ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 278, No. 1, April, pp. 11-20, 1990

Human Glomerular Gareth

J. Thomas,

Epithelial Cell Proteoglycans

Lucy Jenner,

Roger M. Mason,*

and Malcolm

Davies1

Institute of Nephrology, Department of Medicine, University of Wales College of Medicine, Royal Infirmary, Cardiff, CF2 ISZ, Wales, United Kingdom; and *Department of Biochemistry, Charing Cross and Westminster Hospital Medical School, University of London, Fulham Palace Road, London W6 8RF, United Kingdom

Received June 23, 1989, and in revised form October 23,1989

Proteoglycans synthesized by cultures of human glomerular epithelial cells have been isolated and characterized. Three types of heparan sulfate were detected. K,, 6B 0.04) Heparan sulfate proteoglycan I (HSPG-I; was found in the cell layer and medium and accounted for 12% of the total proteoglycans synthesized. HSPGII (K,, 6B 0.25) accounted for 18% of the proteoglycans and was located in the medium and cell layer. A third population (9% of the proteoglycan population), heparan sulfate glycosaminoglycan (HS-GAG; K,, 6B 0.40.8), had properties consistent with single glycosaminoglycan chains or their fragments and was found only in the cell layer. HSPG-I and HSPG-II from the cell layer had hydrophobic properties; they were released from the cell layer by mild trypsin treatment. HS-GAG lacked these properties, consisted of low-molecularmass heparan sulfate oligosaccharides, and were intracellular. HSPG-I and -11 released to the medium lacked hydrophobic properties. The cells also produced three distinct types of chondroitin sulfates. The major species, chondroitin sulfate proteoglycan I (CSPG-I) eluted in the excluded volume of a Sepharose CL-6B column, accounted for 30% of the proteoglycans detected, and was found in both the cell layer and medium. Cell layer CSPG-I bound to octyl-Sepharose. It was released from the cell layer by mild trypsin treatment. CSPG-II (K,, 6B 0.1-0.23) accounted for 10% of the total 35S-labeled macromolecules and was found predominantly in the culture medium. A small amount of CS-GAG (K,, 6B 0.25-0.6) is present in the cell extract and like HSGAG is intracellular. Pulse-chase experiments indicated that HSPG-I and -11 and CSPG-I and -11 are lost from the cell layer either by direct release into the medium or by internalization where they are metabolized to single glycosaminoglycan chains and subsequently to inorganic sulfate. (0 1990 Academic Press, Inc.

’ To whom correspondence 0003-9861/90

should be addressed.

$3.00

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved

Glomerular proteoglycans are the subject of increasing interest because with sialoproteins they make up the polyanionic sites of the glomerular basement membrane and epithelial slit processes and thus serve to form the charge barrier which maintains the selective permeability of the renal filter (1). Removal of the anionic charge with specific enzymes (2, 3) or neutralization of the charge with cationic substances (4, 5) or specific antiheparan sulfate proteoglycan (HSPG)2 antibodies (6-8) induces proteinuria. These observations may have clinical relevance since loss of the charge barrier is associated with several forms of human glomerular disease which are characterized by the leakage of plasma proteins into the urine (g-12). In the renal glomerulus several different forms of proteoglycans have been identified by biochemical analysis (13-15) and immunocytochemical localization (16-N). Although these include chondroitin and dermatan sulfate proteoglycans (CSPG, DSPG), heparan sulfate proteoglycans are the most prevalent. Two immunologically distinct populations of HSPG have been defined (X3,19). The first is located on the cell surface of glomerular epithelial and endothelial cells, while the second is a constituent of the glomerular basement membrane. To date most studies of the biosynthesis of glomerular proteoglycans have been carried out with intact glomeruli isolated from experimental animals by sieving techniques (20-24). While this approach has yielded important information about glomerular proteoglycans in general it does not provide detailed information about the molecules synthesized by the different cell types that

’ Abbreviations used: HSPG, heparan sulfate proteoglycan; CSPG, chondroitin sulfate proteoglycan; Chaps, 3.[(3-cholamidopropyl)dimethylammonio] propanesulfonic acid; EHS, Englebreth-HolmSwarm; CL, cell layer; CM, culture medium; FCS, fetal calf serum; FITC, fluorescein isothiocyonate; DMEM, Dulbecco’s modified Eagle’s medium; CS-GAG, chondroitin sulfate glycosaminoglycan; HSGAG, heparan sulfate glycosaminoglycan; FPLC, fast-protein liquid chromatography. 11

12

THOMAS

make up the glomerulus. There are at least three cell types, mesangial, endothelial, and epithelial (podo@es), each of which has a specialized function (1). The function of the epithelial cells is of particular interest because in the mature animal they probably synthesize the majority of the macromolecules that constitute the glomerular basement membrane (25-27). The proteoglycans of human epithelial cells have not been previously characterized, although it is important to understand their function since epithelial damage is known to occur in human nephropathies (28). The purpose of the present study was to investigate the synthesis and metabolism of 35S-labeled proteoglycans by cultures of human glomerular epithelial cells. METHODS Cell culture and characterization. Tissue was obtained from the nonaffected pole of human kidneys removed at the time of elective nephrectomy from patients with various forms of renal disease. The cortex was dissected away from the medulla and glomeruli were recovered by passage through serially graded sieves with pore diameters 425, 180, and 125 pm. The tubular contamination was less than 1% as determined by phase-contrast microscopy. The glomeruli were washed twice and plated in a minimum of growth medium at 37°C in a humidified atmosphere of 5% CO,, 95% air until attachment. The medium was RPM1 1640, 10% FCS, and supplemented with 2 mM glutamine, 5 pg/ml insulin, 5 pg/ml transferrin, 5 rig/ml selenite, 0.4 rg/ml hydrocortisone, and 1 mM sodium pyruvate (culture medium A). After attachment cellular outgrowths appeared within 12-14 days. Once established, cells were trypsinized, passed through a sterile 90-pm sieve to remove whole glomeruli, replated in 35-mm culture dishes (Falcon), and grown to confluency. Confluent epithelial cells were passaged at a 1:3 split ratio into 35mm petri-culture dishes and used between the second and third passage. Immunofluorescence studies. Cells were fixed in acetone:methanol (1:l) at 4°C for 2 min, air-dried, and incubated with primary antibody for 40 min. After extensive washing with phosphate-buffered saline containing 0.9% (w/v) bovine serum albumin (Sigma) they were incubated with the appropriate fluorescein isothiocyanate (FITC)-conjugated anti-IgG for 1 h in the dark. The antibody was removed and the cells were washed extensively and finally mounted with Hydromount (National Diagnostics, Sommerville, NJ). The following antisera were obtained from commercial sources: anti-myosin and anti-desmin (ICN Biomedicals Ltd., High Wycombe, UK); anti-cytokeratin and anti-vimentin (Dako Ltd., High Wycombe, UK); anti-collagen types I, III, IV, and V (Bio-Nuclear Services Ltd., Reading, UK). Anti-laminin, anti-fibronectin, and a rat monoclonal antibody against EHS sarcoma heparan sulfate proteoglycan (large, low density form) were a gift from Dr. J. Couchman, Birmingham, Alabama. FITC-conjugated anti-Von Willebrand/Factor VIII was a gift from Dr. N. Topley (Hannover, FDR). Monoclonal antibodies to human renal tissues (TNl, TN9, and TNlO) were generously provided by Dr. Gerhard Miiller, Medical University Clinic (Tubingen, FDR). The characterization of these antibodies has been described and allows glomerular visceral epithelial cells to be distinguished from other renal epithelial cells (29). Labeling of cells. At confluency, the culture medium was removed and the cells were washed twice with phosphate-buffered saline (Dulbecco’s formula, without calcium and magnesium, pH 7.2). The cultures were labeled with 50 &i/ml Na,-[35S]sulfate (950-1350 Ci/ mmol, Amersham International, UK) in ‘2 ml of DMEM (Imperial Laboratories, UK) containing 10% of the normal MgSO, concentration, 10% fetal calf serum, and the supplements in culture medium A

ET AL. above. At the end of the labeling period (usually 24 h) the medium was decanted and retained. The cells were quickly washed twice with phosphate-buffered saline, the washings added to culture medium above, and proteinase inhibitors added (final concentrations: phenylmethylsulphonyl fluoride, 0.001 M; N-ethylmaleimide, 0.01 M; benzamidine hydrochloride, 0.005 M; disodium EDTA, 0.01 M; and 6.aminohexanoic acid, 0.1 M). The medium (CM) was stored at -20°C until analyzed. The cell layer (CL) was solubilized with 4.0% (w/v) Chaps and 4.0 M guanidine hydrochloride in the presence of proteinase inhibitors (final concentrations, as above) (30) and stored at -20°C until analyzed. Finally each dish was extracted with 0.5 M NaOH to release residual ?j-labeled glycosaminoglycans. 35S radioactivity in macromolecules was measured by chromatography on prepacked columns of Sephadex G-25 (PD-10 columns; Pharmacia) (24). Pulse-chase experiments. Cell cultures were labeled as above using 50 &i [“‘S]sulfate/ml. After 24 h the cell layers were washed extensively with RPM1 medium and chased for various periods of time up to 6 h in culture medium A. Trypsin treatment of cell layers. Cell layers were labeled with [35S]sulfate (50 &i/ml) as above for 24 h and the culture medium was removed; the cells were washed once with phosphate-buffered saline and the washing was added to the culture medium. The cells were then washed rapidly five times with phosphate-buffered saline to reduce the levels of free [35S]sulfate and finally treated with trypsin (10 fig/ml) in phosphate-buffered saline for 10 min at 20°C. Preliminary experiments showed that this concentration of trypsin was optima1 for the release of minimally degraded “S-labeled macromolecules from the cell surface. After treatment, soya bean trypsin inhibitor (200 pg) was added, the medium was removed, and the cells were washed twice with phosphate-buffered saline containing soya bean trypsin inhibitor (100 pg/ml) prior to extraction with Chaps/guanidine hydrochloride as above. Hydrophobic affinity chromatography. Samples of CM and CL were extracted as above and passed over a Sephadex G-50 column equilibrated with 4 M guanidine hydrochloride, 20 mM Tris-HCl, pH 35S-1abe1ed macromolecules in 7.0, containing proteinase inhibitors. the void volume fraction were applied to a 3-ml column of octyl-Sepharose (Pharmacia) and allowed to equilibrate for 16 h at 4°C. The nonbound and non-specifically-bound material was removed by washing five times with 3-ml portions of the following buffers: 4.0 M guanidine hydrochloride, 20 mM Tris-HCl, pH 7.0; 0.1 M NaCI, 20 mM Tris-HCl, pH 7.0 (31). Bound radiolabeled pH 7.0; 3 M NaCl, 20 mM Tris-HCl, material was eluted in a stepwise fashion using 4.0 M guanidine hydrochloride, 20 mM Tris-HCl, pH 7.0, containing 0.1,0.5, 1.0, and 5% (v/ v) Triton X-100. At each elution step the column was washed five times with 3 ml of eluting buffer. Recovery of 35S-1abeled macromolecules was 85%. Cell layer 35S-labe1ed proteoglycans were inserted into liposomes according to the method of Woods et al. (31). Insertion was demonstrated by a shift in elution profile after chromatography on a Sephacry1 S-500 column (0.006 X 1.5 m) equilibrated in 4 M urea, 50 mM Tris-HCl, pH 8.0, containing 0.35 M NaCl. Analysis ofs5S-labeled macromolecules. In initial studies CM, made 4 M with respect to guanidine hydrochloride, and CL were centrifuged at 10,OOOgfor 10 min and the supernatants eluted under dissociative conditions on a Sepharose CL-6B column (0.006 X 1.5 m) equilibrated with 4 M guanidine hydrochloride buffered with 50 mM sodium acetate, pH 6.0, containing 0.5% (v/v) Triton X-100, 1 mM Na,SO, and 0.05% (W/V) NaN,. Fractions (0.6 ml) were collected and monitored for radioactivity. In addition CM and CL were treated with 0.05 M NaOH containing 1.0 M NaBH, for 20 h at 45°C and then neutralized with glacial acetic acid (32). The “S-labeled glycosaminoglycans released in this reaction were rechromatographed on Sepharose CL-6B as above. For more detailed analysis the CM and CL were eluted on a Sephadex G-50 (fine) column (bed volume 20 ml) equilibrated in 8 M urea, 20 mM Bistris, pH 6.0, 0.15 M NaCl, and 0.5% (w/v) Chaps. The labeled material excluded from the void volume of the column was collected

HUMAN TABLE Immunofuorescence

GLOMERULAR

1

Characteristics of Human Epithelial Cells in Culture

Glomerular

Negative

Positive Collagen type IV Laminin Fibronectin Heparan sulfate proteoglycan Collagen type I Cytokeratin TN10

EPITHELIAL

Collagen type III Collagen type V Von Willebrand/factor Desmin Myosin TNl;

VIII

TN9

Nclte. Cells were fixed and incubated with primary antibody followed by the appropriate FITC-conjugated IgG antibody as detailed under Methods. The stained cells were mounted in Hydromount and examined using a Leitz fluorescence microscope. Monoclonal antibody TN10 is specific for human glomerular visceral epithelial cells.

and further separated by ion-exchange on a Mono Q column (Pharmacia) equilibrated with the same buffer, interfaced with a fast-protein liquid chromatography system (FPLC, Pharmacia). After application, the column was washed with the same buffer (4 ml) and the bound material eluted with a linear NaCl gradient (0.15-1.2 M). Fractions (0.5 ml) were collected and t.he 35S radioactivity was measured. The radiolabeled peaks from the Mono Q were pooled, divided into portions, and precipitated at 4°C with 3 vol of 95% ethanol containing 1.3% (w/v) potassium acetate in the presence of 50 pg/ml chondroitin sulfate and 50 rg/ml heparin. The precipitates were washed with 90% ethanol, dried, and treated in one of two ways: (a) resuspended in water and degraded with freshly prepared ice-cold nitrous acid (33, 34), or (b) resuspended in 50 mM Tris-HCl, 60 mM sodium acetate, 0.01 M sodium fluoride, pH 8.0, anddigested overnight at 37°C with chondroitinase ABC (0.625 unit; Sigma Chemicals) in the presence of proteinase inhibitors (35-37). Untreated material and the final solutions from (a) and (b) were analyzed under dissociative conditions on Sepharose CL-6B as described above. Analysis of susceptibility of the ‘“S-labeled proteoglycans to chondroitinase AC (0.125 units) was in 500 ~1 of 60 mM sodium acetate buffer, 0.01 M sodium fluoride, pH 7.0, at 37°C for 16 h.

CELL

13

PROTEOGLYCANS

Incorporation of Radiolabeled Sulfate into Macromolecules Confluent cultures labeled with 50 PC1 [35S]sulfate/ml for 24 h incorporated 5.45 X lo5 cpm/dish; SD + 1.73 X 105; n = 25 (mean of five separate experiments, five dishes/experiment). Between 58 and 73% (mean 64%) of the 35S-labeled macromolecules were found in culture medium. The remainder were found in the cell layer extract. The amount of 35S radioactivity remaining on the culture dish and removed by 0.5 M NaOH was less than 1% of the total 35S-labeled macromolecules. Analysis of CM and CL under dissociative conditions on Sepharose CL-6B showed that each cell culture produced similar profiles of 35S-labeled macromolecules (Fig. 1). Chromatography after reduction with dithiothreitol (2 mM) and alkylation with iodoacetamide of both the CM and CL gave identical elution profiles indicating no intermolecular disulfide bonding of the proteoglycans (data not

16

--CM1 .

CM11

CM111

12

8

4

0 16

12

RESULTS

Characterization of the Human Glomerular Epithelial Cells The cells used in this study demonstrated no morphological evidence for the presence of renal mesangial cells, macrophages, or fibroblasts. The cells formed a confluent monolayer of polygonal cells. The cells were also reacted with specific antisera and examined by immunofluorescence (Table I). The results indicate fluorescence characteristics of epithelial cells. The use of the monoclonal antibodies TNl, TN9, and TN10 indicated that the cells were derived from glomerular visceral cells and not from Bowmans capsule or tubular epithelia (29). The absence of Von Willebrand/factor VIII and of prominent intracellular fibrillar filaments after immuno staining with anti-myosin further allowed their distinction from endothelial cells and mesangial cells.

8

0

0.75

fl.5 K

Cl.15

1

aV

FIG. 1. Fractionation of Y&labeled macromolecules synthesized by human glomerular epithelial cells in culture. A labeling experiment was carried out as described under Methods. The 35S-labeled macromolecules present in (a) the CM and (b) the CL were analyzed by gel permeation chromatography on a dissociative Sepharose CL-6B column (0). The horizontal bars indicate the fractions which were pooled for further analysis. The elution profiles of the CM and the CL after treatment with 0.05 M NaOH/l.O M NaBH, (0) are also shown.

14

THOMAS

ET AL.

shown). The fractions containing the 35S-labeled material were pooled as indicated in Fig. 1, precipitated with ethanol-acetate, treated with chondroitinase ABC or nitrous acid, and rechromatographed. The results indicated that both heparan sulfate and chondroitin sulfate were present in each fraction (data not shown). The elution patterns of peaks I and II were changed by alkali/borohydride treatment, indicating that the labeled material consisted of multichain proteoglycans containing two or more glycosaminoglycan chains each with K,, (6B) of about 0.45 (Fig. 1). After alkali digestion there was no significant change in the elution profile of either CM111 or CLIII, indicating that they are probably single glycosaminoglycan chains. The same results were observed when the peaks were treated individually. Papain treatment gave essentially the same results (data not shown).

0.8

Characterization of 35S-labeled CM and CL Proteoglycans 35S-labeled proteoglycan material in both the CL and the CM was further characterized by ion-exchange chromatography on a Mono Q column. About 10% of the labeled material in each case did not bind strongly to Mono Q and probably represents 3”S-labeled glycoproteins. The bound material was eluted with a linear gradient of 0.15-1.2 M NaCI as shown in Fig. 2a. Two main peaks of 35S-labeled proteoglycans were separated. In the CM peak A contained 30-40% (n = 5) of the bound labeled material, eluted at 0.8 M NaCl, and consisted of more than 70% heparan sulfate, the rest being accounted for as chondroitin sulfate. Peak B eluted at 0.93 M NaCl and was almost entirely chondroitin sulfate (>90%). The CL peak A (Fig. 2b) accounted for between 50 and 60% (n = 5) of the bound material and contained about 60% heparan sulfate. Peak B was again almost exclusively chondroitin sulfate (90%). To characterize further the types of proteoglycans present, peaks A and B from the CM and CL were fractionatedunder dissociative conditions on Sepharose CL6B and CL-4B after digestion with either chondroitinase ABC or nitrous acid. The results for Sepharose CL-6B are shown in Figs. 3a-3h. Table II summarizes the characteristics of the main proteoglycans distinguished. Heparan sulfates account for about 40% of the total 35S-labeled macromolecules synthesized. Of these a large multichain heparan sulfate proteoglycan (HSPG-I), which eluted at or near the excluded volume of Sepharose CL-6B or Sepharose CL-4B, respectively, accounted for about 12% of the total 35S-labeled macromolecules. Molecules of this size were present in both the CL and CM. A second population of multichain proteoglycans (HSPG-II) eluted in the included volume of Sepharose CL-6B and was clearly separated from HSPG-I (Figs. 3a and 3e). They accounted for about

0.8

0.4

0 0

IO

20

30

FRACTION NUMBER FIG. 2. Anion-exchange liquid chromatography of 35S-labeled macromolecules synthesized by human glomerular epithelial cells in culture. Cells were labeled as described under Methods. The CM and the CL were extracted and the 35S-labeled macromolecules separated from free ?30, on a G-50 Sephadex (fine) column equilibrated in 8 M urea, 20 mM Bistris, pH 6.0,0.15 M NaCI, and 0.5% (w/v) Chaps. The figure shows the Mono Q ion-exchange chromatography profile of the 35Sincorporated material from (a) the CM and (b) the CL. The salt gradient (- -) used to elute the column was generated using a FPLC system. Fractions containing peaks A and B were pooled as indicated by the horizontal bars.

18% and were present in the CL and CM. The third population of 35S-labeled heparan sulfate molecules eluted from the Sepharose CL-6B column with profiles and K,, characteristic of single chain glycosaminoglycans and fragments of glycosaminoglycan chains (HS-GAG). These molecules were found exclusively in the cell layer compartment (Fig. 3e, hatched area). Overall 90% of the total heparan sulfate molecules were separated into peak A of the Mono Q fractionation. Cells incorporated rather more 35S radioactivity into chondroitin sulfates than into heparan sulfates (Table II). Chondroitin-containing molecules, in contrast to the heparan sulfate proteoglycans, were not so clearly sepa-

HUMAN

GLOMERULAR

EPITHELIAL

- a)

16

CELL

8

15

PROTEOGLYCANS

Te)

R HSPG-I

0 16

’

b)

%*8 '

6

CS-GAG -

7

CSPG-I

1 c)

32

8 0 32 24 -

h) CSPG-I

24

CSPG-I

16

L

M

0

0.5

1.0

Kav

FIG. 3. Sepharose CL-6B gel chromatography under dissociative conditions of %+labeled proteoglycans obtained from glomerular epithelial cells by ion-exchange chromatography. Fractions A and B from both the CM and CL (Fig. 2) were chromatographed on an analytical dissociative Sepharose CL-6B column after treatment with either chondroitinase ABC or nitrous acid. For details of the origin of fractions see the legend to Fig. 2. (a, b) CM fraction A; (c, d) CM fraction B; (e, f) CL fraction A; (g, h) CL fraction B. Fractions were chromatographed after digestion with chondroitinase ABC (a, c, e, g) or after treatment with nitrous acid (b, d, f, h). Heparan sulfate proteoglycans (HSPG-I and -11) chondroitin sulfate proteoglycans (CSPG-I and -11) and glycosaminoglycans HS-GAG and CS-GAG are indicated and correspond to the molecules listed in Table II. The hatched area indicates molecules with hydrodynamic size equivalent to or smaller than that of single glycosaminoglycan chains.

rated from one another. However, three populations could be distinguished. CSPG-I accounts for 30% of the total 35S-labeled macromolecules, elutes in the excluded volume of Sepharose CL-6B (Figs. 3d and 3h) and CL4B, and is found in both the CL and CM. CSPG-II is a multichain proteoglycan of small hydrodynamic size, accounts for approximately 10% of the 35S, and is poorly resolved from CSPG-I (Fig. 3d). It is present predominantly in the CM. About 5% of the labeled macromolecules are accounted for as single chondroitin sulfate chains CS-GAG (Fig. 3f, hatched area). These chains are

found only in the CL compartment. CSPG-I and -11 were almost all found in peak B after anion-exchange chromatography whereas CS-GAG were exclusively recovered in peak A. Chondroitinase AC was equally effective in degrading CSPG-I as chondroitinase ABC, demonstrating that this proteoglycan contained chondroitin sulfate rather than dermatan sulfate. CSPG-II was completely digested by chondroitinase ABC while chondroitinase AC released only 80% of its 35S radioactivity as disaccharides. This suggests that CSPG-II contains some L-iduronic acid, further distinguishing it from

16

THOMAS TABLE

II

Hydrodynamic Characteristics of Human Glomerular Epithelial Cell Proteoglycans Proteoglycan HSPG-I HSPG-II HS-GAG CSPG-I CSPG-II CS-GAG

Kav

(6J3)

0.04 0.25 0.4-0.8 VO 0.10-0.23 0.25-0.6

Ka, (4B)

% ?-labeled macromolecules

0.18-0.28 0.44 0.57-0.85 VO 0.29 0.53-0.66

11.5 17.7 9.1 30.0 13.6 5.1

Note. Cells were labeled in culture with Na2s5S04 (50 &i/ml) for 24 h and the 35S-labeled proteoglycans separated from the CM and CL by Mono Q ion-exchange chromatography. After treatment with either chondroitinase ABC or nitrous acid they were analyzed by gel chromatography on Sepharose CL-6B and Sepharose CL-4B. See Figs. 2 and 3 for details.

CSPG-I. This molecule represents containing proteoglycan.

a dermatan

sulfate-

ET AL.

tabolized to inorganic sulfate. Very little of the HS-GAG is released to the medium during the 6-h chase (12% of the initial radioactivity). On the other hand HS-GAG equivalent to 56% of the initial radioactivity is found in the cell layer at the end of the chase, presumably because this fraction is being continuously topped up as an intermediate of the catabolic pathway of the large proteoglycans. The pulse-chase data show that the cell layer chondroitin sulfate proteoglycans undergo a similar fate (Fig. 6). After 6 h of chase about 37% of the initial CSPG-I remains in the cell layer and 41% appears in the medium so the remaining 22%, by extrapolation, must have undergone catabolism. Some CSPG-II appears in the medium (approximately 13%) while 25% remains in the cell layer so for this proteoglycan about 62% must have been catabolized within the 6-h chase. Very little CS-GAG escapes to the medium (6%) and 35S radioactivity equivalent to 56% of the initial amount is found in the cell layer after 6 h of chase, suggesting that, as with the equivalent heparan sulfate, this pool is continuously topped up from the catabolism of larger proteoglycans.

Pulse-Chase Experiments Pulse-chase experiments were carried out to investigate the relationship of 35S-labeled proteoglycans in the cell layer to those in the medium. Cultures were pulsed for 24 h and chased over the next 6 h. Sixty-three percent of the cell layer 35S-labeled macromolecular radioactivity was lost during this period and was matched exactly by the appearance of 35S radioactivity in the chase medium in the form of macromolecules (28%) and inorganic sulfate (34%) (Fig. 4). This suggests that, in common with ovarian granulosa cells (38) and colon carcinoma cells (39), the glomerular epithelial cell layer 35S-labeled proteoglycans have two distinct fates, either internalization and complete catabolism with the subsequent appearance of inorganic sulfate, or release as macromolecules directly to the chase medium. To investigate the fate of each class of proteoglycan, portions of the chase medium and the cell layer extract were digested with chondroitinase ABC or nitrous acid to obtain heparan sulfate and chondroitin sulfate species, respectively, followed by chromatography on Sepharose CL-6B. Three fractions were distinguished in each case: HSPG-I and -11 and HS-GAG; CSPG-I and -11 and CS-GAG as defined in Fig. 3. Results for heparan sulfate are shown in Fig. 5. Fifty-seven percent of HSPG-I appears in the chase medium while 35% remains in the cell layer after a 6-h chase (Fig. 5), indicating that 8% is completely catabolized during this period. HSPG-II also appears to be processed along both routes during the 6-h chase with 32% of the original proteoglycan appearing in the chase medium, 24% remaining in the cell layer, and the balance, 44%, presumably being completely ca-

‘0 -

50

0

1

2

3 Time

4

5

6

(h)

FIG. 4. Release and metabolism of cell layer ?S-labeled proteoglycans. Cells were pulse-labeled as described under Methods, the culture medium was removed, and the cells were rapidly washed with phosphate-buffered saline (X51, and the culture was continued by the addition of fresh culture medium A. At the indicated times the cell medium was removed and the cell layer solubilized with 4% (w/v) Chaps and 4 M guanidine hydrochloride. The total 35S-labeled macromolecules at each time point associated with the cell layer (0 - 0) or released into the chase medium (0 - - - 0) and the inorganic [?S]sulfate released into the chase medium (A - - - A) were quantitated by PD-10 column chromatography as described under Methods.

HUMAN

GLOMERULAR

EPITHELIAL

CELL

17

PROTEOGLYCANS

radioactivity. The 35S-labeled molecules released by trypsin eluted from the Sepharose CL-6B column in positions indicating that they were derived from multichain proteoglycans. No single chain 35S-labeled glycosaminoglycans were released under these conditions, suggesting that HS-GAG and CS-GAG (Figs. 3e and 3f, hatched area) are intracellular. Furthermore, since HSGAG has an overall lower hydrodynamic size (K,, 0.50.7) than the alkali-released glycosaminoglycan chains (K,, 0.45, see Fig. lb) it is likely that it represents glycosaminoglycan fragments in an intracellular compartment undergoing degradation. The elution profile of the CL after trypsin treatment confirmed that the 35S-labeled molecules released by trypsin were derived from HSPGI and -11 and CSPG-I and -11 (data not shown). Octyl-Sepharose Affinity of Proteoglycans FIG. 5. Release and metabolism

of cell layer %-labeled heparan sulfate proteoglycans. Portions of the cell layer and the chase medium from the pulse-chase experiment described in Fig. 4 were treated with chondroitinase ABC to obtain total %labeled heparan sulfate and subjected to chromatography on Sepharose CL-6B under dissociative conditions to obtain HSPG-I, HSPG-II, and HS-GAG. The amounts of %-labeled heparan sulfate remaining in the cell layer (-) and released into chase medium (- - -) at each chase time were obtained by quantitating HSPG-I (O), HSPG-II (A), and HS-GAG (m).

Overall the pulse-chase experiments demonstrate clearly that the epithelial cells catabolize a significant proportion of both newly synthesized heparan sulfate and chondroitin sulfate proteoglycans. The present data summarized in Table III are not adequate to calculate the half-lives of the individual proteoglycan populations. Nevertheless they demonstrate that each pool undergoes some rapid catabolism during the 6 h following synthesis and that the proportions of proteoglycans entering this pathway vary for each proteoglycan population. In both the long pulse (24 h)-chase experiment above and the short pulse (30 min)-chase experiment (data not shown) there was no evidence for the conversion of large proteoglycans (HSPG-I and CSPG-I) to smaller proteoglycans (HSPG-II and CSPG-II).

The hydrodynamic sizes and charge characteristics of the various proteoglycans in the medium, together with the results of the pulse-chase experiments, suggest that they are derived from similar proteoglycans in the cell layer. The quantities of human glomerular epithelial cell 100

0

0

Location of the Cell Layer Proteoglycans and Glycosaminoglycans The labeled material separated in the CL peak A (Fig. 2b) contained a high percentage of glycosaminoglycans (Figs. 3e and 3f). To determine whether this material was intracellular, cells were labeled for 24 h, the culture medium was removed, and the cell layer treated with phosphate-buffered saline containing trypsin (10 pg/ml) or buffered saline alone. Trypsin treatment did not detach the cells but released up to 50% of the cell layer

Chromatography

/ / fL c= f

0

,--* --

1

_

______ __ __ , I

2

__

?J

A A-------__ __ - - -. I I I

4

5

6

Time (h) FIG. 6. Release and metabolism of cell layer ““S-labeled chondroitin sulfate proteoglycans. Portions of the cell layer and chase medium from the pulse-chase experiment described in Fig. 4 were treated with nitrous acid to obtain total %-labeled chondroitin sulfate and subjected to chromatography on Sepharose CL-6B under dissociative conditions to obtain CSPG-I, CSPG-II, and CS-GAG. The amount of “Slabeled chondroitin sulfate remaining in the cell layer (-) and released into chase medium (- - -) were obtained by quantitating CSPGI (O), CSPG-II (A.), and CS-GAG (I).

18

THOMAS TABLE Fate of Cell Layer Proteoglycans Epithelial Cell Cultures

III in Human Glomerular after a 6-h Chase Fate after chase (%)

Proteoglycan

Percentage cell layer a5S-labeled proteoglycans before chase“

HSPG-I HSPG-II CSPG-I CSPG-II

8 13 38 18

Retained in cell layer 35 24 37 25

Release to chase medium

Catabolized to GAG and so,

57 32 41 13

8 44 22 62

ET AL.

described by other workers (40, 41). Since the preparations of isolated glomeruli were decapsulated and free of tubular contamination, the cells are likely to be derived from visceral rather than parietal glomerular epithelial cells. The specific staining with monoclonal antibodies

Note. Cells were pulse-labeled and a chase experiment was carried out as described under Methods. The amount of cell layer “S-labeled proteoglycans present before the chase and their fate after the 6-h chase period were obtained from Figs. 5-7 and as described in the text. a Balance; HS-GAG and CS-GAG.

cultures available so far are insufficient to fully examine the core protein relationship of corresponding cell medium and cell layer proteoglycans. However, differences in the affinity of medium and cell layer proteoglycans for binding octyl-Sepharose and insertion into liposomes are apparent. Table IV shows that over 70% of the cell layer-associated labeled material bound to the octylSepharose and could be eluted with Triton X-100. In contrast only 6% of the culture medium-labeled material bound. Gel chromatography of cell-layer nonbound radiolabeled material (Table IV) revealed that it consisted mainly of single glycosaminoglycan chains and smaller fragments corresponding to CS-GAG and HS-GAG (Fig. 7a, hatched area). The bound radiolabeled cell layer material eluted with 0.1% (v/v) Triton X-100 was CSPG-I (Fig. 7b) while that in the 0.5% (v/v) eluate consisted almost entirely of HSPG-I and -11 (Fig. 7~). Both these eluates bound again to the octyl-Sepharose column on rechromatography but this property was lost by prior mild trypsin treatment (10 pg/ml, 10 min, 2O”C), suggesting that binding was due to a hydrophobic domain in the core protein. The hydrophobic properties of these proteoglycans was further confirmed by the finding that the 35S-labeled proteoglycans of the CL could be inserted into liposomes. Less than 3% of the proteoglycans in the CM could be intercalated under the same conditions (data not shown). The medium CSPG-I and -11 and HSPG-I and -11 may be derived by limited proteolytic cleavage of these proteoglycans in the cell layer with the loss of the latter’s hydrophobic properties. DISCUSSION

The cellular outgrowths used in this study were homogeneous and demonstrated morphological characteristics which resembled those of glomerular epithelial cells

d c) 4

FIG. 7. Sepharose CL-6B chromatography of cell layer ?+labeled proteoglycans isolated on an octyl-Sepharose affinity column. Cultures were labeled as described previously. The cell layer was extracted with 4% Chaps and 4 M guanidine hydrochloride and passed over a Sephadex G-50 (fine) column for buffer exchange and removal of unincorporated isotope. The void volume fraction was equilibrated with an octyl-Sepharose column and subsequently eluted stepwise with buffer alone and buffer containing 0.1, 0.5, 1.0, and 5.0% (v/v) Triton X100. ?S-labeled proteoglycans eluted with (a) buffer alone, (b) buffer containing 0.1% (v/v) Triton X-100, and (c) buffer containing 0.5% (v/v) Triton X-100 were chromatographed on a Sepharose CL-6B column under dissociative conditions. The hatched area indicates molecules with hydrodynamic size equivalent to or smaller than that of single glycosaminoglycan chains.

HUMAN TABLE Binding

of 35S-labeled

GLOMERULAR

EPITHELIAL

IV

Proteoglycans

to Octyl-Sepharose

Elution of ““S-labeled macromolecules (% total recovered) Triton X-100 (%) in eluting buffer

Cell layer

0 0.1 0.5 1.0 5.0

27.0 36.6 28.2 7.6 0.6

Culture

medium 94.0 1.2 3.8 1.0 0.0

Note. Cells were labeled as detailed in Fig. 4 and the cell medium and cell layer prepared for affinity chromatography as described under Methods. Both the CM and the CL were then fractionated on the hydrophobic affinity column and the bound material was eluted stepwise with Triton X-100 as described under Methods. The percentage in each fraction is based on the amount of labeled material recovered. The recovery of labeled material was 85% for each experiment. The results represent the averages of three experiments.

TN10 but not TN1 or TN9 confirms their visceral origin. The prominent staining of intermediate filaments with antibodies to cytokeratin but not desmin provides further evidence for their epithelial origin (40). Antibodies to myosin and Von Willebrand/factor VIII failed to stain the cells, thus distinguishing them from the other two cell types present in the glomerulus-namely mesangial (42) and endothelial (43). When the glomerular epithelial cells were labeled with [35S]sulfate a significant amount of radiolabeled macromolecules was synthesized. These were shown to be mainly proteoglycans and consisted of two distinct groups, those containing heparan sulfate and those containing chondroitin sulfate. Each group consisted of three distinct populations separable by hydrodynamic size. These included large multichain proteoglycans which were excluded from Sepharose CL-GB, smaller multichain proteoglycans which eluted in the included volume of CL-GB, and molecules whose size was consistent with their identity as glycosaminoglycan chains or fragments derived from them. Detailed analysis (see Table III) indicated that the [35S]HSPG and [35S]CSPG were present in both the CL and CM. The data showed that these proteoglycans were not hybrids and that the glycosaminoglycans were located on different core proteins since neither changed their K,, on Sepharose CL6B when they were treated with either chondroitinase ABC or nitrous acid (Fig. 3). Additionally, the cell layer heparan sulfate proteoglycans were well separated from the chondroitin sulfate proteoglycan on octyl-Sepharose chromatography. Pulse-chase experiments indicated the medium proteoglycans were derived from counterparts in the cell. The latter were shown to be accessible to trypsin and therefore extracellular in location whereas glycosami-

CELL

PROTEOGLYCANS

19

noglycan chains in the cell layer were intracellular. Most of the cell layer proteoglycans have hydrophobic properties since they are bound to octyl-Sepharose and can be intercalated into liposomes whereas their culture medium counterparts lacked both these properties. Overall the data are consistent with the release of a proportion of each of the cell layer proteoglycans to the medium by limited proteolytic cleavage with the loss of hydrophobic properties. One possibility is that the cell layer proteoglycans are intercalated with the plasma membrane and are released from it by this cleavage. Additionally a proportion of each of the cell proteoglycans appears to undergo an alternative fate involving uptake into the cell with degradation to single chain glycosaminoglycans and further catabolism with release of inorganic sulfate. The present results compliment and extend previous studies on the biosynthesis of glomerular proteoglycans and in particular those employing isolated glomeruli incubated with [35S]sulfate in vitro (20-24). In the latter prominent CSPG synthesis was observed and attributed to the mesangial cells (20, 22). However, the present work questions this assumption since it shows that [35S]CSPG are not exclusively a product of mesangial cells but are also a product of glomerular epithelial cells. However, a species difference cannot be excluded. Epithelial cells are the major cell type in the glomerulus responsible for the synthesis of proteoglycans (25-27). In addition they are responsible for the synthesis, assembly, and insertion of new basement membrane matrix into the mature glomerular basement membrane (27). Heparan sulfate proteoglycan is the predominant proteoglycan associated with most basement membranes investigated to date, but small amounts of CSPG have also been demonstrated in chemical (15,44,45) and immunohistochemical studies (17). Moreover, 35S-labeled CSPG have been extracted from glomerular basement membrane preparations isolated by osmotic lysis and detergent treatment of whole rat glomeruli labeled in vitro (24). Furthermore, a dermatan sulfate proteoglycan is synthesized by mouse parietal yolk sac (PYS-2) cells (46) which elaborate Reichert’s membrane. These proteoglycans have hydrodynamic and charge properties almost identical to those of CSPG-1 described in the present work (Table II). Similar CSPG molecules have been extracted from isolated rat glomeruli by Kobayashi et al. (22) and Klein et al. (23). Two distinct HSPG have been isolated and characterized from the EHS sarcoma which synthesizes basement membrane molecules (47). Monoclonal antibodies against epitopes on the core protein of the larger of these proteoglycans (1M, approx 650,000) cross-react with mouse tubular and glomerular basement membranes, indicating that a similar proteoglycan might be present in these structures (48). Immunoprecipitation of glomerular extracts with the same antibody identified a large core protein of M, 450,000. Conversely, polyclonal anti-

20

THOMAS

bodies to bovine glomerular basement membrane heparan sulfate proteoglycan immunoprecipitated the precursor protein (n/r, 400,000) synthesized by the EHS sarcoma (49). Our present results indicate that a large heparan sulfate proteoglycan (HSPG-I) is synthesized by human glomerular epithelial cells. However, further experiments will be required to establish their similarity or dissimilarity to the large EHS proteoglycan. ACKNOWLEDGMENT This work was supported by a grant from Smith Kline (1982) Foundation (to M.D.).

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