ECM by a down-regulation of enzymes for which SPARC is a substrate. Together, stabilin-1-mediated endocytosis and regulated expression of enzymes affecting SPARC activity comprise a complex, macrophage-specific mechanism for modulation of the biological activity of SPARC.
In summary, we have identified stabilin-1 as a receptor for SPARC. Stabilin-1 mediates SPARC uptake and endosomal trafficking that result in the lysosomal degradation of SPARC in alternatively activated macrophages. We propose that these cells play a major role in the regulation of the concentration of extracellular SPARC. The signaling and regulatory consequences of stabilin-1-mediated SPARC endocytosis remain to be identified.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by Margarete von Wrangell Habilitationprogramm (to J.K.), Deutsche Forschungsgemeinschaft SFB405, Project B12, and National Institutes of Health Grant GM40711 (to E.H.S.). 
2 Address correspondence and reprint requests to Dr. Julia Kzhyshkowska, Department of Dermatology, University Medical Centre Mannheim, University of Heidelberg, Theodor-Kutzer Ufer 1-3, D-68167 Mannheim, Germany; E-mail address: julia.kzhyshkowska{at}haut.ma.uni-heidelberg.de or Dr. E. Helene Sage, Hope Heart Program, Benaroya Research Institute at Virginia Mason, Seattle, WA 98101; E-mail address: hsage{at}benaroyaresearch.org
3 Abbreviations used in this paper: SPARC, secreted protein acidic and rich in cysteine; ECM, extracellular matrix; EGF, epidermal growth factor; acLDL, acetylated low-density lipoprotein; CHO, Chinese hamster ovary; EEA, early endosome Ag; PFA, paraformaldehyde.
Received for publication January 13, 2006. Accepted for publication March 7, 2006.
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v5 integrin-dependent programmed cell death triggered by a peptide mimic of annexin V. Mol. Cell. 11: 1151-1162. [Medline]This article has been cited by other articles:
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) or an insertless phage control (
). Bars represent the average of three rounds of panning and are mean ± SD. B, A basic local alignment search tool search of the sequence represented in phage no. 54 revealed significant sequence identity with stabilin-1. A diagram of stabilin-1 with its various domains is shown. The sequence identified by phage display is indicated by "S" and spans aa 1362–1367. The expressed fragment containing S and used for ELISA (C) spans aa 1332–1385. C, GST-tagged stabilin fragments S and P9 (aa 2302–2570), as well as GST control protein, were coated onto microtiter wells in triplicate at 50 ng/well and incubated with recombinant human SPARC. Bars represent the average SPARC detected ± SD.
) and CHO-vector control cells (
