JOURNAL OF CELLULAR PHYSIOLOGY 147:45&459 (1991)
Proteoglycan and Glycosaminoglycan Synthesis by Cultured Rat Mesangial Cells GERALD C. GROGGEL,* PETER HOVINGH, AND ALFRED LINKER Departments of Medicine (C.C.C., P.H.), Pathology (A.L.),, and Biochemistry (A.L.), University of Utah School of Medicine and Veterans Administration Medical Center, Salt Lake City, Utah 84132 The synthesis of metabolically labeled proteoglycans and glycosaminoglycans from medium, cell layer and substrate attached material by rat glomerular mesangial cells in culture was characterized. The cellular localization of the labeled proteoglycans and glycosaminoglycans was determined by treating the cells with Flavobacterial heparinase. Of the total sulfated glycosaminoglycans, 33% were heparan sulfate; 55% of the cell layer material was heparan sulfate; 80% of sulfated proteins in the medium were chondroitin sulfateiderrnatan sulfate. Putative glycosaminoglycan free chains of heparan sulfate and chondroitin sulfate were found in both the medium and cell layer; 95% of total proteoglycans and most (90%) of the putative heparan sulfate free chains were removed from the cell layer by the heparinase, whereas only 50% of the chondroitin sulfate and 25% of dermatan sulfate were removed. Large amounts of hyaluronic acid labeled with 3H glucosamine were found in the cell layer. In summary, approximately 60% ot total sulfated glycoproteins was in the form of putative glycosaminoglycan free chains. Thus rat mesangial cells may synthesize large amounts of putative glycosaminoglycan free chains, which may have biological functions in the glomerulus independent of proteoglycans.
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Proteo lycans are important components of the glomerulus w ere they play both structural and functional roles (Kanwar, 1984; Kanwar et al., 1984). In the glomerular basement membrane, they act to maintain both the size and charge selectivity (Kanwar, 1984). Heparan sulfate and chondroitin sulfate are the major proteoglycans found in the glomerulus; dermatan sulfate is present in smaller amounts. Changes in these proteoglycans, particularly heparan sulfate, have been found in a number of glomerular diseases including diabetes (Kanwar et al., 19831, puromycin aminonucleoside nephrosis (Grog el et al., 19871, experimental membranous nephropat y (Groggel et al., 1988), and congenital nephrotic syndrome (Vernier et al., 1983). In membranous nephropathy in the rabbit, changes in heparan sulfate were shown to correlate with changes in the charge-selectivity of the glomerular basement membrane. Recent work has also shown that heparan sulfate glycosaminoglycan is able to inhibit rat mesangial cell growth in culture (Groggel et al., 1990). But for those studies only the glycosaminoglycans were used rather than the intact proteoglycan. Both lomerular mesangial and epithelial cells in culture ave been shown to synthesize proteoglycans (Striker et al., 1980). In these studies, total sulfate incorporation into mesangial cells and supernatants was measured. Recent work has shown that certain cell lines synthesize glycosaminoglycan free chains and these appear to be external plasma membrane components distinct from membrane proteoglycan (Piepkorn et al., 1989).
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0 1991 WILEY-LISS, INC.
As the mesangium serves specific functions in the glomerulus (Mene et al., 1989) and as glycosaminoglycans may contribute to these, the present studies were carried out to determine the pattern of glycosaminoglycans synthesized, the characteristics and roportions of free chains to proteoglycans in the cel layer, and the cellular location of glycosaminoglycans in the cell-associated material.
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METHODS Reagents Media and additives for culture of rat mesangial cells were obtained from Cellgro (Mediatech, Washin on, DC), Irving Scientific (Santa Ana, CA), Sigma C emicals (St. Louis, MO), and Hyclone Laboratories (Logan, UT). Tissue culture plates and dishes were obtained from Falcon (Becton Dickinson, Lincoln Park, NJ). Chondroitin sulfate (shark cartilage) was obtained from Sigma. Crude Flavobacterial "heparinase," which degrades all glycosaminoglycans except keratan sulfate, was prepared as described by Linker and Hovingh (1972). Na2 135S104and D-6-["Hl glucosamine were obtained from New England Nuclear (Boston, MA). Chromatographic media was obtained from Pharmacia (Piscataway, NJ).
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Received May 29, 1990; accepted February 20, 1991. *To whom reprint requestsicorrespondence should be addressed at University of Vermont, D305 Given Bldg., Burlington, VT 05405.
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GROGGEL ET AL.
Mesangial cell culture Rat mesangial cells were cultured from male Sprague-Dawley rats (Simonsen, Gilroy, CA) (Harper et al., 1984; Gro gel et al., 1990). Briefly, the kidneys were perfused b oodless with cold sterile phosphatebuffered saline (PBS; pH 7.4). The kidneys were removed, and the cortex was separated from medulla, minced, and passed through nylon sieves (159,105, and 75 Fm). The resulting glomerular suspension was washed several times with sterile PBS. There was < 3% tubular contamination. The glomerular suspension was plated in 75-cmZ plastic flasks with 12 ml of RPMI-1640 medium enriched with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid buffer (15 mM, pH 7.35), supplemented with regular insulin (0.66 U/ ml), penicillin (100 U/ml), streptomycin (100 pg/ml), amphotericin B (0.25 pgiml), and 20% bovine calf serum. They were kept in 95% air-5% CO, in a humidified incubator at 37"C, 3040% of lomeruli attached, and initially epithelial cells starte to grow. But after 10 days, they were replaced by mesangial cells, which reached confluency after 15-20 days. At this time subcultures were performed by detaching the cells with 0.025% trypsin-0.5 mM EDTA in calcium- and magnesium-free PBS. The harvested cells were suspended in culture medium and lated in the flasks. Mesangial cells grew and reacheL$ confluency in 7-10 days. Cells were fed every 48 h. All experiments were performed using cells between passages 2 and 5 . Several criteria were used to establish the identity of the cultured cells. First, by phase-contrast microsco y using an inverted microscope, the cells had spind eshape morphology with prominent intracellular fibrillar structures. There were no polygonal-shape cells. Second, fibroblast contamination was assessed by growth in L-valine-deficient media supplemented with D-valine (Sigma). Third, immunofluorescent microscopy was performed b growing cells to subconfluency on glass coverslips. $he cells were fixed with 3.7% paraformaldehyde for 10 min at 22°C. After washing, the cells were incubated with specific antibody, washed, and reincubated with fluorescein isothiocyanate-antirabbit immunoglobulin G. The coverslips were mounted and examined by immunofluorescent microscopy. Specific antisera to myosin, actin, desmin, leukocyte common antigen, cytokeratin, and factor VII (ICN ImmunoBiological, Lisk, IL) were used. The results showed positive immunofluorescent staining for actin, myosin, and desmin and negative staining for leukocyte common antigen, cytokeratin, and factor VIII. Fourth, the effect of the aminonucleoside of puromycin (Sigma) was examined. The cells showed no evidence of toxicity upon exposure to puromycin.
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Labeling and extraction of cellular proteoglycans The cells were examined under exponential growth conditions. The endogenous cellular proteoglycans and glycosaminoglycans were metabolically labeled for 24 h using 60 pCiiml of 35S-sulfateand 7 pCi/ml of 3H glucosamine in complete medium. After labeling, the medium was removed and 5 mg of carrier chondroitin sulfate and cetylpyridinium chloride (0.5% final con-
centration) were added to it. The precipitate was collected, washed with NaC1-ethanol to remove the cetylpyridinium chloride, and taken up in 0.1% sodium dodecylsulfate. Labeled products of the cells were extracted as described (Woods et al., 1985; Pie korn et al., 1988). Briefly, the cells were washed wit phosphate-buffered saline and then treated for 30 min at 37°C with 0.2% Triton-X-100 in 2.5 mM Tris-HC1, pH 7.5, containing the following protease inhibitors: 0.2 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, 100 mM E-aminocaproic acid, 10 mM EDTA, and 5 mM benzamidine HC1. Two washes of the flasks were combined with the deter ent extracts. The substratum attached material (SA ) remaining after the above extraction steps was removed from the flask surfaces by treatment with 1%Triton X-100 and 4 M urea with the above-mentioned protease inhibitors for 30 min at 37°C. The surfaces were washed a second time with the solution and this was included. The preparations were added directly to the Sepharose CL-4B column. Isolation of proteoglycans and glycosamino lycans was performed on three separate mesangial ce 1 lines.
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ANALYTICAL METHODS The medium, cell layer, and SAM preparations obtained as above were added to 110 x 1.0 cm columns of Sepharose CL-4B, using 0.1% sodium dodecyl sulfate with 0.35 M NaC1, 50 mM Tris-HC1, pH 7.5, and the aforementioned protease inhibitors, as eluant (Piepkorn et al., 1989). A combination of specific enzymatic degradation, utilizing testicular hyaluronidase and chondroitinase ABC (Piepkorn et al., 19881,and nitrous acid deaminative depolymerization accom lished under the low pH (1.0)conditions described b hively and Conrad (1976),were used to identify speci ic glycosaminoglycans (GAGs).Material resistant to testicular hyaluronidase and nitrous acid degradation but sensitive to degradation by chondroitinase ABC was considered to be dermatan sulfate. Any dermatan sulfate with a low iduronic acid content would be included with the chondroitin sulfates using this procedure. The degradation products were separated on Sephadex G-50 Superfine (50 x 0.4 cm) with 0.2 M NaCl in 10% ethanol as the eluant. The radioactivity of the eluants was monitored and the uronic acid content of the carrier GAGs was used to track the eluants (Blumenkrantz and Asboe-Hansen, 1973). Identification of the products as proteoglycans or as putative GAG free chains was obtained by a combination of elution patterns on Sepharose CL-4B and by the absence or presence of a Kav shift on Sepharose CL-4B following treatment of the peaks with alkali (0.5 M NaOH in 0.025 M Na borohydride) for 24 h at 4°C. This treatment eliminates the GAG chains from the core peptide leading to a change in molecular weight. Procedure for cellular localization of proteoglycans and GAG free chains Cellular localization of the labeled products was probed by the use of GAG-degrading enzymes on the cell layers. The procedure involved the brief treatment of preconfluent cultures, prelabeled for their proteogly-
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GLYCOSAMINOGLYCAN SYNTHESIS BY MESANGIAL CELLS
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T
0-0
0-0
Flavobacter
PBS
6000
4000
2000
0 0.0
1
:
0.2
I
0.4
0.6
1 .o
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Kav Sepharose CL-4B Fig. 1. The elution pattern from Sepharose CL-4B of the cell-associatedGAGs treated with either PBS or Flavobacterial heparinase. After enzyme treatment (closed circles),there was a marked change in the elution profile, Open circles are cells treated with PBS, and closed circles are cells treated with Flavobacterial heparinase.
can and GAG free chain content by 35S sulfate or 3H glucosamine, with crude Flavobacterial heparinase. After removing the media and washing the cells two times with PBS, crude he arinase was added at concentration of 1mg/ml in P S and incubated for 30 min at 37°C or for 60 min at 4°C. Following this incubation, the cells were washed with PBS and extracted as described. The extracts were chromatographed on columns of Sepharose CL-4B to separate proteoglycans and GAG free chains. Fractions were treated with alkaline borohydride and refractionated on Sepharose CL4B. A significant shift in Kav toward a lower molecular weight was taken as evidence for proteoglycans and an absence of shift was used to identify putative GAG free chains. Control cells were treated with PBS alone. RESULTS The elution pattern from Sepharose CL-4B of the 0.2% Triton extract, which represents the cell associated GAGs, is shown in Figure 1 (open circles). The eluates were divided into five fractions as indicated, isolated, treated with alkaline borohydride, and reapplied to Sepharose CL-4B columns. Table 1 shows the molecular weights of the fractions and also the effects of alkali treatment. Fractions A, B, and C can be seen to be proteoglycans differing in size; fractions D and E appear to be putative free GAG chains as no si nificant shift in molecular weight occurred after alka ine degradation (Table 1).The presence of a small peptide cannot, however, be excluded, and so these are referred to as putative free chains. The five fractions were also analyzed for their GAG components. As shown in Table 2, the composition of these fractions is quite variable. The highest molecular weight material (peak A) contains chondroitin sulfates only. Heparan sulfate proteoglycan appears mainly in peak C, whereas the putative free chain fractions contain similar amounts of chondroitin sulfates and
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TABLE 1. Molecular weight distribution of glycosaminoglycans Fraction'
A B C D
E SAM4
Before alkali
After alkali
Kav2
Kav2 0.54 (63)3 0.56 (55) 0.59 (44) 0.63 (32) 0.80 (8) 0.65 (28)
0.0 (>1,000)3 0.11, 0.25 (>1,000,630) 0.40 (200) 0.60 (40) 0.80 (8) 0.19; 0.44 (1,000; 138)
'See Figure 1. 'Obtained by elution from Sepharose CL-4B. 3Numbers in parentheses are Mr (in KDa). 'Substrate attached material (10% of total labeled malerial)
heparan sulfate. Dermatan sulfate is mainly present in the putative free chain fraction. The data show that heparan sulfate constitutes 33% of total sulfated GAGs. Significant amounts of hyaluronic acid were shown to be resent in the void volume fraction by treatment witK testicular hyaluronidase, when labeled glucosamine was used as a precursor. Location of GAGs was determined by incubating the cell layers with Flavobacterial enzymes. As shown in Figure 1 (closed circles), the elution profile on Sepharose was greatly altered after enzyme treatment. The numerical data presented in Table 2 show that 95% of the total proteoglycans (A, B, C) had been removed by the enzyme. The effect on putative free chains varied depending on their type. Most of the heparan sulfate had been digested; only about one-half of the chondroitin sulfate and very little of dermatan sulfate had been removed. This does imply a different location for the dermatan sulfate and half of the chondroitin sulfate free chains than that for the heparan sulfate. In two separate experiments, the hyaluronic acid seemed resistant to enzyme in one experiment; about 40% was removed in the other.
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GROGGEL ET AL
TABLE 2. Identification of glycosaminoglycans in the fractions from seuharose CL-4B
dpm (35,) X After Flavobacter Enzyme'
PBS Fraction2 A B C
D E
Total 12 77 140 202 138
SAM5
62
HS 0 43 126
CS3
69 68
12 34 9 86 44
27
28
DS 0 0 4
47 26 7
Total 0 3 9 90 133 15
HS
CS
DS
0 0 6 0 0
3 0 48 1334
0 0 36
12
3
'Counts remaining after treatment of cell layer with Flavobacterial enzyme. 'See Figure 1. 3No distinction between chondroitin 4 and 6 sulfate is made here. ?The increase includes both CS and DS in this quite low molecular weight fraction and may be due to a shift of material degraded by the enzyme from fraction D to fraction E. 5Substrate attached material.
Although the emphasis here was to determine composition and location of cell-associated material, we also examined the substrate attached material (SAM), obtained by further extraction of the flasks with 1% Triton X-100 and 4M urea; 10% of the total label was found in the SAM. The molecular weight of the proteoglycans is shown in Table 1. No free chains appear to be present. As shown in Table 2, about e ual amounts of chondroitin sulfate and heparan sul ate and small amounts of dermatan sulfate are present. The Flavobacterial enzyme (which had been used on the cell layer) had removed all of the heparan sulfate and more than half of the chondroitin sulfate. Proteoglycans in the medium, 57% of total GAGs, gave two eaks on Sepharose CL-4B with molecular weights o 218 and 100 KDa respectively. After alkaline degradation the GAG chains had 158 and 69 KDa molecular weights, indicating large chains on relatively small protein cores. Over 80% of the total composition consisted of chondroitin sulfate and dermatan sulfate. This material was not further examined. DISCUSSION Glycosaminoglycans, using the most general term, have been implicated in a large variety of biological functions in connective tissue matrices, basement membranes, and also in cell-related functions such as cell-to-cell interaction, cell-to-matrix binding, growth factor attachment, and cell growth regulation (Iozzo, 1985; Ruoslahti, 1988). Six or so closely related but distinct GAGs are widely distributed in most animal organisms. They commonly, but not always, occur in the form of proteoglycans with protein cores varying in size and structure and substituted with GAG chains differing in type, size, structural details, and number of chains er core protein. This high degree of variability raises t e question of specificity versus a more eneral function related to particular cell types an2 their biological role. The kidney glomerulus, a com lex organ, contains three major cell types: endothe ial, mesangial, and epithelial. Each of these plays a different role in the overall processes carried out by the glomerulus. Differences in function may partly be reflected in GAG distribution, structure, and cellular location. This is to
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some extent supported by the published data. Glomerular epithelial cells from mice were reported to primarily synthesize a basement membrane type of heparan sulfate associated with the cell membrane and only small amounts of chondroitin sulfate (Stow et al., 19891, whereas human lomerular epithelial cells produced equal amounts of 0th (Klein et al., 1990a). Free GAG chains were also detected but assumed to be intracellular. Rat glomerular epithelial cells were shown to secrete heparan sulfate and rnesangial cells were reported to synthesize mainly chondroitin sulfate proteoglycans and only minor amounts of heparan sulfate (Striker et al., 1980). Rat glomerular endothelial cells have been shown to secrete heparan sulfate (Castellot et al., 1986). The results of the present study indicate that heparan sulfate constitutes 55% of the cell-associated polymers, whereas the medium GAGs consisted of about 80%chondroitin sulfate and a large component of both these GAGs are in the form of putative free chains. Figure 1 shows the overall size distribution of total GAGs isolated from the cell layer. The data in Table 1 based on the Sepharose elution profile and the effects of alkali treatment show that fractions A, B and C are roteoglycans, whereas D and E appear to be putative free chains or chains attached t o a very small peptide. It is of interest that the size of the polysaccharide in the major putative free chain fraction (D) is smaller than the size of the chains attached to the rotein core in the proteoglycans. Table 2 shows the istribution of the component GAGs. These data are in contradiction to a recent publication by Yaoita et al. (1990), which reported that cultured rat mesangial cells synthesized mainly chondroitin sulfates, more than 90% of total GAGs. But for these studies mesangial cells in a superconfluent state were used. It is possible that in this superconfluent state, the pattern of GAGS synthesis is changed. It may be of interest in this respect that Beavan et al. (1988) reported that when synthesis of proteoglycans by rat renal glomeruli was examined in vivo, mostly heparan sulfate was synthesized, whereas in vitro mainly chondroitin sulfate was found. A recent work by Klein et al. (1990) reports that chondroitin sulfates are the major GAG component synthesized by human mesangial cells. Lelonget et al. (1988) have demonstrated synthesis of large-size chondroitin sulfates by embryonic mouse kidneys. It can also be seen that the ratio of putative free chains (D and E) to proteoglycans equals about 1.5. That is, more material is present in the putative free chain forms. This compares with a ratio of about 2:l in BALBic 3T3 cells (Piepkorn et al., 1989) and in keratinocytes (Piepkorn et al., 1987). As pointed out above, it is possible that the putative free chains may be attached to a small peptide. This is difficult to determine, as a change in molecular weight after alkaline borohydride treatment would not be apparent, and as in our ex erience and that of others (Funderburgh and Conra , 1990; Klein et al., 19901, contaminating proteins that appear to accompany proteoglycans are very difficult to remove. Regardless, as pointed out below, fairly small GAGs are usually assigned to the cell interior, whereas we here show a cell surface location.
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GLYCOSAMINOGLYCANSYNTHESIS BY MESANGlAL CELLS
Cellular location of GAGs was determined by treatment of cell layers with Flavobacterial enzymes. As shown in Figure 1 and Table 2, essentially all proteolycans were removed by the enzymes, whereas a Sifferential effect was seen for putative free chains, about 90% of the heparan sulfate was removed, but only about 55% of the chondroitin sulfate and 25% of the dermatan sulfate. This is of considerable interest as removal by the enzymes indicates cell surface location (Piepkorn et al., 19891, whereas resistance may indicate a location in the cell interior, on the inside of the membrane, or blocking of enzyme activity. This is more conclusive than the usual trypsin treatment. The differential effect of the enzyme on heparan sulfate and chondroitin sulfate may, therefore, be quite significant, as it implies different location, and location certainly is likely to play a role in function. Specificity related to location is also indicated by variability when different cells are compared. In BALBic 3T3 cells, most of the total free GAG chains appear to be present on the plasma membrane surface (Piepkorn et al., 1989), whereas in keratinocytes free chains appeared not to be accessible (Piepkorn et al., 1987) probably indicating a different location. The substrate attached material (SAM) is of interest as in this case it represents a fairly large percentage of total GAGs and as it is reputed to play a role in cytoskeletal-matrix interaction (Woodset al., 1985).As our emphasis was on cell-associated GAGs, we did not examine the SAM fraction in detail but as indicated under Results, most of total proteoglycans and putative free chains were degraded by the Flavobacterial enzyme, and both polymers appeared to be of somewhat larger size than the corresponding material associated with the cells. The significance of these observations may be related to recent observations that certain heparan sulfate GAGs were able to inhibit the growth of rat mesan ial cells in vitro (Groggel et al., 1990).The present stu ies demonstrate that putative GAG free chains are synthesized by mesangial cells. These putative free chains may play a role in the regulation of mesangial cell growth. The presence of these putative GAG free chains suggests that they may have biological roles separate from those of proteoglycans.
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ACKNOWLEDGMENTS G.C.G. is a recipient of the National Institutes of Health Clinical Investigator Award DK-01603. This work was supported by the Veterans Administration.
LITERATURE CITED Beavan, L.A., Davies, M., and Mason, R.M. (1988) Renal glomerular proteoglycans. Biochem. J., 251 ;411418. Blumenkrantz, N., and Asboe-Hansen, G. (1973) New method for quantitative determination of uronic acids. Biochem., 54:484-489. Castellot, J.J.,Hoover, R.L., and Karnovsky, M.J. (1986) Glomerular endothelial cells secrete a heparinlike inhibitor and a peptide stimulator of mesangial cell proliferation. Am. J. Pathol., 125t493500.
Funderburgh, J.L., and Conrad, G.W. (1990) Isoforms of corneal heparan sulfate proteoglycan. J. Biol. Chem., 265.S297-8303. Groggel, G,C., Hovingh, P., Border, W.A., and Linker, A. (1987) Changes in glornerular heparan sulfate in puromycin aminonucleoside nephrosis. Amer. J. Path., 128.521-527. Groggel, G.C., Stevenson, J., Hovingh, P., Linker, A., and Border, W.A. (1988) Changes in heparan sulfate correlate with increased glomerular permeability. Kidney Int., 33.517-523. Groggel, G.C., Marinedes, G.M., Hovingh, P., Hammond, E., and Linker, A. (1990)Inhibition of rat mesangial cell growth by heparan sulfate. Am. J. Physiol., 258 (Renal Fluid Electrolyte Physiol. 27):F259-F265. Harper, P.A., Robinson, J.M., Hoover, R.L., Wright, T.C., and Karnovsky, M.J. (1984) Improved methods for culturing rat glomerular cells. Kidney Int., 26:875-880. Iozzo, R.U. 11985) Proteoglycans: Structure, function and role in neoplasia. Lab. Invest., 53:373-395. Kanwar, Y.S. (1984) Biophysiology of glomerular filtration and proteinuria. Lab. Invest., fil:7-21. Kanwar, Y.S., Rosenzweig, L.J., Linker, A., and Jakubowski, M.L. (1983) Decreased de novo synthesis of glomerular proteoglycans in diabetes. Biochemical and autoradiographic evidence. Proc. Natl. Acad. Sci. USA, 80:2272-2275. Kanwar, Y.S., Veis, A., Kimura, J.H., and Jakubowski, M.L. (1984) Characterization of heparan sulfate-proteoglycan of glomerular basement membranes. Proc. Natl. Acad. Sci. USA, 81:762-766. Klein, D.J., Brown, D.M., Kim, Y., and Oegema, T.R. (1990) Proteonlvcans svnthesized bv human elomerular mesannial cells in cul;ire. J . B h . Chem., i65:9533-$543. Klein, D.J., Oegema, T.R., Fredeen, T.S., Van der Wurde, F., Kim, Y . , and Brown. D.M. 11990ai Partial characterization of Droteodvcans synthesized by human glomerular epithelial cells in culture. ‘krch. Biochem. Biophys., 277:389401. Leloneet. B.. Makino. H.. Dalecki. T.M.. and Kanwar. Y.S. (1988)Role of Goteoglycans in renal development. Dev. Biol.,‘ 128:256-276. Linker, A,, and Hovingh, P. (1972) Heparinase and Heparitinase from Flavobacteria. In: Methods in Enzymology. U.Ginsburg, ed. Academic Press, New York, pp. 902-911. Mene, P., Simonson, M.S., and Dunn, M.J. (1989) Physiology of the mesangial cell. Physiol. Reviews, 69:1347-1424. Piepkorn, M.! Fleckman, P., Carney, H., and Linker, A. (19871 Glycosaminoglycan synthesis by proliferating and differentiated human keratinocytes in culture. J. Invest. Dermatol., 88:215-219. Piepkorn, M., Hovingh, P., and Linker, A. (1988) Evidence for independent metabolism and cell surface localization of cellular proteoglycans and glycosaminoglycan free chains. J. Cell Physiol., 135r189-199. Piepkorn, M., Hovingh, P., and Linker, A. (1989) Glycosaminoglycan free chains. External plasma membrane components distinct from the membrane proteoglycans. J. Biol. Chem., 264:866243669. Shively, J.E., and Conrad, H.E. (1976)Formation of anhydro-sugars in the chemical depolymerization of heparin. Biochemistry, 15:39323942. Stow, J.L., Soroka, C.J., MacKay, K., Striker, L., Striker, G., and Farquhar, M.G. (1989) Basement membrane heparan sulfate proteoglycan is the main proteoglycan synthesized by glomerular epithelial cells in culture. Am. J. Pathol., 135:637-646. Striker, G.E., Killen, P.D., and Farin, F.M. (1980) Human glomurular cells in vitro: Isolation and characterization. Transpl. Proceed. 12:88-99. Rusolahti, E. (1988)Structure and biology of proteoglycanu. Ann. Rev. Cell Biol., 4:22%255. Vernier, R.L., Klein, D.J., Sisson, S.P., Mahan, J.D., Oegema, T.R., and Brown, D.M. (1983) Heparan sulfate-rich anionic sites in the human glomerular basement membrane. Decreased concentration in congenital nephrotic syndrome. N. Engl. J. Med.. 309:lOOl-1008. Woods, A., Couchrnan, J.R., and Hook, M. (1985) Heparan sulfate proteoglycans of rat. embryo fibroblasts. A hydrophobic form may link cytoskeleton and matrix components. J. Biol. Chem., 260:10872-10879. Yaoita, E., Oguri, K., Okayama, E., Kawasaki, K., Kobayashi, S., Kihara, I., and Okavama, M. (1990) Isolation and characterization of proteoglycans synthesized by cultured mesangial cells. J. Biol. Chem., 265:522-531. v
Proteoglycan and Glycosaminoglycan Synthesis by Cultured Rat Mesangial Cells GERALD C. GROGGEL,* PETER HOVINGH, AND ALFRED LINKER Departments of Medicine (C.C.C., P.H.), Pathology (A.L.),, and Biochemistry (A.L.), University of Utah School of Medicine and Veterans Administration Medical Center, Salt Lake City, Utah 84132 The synthesis of metabolically labeled proteoglycans and glycosaminoglycans from medium, cell layer and substrate attached material by rat glomerular mesangial cells in culture was characterized. The cellular localization of the labeled proteoglycans and glycosaminoglycans was determined by treating the cells with Flavobacterial heparinase. Of the total sulfated glycosaminoglycans, 33% were heparan sulfate; 55% of the cell layer material was heparan sulfate; 80% of sulfated proteins in the medium were chondroitin sulfateiderrnatan sulfate. Putative glycosaminoglycan free chains of heparan sulfate and chondroitin sulfate were found in both the medium and cell layer; 95% of total proteoglycans and most (90%) of the putative heparan sulfate free chains were removed from the cell layer by the heparinase, whereas only 50% of the chondroitin sulfate and 25% of dermatan sulfate were removed. Large amounts of hyaluronic acid labeled with 3H glucosamine were found in the cell layer. In summary, approximately 60% ot total sulfated glycoproteins was in the form of putative glycosaminoglycan free chains. Thus rat mesangial cells may synthesize large amounts of putative glycosaminoglycan free chains, which may have biological functions in the glomerulus independent of proteoglycans.
a
Proteo lycans are important components of the glomerulus w ere they play both structural and functional roles (Kanwar, 1984; Kanwar et al., 1984). In the glomerular basement membrane, they act to maintain both the size and charge selectivity (Kanwar, 1984). Heparan sulfate and chondroitin sulfate are the major proteoglycans found in the glomerulus; dermatan sulfate is present in smaller amounts. Changes in these proteoglycans, particularly heparan sulfate, have been found in a number of glomerular diseases including diabetes (Kanwar et al., 19831, puromycin aminonucleoside nephrosis (Grog el et al., 19871, experimental membranous nephropat y (Groggel et al., 1988), and congenital nephrotic syndrome (Vernier et al., 1983). In membranous nephropathy in the rabbit, changes in heparan sulfate were shown to correlate with changes in the charge-selectivity of the glomerular basement membrane. Recent work has also shown that heparan sulfate glycosaminoglycan is able to inhibit rat mesangial cell growth in culture (Groggel et al., 1990). But for those studies only the glycosaminoglycans were used rather than the intact proteoglycan. Both lomerular mesangial and epithelial cells in culture ave been shown to synthesize proteoglycans (Striker et al., 1980). In these studies, total sulfate incorporation into mesangial cells and supernatants was measured. Recent work has shown that certain cell lines synthesize glycosaminoglycan free chains and these appear to be external plasma membrane components distinct from membrane proteoglycan (Piepkorn et al., 1989).
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0 1991 WILEY-LISS, INC.
As the mesangium serves specific functions in the glomerulus (Mene et al., 1989) and as glycosaminoglycans may contribute to these, the present studies were carried out to determine the pattern of glycosaminoglycans synthesized, the characteristics and roportions of free chains to proteoglycans in the cel layer, and the cellular location of glycosaminoglycans in the cell-associated material.
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METHODS Reagents Media and additives for culture of rat mesangial cells were obtained from Cellgro (Mediatech, Washin on, DC), Irving Scientific (Santa Ana, CA), Sigma C emicals (St. Louis, MO), and Hyclone Laboratories (Logan, UT). Tissue culture plates and dishes were obtained from Falcon (Becton Dickinson, Lincoln Park, NJ). Chondroitin sulfate (shark cartilage) was obtained from Sigma. Crude Flavobacterial "heparinase," which degrades all glycosaminoglycans except keratan sulfate, was prepared as described by Linker and Hovingh (1972). Na2 135S104and D-6-["Hl glucosamine were obtained from New England Nuclear (Boston, MA). Chromatographic media was obtained from Pharmacia (Piscataway, NJ).
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Received May 29, 1990; accepted February 20, 1991. *To whom reprint requestsicorrespondence should be addressed at University of Vermont, D305 Given Bldg., Burlington, VT 05405.
456
GROGGEL ET AL.
Mesangial cell culture Rat mesangial cells were cultured from male Sprague-Dawley rats (Simonsen, Gilroy, CA) (Harper et al., 1984; Gro gel et al., 1990). Briefly, the kidneys were perfused b oodless with cold sterile phosphatebuffered saline (PBS; pH 7.4). The kidneys were removed, and the cortex was separated from medulla, minced, and passed through nylon sieves (159,105, and 75 Fm). The resulting glomerular suspension was washed several times with sterile PBS. There was < 3% tubular contamination. The glomerular suspension was plated in 75-cmZ plastic flasks with 12 ml of RPMI-1640 medium enriched with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid buffer (15 mM, pH 7.35), supplemented with regular insulin (0.66 U/ ml), penicillin (100 U/ml), streptomycin (100 pg/ml), amphotericin B (0.25 pgiml), and 20% bovine calf serum. They were kept in 95% air-5% CO, in a humidified incubator at 37"C, 3040% of lomeruli attached, and initially epithelial cells starte to grow. But after 10 days, they were replaced by mesangial cells, which reached confluency after 15-20 days. At this time subcultures were performed by detaching the cells with 0.025% trypsin-0.5 mM EDTA in calcium- and magnesium-free PBS. The harvested cells were suspended in culture medium and lated in the flasks. Mesangial cells grew and reacheL$ confluency in 7-10 days. Cells were fed every 48 h. All experiments were performed using cells between passages 2 and 5 . Several criteria were used to establish the identity of the cultured cells. First, by phase-contrast microsco y using an inverted microscope, the cells had spind eshape morphology with prominent intracellular fibrillar structures. There were no polygonal-shape cells. Second, fibroblast contamination was assessed by growth in L-valine-deficient media supplemented with D-valine (Sigma). Third, immunofluorescent microscopy was performed b growing cells to subconfluency on glass coverslips. $he cells were fixed with 3.7% paraformaldehyde for 10 min at 22°C. After washing, the cells were incubated with specific antibody, washed, and reincubated with fluorescein isothiocyanate-antirabbit immunoglobulin G. The coverslips were mounted and examined by immunofluorescent microscopy. Specific antisera to myosin, actin, desmin, leukocyte common antigen, cytokeratin, and factor VII (ICN ImmunoBiological, Lisk, IL) were used. The results showed positive immunofluorescent staining for actin, myosin, and desmin and negative staining for leukocyte common antigen, cytokeratin, and factor VIII. Fourth, the effect of the aminonucleoside of puromycin (Sigma) was examined. The cells showed no evidence of toxicity upon exposure to puromycin.
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Labeling and extraction of cellular proteoglycans The cells were examined under exponential growth conditions. The endogenous cellular proteoglycans and glycosaminoglycans were metabolically labeled for 24 h using 60 pCiiml of 35S-sulfateand 7 pCi/ml of 3H glucosamine in complete medium. After labeling, the medium was removed and 5 mg of carrier chondroitin sulfate and cetylpyridinium chloride (0.5% final con-
centration) were added to it. The precipitate was collected, washed with NaC1-ethanol to remove the cetylpyridinium chloride, and taken up in 0.1% sodium dodecylsulfate. Labeled products of the cells were extracted as described (Woods et al., 1985; Pie korn et al., 1988). Briefly, the cells were washed wit phosphate-buffered saline and then treated for 30 min at 37°C with 0.2% Triton-X-100 in 2.5 mM Tris-HC1, pH 7.5, containing the following protease inhibitors: 0.2 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, 100 mM E-aminocaproic acid, 10 mM EDTA, and 5 mM benzamidine HC1. Two washes of the flasks were combined with the deter ent extracts. The substratum attached material (SA ) remaining after the above extraction steps was removed from the flask surfaces by treatment with 1%Triton X-100 and 4 M urea with the above-mentioned protease inhibitors for 30 min at 37°C. The surfaces were washed a second time with the solution and this was included. The preparations were added directly to the Sepharose CL-4B column. Isolation of proteoglycans and glycosamino lycans was performed on three separate mesangial ce 1 lines.
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ANALYTICAL METHODS The medium, cell layer, and SAM preparations obtained as above were added to 110 x 1.0 cm columns of Sepharose CL-4B, using 0.1% sodium dodecyl sulfate with 0.35 M NaC1, 50 mM Tris-HC1, pH 7.5, and the aforementioned protease inhibitors, as eluant (Piepkorn et al., 1989). A combination of specific enzymatic degradation, utilizing testicular hyaluronidase and chondroitinase ABC (Piepkorn et al., 19881,and nitrous acid deaminative depolymerization accom lished under the low pH (1.0)conditions described b hively and Conrad (1976),were used to identify speci ic glycosaminoglycans (GAGs).Material resistant to testicular hyaluronidase and nitrous acid degradation but sensitive to degradation by chondroitinase ABC was considered to be dermatan sulfate. Any dermatan sulfate with a low iduronic acid content would be included with the chondroitin sulfates using this procedure. The degradation products were separated on Sephadex G-50 Superfine (50 x 0.4 cm) with 0.2 M NaCl in 10% ethanol as the eluant. The radioactivity of the eluants was monitored and the uronic acid content of the carrier GAGs was used to track the eluants (Blumenkrantz and Asboe-Hansen, 1973). Identification of the products as proteoglycans or as putative GAG free chains was obtained by a combination of elution patterns on Sepharose CL-4B and by the absence or presence of a Kav shift on Sepharose CL-4B following treatment of the peaks with alkali (0.5 M NaOH in 0.025 M Na borohydride) for 24 h at 4°C. This treatment eliminates the GAG chains from the core peptide leading to a change in molecular weight. Procedure for cellular localization of proteoglycans and GAG free chains Cellular localization of the labeled products was probed by the use of GAG-degrading enzymes on the cell layers. The procedure involved the brief treatment of preconfluent cultures, prelabeled for their proteogly-
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457
GLYCOSAMINOGLYCAN SYNTHESIS BY MESANGIAL CELLS
8ooo
T
0-0
0-0
Flavobacter
PBS
6000
4000
2000
0 0.0
1
:
0.2
I
0.4
0.6
1 .o
0.8
Kav Sepharose CL-4B Fig. 1. The elution pattern from Sepharose CL-4B of the cell-associatedGAGs treated with either PBS or Flavobacterial heparinase. After enzyme treatment (closed circles),there was a marked change in the elution profile, Open circles are cells treated with PBS, and closed circles are cells treated with Flavobacterial heparinase.
can and GAG free chain content by 35S sulfate or 3H glucosamine, with crude Flavobacterial heparinase. After removing the media and washing the cells two times with PBS, crude he arinase was added at concentration of 1mg/ml in P S and incubated for 30 min at 37°C or for 60 min at 4°C. Following this incubation, the cells were washed with PBS and extracted as described. The extracts were chromatographed on columns of Sepharose CL-4B to separate proteoglycans and GAG free chains. Fractions were treated with alkaline borohydride and refractionated on Sepharose CL4B. A significant shift in Kav toward a lower molecular weight was taken as evidence for proteoglycans and an absence of shift was used to identify putative GAG free chains. Control cells were treated with PBS alone. RESULTS The elution pattern from Sepharose CL-4B of the 0.2% Triton extract, which represents the cell associated GAGs, is shown in Figure 1 (open circles). The eluates were divided into five fractions as indicated, isolated, treated with alkaline borohydride, and reapplied to Sepharose CL-4B columns. Table 1 shows the molecular weights of the fractions and also the effects of alkali treatment. Fractions A, B, and C can be seen to be proteoglycans differing in size; fractions D and E appear to be putative free GAG chains as no si nificant shift in molecular weight occurred after alka ine degradation (Table 1).The presence of a small peptide cannot, however, be excluded, and so these are referred to as putative free chains. The five fractions were also analyzed for their GAG components. As shown in Table 2, the composition of these fractions is quite variable. The highest molecular weight material (peak A) contains chondroitin sulfates only. Heparan sulfate proteoglycan appears mainly in peak C, whereas the putative free chain fractions contain similar amounts of chondroitin sulfates and
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TABLE 1. Molecular weight distribution of glycosaminoglycans Fraction'
A B C D
E SAM4
Before alkali
After alkali
Kav2
Kav2 0.54 (63)3 0.56 (55) 0.59 (44) 0.63 (32) 0.80 (8) 0.65 (28)
0.0 (>1,000)3 0.11, 0.25 (>1,000,630) 0.40 (200) 0.60 (40) 0.80 (8) 0.19; 0.44 (1,000; 138)
'See Figure 1. 'Obtained by elution from Sepharose CL-4B. 3Numbers in parentheses are Mr (in KDa). 'Substrate attached material (10% of total labeled malerial)
heparan sulfate. Dermatan sulfate is mainly present in the putative free chain fraction. The data show that heparan sulfate constitutes 33% of total sulfated GAGs. Significant amounts of hyaluronic acid were shown to be resent in the void volume fraction by treatment witK testicular hyaluronidase, when labeled glucosamine was used as a precursor. Location of GAGs was determined by incubating the cell layers with Flavobacterial enzymes. As shown in Figure 1 (closed circles), the elution profile on Sepharose was greatly altered after enzyme treatment. The numerical data presented in Table 2 show that 95% of the total proteoglycans (A, B, C) had been removed by the enzyme. The effect on putative free chains varied depending on their type. Most of the heparan sulfate had been digested; only about one-half of the chondroitin sulfate and very little of dermatan sulfate had been removed. This does imply a different location for the dermatan sulfate and half of the chondroitin sulfate free chains than that for the heparan sulfate. In two separate experiments, the hyaluronic acid seemed resistant to enzyme in one experiment; about 40% was removed in the other.
458
GROGGEL ET AL
TABLE 2. Identification of glycosaminoglycans in the fractions from seuharose CL-4B
dpm (35,) X After Flavobacter Enzyme'
PBS Fraction2 A B C
D E
Total 12 77 140 202 138
SAM5
62
HS 0 43 126
CS3
69 68
12 34 9 86 44
27
28
DS 0 0 4
47 26 7
Total 0 3 9 90 133 15
HS
CS
DS
0 0 6 0 0
3 0 48 1334
0 0 36
12
3
'Counts remaining after treatment of cell layer with Flavobacterial enzyme. 'See Figure 1. 3No distinction between chondroitin 4 and 6 sulfate is made here. ?The increase includes both CS and DS in this quite low molecular weight fraction and may be due to a shift of material degraded by the enzyme from fraction D to fraction E. 5Substrate attached material.
Although the emphasis here was to determine composition and location of cell-associated material, we also examined the substrate attached material (SAM), obtained by further extraction of the flasks with 1% Triton X-100 and 4M urea; 10% of the total label was found in the SAM. The molecular weight of the proteoglycans is shown in Table 1. No free chains appear to be present. As shown in Table 2, about e ual amounts of chondroitin sulfate and heparan sul ate and small amounts of dermatan sulfate are present. The Flavobacterial enzyme (which had been used on the cell layer) had removed all of the heparan sulfate and more than half of the chondroitin sulfate. Proteoglycans in the medium, 57% of total GAGs, gave two eaks on Sepharose CL-4B with molecular weights o 218 and 100 KDa respectively. After alkaline degradation the GAG chains had 158 and 69 KDa molecular weights, indicating large chains on relatively small protein cores. Over 80% of the total composition consisted of chondroitin sulfate and dermatan sulfate. This material was not further examined. DISCUSSION Glycosaminoglycans, using the most general term, have been implicated in a large variety of biological functions in connective tissue matrices, basement membranes, and also in cell-related functions such as cell-to-cell interaction, cell-to-matrix binding, growth factor attachment, and cell growth regulation (Iozzo, 1985; Ruoslahti, 1988). Six or so closely related but distinct GAGs are widely distributed in most animal organisms. They commonly, but not always, occur in the form of proteoglycans with protein cores varying in size and structure and substituted with GAG chains differing in type, size, structural details, and number of chains er core protein. This high degree of variability raises t e question of specificity versus a more eneral function related to particular cell types an2 their biological role. The kidney glomerulus, a com lex organ, contains three major cell types: endothe ial, mesangial, and epithelial. Each of these plays a different role in the overall processes carried out by the glomerulus. Differences in function may partly be reflected in GAG distribution, structure, and cellular location. This is to
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some extent supported by the published data. Glomerular epithelial cells from mice were reported to primarily synthesize a basement membrane type of heparan sulfate associated with the cell membrane and only small amounts of chondroitin sulfate (Stow et al., 19891, whereas human lomerular epithelial cells produced equal amounts of 0th (Klein et al., 1990a). Free GAG chains were also detected but assumed to be intracellular. Rat glomerular epithelial cells were shown to secrete heparan sulfate and rnesangial cells were reported to synthesize mainly chondroitin sulfate proteoglycans and only minor amounts of heparan sulfate (Striker et al., 1980). Rat glomerular endothelial cells have been shown to secrete heparan sulfate (Castellot et al., 1986). The results of the present study indicate that heparan sulfate constitutes 55% of the cell-associated polymers, whereas the medium GAGs consisted of about 80%chondroitin sulfate and a large component of both these GAGs are in the form of putative free chains. Figure 1 shows the overall size distribution of total GAGs isolated from the cell layer. The data in Table 1 based on the Sepharose elution profile and the effects of alkali treatment show that fractions A, B and C are roteoglycans, whereas D and E appear to be putative free chains or chains attached t o a very small peptide. It is of interest that the size of the polysaccharide in the major putative free chain fraction (D) is smaller than the size of the chains attached to the rotein core in the proteoglycans. Table 2 shows the istribution of the component GAGs. These data are in contradiction to a recent publication by Yaoita et al. (1990), which reported that cultured rat mesangial cells synthesized mainly chondroitin sulfates, more than 90% of total GAGs. But for these studies mesangial cells in a superconfluent state were used. It is possible that in this superconfluent state, the pattern of GAGS synthesis is changed. It may be of interest in this respect that Beavan et al. (1988) reported that when synthesis of proteoglycans by rat renal glomeruli was examined in vivo, mostly heparan sulfate was synthesized, whereas in vitro mainly chondroitin sulfate was found. A recent work by Klein et al. (1990) reports that chondroitin sulfates are the major GAG component synthesized by human mesangial cells. Lelonget et al. (1988) have demonstrated synthesis of large-size chondroitin sulfates by embryonic mouse kidneys. It can also be seen that the ratio of putative free chains (D and E) to proteoglycans equals about 1.5. That is, more material is present in the putative free chain forms. This compares with a ratio of about 2:l in BALBic 3T3 cells (Piepkorn et al., 1989) and in keratinocytes (Piepkorn et al., 1987). As pointed out above, it is possible that the putative free chains may be attached to a small peptide. This is difficult to determine, as a change in molecular weight after alkaline borohydride treatment would not be apparent, and as in our ex erience and that of others (Funderburgh and Conra , 1990; Klein et al., 19901, contaminating proteins that appear to accompany proteoglycans are very difficult to remove. Regardless, as pointed out below, fairly small GAGs are usually assigned to the cell interior, whereas we here show a cell surface location.
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459
GLYCOSAMINOGLYCANSYNTHESIS BY MESANGlAL CELLS
Cellular location of GAGs was determined by treatment of cell layers with Flavobacterial enzymes. As shown in Figure 1 and Table 2, essentially all proteolycans were removed by the enzymes, whereas a Sifferential effect was seen for putative free chains, about 90% of the heparan sulfate was removed, but only about 55% of the chondroitin sulfate and 25% of the dermatan sulfate. This is of considerable interest as removal by the enzymes indicates cell surface location (Piepkorn et al., 19891, whereas resistance may indicate a location in the cell interior, on the inside of the membrane, or blocking of enzyme activity. This is more conclusive than the usual trypsin treatment. The differential effect of the enzyme on heparan sulfate and chondroitin sulfate may, therefore, be quite significant, as it implies different location, and location certainly is likely to play a role in function. Specificity related to location is also indicated by variability when different cells are compared. In BALBic 3T3 cells, most of the total free GAG chains appear to be present on the plasma membrane surface (Piepkorn et al., 1989), whereas in keratinocytes free chains appeared not to be accessible (Piepkorn et al., 1987) probably indicating a different location. The substrate attached material (SAM) is of interest as in this case it represents a fairly large percentage of total GAGs and as it is reputed to play a role in cytoskeletal-matrix interaction (Woodset al., 1985).As our emphasis was on cell-associated GAGs, we did not examine the SAM fraction in detail but as indicated under Results, most of total proteoglycans and putative free chains were degraded by the Flavobacterial enzyme, and both polymers appeared to be of somewhat larger size than the corresponding material associated with the cells. The significance of these observations may be related to recent observations that certain heparan sulfate GAGs were able to inhibit the growth of rat mesan ial cells in vitro (Groggel et al., 1990).The present stu ies demonstrate that putative GAG free chains are synthesized by mesangial cells. These putative free chains may play a role in the regulation of mesangial cell growth. The presence of these putative GAG free chains suggests that they may have biological roles separate from those of proteoglycans.
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ACKNOWLEDGMENTS G.C.G. is a recipient of the National Institutes of Health Clinical Investigator Award DK-01603. This work was supported by the Veterans Administration.
LITERATURE CITED Beavan, L.A., Davies, M., and Mason, R.M. (1988) Renal glomerular proteoglycans. Biochem. J., 251 ;411418. Blumenkrantz, N., and Asboe-Hansen, G. (1973) New method for quantitative determination of uronic acids. Biochem., 54:484-489. Castellot, J.J.,Hoover, R.L., and Karnovsky, M.J. (1986) Glomerular endothelial cells secrete a heparinlike inhibitor and a peptide stimulator of mesangial cell proliferation. Am. J. Pathol., 125t493500.
Funderburgh, J.L., and Conrad, G.W. (1990) Isoforms of corneal heparan sulfate proteoglycan. J. Biol. Chem., 265.S297-8303. Groggel, G,C., Hovingh, P., Border, W.A., and Linker, A. (1987) Changes in glornerular heparan sulfate in puromycin aminonucleoside nephrosis. Amer. J. Path., 128.521-527. Groggel, G.C., Stevenson, J., Hovingh, P., Linker, A., and Border, W.A. (1988) Changes in heparan sulfate correlate with increased glomerular permeability. Kidney Int., 33.517-523. Groggel, G.C., Marinedes, G.M., Hovingh, P., Hammond, E., and Linker, A. (1990)Inhibition of rat mesangial cell growth by heparan sulfate. Am. J. Physiol., 258 (Renal Fluid Electrolyte Physiol. 27):F259-F265. Harper, P.A., Robinson, J.M., Hoover, R.L., Wright, T.C., and Karnovsky, M.J. (1984) Improved methods for culturing rat glomerular cells. Kidney Int., 26:875-880. Iozzo, R.U. 11985) Proteoglycans: Structure, function and role in neoplasia. Lab. Invest., 53:373-395. Kanwar, Y.S. (1984) Biophysiology of glomerular filtration and proteinuria. Lab. Invest., fil:7-21. Kanwar, Y.S., Rosenzweig, L.J., Linker, A., and Jakubowski, M.L. (1983) Decreased de novo synthesis of glomerular proteoglycans in diabetes. Biochemical and autoradiographic evidence. Proc. Natl. Acad. Sci. USA, 80:2272-2275. Kanwar, Y.S., Veis, A., Kimura, J.H., and Jakubowski, M.L. (1984) Characterization of heparan sulfate-proteoglycan of glomerular basement membranes. Proc. Natl. Acad. Sci. USA, 81:762-766. Klein, D.J., Brown, D.M., Kim, Y., and Oegema, T.R. (1990) Proteonlvcans svnthesized bv human elomerular mesannial cells in cul;ire. J . B h . Chem., i65:9533-$543. Klein, D.J., Oegema, T.R., Fredeen, T.S., Van der Wurde, F., Kim, Y . , and Brown. D.M. 11990ai Partial characterization of Droteodvcans synthesized by human glomerular epithelial cells in culture. ‘krch. Biochem. Biophys., 277:389401. Leloneet. B.. Makino. H.. Dalecki. T.M.. and Kanwar. Y.S. (1988)Role of Goteoglycans in renal development. Dev. Biol.,‘ 128:256-276. Linker, A,, and Hovingh, P. (1972) Heparinase and Heparitinase from Flavobacteria. In: Methods in Enzymology. U.Ginsburg, ed. Academic Press, New York, pp. 902-911. Mene, P., Simonson, M.S., and Dunn, M.J. (1989) Physiology of the mesangial cell. Physiol. Reviews, 69:1347-1424. Piepkorn, M.! Fleckman, P., Carney, H., and Linker, A. (19871 Glycosaminoglycan synthesis by proliferating and differentiated human keratinocytes in culture. J. Invest. Dermatol., 88:215-219. Piepkorn, M., Hovingh, P., and Linker, A. (1988) Evidence for independent metabolism and cell surface localization of cellular proteoglycans and glycosaminoglycan free chains. J. Cell Physiol., 135r189-199. Piepkorn, M., Hovingh, P., and Linker, A. (1989) Glycosaminoglycan free chains. External plasma membrane components distinct from the membrane proteoglycans. J. Biol. Chem., 264:866243669. Shively, J.E., and Conrad, H.E. (1976)Formation of anhydro-sugars in the chemical depolymerization of heparin. Biochemistry, 15:39323942. Stow, J.L., Soroka, C.J., MacKay, K., Striker, L., Striker, G., and Farquhar, M.G. (1989) Basement membrane heparan sulfate proteoglycan is the main proteoglycan synthesized by glomerular epithelial cells in culture. Am. J. Pathol., 135:637-646. Striker, G.E., Killen, P.D., and Farin, F.M. (1980) Human glomurular cells in vitro: Isolation and characterization. Transpl. Proceed. 12:88-99. Rusolahti, E. (1988)Structure and biology of proteoglycanu. Ann. Rev. Cell Biol., 4:22%255. Vernier, R.L., Klein, D.J., Sisson, S.P., Mahan, J.D., Oegema, T.R., and Brown, D.M. (1983) Heparan sulfate-rich anionic sites in the human glomerular basement membrane. Decreased concentration in congenital nephrotic syndrome. N. Engl. J. Med.. 309:lOOl-1008. Woods, A., Couchrnan, J.R., and Hook, M. (1985) Heparan sulfate proteoglycans of rat. embryo fibroblasts. A hydrophobic form may link cytoskeleton and matrix components. J. Biol. Chem., 260:10872-10879. Yaoita, E., Oguri, K., Okayama, E., Kawasaki, K., Kobayashi, S., Kihara, I., and Okavama, M. (1990) Isolation and characterization of proteoglycans synthesized by cultured mesangial cells. J. Biol. Chem., 265:522-531. v
