Production membrane
and polarized secretion of basement components by glomerular epithelial cells
YASUHIRO NATORI, YVONNE M. O’MEARA, ERIC C. MANNING, ANDREW W. M. MINTO, JERROLD S. LEVINE, WOLFGANG J. WEISE, AND DAVID J. SALANT Evans Memorial Department of Clinical Research and Department of Medicine, University Hospital, Boston University Medical Center, Boston, Massachusetts 02118 Natori, Yasuhiro, Yvonne M. O’Meara, Eric C. Manning, Andrew W. M. Minto, Jerrold S. Levine, Wolfgang J. Weise, and David J. Salant. Production and polarized secretion of basement membrane components by glomerular epithelial cells. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F131-F13’7, 1992.-To study the formation of basement membrane by glomerular epithelial cells (GECs), production and secretion of type IV collagen and laminin by rat GECs in culture were evaluated. GECs produced two chains of type IV collagen (180 and 170 kDa) in the ratio of approximately 2 to 1, when immunoprecipitated with antibody to type IV collagen of mouse Engelbreth-Holm-Swarm (EHS) sarcoma. GECs also produced proteins that were precipitated by antibody to EHS laminin, i.e., two bands each in the positions of the A and B chains of mouse laminin. On enzyme-linked immunosorbent assay (ELISA), type IV collagen and laminin were found mainly in the cell-associated fraction and in the subepithelial culture medium. Confluent GECs on membrane filters formed a tight barrier against the flux of macromolecules. Under these conditions, 80% of newly synthesized and secreted matrix proteins were detected in the basolateral medium. Moreover, treatment with ammonium chloride, which is known to affect polarized secretion, caused both type IV collagen and laminin to be secreted via the basolateral and apical surfaces in similar amounts. These results indicate that cultured GECs are polarized and that they produce and secrete basement membrane components via the basolateral side.
able heterogeneity between BMs derived from different tissues and that the molecular composition of the constituent BM matrix components may differ from those produced by tumor cells (26). Therefore tumor cells may not provide information on the structure of native, organ-specific BMs. Such information may be better obtained from cells derived from specific tissues if it can be shown that they exhibit characteristics similar to those that exist in vivo. The structure and biochemical composition of the GBM have been studied extensively, because it is relatively easy to obtain substantial quantities of pure GBM. The GBM is a composite BM, produced by and located between glomerular endothelial and epithelial cells (27). In addition to its structural role, it plays an important part in restricting the glomerular filtration of macromolecules (15). Glomerular epithelial cells (GECs) in culture have been shown to produce collagen (4, 18, 34) and proteoglycans (29). However, the type and subunit structure of the collagen produced by GECs is still obscure, and the production of laminin by cultured GECs has not yet been shown. In this study, we used monospecific antibodies and immunoprecipitation and enzyme-linked immunosorbent assay (ELISA) methods to study the production type IV collagen; laminin; biosynthesis and secretion of type IV collagen and laminin by rat GECs in culture. Our results show that GECs produce BASEMENT MEMBRANE (BM) is an extracellular matrix two a-chains of type IV collagen (possibly cyl and c&, composed of collagenous and noncollagenous glycoproand A and B chains of laminin. In addition, we found teins and proteoglycans (30). Early biochemical studies that these molecules are mostly secreted via the basolatof BM isolated from normal tissues including glomeruli era1 side. identified type IV collagen (17) and heparan sulfate proteoglycan (16) as a nonfibrous collagen and a major AND METHODS anionic component of glomerular BM (GBM), respec- MATERIALS tively. Difficulties in solubilizing components of native Culture of GECs. GECs were obtained from Sprague-Dawley BM without proteolytic degradation (31) were overcome rats (CD; Charles River Breeding Laboratories, Wilmington, through the use of BM-producing tumor cell lines, such MA) and cultured as detailed previously (24). In brief, glomeruli as Engelbreth-Holm-Swarm (EHS) mouse sarcoma (ZZ), were isolated from young rats and plated on collagen gel matrix (Vitrogen 100; Collagen, Palo Alto, CA) in a 1:l mix of condiwhich greatly facilitated further progress. Novel glycoproteins such as laminin (32) and entactin/nidogen (8) tioned medium from NIH 3T3 fibroblasts and Kl medium Dulbecco’s modified Eagle’s medium (DMEM) were identified in the matrix of such tumor cells and containing were also shown in authentic BM by use of antibodies to Ham’s F-10 (1:l) (GIBCO, Grand Island, NY) with 5% NuSerum (Collaborative Research, Bedford, MA) and hormone the tumor-derived matrix proteins (8, 32). mixture. After -7-9 days, clearly defined epithelioid colonies The molecular structures of type IV collagen and lam- were excised from the gel and plated in collagen-coated wells. inin were derived mainly from tumor cell lines. Type IV Those wells exhibiting growth of pure epithelial-like colonies collagen was found to be a heterotrimer of two al-chains without mesangial cell contamination were selected for further and one Q2-chain, and EHS laminin was shown to consist expansion in Kl medium. For these studies, cells were studied of three subunits, an A chain with a molecular mass of between passages 15 and 35 and were shown to have the same 400 kDa and B1 and Bz chains of 200-220 kDa (30). characteristics as early passage cells, namely, cobblestone apRecently, it has become apparent that there is consider- pearance at confluence, positive cytokeratin staining, sensitiv0363-6127/92
$2.00 Copyright
0 1992 the American
Physiological
Society
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ity to low concentrations of purine aminonucleoside (25-100 pg/ml), ultrastructural features of epithelial cells such as apical microvilli and junctional complexes, and susceptibility to the cytotoxic effects of anti-FxlA and complement (24). Biosynthetic labeling of GECs with r”‘S]methionine. Subconfluent GECs grown on collagen gel (60 mm dish) were washed three times with methionine-free DMEM and incubated for 60 min with methionine-free DMEM containing 5% heat-inactivated and dialyzed NuSerum and 0.5% hormone mixture. Then they were washed twice with methionine-free DMEM and incubated for 15 h with 0.5 mCi L-[““Slmethionine (1,134 Ci/ mmol; New England Nuclear, Boston, MA) in the methioninefree medium described above containing 50 lug/ml of L-ascorbic acid and 50 pg/ml of ,&aminopropionitrile. The medium was harvested, and the cell layer and adherent collagen gel were washed three times with phosphate-buffered saline (PBS) and solubilized with 0.4 ml of 2% sodium dodecyl sulfate (SDS)-50 mM tris(hydroxymethyl)aminomethane (Tris) hydrochloride (pH 7.6). Protease inhibitors and detergents were added immediately to both fractions. The final concentrations of the inhibitors and the detergents were 20 mM EDTA, 10 mM 1vethylmaleimide, 1 pg/ml leupeptin, 10 pg/ml benzamidine, 2 pg/ml antipain, 1 pg/ml aprotinin, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS. Washing buffer for immunoprecipitation contained the same concentrations of these inhibitors and detergents. The entire volume of both fractions (medium and cell layer) was divided into equal aliquots and analyzed by immunoprecipitation. Immunoprecipitation, gel electrophoresis, and autoradiography. Media and cell lysates were pretreated with 0.5 ml of 10% Staphylococcus aureus slurry (IgGsorb; Enzyme Center, Malden, MA) and 20 ~1 of 1 mg/ml normal rabbit immunoglobulin G (IgG) for 60 min. After centrifugation, the supernatants were incubated with 2 pg of affinity-purified rabbit anti-mouse type I or type IV collagen (kindly provided by Dr. H. Furthmayr), normal rabbit IgG, affinity-purified sheep anti-mouse laminin (provided by Dr. D. Abrahamson), or normal sheep IgG for 120 min. Sheep anti-laminin was purified by elution from lamininSepharose (1). Specificity for laminin and nonreactivity with fibronectin and type IV collagen were verified by inhibition ELISA (2). Rabbit anti-mouse type IV collagen was purified by absorption against types I and III collagen and elution from an affinity column of mouse type IV collagen as described (12). Specificity for type IV collagen was confirmed by inhibition ELISA. The reactivity of all antisera with rat antigens was established by immunofluorescence on normal rat kidney and by ELISA. Samples incubated with anti-laminin and normal sheep IgG were further incubated with 30 pug of rabbit antisheep IgG for 60 min, and then all samples were treated with IgGsorb (20 ~1 for anti-collagen and normal rabbit IgG and 120 ~1 for anti-laminin and normal sheep IgG) for 30 min. The precipitates were then washed six times with washing buffer described above and once with 50 mM Tris HCl (pH 6.8), boiled in Laemmli sample buffer (20) for 5 min, and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with 5% acrylamide gels under reducing conditions. Mouse laminin isolated from EHS sarcoma (Collaborative Research), 14C-labeled type I collagen (provided by Dr. B. Smith) and molecular weight standards (Bio-Rad Laboratories, Richmond, CA) were used as molecular weight markers. Gels were stained, destained, treated with Enlightning (New England Nuclear) for 30 min, dried, and autoradiographed with preflashed Kodak X-Omat AR X-ray films (Eastman Kodak, Rochester, NY) for 2 wk. Photographs of exposed X-ray films were used for densitometry. To count S, dried gels were cut at the position of the bands, soaked in a few drops of water, dissolved in 5% Protosol in Econofluor (New England Nuclear) at 37°C overnight, and
MEMBRANE
COMPONENTS
counted in a ,&scintillation counter. ELISA for type IV colZagen and laminin. Nearly confluent GECs cultured on collagen gel matrix were used. To measure laminin, after the culture medium was harvested, the GECs on collagen gel were washed with PBS, pH 7.2, incubated with 0.2% collagenase (CLS IV; Cooper Biomedical, Malvern, PA) for 45 min at 37OC, and centrifuged at 800 g for 3 min. Then the cell pellet was treated with 0.1 mg/ml pepsin in 0.5 M acetic acid at 4°C overnight, and the pepsin-solubilized fraction was neutralized by adding NaOH. The sum of the collagenase- and pepsin-solubilized fractions was expressed as the cell-associated fraction. To measure type IV collagen, the collagen gel matrix was melted in PBS by incubation at 47°C for 30 min (ll), instead of digestion with collagenase. GECs were solubilized with 2% SDS-50 mM Tris HCl (pH 7.6) and used for ELISA. Lysates in 2% SDS-containing buffer were diluted more than 20 times to lessen the effect of SDS on ELISA. The sum of the melted collagen fraction and SDS lysate was expressed as the cell-associated fraction. To obtain apical or basal medium separately, the following procedure was followed. The medium over the cells was harvested first after GECs became confluent. Then the GECs with gel matrix were taken out and centrifuged. The medium remaining in the culture plates and the supernatant of the centrifuged GECs were used as medium under the cells. For ELISA, wells of microtitration plates (Falcon, BecktonDickinson, Oxnard, CA) were coated with 100 ~1 of 2 pg/ml affinity-purified sheep anti-mouse laminin or affinity-purified rabbit anti-mouse type IV collagen antibodies diluted in PBS at 4°C overnight. Wells were blocked with 200 ~1 of blocking buffer (1% bovine serum albumin-O.15 M Tris HCl, pH 7.6) for 60 min, washed with washing buffer [0.05% Tween-20-15 mM Tris HCl (pH 7.6)-135 mM NaCl] three times, and incubated with sample solutions (100 ~1) diluted in blocking buffer overnight. After washing three times, the wells were incubated overnight with the corresponding biotin-conjugated antibodies (1 pg/ml in blocking buffer), followed by incubation with avidin-peroxidase (2.5 pg/ml in blocking buffer) (Sigma Chemical, St. Louis, MO) for 30 min and substrate solution (0.4 mg/ml o-phenylenediamine-0.012% H202-50 mM phosphate citrate buffer, pH 5.0) for 15-30 min. The reaction was stopped with 50 ~1 of 2.5 M H,SO,, and absorbance at 490 nm was read by ELISA reader. As standards, mouse type IV collagen and mouse laminin (Collaborative Research) were used. Standards were diluted in the same buffer as that for samples; for example, diluted lysis buffer was used for measuring the collagen content in cell lysate. Bovine type I collagen (Vitrogen), the substrate used for cell culture, did not interfere with these ELISA systems. NuSerum contains low levels of immunoreactive laminin so the observed laminin content in conditioned medium was subtracted. The sensitivity of these ELISA assays is 100 rig/ml of mouse type IV collagen and 1 rig/ml of mouse laminin. Culture of GECs on membrane filters. GECs were also cultured on collagen-coated filters (Transwell-COL, 24.5 mm diameter, 3 pm pore size; Costar, Cambridge, MA) for 3-4 days in Kl medium until they became completely confluent. To confirm that there was a low level of transepithelial leakage of proteins, the barrier function of the cell layers on filters was measured in all experiments. Rat serum albumin (50 pg/ml, Sigma) was added to the lower medium, and the concentration of rat serum albumin in upper and lower media after incubation was measured by ELISA. Only those filters that were completely (>95%) impermeable to rat albumin were used. Biosynthetic labeling of GECs with L- [‘“Slmethionine, immunoprecipitation, SDS-PAGE, and autoradiography were performed as described above. GECs on filters were labeled for 15-U h with 0.25 mCi L-[‘“‘Slmethionine added to the lower
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compartment. Then the entire upper (1.5 ml) and lower media (2.6 ml) were harvested, centrifuged, added to inhibitors and detergents, divided into four equal aliquots and analyzed by immunoprecipitation with antiserato laminin, type IV collagen, and their respectivecontrols. NH,Cl (final concentration of 10 mM) was added to the culture medium in someexperiments. This concentration of NH&l did not disturb the GEC barrier to rat serumalbumin flux, which is consistent with the observation by Caplan et al. (6). GECs on filters were also labeled with L-[U-‘Clproline (286.1 mCi/mmol, New England Nuclear). Confluent GECs grown on filters were incubated with 12.3 &i L-[‘Clproline in DMEM containing 5% heat-inactivated and dialyzed NuSerum and hormone mixture for 16 h in the presenceof ascorbicacid and @-aminopropionitrile. Upper and lower media were harvested, and proteins including collagen were precipitated with 80% ethanol at -20°C overnight and washedwith cold 80% ethanol. After lyophilizing, the precipitates were dissolved in 0.1 M N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid buffer (pH 7.3) and divided into two aliquots. One was exposed to a collagenasesolution containing 10 units collagenase(Form III; Advance Biofactures, Lynbrook, NY), 25 mM Tris.HCl (pH 7.5), and 100 mM CaCl,, and the other was addedto the same solution excluding collagenase.Both were incubated at 37°C for 24 h. Proteins in each samplewere precipitated again with 80% ethanol at -20°C overnight, washed, and analyzed by SDS-PAGE and autoradiography asdescribedabove. RESULTS
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lated from the mobilities of a, 0, and y chains of bovine type I collagen. The ratio of the two bands determined by densitometry was approximately 2:l (Fig. 1B). In the cell lysate, the same two bands were observed in addition to several nonspecific bands (Fig. lA, lanes 3 and 4). On reaction with a sheep anti-mouse laminin antibody, two specific bands, each in the positions of A and B chains of laminin isolated from EHS sarcoma, were observed in the culture medium (Fig. 2, lanes 1 and 2). In the cell lysate fraction, bands were seen in the same positions, although they were not as clear because of nonspecific bands (lanes 3 and 4). These results show that cultured GECs produce and secrete A and B chains of laminin. Type I collagen could not be detected in the medium nor in the cell lysate fraction of these unstimulated cells. Identical results for type IV collagen and laminin were obtained on three occasions with cells from different passages. Quantitative determination of type IV collagen and laminin of cultured GECs. To estimate the distribution
of these proteins produced by GECs, sandwich ELISA systems were established. As shown in Table 1, type IV collagen and laminin were found mainly in the cellassociated fraction but were also detected in the culture medium. The cells were initially cultured on collagen gel in plastic wells so that there were two layers of media;
Production of type IV collagen and laminin by cultured GECs. GECs were labeled with [35S]methionine and used
Medium
for immunoprecipitation. When the culture medium was analyzed with anti-type IV collagen antibody, two bands were observed (Fig. lA, lanes 1 and 2). The molecular masses of the bands were 180 and 170 kDa when calcuMedium
F133
COMPONENTS
Lysate
Lysate
200 -
116
-
-
al
-
a2
116 !Y7-
9l-
a-C
n
a-C
n
Fig. 1. Autoradiogram of ,“S-labeled proteins in medium and cell lysate of cultured elomerular enithelial cells (GECs). Samnles were immunoprecipitated-with rabbit anti-type IV collagen’antibddy (a-C) or normal rabbit IgG (n). Positions of globular marker proteins (molecular masses in kDa) are shown on left, and positions of the cx-,p-, and y-chains of bovine type I collagen are on the right. Also shown is a densitogram of lane 1 (medium precipitated with a-C).
a-L
n
a-L
n
Fig. 2. Autoradiogram of “‘S-labeled proteins in medium and cell lysate ofcultured GECs.-Samples were immunoprecipitated with sheep-antilaminin antibody (a-L) or normal sheep IgG (n). Positions of marker proteins are shown on left, and positions of A and B chains of Engelbreth-Holm-Swarm (EHS) sarcoma laminin are on right.
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Table
1. Quantitative ELISA measurement of type IV collagen and laminin produced by cultured GECs
Medium Cell-associated
fraction
Type IV Collagen, b%/well
Laminin, rig/well
2.7kO.2 4.8rt3.2
5.6k0.6 23.3rtl.7
Values are means + SD. Glomerular epithelial cells (GECs) were cultured in a 6-well plate for 5-6 days (n = 6) for collagen studies and were cultured in a 12-well plate for 4-5 days (n = 7) for laminin studies.
Table 2. Distribution of type IV collagen and laminin GEC-conditioned media
Medium Medium
Type IV Collagen, !&ml
Laminin, w/ml
ND 3.2kO.6
18+2 63+7
over cells under cells
Values are means + SD; n = 3 experiments. 60-mm dishes for 7 days. ND, not detected.
GECs
were
cultured
in
in
120
MEMBRANE
COMPONENTS
basolateral medium than in the upper, apical medium (Fig. 4, lanes 1-4). Each band of laminin was also darker in the lower medium than in the upper medium (lanes 5-8). On the basis on the counts of 35S in the specific bands, 79.6 + 9.3% (n = 6) of type IV collagen and 82.1 + 5.7% (n = 7) of laminin secreted by the cells were found in the lower medium. When the cells were labeled with [14C]proline, two bands of type IV collagen were also seen in the lower medium with several additional collagenase-sensitive bands on SDS-PAGE (Fig. 5). NH&l, which is considered to increase pH in certain intracellular organelles, has been shown by Caplan et al. (7) to abolish the polarized secretion of laminin and heparan sulfate proteoglycan by MDCK cells. In the presence of 10 mM of NH4C1, similar amounts of type IV collagen and laminin were detected in both lower and upper media of GECs (Fig. 6), indicating that the polarized secretion of type IV collagen and laminin by GECs was abolished by NH4C1. DISCUSSION
T
T
wrhGEC.s 12
Incubation
16
20
24
Time (hours)
Fig. 3. Barrier function of GECs grown on collagen-coated membrane filters against flux of rat serum albumin. Albumin flux was calculated from concentration of albumin in upper medium at various time points and is expressed as a percentage of concentration in lower medium; o, membrane alone; l , membrane with confluent GECs.
medium over the cells and medium in the collagen gel under the cells. When these two culture media were harvested and analyzed separately, the concentrations of type IV collagen and laminin were substantially higher in the medium under the collagen gel (Table 2). This result was the first indication that the secretion of these proteins by GECs is polarized. Culture of GECs on filters. To further analyze the polarity of the secretion of the proteins, GECs were cultured on collagen-coated filters. Confluent GECs formed a tight barrier against the flux of rat serum albumin (Fig. 3). Twenty-four hours after adding rat serum albumin to the lower medium, the concentration of albumin in the upper medium was -1% of that in the lower medium, Without cells there was complete equilibration of albumin within 24 h, equivalent to 100% leakage. To study the polarized secretion of type IV collagen and laminin, the cells on filters were labeled with [35S]methionine, and the upper and lower media were analyzed by immunoprecipitation. The two bands of cychains of type IV collagen were much darker in the lower,
In this report we have shown that cultured rat GECs produce two a-chains of type IV collagen and subunits of laminin and secrete these components in a polarized fashion. The a-chains of type IV collagen produced by cultured GECs were detected by immunoprecipitation with antibody to type IV collagen of mouse EHS sarcoma. Their molecular masses, based on the mobility of type I collagen, were 180 and 170 kDa, which is consistent with those of proal- and proa2-chains of type IV collagen (35). The ratio of the density of the two bands was approximately 2:1, and the results of [14C]proline labeling and collagenase digestion confirmed that they are both collagenous peptides. These results suggest that type IV collagen produced by cultured GECs consists of two aland one az-chains, which is the predominant composition
Upper
Lower
Upper
Lower
200 -
116 97 66-
a-C
n
a-C
n
a-L
n
a-L
n
Fig. 4. Autoradiogram of ““S-labeled proteins in upper and lower media of confluent GECs after immunoprecipitation with rabbit anti-type IV collagen (a-C), sheep anti-laminin (a-L), and normal rabbit or sheep IgG (n). Positions of marker proteins are shown on left.
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Lower
+NH&I Lower
Upper
Lower
Upper
-
a-C
n
a-C
n
a-C
n
a-C
n
a-1,
n
a-L
n
a-1,
n
a-L
n
- al -a2
116 97 66
t-1
(+)
(4
(+I
Collagenase
Fig. ^._ 6. Autoradiogram of proteins in medium of GECs labeled with [‘“Slmethionine in absence (-NH&l) or presence (+NH,Cl) of 10 mM NH&l. Media were harvested from upper or lower chambers and were immunoprecipitated with anti-type IV collagen (a-C), normal rabbit IgG (n), anti-laminin (a-L), or normal sheep IgG (n). Identity of band above type IV collagen is unknown, and its density was variable from sample to sample (compare Figs. 1, 5, and 6).
Fig. 5. Autoradiogram of “C-labeled proteins in upper or lower medium of GECs. Samples were incubated without (-) or with (+) collagenase before electrophoresis. Positions of marker proteins are shown on left, and positions of the cy-,/3-, and -r-chains of type I collagen are shown on right.
different BMs. Laminin composition appears to be tissue specific, and even the same cells may vary their usage of the A and B chains with different external stimuli (33). In this study, we found that laminin produced by cultured GECs consists of A and B chain doublets, which suggests of native type IV collagen isolated from native BM. the presence of an additional A chain. Tokida et al. (33) Recent studies have revealed novel chains (Q, a4, and reported that doublet bands of the A chain of laminin as) of type IV collagen in native human and bovine BM were observed in the intracellular fraction of cultured (13, 23, 28). The target antigen of anti-GBM antibody endothelial cells and demonstrated their precursor-prodin patients with Goodpasture’s syndrome has been iden- uct relationship. The doublet of A chain of GECs seems tified in the noncollagenous portion (NCl) of the (Ye- different from that of endothelial cells, because we found chain of type IV collagen (28). The defect responsible for them in the culture medium of GECs as well as in the several kindreds of Alport’s syndrome resides in the LYE- cell lysate fraction. Whether or not these represent two chain of type IV collagen (5, 23). These novel chains distinct A chains or alternate splicing of a single A chain were found in restricted BM of tissues including GBM gene remains to be determined. and were not detected in the EHS tumor (19). Because In this study we found type IV collagen and laminin the electrophoretic mobilities of the intact molecules of produced by GECs to be associated mainly with the cellthese novel a-chains have not been defined and because associated fraction (presumably in a cell-associated basal the anti-type IV collagen used in this study was prepared lamina). Additional amounts of each matrix protein were with EHS type IV collagen, we are not able to determine found in soluble form in the media. Collagenous molewhether the type IV collagen bands on SDS-PAGE in cules have been previously demonstrated in the media this study include these novel a-chains. Monospecific and substrata of human and mouse GECs in culture (4, antibodies reactive with the rat equivalents of the LYE-, 10, 18, 34); however, this study with immunoprecipitaa4-, and as-chains of type IV collagen or more information and ELISA is the first quantitative analysis of tion on their electrophoretic mobilities are necessary to laminin chains produced by GECs in culture. These establish this fact. quantitative techniques also permitted us to document Laminin, as originally identified in mouse tumor cell the polarized secretion of type IV collagen and laminin, lines (32), consists of three chains, A, B1, and B2 (30). which is of relevance to the role of GECs in GBM Recent studies have shown the existence of laminin A assembly. chain- and B chain-related proteins in normal tissues or During embryogenesis of the developing glomerulus, GECs are cuboidal in shape and adjacent cells are linked normal cultured cells (9, 14, 21, 33), but it is important to point out that there is heterogeneity of laminin in by a subapical tight junction that migrates basally to
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form the slit diaphragm as the developing GEC matures into a differentiated podocyte (3, 25). These features of the immature GEC are similar to the morphological characteristics of our cultured rat GECs, which are cuboidal and exhibit apical microvilli, junctional complexes (24), and the functional characteristics of a tight epithelium (present study). Moreover, morphological studies and immunohistology have clearly established that GECs play an important part in the assembly of the developing GBM and for the insertion of type IV collagen and laminin (3, 27). A subject of active current investigation is to determine whether GECs in mature glomeruli continue to fulfill this function and are stimulated to increase matrix synthesis after injury. Thus our present studies, showing that GECs grown on collagen-coated membrane filters form a tight barrier to the flux of macromolecules and secrete type IV collagen and laminin in a polarized fashion, demonstrate that cultured GECs retain several features of the developing glomerular epithelium. This provides a useful model system to study BM assembly in vitro as well as the opportunity to examine the effects of various mediators of injury on polarized matrix production by GECs. Our findings are also in close agreement with those of Caplan et al. (7), who demonstrated polarized secretion of laminin and heparan sulfate proteoglycans by MDCK cells, a cell line that has been used extensively for studies of cell polarity. Caplan et al. (7) also showed that the polarized secretion was abolished by NH&l, suggesting the importance of an acidic intracellular compartment for the sorting process that mediates the basolateral secretion of certain matrix components. Our studies indicate that GECs appear to utilize a similar sorting system for the polarized secretion of laminin and type IV collagen. Address Received
reprint 20 May
requests
to D. J. Salant.
1991; accepted
in final
form
26 August
1991.
REFERENCES D. R., and J. P. Caulfield. Proteinuria and 1. Abrahamson, structural alterations in rat glomerular basement membranes induced by intravenously injected anti-laminin immunoglobulin G. J. Exp. Med. 156: 128~145,1982. 2. Abrahamson, D. R. Origin of the glomerular basement membrane visualized after in vivo labeling of laminin in newborn rat kidneys. J. Cell Biol. 100: 1988-2000, 1985. 3. Abrahamson, D. R. Structure and development of the glomerular capillary wall and basement membrane. Am. J. Physiol. 253 (Renal Fluid Electrolyte Physiol. 22): F783-F794, 1987. 4. Ardaillou, N., G. Bellon, M.-P. Nivez, S. Rakotoarison, and R. Ardaillou. Quantification of collagen synthesis by cultured human glomerular cells. Biochim. Biophys. Acta 991: 445-452,1989. 5. Barker, D. F., S. L. Hostikka, J. Zhou, L. T. Chow, A. R. Oliphant, S. C. Gerken, M. C. Gregory, M. H. Skolnick, C. L. Atkin, and K. Tryggvason. Identification of mutations in the COL4A5 collagen gene in Alport syndrome. Science Wash. DC 248: 1224-1226, 1990. 6. Caplan, M. J., H. C. Anderson, G. E. Palade, and J. D. Jamieson. Intracellular sorting and polarized cell surface delivery of (Na’,K’)ATPase, an endogenous component of MDCK cell basolateral plasma membranes. Cell 46: 623-631, 1986. 7. Caplan, M. J., J. L. Stow, A. P. Newman, J. Madri, H. C. Anderson, M. G. Farquhar, G. E. Palade, and J. D. Jamieson. Dependence on pH of polarized sorting of secreted proteins. Nature Lond. 329: 632-635, 1987.
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8. Carlin, B., R. Jaffe, B. Bender, and A. E. Chung. Entactin, a novel basal lamina-associated sulfated glycoprotein. J. Biol. Chem. 256: 5209-5214,198l. 9. Ehrig, K., I. Leivo, W. S. Argraves, E. Ruoslahti, and E. Engvall. Merosin, a tissue-specific basement membrane protein, is a laminin-like protein. Proc. Natl. Acad. Sci. USA 87: 3264-3268, 1990. 10. Foidart, J. M., J. B. Foidart, and P. R. Mahieu. Synthesis of collagen and fibronectin by glomerular cells in culture. Renal Physiol. Base1 3: 183-192, 1980. 11. Frenette, G. P., R. W. Ruddon, R. F. Krzesicki, J. A. Naser, and B. P. Peters. Biosynthesis and deposition of a noncovalent laminin-heparan sulfate proteoglycan complex and other basal lamina components by human malignant cell line. J. Biol. Chem. 264: 3078-3088,1989. 12. Furthmayr, H. Immunization procedures, isolation by affinity chromatography, and serological and immunochemical characterization of collagen specific antibodies. In: Immunochemistry of the Extracellular Matrix, edited by H. Furthmayr. Boca Raton, FL: CRC, 1982, p. 143-171. 13. Gunwar, S., J. Saus, M. E. Noelken, and B. G. Hudson. Glomerular basement membrane. Identification of a fourth chain, cy4, of type IV collagen. J. Biol. Chem. 265: 5466-5469, 1990. 14. Hunter, D. D., V. Shah, J. P. Merlie, and J. R. Sanes. A laminin-like adhesive protein concentrated in the synaptic cleft of the neuromuscular junction. Nature Lond. 338: 229-234, 1989. 15. Kanwar, Y. S. Biophysiology of glomerular filtration and proteinuria. Lab. Invest. 51: 7-21, 1984. 16. Kanwar, Y. S., and M. G. Farquhar. Isolation of glycosaminoglycans (heparan sulfate) from glomerular basement membranes. Proc. Natl. Acad. Sci. USA 76: 4493-4497, 1979. 17. Kefalides, N. A. Isolation of collagen from basement membrane containing three identical a-chains. Biochem. Biophys. Res. Commun. 45: 226-232, 1971. 18. Killen, P. D., and G. E. Striker. Human glomerular visceral epithelial cells synthesize a basal lamina collagen in vitro. Proc. Natl. Acad. Sci. USA 76: 3518-3522, 1979. 19. Kleppel, M. M., A. F. Michael, and A. J. Fish. Antibody specificity of human glomerular basement membrane type IV collagen NC1 subunits. Species variation in subunit composition. J. Biol. Chem. 261: 16547-16552,1986. 20. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature Lond. 227: 680-685, 1970. 21. Ohno, M., A. Martinez-Hernandez, N. Ohno, and N. A. Kefalides. Isolation of laminin from human placental basement membranes: amnion, chorion and chorionic microvessels. Biochem. Biophys. Res. Commun. 112: 1091-1098, 1983. 93 Orkin, R. W., P. Gehron, E. B. McGoodwin, G. R. Martin, T. Valentine, and R. Swarm. A murine tumor producing a matrix of basement membrane. J. Exp. Med. 145: 204-220, 1977. T., E. R. Pohjolainen, and J. C. Myers. Com23* Pihlajaniemi, plete primary structure of the triple-helical region and the carboxyl-terminal domain of a new type-IV collagen chain, cu-5(IV). J. Biol. Chem. 265: 13758-13766, 1990. 24, Quigg, R. J., A. V. Cybulsky, J. B. Jacobs, and D. J. Salant. Anti-FxlA produces complement-dependent cytotoxicity of glomerular epithelial cells. Kidney Int. 34: 43-52, 1988. 25. Reeves, W., J. P. Caulfield, and M. G. Farquhar. Differentiation of epithelial foot processes and filtration slits. Sequential appearance of occluding junctions, epithelial polyanion, and slit membranes in developing glomeruli. Lab. Invest. 39: 90-100, 1978. R. Butkowski, and D. D. Hunter. 26. Sanes, J. R., E. Engvall, Molecular heterogeneity of basal laminae-isoforms of laminin and collagen-IV at the neuromuscular junction and elsewhere. J. Cell Biol. 111: 1685-1699, 1990. 27. Sariola, H., R. Timpl, K. Von der Mark, R. Mayne, J. M. Fitch, T. F. Linsenmayer, and P. Ekblom. Dual origin of glomerular basement membrane. Dev. Biol. 101: 86-96, 1984. J. P. M. Langeveld, S. Quinones, 28. Saus, J., J. Wieslander, and B. G. Hudson. Identification of the Goodpasture’s antigen as the CY:%(IV) chain of collagen IV. J. Biol. Chem. 263: 1337413380,1988. K. MacKay, L. Striker, G. Striker, 29. Stow, J. L., C. J. Soroka, and M. G. Farquhar. Basement membrane heparan sulfate pro-
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30. 31. 32.
33.
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teoglycan is the main proteoglycan synthesized by glomerular epithelial cells in culture. Am. J. PathoZ. 135: 637-646, 1989. Timpl, R. Structure and biological activity of basement membrane proteins. Eur. J. Biochem. 180: 487-502, 1989. Timpl, R., M. Paulsson, M. Dziadek, and S. Fujiwara. Basement membranes. Methods Enzymol. 145: 363-391, 1987. Timpl, R-, H. Rohde, P. G. Robey, S. I. Rennard, J. M. Foidart, and G. R. Martin. Laminin, a glycoprotein from basement membranes. J. BioZ. Chem. 254: 9933-9937,1979. Tokida, Y., Y. Aratani, A. Morita, and Y. Kitagawa. Pro-
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duction of two variant laminin forms by endothelial cells and shift of their relative levels by angiostatic steroids. J. BioZ. Chem. 265: 18123-18129,199O. 34. Torbohm, I., B. Berger, M. Schonermark, J. Von Kempis, K. Rother, and G. M. Hansch. Modulation of collagen synthesis in human glomerular epithelial cells by interleukin 1. CZin. Exp. ImmunoZ. 75: 427-431,1989. 35. Tryggvason, K., P. Gehron Robey, and G. R. Martin. Biosynthesis of type IV procollagens. Biochemistry 19: 1284-1289, 1980.
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and polarized secretion of basement components by glomerular epithelial cells
YASUHIRO NATORI, YVONNE M. O’MEARA, ERIC C. MANNING, ANDREW W. M. MINTO, JERROLD S. LEVINE, WOLFGANG J. WEISE, AND DAVID J. SALANT Evans Memorial Department of Clinical Research and Department of Medicine, University Hospital, Boston University Medical Center, Boston, Massachusetts 02118 Natori, Yasuhiro, Yvonne M. O’Meara, Eric C. Manning, Andrew W. M. Minto, Jerrold S. Levine, Wolfgang J. Weise, and David J. Salant. Production and polarized secretion of basement membrane components by glomerular epithelial cells. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F131-F13’7, 1992.-To study the formation of basement membrane by glomerular epithelial cells (GECs), production and secretion of type IV collagen and laminin by rat GECs in culture were evaluated. GECs produced two chains of type IV collagen (180 and 170 kDa) in the ratio of approximately 2 to 1, when immunoprecipitated with antibody to type IV collagen of mouse Engelbreth-Holm-Swarm (EHS) sarcoma. GECs also produced proteins that were precipitated by antibody to EHS laminin, i.e., two bands each in the positions of the A and B chains of mouse laminin. On enzyme-linked immunosorbent assay (ELISA), type IV collagen and laminin were found mainly in the cell-associated fraction and in the subepithelial culture medium. Confluent GECs on membrane filters formed a tight barrier against the flux of macromolecules. Under these conditions, 80% of newly synthesized and secreted matrix proteins were detected in the basolateral medium. Moreover, treatment with ammonium chloride, which is known to affect polarized secretion, caused both type IV collagen and laminin to be secreted via the basolateral and apical surfaces in similar amounts. These results indicate that cultured GECs are polarized and that they produce and secrete basement membrane components via the basolateral side.
able heterogeneity between BMs derived from different tissues and that the molecular composition of the constituent BM matrix components may differ from those produced by tumor cells (26). Therefore tumor cells may not provide information on the structure of native, organ-specific BMs. Such information may be better obtained from cells derived from specific tissues if it can be shown that they exhibit characteristics similar to those that exist in vivo. The structure and biochemical composition of the GBM have been studied extensively, because it is relatively easy to obtain substantial quantities of pure GBM. The GBM is a composite BM, produced by and located between glomerular endothelial and epithelial cells (27). In addition to its structural role, it plays an important part in restricting the glomerular filtration of macromolecules (15). Glomerular epithelial cells (GECs) in culture have been shown to produce collagen (4, 18, 34) and proteoglycans (29). However, the type and subunit structure of the collagen produced by GECs is still obscure, and the production of laminin by cultured GECs has not yet been shown. In this study, we used monospecific antibodies and immunoprecipitation and enzyme-linked immunosorbent assay (ELISA) methods to study the production type IV collagen; laminin; biosynthesis and secretion of type IV collagen and laminin by rat GECs in culture. Our results show that GECs produce BASEMENT MEMBRANE (BM) is an extracellular matrix two a-chains of type IV collagen (possibly cyl and c&, composed of collagenous and noncollagenous glycoproand A and B chains of laminin. In addition, we found teins and proteoglycans (30). Early biochemical studies that these molecules are mostly secreted via the basolatof BM isolated from normal tissues including glomeruli era1 side. identified type IV collagen (17) and heparan sulfate proteoglycan (16) as a nonfibrous collagen and a major AND METHODS anionic component of glomerular BM (GBM), respec- MATERIALS tively. Difficulties in solubilizing components of native Culture of GECs. GECs were obtained from Sprague-Dawley BM without proteolytic degradation (31) were overcome rats (CD; Charles River Breeding Laboratories, Wilmington, through the use of BM-producing tumor cell lines, such MA) and cultured as detailed previously (24). In brief, glomeruli as Engelbreth-Holm-Swarm (EHS) mouse sarcoma (ZZ), were isolated from young rats and plated on collagen gel matrix (Vitrogen 100; Collagen, Palo Alto, CA) in a 1:l mix of condiwhich greatly facilitated further progress. Novel glycoproteins such as laminin (32) and entactin/nidogen (8) tioned medium from NIH 3T3 fibroblasts and Kl medium Dulbecco’s modified Eagle’s medium (DMEM) were identified in the matrix of such tumor cells and containing were also shown in authentic BM by use of antibodies to Ham’s F-10 (1:l) (GIBCO, Grand Island, NY) with 5% NuSerum (Collaborative Research, Bedford, MA) and hormone the tumor-derived matrix proteins (8, 32). mixture. After -7-9 days, clearly defined epithelioid colonies The molecular structures of type IV collagen and lam- were excised from the gel and plated in collagen-coated wells. inin were derived mainly from tumor cell lines. Type IV Those wells exhibiting growth of pure epithelial-like colonies collagen was found to be a heterotrimer of two al-chains without mesangial cell contamination were selected for further and one Q2-chain, and EHS laminin was shown to consist expansion in Kl medium. For these studies, cells were studied of three subunits, an A chain with a molecular mass of between passages 15 and 35 and were shown to have the same 400 kDa and B1 and Bz chains of 200-220 kDa (30). characteristics as early passage cells, namely, cobblestone apRecently, it has become apparent that there is consider- pearance at confluence, positive cytokeratin staining, sensitiv0363-6127/92
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ity to low concentrations of purine aminonucleoside (25-100 pg/ml), ultrastructural features of epithelial cells such as apical microvilli and junctional complexes, and susceptibility to the cytotoxic effects of anti-FxlA and complement (24). Biosynthetic labeling of GECs with r”‘S]methionine. Subconfluent GECs grown on collagen gel (60 mm dish) were washed three times with methionine-free DMEM and incubated for 60 min with methionine-free DMEM containing 5% heat-inactivated and dialyzed NuSerum and 0.5% hormone mixture. Then they were washed twice with methionine-free DMEM and incubated for 15 h with 0.5 mCi L-[““Slmethionine (1,134 Ci/ mmol; New England Nuclear, Boston, MA) in the methioninefree medium described above containing 50 lug/ml of L-ascorbic acid and 50 pg/ml of ,&aminopropionitrile. The medium was harvested, and the cell layer and adherent collagen gel were washed three times with phosphate-buffered saline (PBS) and solubilized with 0.4 ml of 2% sodium dodecyl sulfate (SDS)-50 mM tris(hydroxymethyl)aminomethane (Tris) hydrochloride (pH 7.6). Protease inhibitors and detergents were added immediately to both fractions. The final concentrations of the inhibitors and the detergents were 20 mM EDTA, 10 mM 1vethylmaleimide, 1 pg/ml leupeptin, 10 pg/ml benzamidine, 2 pg/ml antipain, 1 pg/ml aprotinin, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS. Washing buffer for immunoprecipitation contained the same concentrations of these inhibitors and detergents. The entire volume of both fractions (medium and cell layer) was divided into equal aliquots and analyzed by immunoprecipitation. Immunoprecipitation, gel electrophoresis, and autoradiography. Media and cell lysates were pretreated with 0.5 ml of 10% Staphylococcus aureus slurry (IgGsorb; Enzyme Center, Malden, MA) and 20 ~1 of 1 mg/ml normal rabbit immunoglobulin G (IgG) for 60 min. After centrifugation, the supernatants were incubated with 2 pg of affinity-purified rabbit anti-mouse type I or type IV collagen (kindly provided by Dr. H. Furthmayr), normal rabbit IgG, affinity-purified sheep anti-mouse laminin (provided by Dr. D. Abrahamson), or normal sheep IgG for 120 min. Sheep anti-laminin was purified by elution from lamininSepharose (1). Specificity for laminin and nonreactivity with fibronectin and type IV collagen were verified by inhibition ELISA (2). Rabbit anti-mouse type IV collagen was purified by absorption against types I and III collagen and elution from an affinity column of mouse type IV collagen as described (12). Specificity for type IV collagen was confirmed by inhibition ELISA. The reactivity of all antisera with rat antigens was established by immunofluorescence on normal rat kidney and by ELISA. Samples incubated with anti-laminin and normal sheep IgG were further incubated with 30 pug of rabbit antisheep IgG for 60 min, and then all samples were treated with IgGsorb (20 ~1 for anti-collagen and normal rabbit IgG and 120 ~1 for anti-laminin and normal sheep IgG) for 30 min. The precipitates were then washed six times with washing buffer described above and once with 50 mM Tris HCl (pH 6.8), boiled in Laemmli sample buffer (20) for 5 min, and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with 5% acrylamide gels under reducing conditions. Mouse laminin isolated from EHS sarcoma (Collaborative Research), 14C-labeled type I collagen (provided by Dr. B. Smith) and molecular weight standards (Bio-Rad Laboratories, Richmond, CA) were used as molecular weight markers. Gels were stained, destained, treated with Enlightning (New England Nuclear) for 30 min, dried, and autoradiographed with preflashed Kodak X-Omat AR X-ray films (Eastman Kodak, Rochester, NY) for 2 wk. Photographs of exposed X-ray films were used for densitometry. To count S, dried gels were cut at the position of the bands, soaked in a few drops of water, dissolved in 5% Protosol in Econofluor (New England Nuclear) at 37°C overnight, and
MEMBRANE
COMPONENTS
counted in a ,&scintillation counter. ELISA for type IV colZagen and laminin. Nearly confluent GECs cultured on collagen gel matrix were used. To measure laminin, after the culture medium was harvested, the GECs on collagen gel were washed with PBS, pH 7.2, incubated with 0.2% collagenase (CLS IV; Cooper Biomedical, Malvern, PA) for 45 min at 37OC, and centrifuged at 800 g for 3 min. Then the cell pellet was treated with 0.1 mg/ml pepsin in 0.5 M acetic acid at 4°C overnight, and the pepsin-solubilized fraction was neutralized by adding NaOH. The sum of the collagenase- and pepsin-solubilized fractions was expressed as the cell-associated fraction. To measure type IV collagen, the collagen gel matrix was melted in PBS by incubation at 47°C for 30 min (ll), instead of digestion with collagenase. GECs were solubilized with 2% SDS-50 mM Tris HCl (pH 7.6) and used for ELISA. Lysates in 2% SDS-containing buffer were diluted more than 20 times to lessen the effect of SDS on ELISA. The sum of the melted collagen fraction and SDS lysate was expressed as the cell-associated fraction. To obtain apical or basal medium separately, the following procedure was followed. The medium over the cells was harvested first after GECs became confluent. Then the GECs with gel matrix were taken out and centrifuged. The medium remaining in the culture plates and the supernatant of the centrifuged GECs were used as medium under the cells. For ELISA, wells of microtitration plates (Falcon, BecktonDickinson, Oxnard, CA) were coated with 100 ~1 of 2 pg/ml affinity-purified sheep anti-mouse laminin or affinity-purified rabbit anti-mouse type IV collagen antibodies diluted in PBS at 4°C overnight. Wells were blocked with 200 ~1 of blocking buffer (1% bovine serum albumin-O.15 M Tris HCl, pH 7.6) for 60 min, washed with washing buffer [0.05% Tween-20-15 mM Tris HCl (pH 7.6)-135 mM NaCl] three times, and incubated with sample solutions (100 ~1) diluted in blocking buffer overnight. After washing three times, the wells were incubated overnight with the corresponding biotin-conjugated antibodies (1 pg/ml in blocking buffer), followed by incubation with avidin-peroxidase (2.5 pg/ml in blocking buffer) (Sigma Chemical, St. Louis, MO) for 30 min and substrate solution (0.4 mg/ml o-phenylenediamine-0.012% H202-50 mM phosphate citrate buffer, pH 5.0) for 15-30 min. The reaction was stopped with 50 ~1 of 2.5 M H,SO,, and absorbance at 490 nm was read by ELISA reader. As standards, mouse type IV collagen and mouse laminin (Collaborative Research) were used. Standards were diluted in the same buffer as that for samples; for example, diluted lysis buffer was used for measuring the collagen content in cell lysate. Bovine type I collagen (Vitrogen), the substrate used for cell culture, did not interfere with these ELISA systems. NuSerum contains low levels of immunoreactive laminin so the observed laminin content in conditioned medium was subtracted. The sensitivity of these ELISA assays is 100 rig/ml of mouse type IV collagen and 1 rig/ml of mouse laminin. Culture of GECs on membrane filters. GECs were also cultured on collagen-coated filters (Transwell-COL, 24.5 mm diameter, 3 pm pore size; Costar, Cambridge, MA) for 3-4 days in Kl medium until they became completely confluent. To confirm that there was a low level of transepithelial leakage of proteins, the barrier function of the cell layers on filters was measured in all experiments. Rat serum albumin (50 pg/ml, Sigma) was added to the lower medium, and the concentration of rat serum albumin in upper and lower media after incubation was measured by ELISA. Only those filters that were completely (>95%) impermeable to rat albumin were used. Biosynthetic labeling of GECs with L- [‘“Slmethionine, immunoprecipitation, SDS-PAGE, and autoradiography were performed as described above. GECs on filters were labeled for 15-U h with 0.25 mCi L-[‘“‘Slmethionine added to the lower
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compartment. Then the entire upper (1.5 ml) and lower media (2.6 ml) were harvested, centrifuged, added to inhibitors and detergents, divided into four equal aliquots and analyzed by immunoprecipitation with antiserato laminin, type IV collagen, and their respectivecontrols. NH,Cl (final concentration of 10 mM) was added to the culture medium in someexperiments. This concentration of NH&l did not disturb the GEC barrier to rat serumalbumin flux, which is consistent with the observation by Caplan et al. (6). GECs on filters were also labeled with L-[U-‘Clproline (286.1 mCi/mmol, New England Nuclear). Confluent GECs grown on filters were incubated with 12.3 &i L-[‘Clproline in DMEM containing 5% heat-inactivated and dialyzed NuSerum and hormone mixture for 16 h in the presenceof ascorbicacid and @-aminopropionitrile. Upper and lower media were harvested, and proteins including collagen were precipitated with 80% ethanol at -20°C overnight and washedwith cold 80% ethanol. After lyophilizing, the precipitates were dissolved in 0.1 M N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid buffer (pH 7.3) and divided into two aliquots. One was exposed to a collagenasesolution containing 10 units collagenase(Form III; Advance Biofactures, Lynbrook, NY), 25 mM Tris.HCl (pH 7.5), and 100 mM CaCl,, and the other was addedto the same solution excluding collagenase.Both were incubated at 37°C for 24 h. Proteins in each samplewere precipitated again with 80% ethanol at -20°C overnight, washed, and analyzed by SDS-PAGE and autoradiography asdescribedabove. RESULTS
MEMBRANE
lated from the mobilities of a, 0, and y chains of bovine type I collagen. The ratio of the two bands determined by densitometry was approximately 2:l (Fig. 1B). In the cell lysate, the same two bands were observed in addition to several nonspecific bands (Fig. lA, lanes 3 and 4). On reaction with a sheep anti-mouse laminin antibody, two specific bands, each in the positions of A and B chains of laminin isolated from EHS sarcoma, were observed in the culture medium (Fig. 2, lanes 1 and 2). In the cell lysate fraction, bands were seen in the same positions, although they were not as clear because of nonspecific bands (lanes 3 and 4). These results show that cultured GECs produce and secrete A and B chains of laminin. Type I collagen could not be detected in the medium nor in the cell lysate fraction of these unstimulated cells. Identical results for type IV collagen and laminin were obtained on three occasions with cells from different passages. Quantitative determination of type IV collagen and laminin of cultured GECs. To estimate the distribution
of these proteins produced by GECs, sandwich ELISA systems were established. As shown in Table 1, type IV collagen and laminin were found mainly in the cellassociated fraction but were also detected in the culture medium. The cells were initially cultured on collagen gel in plastic wells so that there were two layers of media;
Production of type IV collagen and laminin by cultured GECs. GECs were labeled with [35S]methionine and used
Medium
for immunoprecipitation. When the culture medium was analyzed with anti-type IV collagen antibody, two bands were observed (Fig. lA, lanes 1 and 2). The molecular masses of the bands were 180 and 170 kDa when calcuMedium
F133
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Lysate
Lysate
200 -
116
-
-
al
-
a2
116 !Y7-
9l-
a-C
n
a-C
n
Fig. 1. Autoradiogram of ,“S-labeled proteins in medium and cell lysate of cultured elomerular enithelial cells (GECs). Samnles were immunoprecipitated-with rabbit anti-type IV collagen’antibddy (a-C) or normal rabbit IgG (n). Positions of globular marker proteins (molecular masses in kDa) are shown on left, and positions of the cx-,p-, and y-chains of bovine type I collagen are on the right. Also shown is a densitogram of lane 1 (medium precipitated with a-C).
a-L
n
a-L
n
Fig. 2. Autoradiogram of “‘S-labeled proteins in medium and cell lysate ofcultured GECs.-Samples were immunoprecipitated with sheep-antilaminin antibody (a-L) or normal sheep IgG (n). Positions of marker proteins are shown on left, and positions of A and B chains of Engelbreth-Holm-Swarm (EHS) sarcoma laminin are on right.
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Table
1. Quantitative ELISA measurement of type IV collagen and laminin produced by cultured GECs
Medium Cell-associated
fraction
Type IV Collagen, b%/well
Laminin, rig/well
2.7kO.2 4.8rt3.2
5.6k0.6 23.3rtl.7
Values are means + SD. Glomerular epithelial cells (GECs) were cultured in a 6-well plate for 5-6 days (n = 6) for collagen studies and were cultured in a 12-well plate for 4-5 days (n = 7) for laminin studies.
Table 2. Distribution of type IV collagen and laminin GEC-conditioned media
Medium Medium
Type IV Collagen, !&ml
Laminin, w/ml
ND 3.2kO.6
18+2 63+7
over cells under cells
Values are means + SD; n = 3 experiments. 60-mm dishes for 7 days. ND, not detected.
GECs
were
cultured
in
in
120
MEMBRANE
COMPONENTS
basolateral medium than in the upper, apical medium (Fig. 4, lanes 1-4). Each band of laminin was also darker in the lower medium than in the upper medium (lanes 5-8). On the basis on the counts of 35S in the specific bands, 79.6 + 9.3% (n = 6) of type IV collagen and 82.1 + 5.7% (n = 7) of laminin secreted by the cells were found in the lower medium. When the cells were labeled with [14C]proline, two bands of type IV collagen were also seen in the lower medium with several additional collagenase-sensitive bands on SDS-PAGE (Fig. 5). NH&l, which is considered to increase pH in certain intracellular organelles, has been shown by Caplan et al. (7) to abolish the polarized secretion of laminin and heparan sulfate proteoglycan by MDCK cells. In the presence of 10 mM of NH4C1, similar amounts of type IV collagen and laminin were detected in both lower and upper media of GECs (Fig. 6), indicating that the polarized secretion of type IV collagen and laminin by GECs was abolished by NH4C1. DISCUSSION
T
T
wrhGEC.s 12
Incubation
16
20
24
Time (hours)
Fig. 3. Barrier function of GECs grown on collagen-coated membrane filters against flux of rat serum albumin. Albumin flux was calculated from concentration of albumin in upper medium at various time points and is expressed as a percentage of concentration in lower medium; o, membrane alone; l , membrane with confluent GECs.
medium over the cells and medium in the collagen gel under the cells. When these two culture media were harvested and analyzed separately, the concentrations of type IV collagen and laminin were substantially higher in the medium under the collagen gel (Table 2). This result was the first indication that the secretion of these proteins by GECs is polarized. Culture of GECs on filters. To further analyze the polarity of the secretion of the proteins, GECs were cultured on collagen-coated filters. Confluent GECs formed a tight barrier against the flux of rat serum albumin (Fig. 3). Twenty-four hours after adding rat serum albumin to the lower medium, the concentration of albumin in the upper medium was -1% of that in the lower medium, Without cells there was complete equilibration of albumin within 24 h, equivalent to 100% leakage. To study the polarized secretion of type IV collagen and laminin, the cells on filters were labeled with [35S]methionine, and the upper and lower media were analyzed by immunoprecipitation. The two bands of cychains of type IV collagen were much darker in the lower,
In this report we have shown that cultured rat GECs produce two a-chains of type IV collagen and subunits of laminin and secrete these components in a polarized fashion. The a-chains of type IV collagen produced by cultured GECs were detected by immunoprecipitation with antibody to type IV collagen of mouse EHS sarcoma. Their molecular masses, based on the mobility of type I collagen, were 180 and 170 kDa, which is consistent with those of proal- and proa2-chains of type IV collagen (35). The ratio of the density of the two bands was approximately 2:1, and the results of [14C]proline labeling and collagenase digestion confirmed that they are both collagenous peptides. These results suggest that type IV collagen produced by cultured GECs consists of two aland one az-chains, which is the predominant composition
Upper
Lower
Upper
Lower
200 -
116 97 66-
a-C
n
a-C
n
a-L
n
a-L
n
Fig. 4. Autoradiogram of ““S-labeled proteins in upper and lower media of confluent GECs after immunoprecipitation with rabbit anti-type IV collagen (a-C), sheep anti-laminin (a-L), and normal rabbit or sheep IgG (n). Positions of marker proteins are shown on left.
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+NH&I Lower
Upper
Lower
Upper
-
a-C
n
a-C
n
a-C
n
a-C
n
a-1,
n
a-L
n
a-1,
n
a-L
n
- al -a2
116 97 66
t-1
(+)
(4
(+I
Collagenase
Fig. ^._ 6. Autoradiogram of proteins in medium of GECs labeled with [‘“Slmethionine in absence (-NH&l) or presence (+NH,Cl) of 10 mM NH&l. Media were harvested from upper or lower chambers and were immunoprecipitated with anti-type IV collagen (a-C), normal rabbit IgG (n), anti-laminin (a-L), or normal sheep IgG (n). Identity of band above type IV collagen is unknown, and its density was variable from sample to sample (compare Figs. 1, 5, and 6).
Fig. 5. Autoradiogram of “C-labeled proteins in upper or lower medium of GECs. Samples were incubated without (-) or with (+) collagenase before electrophoresis. Positions of marker proteins are shown on left, and positions of the cy-,/3-, and -r-chains of type I collagen are shown on right.
different BMs. Laminin composition appears to be tissue specific, and even the same cells may vary their usage of the A and B chains with different external stimuli (33). In this study, we found that laminin produced by cultured GECs consists of A and B chain doublets, which suggests of native type IV collagen isolated from native BM. the presence of an additional A chain. Tokida et al. (33) Recent studies have revealed novel chains (Q, a4, and reported that doublet bands of the A chain of laminin as) of type IV collagen in native human and bovine BM were observed in the intracellular fraction of cultured (13, 23, 28). The target antigen of anti-GBM antibody endothelial cells and demonstrated their precursor-prodin patients with Goodpasture’s syndrome has been iden- uct relationship. The doublet of A chain of GECs seems tified in the noncollagenous portion (NCl) of the (Ye- different from that of endothelial cells, because we found chain of type IV collagen (28). The defect responsible for them in the culture medium of GECs as well as in the several kindreds of Alport’s syndrome resides in the LYE- cell lysate fraction. Whether or not these represent two chain of type IV collagen (5, 23). These novel chains distinct A chains or alternate splicing of a single A chain were found in restricted BM of tissues including GBM gene remains to be determined. and were not detected in the EHS tumor (19). Because In this study we found type IV collagen and laminin the electrophoretic mobilities of the intact molecules of produced by GECs to be associated mainly with the cellthese novel a-chains have not been defined and because associated fraction (presumably in a cell-associated basal the anti-type IV collagen used in this study was prepared lamina). Additional amounts of each matrix protein were with EHS type IV collagen, we are not able to determine found in soluble form in the media. Collagenous molewhether the type IV collagen bands on SDS-PAGE in cules have been previously demonstrated in the media this study include these novel a-chains. Monospecific and substrata of human and mouse GECs in culture (4, antibodies reactive with the rat equivalents of the LYE-, 10, 18, 34); however, this study with immunoprecipitaa4-, and as-chains of type IV collagen or more information and ELISA is the first quantitative analysis of tion on their electrophoretic mobilities are necessary to laminin chains produced by GECs in culture. These establish this fact. quantitative techniques also permitted us to document Laminin, as originally identified in mouse tumor cell the polarized secretion of type IV collagen and laminin, lines (32), consists of three chains, A, B1, and B2 (30). which is of relevance to the role of GECs in GBM Recent studies have shown the existence of laminin A assembly. chain- and B chain-related proteins in normal tissues or During embryogenesis of the developing glomerulus, GECs are cuboidal in shape and adjacent cells are linked normal cultured cells (9, 14, 21, 33), but it is important to point out that there is heterogeneity of laminin in by a subapical tight junction that migrates basally to
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form the slit diaphragm as the developing GEC matures into a differentiated podocyte (3, 25). These features of the immature GEC are similar to the morphological characteristics of our cultured rat GECs, which are cuboidal and exhibit apical microvilli, junctional complexes (24), and the functional characteristics of a tight epithelium (present study). Moreover, morphological studies and immunohistology have clearly established that GECs play an important part in the assembly of the developing GBM and for the insertion of type IV collagen and laminin (3, 27). A subject of active current investigation is to determine whether GECs in mature glomeruli continue to fulfill this function and are stimulated to increase matrix synthesis after injury. Thus our present studies, showing that GECs grown on collagen-coated membrane filters form a tight barrier to the flux of macromolecules and secrete type IV collagen and laminin in a polarized fashion, demonstrate that cultured GECs retain several features of the developing glomerular epithelium. This provides a useful model system to study BM assembly in vitro as well as the opportunity to examine the effects of various mediators of injury on polarized matrix production by GECs. Our findings are also in close agreement with those of Caplan et al. (7), who demonstrated polarized secretion of laminin and heparan sulfate proteoglycans by MDCK cells, a cell line that has been used extensively for studies of cell polarity. Caplan et al. (7) also showed that the polarized secretion was abolished by NH&l, suggesting the importance of an acidic intracellular compartment for the sorting process that mediates the basolateral secretion of certain matrix components. Our studies indicate that GECs appear to utilize a similar sorting system for the polarized secretion of laminin and type IV collagen. Address Received
reprint 20 May
requests
to D. J. Salant.
1991; accepted
in final
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26 August
1991.
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