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Regulated Shedding of Syndecan-1 and -4 Ectodomains by Thrombin and Growth Factor Receptor Activation

1997; Elsevier BV; Volume: 272; Issue: 23 Linguagem: Inglês

10.1074/jbc.272.23.14713

1083-351X

Sukanya Subramanian, Marilyn L. Fitzgerald, Merton Bernfield,

Blood Coagulation and Thrombosis Mechanisms

The syndecan family of transmembrane heparan sulfate proteoglycans is abundant on the surface of all adherent mammalian cells. Syndecans bind and modify the action of various growth factors/cytokines, proteases/antiproteases, cell adhesion molecules, and extracellular matrix components. Syndecan expression is highly regulated during wound repair, a process orchestrated by many of these effectors. Each syndecan ectodomain is shed constitutively by cultured cells, but the mechanism and significance of this shedding are not understood. Therefore, we examined (i) whether physiological agents active during wound repair influence syndecan shedding, and (ii) whether wound fluids contain shed syndecan ectodomains.Using SVEC4–10 endothelial cells we find that certain proteases and growth factors accelerate shedding of the syndecan-1 and -4 ectodomains. Protease-accelerated shedding is completely inhibited by serum-containing media. Thrombin activity is duplicated by the 14-amino acid thrombin receptor agonist peptide that directly activates the thrombin receptor and is not inhibited by serum. Epidermal growth factor family members accelerate shedding but FGF-2, platelet-derived growth factor-AB, transforming growth factor-β, tumor necrosis factor-α, and vascular endothelial cell growth factor 165 do not. Shed ectodomains are soluble, stable in the conditioned medium, have the same size core proteins regardless whether shed at a basal rate, or accelerated by thrombin or epidermal growth factor-family members and are found in acute human dermal wound fluids. Thus, shedding is accelerated by activation of at least two distinct receptor classes, G protein-coupled (thrombin) and protein tyrosine kinase (epidermal growth factor). Proteases and growth factors active during wound repair can accelerate syndecan shedding from cell surfaces. Regulated shedding of syndecans suggests physiological roles for the soluble proteoglycan ectodomains. The syndecan family of transmembrane heparan sulfate proteoglycans is abundant on the surface of all adherent mammalian cells. Syndecans bind and modify the action of various growth factors/cytokines, proteases/antiproteases, cell adhesion molecules, and extracellular matrix components. Syndecan expression is highly regulated during wound repair, a process orchestrated by many of these effectors. Each syndecan ectodomain is shed constitutively by cultured cells, but the mechanism and significance of this shedding are not understood. Therefore, we examined (i) whether physiological agents active during wound repair influence syndecan shedding, and (ii) whether wound fluids contain shed syndecan ectodomains. Using SVEC4–10 endothelial cells we find that certain proteases and growth factors accelerate shedding of the syndecan-1 and -4 ectodomains. Protease-accelerated shedding is completely inhibited by serum-containing media. Thrombin activity is duplicated by the 14-amino acid thrombin receptor agonist peptide that directly activates the thrombin receptor and is not inhibited by serum. Epidermal growth factor family members accelerate shedding but FGF-2, platelet-derived growth factor-AB, transforming growth factor-β, tumor necrosis factor-α, and vascular endothelial cell growth factor 165 do not. Shed ectodomains are soluble, stable in the conditioned medium, have the same size core proteins regardless whether shed at a basal rate, or accelerated by thrombin or epidermal growth factor-family members and are found in acute human dermal wound fluids. Thus, shedding is accelerated by activation of at least two distinct receptor classes, G protein-coupled (thrombin) and protein tyrosine kinase (epidermal growth factor). Proteases and growth factors active during wound repair can accelerate syndecan shedding from cell surfaces. Regulated shedding of syndecans suggests physiological roles for the soluble proteoglycan ectodomains. The response to tissue injury is orchestrated by multiple soluble effectors. These are derived from the blood plasma, immigrant cells from the circulation and resident cells at the wound site, and include proteases, antiproteases, growth factors, cytokines, and chemokines (1Bourin M.C. Lindahl U. Biochem. J. 1993; 289: 313-330Google Scholar). Heparin modifies the action of several of these effector molecules such as thrombin, antithrombin III, heparin-binding epidermal growth factor-like growth factor (HB-EGF), 1The abbreviations used are: HB-EGF, heparin-binding epidermal growth factor-like growth factor; EGF, epidermal growth factor; FGF-2, fibroblast growth factor-2; TGF-α, transforming growth factor-α; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α; FCS, fetal calf serum; GAG, glycosaminoglycan; PMA, phorbol 12-myristate 13-acetate; TRAP, thrombin receptor agonist peptide; TPCK,l-1-tosylamido-2-phenylethyl chloromethyl ketone; CM, conditioned medium; HSE-1, serum antibody against human syndecan-1 ectodomain; AU, absorbance unit(s); PAGE, polyacrylamide gel electrophoresis; mAbs, monoclonal antibody. vascular endothelial cell growth factor, basic fibroblast growth factor, and interleukin-8 (2Bernfield M. Hinkes M.T. Biochem. J. 1993; Google Scholar, 3Bourin M.C. Lindahl U. Biochem. J. 1993; 289: 313-330Google Scholar, 4Gallo R.L. Bernfield M. Clark R.A. Molecular and Cellular Biology of Wound Repair. Plenum, New York1995: 475-492Google Scholar). These interactions focus attention on the potential regulatory role of the heparan sulfate that is at the surface of all adherent cells. Much of the heparan sulfate at the cell surface is derived from the syndecan family of transmembrane proteoglycans. These four gene products consist of single polypeptides which comprise at their COOH termini a short cytoplasmic domain (28–34 amino acids) containing three invariant tyrosines, and at their NH2 termini an extracellular domain (ectodomain) that places heparan sulfate chains distal from the plasma membrane. The syndecans bind a variety of growth factors, cytokines, proteases, antiproteases, and cell adhesion molecules (5Jalkanen M. Jalkanen S. Bernfield M. Mecham R.P. McDonald J.A. Biology of Extracellular Matrix. Receptors for Extracellular Matrix Proteins. Academic Press, Inc., Orlando, FL1991: 1-37Google Scholar, 6Bernfield M. Kokenyesi R. Kato M. Hinkes M.T. Spring J. Gallo R.L. Lose E.J. Annu. Rev. Cell Biol. 1992; 8: 365-398Google Scholar), are individually expressed in distinct cell-, tissue-, and development-specific patterns (7Kim C.W. Goldberger O.A. Gallo R.L. Bernfield M. Mol. Biol. Cell. 1994; 5: 797-805Google Scholar), and show cell-specific variations in the structure of their heparan sulfate chains (8Kato M. Wang H. Bernfield M. Gallagher J.T. Turnbull J.E. J. Biol. Chem. 1994; 269: 18881-18890Google Scholar, 9Sanderson R.D. Turnbull J.E. Gallagher J.T. Lander A.D. J. Biol. Chem. 1994; 269: 13100-13106Google Scholar). Syndecan expression is highly regulated during development, neoplasia, and wound repair (2Bernfield M. Hinkes M.T. Biochem. J. 1993; Google Scholar, 4Gallo R.L. Bernfield M. Clark R.A. Molecular and Cellular Biology of Wound Repair. Plenum, New York1995: 475-492Google Scholar, 6Bernfield M. Kokenyesi R. Kato M. Hinkes M.T. Spring J. Gallo R.L. Lose E.J. Annu. Rev. Cell Biol. 1992; 8: 365-398Google Scholar, 10Inki P. Jalkanen M. Ann. Med. 1996; 28: 63-67Google Scholar). Following skin wounds in mice, the keratinocytes at the leading edge migrating into the wound show a loss of cell surface syndecan-1. Concomitantly, syndecan-1 expression increases on the endothelial cells and syndecan-4 expression increases on the dermal fibroblasts that comprise the forming granulation tissue (11Elenius K. Vainio S. Laato M. Salmivirta M. Thesleff I. Jalkanen M. J. Cell Biol. 1991; 114: 585-595Google Scholar, 12Gallo R. Kim C. Kokenyesi R. Adzick N.S. Bernfield M. J. Invest. Dermatol. 1996; 107: 676-683Google Scholar). Cell surface syndecan-1 and -4 and their transcripts are induced in cultured endothelia and fibroblasts by PR-39, a pig neutrophil-derived antimicrobial peptide, and analogous inductive activity is found in human wound fluid (13Gallo R.L. Ono M. Povsic T. Page C. Eriksson E. Klagsbrun M. Bernfield M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11035-11039Google Scholar). While syndecan expression can be regulated at or following transcription, depending on the cell type or pathophysiological situation, the precise mechanism(s) underlying these changes is unknown (11Elenius K. Vainio S. Laato M. Salmivirta M. Thesleff I. Jalkanen M. J. Cell Biol. 1991; 114: 585-595Google Scholar, 14Sanderson R.D. Bernfield M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9562-9566Google Scholar, 15Vainio S. Jalkanen M. Vaahtokari A. Sahlberg C. Mali M. Bernfield M. Thesleff I. Dev. Biol. 1991; 147: 322-333Google Scholar, 16Sanderson R.D. Hinkes M.T. Bernfield M. J. Invest. Dermatol. 1992; 99: 1-7Google Scholar, 17Yeaman C. Rapraeger A. J. Cell Biol. 1993; 122: 941-950Google Scholar). A diverse group of transmembrane proteins is regulated by proteolytic cleavage of their ectodomains which are then released into the surrounding milieu (18Ehlers M.R. Riordan J.F. Biochemistry. 1991; 30: 10065-10074Google Scholar, 19Massagué J. Pandiella A. Annu. Rev. Biochem. 1993; 62: 515-541Google Scholar, 20Rose-John S. Heinrich P.C. Biochem. J. 1994; 300: 281-290Google Scholar). These cell surface proteins often have soluble counterparts in vivo, and can be detected in various body fluids (18Ehlers M.R. Riordan J.F. Biochemistry. 1991; 30: 10065-10074Google Scholar, 21Marikovsky M. Brueing K. Liu P.Y. Eriksson E. Higashiyama S. Farber P. Abraham J. Klagsbrun M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3889-3893Google Scholar). This shedding can result in solubilization of the functional domains of the cell surface proteins. Recently, this shedding has been shown to be a highly regulated process and a common system has been proposed for regulating the shedding of several transmembrane proteins released by the action of calcium ionophores or protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA) (22Pandiella A. Massagué J. J. Biol. Chem. 1991; 266: 5769-5773Google Scholar, 23Arribas J. Massagué J. J. Cell Biol. 1995; 128: 433-441Google Scholar, 24Arribas J. Coodly L. Vollmer P. Kishimoto T.K. Rose-John S. Massagué J. J. Biol. Chem. 1996; 271: 11376-11382Google Scholar). It has been known for many years that syndecan-1 is shed constitutively by cultured cells (25Jalkanen M. Rapraeger A. Saunders S. Bernfield M. J. Cell Biol. 1987; 105: 3087-3096Google Scholar) and that this shedding involves release of the soluble ectodomain (26Saunders S. Jalkanen M. O'Farrell S. Bernfield M. J. Cell Biol. 1989; 108: 1547-1556Google Scholar). Indeed, each of the syndecan family members is shed (7Kim C.W. Goldberger O.A. Gallo R.L. Bernfield M. Mol. Biol. Cell. 1994; 5: 797-805Google Scholar) and the site of cleavage has been suggested to be a dibasic sequence (syndecan-1, -2, -3) or a basic residue (syndecan-4) adjacent to the plasma membrane (6Bernfield M. Kokenyesi R. Kato M. Hinkes M.T. Spring J. Gallo R.L. Lose E.J. Annu. Rev. Cell Biol. 1992; 8: 365-398Google Scholar, 25Jalkanen M. Rapraeger A. Saunders S. Bernfield M. J. Cell Biol. 1987; 105: 3087-3096Google Scholar). We have recently found that features of syndecan shedding are similar to those of the common system proposed to be responsible for shedding of several membrane-anchored growth factors, growth factor receptors, cell adhesion, and other membrane proteins. As with the common system, syndecan-1 and -4 are shed at basal levels, but this shedding is accelerated within minutes of treating cells with PMA (27Saunders S. Jalkanen M. O'Farrell S. Bernfield M. J. Cell Biol. 1989; 108: 1547-1556Google Scholar).Drosophila syndecan lacks basic residues adjacent to the plasma membrane, yet is readily shed from cultured cells (28Spring J. Paine-Saunders S.E. Hynes R.O. Bernfield M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3334-3338Google Scholar). Thus, as with cleavage by the common system, the amino acid sequence may not be of primary importance to the cleavage process. Finally, the apparent loss of cell surface syndecan-1 during wound repair, a process involving various proteases and growth factors, recalled the findings that receptor stimulation can activate the common shedding system (29Roark E.F. Paradies N.E. Lagunowich L.A. Grunwald G.B. Development. 1992; 114: 973-984Google Scholar, 30Cabrera N. Diaz-Rodriguez E. Becker E. Martin-Zanca D. Pandiella A. J. Cell Biol. 1996; 132: 427-436Google Scholar, 31Levine S.L. Logun C. Chopra D.P. Rhim J.S. Shelhamner J.H. Am. J. Respir. Cell Mol. Biol. 1996; 14: 254-261Google Scholar). Therefore, we examined whether (i) agents that are active during acute wound repair influence syndecan shedding and (ii) dermal wound fluids contain shed syndecans. We find that plasmin, thrombin, and epidermal growth factor (EGF) family members accelerate the shedding of the syndecan-1 and -4 ectodomains from cultured endothelial cell surfaces, that activation of at least two distinct receptor classes (G protein-coupled and protein tyrosine kinase) accelerates shedding, and that the syndecan -1 and -4 ectodomains are in acute human dermal wound fluids. Regulated shedding of the syndecans suggests a physiological role for the soluble proteoglycan ectodomains. Recombinant human (rHu) HB-EGF was obtained from Dr. J. Abraham (Scios-Nova, Mountain View, CA); rHu EGF, TGF-α, and FGF-2 from Intergen (Purchase, NY); rHu platelet derived growth factor AB from Upstate Biotechnology (Lake Placid, NY); rHu vascular endothelial cell growth factor 165 from Dr. G. Neufeld (Israel Institute of Technology, Haifa, Israel); rHu TNF-α and porcine platelet TGF-β1 from R & D Systems (Minneapolis, MN). Plasmin (human plasma), thrombin (human plasma), thrombin receptor agonist peptide (TRAP), soybean trypsin inhibitor, and genistein were from Calbiochem (La Jolla, CA). Tyrphostin 25 and methyl 2,5-dihydroxycinnamate were from Toronto Research Chemicals (Ontario, Canada). Urokinase-type plasminogen activator, PMA, and TPCK-treated trypsin were from Sigma. Heparan sulfate lyase (heparatinase, EC 4.2.2.8) and chondroitin sulfate ABC lyase (chondroitinase ABC, EC 4.2.2.4) were from Seikagaku America Inc. (Rockville, MD). Horseradish peroxidase-conjugated goat anti-rat IgG and horseradish peroxidase goat anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA) or Amersham Life Sciences. Antibodies specific to syndecan ectodomains included monoclonal antibodies 281-2 against the mouse syndecan-1 ectodomain (32Jalkanen M. Nguyen H. Rapraeger A. Kurn N. Bernfield M. J. Cell Biol. 1985; 101: 976-984Google Scholar), 5G9 and 8C7 against the syndecan-4 ectodomain (12Gallo R. Kim C. Kokenyesi R. Adzick N.S. Bernfield M. J. Invest. Dermatol. 1996; 107: 676-683Google Scholar), polyclonal antisera MSE-2, MSE-3, and MSE-4, against their respective recombinant mouse syndecan ectodomains (7Kim C.W. Goldberger O.A. Gallo R.L. Bernfield M. Mol. Biol. Cell. 1994; 5: 797-805Google Scholar) and polyclonal antisera HSE-1, against the recombinant human syndecan-1 ectodomain (12Gallo R. Kim C. Kokenyesi R. Adzick N.S. Bernfield M. J. Invest. Dermatol. 1996; 107: 676-683Google Scholar). Serum antibodies prepared against the syndecan-1 and -4 cytoplasmic domains, which are identical in sequence across species, detect both mouse and human syndecan-1 and -4, respectively. Polyclonal antisera S7C, against a 7-amino acid synthetic peptide (KQEEFYA) corresponding to the COOH terminus of syndecan-1, has been described (26Saunders S. Jalkanen M. O'Farrell S. Bernfield M. J. Cell Biol. 1989; 108: 1547-1556Google Scholar). Antibodies specific for the syndecan-4 cytoplasmic domain were prepared against a 13-amino acid peptide (LGKKPIYKKAPTN) unique to the syndecan-4 cytoplasmic domain. The immunogen was synthesized using the multiple antigen peptide system and used for immunization and boosts of rabbits at Quality Controlled Biochemicals Inc. (Hopkinton, MA). The antisera was called SCD-4 for syndecan cytoplasmic domain followed by the number corresponding to the specific syndecan family member. Specificity of SCD-4 was determined by immunoprecipitation of [35S]S04-labeled SVEC4–10 cell lysates prepared in RIPA buffer (RIPA: 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 1% deoxycholate, 0.1% SDS, 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride) with antisera specific for each of the four syndecan ectodomains (281–2, MSE-2, MSE-3, and MSE-4). Each antigen-antibody complex contained radioactivity, indicating that these cells express all four syndecans. The complexes were eluted from protein A beads by boiling in 1:1 (v/v) of 2% SDS in 100 mm Tris-HCl, pH 7.4, the eluate was diluted to 0.1% SDS with RIPA buffer without SDS and equal portions were re-immunoprecipitated with SCD-4 sera and preimmune sera as a control. [35S]S04 counts in the resulting immune complexes were found only in the complex obtained with MSE-4. Thus, SCD-4 is selective for the cytoplasmic domain of syndecan-4. The shed form of syndecan-1 and -4 was partially purified from the conditioned media (CM) of confluent cells cultured for 3 days by QAE-Sephadex A-25 (Pharmacia) and cesium chloride density gradient separation similar to the method of Rapraeger and Bernfield (33Rapraeger A. Bernfield M. J. Biol. Chem. 1985; 260: 4103-4109Google Scholar). One μl of this partially purified CM represents the amount of heparan sulfate proteoglycan purified from 1 ml of SVEC4–10 CM. The native transmembrane form of syndecan-1 and -4 was prepared from a RIPA lysate of confluent cells in a 75-cm2 flask (Falcon). The lysate was partially purified on a DEAE-Sephacel column, precipitated with 95% ethanol containing 1.3% potassium acetate, and dissolved in deionized water. Five hundred μl of this partially purified lysate represents the amount of heparin sulfate proteoglycan in the lysate collected from 2.5 × 107 cells. Acute human dermal wound fluids were obtained and treated essentially as described by Grinnellet al. (34Grinnell F. Ho C. Wysocki A. J. Invest. Dermatol. 1992; 98: 410-416Abstract Full Text PDF Google Scholar) and Wysocki et al. (35Wysocki A.B. Staiano-Coico L. Grinnell F. J. Invest. Dermatol. 1993; 101: 64-68Google Scholar). Briefly, wound fluid was collected at 1-day intervals for three consecutive days from sterile closed-suction drains routinely placed in the subcutaneous space following mammoplasty. Fluids were centrifuged at 200 ×g for 15 min to pellet cells and debri, and further clarified at 3300 × g and stored frozen (−70 °C). Wound fluids were kindly provided by Dr. E. Eriksson, Brigham and Women's Hospital, Boston, MA. The use of this anonymous discarded material was approved by the Human Research Committee of the Brigham and Women's Hospital, protocol number 92–5416-01. Blood plasma was collected from human blood diluted 1:1 (v/v) with phosphate-buffered saline, pH 7.4, at 37 °C following separation of cellular components on a Ficoll (Histopaque 1083, Sigma) gradient by centrifugation at 1000 × g for 30 min at room temperature and stored frozen (−70 °C). SVEC4–10 cells were cultured in 96-well and 6-well tissue culture plates (Costar or Falcon) for 16 and 8 h assays, respectively. Cells were grown to confluence in Dulbecco's modified Eagle's medium containing glucose at 4.5 g/liter (Life Technologies, Inc., Grand Island, NY), supplemented with 10% fetal calf serum (FCS, Intergen, Purchase, NY) and l-glutamine. At the time of treatment, culture media was replaced with fresh media containing the indicated amounts of FCS and test agents. Following incubation for the indicated times, cells were examined by phase microscopy for survival and morphology and the conditioned media harvested for dot blot analysis. Cells in 96-well plates were fixed with 2% paraformaldehyde (in Hepes-buffered saline, pH 7.4, with 1 mm CaCl2, 0.5 mmMgCl2), washed with Tris-buffered saline (TBS) and extracted with 0.1 n NaOH or 1% Triton X-100 in 50 mm Tris-HCl, 1 mm EDTA, pH 8.0. Protein was measured using the BCA protein assay (Pierce) with bovine serum albumin as the standard. The 96-well plate (16 h) assays were performed in triplicate with 100 μl of media per well. The 6-well plate (8 h) assays yielded 600 μl of media per well, which was divided into three equal portions for dot blot analysis. All assays were performed at least three times. Trypsinization of cell surface syndecans from SVEC4–10 cell monolayers in 6-well plates was performed essentially as described (25Jalkanen M. Rapraeger A. Saunders S. Bernfield M. J. Cell Biol. 1987; 105: 3087-3096Google Scholar). Briefly, following harvesting of the conditioned media (600 μl), cell layers were washed twice with ice-cold 0.5 mm EDTA-TBS and incubated with 600 μl of 10 μg/ml TPCK-treated trypsin in the same buffer for 10 min on ice. After incubation, soybean trypsin inhibitor was added to 50 μg/ml, cells were counted with a hemocytometer and centrifuged (200 ×g) for 5 min at 4 °C. Supernatants containing the ectodomains were used immediately or stored at 4 °C for protein determination and dot immunoassay. Total RNA was extracted from pellets as described below. SVEC4–10 cell conditioned media, lysates, and trypsinates, and human dermal wound fluids were diluted in Buffer A (0.15 m NaCl buffered to pH 4.5 with 50 mmNaOAc, with 0.1% Triton X-100), and applied to cationic polyvinylidene difluoride-based membranes (Immobilon-N, Millipore, Bedford, MA) under mild vacuum in an immunodot apparatus (V&P Scientific, San Diego, CA). The membranes were washed twice with Buffer A, and then blocked by a 1-h incubation in Blotto (3% Carnation instant nonfat dry milk, 0.5% bovine serum albumin, 0.15 m NaCl in 10 mmTris, pH 7.4). Membranes were incubated with the indicated concentration of primary antibody specific for a syndecan ectodomain or cytoplasmic domain, washed in TBS containing 0.3% Tween 20, and incubated with a 5000-fold dilution of the appropriate horseradish peroxidase-conjugated secondary. All antibodies were diluted in Blotto with 0.3% Tween 20. Detection was by the ECL system (Amersham) as described by the manufacturer, and quantitation was by laser densitometry using the LKB UltroScan XL densitometer (Pharmacia Biotech Inc.) running the GelScan XL software package (Pharmacia). Assay specificity was confirmed by eluting the membranes containing conditioned media by boiling in 2% SDS-PAGE sample buffer. The only immunoreactive materials detected on Western blots were the respective proteoglycans. Assay quantitation was provided by including known standard amounts of the purified soluble syndecan-1 ectodomain (25Jalkanen M. Rapraeger A. Saunders S. Bernfield M. J. Cell Biol. 1987; 105: 3087-3096Google Scholar) on each dot blot as an internal control. These showed a linear increase of absorbance units (AU) between 0.2 and 3.2 ng of syndecan-1. Standard amounts of SVEC4–10 cell conditioned media (day 3) were used as an internal control for the shed syndecan-4 ectodomain, and these also showed a linear increase of AU. In each case experimental values for dot blot quantitation were within these linear ranges. AU varied between experiments, in part, because of differences in exposure times and, in part, because of differences in treatment parameters. Thus, absorbance units were not compared between experiments. Results are expressed as the amount of syndecan shed in AU; for 16 h assays the values are normalized for micrograms of cellular protein. Each point represents the mean ± S.D. of triplicate determinations. Statistical significance was calculated using the Student's t test with the Instat biostatistic program. SVEC4–10 cells at confluence in 150-mm plates (Falcon, Lincoln Park, NJ) were incubated in 12 ml of serum-free medium for 16 h. Proteoglycans in the conditioned medium were precipitated twice with 95% ethanol containing 3% potassium acetate and resuspended in 100 mm Tris-HCl, pH 8.0, containing 0.1% Triton X-100, 5 mm EDTA, and 1 mmphenylmethylsulfonyl fluoride. Half of each sample was incubated with 10 milliunits/ml heparan sulfate lyase and 25 milliunits/ml chondroitin sulfate lyase ABC (Seikagaku America Inc., Rockville, MD) for 2 h at 37 °C. Enzyme-treated samples (0.5 and 2.5 ml of conditioned media equivalent for syndecan-1 and -4, respectively) were subjected to SDS-PAGE (3.5–20% gradient gel) with a discontinuous buffer system (69Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar), and electrotransferred to Immobilon-N. The membrane was processed as for the dot immunoassay; syndecan-1 and -4 core proteins were detected using mAb 281-2 and antisera MSE-4, respectively, followed by ECL detection. Wound fluid (day 1 and 3) was immunoprecipitated using antiserum against the human syndecan-1 ectodomain (HSE-1), or a mixture of mAbs against the human syndecan-4 ectodomain (5G9 and 8C7). The protein A immune complexes were subjected to SDS-PAGE (3.5–20% gradient gel) and electrotransferred to Immobilon-N. A431 human squamous cell carcinoma cell conditioned media (day 3), partially purified by QAE-Sephadex A-25 (Pharmacia) and cesium chloride density gradient separation (33Rapraeger A. Bernfield M. J. Biol. Chem. 1985; 260: 4103-4109Google Scholar), was run as a control. Syndecan-1 and -4 were detected by ECL using HSE-1 or mAbs 5G9 and 8C7, respectively. Total RNA was extracted from approximately 1 × 106 cells by resuspension in guanidinium denaturation solution, phenol/chloroform extraction, and isopropyl alcohol precipitation as described in the Ambion Totally RNA isolation kit (Austin, TX). RNA was separated in a 1% agarose, 0.6% formaldehyde gel, transferred to Nytran membranes using the Turboblotter (Schleicher & Schuell Inc.), and UV cross-linked. Membranes were prehybridized and hybridized in QuikHyb (Stratagene, La Jolla, CA) at 68 °C for 2 h with 1–2 × 106cpm/ml of a BamHI-EcoRI fragment of the mouse syndecan-1 cDNA (26Saunders S. Jalkanen M. O'Farrell S. Bernfield M. J. Cell Biol. 1989; 108: 1547-1556Google Scholar). Blots were washed twice at room temperature with 2 × SSPE, 0.1% SDS followed by two washes at 65 °C in 0.2 × SSPE with 0.5% SDS and exposed to film (X-Omat AR; Eastman Kodak) with an intensifying screen at −80 °C. The membranes were rehybridized with a 32P-labeled 800-base pairPstI fragment of the mouse β-actin probe provided by Dr. Stella Kourembanas (Childrens Hospital, Boston, MA) to quantitate the relative amount of RNA per lane. Results were quantified by densitometric scanning (Hewlett Packard Scan Jet IIC) using area measurements from NIH Image, version 1.57. We assessed whether agents known to be involved in wound repair regulate the shedding of syndecan ectodomains from the cell surface. SVEC4–10 cells, a mouse SV40 transformed endothelial cell line, were used as a test cell line (36O'Connell K.A. Edidin M. J. Immunol. 1990; 144: 521-525Google Scholar). These cells were chosen because they remain viable in the presence or absence of serum, express both syndecan-1 and -4, and shed the ectodomains of these syndecans at a low basal rate. Shedding was assayed by measuring the appearance in the culture media of immunoreactive syndecan ectodomains. Shed proteoglycans are not endocytosed by these cells during a 16-h incubation and thus are stable within the conditioned medium (data not shown). NMuMG mouse mammary epithelial cells were previously shown to shed the syndecan-1 ectodomain by demonstrating that the material in conditioned media (i) migrated as a GAG-free core protein at the sameMr as the trypsin-released ectodomain (25Jalkanen M. Rapraeger A. Saunders S. Bernfield M. J. Cell Biol. 1987; 105: 3087-3096Google Scholar) but smaller than the transmembrane proteoglycan (37Rapraeger A. Jalkanen M. Bernfield M. Wight R.N. Mecham R.P. Biology of Extracellular Matrix, Biology of Proteoglycans. Mercel Dekker, Inc., Academic Press, New York1987: 129-154Google Scholar), and (ii) failed to react with an antibody directed against the cytoplasmic domain (26Saunders S. Jalkanen M. O'Farrell S. Bernfield M. J. Cell Biol. 1989; 108: 1547-1556Google Scholar). We confirmed these results for syndecan-1 and -4 shed into conditioned media by SVEC4–10 cells in the presence or absence of serum. Proteoglycans in conditioned media and RIPA cell lysates were partially purified as described under "Experimental Procedures," treated with lyases to remove GAG chains, and subjected to SDS-PAGE and transferred to cationic polyvinylidene difluoride membranes. As expected, the glycosaminoglycan-free core proteins derived from the conditioned media were smaller than those from lysates (data not shown). The proteoglycans were immobilized on cationic membranes and probed with core protein-specific antibodies directed against the ectodomains and the cytoplasmic domains (Fig. 1). Antibodies against the syndecan-1 and -4 ectodomains detected these proteoglycans in cell lysates and conditioned media, as expected. Antibodies against the cytoplasmic domains detected proteoglycans in the cell lysates, but failed to detect proteoglycans in conditioned media, indicating that these are the ectodomains. We first examined the effect of various agents on syndecan shedding by SVEC4–10 cells during a 16-h incubation in the presence and absence of 5% fetal calf serum. Basal shedding of syndecan-1 and -4 was barely detectable, but shedding was markedly increased by PMA treatment (Fig.2), as described previously (27Saunders S. Jalkanen M. O'Farrell S. Bernfield M. J. Cell Biol. 1989; 108: 1547-1556Google Scholar). Both EGF and HB-EGF (10 ng/ml) accelerated shedding, however, vascular endothelial cell growth factor 165, FGF-2, platelet-derived growth factor AB, TGFβ, and TNFα (10 ng/ml)