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SPARC Regulates Extracellular Matrix Organization through Its Modulation of Integrin-linked Kinase Activity

2005; Elsevier BV; Volume: 280; Issue: 43 Linguagem: Inglês

10.1074/jbc.m504663200

ISSN

1083-351X

Autores

Thomas H. Barker, Gretchen Baneyx, Marina Cardó‐Vila, Gail Workman, Matt Weaver, Priya M. Menon, Shoukat Dedhar, Sandra A. Rempel, Wadih Arap, Renata Pasqualini, Viola Vogel, E. Helene Sage,

Tópico(s)

Connective tissue disorders research

Resumo

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. 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. Matricellular proteins such as SPARC function as modulators of cell-extracellular matrix (ECM) 2The abbreviations used are:ECMextracellular matrixAdadenovirusAd.SPadenovirus containing mouse SPARC cDNAAd.RLucadenovirus containing Renilla luciferase cDNAAFAlexaFluor©FnfibronectinFn-D/Adoubly labeled fibronectinGdnHClguanidine hydrochlorideHAhemagglutininIAintensity of acceptor fluoreIDintensity of donor fluoreILKintegrin-linked kinaseMBPmyelin basic proteinMLCmyosin light chainMLCPmyosin light chain phosphataseS-/-SPARC-nullWTwild-typeTRITCtetramethylrhodamine isothiocyanateFRETfluorescence resonance energy transferFBSfetal bovine serumPBSphosphate-buffered salinerhuSPARCrecombinant human SPARCrhuILKrecombinant human ILKBSAbovine serum albumin 2The abbreviations used are:ECMextracellular matrixAdadenovirusAd.SPadenovirus containing mouse SPARC cDNAAd.RLucadenovirus containing Renilla luciferase cDNAAFAlexaFluor©FnfibronectinFn-D/Adoubly labeled fibronectinGdnHClguanidine hydrochlorideHAhemagglutininIAintensity of acceptor fluoreIDintensity of donor fluoreILKintegrin-linked kinaseMBPmyelin basic proteinMLCmyosin light chainMLCPmyosin light chain phosphataseS-/-SPARC-nullWTwild-typeTRITCtetramethylrhodamine isothiocyanateFRETfluorescence resonance energy transferFBSfetal bovine serumPBSphosphate-buffered salinerhuSPARCrecombinant human SPARCrhuILKrecombinant human ILKBSAbovine serum albumin interactions (1Bornstein P. Sage E.H. Curr. Opin. Cell Biol. 2002; 14: 608-616Crossref PubMed Scopus (765) Google Scholar, 2Brekken R.A. Sage E.H. Matrix Biol. 2001; 19: 569-580Crossref Scopus (230) Google Scholar). 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 (3Murphy-Ullrich J.E. Lane T.F. Pallero M.A. Sage E.H. J. Cell. Biochem. 1995; 57: 341-350Crossref PubMed Scopus (146) Google Scholar, 4Sage E.H. Biochem. Cell Biol. 1992; 70: 56-62Crossref PubMed Scopus (14) Google Scholar, 5Sage H. Vernon R.B. Funk S.E. Everitt E.A. Angello J. J. Cell Biol. 1989; 109: 341-356Crossref PubMed Scopus (314) Google Scholar). The induction of an intermediate state of cell adhesion by SPARC (6Murphy-Ullrich J.E. J. Clin. Invest. 2001; 107: 785-790Crossref PubMed Scopus (401) Google Scholar) 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 (7Bradshaw A.D. Puolakkainen P. Dasgupta J. Davidson J.M. Wight T.N. Sage E.H. J. Invest. Dermatol. 2003; 120: 949-955Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 8Yan Q. Clark J.I. Wight T.N. Sage E.H. J. Cell Sci. 2002; 115: 2747-2756Crossref PubMed Google Scholar, 9Bradshaw A.D. Graves D.C. Motamed K. Sage E.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6045-6050Crossref PubMed Scopus (180) Google Scholar) and show accelerated closure of dermal wounds (10Bradshaw A.D. Reed M.J. Carbon J.G. Pinney E. Brekken R.A. Sage E.H. Wound Repair Regen. 2001; 9: 522-530Crossref PubMed Scopus (55) Google Scholar, 11Bradshaw A.D. Reed M.J. Sage E.H. J. Histochem. Cytochem. 2002; 50: 1-10Crossref PubMed Scopus (146) Google Scholar), diminished foreign body response (12Puolakkainen P. Bradshaw A.D. Kyriakides T.R. Reed M. Brekken R. Wight T. Bornstein P. Ratner B. Sage E.H. Am. J. Pathol. 2003; 162: 627-635Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), and enhanced tumor growth (13Brekken R.A. Puolakkainen P. Graves D.C. Workman G. Lubkin S.R. Sage E.H. J. Clin. Invest. 2003; 111: 487-495Crossref PubMed Scopus (172) Google Scholar). 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 (7Bradshaw A.D. Puolakkainen P. Dasgupta J. Davidson J.M. Wight T.N. Sage E.H. J. Invest. Dermatol. 2003; 120: 949-955Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). However, the mechanism by which SPARC directs ECM assembly has not been identified. extracellular matrix adenovirus adenovirus containing mouse SPARC cDNA adenovirus containing Renilla luciferase cDNA AlexaFluor© fibronectin doubly labeled fibronectin guanidine hydrochloride hemagglutinin intensity of acceptor fluore intensity of donor fluore integrin-linked kinase myelin basic protein myosin light chain myosin light chain phosphatase SPARC-null wild-type tetramethylrhodamine isothiocyanate fluorescence resonance energy transfer fetal bovine serum phosphate-buffered saline recombinant human SPARC recombinant human ILK bovine serum albumin extracellular matrix adenovirus adenovirus containing mouse SPARC cDNA adenovirus containing Renilla luciferase cDNA AlexaFluor© fibronectin doubly labeled fibronectin guanidine hydrochloride hemagglutinin intensity of acceptor fluore intensity of donor fluore integrin-linked kinase myelin basic protein myosin light chain myosin light chain phosphatase SPARC-null wild-type tetramethylrhodamine isothiocyanate fluorescence resonance energy transfer fetal bovine serum phosphate-buffered saline recombinant human SPARC recombinant human ILK bovine serum albumin 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 (14George E.L. Georges-Labouesse E.N. Patel-King R.S. Rayburn H. Hynes R.O. Development. 1993; 119: 1079-1091Crossref PubMed Google Scholar). In response to challenge, Fn serves as an intermediate or provisional matrix (15Magnusson M.K. Mosher D.F. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1363-1370Crossref PubMed Scopus (254) Google Scholar, 16Robinson E.E. Foty R.A. Corbett S.A. Mol. Biol. Cell. 2004; 15: 973-981Crossref PubMed Scopus (106) Google Scholar) 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 (17Sottile J. Hocking D.C. Mol. Biol. Cell. 2002; 13: 3546-3559Crossref PubMed Scopus (444) Google Scholar, 18Velling T. Risteli J. Wennerberg K. Mosher D.F. Johansson S. J. Biol. Chem. 2002; 277: 37377-37381Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Fn matrix assembly by cells requires integrin (α5β1) binding to Fn molecules and active extension of the Fn molecule through the actinomyosin contractile machinery (19Schwarzbauer J.E. Sechler J.L. Curr. Opin. Cell Biol. 1999; 11: 622-627Crossref PubMed Scopus (247) Google Scholar). Partial unfolding of Fn is thought to expose binding sites for other Fn molecules and thus promotes the formation of Fn fibers (20Baneyx G. Baugh L. Vogel V. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14464-144648Crossref PubMed Scopus (161) Google Scholar, 21Baneyx G. Baugh L. Vogel V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5139-5143Crossref PubMed Scopus (295) Google Scholar, 22Gao M. Craig D. Lequin O. Vogel V. Schulten K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14784-14789Crossref PubMed Scopus (162) Google Scholar, 23Sechler J.L. Corbett S.A. Schwarzbauer J.E. Mol. Biol. Cell. 1997; 8: 2563-2573Crossref PubMed Scopus (125) Google Scholar, 24Sechler J.L. Rao H. Cumiskey A.M. Vega-Colon I. Smith M.S. Murata T. Schwarzbauer J.E. J. Cell Biol. 2001; 154: 1081-1088Crossref PubMed Scopus (97) Google Scholar). 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 β1 integrin immediately adjacent to the plasma membrane and is activated by β1 integrins and growth factors, has been shown to control the intracellular signaling cascades that influence cellular contractile elements (25Wu C. Dedhar S. J. Cell Biol. 2001; 155: 505-510Crossref PubMed Scopus (350) Google Scholar). ILK interacts directly with actin and α-actinin-binding proteins such as the parvins, affixin and paxillin (26Zhang Y. Chen K. Tu Y. Wu C. J. Biol. Chem. 2004; 279: 41695-41705Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 27Yamaji S. Suzuki A. Sugiyama Y. Koide Y. Yoshida M. Kanamori H. Mohri H. Ohno S. Ishigatsubo Y. J. Cell Biol. 2001; 153: 1251-1264Crossref PubMed Scopus (169) Google Scholar, 28Nikolopoulos S.N. Turner C.E. J. Biol. Chem. 2001; 276: 23499-23505Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), and localizes to focal adhesion complexes (29Hannigan G. Troussard A.A. Dedhar S. Nat. Rev. Cancer. 2005; 5: 51-63Crossref PubMed Scopus (507) Google Scholar). ILK phosphorylates, and thereby inactivates, myosin light chain phosphatase (MLCP) (30Deng J.T. Sutherland C. Brautigan D.L. Eto M. Walsh M.P. Biochem. J. 2002; 367: 517-524Crossref PubMed Scopus (116) Google Scholar, 31Kiss E. Muranyi A. Csortos C. Gergely P. Ito M. Hartshorne D.J. Erdodi F. Biochem. J. 2002; 365: 79-87Crossref PubMed Scopus (70) Google Scholar, 32Muranyi A. MacDonald J.A. Deng J.T. Wilson D.P. Haystead T.A. Walsh M.P. Erdodi F. Kiss E. Wu Y. Hartshorne D.J. Biochem. J. 2002; 366: 211-216Crossref PubMed Google Scholar). 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 (33Deng J.T. Van Lierop J.E. Sutherland C. Walsh M.P. J. Biol. Chem. 2001; 276: 16365-16373Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar), an activity that leads to the induction of a contractile state. Thus, by both inhibiting MLCP activity and enhancing MLC activity, ILK activation by β1 integrin leads to the induction of a contractile cell phenotype. Perhaps not surprisingly, ILK has also been shown to modulate Fn matrix assembly (34Wu C. Keightley S.Y. Leung-Hagesteijn C. Radeva G. Coppolino M. Goicoechea S. McDonald J.A. Dedhar S. J. Biol. Chem. 1998; 273: 528-536Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). 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 β1 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. Isolation of Primary Fibroblasts—Fibroblasts from pulmonary tissue of WT and S-/- (C57Bl6/J) mice were isolated and cultured as previously described (35Hagood J.S. Miller P.J. Lasky J.A. Tousson A. Guo B. Fuller G.M. McIntosh J.C. Am. J. Physiol. 1999; 277: L218-L224PubMed Google Scholar). 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 (20Baneyx G. Baugh L. Vogel V. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14464-144648Crossref PubMed Scopus (161) Google Scholar). 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; ×100 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 × 105 cells were stained for integrin α5 and integrin β1 by incubation with fluorescein isothiocyanate-conjugated hamster anti-α5 and hamster anti-β1 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 × 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 × 1010 transducing units. rhuSPARC protein was coated onto microtiter wells as previously described (36Koivunen E. Gay D.A. Ruoslahti E. J. Biol. Chem. 1993; 268: 20205-20210Abstract Full Text PDF PubMed Google Scholar, 37Smith G.P. Scott J.K. Methods Enzymol. 1993; 217: 228-257Crossref PubMed Scopus (697) Google Scholar). Phage binding assays on purified proteins were carried out as described previously (38Giordano R.J. Cardo-Vila M. Lahdenranta J. Pasqualini R. Arap W. Nat. Med. 2001; 7: 1249-1253Crossref PubMed Scopus (236) Google Scholar). SPARC, hevin (39Brekken R.A. Sullivan M.M. Workman G. Bradshaw A.D. Carbon J. Siadek A. Murri C. Framson P.E. Sage E.H. J. Histochem. Cytochem. 2004; 52: 735-748Crossref PubMed Scopus (51) Google Scholar), bovine collagen I (Vitrogen™; 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 × 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 (40Bradshaw A.D. Bassuk J.A. Francki A. Sage E.H. Mol. Cell. Biol. Res. Commun. 2000; 3: 345-351Crossref PubMed Scopus (32) Google Scholar, 41Sage E.H. Yamada K. Current Protocols in Cell Biology. John Wiley & Sons, Inc., Hillsborough, NJ2004: 10.11.1-10.11.23Google Scholar). 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-β-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) (42Sweetwyne M.T. Brekken R.A. Workman G. Bradshaw A.D. Carbon J. Siadak A.W. Murri C. Sage E.H. J. Histochem. Cytochem. 2004; 52: 723-733Crossref PubMed Scopus (38) Google Scholar) 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 Biotechnology, Inc., Lake Placid, NY). Integrin β1 was detected with fluorescein isothiocyanate-conjugated hamster anti-β1 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 × 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 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 Biotechnology) for 60 min prior to Fn stimulation or transfection of inactive ILK or hyper-ILK expression vectors (43Persad S. Attwell S. Gray V. Mawji N. Deng J.T. Leung D. Yan J. Sanghera J. Walsh M.P. Dedhar S. J. Biol. Chem. 2001; 271: 27462-27469Abstract Full Text Full Text PDF Scopus (416) Google Scholar) by Lipofectamine 2000 (Invitrogen) 48 h prior to Fn stimulation. At appropriate intervals, the cells were lysed directly in 2× SDS-PAGE sample buffer containing 2% β-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 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 × 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 (44Delcommenne M. Tan C. Gray V. Rue L. Woodgett J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11211-11216Crossref PubMed Scopus (945) Google Scholar). 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 × 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 2× SDS-PAGE sample buffer containing β-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 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. 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 (6Murphy-Ullrich J.E. J. Clin. Invest. 2001; 107: 785-790Crossref PubMed Scopus (401) Google Scholar), 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

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