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Originally published In Press as doi:10.1074/jbc.M504663200 on August 22, 2005

J. Biol. Chem., Vol. 280, Issue 43, 36483-36493, October 28, 2005
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SPARC Regulates Extracellular Matrix Organization through Its Modulation of Integrin-linked Kinase Activity*

Thomas H. Barker{ddagger}, Gretchen Baneyx§, Marina Cardó-Vila¶, Gail A. Workman{ddagger}, Matt Weaver{ddagger}, Priya M. Menon||, Shoukat Dedhar**, Sandra A. Rempel||, Wadih Arap¶, Renata Pasqualini¶, Viola Vogel§{ddagger}{ddagger}, and E. Helene Sage{ddagger}1

From the {ddagger}Hope Heart Program, Benaroya Research Institute at Virginia Mason, and the §Department of Bioengineering, University of || Washington, Seattle, Washington 98101, the University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, the Barbara Jane Levy Laboratory, Hermelin Brain Tumor Center, Henry Ford Hospital, Detroit, Michigan 48202, the **BC Cancer Agency, Jack Bell Research Centre, Vancouver, British Columbia V5Z 1L3, Canada, and the {ddagger}{ddagger}Swiss Federal Institute of Technology, ETH, CH-8093 Zurich, Switzerland

Received for publication, April 28, 2005 , and in revised form, August 19, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SPARC, a 32-kDa matricellular glycoprotein, mediates interactions between cells and their extracellular matrix, and targeted deletion of Sparc results in compromised extracellular matrix in mice. Fibronectin matrix provides provisional tissue scaffolding during development and wound healing and is essential for the stabilization of mature extracellular matrix. Herein, we report that SPARC expression does not significantly affect fibronectin-induced cell spreading but enhances fibronectin-induced stress fiber formation and cell-mediated partial unfolding of fibronectin molecules, an essential process in fibronectin matrix assembly. By phage display, we identify integrin-linked kinase as a potential binding partner of SPARC and verify the interaction by co-immunoprecipitation and colocalization in vitro. Cells lacking SPARC exhibit diminished fibronectin-induced integrin-linked kinase activation and integrin-linked kinase-dependent cell-contractile signaling. Furthermore, induced expression of SPARC in SPARC-null fibroblasts restores fibronectin-induced integrin-linked kinase activation, downstream signaling, and fibronectin unfolding. These data further confirm the function of SPARC in extracellular matrix organization and identify a novel mechanism by which SPARC regulates extracellular matrix assembly.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Matricellular proteins such as SPARC function as modulators of cell-extracellular matrix (ECM)2 interactions (1, 2). SPARC is considered "antiadhesive," because it does not directly support cell attachment. Moreover, it induces focal adhesion disassembly and cell rounding when the purified protein is added to spread cells (3-5). The induction of an intermediate state of cell adhesion by SPARC (6) implies a role for SPARC in the organization of ECM. Consistent with data acquired in vitro, mice with a targeted disruption of Sparc have marked developmental abnormalities in the dermis, eye, and adipose tissue (7-9) and show accelerated closure of dermal wounds (10-11), diminished foreign body response (12), and enhanced tumor growth (13). These aberrations have been explained, in part, by altered ECM production and assembly. Specifically, tissues from SPARC-null (S-/-) mice contain less collagen than those from wild-type (WT) mice, and the collagen present is less mature (7). However, the mechanism by which SPARC directs ECM assembly has not been identified.

The development of mature ECM requires proper formation of an organized fibronectin (Fn) matrix. The importance of Fn in the morphogenesis and patterning of tissues is established, since Fn-null mice die during early gastrulation as a result of defective cell migration (14). In response to challenge, Fn serves as an intermediate or provisional matrix (15, 16) that has been shown to stabilize early collagen fibrils. The reliance of mature (collagen-based) ECM on Fn assembly has been demonstrated by experiments with Fn-null cells, in which exogenous Fn was an absolute requirement for their generation of collagenous ECM (17, 18). Fn matrix assembly by cells requires integrin ({alpha}5{beta}1) binding to Fn molecules and active extension of the Fn molecule through the actinomyosin contractile machinery (19). Partial unfolding of Fn is thought to expose binding sites for other Fn molecules and thus promotes the formation of Fn fibers (20-24). Fn matrix assembly, which relies on cell binding and contraction, is sensitive to molecules that regulate cell-ECM interactions, such as matricellular proteins.

Integrin-linked kinase (ILK), a serine/threonine kinase that binds to the intracellular domain of {beta}1 integrin immediately adjacent to the plasma membrane and is activated by {beta}1 integrins and growth factors, has been shown to control the intracellular signaling cascades that influence cellular contractile elements (25). ILK interacts directly with actin and {alpha}-actinin-binding proteins such as the parvins, affixin and paxillin (26-28), and localizes to focal adhesion complexes (29). ILK phosphorylates, and thereby inactivates, myosin light chain phosphatase (MLCP) (30-32). MLCP specifically dephosphorylates and inactivates the regulatory myosin light chain (MLC) and thereby predisposes the cell toward a noncontractile state. Furthermore, ILK can also act as a calcium-independent myosin light chain kinase (33), an activity that leads to the induction of a contractile state. Thus, by both inhibiting MLCP activity and enhancing MLC activity, ILK activation by {beta}1 integrin leads to the induction of a contractile cell phenotype. Perhaps not surprisingly, ILK has also been shown to modulate Fn matrix assembly (34).

In this report, we demonstrate that ILK, through its regulation of contractile signaling, participates in the mechanism by which SPARC regulates Fn matrix assembly. SPARC exhibited saturable binding to ILK and was coincident with ILK and integrin {beta}1 on the cell surface. Moreover, we show that SPARC was required for Fn-induced ILK activation and downstream MLCP inactivation. These data reinforce the role of SPARC in the regulation of ECM organization and provide a molecular mechanism for this activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of Primary Fibroblasts—Fibroblasts from pulmonary tissue of WT and S-/- (C57Bl6/J) mice were isolated and cultured as previously described (35). Fibroblast populations were enriched by the removal of CD31- and CD45-positive cells with Dynabeads (Dynal Biotech, Brown Deer, WI). Genotypes were verified by PCR, and the absence of SPARC protein in S-/- fibroblasts was confirmed. Fibroblasts were cultured in Dulbecco's modified Eagle's medium containing 10% FBS, 10 units/ml penicillin G, and 10 µg/ml streptomycin SO4 (growth medium).

Adenoviral Constructs—Ad vectors containing Renilla luciferase and mouse SPARC transgenes were produced by the use of the Transpose Ad vector system (Qbiogene, Carlsbad, CA). A fragment of mouse SPARC cDNA (931 bp) was prepared by reverse transcription-PCR of purified MEF1 (ATCC, Manassas, VA) mRNA with mouse SPARC-specific primers, both (5' and 3') containing NheI restriction sites, which were subsequently cloned into the NheI restriction site of the shuttle vector pCR259. RLuc transgene cDNA was prepared by double digestion of pRL-SV40 (Promega, Madison, WI) with NheI and XbaI to generate a 947-bp fragment that was ligated into the NheI restriction site of pCR259. Transgene orientation was verified by analytical PCR.

Fluorescence Resonance Energy Transfer (FRET)—Human plasma Fn (>95% purity; Chemicon, Temecula, CA) in PBS was doubly labeled (Fn-D/A) with AlexaFluor 488 (AF-488) and AF-564 (Molecular Probes, Inc., Eugene, OR) for FRET by a two-step process described previously (20). Sterile LabTek 4-well chamber slides (Nalge Nunc International, Rochester, NY) were coated with unlabeled Fn by adsorption from a 25 µg/ml Fn solution in PBS for 60 min at 37 °C. Thirty min after plating, the medium containing any nonadherent cells was removed and was replaced with growth medium containing a mixture of Fn-D/A and a 10-fold excess of unlabeled Fn to yield a final Fn concentration of 100 µg/ml (exclusive of Fn from FBS). Samples were incubated for 4 h (early matrix assembly) or for 4 days (long term matrix assembly). Samples for 4 day time points were prepared identically, with medium replaced at 48 h (growth medium with 10% Fn-D/A, 90% Fn mixture). Live cultures were prepared for examination by washing with PBS followed by treatment with PBS containing 1.5 mM Trolox (Sigma). Imaging and spectroscopy of Fn-D/A in cell matrices were performed with an inverted epifluorescence microscope (TE 2000E, Nikon; x100 oil PlanFluor objective, 1.3 numerical aperture; Nikon) attached to a spectrometer (Acton 150; Roper Scientific, Acton, MA). Spectra were analyzed by a custom software program created in IGOR PRO (WaveMetrics, Lake Oswego, OR). FRET values are reported as IA/ID.

Fluorescence-activated Cell Sorting—Fibroblasts were released with trypsin, counted, and washed in PBS containing 1% FBS. 1 x 105 cells were stained for integrin {alpha}5 and integrin {beta}1 by incubation with fluorescein isothiocyanate-conjugated hamster anti-{alpha}5 and hamster anti-{beta}1 IgG (1:50 dilution in PBS plus 1% FBS solution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 30 min at 4 °C. Fluorescein isothiocyanate-conjugated hamster IgG (Santa Cruz Biotechnology) was used as a negative control. Stained cells were washed and stored in PBS containing 1% FBS, 1.5% paraformaldehyde, and 0.005% sodium azide. Data acquisition was performed on a BD FACStar Plus flow cytometer (BD Biosciences), and analysis was performed with the CellQuest Pro software package (BD Biosciences).

Electric Cell-substrate Impedance Sensing Attachment Assay—Electric cell-substrate impedance sensing (Applied Biophysics Inc., Troy, NY) was used to assess cell attachment and spreading dynamics. Wells of the gold-plated electrode arrays were coated with Fn (Chemicon) by adsorption from a Fn solution (100 µg/ml in PBS) for 30 min at room temperature, rinsed with PBS, and filled with 300 µl of growth medium. The arrays were equilibrated at 37 °C for 10 min in the unit incubator prior to connection to the electrodes. Electrode connections were verified, and the base-line resistance (ohm) readings for medium only were automatically graphed over time. The cells were released with trypsin, counted, and resuspended in growth medium. Cells (200 µl of 5 x 105/ml) were added to each well, and the electrode resistance was recorded. Complete single layer cell coverage was confirmed microscopically at the end of the run. For analysis, trace resistance values were adjusted by subtraction of the medium only base line. Corrected adhesion/spreading profiles were generated in Microsoft Excel (Microsoft Corp., Redmond, WA). NIH3T3 cells were used as internal standards but are not reported.

Phage Display Screening—A phage display random peptide library displaying the insert CX7C was used in the screenings; phage input was 3 x 1010 transducing units. rhuSPARC protein was coated onto microtiter wells as previously described (36, 37). Phage binding assays on purified proteins were carried out as described previously (38). SPARC, hevin (39), bovine collagen I (VitrogenTM; Collaborative Biomedical Products, Bedford, MA), and BSA (Pierce) at 1 µgin50 µl of PBS plus 0.1 mM Ca2+ were immobilized on microtiter wells overnight at 4 °C. Wells were washed twice with PBS, blocked with PBS plus 3% BSA for 2 h at room temperature, and incubated with 2 x 109 transducing units of each phage (CWVAGLVPC, CFRPYGSAC) or fd-tet phage (control) in 50 µl of PBS plus 1.5% BSA. After 1 h at room temperature, wells were washed 10 times with PBS, and phage were recovered by bacterial infection. Three rounds of panning were performed.

In Vitro Binding Assay—rhuSPARC was generated in SF9 cells and was purified by gel chromatography (40, 41). Purified rhuSPARC was labeled with AF-488 or AF-568 succinimidyl ester (Molecular Probes) for direct detection of SPARC binding. Free probe was cleared by chromatography on a PD-10 column (Amersham Biosciences). The activity of the labeled rhuSPARC was verified prior to binding assays. Labeled SPARC was used for both in vitro binding assays and localization of exogenous SPARC. Wild type ILK, produced in E. coli in the presence of isopropyl 1-thio-{beta}-D-galactopyranoside and purified from inclusion bodies following the Novagen protein refolding kit (EMD Biosciences, San Diego, CA), was coated (400 ng in Hanks' buffered saline solution) in wells of a standard protein high binding 96-well plate (Nalge Nunc International); subsequently, the wells were washed with Hanks' buffered saline solution containing 0.05% Tween 20. Solid phase controls (e.g. BSA) were performed at molar equivalence to ILK. Wells were blocked with 5% casein, 0.1% Tween 20, Hanks' buffered saline solution and incubated with increasing concentrations of AF-568-labeled rhuSPARC (0.5, 1, 2, 4, 8, 12, 16, and 20 µg in Hanks' buffered saline solution). AF-568-labeled rabbit IgG was tested to ensure that nonspecific binding due to ILK immobilization did not occur. Unbound AF-568-labeled protein was removed, the wells were washed, and bound protein was detected at an excitation wavelength of 485 nm and emission wavelength of 535 nm on a Fusion universal microplate analyzer (Packard Instrument Co.).

Fluorescence Microscopy—Cells were plated on Fn-adsorbed coverslips for appropriate times and were fixed with methanol-free EM grade 3% formaldehyde (Tousimis, Rockville, MD) in PBS for 10 min at 4 °C to maintain cell membrane integrity. Cells were either rendered permeable with 0.1% saponin (Sigma) throughout the staining protocol, or cell surface integrity was preserved. Actin stress fibers were detected with AF-488-phalloidin (Molecular Probes). SPARC was detected with anti-SPARC monoclonal antibody (clone 293) (42) and anti-mouse SPARC goat polyclonal IgG (R&D Systems), and ILK was detected with anti-ILK rabbit polyclonal IgG and anti-ILK monoclonal antibody (Upstate%20Biotechnology">Upstate Biotechnology, Inc., Lake Placid, NY). Integrin {beta}1 was detected with fluorescein isothiocyanate-conjugated hamster anti-{beta}1 IgG (Santa Cruz). TRITC-conjugated donkey anti-rabbit IgG, fluorescein isothiocyanate-conjugated donkey anti-goat IgG (Jackson Laboratories, West Grove, PA), and AF-350-conjugated donkey anti-goat IgG (Molecular Probes) were used as indicated. Appropriate isotype controls were performed to evaluate nonspecific binding. Brightness adjustment and image merging were performed in Adobe Photoshop (version 7.0; Adobe Systems) in a nonbiased manner by adjusting all images by identical values.

Immunoprecipitation and Immunoblotting—For immunoprecipitation, 1 x 106 cells were lysed in immunoprecipitation buffer containing 1% Brij 98, 50 mM HEPES, 150 mM NaCl, 5 mM Na3VO4, 5 mM NaF, and protease inhibitor mixture (Roche Applied Science). Lysates were precleared with Protein A/G-Sepharose (Santa Cruz Biotechnology) and were incubated with rabbit anti-ILK polyclonal IgG at 4 °C for 16 h. Immune complexes were purified on Protein A/G-Sepharose with sequential washes of lysis buffer. Rabbit anti-hemagglutinin (HA) IgG was used to control for nonspecific antibody interactions. Samples were resolved by SDS-PAGE, transferred onto polyvinylidene difluoride-plus membranes, and probed with mouse anti-SPARC IgG (clone 303) and mouse anti-ILK (Upstate%20Biotechnology">Upstate Biotechnology). For immunoblotting of signaling proteins, cells were plated on Fn-adsorbed tissue culture plastic; some cultures were treated with inhibitors of ILK (KP074728; BC Cancer Agency, Vancouver, Canada) and Rho-associated kinase (Y27632; Upstate%20Biotechnology">Upstate Biotechnology) for 60 min prior to Fn stimulation or transfection of inactive ILK or hyper-ILK expression vectors (43) by Lipofectamine 2000 (Invitrogen) 48 h prior to Fn stimulation. At appropriate intervals, the cells were lysed directly in 2x SDS-PAGE sample buffer containing 2% {beta}-mercaptoethanol. Samples were boiled, and proteins were resolved by SDS-PAGE, transferred onto polyvinylidene difluoride-plus membranes, probed with appropriate antibodies (anti-MYPT1, Covance, Princeton, NJ; anti-phospho-MLCP, Upstate%20Biotechnology">Upstate Biotechnology), and detected with appropriate horseradish peroxidase-conjugated secondary antibodies and ECL reagent (Amersham Biosciences). Exposed film was scanned into Adobe Photoshop (Adobe Systems, San Jose, CA), and the film background was subtracted from the images. In some cases, lanes were moved (in the horizontal plane only) to conserve space for image presentation. Experiments were performed in triplicate. All images presented represent data from a single experiment with a single exposure.

Biotinylation of Cell Surface Proteins—Fibroblasts were plated in Dulbecco's modified Eagle's medium on Fn-coated 100-mm tissue culture plates (10 µg/ml) for 120 min to allow full spreading. Spread cells were washed extensively with warm PBS containing 1 mM CaCl2 and 1 mM MgCl2 to remove excessive reactive species and primary amine-containing buffers. Approximately 5 x 105 live cells were incubated witha5mM solution of EZ-link biotinylation reagent (Pierce) for 30 min at room temperature in PBS, pH 7.4, containing 1 mM CaCl2 and 1 mM MgCl2. The biotinylation reaction was quenched by three successive washes in Tris-buffered saline, pH 7.4. Cells were lysed in immunoprecipitation buffer, and ILK immunocomplexes were produced as described above with rabbit anti-ILK polyclonal IgG. Appropriate controls, rabbit anti-HA for nonspecific IgG interactions and mouse anti-lamin A/C for biotinylation of intracellular proteins, were performed. Biotinylated proteins were detected by Western blot with avidin-horseradish peroxidase (DAKO Cytomation, Carpinteria, CA) and ECL reagent (Amersham Biosciences).

ILK Activity Assay—Fn-induced ILK activity was determined as previously published with minor modifications (44). Cells that were serum-starved (Dulbecco's modified Eagle's medium containing 0.1% FBS) for 16 h were removed by trypsin and were plated (1.3 x 104 cells/cm2) on tissue culture plates coated with Fn by adsorption from a Fn/PBS solution (10 µg/ml). At appropriate intervals, cells were lysed in ILK lysis buffer (1% Nonidet P-40, 50 mM HEPES, 150 mM NaCl, 5 mM Na3VO4, 5 mM NaF, 400 µg/ml DNase, and protease inhibitor mixture (Complete; Roche Applied Science)). Lysate (250 µg) was incubated with 5 µg of rabbit anti-ILK polyclonal or rabbit anti-HA tag (negative control) IgG for 16 h at 4 °C with gentle agitation. Immune complexes were purified by incubation with Protein A/G-Sepharose (Santa Cruz Biotechnology) for 1 h at 4°C, followed by two sequential washes in ILK lysis buffer and two sequential washes in ILK kinase buffer (50 mM HEPES, 10 mM MgCl2, 2 mM MnCl2, 5 mM Na3VO4, and 5 mM NaF). The final wash was removed, and bead-protein complexes were subsequently incubated with kinase buffer containing 200 mM ATP and 5 µg of myelin basic protein (MBP) at 30 °C for 25 min. Reactions were quenched with 2x SDS-PAGE sample buffer containing {beta}-mercaptoethanol (2% by volume) and were boiled for 5 min. Samples were resolved by SDS-PAGE, transferred onto polyvinylidene difluoride-plus membranes, and immunoblotted with horseradish peroxidase-conjugated mouse anti-phospho-MBP monoclonal antibody (Upstate%20Biotechnology">Upstate Biotechnology).

Statistics—Quantification of Western blot band intensities was performed on unmodified images by the use of Image J software (version 1.33, National Institutes of Health, Bethesda, MD). Student's t test was used to determine statistical significance (n = 3, unless otherwise stated under "Results"). All data are reported as the average ± S.E. (unless otherwise stated under "Results"). p values, where statistical significance was achieved, are reported.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Recent reports that S-/- mice exhibited significantly less collagen than WT counterparts prompted us to question the role of SPARC in the regulation of ECM assembly. Because Fn matrix assembly precedes collagen accumulation and is a requirement for collagen stability, we addressed the function of SPARC in the assembly of Fn. Although SPARC has been shown to disassemble focal adhesions and to induce a state of intermediate adhesion (6), a process that influences Fn matrix assembly, a molecular mechanism whereby this activity is mediated is unknown and was therefore the primary goal of this study.

S-/- Fibroblast Cultures Exhibit Deficient Organization of Fn Matrix—Mice with a targeted disruption of Sparc display altered ECM. Because Fn plays a critical role in the progression from early/provisional to mature ECM, we asked whether S-/- fibroblasts were inhibited in their capacity to generate a Fn matrix. We used primary fibroblasts isolated from lung tissue because they can be cultured for several passages without transformation or senescence. When plated on Fn-adsorbed coverslips and grown in Dulbecco's modified Eagle's medium supplemented with AF-488-labeled Fn (25 µg/ml), S-/- fibroblasts generated a Fn matrix that was qualitatively less (fewer, thicker fibers) than that produced by WT fibroblasts (initial data not shown; see Fig. 1, d (WT) and e (S-/-), for subsequent representative images).

S-/- Fibroblasts Show Reduced Capacity to Unfold Fn Molecules during Fn Matrix Assembly—SPARC exhibits antiadhesive properties and regulates cell-ECM interactions (2). We therefore asked whether the difference observed between S-/- and WT fibroblasts reflected their respective capacities to unfold Fn molecules via cellular contraction, an important step in the progression of Fn fibril formation. We used a recently published technique employing intramolecular FRET to investigate the structural state of Fn molecules during matrix assembly by fibroblasts plated on Fn-adsorbed rigid surfaces (20, 21). Fn molecules are labeled with acceptor and donor fluorophores at different locations along the peptide backbone to generate doubly labeled Fn. As the cells incorporate the Fn-D/A into the Fn matrix, they partially unfold the Fn-D/A, resulting in an increase in the average distance between donor and acceptor fluorophores and, consequently, a decrease in the FRET signal. Thus, by examining the intensity of the acceptor (IA) and donor (ID), we can indirectly assess the cell-mediated mechanical stretch of Fn during matrix assembly. Because the FRET characteristics change each time the labeling is performed, a calibration curve is generated for each batch of Fn-D/A after the use of guanidine hydrochloride (GdnHCl) to denature the protein (Fig. 1a). For correlation of FRET with changes in the secondary structure of Fn, changes in the acceptor to donor emission, IA/ID, were measured in solution for each Fn-D/A batch as a function of the denaturant GdnHCl. As described elsewhere, Fn begins to show significant loss of secondary structure (>10%) above 2 M GdnHCl (45). Here we refer to Fn as extended if the dimeric arms of Fn open from a compact conformation without the loss of secondary structure, and we refer to Fn as partially unfolded when solution data indicate the loss of secondary structure. The data derived from FRET show that S-/- fibroblasts plated on surface-adsorbed Fn and in the presence of Fn-D/A displayed significantly less unfolding of Fn molecules, in comparison with WT fibroblasts (p < 0.00005, n (WT) = 200, n (S-/-) = 100) within 4 h (Fig. 1b). Although these differences in Fn unfolding were most obvious during early time points, the differences were persistent and statistically significant up to 4 days in culture (Fig. 1c; p < 0.00005, n (WT) = 2000, n (S-/-) = 500). Furthermore, gross examination of the Fn matrix at 4 days revealed that, qualitatively, WT fibroblasts produced a greater number of fibers in a more extensive matrix, whereas S-/- fibroblasts generated fewer fibers (Fig. 1, compare d and e). We verified that substrate-adsorbed Fn, rather than a component of serum, was responsible for the initial signaling required to generate the contractile forces necessary for cell-mediated Fn unfolding by demonstrating that the removal of serum did not alter the Fn FRET profiles of WT fibroblasts (p = 0.10, n (WT) = 100, n (WT without serum) = 100) or S-/- fibroblasts (p = 0.25, n (S-/-) = 100, n (S-/- without serum) = 50) at the 4-h time point (data not shown).



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  		FIGURE 1.
S-/- fibroblasts show reduced capacity to extend Fn molecules during Fn matrix assembly. The capacity of WT and S-/- fibroblasts to assemble Fn matrix was examined by intramolecular FRET. a, a calibration curve of Fn-A/D unfolding, showing compact, extended, and partially unfolded states of the labeled protein in solution. This batch of Fn-A/D labeled with 11.7 donors and 4.0 acceptors per Fn molecule exhibited an IA/ID ratio of 1.03 in 2 M GdnHCl. b, Fn matrix at 4 h exhibited the greatest difference between the fibroblast populations (black line, WT; gray line, S-/-; p < 0.00005; 4 and 2 M GdnHCl values are denoted by dashed vertical lines), although the differences in Fn unfolding persisted through 4 days (c, black line, WT; gray line, S-/-; p < 0.00005; 2 and 4 M GdnHCl values are denoted by vertical dotted lines). Images of the Fn matrix at 4 days indicate that WT fibroblasts (d) produce a more extensive matrix compared with S-/- fibroblasts (e), which produced fewer fibers. Scale bars, 10 µm.

 
Lack of SPARC Expression Does Not Significantly Affect Cell Surface Levels of {alpha}5 or {beta}1 Integrin but Alters Cell Spreading Dynamics and Prevents Formation of Actin Stress Fibers—Fn matrix assembly requires binding to integrin {alpha}5{beta}1 and subsequent cell contraction. For these reasons, we asked whether the differences in the assembly of Fn as seen in Fig. 1 were due to variable levels of cell surface integrin {alpha}5 or {beta}1. Fluorescence-activated cell sorting analysis of S-/- and WT fibroblast cell surface {alpha}5 and {beta}1 integrins indicated no significant, quantitative differences in the mean fluorescence intensity between the two fibroblast populations ({beta}1 mean fluorescence intensity, WT = 95, S-/-= 130; {alpha}5 mean fluorescence intensity, WT = 20, S-/-= 29); the weak {alpha}5 staining of cells in suspension observed in all experiments was above background levels. Furthermore, because SPARC is known to inhibit cell spreading, we asked whether the different FRET profiles obtained for WT and S-/- fibroblasts (Fig. 1, b and c) resulted from intrinsic differences in the spread state of the two fibroblast populations at the time when FRET data were acquired. Using electric cell-substrate impedance sensing (46, 47), we analyzed attachment and spreading dynamics of WT and S-/- fibroblasts. Under conditions identical to those used for FRET, we found that both populations had reached their respective 95% maximal spread state within the time frame of the FRET experimentation (WT = 84.0 min, S-/-= 68.3 min; p = 0.39; Fig. 2a); therefore, the differences in FRET shown in Fig. 1, b and c, were not attributed to variability in cell spreading. However, minor differences in the spreading dynamics between the two genotypes were observed. Specifically, S-/- fibroblasts consistently displayed a short delay in the onset of spreading (ranging from 3.5 to 5 min) after attachment. The slightly increased spreading rate, although not statistically significant, and the decreased maximum resistance (WT = 8167 ohms, S-/- = 6133 ohms; p < 0.05; Fig. 2b) exhibited by S-/- fibroblasts accounted in large part for their reaching a maximal spread state before that of WT fibroblasts. These results are in agreement with parallel spreading experiments performed on ILK-/- cells. After an extended interval, ILK-/- cells have shown a more rapid spreading, but to a smaller final area, compared with WT controls.3 In addition to the dissimilar spreading characteristics, staining for F-actin indicated that S-/- fibroblasts formed fewer stress fibers and a less extensive cytoskeletal network in response to substrate-adsorbed Fn (Fig. 2, c-f, WT, and g-j, S-/- Fibroblasts). These data indicate a possible interruption in signaling cascades that direct internal cell tension and stress fiber formation. Significant signaling proteins that regulate cell contraction and stress fiber formation are those involved in the phosphorylation/dephosphorylation of the regulatory MLC. We therefore screened for SPARC-binding partners and identified a candidate that is a known mediator of cell contraction signaling.



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  		FIGURE 2.
Lack of SPARC does not significantly alter the time required for maximal cell spreading but prevents formation of actin stress fibers. a, WT (black line) and S-/- (gray line) fibroblasts were evaluated by electric cell-substrate impedance sensing for cell spreading dynamics. Despite a few differences in the spreading dynamics of WT and S-/- fibroblasts on Fn-adsorbed surfaces, both reached their maximal spread state at nearly equivalent times (WT = 84.0 min, S-/-= 68.3 min; p = 0.39) and prior to the time when FRET data were acquired. b, WT (black bar) and S-/- (gray bar) fibroblasts display different maximal resistance on Fn-adsorbed electrodes (WT = 8167 ohms, S-/-= 6133 ohms; *, p < 0.05). WT (c-f) and S-/- (g-j) fibroblasts plated on Fn-adsorbed coverslips in the absence of serum (c and g, 5 min; d and h, 30 min; e and i, 60 min; f and j, 180 min; scale bars, 10 µm) were stained for F-actin with AF-488-labeled phalloidin. The data indicate that S-/- fibroblasts were inhibited in their capacity to form actin stress fibers in response to Fn.

 
The Identification of ILK as a Potential SPARC Binding Partner by Phage Display Is Verified by Solid Phase Binding Assays—We isolated SPARC-binding peptides by screening a phage library displaying random CX7C peptide inserts on purified rhuSPARC that was immobilized on 96-well plates. The SPARC homolog hevin, collagen I, and BSA were used as negative controls for enrichment during consecutive rounds of panning. Phage were sequenced from randomly selected clones after three rounds of selection (data not shown). Two different phage displaying the sequences CPVLGAVWC (clone 47) and CFRPYGSAC (clone 51) were found to bind to rhuSPARC (p < 0.01 and p < 0.05 compared with fd-tet, respectively) but not to hevin, collagen I, or BSA (Fig. 3a). A BLAST search based on phage 47 and 51 peptide sequences revealed that both sequences have similarity to ILK, with each sequence mapping to a 24-amino acid region (clone 47, PVLGA, amino acids 252-256; clone 51, PYGS, amino acids 273-276) in the kinase domain of ILK1 and ILK2 (ILK1 sequence shown in Fig. 3b; blue text indicates ILK activation site, boldface text indicates the kinase domain, and red text indicates the location of the phage sequences that matched to ILK). Although phage display is useful for identification of potential binding partners, validation of binding between the two full-length proteins is necessary. Assays for binding between SPARC and ILK were performed in solid phase with AF-546-labeled purified rhuSPARC and rhuILK expressed in E. coli (purified rhuILK; Fig. 3c). As shown in Fig. 3d, at saturation, rhuSPARC bound rhuILK (solid black line; *, p < 0.05; **, p < 0.01) at a 0.94 molar ratio (SPARC·ILK). SPARC did show minor binding to BSA (Fig. 3d, gray line, as reported previously (63). The lack of binding between AF-488-labeled IgG and rhuILK (Fig. 3d, dashed black line) indicated that immobilized rhuILK supports minimal (if any) nonspecific binding. All values were normalized to values derived from the binding of SPARC to the blocking protein (casein). Furthermore, preincubation of SPARC with soluble ILK, but not BSA, resulted in a significant decrease in the binding of SPARC to immobilized ILK, demonstrating competitive binding and the specific nature of SPARC·ILK binding (Fig. 3e; **, p < 0.01).

The Presence of a Membrane-associated SPARC-ILK Complex in WT Fibroblasts Is Supported by Immunofluorescence, Co-immunoprecipitation, and Cell Surface Protein Biotinylation—Validation of the interaction between SPARC and ILK in a physiological context was demonstrated by the colocalization and co-immunoprecipitation of a SPARC-ILK complex. SPARC and ILK appeared coincident in WT fibroblasts when nonpermeabilized cells were stained with multiple combinations of antibodies to detect the two proteins (mouse anti-SPARC and rabbit anti-ILK, Fig. 4, a-d; goat anti-SPARC and mouse anti-ILK, Fig. 4, e-h). ILK appeared as small clusters (Fig. 4, b and f, white arrows), where overlapping with SPARC did not occur, and in larger domains (Fig. 4, b and f, open arrows), where coincidence with SPARC (Fig. 4, a and e, open arrows) was observed. Some of the SPARC-ILK clusters also appeared coincident with integrin {beta}1 (Fig. 4, g and h, open arrows; Fig. 4g, gray arrows indicate fibrillar adhesion structures). Based on the indication of a membrane-associated SPARC-ILK cluster in WT fibroblasts, we performed cell surface biotinylation and immunoprecipitation of S-/- and WT fibroblasts plated on Fn with rabbit anti-ILK antibodies. Detection of immunocomplexes with mouse anti-SPARC antibody (Fig. 4j; clone 303), mouse anti-ILK (Fig. 4k), and avidin-horseradish peroxidase (Fig. 4l) indicates that SPARC coprecipitates with ILK in WT, but not S-/-, fibroblasts; that ILK is present and can be immunoprecipitated from both WT and S-/- fibroblasts; and that both proteins are biotinylated (Fig. 4l, black arrows) by cell-impermeable reagents. Biotinylation of the intracellular proteins lamin A/C was not apparent (Fig. 4, j-l). Additional bands do appear in the biotinylated ILK·SPARC complex, but one anticipates that many bands will be biotinylated. The additional bands could be associated with the SPARC·ILK complex, either specifically or nonspecifically, or may represent partially degraded SPARC (gray arrow denotes the possible presence of the 30-kDa fragment of SPARC). These data support the presence of a SPARC-ILK complex in intact WT fibroblasts plated on Fn, and with the solid phase binding assay, they provide substantive evidence that direct interaction between SPARC and ILK occurs.



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  		FIGURE 3.
Identification of ILK as a potential SPARC-binding partner by phage display is confirmed by solid substrate binding assays. a, rhuSPARC, hevin, collagen I, or BSA was coated onto microtiter wells at 10 µg/ml and incubated with phage clones 47 (white bars), 51 (gray bars), and fd-tet (insertless; black bars; *, p < 0.05; **, p < 0.01). b, a BLAST search of the two phage peptide sequences indicated significant sequence identity with ILK (blue text, ILK activation site; boldface text, ILK kinase domain; red text, mapped phage sequences). c, rhuILK was produced in E. coli and was purified to >95% from inclusion bodies (left lane, reducing (R) conditions; right lane, nonreducing (NR) conditions; molecular mass standards in kDa on left). d, in vitro binding assay (enzyme-linked immunosorbent assay format) with AF-568-labeled rhuSPARC demonstrated saturable binding to rILK (black line). The binding of SPARC to ILK was significantly greater than that of SPARC to BSA (*, p < 0.05; **, p < 0.01), although minor binding of SPARC to BSA (gray line) was observed. AF-568-labeled IgG showed comparable levels of binding to immobilized ILK (dashed line) as was seen with the binding of SPARC to the blocking protein casein (base line subtracted from all data points). e, SPARC was preincubated with ILK or BSA at increasing molar ratios overnight at 4 °C prior to incubation on ILK-coated plates. Soluble ILK, but not BSA, significantly competed for the binding of SPARC to immobilized ILK (**, p < 0.01).

 
Fn-induced ILK Activation and ILK-mediated Phosphorylation of MLCP Are Inhibited in S-/- Fibroblasts—ILK is known to interact directly with the cytoplasmic tail of {beta}1 integrin immediately adjacent to the plasma membrane (for a review, see Ref. 48), inactivate MLCP, and activate MLC. These activities indicate an important role for ILK in the regulation of contractile signaling, in cell-mediated, force-induced partial unfolding of Fn, and Fn matrix assembly. In consideration of these known activities of ILK, we asked whether the lack of SPARC in S-/- fibroblasts affects ILK activation and downstream signaling, results that could explain, in part, the observed contractile defect of S-/- fibroblasts.

Using Fn-adsorbed plates to induce {beta}1 integrin clustering and activation of ILK during cell attachment and spreading, we found that S-/- fibroblasts exhibited no detectable level of ILK activation (Fig. 5a, lanes 2-6). The level of ILK activation in the S-/- fibroblasts was similar to that of the control (WT fibroblast lysates from cells stimulated for 60 min on Fn-adsorbed plates immunoprecipitated with anti-HA IgG; Fig. 5a, lane 1). In contrast, WT fibroblasts exhibited a robust activation of ILK upon seeding onto an Fn surface (Fig. 5a, lanes 7-11). As reported previously, Fn induced a maximal ILK activation in WT fibroblasts at 60 min (Fig. 5a, lane 9), which persisted through 240 min. S-/- fibroblasts also displayed diminished phosphorylation of MLCP, a known downstream target of ILK and a direct mediator of cell contraction through its dephosphorylation of MLC (Fig. 5b). The role of ILK in the phosphorylation of MLCP in WT fibroblasts was first demonstrated by inhibition of ILK activity with the pharmacological reagent KP-074728 (denoted KP07 in Fig. 5c). Inhibition of ILK resulted in a significant decrease (p < 0.05, n = 4) in the Fn-induced phospho-MLCP signal in WT fibroblasts, data indicating a primary role for this kinase in FN-induced MLCP signaling in fibroblasts (Fig. 5c, lane 1, control; lane 2, KP-074728-treated at 240 min; bar graph with quantification on right; black bar, control; white bar, KP-074728-treated). Inhibition of Rho-associated kinase with Y27632 resulted in a nonsignificant decrease in the level of phosphorylated MLCP (Fig. 5c, lane 3, control; lane 4, Y27632-treated at 240 min; bar graph with quantification on right; black bar, control; white bar, Y27632-treated), a result that further implicates ILK in early (0-4-h) Fn-induced contractile signaling. Hyper-ILK vectors were transfected into S-/- cells to determine whether ILK rescue could restore MLCP signaling in S-/- fibroblasts. Hyper-ILK-expressing S-/- fibroblasts demonstrated a significant increase (p < 0.05) in Fn-induced phosphorylation of MLCP (Fig. 5d, lane 1, S-/- cells; lane 2, inactive ILK-transfected S-/- cells; lane 3, hyper-ILK-transfected S-/- cells on Fn-adsorbed plates for 240 min; bar graph with quantification on right). These data indicate that ILK activation is required for Fn-induced contractile signaling and that SPARC is required for ILK activation by Fn. These data also provide a partial explanation for the differences in Fn unfolding observed between S-/- and WT fibroblasts.



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  		FIGURE 4.
SPARC and ILK appear coincident by immunolocalization on fibroblasts, co-precipitate from fibroblast lysates, and can be detected by membrane-impermeable biotinylation. a-d, SPARC and ILK were identified by immunofluorescence after exposure to mouse anti-SPARC and rabbit anti-ILK polyclonal IgG on WT fibroblasts. White arrows (b) denote small ILK-positive clusters, and open arrows (a and b) denote ILK-positive clusters that appeared coincident with SPARC. d, zoom out of single cell with a box indicating the region shown in a-c. e-h, the coincidence of ILK with SPARC in WT cells was further confirmed using goat anti-SPARC IgG (e) and mouse anti-ILK IgG (f). White arrows denote ILK clusters (f), and open arrows (e and f) indicate clusters of ILK and SPARC coincidence. g, several, but not all, SPARC-ILK clusters also appeared coincident with integrin {beta}1 (open arrows). SPARC-ILK clusters were not observed in fibrillar adhesions (gray arrows). h, a merge and zoom out of the cell shown in e-g (box indicates the region displayed). i, control antibodies (goat IgG, mouse IgG, and hamster IgG with appropriate secondary antibodies) show no significant staining. Scale bar, 10 µm (d, h, and i) and 1 µm (a-c and e-g). j-l, immunocomplexes from whole cell lysates of cell surface-biotinylated fibroblasts seeded on Fn and probed with mouse anti-SPARC (j), mouse anti-ILK (k), and avidin-horseradish peroxidase (l). Lanes in j-l from left to right, ILK immunocomplexes from S-/- lysate, ILK immunocomplexes from WT lysate, lamin A/C (control) immunocomplexes from WT lysate, and HA-tag (control) immunocomplexes from WT lysate. Molecular mass markers are denoted in kDa on the left. I.P., immunoprecipitation.

 
Expression of SPARC in S-/- Fibroblasts Rescues Fn-induced ILK Activation, MLCP Deactivation, and Fn Unfolding during Assembly—Expression of SPARC was induced in primary S-/- fibroblasts by Ad delivery. Fibroblasts infected with supernatants from Ad-producing 293 cells showed an infection efficiency (as seen by immunofluorescent staining of SPARC) (Fig. 6a) of 60-80%. Localization and relative levels of SPARC in Ad-infected fibroblasts appeared similar to those seen in WT fibroblasts (Fig. 6b). Fn-induced ILK activation was restored in S-/- fibroblasts after infection with Ad-containing mouse SPARC cDNA (Ad.SP) (Fig. 6c, lane 1, S-/- fibroblasts; lane 3, Ad.SP-infected S-/- fibroblasts), but not with a control Ad-containing Renilla luciferase cDNA (Ad.RLuc) (Fig. 6c, lane 2, and quantification in Fig. 6d; black bar, S-/- cells; white bar, Ad.RLuc-infected S-/- cells; gray bar, Ad.SP-infected S-/-cells). The phosphorylation of MLCP in response to Fn was also restored (Fig. 6e, compare lanes 1-4 (Ad.RLuc-infected S-/- cells) with lanes 5-8 (Ad.SP-infected S-/- cells)). The phospho-MLCP signal resulting from the reexpression of SPARC was sensitive to the ILK inhibitor KP-074728 (Fig. 6, e, lanes 9-12, and f), a result that verifies this event was ILK-dependent. Finally, the capacity of S-/- fibroblasts to unfold Fn molecules during Fn matrix assembly was restored to WT levels after infection by Ad.SP (Fig.6g; WT (black line) versus Ad.SP (red line), p = 0.10; Ad.SP versus S-/- (gray line), p < 0.00005, n = 150 each set), where as Ad.Rluc-infected S-/- fibroblasts (Fig.6g, blue line) exhibited unfolding of Fn similar to that of S-/- fibroblasts (Fig. 6g; S-/- versus Ad.RL, p = 0.20; Ad.RL versus WT, p < 0.00005). These data confirm that SPARC is required for Fn-induced activation of the ILK-MLCP signaling pathway and for the partial unfolding of Fn mediated by cell contraction.

Exogenously Added SPARC Displays Altered Cellular Localization and Fails to Rescue S-/- Fibroblasts—SPARC has been previously described as a secreted protein; we have shown that it clusters with ILK and that this cluster is accessible to extracellular reagents, suggesting that extracellular SPARC might interact with an extracellularly exposed ILK. However, when AF-488-labeled rhuSPARC, verified as active in proliferation assays, was added to S-/- fibroblasts, it was localized to small intracellular endosomes or endosome-like structures (Fig. 7a), as opposed to the normal diffuse punctate intracellular staining pattern observed in WT fibroblasts (Fig. 7b). Consistent with the altered cellular localization, exogenous rhuSPARC failed to rescue any of the S-/- diminished activities, including Fn-induced ILK activity (Fig. 7c), spreading characteristics (Fig. 7d), and Fn unfolding during Fn matrix assembly (Fig. 7e). Consistent with previous reports, exogenous SPARC was antiadhesive as seen by the decrease in electric cell-substrate impedance sensing resistance and decreased Fn unfolding (demonstrated by a shift of the curve to the right) in FRET experiments, following the addition of rhuSPARC to S-/- fibroblasts.



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  		FIGURE 5.
Fn-induced ILK activation and phosphorylation of MLCP are inhibited in S-/- fibroblasts, and MLCP phosphorylation is dependent on Fn-induced ILK activation. a, in vitro kinase assays indicate that S-/- fibroblasts exhibited no increase in the activation state of ILK above control after stimulation by plating on an Fn substrate, in comparison with WT cells that showed a normal response. Lane 1, anti-HA tag control (WT cells at 60 min); lanes 2-6, S-/- cells at 15, 30, 60, 120, and 240 min, respectively; lanes 7-11, WT cells at 15, 30, 60, 120, and 240 min, respectively (p-MBP, phosphorylated myelin basic protein). b, Fn-integrin engagement resulted in no increase in MLCP phosphorylation in S-/- fibroblasts, whereas WT fibroblasts showed a typical response (lanes 1-4, S-/- cells at 30, 60, 120, and 240 min, respectively; lanes 5-8, WT cells at 30, 60, 120, and 240 min, respectively). c, blocking ILK activity in WT fibroblasts with the inhibitor KP-074728 (KP07) results in the inhibition of p-MLCP (lane 1, WT control at 240 min; lane 2, WT fibroblasts + KP-074728 at 240 min), whereas inhibiting Rho-associated kinase activity with inhibitor Y27632 (Y27; lane 3, WT at 240 min; lane 4, WT fibroblasts + Y27632 at 240 min) resulted in a nonsignificant decrease in the levels of p-MLCP (bar graph on right; black bars, buffer controls; white bars, inhibitor-treated; *, p < 0.05). d, transfection of S-/- fibroblasts with a vector driving expression of a constitutively active ILK (hyper-ILK) rescues Fn-induced MLCP phosphorylation in S-/- fibroblasts (lane 1, S-/- cells on Fn at 240 min; lane 2, S-/- cells transfected with mock vector on Fn at 240 min; lane 3, S-/- cells transfected with hyper-ILK vector on Fn at 240 min; graph shown on right, ***, p < 0.001, n = 4). Molecular mass markers are denoted in kDa on the left of each blot.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our interest in understanding the role of SPARC in the organization of ECM during development and in response to injury led us to ask whether SPARC influenced Fn matrix assembly, since Fn plays a pivotal role in the transition from provisional ECM to mature ECM (17, 18). In this report, we demonstrate that fibroblasts lacking SPARC are substantially reduced in their capacity to assemble Fn into fibrils and identify a mechanism responsible for this diminished activity: inhibition of cell contraction. We identify ILK as a novel SPARC-binding partner by phage display, verify the binding by solid phase binding assay and coimmunoprecipitation, and demonstrate the SPARC·ILK complex in cells by immunofluorescent staining of nonpermeabilized WT fibroblasts and cell surface protein biotinylation of live WT fibroblasts. Through both pharmacological inhibitors and gene transfer of mutant ILK in both WT and S-/- fibroblasts, we demonstrate the central role of ILK in the observed differences between S-/- and WT fibroblasts. We also show, by gene rescue, that SPARC expression is required for optimal Fn-induced ILK activation and downstream inactivation of MLCP, modifications leading to increased cell contraction (Fig. 8). The altered localization and lack of rescue by exogenous SPARC indicate that the cellular localization of SPARC is regulated and important for ILK activity. However, the inability of exogenous rhuSPARC to activate immunoprecipitated ILK (Fig. 7c, lane 5) suggests that SPARC presented in this manner is not sufficient for productive activation of ILK.

Despite substantive data describing the antiadhesive activity of purified SPARC added to cells in culture (3-5), we describe a new activity for SPARC, enhancement of cell-contractile activity. These new data are consistent with the markedly diminished ECM observed in S-/- mice, specifically with the observations that S-/- mice exhibit more immature and less overall collagen in the dermis (7), reduced foreign body encapsulation (12), a highly permeable lens capsule (8), and enhanced ectopic tumor growth due in part to diminished tumor stroma and capsule formation (13). Furthermore, our data identifying a role for SPARC in the activation of ILK are consistent with, and may explain, a recent report that S-/-/hairless mice are protected against UV-induced papilloma formation (49) and may provide significant insight into a recent finding that implicates SPARC in breast cancer metastasis to lung (50). ILK, an activator of the cell survival signaling molecule Akt, has been shown to be important in the oncogenic transformation conversion stage of skin tumors (51). Our data, indicating that SPARC is required for proper ILK activation, taken together with the report that SPARC enhances Akt activation-dependent cell survival (52), provides evidence for the protective effect exhibited by S-/- mice in skin tumor models. Whether the protection against skin cancer that S-/- mice exhibit extends to other cancer models has not been determined, but the link between SPARC and ILK indicates that SPARC could play an important role in oncogenic transformation.

Although ILK has been characterized as an intracellular focal adhesion-associated protein (25, 29), we demonstrate by immunofluorescence of nonpermeabilized WT fibroblasts and cell surface protein biotinylation of live cells that SPARC and ILK are a part of a membrane-associated complex that is accessible to extracellular reagents. Although these results suggest a cell surface location for this complex, the data are not conclusive on the exact localization with respect to the membrane. Recent papers describing conformational changes within the plasma membrane immediately adjacent to integrins undergoing clustering and activation (53-55) indicate that the reactivity of the SPARC-ILK complex with membrane-impermeable reagents (i.e. IgGs and N-hydroxy-succinimide-biotin) could be interpreted as resulting from local perturbations in the plasma membrane adjacent to the integrin cluster. Receptors have been shown to exhibit significant localized motion (both scissoring and piston-like) within the membrane during ligand binding (56, 57), resulting in perturbations that could facilitate the exposure of intracellular proteins on the outer cell surface (and vice versa). Additionally, certain phospholipids retain the capacity to flip from the inner to the outer leaflet of the plasma membrane during cell activation (58). Whether such events result in alterations in the local membrane permeability or whether such perturbations would be sufficient to facilitate the exposure of cytoplasmic proteins to the extracellular space is unknown. Little is known about the molecular dynamics in and around integrin adhesion clusters and whether these clusters could facilitate the partial exposure of intracellular proteins to the extracellular space. Additional experimentation is therefore required to elucidate the details of this new interaction.



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  		FIGURE 6.
Expression of SPARC in S-/- Fibroblasts rescues Fn-induced ILK activation, MLCP phosphorylation, and cell-mediated Fn unfolding. a, mouse SPARC expression was restored in S-/- fibroblasts by Ad delivery of mouse SPARC cDNA (infection efficiency 60-80% by immunofluorescence staining of SPARC). White arrows indicate Ad.SP-infected S-/- cells demonstrating a perinuclear/cytoplasmic localization of SPARC similar to that seen in WT fibroblasts (b). Scale bar, 50 µm (a) and 10 µm (b). c, Ad delivery of mouse SPARC cDNA to S-/- fibroblasts restored Fn-induced ILK activation. Lane 1, S-/- fibroblasts at 60 min; lane 2, Ad.RLuc-infected S-/- fibroblasts at 60 min; lane 3, Ad.SP-infected S-/- fibroblasts at 60 min. Graph shown on right (d; Ad.SP versus S-/- and Ad.SP versus Ad.RLuc; **, p < 0.01; black, S-/- cells; white, Ad.RLuc-infected S-/- cells; gray, Ad.SP-infected S-/- cells). e, phospho-MLCP immunoblots of Fn-stimulated fibroblasts show that Ad delivery of mouse SPARC rescued ILK-dependent Fn-induced MLCP phosphorylation. Lanes 1-4, Ad.RLuc-infected S-/- fibroblasts at 30, 60, 120, and 240 min, respectively; lanes 5-8, Ad.SP-infected S-/- fibroblasts at 30, 60, 120, and 240 min, respectively, and lanes 9-12, Ad.SP-infected S-/- fibroblasts + KP-074728 at 30, 60, 120, and 240 min, respectively. f, graph shown on right (black, Ad.RLuc-infected S-/- cells; white, Ad.SP-infected S-/- cells; gray, KP-074728-treated Ad.SP-infected S-/- cells; *, p > 0.05; ***, p > 0.001). Molecular mass markers are denoted in kDa on the left of c and e. g, Fn molecular unfolding as determined by intramolecular Fn FRET using Fn labeled with 11.0 donor fluores and 5.2 acceptor fluores (black, WT cells; gray, S-/- cells; red, Ad.SP-infected S-/- cells; blue, Ad.RLuc-infected S-/-cells; 2 and 4 M GdnHCl values are denoted by vertical dotted lines on the graph). Differences in Fn FRET are significant between 1) WT cells and S-/- cells, 2) WT cells and Ad.RLuc-infected S-/- cells, 3) S-/- cells and Ad.SP-infected S/- cells, and 4) Ad.SP-infected S-/- cells and Ad.RLuc-infected S-/- cells (p < 0.00005; n = 150, each data set).

 
Recently, another secreted cysteine-rich matricellular protein, Cyr61, has been linked to ILK. Although coprecipitation of Cyr61 with ILK was not reported, its overexpression resulted in increased activation of ILK in U343 glioma cells (59). Whether the coincident localization of SPARC and ILK is a consequence of integrin clustering or activation and/or a requirement for the activation of ILK is under study. However, we have shown that SPARC binds ILK and that its expression appears to be a requirement for either the activation of ILK by Fn or ILK kinase activity following activation by Fn. Furthermore, this new function of SPARC appears not to be restricted to fibroblasts, since we have similar findings in lens epithelial cells (64).

Ad-mediated gene transfer of mouse SPARC cDNA rescued S-/- cells. It is, however, significant that exogenous rhuSPARC was unable to restore the WT phenotype, especially given that SPARC is a secreted protein. Although purified rhuSPARC exhibits activities similar to those of mouse SPARC purified from mouse parietal yolk sac carcinoma cells (e.g. counteradhesive and antiproliferative) (5, 40, 41), this comparison does not extend to the newly identified activity of cell contraction. That subconfluent WT cells secrete SPARC but remain spread, despite the reported decrease in focal adhesions at concentrations of SPARC as low as 30 nM (3) (below the concentrations normally seen in culture or at sites of tissue remodeling or injury (60)), is an intriguing observation. Our new data may begin to explain why such a phenomenon occurs. Valid technical explanations include improper protein glycosylation, posttranslational modifications, and folding for the different activities observed between ectopic (and endogenous) SPARC expression and the addition of a purified rhuSPARC. However, we believe that the cellular location of SPARC, an understudied topic, may be a more definitive factor in explaining these differences. For example, if SPARC acts as a chaperone for certain ECM proteins, its localization would become a significant determinant in predicting its activity (65). Both the matricellular protein thrombospondin-2 and the proteoglycan decorin have also been shown to exhibit different characteristics, depending on their contextual presentation to cells (61, 62).



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  		FIGURE 7.
rhuSPARC added to S-/- fibroblasts localizes to endosomes within the cell and does not restore Fn-induced ILK activation or cell-mediated Fn unfolding. Exogenous AF-488-labeled rhuSPARC (a), added to the culture media of S-/- fibroblasts, displays an altered cellular localization, in comparison with than that of SPARC produced by WT fibroblasts (b). Magnification bars, 10 µm. c, in vitro kinase assays show that the addition of rhuSPARC (20 µg/ml) to the culture media of S-/- fibroblasts or to the post-lysate ILK immunocomplexes of S-/- fibroblasts did not restore Fn-induced ILK activation. Lane 1, WT cells stimulated 60 min (anti-HA tag control); lane 2, WT cells stimulated 60 min; lane 3, S-/- cells stimulated 60 min; lane 4, S-/- cells stimulated 60 min with 20 µg/ml rhuSPARC added during stimulation; lane 5, S-/- cells stimulated 60 min with 20 µg/ml rhuSPARC added during immunoprecipitation. Lanes 2-5 show immunoprecipitates with anti-ILK IgG. Reaction mixtures were resolved by SDS-PAGE and were immunoblotted for pMBP. Hatch marks indicate where the scan of the immunoblot was spliced. A molecular mass marker is denoted in kDa on the left. d, rhuSPARC (20 µg/ml) added to S-/- fibroblasts induces a less adherent state (the graph represents the relative average resistance (with respect to WT fibroblasts ± S.D.). e, the addition of rhuSPARC (20 µg/ml) did not result in a restoration of the capacity of S-/- fibroblasts to unfold Fn molecules (as determined by intramolecular Fn FRET; solid line, S-/- fibroblasts; dotted line, S-/- fibroblasts with 20 µg/ml rhuSPARC; 2 and 4 M GdnHCl values are denoted by vertical dotted lines on the graph).

 



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  		FIGURE 8.
SPARC-mediated cellular adhesion and contraction: A model. ILK is activated following {beta}1 integrin engagement by Fn and stimulates cell contractility via phosphorylation of MLCP (inhibitory) and MLC (stimulatory). SPARC (yellow) binds to ILK and is necessary for ILK-mediated cell-contractile signaling leading to partial unfolding of Fn during Fn-matrix assembly. Although data shown in this study are suggestive that the ILK·SPARC complex is exposed extracellularly, they do not identify the exact location, and further investigation is required before defining a cellular localization of this complex. Akt, a target of ILK (Wu and Dedhar (25)), has recently been shown to be activated by SPARC (Shi et al. (52)) and, with our data, provides additional evidence for the interaction between SPARC and ILK. {alpha} and {beta}, {alpha}5 and {beta}1 integrins; green balls, phosphorylation.

 
In this report, we have identified a novel activity for SPARC, specifically the promotion of cell contraction. This activity appears central to the capacity of normal cells to assemble Fn matrix efficiently.5 Furthermore, we have uncovered an intracellular signaling cascade engaged by SPARC that partially explains this function. The data provide evidence for a new paradigm regarding SPARC, its localization, and activity and offer an explanation for the function of SPARC in the development of ECM and regulation of tissue architecture.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants GM40711 and EY13180 (to E. H. S.) and CA 086997 (to S. A. R.) and National Science Foundation Grant EEC-9529161 (to E. H. S. and V. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Hope Heart Program, Benaroya Research Institute at Virginia Mason, 1201 9th Ave. Seattle, WA 98101. Tel.: 206-341-1314; Fax: 206-341-1375; E-mail: hsage{at}benaroyaresearch.org.

2 The abbreviations used are: ECM, extracellular matrix; Ad, adenovirus; Ad.SP, adenovirus containing mouse SPARC cDNA; Ad.RLuc, adenovirus containing Renilla luciferase cDNA; AF, AlexaFluor©; Fn, fibronectin; Fn-D/A, doubly labeled fibronectin; GdnHCl, guanidine hydrochloride; HA, hemagglutinin; IA, intensity of acceptor fluore; ID, intensity of donor fluore; ILK, integrin-linked kinase; MBP, myelin basic protein; MLC, myosin light chain; MLCP, myosin light chain phosphatase; S-/-, SPARC-null; WT, wild-type; TRITC, tetramethylrhodamine isothiocyanate; FRET, fluorescence resonance energy transfer; FBS, fetal bovine serum; PBS, phosphate-buffered saline; rhuSPARC, recombinant human SPARC; rhuILK, recombinant human ILK; BSA, bovine serum albumin. Back

3 H. Xenias, R. Faessler, and M. Sheetz, personal communication. Back

5 M. Antia, G. Baneyx, G. Young, and V. Vogel, submitted for publication. Back


   ACKNOWLEDGMENTS
 
We thank Virginia Gray for assistance with ILK reagents and protocols, Drs. Irene Newsham and Oliver Bögler for technical advice and valuable comments, Christopher K. Yunker for assistance with electric cell-substrate impedance sensing controls, Sarah E. Funk for animal husbandry, Dr. Qi Yan for valuable discussion, and Dr. T. Segura, E. Neligan, and S. E. Funk for assistance with the manuscript and graphics.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bornstein, P., and Sage, E. H. (2002) Curr. Opin. Cell Biol. 14, 608-616[CrossRef][Medline] [Order article via Infotrieve]
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