Biglycan, a Nitric Oxide-regulated Gene, Affects Adhesion, Growth, and Survival of Mesangial Cells
2003; Elsevier BV; Volume: 278; Issue: 28 Linguagem: Inglês
10.1074/jbc.m210574200
ISSN1083-351X
AutoresLiliana Schaefer, Karl‐Friedrich Beck, Igor Raslik, Sebastian Walpen, Daniel Mihálik, Miroslava Micegova, Katarína Mačáková, Elke Schönherr, Daniela G. Seidler, Georg Varga, Roland M. Schaefer, Hans Kresse, Josef Pfeilschifter,
Tópico(s)Polysaccharides and Plant Cell Walls
ResumoDuring glomerular inflammation mesangial cells are the major source and target of nitric oxide that pro-foundly influences proliferation, adhesion, and death of mesangial cells. The effect of nitric oxide on the mRNA expression pattern of cultured rat mesangial cells was therefore investigated by RNA-arbitrarily-primed polymerase chain reaction. Employing this approach, biglycan expression turned out to be down-regulated time- and dose-dependently either by interleukin-1β-stimulated endogenous nitric oxide production or by direct application of the exogenous nitric oxide donor, diethylenetriamine nitric oxide. There was a corresponding decline in the rate of biglycan biosynthesis and in the steady state level of this proteoglycan. In vivo, in a model of mesangioproliferative glomerulonephritis up-regulation of inducible nitric-oxide synthase mRNA was associated with reduced expression of biglycan in isolated glomeruli. Biglycan expression could be normalized, both in vitro and in vivo, by using a specific inhibitor of the inducible nitric-oxide synthase, l-N 6-(l-iminoethyl)-l-lysine dihydrochloride. Further studies showed that biglycan inhibited cell adhesion on type I collagen and fibronectin because of its binding to these substrates. More importantly, biglycan protected mesangial cells from apoptosis by decreasing caspase-3 activity, and it counteracted the proliferative effects of platelet-derived growth factor-BB. These findings indicate a signaling role of biglycan and describe a novel pathomechanism by which nitric oxide modulates the course of renal glomerular disease through regulation of biglycan expression. During glomerular inflammation mesangial cells are the major source and target of nitric oxide that pro-foundly influences proliferation, adhesion, and death of mesangial cells. The effect of nitric oxide on the mRNA expression pattern of cultured rat mesangial cells was therefore investigated by RNA-arbitrarily-primed polymerase chain reaction. Employing this approach, biglycan expression turned out to be down-regulated time- and dose-dependently either by interleukin-1β-stimulated endogenous nitric oxide production or by direct application of the exogenous nitric oxide donor, diethylenetriamine nitric oxide. There was a corresponding decline in the rate of biglycan biosynthesis and in the steady state level of this proteoglycan. In vivo, in a model of mesangioproliferative glomerulonephritis up-regulation of inducible nitric-oxide synthase mRNA was associated with reduced expression of biglycan in isolated glomeruli. Biglycan expression could be normalized, both in vitro and in vivo, by using a specific inhibitor of the inducible nitric-oxide synthase, l-N 6-(l-iminoethyl)-l-lysine dihydrochloride. Further studies showed that biglycan inhibited cell adhesion on type I collagen and fibronectin because of its binding to these substrates. More importantly, biglycan protected mesangial cells from apoptosis by decreasing caspase-3 activity, and it counteracted the proliferative effects of platelet-derived growth factor-BB. These findings indicate a signaling role of biglycan and describe a novel pathomechanism by which nitric oxide modulates the course of renal glomerular disease through regulation of biglycan expression. Biglycan (BGN) 1The abbreviations used are: BGN, biglycan; NO, nitric oxide; DETA-NO, diethylenetriamine-NO; FACS, fluorescence-activated cell scanning; IL-1β, interleukin-1β; iNOS, inducible NO synthase; MC, mesangial cell; l-NIL, l-N 6-(l-iminoethyl)-l-lysine dihydrochloride; l-NMMA, N G-monomethyl-l-arginine; PDGF-BB, platelet-derived growth factor-BB; RAP-PCR, RNA-arbitrarily-primed PCR; SLRPs, small leucine-rich proteoglycans; SNAP, S-nitroso-N-acetyl-dl-penicillamine; TGF-β, transforming growth factor-β; TNFα, tumor necrosis factor-α; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GN, glomerulonephritis; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling; ELISA, enzyme-linked immunosorbent assay. belongs to the family of small, leucine-rich repeat glycoproteins/proteoglycans (SLRPs), which are characterized by core proteins with centrally located leucine-rich repeat motifs flanked by cysteine clusters. Near the N-terminal end BGN carries two (or sometimes only one) chondroitin/dermatan sulfate chain(s). Despite its abundance in different tissues, the precise biological role of BGN is still a matter of debate. SLRPs are primarily considered to play a role as organizers of extracellular matrices. BGN, decorin, and other members of the family of SLRPs interact with fibrillar collagens, thereby modulating fibril formation and stability (reviewed in Refs. 1Iozzo R.V. Annu. Rev. Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1349) Google Scholar, 2Iozzo R.V. J. Biol. Chem. 1999; 274: 18843-18846Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar, 3Kresse H. Schönherr E. J. Cell. Physiol. 2001; 189: 266-274Crossref PubMed Scopus (344) Google Scholar). Targeted disruption of the BGN gene resulted in abnormal collagen fibril morphology (4Ameye L. Aria D. Jepsen K. Oldberg Å. Xu T. Young M.F FASEB J. 2002; 16: 673-680Crossref PubMed Scopus (277) Google Scholar) and in an osteoporosis-like phenotype (5Xu T. Bianco P. Fisher L.W. Longenecker G. Smith E. Goldstein S. Bonadio J. Boskey A. Heegaard A.M. Sommer B. Satomura K. Dominguez P. Zhao C. Kulkarni A.B. Robey P.G. Young M.F. Nat. Genet. 1998; 20: 78-82Crossref PubMed Scopus (389) Google Scholar), possibly because of defects in bone marrow stromal cells (6Chen X.D. Shi S. Xu T. Robey P.G. Young M.F. J. Bone Miner. Res. 2002; 17: 331-340Crossref PubMed Scopus (134) Google Scholar). Additionally, BGN, as well as decorin, modulates adhesion of cells to matrix glycoproteins like fibronectin and thrombospondin (7Winnemöller M. Schmidt G. Kresse H. Eur. J. Cell Biol. 1991; 54: 10-17PubMed Google Scholar, 8Bidanset D.J. LeBaron R. Rosenberg L. Murphy-Ullrich J.E. Hook M. J. Cell Biol. 1992; 118: 1523-1531Crossref PubMed Scopus (90) Google Scholar). Interactions with cell membrane components have also to be taken into account, because BGN is most abundant near the cell surface (9Bianco P. Fisher L.W. Young M.F. Termine J.D. Robey P.G. J. Histochem. Cytochem. 1990; 38: 1549-1563Crossref PubMed Scopus (552) Google Scholar). Several SLRPs were shown to form complexes with TGF-β (10Hildebrand A. Romaris M. Rasmussen L.M. Heinegård D. Twardzik D.R. Border W.A. Ruoslahti E. Biochem. J. 1994; 302: 527-534Crossref PubMed Scopus (866) Google Scholar), which in the case of decorin affects the biological activity of the cytokine (11Border W.A. Noble N.A. Yamamoto T. Harper J.R. Yamaguchi Y. Pierschbacher M.D. Ruoslahti E. Nature. 1992; 360: 361-364Crossref PubMed Scopus (928) Google Scholar, 12Kolb M. Margetts P.J. Sime P.J. Gauldie J. Am. J. Physiol. Lung Cell Mol. Physiol. 2001; 280: L1327-L1334Crossref PubMed Google Scholar). No such properties have been reported so far for BGN·TGF-β complexes (12Kolb M. Margetts P.J. Sime P.J. Gauldie J. Am. J. Physiol. Lung Cell Mol. Physiol. 2001; 280: L1327-L1334Crossref PubMed Google Scholar). It is becoming increasingly clear that, in addition to their interaction with TGF-β, small proteoglycans are also directly involved in cell signaling (3Kresse H. Schönherr E. J. Cell. Physiol. 2001; 189: 266-274Crossref PubMed Scopus (344) Google Scholar). Decorin has been shown to interact with members of the ErbB receptor family (13Santra M. Eichstetter I. Iozzo R.V. J. Biol. Chem. 2000; 275: 35153-35161Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), which leads to the induction of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (14De Luca A. Santra M. Baldi A. Giordano A. Iozzo R.V. J. Biol. Chem. 1997; 271: 18961-18965Abstract Full Text Full Text PDF Scopus (226) Google Scholar) and growth arrest of certain tumor cells. In endothelial cells (15Schönherr E. Levkau B. Schaefer L. Kresse H. Walsh K. J. Biol. Chem. 2001; 276: 40687-40692Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), renal tubular epithelial cells (16Schaefer L. Macakova K. Raslik I. Micegova M. Gröne H.J. Schönherr E. Robenek H. Echtermeyer F.G. Grässel S. Bruckner P. Schaefer R.M. Iozzo R.V. Kresse H. Am. J. Pathol. 2002; 160: 1181-1191Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), and macrophages (17Xaus J. Comalada M. Cardo M. Valledor A.F. Celada A. Blood. 2001; 98: 2124-2133Crossref PubMed Scopus (112) Google Scholar) decorin protects against apoptosis and induces p27KIP1. BGN seems to be required for endothelial cell migration (18Kinsella M.G. Tsoi C.K. Järveläinen H.T. Wight T.N. J. Biol. Chem. 1997; 272: 318-325Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). It may also affect signal transduction during growth and differentiation via induction of p27KIP1 (19Weber C.K. Sommer G. Michl P. Fensterer H. Weimer M. Gansauge F. Leder G. Adler G. Gress T.M. Gastroenterology. 2001; 121: 657-667Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Further signaling functions may be deduced from the capability of BGN to interact via its glycosaminoglycan chains with dystroglycan (20Bowe M.A. Mendis D.B. Fallon J.R. J. Cell Biol. 2000; 148: 801-810Crossref PubMed Scopus (134) Google Scholar). BGN has been shown to stimulate growth and differentiation of monocytic lineage cells from various lymphatic organs (21Kikuchi A. Tomoyasu H. Kido I. Takahashi K. Tanaka A. Nonaka I. Iwakami N. Kamo I. J. Neuroimmunol. 2000; 106: 78-86Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). In the normal kidney BGN is found primarily in the tubulointerstitium. The normal glomerulus contains trace amounts of BGN produced by mesangial and endothelial cells, as well as podocytes (22Schaefer L. Gröne H.J. Raslik I. Robenek H. Ugorcakova J. Budny S. Schaefer R.M. Kresse H. Kidney Int. 2000; 58: 1557-1568Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In contrast, in advanced stages of glomerulosclerosis high amounts of BGN are deposited in the mesangial matrix (23Stokes M.B. Holler S. Cui Y. Hudkins K.L. Eitner F. Fogo A. Alpers C.E. Kidney Int. 2000; 57: 487-498Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 24Schaefer L. Raslik I. Gröne H.J. Schönherr E. Macakova K. Ugorcakova J. Budny S. Schaefer R.M. Kresse H. FASEB J. 2001; 15: 559-561Crossref PubMed Scopus (118) Google Scholar). In obstructive nephropathy BGN becomes up-regulated, an effect that is dramatically enhanced in decorin-deficient mice. This increase is primarily because of the appearance of BGN-expressing macrophages (16Schaefer L. Macakova K. Raslik I. Micegova M. Gröne H.J. Schönherr E. Robenek H. Echtermeyer F.G. Grässel S. Bruckner P. Schaefer R.M. Iozzo R.V. Kresse H. Am. J. Pathol. 2002; 160: 1181-1191Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Taken together, these data suggest a regulatory role of BGN during the development of renal diseases (23Stokes M.B. Holler S. Cui Y. Hudkins K.L. Eitner F. Fogo A. Alpers C.E. Kidney Int. 2000; 57: 487-498Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 24Schaefer L. Raslik I. Gröne H.J. Schönherr E. Macakova K. Ugorcakova J. Budny S. Schaefer R.M. Kresse H. FASEB J. 2001; 15: 559-561Crossref PubMed Scopus (118) Google Scholar). Nitric oxide, either produced in physiological amounts by endothelial cells and macrophages or overproduced by the inducible isoform of NO synthase (iNOS), has been shown to be an important regulatory factor in a number of inflammatory diseases (reviewed in Refs. 25Moncada S. J. R. Soc. Med. 1999; 92: 164-169Crossref PubMed Scopus (293) Google Scholar and 26Nathan C. J. Clin. Invest. 1997; 100: 2417-2423Crossref PubMed Scopus (845) Google Scholar). In the kidney, NO triggers the expression of proinflammatory and protective gene products in various types of glomerulonephritis (27Pfeilschifter J. Nephrol. Dial. Transplant. 2002; 17: 347-348Crossref PubMed Scopus (29) Google Scholar, 28Pfeilschifter J. Beck K.F. Eberhardt W. Huwiler A. Kidney Int. 2002; 61: 809-815Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Besides infiltrating cells, renal mesangial cells (MCs), exposed to inflammatory cytokines such as interleukin-1β (IL-1β) or tumor necrosis factor-α (TNFα), start to express iNOS followed by enhanced generation of NO (29Pfeilschifter J. Schwarzenbach H. FEBS Lett. 1990; 273: 185-187Crossref PubMed Scopus (133) Google Scholar). NO exerts complex regulatory actions on proliferation (30Raij L. Shultz P.J. J. Am. Soc. Nephrol. 1993; 3: 1435-1441PubMed Google Scholar, 31Rupprecht H.D. Akagi Y. Keil A. Hofer G. Kidney Int. 2000; 57: 70-82Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), adhesion (32Yao J. Schoecklmann H.O. Pröls F. Gauer S. Sterzel R.B. Kidney Int. 1998; 53: 598-608Abstract Full Text PDF PubMed Scopus (44) Google Scholar), and death of MCs (33Mühl H. Sandau K. Brune B. Briner V.A. Pfeilschifter J. Eur. J. Pharmacol. 1996; 317: 137-149Crossref PubMed Scopus (90) Google Scholar, 34Sandau K. Pfeilschifter J. Brune B. J. Immunol. 1997; 158: 4938-4946PubMed Google Scholar). Here we show for the first time, using PCR-based analysis of differential mRNA expression patterns of MCs exposed to exogenously or endogenously produced NO, that BGN is an NO-regulated gene in MCs both in vitro and in vivo and that it is involved in the modulation of the extent of adhesion, proliferation, and survival of MCs. Reagents—Radiochemicals and a Recliprime DNA Labeling System were obtained from Amersham Biosciences. Nylon blotting membranes were from Schleicher & Schüll or Millipore. Tissue culture plastic was from Falcon (BD Biosciences), and media and sera were from Invitrogen. The NO donors DETA-NO and SNAP, as well as the inhibitors of iNOS, l-NMMA and l-NIL, were from Alexis (Grünberg, Germany). Chemicals for reverse transcriptase-PCR were obtained from Stratagene. All other chemicals were purchased from Sigma. Cell Culture and Stimulation—Rat MCs were cultured as described previously (35Beck K.F. Walpen S. Eberhardt W. Pfeilschifter J. Life Sci. 2001; 69: 2945-2955Crossref PubMed Scopus (12) Google Scholar). They were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mm glutamine, 5 ng/ml insulin, 100 units/ml penicillin, and 100 μg/ml streptomycin for eight to 19 passages. To obtain quiescent cells, MCs were maintained in serum-free Dulbecco's modified Eagle's medium supplemented with 0.1 mg/ml fatty acid-free bovine serum albumin for 24 h prior to the addition of buffer, DETA-NO (0.063–1 mm), l-NIL (0.3–3 mm), or IL-1β (2 nm). Viability of MCs was not altered under the conditions used for the experiments, as determined by lactate dehydrogenase release into the culture medium using a cytotoxicity detection kit (Roche Applied Science). Analysis of the mRNA Expression Pattern in MCs by RAP-PCR—In the present work BGN was identified as a nitric oxide-regulated gene in a set of experiments performed analogously as in a previous study on NO-mediated regulation of macrophage inflammatory protein 2, using the same conditions and primers (36Walpen S. Beck K.F. Schaefer L. Raslik I. Eberhardt W. Schaefer R.M. Pfeilschifter J. FASEB J. 2001; 3: 571-573Crossref Scopus (52) Google Scholar). Briefly, mRNA from MCs was prepared using an mRNA isolation kit (Stratagene) followed by the low stringency RAP-PCR protocol provided by the manufacturer and using [α-33P]dCTP as radioactive precursor. As an internal control the reverse transcription step was performed additionally using the primer G5 that matched with the reverse strand of rat GAPDH cDNA at position 705–724 (GenBank™ accession number M17701), whereas during the PCR steps primer G3, which matched with the coding strand of rat GAPDH cDNA at position 131–150, was also present. The PCR was performed for one cycle at an annealing temperature of 35 °C and for 40 further cycles at 53 °C. The products were separated on a 4% sequencing gel. After an overnight exposition to x-ray film, bands of interest were excised, reamplified, and blunted using Pfu polymerase (Promega) according to the high stringency protocol mentioned above and sequenced. For further analysis, the PCR fragments were cloned into EcoRV sites of pBluescript KS+ (Stratagene). Nitrite Analysis—To verify endogenous NO production, nitrite as a stable end product of NO metabolism was measured routinely in culture media using the Griess reagent (Merck). Rat Model of Glomerulonephritis—The anti-Thy 1-glomerulonephritis (anti-Thy 1-GN) was induced as described before (36Walpen S. Beck K.F. Schaefer L. Raslik I. Eberhardt W. Schaefer R.M. Pfeilschifter J. FASEB J. 2001; 3: 571-573Crossref Scopus (52) Google Scholar). l-NIL, a selective inhibitor of iNOS, was administered intravenously at a dose of 5 mg/kg body weight to control and nephritic rats 45 min before and 8 h after anti-Thy 1 injection. Kidneys were harvested 16 h (n = 5 animals per group) after injection of the anti-Thy 1.1 antibody. Monitoring of systolic blood pressure and isolation of glomeruli were performed as described (36Walpen S. Beck K.F. Schaefer L. Raslik I. Eberhardt W. Schaefer R.M. Pfeilschifter J. FASEB J. 2001; 3: 571-573Crossref Scopus (52) Google Scholar, 37Schaefer L. Hausser H. Altenburger M. Ugorcakova J. August C. Fisher L.W. Schaefer R.M. Kresse H. Kidney Int. 1998; 54: 1529-1541Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Northern Blot Analysis and In Situ Hybridization—Total RNA was extracted from isolated glomeruli using TRIzol (Invitrogen). Northern blots were performed and analyzed as described previously (37Schaefer L. Hausser H. Altenburger M. Ugorcakova J. August C. Fisher L.W. Schaefer R.M. Kresse H. Kidney Int. 1998; 54: 1529-1541Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In situ hybridization of rat renal sections was performed in parallel with the sense and antisense probes for rat BGN (24Schaefer L. Raslik I. Gröne H.J. Schönherr E. Macakova K. Ugorcakova J. Budny S. Schaefer R.M. Kresse H. FASEB J. 2001; 15: 559-561Crossref PubMed Scopus (118) Google Scholar, 37Schaefer L. Hausser H. Altenburger M. Ugorcakova J. August C. Fisher L.W. Schaefer R.M. Kresse H. Kidney Int. 1998; 54: 1529-1541Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Determination of BGN in Isolated Rat Glomeruli and MC Cultures— Cell culture supernatants from stimulated (30 h) and control MCs were collected. MCs were washed three times with Hanks' solution before cell protein was extracted with 50 mm sodium acetate, pH 6.0, 4 m guanidinium chloride, 0.1% Triton X-100, and protease inhibitors (38Glössl J. Beck M. Kresse H. J. Biol. Chem. 1984; 259: 14144-14150Abstract Full Text PDF PubMed Google Scholar). Five percent of the total volume of each cellular or glomerular extract was collected for Western blot analysis of β-tubulin and for the determination of protein content. After centrifugation, cell lysates (diluted to give 0.2 m guanidinium chloride as final concentration and made 7 m with respect to urea by adding solid substance), cell culture supernatants, and the appropriate standard solutions were loaded on 0.5 ml columns of DEAE-Trisacryl M (Invitrogen), prepared in Pasteur pipettes, and equilibrated with Buffer 1 (20 mm Tris/HCl, pH 7.4, containing 0.15 m NaCl, 0.1% Triton X-100, 7 m urea, and protease inhibitors) and processed as described (24Schaefer L. Raslik I. Gröne H.J. Schönherr E. Macakova K. Ugorcakova J. Budny S. Schaefer R.M. Kresse H. FASEB J. 2001; 15: 559-561Crossref PubMed Scopus (118) Google Scholar). Glomerular homogenates containing equal amounts of glomeruli were mixed with DEAE-Trisacryl M (100 mg wet weight), equilibrated with Buffer 1, and mixed by rotation for 1 h at 4 °C. The samples were washed sequentially with 3 ml of Buffer 1, 3 ml of urea-free Buffer 1, and 3 ml of urea-free Buffer 1 containing 0.3 m NaCl. Elution was achieved with 1.5 ml of urea-free Buffer 1 containing 1 m NaCl. Upon adding 5 volumes of methanol and 1 volume of chloroform followed by freezing on dry ice, proteoglycans were collected after thawing at the interphase between chloroform and aqueous methanol. The upper phase was removed, and proteoglycans were precipitated to the bottom of the tube by adding again 5 volumes of methanol. The proteoglycans from cell culture supernatants, MCs, or glomerular homogenates were digested with chondroitin ABC lyase (Seikagaku Kogyo, Tokyo, Japan) to remove chondroitin sulfate and dermatan sulfate chains. BGN from plasma or urine samples was semipurified as described previously (24Schaefer L. Raslik I. Gröne H.J. Schönherr E. Macakova K. Ugorcakova J. Budny S. Schaefer R.M. Kresse H. FASEB J. 2001; 15: 559-561Crossref PubMed Scopus (118) Google Scholar). According to the analysis of [35S]sulfate-labeled biglycan from fibroblast secretions as an internal standard, the recovery after the ion exchange chromatography step varied by 85 ± 10%. The presence of 0.1% Triton X-100 was the critical component for achieving good recovery. Additional control experiments, yielding the expected results, were performed by adding known quantities of BGN to the culture medium prior to loading on the DEAE column. Untreated and chondroitin ABC lyase-treated samples from MCs and their culture supernatants were subjected to SDS-PAGE followed by Western blotting (38Glössl J. Beck M. Kresse H. J. Biol. Chem. 1984; 259: 14144-14150Abstract Full Text PDF PubMed Google Scholar). Plasma, urine, and glomerular samples were transferred to nitrocellulose membranes using the Bio-Dot microfiltration apparatus (Bio-Rad). The membranes were blocked with 3% casein, 1% goat serum, and 0.002% Tween 20 in 10 mm Tris/HCl, pH 7.4, 0.15 m NaCl. Western and dot blot membranes were incubated with chicken anti-rat BGN (37Schaefer L. Hausser H. Altenburger M. Ugorcakova J. August C. Fisher L.W. Schaefer R.M. Kresse H. Kidney Int. 1998; 54: 1529-1541Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) or with rabbit anti-mouse BGN (LF-106) antibodies (dilution 1:500 with 10 mm Tris/HCl, pH 7.4, 0.15 m NaCl/1% bovine serum albumin) for 90 min at 37 °C, whereas the second antibody, horseradish peroxidase-coupled goat anti-rabbit (enzyme immunoassay grade; Bio-Rad) was applied for 90 min at ambient temperature. Additionally, β-tubulin content in cell extracts was quantified by Western blot analysis (rabbit anti-β-tubulin; 1:500; Santa Cruz, Biotechnology, Inc.) as a control for loading. The samples were visualized by using the ECL Western blotting reagent kit (Amersham Biosciences), and analysis was performed with IQ Solutions ImageQuant software (Amersham Biosciences). Metabolic Labeling of MCs and Determination of Newly Synthesized BGN—Metabolic labeling of MCs was performed either with [4,5-3H]leucine or with [35S]sulfate. Quiescent MCs were treated in the presence and absence of DETA-NO (1.0 mm) for 24 h followed by preincubation with leucine-free Weymouth medium for 1 h (7 ml/75-cm2 culture flask) and subsequently labeled with 40 μCi/ml [4,5-3H]leucine for 5 h with and without NO donor, respectively. Metabolic labeling of MCs with [35S]sulfate was performed as described for fibroblasts (38Glössl J. Beck M. Kresse H. J. Biol. Chem. 1984; 259: 14144-14150Abstract Full Text PDF PubMed Google Scholar). The culture medium was supplemented with proteinase inhibitors and made 70% saturated with (NH4)2SO4. After centrifugation, the pellet was dissolved in Buffer 1 and processed as described above. MCs were harvested in Buffer 1 and treated identically as the culture medium samples. Because an immunoprecipitating anti-rat BGN antibody was not available, and because neither intact decorin and BGN nor their respective core proteins can reliably be separated by gel filtration, decorin was first removed by immunoprecipitation with an immobilized rabbit antibody against rat decorin (37Schaefer L. Hausser H. Altenburger M. Ugorcakova J. August C. Fisher L.W. Schaefer R.M. Kresse H. Kidney Int. 1998; 54: 1529-1541Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). After immunoprecipitation, supernatants of the samples labeled with [35S]sulfate were concentrated and then fractionated on a Superose 6 column (Amersham Biosciences) equilibrated with 4 m guanidinium chloride, 0.05 m sodium acetate, pH 6.0, 1% Triton X-100, and proteinase inhibitors at a flow rate of 300 μl/min. [35S]Sulfate-labeled human BGN from stably transfected 293 cells was used as a control (39Kresse H. Seidler D.G. Müller M. Breuer E. Hausser H. Roughley P.J. Schönherr E. J. Biol. Chem. 2001; 276: 13411-13416Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Appropriate radioactive fractions were tested by a dot blot assay with the anti-biglycan antibody (LF-106) as described above. Biglycan was eluted at a K av value of 0.27. Supernatants of the samples labeled with [4,5-3H]leucine were purified on a concanavalin A-Sepharose column (Fluka) and treated with chondroitin ABC lyase prior to SDS-PAGE (12.5% total acrylamide in the separation gel) and fluorography (38Glössl J. Beck M. Kresse H. J. Biol. Chem. 1984; 259: 14144-14150Abstract Full Text PDF PubMed Google Scholar). Assessment of Adhesion, Proliferation, and Survival of Cultured MCs—For determination of cell adhesion MCs were prepared using enzyme-free cell dissociation buffer (Invitrogen). Quantitative determination of adhesion was performed by using 96-well CytoMatrix cell adhesion strips (Chemicon, Hofheim, Germany) coated with fibronectin, acid-solubilized type I collagen, or bovine serum albumin, respectively. Briefly, 2 × 106 MCs/ml were seeded on the coated substrates under serum-free conditions with or without recombinant BGN (2.5–50 μg/ml) from 293 cells (39Kresse H. Seidler D.G. Müller M. Breuer E. Hausser H. Roughley P.J. Schönherr E. J. Biol. Chem. 2001; 276: 13411-13416Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) for 1 h. Adherent cells were fixed and stained, and the relative attachment was calculated from the absorbance at 540 nm according to the manufacturer's protocol. The percentage of non-adherent MCs cultured in the absence of BGN was taken as baseline value. Additionally, 96-well culture plates, coated as described previously (7Winnemöller M. Schmidt G. Kresse H. Eur. J. Cell Biol. 1991; 54: 10-17PubMed Google Scholar) with fibronectin peptides in concentrations equimolar to a fibronectin concentration of 10 μg/ml: F120 containing the cell-binding domain of fibronectin (FN CBD; Chemicon), F30 containing the N-terminal heparin-binding domain (FN N-term HBD; Sigma), F1977–1991 containing the C-terminal heparin-binding domain (FN C-term HBD; Sigma), or commercially coated wells with human fibronectin or type I collagen (BD Biosciences) dissolved in 7 mm acetic acid were used for the assessment of adhesion or in a solid-phase assay. The solid-phase assay was performed as described previously (40Winnemöller M. Schon P. Vischer P. Kresse H. Eur. J. Cell Biol. 1992; 59: 47-55PubMed Google Scholar) using [35S]sulfate- or [35S]methionine-labeled human BGN from stably transfected 293 cells (39Kresse H. Seidler D.G. Müller M. Breuer E. Hausser H. Roughley P.J. Schönherr E. J. Biol. Chem. 2001; 276: 13411-13416Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) in the presence or absence of heparin from porcine intestinal mucosa (Sigma) in a final concentration of 25 μg/ml. When required, the glycosaminoglycan chains of BGN were cleaved immediately before the experiment by chondroitin ABC lyase (Seikagaku Kogyo). Data are given as means of duplicates of three independent measurements in each group. Expression of BGN on the cell surface of MCs was examined by FACS analysis using rabbit anti-human BGN (24Schaefer L. Raslik I. Gröne H.J. Schönherr E. Macakova K. Ugorcakova J. Budny S. Schaefer R.M. Kresse H. FASEB J. 2001; 15: 559-561Crossref PubMed Scopus (118) Google Scholar) and affinity-purified goat anti-rabbit, fluorescein isothiocyanate-labeled antibodies (Dianova, Hamburg, Germany). In brief, MCs were incubated for 30 min at 37 °C with or without 10 μg of BGN or BGN core protein. Thereafter, cells were washed with phosphate-buffered saline/1% FCS. Cells were resuspended in 100 μl phosphate-buffered saline/1% FCS, and the primary antibody (rabbit anti-human BGN; 1:200) was added and incubated for 30 min at room temperature. Cells were washed twice, and the fluorescein isothiocyanate-conjugated secondary antibody (goat anti-rabbit; 1:200) was added for another 30 min at room temperature. Subsequently cells were washed twice and resuspended in 500 μl of phosphate-buffered saline for analysis. Cells were evaluated with a FACS-calibur flow cytometer using CellQuestPro software (BD Biosciences). For flow cytometric analysis 5 × 105 MCs were used, and all experiments were performed at least three times. Additionally, MCs were cultured for 18 h, plated on 8-well fibronectin-coated chamber slides (BD Biosciences) in the presence or absence of 10 μg of BGN, and subsequently immunostained for BGN with alkaline phosphatase anti-alkaline phosphatase (24Schaefer L. Raslik I. Gröne H.J. Schönherr E. Macakova K. Ugorcakova J. Budny S. Schaefer R.M. Kresse H. FASEB J. 2001; 15: 559-561Crossref PubMed Scopus (118) Google Scholar). To assess the effect of BGN on MC proliferation, subconfluent cells (2 × 104 MCs/well) were cultured in 96-well microtiter for 24 h under serum-free conditions in the presence or absence of recombinant BGN (2.5–25 μg/ml). Alternatively, MCs were deprived of serum for 24 h and then treated simultaneously with 10% FCS and BGN (5 μg/ml) or with rat recombinant PDG
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