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• W2007274239 abstract "The matricellular protein SPARC is expressed at high levels in cells that participate in tissue remodeling and is thought to regulate mesangial cell proliferation and extracellular matrix production in the kidney glomerulus in a rat model of glomerulonephritis (Pichler, R. H., Bassuk, J. A., Hugo, C., Reed, M. J., Eng, E., Gordon, K. L., Pippin, J., Alpers, C. E., Couser, W. G., Sage, E. H., and Johnson, R. J. (1997) Am. J. Pathol. 148, 1153–1167). A potential mechanism by which SPARC controls both cell cycle and matrix production has been attributed to its regulation of a pleiotropic growth factor. In this study we used primary mesangial cell cultures from wild-type mice and from mice with a targeted disruption of the SPARCgene. SPARC-null cells displayed diminished expression of collagen type I mRNA and protein, relative to wild-type cells, by the criteria of immunocytochemistry, immunoblotting, and the reverse transcription-polymerase chain reaction. The SPARC-null cells also showed significantly decreased steady-state levels of transforming growth factor-β1 (TGF-β1) mRNA and secreted TGF-β1 protein. Addition of recombinant SPARC to SPARC-null cells restored the expression of collagen type I mRNA to 70% and TGF-β1 mRNA to 100% of wild-type levels. We conclude that SPARC regulates the expression of collagen type I and TGF-β1 in kidney mesangial cells. Since increased mitosis and matrix deposition by mesangial cells are characteristics of glomerulopathies, we propose that SPARC is one of the factors that maintains the balance between cell proliferation and matrix production in the glomerulus. The matricellular protein SPARC is expressed at high levels in cells that participate in tissue remodeling and is thought to regulate mesangial cell proliferation and extracellular matrix production in the kidney glomerulus in a rat model of glomerulonephritis (Pichler, R. H., Bassuk, J. A., Hugo, C., Reed, M. J., Eng, E., Gordon, K. L., Pippin, J., Alpers, C. E., Couser, W. G., Sage, E. H., and Johnson, R. J. (1997) Am. J. Pathol. 148, 1153–1167). A potential mechanism by which SPARC controls both cell cycle and matrix production has been attributed to its regulation of a pleiotropic growth factor. In this study we used primary mesangial cell cultures from wild-type mice and from mice with a targeted disruption of the SPARCgene. SPARC-null cells displayed diminished expression of collagen type I mRNA and protein, relative to wild-type cells, by the criteria of immunocytochemistry, immunoblotting, and the reverse transcription-polymerase chain reaction. The SPARC-null cells also showed significantly decreased steady-state levels of transforming growth factor-β1 (TGF-β1) mRNA and secreted TGF-β1 protein. Addition of recombinant SPARC to SPARC-null cells restored the expression of collagen type I mRNA to 70% and TGF-β1 mRNA to 100% of wild-type levels. We conclude that SPARC regulates the expression of collagen type I and TGF-β1 in kidney mesangial cells. Since increased mitosis and matrix deposition by mesangial cells are characteristics of glomerulopathies, we propose that SPARC is one of the factors that maintains the balance between cell proliferation and matrix production in the glomerulus. SPARC (secreted protein acidic and rich in cysteine), a matricellular glycoprotein also known as BM-40, osteonectin, or 43-kDa protein, modulates the interaction of cells with the extracellular matrix through its regulation of cell adhesion and binding of growth factors (1Lane T.F. Sage E.H. FASEB J. 1994; 8: 163-173Crossref PubMed Scopus (479) Google Scholar, 2Bornstein P. J. Cell Biol. 1995; 130: 503-506Crossref PubMed Scopus (586) Google Scholar). It has been shown to inhibit proliferation, disrupt focal adhesions, and prevent cell spreading in vitro (3Motamed K. Sage E.H. J. Cell. Biochem. 1998; 70: 543-552Crossref PubMed Scopus (74) Google Scholar). In addition, SPARC is known to bind to certain growth factors, for example platelet-derived growth factor (PDGF) 1The abbreviations used are:PDGFplatelet-derived growth factorTGF-β1transforming growth factor-β1rhrecombinant humanELISAenzyme-linked immunosorbent assayPAGEpolyacrylamide gel electrophoresisRT-PCRreverse-transcribed polymerase chain reactionrpribosomal proteinFGFfibroblast growth factorEtBrethidium bromideVEGFvascular endothelial growth factor (4Raines E.W. Lane T.F. Iruela-Arispe M.L. Ross R. Sage E.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1281-1285Crossref PubMed Scopus (328) Google Scholar), and to bind extracellular matrix proteins such as collagen type I (5Sasaki T. Hohenester E. Göhring W. Timpl R. EMBO J. 1998; 17: 1625-1634Crossref PubMed Scopus (125) Google Scholar). SPARC regulates the expression of a number of secreted proteins (6Lane T.F. Iruela-Arispe M.L. Sage E.H. J. Biol. Chem. 1992; 267: 16736-16745Abstract Full Text PDF PubMed Google Scholar) as well as matrix metalloproteinases (7Tremble P.M. Lane T.F. Sage E.H. Werb Z. J. Cell Biol. 1993; 121: 1433-1444Crossref PubMed Scopus (250) Google Scholar) in certain cell types and is thought to modulate the interactions between cells and the surrounding extracellular matrix at least partially through this activity. In vivo, it is expressed during development (8Engelmann G.L. Cardiovasc. Res. 1993; 27: 1598-1605Crossref PubMed Scopus (27) Google Scholar) and is produced at sites of wound repair (9Reed M.J. Puolakkainen P. Lane T.F. Dickerson D. Bornstein P. Sage E.H. J. Histochem. Cytochem. 1993; 41: 1467-1477Crossref PubMed Scopus (195) Google Scholar) and tissue remodeling (10Shankavaram U.T. DeWitt D.L. Funk S.E. Sage E.H. Wahl L.M. J. Cell. Physiol. 1997; 173: 327-334Crossref PubMed Scopus (100) Google Scholar). Furthermore, the production of SPARC mRNA is increased in certain types of carcinoma (11Porte H. Triboulet J.P. Kotelevets L. Carrat F. Prévot S. Nordlinger B. DiGioia Y. Wurtz A. Comoglio P. Gespach C. Chastre E. Clin. Cancer Res. 1998; 4: 1375-1382PubMed Google Scholar), in scleroderma (12Unemori E.N. Amento E.P. Curr. Opin. Rheumatol. 1991; 3: 953-959Crossref PubMed Scopus (12) Google Scholar), atherosclerotic lesions (4Raines E.W. Lane T.F. Iruela-Arispe M.L. Ross R. Sage E.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1281-1285Crossref PubMed Scopus (328) Google Scholar), passive Heymann nephritis (13Floege J. Johnson R.J. Alpers C.E. Fatemi-Nainie S. Richardson C.A. Gordon K. Couser W.G. Am. J. Pathol. 1993; 142: 637-650PubMed Google Scholar), and mesangioproliferative glomerulonephritis (14Pichler R.H. Bassuk J.A. Hugo C. Reed M.J. Eng E. Gordon K.L. Pippin J. Alpers C.E. Couser W.G. Sage E.H. Johnson R.J. Am. J. Pathol. 1996; 148: 1153-1167PubMed Google Scholar). For example, SPARC has been shown to be involved in the resolution of mesangioproliferative glomerulonephritis in the Thy 1.1 model in the rat and to inhibit PDGF-induced proliferation of mesangial cellsin vitro (14Pichler R.H. Bassuk J.A. Hugo C. Reed M.J. Eng E. Gordon K.L. Pippin J. Alpers C.E. Couser W.G. Sage E.H. Johnson R.J. Am. J. Pathol. 1996; 148: 1153-1167PubMed Google Scholar). platelet-derived growth factor transforming growth factor-β1 recombinant human enzyme-linked immunosorbent assay polyacrylamide gel electrophoresis reverse-transcribed polymerase chain reaction ribosomal protein fibroblast growth factor ethidium bromide vascular endothelial growth factor Transforming growth factor-β1 (TGF-β1) is also produced by mesangial cells during mesangioproliferative glomerulonephritis (15Iwano M. Akai Y. Fujii Y. Dohi Y. Matsumura N. Dohi K. Clin. Exp. Immunol. 1994; 97: 309-314Crossref PubMed Scopus (51) Google Scholar). A multifunctional growth factor that belongs to a family of proteins, TGF-β1 functions in various physiological processes such as growth, differentiation, proliferation, tissue remodeling, and wound healing (16Pepper M.S. Cytokine Growth Factor Rev. 1997; 8: 21-43Crossref PubMed Scopus (605) Google Scholar). Although specific receptors have been found on nearly all mammalian cells, the effects of TGF-β1 differ according to cell type, growth conditions, and concentration of growth factor (17Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3998) Google Scholar). TGF-β1 has been implicated in development and in the remodeling of tissues that takes place during adult life (18Frank R. Adelmann-Grill B.C. Herrmann K. Haustein U.F. Petri J.B. Heckmann M. J. Invest. Dermatol. 1996; 106: 36-41Abstract Full Text PDF PubMed Scopus (25) Google Scholar), although its effects on proliferation and differentiation can be stimulatory or inhibitory (19Floege J. Topley N. Resch K. Am. J. Kidney Dis. 1991; 17: 673-676Abstract Full Text PDF PubMed Scopus (47) Google Scholar). TGF-β1 mediates the formation of extracellular matrix via its stimulation of the synthesis of components such as collagen type I. Moreover, it inhibits the degradation of extracellular matrix by suppression of matrix metalloproteinases and induction of tissue inhibitors of these enzymes (20Poncelet A.C. Schnaper H.W. Am. J. Physiol. 1998; 275: F458-F466Crossref PubMed Google Scholar). A number of publications have identified TGF-β1 as a critical factor in kidney diseases such as glomerulosclerosis (21Gilbert R.E. Wilkinson-Berka J.L. Johnson D.W. Cox A. Soulis T. Wu L.-L. Kelly D.J. Jerums G. Pollock C.A. Cooper M.E. Kidney Int. 1998; 54: 1052-1062Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) and mesangioproliferative glomerulonephritis (22Yamamoto T. Noble N.A. Cohen A.H. Nast C.C. Hishida A. Gold L.I. Border W.A. Kidney Int. 1996; 49: 461-469Abstract Full Text PDF PubMed Scopus (422) Google Scholar). Furthermore, it has been shown that TGF-β1 augments the accumulation of glomerular matrix through its induction of collagen type I (23Kagami S. Kuhara T. Yasutomo K. Okada K. Loster K. Reutter W. Kuroda Y. Exp. Cell Res. 1996; 229: 1-6Crossref PubMed Scopus (64) Google Scholar). The most abundant fibrillar collagen expressed by a variety of cell types, collagen type I maintains the structural integrity of tissues such as bone, skin, organ capsules, and blood vessels (24Iruela-Arispe M.L. Vernon R.B. Wu H. Jaenisch R. Sage E.H. Dev. Dyn. 1996; 207: 171-183Crossref PubMed Scopus (54) Google Scholar). A number of factors modulate expression of the collagen genes during development (24Iruela-Arispe M.L. Vernon R.B. Wu H. Jaenisch R. Sage E.H. Dev. Dyn. 1996; 207: 171-183Crossref PubMed Scopus (54) Google Scholar), wound healing (25Reed M.J. Vernon R.B. Abrass I.B. Sage E.H. J. Cell. Physiol. 1994; 158: 169-179Crossref PubMed Scopus (162) Google Scholar), inflammation (26Lloyd C.M. Minto A.W. Dorf M.E. Proudfoot A. Wells T.N. Salant D.J. Gutierrez-Ramos J.C. J. Exp. Med. 1997; 185: 1371-1380Crossref PubMed Scopus (440) Google Scholar), cancer (27Sato N. Beitz J.G. Kato J. Yamamoto M. Clark J.W. Calabresi P. Raymond A. Frackelton A.R. Am. J. Pathol. 1993; 142: 1119-1130PubMed Google Scholar), and glomerulonephritis (20Poncelet A.C. Schnaper H.W. Am. J. Physiol. 1998; 275: F458-F466Crossref PubMed Google Scholar). Numerous studies have shown that collagen synthesis and deposition are regulated by TGF-β1 (28Grande J.P. Melder D.C. Zinsmeister A.R. J. Lab. Clin. Med. 1997; 130: 476-486Abstract Full Text PDF PubMed Scopus (68) Google Scholar) and by alterations in cell-extracellular matrix interactions that are accompanied by reorganization of the cytoskeletal network (29Varedi M. Ghahary A. Scott P.G. Tredget E.E. J. Cell. Physiol. 1997; 172: 192-199Crossref PubMed Scopus (43) Google Scholar). Under pathological conditions, changes in regulatory pathways occur that can lead to the elevated expression of collagen type I (30Lenz O. Striker L.J. Jacot T.A. Elliot S.J. Killen P.D. Striker G.E. J. Am. Soc. Nephrol. 1998; 9: 2040-2047PubMed Google Scholar), with eventual fibrosis or sclerosis and impaired organ function. Thus, it is critical to understand the different factors involved in the regulation of this predominant collagen. To investigate the function of SPARC in the regulation of collagen type I and TGF-β1, we chose a model in which we could study interactions among SPARC, collagen type I, and TGF-β1 in primary mesangial cell cultures from wild-type and SPARC-null mice. We present evidence that SPARC regulates the expression of both collagen type I and TGF-β1 in mouse mesangial cells. SPARC-null cells exhibited a significantly diminished expression of collagen type I and TGF-β1. After treatment of these cells with recombinant human (rh) SPARC, the levels of collagen type I and TGF-β1 were restored to 70% and 100%, respectively, of those produced by wild-type cells. Furthermore, we show that SPARC exhibits some of its effects on collagen type I expression via a TGF-β1-dependent pathway. Since TGF-β1 can induce the expression of SPARC under certain conditions (26Lloyd C.M. Minto A.W. Dorf M.E. Proudfoot A. Wells T.N. Salant D.J. Gutierrez-Ramos J.C. J. Exp. Med. 1997; 185: 1371-1380Crossref PubMed Scopus (440) Google Scholar), it is likely that SPARC and TGF-β1 participate in a reciprocal, positive autocrine feedback loop that is especially prominent in mesangial cells. 129/SvJ × C57BL/6J wild-type and SPARC-null mice (31Norose K. Clark J.I. Syed N.A. Basu A. Heber-Katz E. Sage E.H. Howe C.C. Invest. Ophthalmol. & Visual Sci. 1998; 39: 2674-2680PubMed Google Scholar) were maintained in a specific pathogen-free facility. Mice were euthanized at 3–6 months of age, and the kidneys were removed. The method for the preparation of primary mesangial cells is based on a partial collagenase digestion of isolated glomeruli (32Radeke H.H. Resch K. Clin. Invest. 1992; 70: 825-842Crossref PubMed Scopus (68) Google Scholar). The cortex was removed and was kept in sterile ice-cold phosphate-buffered saline (PBS, 120 mm NaCl, 2.7 mm KCl, and 10 mm phosphate-buffered saline, pH 7.5). The following procedures were performed on ice. 1) The cortex was minced finely with a scalpel, and the homogenate was passed over sieves with meshes of 180 and 106 μm. 2) The next sieve, with a mesh of 45 μm, retained 98% of the glomeruli, which were washed 3 times with sterile PBS. 3) The glomeruli were digested for 15–25 min at 37 °C in a solution of collagenase (Worthington) (50 mg of collagenase type CLS 4, 184 units/ml, dissolved in 50 ml of PBS). The suspension was shaken every 3–5 min, and the digestion was stopped when thread-like collagen-containing fibers began to form on the glomeruli. The glomeruli were washed 2 times (4 °C, 150 ×g) in growth medium (Dulbecco's modified Eagle's medium (55%), F-12 Nutrient Mixture (20%) (Life Technologies, Inc.), fetal bovine serum (20%) (Summit Biotechnologies, Stoughton, MA), trace elements (1%) (Biofluids, Inc., Rockville, MD),l-glutamine (2 mm), transferrin (5 μg/ml), insulin (125 units/ml), penicillin G, (500 units/ml), streptomycin sulfate (500 units/ml), and amphotericin B (2 μg/ml) (Sigma)), placed in small culture flasks in 2 ml, and incubated at 37 °C in a humidified atmosphere of 5% CO2. After 10–14 days, the preparation was checked microscopically. All cell populations except the mesangial cells were marked on the outside of the flask and were subsequently scraped off. For passages 0–3, the mesangial cells were grown to confluence in 50% fresh growth medium and 50% sterile-filtered conditioned medium. In these enriched populations of mesangial cells, there were no macrophages, endothelial- or epithelial-like cells, or fibroblasts, according to morphological criteria. In contrast to other glomerular cells, mesangial cells exhibited immunofluorescent staining for myosin, α-smooth muscle actin, desmin, vimentin, fibronectin, collagen type IV, and major histocompatibility complex class I antigen. The absence of von Willebrand factor and cytokeratins 18 and 19 indicated that endothelial and epithelial cell contamination was minimal. Furthermore, the use of mesangial cells after passage 3 essentially eliminates contamination by macrophages and endothelial cells, as these cell types do not survive multiple passaging under the culture conditions described above. Experiments were performed on five independent preparations of mesangial cells isolated from pools of 8 kidneys each. All experiments were repeated three times if not stated otherwise. Mesangial cells were detached in a solution of trypsin/EDTA (0.125%/0.010%, w/v) (Life Technologies, Inc.) and were replated at a split ratio of 1:3. The cells were used between passages 3 and 8. For the TGF-β1 assays, wild-type and SPARC-null cells were grown to 80% confluence in growth medium, as described above. The cells were washed 2 times with PBS and were changed into fresh growth medium for 96 h. To measure the amounts of TGF-β1 protein in the conditioned culture media of wild-type and SPARC-null cells, we used an enzyme-linked immunosorbent assay (ELISA) kit (R & D Systems Inc., Minneapolis, MN) according to the manufacturer's instructions. The assay was repeated five times. For the mRNA preparation, wild-type and SPARC-null cells were grown to 80% confluence in growth medium. Total cellular RNA was prepared from mesangial cells by a modified method (33Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63231) Google Scholar) that incorporated TRI-reagent (Molecular Research Center Inc., Cincinnati, OH). To increase the purity of the RNA samples, we added an additional step with 4m LiCl to eliminate residual contaminating polysaccharides, a DNase digestion step to eliminate DNA, and an additional precipitation of the RNA with ethanol. For preparation of cellular protein, wild-type and SPARC-null cells were grown to 80% confluence in growth medium. The insoluble (extracellular matrix proteins and membranes) and soluble cellular protein fractions were prepared either with the TRI-reagent or by dissolution of the cells in 1% SDS. The protein concentrations of the cell fractions were determined by the Bradford protein assay (34Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217450) Google Scholar). rhSPARC was prepared in SF9 cells by the use of the baculovirus protein expression system (35Bradshaw A.D. Francki A. Motamed K. Howe C.C. Sage E.H. Mol. Biol. Cell. 1999; 10: 1569-1579Crossref PubMed Scopus (88) Google Scholar) and was collected in serum-free medium. rhSPARC was isolated by anion-exchange chromatography and was identified by SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting with a specific monoclonal anti-SPARC antibody (Haemotological Technologies, Essex Junction, VT). 2A. D. Bradshaw, J. A. Bassuk, and E. H. Sage, manuscript in preparation. The rhSPARC had activity similar to that of recombinant SPARC expressed inEscherichia coli (36Bassuk J.A. Baneyx F. Vernon R.B. Funk S.E. Sage E.H. Arch. Biochem. Biophys. 1996; 325: 8-19Crossref PubMed Scopus (33) Google Scholar) and to SPARC synthesized by cultured mammalian cells (37Funk S.E. Sage E.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2648-2652Crossref PubMed Scopus (200) Google Scholar), as measured by inhibition of proliferation and spreading (35Bradshaw A.D. Francki A. Motamed K. Howe C.C. Sage E.H. Mol. Biol. Cell. 1999; 10: 1569-1579Crossref PubMed Scopus (88) Google Scholar). RT-PCR reactions containing 1 μg of total RNA were performed with the Access RT-PCR SystemTM (Promega) with oligonucleotide primers complementary to mouse β-tubulin, mouse ribosomal protein (rp) S6, mouse SPARC, mouse collagen α1(I) and α2(I), mouse collagen α1 (III), mouse collagen α1 (IV) and α2 (IV), mouse collagen α1 (VIII) and α2 (VIII), mouse fibroblast growth factor 1 (FGF-1), fibroblast growth factor 2 (FGF-2), PDGF-A and -B chain, and TGF-β1. The primers were designed according to the Entrez nucleotide query program to retrieve the appropriate cDNAs from GenBankTM and the Primer selection TM 3 oligonucleotide search program. Furthermore, with the Amplify 1.2 program, the primer pairs were tested for “cross-annealing” such that up to three primer pairs could be used together in one PCR reaction (38Goswami P.C. Albee L.D. Spitz D.R. Ridnour L.A. Cell Proliferation. 1997; 30: 271-282Crossref PubMed Scopus (13) Google Scholar). To establish conditions that allow comparison of the amounts of cDNA produced by RT-PCR, we varied the number of cycles from 24 to 40. For an internal standard, we either amplified β-tubulin mRNA or rpS6 mRNA, two ubiquitously expressed genes. After electrophoresis of 1/10 of the PCR reaction (5 μl), the bands (stained with ethidium bromide (EtBr), 0.5 μg/ml) became visible after 22 PCR cycles, and the staining reached saturation after 28 cycles. Therefore, a cycle number of 24 was chosen to compare the different levels of expression of the various mRNAs and to avoid saturation of the PCR DNA product and EtBr staining. The amounts of β-tubulin or rpS6 appeared to be unchanged between wild-type and SPARC-null cells. For quantification, values obtained from scanning densitometry of the cDNA bands generated from the respective mRNAs were normalized to the β-tubulin or rpS6 band. Since the β-tubulin/rpS6 and the other cDNAs were synthesized in the same tube, a direct comparison of the levels of expression is reasonable. Amplification of the newly synthesized first strand cDNA was performed in a Thermolyne Temptronic Thermal CyclerTM. Equivalent aliquots of each amplification reaction were separated on a 1.2% agarose gel containing 0.5 μg/ml EtBr in 0.04 mTris acetate, 0.001 m EDTA, pH 7.6. The gels were subjected to electrophoresis for 3 h at 100 V and were subsequently photographed. Primary mesangial cell cultures from wild-type and SPARC-null mice were grown to 80% confluence in the presence of 50 μg/ml sodium ascorbate for the final 24 h, and protein was prepared as described above. Equal amounts of protein per lane were resolved by SDS-PAGE (7% gels) under reducing conditions and were electrotransferred onto nitrocellulose membranes, which were subsequently blocked for 1 h with 5% nonfat dry milk and 0.05% Tween 20 (Sigma) in PBS. The blots were incubated with antibodies against collagen I (guinea pig anti-rat collagen I that cross-reacts with mouse collagen type I) (24Iruela-Arispe M.L. Vernon R.B. Wu H. Jaenisch R. Sage E.H. Dev. Dyn. 1996; 207: 171-183Crossref PubMed Scopus (54) Google Scholar) for 1 h. Immunoreactivity was detected by incubation of the blot with goat anti-guinea pig IgG coupled to horseradish peroxidase (Vector Laboratories Inc., Burlingame, CA), followed by enhanced chemiluminescence (Amersham Pharmacia Biotech). For assessment of differences in protein loading, the blot was incubated with rabbit anti-human α-enolase IgG (gift from Dr. E. Plow, Cleveland Clinic, Cleveland, OH) that cross-reacts with mouse α-enolase, followed by incubation with goat anti-rabbit IgG conjugated to horseradish peroxidase. For metabolic labeling, the mesangial cells were grown to 80% confluence and were incubated with sodium ascorbate (50 μg/ml) for 24 h. Cultures were subsequently incubated in fresh growth medium containing 50 μCi/ml l-[2,3,4,5-3H]proline (100 Ci/mmol, NEN Life Science Products). After 18 h, media containing radiolabeled proteins were removed and mixed with a proteinase inhibitor mixture (CompleteTM, Roche Molecular Biochemicals), centrifuged to remove cell debris, and dialyzed against 0.1 n acetic acid to remove unincorporated isotope. Incorporation of [3H]proline was measured in a scintillation counter, prior to lyophilization of the supernatants. The proteins were resuspended in collagenase buffer (50 mmTris-HCl, pH 7.5, 0.15 NaCl, 5 mm CaCl2) to reflect equal amounts of cpm/ml. Aliquots of the supernatants were digested for 16 h with bacterial collagenase (Worthington). Equal volumes of supernatants were resolved by SDS-PAGE (8% gels), stained with Coomassie Brilliant Blue R, and incubated in EnhanceTM(NEN Life Science Products) prior to fluorography. Primary mesangial cell cultures from wild-type and SPARC-null mice were plated on glass coverslips, incubated with sodium ascorbate (50 μg/ml) for 24 h, and fixed in 2% paraformaldehyde for 30 min. Paraformaldehyde was removed by three 5-min washes each with PBS. The coverslips were incubated for 30 min in 2% normal goat serum to block nonspecific binding and 0.5% Triton X-100 (Sigma) to solubilize the cell membranes. Subsequently, the cells were incubated for 1 h with antibodies against collagen type I. After three rinses in PBS, the coverslips were incubated for 30 min with rhodamine-conjugated goat anti-guinea pig IgG. Immunoreactivity was visualized by fluorescence microscopy (Nikon, Inc., Garden City, NY). To determine whether the effects of exogenous SPARC were exerted through a TGF-β1-dependent pathway, we cultured cells as described above and treated them with or without the following: (i) anti-TGF-β1-blocking antibodies (polyclonal goat anti-human IgG, R & D Systems, Inc., Minneapolis, MN) at a final concentration of 30 μg/ml; (ii) rhSPARC (30 μg/ml, 0.9 μm); (iii) rhTGF-β1 (1, 5, and 10 ng/ml); and (iv) either rhSPARC or rhTGF-β1 together with anti-TGF-β1-blocking antibodies for 0–6 h. As a control we used an irrelevant polyclonal goat anti-rabbit IgG (Vector). Total RNA was prepared as described above, and the levels of mouse α1(I) and mouse TGF-β1 mRNA were determined. All immunocytochemistry slides, autoradiograms, immunoblots, and agarose gels were photographed and converted to digital computer files with a UMAX S-6E scannerTM and Adobe Photoshop softwareTM. Files were processed and analyzed by NIH Image softwareTM and are presented as composite figures. SPARC has been implicated as a modulator of interactions between cells and the extracellular matrix. It is known to bind to growth factors and to matricellular proteins as well as to matrix proteins. Alterations in cell-matrix interactions usually occur during wound healing, tissue remodeling, and fibrosis (29Varedi M. Ghahary A. Scott P.G. Tredget E.E. J. Cell. Physiol. 1997; 172: 192-199Crossref PubMed Scopus (43) Google Scholar). Since SPARC (14Pichler R.H. Bassuk J.A. Hugo C. Reed M.J. Eng E. Gordon K.L. Pippin J. Alpers C.E. Couser W.G. Sage E.H. Johnson R.J. Am. J. Pathol. 1996; 148: 1153-1167PubMed Google Scholar), collagen type I (20Poncelet A.C. Schnaper H.W. Am. J. Physiol. 1998; 275: F458-F466Crossref PubMed Google Scholar), and TGF-β1 (22Yamamoto T. Noble N.A. Cohen A.H. Nast C.C. Hishida A. Gold L.I. Border W.A. Kidney Int. 1996; 49: 461-469Abstract Full Text PDF PubMed Scopus (422) Google Scholar) were among the proteins shown to be augmented during mesangioproliferative glomerulonephritis, we examined the effect of SPARC on the expression of collagen type I and TGF-β1 in mesangial cells cultured from wild-type and SPARC-null mice. The following results were observed in five independent preparations of mesangial cells isolated from pools of eight kidneys each. The experiments were performed four times with all five preparations. Means ± S.D. were calculated for all experiments. One of the first observations we made was that SPARC-null mesangial cells proliferated faster and exhibited a more rounded, cobblestone-like cell shape in comparison to their wild-type counterparts (35Bradshaw A.D. Francki A. Motamed K. Howe C.C. Sage E.H. Mol. Biol. Cell. 1999; 10: 1569-1579Crossref PubMed Scopus (88) Google Scholar). Confluent monolayers of SPARC-null mesangial cells displayed very few of the hillocks (localized accumulations of extracellular matrix) that typify mesangial cell cultures. Mesangial cells also produced diminished levels of collagen type I, as shown in Fig. 1. Wild-type cells incubated with anti-collagen type I antibody exhibited a typical granular staining pattern throughout the cytoplasm (A), whereas SPARC-null cells showed significantly less staining for collagen type I (B). Immunoreactivity with an irrelevant antibody or the secondary antibody alone was negative (data not shown). The amount of procollagen type I secreted by the SPARC-null cells was also considerably diminished, relative to levels produced by wild-type cells (Fig. 2, lanes 1 and3). Digestion of the secreted protein with collagenase (Fig.2, lanes 2 and 4) prior to SDS-PAGE confirmed the identity of these bands as procollagen and its processed α chains. Under the conditions used for SDS-PAGE, the α1(I) and the α2(I) chains comigrated in our metabolic labeling and immunoblotting experiments. Collagen type I mRNA was detectable in mesangial cells by RT-PCR. The initial amounts of reverse-transcribed mRNAs for α1(I) were significantly lower in the SPARC-null cells (lane 2) in comparison with those in wild-type cells (lane 1) (Fig.3 A). By scanning densitometry with β-tubulin or rpS6 as an internal control, we found consistently diminished levels of α1(I) mRNA relative to that of wild-type cells, with a mean value of 44 ± 3%, a decrease of 2.2-fold (Table I). Similar decreases in α2(I) mRNA were seen in SPARC-null cells by RT-PCR. Relative to wild-type cells, levels of α2(I) mRNA in SPARC-null cells were diminished by 53 ± 4% (1.9-fold). Since the changes in expression of α2(I) were quantitatively similar to those of the α1(I) chain, only the latter has been shown.Table IExpression of collagen type I mRNA in wild-type and SPARC-null mesangial cellsWild-typeSPARC-null1. Untreated control100%44 ± 3% (2.2-fold decrease)AdditionChanges in α1(I) mRNA expression2. rhSPARC (0.9 μm)1.3-FoldaLevel of α1(I) mRNA in treated cells relative to that of untreated cells (untreated wild-type cells are set at 100%, in bold). In comparison to wild-type cells, SPARC-null cells expressed 44% collagen type I mRNA (2.2-fold decrease, Row 1). Treatment with rhSPARC or rhTGF-β1 increased the expression of collagen type I mRNA by ∼1.3- and 1.4-fold, respectively (Rows 2 and 3) in wild-type cells, whereas in SPARC-null cells the expressio" @default.
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