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Complement (C5b-9) induces glomerular epithelial cell DNA synthesis but not proliferation in vitro
Kidney International, Vol. 56 (1999), pp. 538–548
CELL BIOLOGY – IMMUNOLOGY – PATHOLOGY
Complement (C5b-9) induces glomerular epithelial cell DNA synthesis but not proliferation in vitro STUART J. SHANKLAND, JEFFREY W. PIPPIN, and WILLIAM G. COUSER Department of Medicine, Division of Nephrology, University of Washington, Seattle, Washington, USA
Complement (C5b-9) induces glomerular epithelial cell DNA synthesis but not proliferation in vitro. Background. The C5b-9 membrane attack complex of complement is the principal mediator of injury induced experimentally by antibodies directed at glomerular cell membranes. In experimental membranous nephropathy, C5b-9 induced injury to the glomerular visceral epithelial cell (VEC) is associated with DNA synthesis, but not cytokinesis. In the current study we determined if C5b-9 increases DNA synthesis in VEC in vitro, and defined the mechanisms involved. Methods. Rat VEC in vitro were divided into three groups: (1) sensitized with anti-VEC antibody and exposed to sublytic concentrations of C 1/PVG serum (normal complement components); (2) anti-VEC antibody and control C-/PVG serum (C6 deficient); (3) no anti-VEC antibody. DNA synthesis (BrdU staining), mitosis (mitotic figures) and cytokinesis (cell counts) were measured at 24 and 48 hours. To examine the expression of specific S-phase and M-phase cell cycle regulatory proteins and their inhibitors, immunostaining and Western blot analysis was performed for cyclin A, CDK2, p21 and p27, cyclin B and cdc2. Results. In the absence of growth factors, sublytic C5b-9 attack did not increase proliferation. In contrast, sublytic C5b-9 attack (group 1) augmented growth factor induced DNA synthesis by 50% compared to controls (groups 2 and 3; P , 0.001), and was accompanied by increased levels of cyclin A and CDK2, and a decrease in the cyclin kinase inhibitor p27 (but not p21). Sublytic C5b-9 attack reduced the expression of the M phase cell cycle proteins, cyclin B and cdc2, accompanied by reduced mitosis (mitotic figures) and cytokinesis (cell number). Conclusions. Our results show that the C5b-9 augmented growth factor entry into the S phase in VEC is regulated by changes in specific cell cycle regulatory proteins. However, antibody and complement decreased the M phase cell cycle proteins, and prevented VEC mitosis and cytokinesis, suggesting a delay or arrest at the G2/M phase.
Complement is a critical mediator of antibody-induced renal disease [1]. Established effects include generation of chemotactic factors such as C5a leading to recruitment of circulating inflammatory cells and formation of the C5b-9 membrane attack complex of complement which can insert into and activate renal cells to make other inflammatory mediators [2, 3]. The role of C5b-9 has been best established in renal diseases induced experimentally by antibodies to glomerular cell membrane antigens. Thus, C5b-9 is the principal mediator of injury induced by antibody to the glomerular visceral epithelial cell (VEC) (passive Heymann nephritis; PHN) [4–7], mesangial cell (experimental mesangial proliferative glomerulonephritis, Thy1 model) [8], and endothelial cell (thrombotic microangiopathy model) [9]. Of note, however, is that the consequences of C5b-9 attack appear to differ depending on which cell type is involved. In the mesangium, antibody and complement induce mesangial cell lysis, followed by a phase of proliferation [8, 10]. In contrast, C5b-9 attack on the VEC in vivo does not cause lysis and is followed by cell activation and DNA synthesis, occasional mitosis and ploidy [11– 13]. However, the VEC does not readily undergo cytokinesis (cell division) and the VEC number does not increase in vivo [14]. It is likely that this failure of the VEC to proliferate in some forms of glomerular disease contributes to glomerular sclerosis and renal failure [15–17]. Cell proliferation is ultimately controlled in the nucleus at the level of the cell cycle, and requires a transition through specific phases of the cell cycle [18]. Each phase of the cell cycle serves a unique role in preparing a cell to proliferate (increase in cell number) [19]. Thus, the S phase is required for DNA synthesis, and the M phase is required for mitosis and cell division (cytokinesis) [20]. Each phase of the cell cycle is governed by specific positive cell cycle regulatory proteins called cyclins and cyclin dependent kinases (CDK) [21]. DNA synthesis (S phase) requires that CDK2 be activated by its partner cyclin A [22]. Mitosis and cytokinesis
Key words: p27, cyclins, cell cycle, proliferation, podocyte, membrane attack complex, visceral epithelial cell. Received for publication July 31, 1998 and in revised form February 5, 1999 Accepted for publication March 8, 1999
1999 by the International Society of Nephrology
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(M phase) require that cdc2 (formerly called CDK1) be activated by cyclin B [23]. In contrast, cyclin kinase inhibitors such as p21 and p27 inhibit cell cycle progression by inactivating specific cyclin-CDK complexes [24]. To better understand the consequences of C5b-9 induced injury in diseases of the VEC, we have examined the effects of sublytic C5b-9 attack on VEC proliferation by examining DNA synthesis, mitosis and cytokinesis in vitro as they relate to changes in expression of cell cycle regulatory proteins. To our knowledge, our results provide the first evidence that C5b-9 increases DNA synthesis in a resident glomerular cell, suggest that this effect is mediated in part by additional cell-derived mitogen(s), and establish a role for cell cycle regulatory proteins in the response of the VEC to complement-mediated injury. METHODS Glomerular epithelial cell culture and experimental design Rat visceral glomerular epithelial cells (VEC) (podocytes) were established in culture as previously described [25]. In brief, glomeruli were isolated from 60 to 100 g male Sprague-Dawley rats (Simonsen, Gilroy, CA, USA) by differential sieving, and placed in culture dishes containing Vitrogen (Celtrix, Santa Clara, CA, USA) and K-1 Media consisting of Ham’s F-12 and DME-low glucose 1:1 (Gibco, Grand Island, NY, USA) with 2% NuSerum (Collaborative Biomedical Products, Bedford, MA, USA) and ITS Premix (Collaborative Biomedical Products). After five to seven days, colonies of VEC were marked in the culture dish, excised, and replated. Initially, cells were passaged every four to five days onto vitrogen and after 20 passages they were passaged onto plastic. VEC were characterized by established criteria, which included a polygonal shape and cobblestone appearance at confluency, and positive staining with antibodies to Fx1A, cytokeratin, and podocalyxin and negative staining with antibodies directed against the mesangial cell (Thy-1) and endothelial cell (Factor VIII) [25]. Antibody-C5b-9 stimulation of VEC To induce sublytic C5b-9 attack, VEC were split 1:3, plated overnight in K-1 media, and allowed to grow to 50% confluency. VEC were washed three times with serum-free K-1 media, and divided into three groups. Group 1. VEC were sensitized with 3 mg/ml of sheepanti-Fx1A IgG at 378C for 30 minutes [26]. Unbound antibody was removed by washing three times with serum free K-1 media. Sensitized VEC were then exposed to a sublytic complement source (PVG C1 serum, see below) in the media for 60 minutes. VEC were washed
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three times, and the cells were then incubated in serumfree media or media with serum. Group 2. VEC served as a control for C5b-9 attacked cells. VEC were sensitized with anti-Fx1A IgG and then exposed to serum for 60 minutes that lacked C6 (PVG C2 serum, see below), washed, and incubated in serum free media or media with serum. Group 3. VEC served as a control for anti-Fx1A IgG: VEC were exposed to media at 378C for 30 minutes in the absence of anti-Fx1A. Experimental design Two experimental conditions were used to answer specific questions. To determine if sublytic C5b-9 attack was sufficient to increase DNA synthesis, mitosis and cytokinesis in the absence of a source of growth factors (Nuserum), VEC from each group were incubated in serum free media for 18 to 48 hours. To determine if sublytic C5b-9 attack augmented DNA synthesis and mitosis induced by growth factors, VEC from each group where grown in Nuserum for 18 to 48 hours. Preparing sublytic C5b-9 attack Serum from normocomplementemic PVG rats (C1; Harlan Sprague-Dawley, Indianapolis, IN, USA) was utilized as a complement source to induce sublytic C5b-9 attack. Serum from C6 deficient-PVG rats (C2; Bantin and Kingman Universal, Edmonds, WA, USA) was used as control. C2 rats exhibit a complete absence of C6, inherited in an autosomal recessive pattern, and are therefore unable to assemble C5b-9 [27]. Serum was obtained from both rat strains by aortic puncture and allowed to clot. The serum was separated by centrifugation, sterile filtered on ice and stored at 2708C. The serum was tested in a CH50 hemolytic assay to establish the complement activity [28]. C1 serum exhibited complement activity equivalent to normal Sprague-Dawley rat serum. C2 serum had no hemolytic activity. In select experiments, serum from Sprague-Dawley rats was also used as a source of complement, and heat-inactivated Sprague-Dawley rat serum was used as a control. The selection of the antibody and complement concentrations used in the current study was based on checkerboard titration studies to define the maximal amounts of antibody and complement that could be used without producing VEC injury or lysis (defined as .5% LDH release; see below). Serum was diluted to 4% in serum free K-1 media and applied to the antibody sensitized VEC for one hour at 378C to allow C5b-9 insertion. VEC were then washed with serum free K-1 media to remove any unbound complement. Lactate dehydrogenase assay To ensure that C5b-9 attack was not inducing significant cell lysis, lactate dehydrogenase (LDH) was mea-
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sured in the supernatants using the Lactate Dehydrogenase Kinetic Assay Kit (Sigma Diagnostics, St. Louis, MO, USA) [29]. Conditioned media was removed one hour after the addition of the complement source, and the remaining cells were lyzed with 2% triton. The conditioned media or the lyzed cells were diluted 1:60 in nicotinamide adenine dinucleotide reduced form (NADH) 1 mg/ml. Sodium pyruvate solution was then added, and the reduction of NADH to NAD by the LDH in the media or the lyzed cells was measured by a drop in absorbance at 340 nm by a Lambda Bio Spectrophotometer (Perkin Elmer, Norwalk, CT, USA). Lysis was expressed as a percentage of the change in the absorbance of the media compared to changes in absorbance of the triton lyzed cells and the media. Less than 5% LDH release was regarded as sublytic. S phase measured by BrdU staining To determine if attack by antibody and C5b-9 was sufficient to augment DNA synthesis in VEC grown in the absence of growth factors, VEC from groups 1, 2 and 3 were incubated in Nuserum-free media for 18 hours. To determine if sublytic C5b-9 attack augmented DNA synthesis induced by growth factors, VEC from each group were incubated in media containing serum and other growth factors (2% Nu serum) for 18 hours after the C5b-9 attack. Comparisons were made to group 2 and group 3 VEC grown in 2% Nu serum for 18 hours. In each experiment, entry to the S phase was measured by BrdU immunostaining, a marker of DNA synthesis. BrdU staining (5-bromo-29-deoxyuridine) was measured with a cell proliferation kit (Amersham Life Science, Arlington, IL, USA) as previously described [30]. In brief, VEC were pulsed with BrdU (1:500 dilution) in media with or without Nuserum for 18 hours, and immunostaining was performed according to the manufacturer’s directions. BrdU was detected using a monoclonal antibody to BrdU, followed by a peroxidase conjugated sheep-antimouse IgG2a antibody and Diaminobenzidine with cobalt and nickel. Camco quick stain II (Baxter Scientific Products, McGaw Park, IL, USA) was used as a nuclear counterstain. To determine the percentage of cells staining positive for BrdU, 200 VEC were counted, and the percentage of VEC staining positive for BrdU was determined. All the experiments reported in this study were performed a minimum of six times. Measuring the M phase of the cell cycle Cell proliferation requires that a cell complete both the S phase and M phase of the cell cycle. To determine if C5b-9 increased entry into the M phase, mitotic figures [23], a marker of the M phase, were counted in group 1 (sublytic C5b-9 attack) and group 2 (anti-Fx1A without C5b-9) VEC grown in Nuserum for 24 hours. The num-
ber of mitotic figures/HPF were counted in 20 fields, and were expressed as a percentage of the total cell count. To determine if C5b-9 increased cell division (cytokinesis), the cell number was measured prior to exposure to anti-Fx1A antibody, and at 24 and 48 hours in group 1 and group 2 VEC grown in Nuserum. Cell number was reported as the percentage increase from the start of the experiment at each of these time points. Determining if mitogens are released following sublytic C5b-9 attack To determine if sublytic C5b-9 attack was associated with the release of mitogens, group 1 and 2 VEC were incubated in Nuserum-free media for 18 hours after exposure to anti-Fx1A antibody and C1 serum or C2 serum. The supernatants were collected after 18 hours, and transferred to VEC that were growing at 50% confluency in Nuserum-containing media, and BrdU immunostaining was measured in these cells after 18 hours. Basic fibroblast growth factor and TGF-b1 blocking studies To test the hypothesis that C5b-9-induced DNA synthesis resulted in part from the release of mitogenic growth factors, a neutralizing antibody to basic fibroblast growth factor (bFGF) was used to block bFGF activity following complement stimulation. Goat anti-bFGF (obtained from Dr. Michael A. Reidy, Dept. of Pathology, University of Washington, Seattle, WA, USA) or control goat IgG was added at 1 mg/ml to the K-1 media following the addition of a complement source. VEC were incubated with anti-bFGF antibody for 18 hours, and DNA synthesis was measured. To ensure that the concentration of anti-bFGF used in this study inhibited bFGF, additional experiments were performed where recombinant bFGF (10 ng/ml) was added to quiescent VEC in the presence of anti-bFGF or control goat IgG. Because transforming growth factor-b1 (TGF-b1) has been shown to reduce growth factor-induced proliferation in many cell types including VEC [31], the effect of TGF-b1 on C5b-9 stimulated DNA synthesis was also assessed. TGF-b1 (10 ng/ml; R&D Systems, Minneapolis, MN, USA) was activated in 4 mm HCl containing 0.1% BSA and added to VEC at a concentration of 10 ng/ml following exposure to C5b-9. DNA synthesis was measured 18 hours later. Determining the expression of cell-cycle regulatory proteins in VEC The levels of S phase and M phase cell-cycle regulatory proteins were determined by immunostaining and Western blot analysis [10, 30] in group I (C5b-9 attack) and 2 (control) cells after 18 hours of exposure to Nuserum. Immunostaining. VEC were grown on 24-well plates, and fixed in 1:1 methanol/acetone for 15 minutes at
Shankland et al: C5b-9 and podocyte proliferation
2208C, then washed three times in cold PBS. The cells were permeabilized with 0.5% NP-40 buffer for 10 minutes at room temperature. Following a wash in PBS, primary affinity purified rabbit polyclonal antibodies [10, 30] to cyclin A (dilution 1:1000; provided by J.M. Roberts, Fred Hutchinson Cancer Research Center, Seattle, WA, USA), CDK2 (dilution 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), cyclin B1 (dilution 1:100; Santa Cruz Biotechnology) cdc2 (dilution 1:100; Santa Cruz Biotechnology), p21 (dilution 1:1000; Santa Cruz Biotechnology) and p27 (dilution 1:1000; provided by J.M. Roberts), were incubated overnight at 48C. A secondary biotinylated goat antirabbit antibody was added (1:400 dilution, Vector Laboratories) for 60 minutes at room temperature, followed by streptavidin FITC (1:32 dilution; Cappel Research Products, Durham, NC, USA) or Avidin D horseradish peroxidase (1:2000) for 60 minutes at room temperature, followed by 49, 6-diamidino-2phenylindole (Boehringer Mannheim, Indianapolis, IN, USA). Nuclei were counterstained with Camco Quick Stain II (Baxter Scientific Products). The number of VEC staining positive for each cell cycle protein was expressed as a percentage of the total number of cells. Western blot analysis. Cell protein was obtained from group 1 (C5b-9 attacked) and group 2 (control) VEC after an 18 hour exposure to Nuserum. At each time point VEC were washed three times with cold PBS, trypsinized, and following centrifugation, the pellet was resuspended in a buffer containing 1% triton, 10% glycerol, 20 mm HEPES, 100 mm NaCl with 10 mg/ml leupeptin, 10 mg/ml antipain, 10 mg/ml pepstatin, 0.1 mm sodium orthovanadate and 50 mm sodium fluoride (reagents purchased from Sigma Biosciences). Following a vortex, the VEC were placed on ice for 15 minutes, centrifuged at 14,000 r.p.m. for 5 minutes, and the protein concentration of the supernatant was measured by a BCA protein assay (Pierce, Rockford, IL, USA). For Western blot analysis, 20 mg of VEC protein extract was loaded in each lane, separated under reducing conditions on a 15% SDSPAGE gel, and transferred to PVDF membranes (Millipore, Bedford, MA, USA) by electroblotting. To reduce nonspecific antibody binding, the membranes were first blocked with 5% nonfat dried milk for 30 minutes at room temperature. This was followed by an overnight incubation at 48C with antibodies to either cyclin A, CDK2, p21, p27, cyclin B or cdc2. An alkaline phosphatase-conjugated secondary antibody (Promega, Madison, WI, USA) was used for detection with the chromagen, 5-Bromo-4-Chloro-3-Indolyl phosphate/nitro blue tetrazolium (Sigma Biosciences). The gels were stained with Coomassie Blue to check for complete protein transfer. Following Western blot analysis, filters were also stained with Coomassie Blue to ensure that protein loading was equal. Controls for immunostaining and Western blot analy-
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sis included omitting the primary antibody, replacing the primary antibody with an irrelevant antibody of the same species and peptide absorption (the source of peptides was the same as the primary antibody). Statistics Values were reported as mean 6 sem, unless otherwise stated. Statistical significance (P , 0.05) was evaluated using the Student’s t-test, and a modified t-test was performed using the Bonferroni-Dunn correction. RESULTS C5b-9 augments growth factor-induced DNA synthesis in VEC in vitro Entry into the S phase of the cell cycle was measured by BrdU staining, a marker of DNA synthesis. A first question was to determine if sublytic C5b-9 attack increased DNA synthesis in the absence of growth factors (in this study Nuserum was used as a source of growth factors). Accordingly, BrdU staining was quantitated in each VEC group grown in Nuserum-free media for 18 hours, and the results are shown in Table 1. In the absence of a source of growth factors (Nuserum-free media) there was no difference in BrdU staining in each group. Thus, the sublytic C5b-9 attack alone did not increase DNA synthesis. A second question asked if the sublytic C5b-9 attack augmented DNA synthesis induced by growth factors. Table 1 shows there was an increase in BrdU staining in group 2 (anti-Fx1A without C5b-9) and group 3 (no anti-Fx1A antibody) VEC in the presence of serum compared to Nuserum-free conditions. There was no difference in BrdU staining in group 2 and 3 VEC at 18 hours. In contrast, there was approximately a twofold increase in BrdU staining in group 1 (sublytic C5b-9 attack) VEC compared to control (group 2 and group 3) VEC. In separate studies, sublytic C5b-9 attack induced with Sprague-Dawley serum also increased BrdU staining twofold compared to VEC exposed to antibody and heatinactivated Sprague-Dawley serum (P , 0.0001; data not shown). These results show that sublytic C5b-9 augmented growth factor induced DNA synthesis in VEC in vitro, and show that sublytic C5b-9 attack by itself was not sufficient to engage VEC into the S phase (DNA synthesis) of the cell cycle in the absence of growth factors. VEC arrest at G2/M following C5b-9 attack Entry into the M phase of the cell cycle was measured by counting mitotic figures. Because DNA synthesis was increased by C5b-9 only in the presence of serum, mitosis was assessed at 24 hours following the sublytic C5b-9 attack in VEC grown in media with growth factors (Nuserum). There were 4.43 6 0.26 mitotic figures/HPF in
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Shankland et al: C5b-9 and podocyte proliferation Table 1. Quantitation of BrdU staining Nuserum free (18h)
Group 1 2 3
Experiment Anti-Fx1A and C5b-9 Anti-Fx1A and no C5b-9 No anti-Fx1A
Nuserum (18h)
% positive BrdU cells 19 6 1.1 18 6 1.0 16 6 0.9
63 6 1.0 27 6 2.8 30.3 6 3.8 a,b a
a
P , 0.001 vs. Nuserum free P , 0.001 vs. groups 2 and 3 Nuserum Visceral epithelial cells (VEC) were divided into three groups based on the presence or absence of anti-VEC antibody and C5b-9, and were then grown in media with or without Nuserum for 18 hours. Nuserum, a source of growth factors increased the percentage of cells that stained positive for BrdU, a marker of DNA synthesis, was doubled by sublytic C5b-9. a
b
Group 1 VEC, but the increase was less than 50% compared to controls. These results show that despite increased entry into the S phase (DNA synthesis) at 18 hours induced by C5b-9 attack, there was no corresponding increase in entry into (mitosis) or progression through (cytokinesis) the M phase of the cell cycle. Thus, sublytic C5b-9 attack either causes VEC to arrest at the G2/M phase or delays G2/M phase progression and entry into M phase, despite the presence of growth factors (Nuserum) to which they can respond. C5b-9-induced VEC proliferation is induced in part by a secreted mitogen
Fig. 1. Quantitation of cell number. Glomerular visceral epithelial cells (VEC) were divided into three groups and cells were counted when placed in media with Nuserum (0 hr) and at 24 and 48 hours. The cell number was increased in control VEC [groups 2 (e) and 3 (s)] compared to VEC exposed to sublytic C5b-9 (group 1; h) at 24 and 48 hours. There was no difference in cell number in control VEC at 24 and 48 hours. aP , 0.0010 vs. groups 2 and 3; bP , 0.0007 vs. groups 2 and 3; cP , 0.099 vs. group 3; dP , 0.24 vs. group 3.
Group 2 (anti-Fx1A without C5b-9) VEC. In contrast, there were only 0.52 6 0.73 mitotic figures/HPF in group 1 (sublytic C5b-9 attack) VEC (P , 0.001 vs. group 2) To determine the effect of sublytic C5b-9 on cytokinesis (cell division), cell number was measured at 24 and 48 hours following exposure to antibody with or without a complement source in VEC grown in the presence of Nuserum. Figure 1 shows that in control VEC (groups 2 and 3) grown in Nuserum, there was a progressive increase in cell number at 24 hours (P , 0.001 vs. 0 hr) and 48 hours (P , 0.001 vs. 24 hr). In contrast, cell number did not increase in VEC following sublytic C5b-9 attack (group 1) at 24 hours (P . 0.05 vs. groups 2 and 3). However, cell number did increase at 48 hours in
To determine if the increase in DNA synthesis (BrdU staining) induced by C5b-9 and growth factors was due to the release of mitogens from VEC, supernatants were collected after 18 hours from each group of VEC and transferred to normal VEC growing in Nuserum. DNA synthesis was measured by BrdU incorporation into DNA at 18 hours after the transfer. When VEC were incubated with supernatants from control VEC (groups 2 and 3), BrdU staining was not increased (19 6 1 vs. 18 6 0.7, P . 0.05). In contrast, incubating VEC with supernatants derived from VEC subjected to sublytic C5b-9 attack (group 1) increased BrdU staining 1.5-fold (28 6 0.1 vs. 18 6 0.7, P , 0.001). Taken together, these results suggest that the increase in DNA synthesis induced by C5b-9 attack was due in part to mitogen(s) “released” following exposure of VEC to sublytic C5b-9. Complement-induced VEC DNA synthesis is not due to bFGF The cytokine bFGF is increased in experimental forms of VEC injury in vivo and bFGF is mitogenic for VEC [31, 32]. To determine if C5b-9 induced DNA synthesis was due to the release of bFGF from injured cells, DNA synthesis was measured in the presence of a specific neutralizing anti-bFGF antibody. Anti-bFGF antibody did not reduce BrdU staining induced by C5b-9 stimulation compared to control IgG (67 6 2.5 vs. 62 6 1.5%,
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P . 0.05). The concentration of anti-bFGF antibody used was effective as judged by a reduction in DNA synthesis to baseline when bFGF was used as a mitogen in the presence of antibody (results not shown). Taken together, these results show that the increase in DNA synthesis induced by C5b-9 was not due to bFGF. Complement-induced VEC DNA synthesis is not inhibited by TGF-b1 Because the cytokine TGF-b1 has been shown to inhibit specific mitogen-induced VEC proliferation in vitro [31], we asked if TGF-b1 would inhibit complement-induced VEC proliferation. The concentration of TGF-b1 used in the current study reduced BrdU staining significantly in VEC exposed to growth factors to stimulate mitogenesis (26.5 6 0.5 vs. 9 6 1, P , 0.05). In contrast, DNA synthesis induced by sublytic C5b-9 was not reduced by TGF-b1 (52 6 1 vs. 49 6 0, P . 0.05). C5b-9 increases cell cycle proteins required for S phase To better understand the effects of sublytic C5b-9 on the VEC cell cycle, we measured the expression of the S phase cyclin A and its partner CDK2 in Group 1 (sublytic C5b-9 attack), and control group 2 (anti-Fx1A without C5b-9) and group 3 (no anti-Fx1A) VEC at 18 hours, the peak of DNA synthesis in this study when VEC were grown in Nuserum-containing media. These cell cycle regulatory proteins are required for DNA synthesis (S phase) in other cell types [21], including mesangial cells [30]. Less than 10% of control VEC stained positive for CDK2 (results not shown). There was a marked increase in the number of VEC staining positive for CDK2 in C5b-9 attacked VEC (38%, P , 0.001 vs. controls; not shown) at 18 hours. The increase in CDK2 protein levels was confirmed by Western blot analysis (Fig. 2). Figure 2 shows that cyclin A was also detected in low levels by Western blot analysis in control VEC (groups 2 and 3) grown in Nuserum for 18 hours. There was also an increase in cyclin A protein levels in C5b-9 attacked VEC (group 1) compared to controls at 18 hours (Fig. 1). These results demonstrate that expression of the S phase cell cycle proteins, CDK2 and cyclin A is increased in VEC exposed to sublytic C5b-9, data which demonstrate that these cells do exhibit the changes in cell cycle proteins required to proliferate. C5b-9 causes a differential expression of cyclin kinase inhibitors To further assess why DNA synthesis occurred in response to C5b-9 but proliferation did not, the levels of the cyclin kinase inhibitors (CKI), p21 and p27, were measured by Western blot analysis. These CKI have been shown to inhibit DNA synthesis by binding to cyclin A-CDK2 complexes. There was no change in p21 levels
Fig. 2. Western blot of S phase cell cycle regulatory proteins. Glomerular visceral epithelial cells (VEC) were sensitized to anti-Fx1A antibody and C5b-9 (group 1, lane 2), and grown in Nuserm-containing media for 18 hours with the antibody. Controls included VEC exposed to antibody without C5b-9 (group 2, lane 3) or no antibody (group 3, lane 1). Cyclin dependent kinase-2 (CDK2) and cyclin A levels increased in VEC subjected to sublytic C5b-9 injury compared to controls, and this coincided with the increase in DNA synthesis in these cells. C5b-9 caused a decrease in the levels of the cyclin kinase inhibitor p27, but did not alter p21 levels.
by Western blot analysis (Fig. 2) in VEC exposed to sublytic C5b-9 compared to control in the presence of Nuserum. In control VEC (groups 2 and 3) grown in Nuserumcontaining media where less than 20% of cells were undergoing DNA synthesis, p27 protein was readily detected by Western blot analysis (Fig. 2). In contrast, there was a marked decrease in p27 levels in VEC following exposure to sublytic C5b-9 (group 1) compared to controls (groups 2), and this coincided with the increase in DNA synthesis in these cells. These findings are also
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Fig. 3. Protein levels for cyclin B. The number of VEC staining positive for cyclin B (A) were higher in control VEC exposed to anti-Fx1A antibody without C5b-9 (group 2) compared to VEC attacked by anti-Fx1A with C5b-9 (group 1) (B). Western blot analysis for cyclin B, confirming a decrease in cyclin B levels in VEC exposed to sublytic C5b-9 (lane 2) compared to controls (lanes 1 and 3).
consistent with the cells exhibiting increased DNA synthesis in response to C5b-9. M phase cell cycle proteins are altered by C5b-9 Mitosis and cytokinesis are regulated by the M phase cell cycle proteins, cdc2 and its partner cyclin B [23]. Cyclin B protein was barely detected by immunostaining or Western blot analysis in VEC prior to sensitization with anti-Fx1A (results not shown). Cyclin B protein was detected by immunostaining and Western blot analysis in control VEC grown in serum for 24 hours after exposure to anti-Fx1A without C5b-9 (group 2) and control VEC not exposed to anti-Fx1A (group 3) (Fig. 3). In contrast, cyclin B was barely detected by immunostaining and Western blot analysis (Fig. 3) in VEC grown in Nuserum for 24 hours after exposure to sublytic C5b-9 (group 1), and the levels were lower in C5b-9 attacked VEC compared to controls.
cdc2 was barely detected by immunostaining and Western blot in VEC prior to exposure of Anti-Fx1A and C5b-9 (results not shown). Immunostaining (Fig. 4) and protein levels (Fig. 4) were readily detected in control VEC (groups 2 and 3) grown in Nuserum-containing media for 24 hours. In contrast, cdc2 immunostaining (Fig. 4) and protein levels by Western blot analysis (Fig. 3) were lower in C5b-9 attacked VEC (group 1) grown in Nuserum for 24 hours compared to controls. These results show that the failure of VEC to undergo mitosis and cytokinesis following sublytic C5b-9 attack was associated with decreased expression of the M phase cell cycle proteins, cyclin B and cdc2. DISCUSSION In the current study we have shown that VEC in vitro sensitized by incubation with low concentrations of anti-
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Fig. 4. Expression for cdc2. There was a decrease in immunostaining for cdc2 in VEC exposed to sublytic C5b-9 (group 1) (A) compared to control (group 2) (B). The Western blot analysis for cdc2 demonstrates that cdc2 levels decreased in C5b-9 stimulated VEC (lane 2) compared to control VEC that did (group 2, lane 3) or did not receive anti-VEC antibody (group 3, lane 1).
VEC IgG (anti-Fx1A) and then exposed to sublytic concentrations of fresh serum (C1) to activate complement and induce formation of membrane-inserted C5b-9 complexes, increase the expression of cyclin A and CDK2 and enter S phase in the presence of growth factors. However, C5b-9 decreases cyclin B and cdc2 expression, resulting in the delay in G2/M progression. In experimental membranous nephropathy induced by antibody to the visceral glomerular epithelial cell (VEC) or podocyte, glomerular injury that occurs as a consequence of C5b-9 attack on VEC results in cell activation and production of oxidants and proteases that damage underlying GBM [4, 33, 34]. Accompanying this process is a low-grade increase in DNA synthesis, which is complement-dependent [11]. However, there is no apparent increase in VEC number [11, 14]. This is in marked contrast to the mesangial cell [35], which also makes oxidants and proteases in response to antibody/complement attack in vivo [33], but exhibits a dramatic proliferative response as well, mediated by PDGF, with a substan-
tial increase in cell number [8]. To better understand the response of the VEC to sublytic C5b-9 and relate it to proliferation and cell cycle control, the present study was undertaken to examine the effects of sublytic C5b-9 on VEC proliferation in vitro where other variables can be better controlled. Earlier studies by our group [11] and others [31, 32, 36–38] have focused on the cytokines responsible for VEC proliferation. In the current study, VEC were briefly (30 min) exposed to an antibody directed to antigens on the VEC (anti-Fx1A) followed by sublytic concentrations of fresh, complement sufficient serum (C1) or control serum which lacked C6 (C2). The first finding in this study was that the sublytic C5b-9 attack (in the absence of a source of growth factors) was not sufficient by itself to increase DNA synthesis in vitro. However, prior sublytic injury to VEC by C5b-9 significantly augmented entry into the S phase of the cell cycle (DNA synthesis) induced by growth factors (Nuserum). In contrast, antibody binding to the VEC in the absence of
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C5b-9 (C2 serum) did not increase DNA synthesis compared to VEC grown in media containing Nuserum that had not been exposed to antibody. The results of this study differ from findings in other cell types. C5b-9 alone (in the absence of other growth factors) is sufficient to increase DNA synthesis in certain nonrenal cells, including 3T3 [39], B cells [40] and oligodendrocytes [41]. Thus, in contrast to these studies, we were unable to show that the sublytic C5b-9 attack by itself was sufficient to induce DNA synthesis in the absence of other mitogens. However, C5b-9 did increase DNA synthesis in the presence of growth factors. These results are the first, to our knowledge, to establish that sublytic C5b-9 can augment DNA synthesis in renal cells in vitro. To determine whether or not the sublytic C5b-9 effect on DNA synthesis after exposure to serum was a direct or indirect one, we exposed normal VEC to supernatants of C5b-9 stimulated VEC and found that these supernatants contained mitogenic activity. However, the increase in DNA synthesis was only 50% of that induced by direct C5b-9 stimulation. This discrepancy could reflect a loss of growth factors during culture or the transferring of supernatants. However, the results are also consistent with the possibility that C5b-9 has both a direct effect on proliferation independent of other mitogens as well as an indirect effect resulting from release of mitogens. The indirect effect is consistent with other studies that document the release of several potential mitogens from cells following C5b-9 attack, including reactive oxygen species, interleukin (IL)-1, IL-6 and tumor necrosis factor-a (TNF-a) [3, 42, 43]. Because bFGF is a mitogenic growth factor for VEC in vitro [32] and bFGF has been shown to be up-regulated following C5b-9 mediated VEC injury in experimental membranous nephropathy (PHN model) [12], we next examined if bFGF was the mitogen released following C5b-9 induced VEC stimulation in vitro. Our results show that neutralizing bFGF with a specific anti-bFGF antibody did not reduce C5b-9 induced DNA synthesis. The concentration of anti-bFGF antibody used reduced bFGF-stimulated DNA synthesis in VEC. However, we did not measure the concentration of bFGF in the supernatants. This indirect method used in this study showing that bFGF did not mediate C5b-9 induced DNA synthesis also differs from results reported in nonrenal cells [44]. These studies suggest that the mechanisms by which C5b-9 induces DNA synthesis may be cell type specific. PDGF has been shown to mediate C5b-9 induced mitogenesis in certain nonrenal cells [39]. Although we have previously shown an increase in PDGF synthesis by C5b-9 stimulated VEC [11], PDGF is not a likely mitogen for these cells since VEC lack PDGF receptors [11]. Further studies are needed to better define the nature of the mitogen released by VEC in response to C5b-9.
The postulate that C5b-9 may have a direct effect on DNA synthesis is consistent with what is known of the activation of cell signaling pathways by C5b-9, many of which participate in receptor mediated processes including the response to growth factors. Thus, sublytic C5b-9 can induce a transient increase in cytosolic calcium [40], activation of protein kinase C [45], phospholipase A2 [46], PGI2 formation [47] and the MAPK pathway [48, 49], and increased expression of certain transcription factors [41] and nuclear factor-kB (NF-kB) [50]. In this study we explored the possibility that the effect of C5b-9 on VEC proliferation occurs at the level of the cell cycle. Studies of C5b-9-induced effects on cell cycle in VEC in vitro are of particular interest if they help to understand events that occur in vivo. In both human and experimental membranous nephropathy the C5b-9 attack on VEC is an established (experimental) or likely (human) pathogenic mechanism, but mitotic arrest apparently occurs and no increase in VEC numbers is seen [14]. In other models of VEC injury, Rennke and Kritz have proposed that the inability of the VEC to proliferate in vivo may underlie the development of progressive glomerulosclerosis [14–17, 51]. In PHN, we have established that some increase in DNA synthesis assessed by PCNA staining occurs, mitotic figures are seen and S phase cyclins and the CDK-inhibitors p21 and p27 are increased [11, 52]. This increased expression of CKI may prevent progression through the S phase in vivo [52]. Our results in vitro are similar in some respects but differ in others. In vitro, C5b-9 also induced an increase in BrdU staining (in the presence of Nuserum, a source of growth factors) and an increase in S phase cyclin and CDK. However, the expression of the CKI’s p21 and p27 was reduced rather than increased. Despite this, progression through M phase and cell division did not occur, apparently due to reduced expression of M phase cyclins B and CDC2. Thus, the failure of VEC to proliferate in response to C5b-9 in vitro appears to reflect the effects on M phase cell cycle proteins rather than the effect of CKI. Another explanation may be that C5b-9 delays G2/M phase progression. The mechanisms were not determined in the current study. The reasons for this discrepancy in CKI expression in vivo and in vitro are not clear, but could reflect differences in the environment in which the VEC lives in vivo versus in vitro, differences in cell-matrix attachments, differences in the amount of C5b-9 to which they are exposed (there is likely more in vitro), and differences in the timing required for the anti-Fx1A antibody to be shed. Moreover, since M phase cyclins were not studied in vivo, it is quite possible that a similar block in the M phase occurs in this setting as well. In any event, together these studies show that effects of C5b-9 on cell cycle regulatory proteins may in large part explain the failure
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of VEC to divide and proliferate in response to antibodycomplement mediated injury. An alternative explanation for our results would be that the cell number did not increase in response to C5b-9 because cell death was increased. We believe this is unlikely for two reasons. First, in pilot studies we showed that although there was C5b-9 induced low-grade apoptosis in VEC, this occurred within the first three hours of attack, and not thereafter. Second, there was no decrease in cell number at 24 and 48 hours following the C5b-9 attack, which occurred after the increase in DNA synthesis (at 18 hr) in this study. Previous studies have shown that TGF-b limits DNA synthesis in many renal cell types, including mitogeninduced VEC proliferation in vitro [31]. Furthermore, we have shown that TGF-b is increased following complement-induced injury to the VEC in experimental membranous nephropathy [53]. Therefore, we examined whether or not TGF-b reduced C5b-9 induced DNA synthesis in VEC in vitro. We found that C5b-9 induced VEC DNA synthesis was not inhibited by TGF-b1. In contrast, TGF-b1 did reduce DNA synthesis in control VEC, indicating that the concentration of TGF-b1 used in this study could inhibit VEC proliferation. Of note, although we studied TGF-b1 here, our in vivo studies demonstrate up-regulation of the TGF-b2 and TGF-b3 isoforms in C5b-9 mediated VEC injury, while no changes are detected in TGF-b1 [53]. Although most studies suggest that all TGF-b isoforms have similar biological effects, we have not excluded the possibility that TGF-b2 or -b3 might inhibit C5b-9 induced VEC proliferation. In summary, our study provides the first evidence that sublytic C5b-9 augments growth factor induced DNA synthesis in VEC. However, C5b-9 alone is not sufficient to engage VEC in the S phase of the cell cycle. Furthermore, sublytic C5b-9 attack arrests VEC at G2/M phase, thereby preventing mitosis and cytokinesis in vitro. Thus, the ability of VEC to enter the S phase of the cell cycle, and to undergo low grade DNA synthesis but not divide and produce an increase in cell number following C5b-9 mediated injury is similar to C5b-9 mediated injury in experimental and human membranous nephropathy. ACKNOWLEDGMENTS This study was supported by Public Health Service grants DK34198, DK47659, DK52121, DK51096. We thank J.M. Roberts for the generous supply of antibodies to cyclin A. No reprints are available. Correspondence to Stuart J. Shankland, M.D., Division of Nephrology, University of Washington, P.O. Box 356521, Seattle, Washington 98195, USA. E-mail: [email protected]
APPENDIX Abbreviations used in this article are: bFGF, basic fibroblast growth factor; BrdU, 5-bromo-29-deoxyuridine; C1, complement sufficient
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PVG rats; C2, complement deficient PVG rats; CDK, cyclin dependent kinase; CKI, cyclin kinase inhibitors; GBM, gomerular basement membrane; IL, interleukin; LDH, lactate dehydrogenase; NADH, nicotinamide adenine dinucleotide; p21, p21WAF1,CIP1; p27, p27Kip1; TGF-b, transforming growth factor-b; TNF-a, tumor necrosis factor-alpha; VEC, glomerular visceral epithelial cell.
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39. Halperin JA, Taratuska A, Nicholson-Weller A: Terminal complement complex C5b-9 stumulates mitogenesis in 3T3 cells. J Clin Invest 91:1974–1978, 1993 40. Hivroz C, Fischer E, Kazatchkine MD, Grillot-Courvalin C: Differential effects of the stimulation of complement receptors CR1 (CD35) and CR2 (CD21) on cell prolieration and intracellular Ca21 mobilation of chronic lymphocytic leukemia B cells. J Immunol 146:1766–1772, 1991 41. Rus HG, Niculescu F, Shin ML: Sublytic complement attack induces cell cycle in oligodendrocytes. J Immunol 156:4892–4900, 1996 42. Schonermark M, Deppisch R, Rother K, Hansch GM: Induction of mediator release from human glomerular mesangial cells by the terminal complement components C5b-9. Int Arch Allergy Appl Immunol 96:331–337, 1991 43. Hansch GM, Seitz M, Betz M: Effects of the late complement components C5b-9 on human monocytes: Release of prostanoids, oxygen radicals and of a factor inducing cell prolieration. Int Archs Allergy Appl Immun 82:317–320, 1987 44. Benzaquen LR, Nicholson-Weller A, Halperin JA: Terminal complement proteins C5b-9 release basic fibroblast growth factor and platelet-derived growth factor from endothelial cells. J Exp Med 179:985–992, 1994 45. Carney DF, Hammer C, Shin ML: Multiple signal messengers generated by terminal complement complexes and their role in terminal complement complex elimination. J Immunol 145:623– 629, 1990 46. Cybulsky AV, Monge JC, Papillon J, Mctavish AJ: Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells. Am J Physiol 269:F739–F749, 1995 47. Suttorp N, Seeger W, Zinsky S, Bhakdi S: Complement complex C5b-8 induces PGI2 formation in cultured endothelial cells. Am J Physiol 253:C13–C21, 1987 48. Niculescu F, Rus H, van Biesen T, Shin ML: Activation of Ras and mitogen-activated protein kinase pathway by terminal complement complexes is G protein dependent. J Immunol 158:4405–4412, 1997 49. Rus H, Niculescu F, Badea T, Ml S: Terminal complement complexes induce cell cycle entry in oligodendrocytes through mitogen activated protein kinase pathway. Immunopharmacol 38:177–187, 1997 50. Kilgore KS, Schmid E, Shanley TP, Flory CM, Maheswari V, Tramontini NL, Cohen H, Ward PA, Friedl HP, Warren JS: Sublytic concentrations of the membrane attack complex of complement induce endothelial interleukin-8 and monocyte chemoattractant protein-1 through nucleur factor-kB activation. Am J Pathol 150:2019–2012, 1997 51. Kriz W, Kretzler M, Nagata M, Provoost AP, Shirato I, Uiker S, Sakai T, Lemley KV: A frequent pathway to glomerulosclerosis: Deterioration of tuft architecture-podocyte damage-segmental sclerosis. Kidney Blood Press Res 19:245–253, 1996 52. Shankland SJ, Floege J, Thomas SE, Nangaku M, Hugo C, Pippin J, Henne K, Hockenberry DM, Johnson RJ, Couser WG: Cyclin kinase inhibitors are increased during experimental membranous nephropathy: Potential role in limiting glomerular epithelial cell proliferation in vivo. Kidney Int 52:404–413, 1997 53. Shankland SJ, Pippin J, Pichler RH, Gordon KL, Friedman S, Gold LI, Johnson RJ, Couser WG: Differential expression of transforming growth factor-b isoforms and receptors in experimental membranous nephropathy. Kidney Int 50:116–124, 1996
CELL BIOLOGY – IMMUNOLOGY – PATHOLOGY
Complement (C5b-9) induces glomerular epithelial cell DNA synthesis but not proliferation in vitro STUART J. SHANKLAND, JEFFREY W. PIPPIN, and WILLIAM G. COUSER Department of Medicine, Division of Nephrology, University of Washington, Seattle, Washington, USA
Complement (C5b-9) induces glomerular epithelial cell DNA synthesis but not proliferation in vitro. Background. The C5b-9 membrane attack complex of complement is the principal mediator of injury induced experimentally by antibodies directed at glomerular cell membranes. In experimental membranous nephropathy, C5b-9 induced injury to the glomerular visceral epithelial cell (VEC) is associated with DNA synthesis, but not cytokinesis. In the current study we determined if C5b-9 increases DNA synthesis in VEC in vitro, and defined the mechanisms involved. Methods. Rat VEC in vitro were divided into three groups: (1) sensitized with anti-VEC antibody and exposed to sublytic concentrations of C 1/PVG serum (normal complement components); (2) anti-VEC antibody and control C-/PVG serum (C6 deficient); (3) no anti-VEC antibody. DNA synthesis (BrdU staining), mitosis (mitotic figures) and cytokinesis (cell counts) were measured at 24 and 48 hours. To examine the expression of specific S-phase and M-phase cell cycle regulatory proteins and their inhibitors, immunostaining and Western blot analysis was performed for cyclin A, CDK2, p21 and p27, cyclin B and cdc2. Results. In the absence of growth factors, sublytic C5b-9 attack did not increase proliferation. In contrast, sublytic C5b-9 attack (group 1) augmented growth factor induced DNA synthesis by 50% compared to controls (groups 2 and 3; P , 0.001), and was accompanied by increased levels of cyclin A and CDK2, and a decrease in the cyclin kinase inhibitor p27 (but not p21). Sublytic C5b-9 attack reduced the expression of the M phase cell cycle proteins, cyclin B and cdc2, accompanied by reduced mitosis (mitotic figures) and cytokinesis (cell number). Conclusions. Our results show that the C5b-9 augmented growth factor entry into the S phase in VEC is regulated by changes in specific cell cycle regulatory proteins. However, antibody and complement decreased the M phase cell cycle proteins, and prevented VEC mitosis and cytokinesis, suggesting a delay or arrest at the G2/M phase.
Complement is a critical mediator of antibody-induced renal disease [1]. Established effects include generation of chemotactic factors such as C5a leading to recruitment of circulating inflammatory cells and formation of the C5b-9 membrane attack complex of complement which can insert into and activate renal cells to make other inflammatory mediators [2, 3]. The role of C5b-9 has been best established in renal diseases induced experimentally by antibodies to glomerular cell membrane antigens. Thus, C5b-9 is the principal mediator of injury induced by antibody to the glomerular visceral epithelial cell (VEC) (passive Heymann nephritis; PHN) [4–7], mesangial cell (experimental mesangial proliferative glomerulonephritis, Thy1 model) [8], and endothelial cell (thrombotic microangiopathy model) [9]. Of note, however, is that the consequences of C5b-9 attack appear to differ depending on which cell type is involved. In the mesangium, antibody and complement induce mesangial cell lysis, followed by a phase of proliferation [8, 10]. In contrast, C5b-9 attack on the VEC in vivo does not cause lysis and is followed by cell activation and DNA synthesis, occasional mitosis and ploidy [11– 13]. However, the VEC does not readily undergo cytokinesis (cell division) and the VEC number does not increase in vivo [14]. It is likely that this failure of the VEC to proliferate in some forms of glomerular disease contributes to glomerular sclerosis and renal failure [15–17]. Cell proliferation is ultimately controlled in the nucleus at the level of the cell cycle, and requires a transition through specific phases of the cell cycle [18]. Each phase of the cell cycle serves a unique role in preparing a cell to proliferate (increase in cell number) [19]. Thus, the S phase is required for DNA synthesis, and the M phase is required for mitosis and cell division (cytokinesis) [20]. Each phase of the cell cycle is governed by specific positive cell cycle regulatory proteins called cyclins and cyclin dependent kinases (CDK) [21]. DNA synthesis (S phase) requires that CDK2 be activated by its partner cyclin A [22]. Mitosis and cytokinesis
Key words: p27, cyclins, cell cycle, proliferation, podocyte, membrane attack complex, visceral epithelial cell. Received for publication July 31, 1998 and in revised form February 5, 1999 Accepted for publication March 8, 1999
1999 by the International Society of Nephrology
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(M phase) require that cdc2 (formerly called CDK1) be activated by cyclin B [23]. In contrast, cyclin kinase inhibitors such as p21 and p27 inhibit cell cycle progression by inactivating specific cyclin-CDK complexes [24]. To better understand the consequences of C5b-9 induced injury in diseases of the VEC, we have examined the effects of sublytic C5b-9 attack on VEC proliferation by examining DNA synthesis, mitosis and cytokinesis in vitro as they relate to changes in expression of cell cycle regulatory proteins. To our knowledge, our results provide the first evidence that C5b-9 increases DNA synthesis in a resident glomerular cell, suggest that this effect is mediated in part by additional cell-derived mitogen(s), and establish a role for cell cycle regulatory proteins in the response of the VEC to complement-mediated injury. METHODS Glomerular epithelial cell culture and experimental design Rat visceral glomerular epithelial cells (VEC) (podocytes) were established in culture as previously described [25]. In brief, glomeruli were isolated from 60 to 100 g male Sprague-Dawley rats (Simonsen, Gilroy, CA, USA) by differential sieving, and placed in culture dishes containing Vitrogen (Celtrix, Santa Clara, CA, USA) and K-1 Media consisting of Ham’s F-12 and DME-low glucose 1:1 (Gibco, Grand Island, NY, USA) with 2% NuSerum (Collaborative Biomedical Products, Bedford, MA, USA) and ITS Premix (Collaborative Biomedical Products). After five to seven days, colonies of VEC were marked in the culture dish, excised, and replated. Initially, cells were passaged every four to five days onto vitrogen and after 20 passages they were passaged onto plastic. VEC were characterized by established criteria, which included a polygonal shape and cobblestone appearance at confluency, and positive staining with antibodies to Fx1A, cytokeratin, and podocalyxin and negative staining with antibodies directed against the mesangial cell (Thy-1) and endothelial cell (Factor VIII) [25]. Antibody-C5b-9 stimulation of VEC To induce sublytic C5b-9 attack, VEC were split 1:3, plated overnight in K-1 media, and allowed to grow to 50% confluency. VEC were washed three times with serum-free K-1 media, and divided into three groups. Group 1. VEC were sensitized with 3 mg/ml of sheepanti-Fx1A IgG at 378C for 30 minutes [26]. Unbound antibody was removed by washing three times with serum free K-1 media. Sensitized VEC were then exposed to a sublytic complement source (PVG C1 serum, see below) in the media for 60 minutes. VEC were washed
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three times, and the cells were then incubated in serumfree media or media with serum. Group 2. VEC served as a control for C5b-9 attacked cells. VEC were sensitized with anti-Fx1A IgG and then exposed to serum for 60 minutes that lacked C6 (PVG C2 serum, see below), washed, and incubated in serum free media or media with serum. Group 3. VEC served as a control for anti-Fx1A IgG: VEC were exposed to media at 378C for 30 minutes in the absence of anti-Fx1A. Experimental design Two experimental conditions were used to answer specific questions. To determine if sublytic C5b-9 attack was sufficient to increase DNA synthesis, mitosis and cytokinesis in the absence of a source of growth factors (Nuserum), VEC from each group were incubated in serum free media for 18 to 48 hours. To determine if sublytic C5b-9 attack augmented DNA synthesis and mitosis induced by growth factors, VEC from each group where grown in Nuserum for 18 to 48 hours. Preparing sublytic C5b-9 attack Serum from normocomplementemic PVG rats (C1; Harlan Sprague-Dawley, Indianapolis, IN, USA) was utilized as a complement source to induce sublytic C5b-9 attack. Serum from C6 deficient-PVG rats (C2; Bantin and Kingman Universal, Edmonds, WA, USA) was used as control. C2 rats exhibit a complete absence of C6, inherited in an autosomal recessive pattern, and are therefore unable to assemble C5b-9 [27]. Serum was obtained from both rat strains by aortic puncture and allowed to clot. The serum was separated by centrifugation, sterile filtered on ice and stored at 2708C. The serum was tested in a CH50 hemolytic assay to establish the complement activity [28]. C1 serum exhibited complement activity equivalent to normal Sprague-Dawley rat serum. C2 serum had no hemolytic activity. In select experiments, serum from Sprague-Dawley rats was also used as a source of complement, and heat-inactivated Sprague-Dawley rat serum was used as a control. The selection of the antibody and complement concentrations used in the current study was based on checkerboard titration studies to define the maximal amounts of antibody and complement that could be used without producing VEC injury or lysis (defined as .5% LDH release; see below). Serum was diluted to 4% in serum free K-1 media and applied to the antibody sensitized VEC for one hour at 378C to allow C5b-9 insertion. VEC were then washed with serum free K-1 media to remove any unbound complement. Lactate dehydrogenase assay To ensure that C5b-9 attack was not inducing significant cell lysis, lactate dehydrogenase (LDH) was mea-
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sured in the supernatants using the Lactate Dehydrogenase Kinetic Assay Kit (Sigma Diagnostics, St. Louis, MO, USA) [29]. Conditioned media was removed one hour after the addition of the complement source, and the remaining cells were lyzed with 2% triton. The conditioned media or the lyzed cells were diluted 1:60 in nicotinamide adenine dinucleotide reduced form (NADH) 1 mg/ml. Sodium pyruvate solution was then added, and the reduction of NADH to NAD by the LDH in the media or the lyzed cells was measured by a drop in absorbance at 340 nm by a Lambda Bio Spectrophotometer (Perkin Elmer, Norwalk, CT, USA). Lysis was expressed as a percentage of the change in the absorbance of the media compared to changes in absorbance of the triton lyzed cells and the media. Less than 5% LDH release was regarded as sublytic. S phase measured by BrdU staining To determine if attack by antibody and C5b-9 was sufficient to augment DNA synthesis in VEC grown in the absence of growth factors, VEC from groups 1, 2 and 3 were incubated in Nuserum-free media for 18 hours. To determine if sublytic C5b-9 attack augmented DNA synthesis induced by growth factors, VEC from each group were incubated in media containing serum and other growth factors (2% Nu serum) for 18 hours after the C5b-9 attack. Comparisons were made to group 2 and group 3 VEC grown in 2% Nu serum for 18 hours. In each experiment, entry to the S phase was measured by BrdU immunostaining, a marker of DNA synthesis. BrdU staining (5-bromo-29-deoxyuridine) was measured with a cell proliferation kit (Amersham Life Science, Arlington, IL, USA) as previously described [30]. In brief, VEC were pulsed with BrdU (1:500 dilution) in media with or without Nuserum for 18 hours, and immunostaining was performed according to the manufacturer’s directions. BrdU was detected using a monoclonal antibody to BrdU, followed by a peroxidase conjugated sheep-antimouse IgG2a antibody and Diaminobenzidine with cobalt and nickel. Camco quick stain II (Baxter Scientific Products, McGaw Park, IL, USA) was used as a nuclear counterstain. To determine the percentage of cells staining positive for BrdU, 200 VEC were counted, and the percentage of VEC staining positive for BrdU was determined. All the experiments reported in this study were performed a minimum of six times. Measuring the M phase of the cell cycle Cell proliferation requires that a cell complete both the S phase and M phase of the cell cycle. To determine if C5b-9 increased entry into the M phase, mitotic figures [23], a marker of the M phase, were counted in group 1 (sublytic C5b-9 attack) and group 2 (anti-Fx1A without C5b-9) VEC grown in Nuserum for 24 hours. The num-
ber of mitotic figures/HPF were counted in 20 fields, and were expressed as a percentage of the total cell count. To determine if C5b-9 increased cell division (cytokinesis), the cell number was measured prior to exposure to anti-Fx1A antibody, and at 24 and 48 hours in group 1 and group 2 VEC grown in Nuserum. Cell number was reported as the percentage increase from the start of the experiment at each of these time points. Determining if mitogens are released following sublytic C5b-9 attack To determine if sublytic C5b-9 attack was associated with the release of mitogens, group 1 and 2 VEC were incubated in Nuserum-free media for 18 hours after exposure to anti-Fx1A antibody and C1 serum or C2 serum. The supernatants were collected after 18 hours, and transferred to VEC that were growing at 50% confluency in Nuserum-containing media, and BrdU immunostaining was measured in these cells after 18 hours. Basic fibroblast growth factor and TGF-b1 blocking studies To test the hypothesis that C5b-9-induced DNA synthesis resulted in part from the release of mitogenic growth factors, a neutralizing antibody to basic fibroblast growth factor (bFGF) was used to block bFGF activity following complement stimulation. Goat anti-bFGF (obtained from Dr. Michael A. Reidy, Dept. of Pathology, University of Washington, Seattle, WA, USA) or control goat IgG was added at 1 mg/ml to the K-1 media following the addition of a complement source. VEC were incubated with anti-bFGF antibody for 18 hours, and DNA synthesis was measured. To ensure that the concentration of anti-bFGF used in this study inhibited bFGF, additional experiments were performed where recombinant bFGF (10 ng/ml) was added to quiescent VEC in the presence of anti-bFGF or control goat IgG. Because transforming growth factor-b1 (TGF-b1) has been shown to reduce growth factor-induced proliferation in many cell types including VEC [31], the effect of TGF-b1 on C5b-9 stimulated DNA synthesis was also assessed. TGF-b1 (10 ng/ml; R&D Systems, Minneapolis, MN, USA) was activated in 4 mm HCl containing 0.1% BSA and added to VEC at a concentration of 10 ng/ml following exposure to C5b-9. DNA synthesis was measured 18 hours later. Determining the expression of cell-cycle regulatory proteins in VEC The levels of S phase and M phase cell-cycle regulatory proteins were determined by immunostaining and Western blot analysis [10, 30] in group I (C5b-9 attack) and 2 (control) cells after 18 hours of exposure to Nuserum. Immunostaining. VEC were grown on 24-well plates, and fixed in 1:1 methanol/acetone for 15 minutes at
Shankland et al: C5b-9 and podocyte proliferation
2208C, then washed three times in cold PBS. The cells were permeabilized with 0.5% NP-40 buffer for 10 minutes at room temperature. Following a wash in PBS, primary affinity purified rabbit polyclonal antibodies [10, 30] to cyclin A (dilution 1:1000; provided by J.M. Roberts, Fred Hutchinson Cancer Research Center, Seattle, WA, USA), CDK2 (dilution 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), cyclin B1 (dilution 1:100; Santa Cruz Biotechnology) cdc2 (dilution 1:100; Santa Cruz Biotechnology), p21 (dilution 1:1000; Santa Cruz Biotechnology) and p27 (dilution 1:1000; provided by J.M. Roberts), were incubated overnight at 48C. A secondary biotinylated goat antirabbit antibody was added (1:400 dilution, Vector Laboratories) for 60 minutes at room temperature, followed by streptavidin FITC (1:32 dilution; Cappel Research Products, Durham, NC, USA) or Avidin D horseradish peroxidase (1:2000) for 60 minutes at room temperature, followed by 49, 6-diamidino-2phenylindole (Boehringer Mannheim, Indianapolis, IN, USA). Nuclei were counterstained with Camco Quick Stain II (Baxter Scientific Products). The number of VEC staining positive for each cell cycle protein was expressed as a percentage of the total number of cells. Western blot analysis. Cell protein was obtained from group 1 (C5b-9 attacked) and group 2 (control) VEC after an 18 hour exposure to Nuserum. At each time point VEC were washed three times with cold PBS, trypsinized, and following centrifugation, the pellet was resuspended in a buffer containing 1% triton, 10% glycerol, 20 mm HEPES, 100 mm NaCl with 10 mg/ml leupeptin, 10 mg/ml antipain, 10 mg/ml pepstatin, 0.1 mm sodium orthovanadate and 50 mm sodium fluoride (reagents purchased from Sigma Biosciences). Following a vortex, the VEC were placed on ice for 15 minutes, centrifuged at 14,000 r.p.m. for 5 minutes, and the protein concentration of the supernatant was measured by a BCA protein assay (Pierce, Rockford, IL, USA). For Western blot analysis, 20 mg of VEC protein extract was loaded in each lane, separated under reducing conditions on a 15% SDSPAGE gel, and transferred to PVDF membranes (Millipore, Bedford, MA, USA) by electroblotting. To reduce nonspecific antibody binding, the membranes were first blocked with 5% nonfat dried milk for 30 minutes at room temperature. This was followed by an overnight incubation at 48C with antibodies to either cyclin A, CDK2, p21, p27, cyclin B or cdc2. An alkaline phosphatase-conjugated secondary antibody (Promega, Madison, WI, USA) was used for detection with the chromagen, 5-Bromo-4-Chloro-3-Indolyl phosphate/nitro blue tetrazolium (Sigma Biosciences). The gels were stained with Coomassie Blue to check for complete protein transfer. Following Western blot analysis, filters were also stained with Coomassie Blue to ensure that protein loading was equal. Controls for immunostaining and Western blot analy-
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sis included omitting the primary antibody, replacing the primary antibody with an irrelevant antibody of the same species and peptide absorption (the source of peptides was the same as the primary antibody). Statistics Values were reported as mean 6 sem, unless otherwise stated. Statistical significance (P , 0.05) was evaluated using the Student’s t-test, and a modified t-test was performed using the Bonferroni-Dunn correction. RESULTS C5b-9 augments growth factor-induced DNA synthesis in VEC in vitro Entry into the S phase of the cell cycle was measured by BrdU staining, a marker of DNA synthesis. A first question was to determine if sublytic C5b-9 attack increased DNA synthesis in the absence of growth factors (in this study Nuserum was used as a source of growth factors). Accordingly, BrdU staining was quantitated in each VEC group grown in Nuserum-free media for 18 hours, and the results are shown in Table 1. In the absence of a source of growth factors (Nuserum-free media) there was no difference in BrdU staining in each group. Thus, the sublytic C5b-9 attack alone did not increase DNA synthesis. A second question asked if the sublytic C5b-9 attack augmented DNA synthesis induced by growth factors. Table 1 shows there was an increase in BrdU staining in group 2 (anti-Fx1A without C5b-9) and group 3 (no anti-Fx1A antibody) VEC in the presence of serum compared to Nuserum-free conditions. There was no difference in BrdU staining in group 2 and 3 VEC at 18 hours. In contrast, there was approximately a twofold increase in BrdU staining in group 1 (sublytic C5b-9 attack) VEC compared to control (group 2 and group 3) VEC. In separate studies, sublytic C5b-9 attack induced with Sprague-Dawley serum also increased BrdU staining twofold compared to VEC exposed to antibody and heatinactivated Sprague-Dawley serum (P , 0.0001; data not shown). These results show that sublytic C5b-9 augmented growth factor induced DNA synthesis in VEC in vitro, and show that sublytic C5b-9 attack by itself was not sufficient to engage VEC into the S phase (DNA synthesis) of the cell cycle in the absence of growth factors. VEC arrest at G2/M following C5b-9 attack Entry into the M phase of the cell cycle was measured by counting mitotic figures. Because DNA synthesis was increased by C5b-9 only in the presence of serum, mitosis was assessed at 24 hours following the sublytic C5b-9 attack in VEC grown in media with growth factors (Nuserum). There were 4.43 6 0.26 mitotic figures/HPF in
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Shankland et al: C5b-9 and podocyte proliferation Table 1. Quantitation of BrdU staining Nuserum free (18h)
Group 1 2 3
Experiment Anti-Fx1A and C5b-9 Anti-Fx1A and no C5b-9 No anti-Fx1A
Nuserum (18h)
% positive BrdU cells 19 6 1.1 18 6 1.0 16 6 0.9
63 6 1.0 27 6 2.8 30.3 6 3.8 a,b a
a
P , 0.001 vs. Nuserum free P , 0.001 vs. groups 2 and 3 Nuserum Visceral epithelial cells (VEC) were divided into three groups based on the presence or absence of anti-VEC antibody and C5b-9, and were then grown in media with or without Nuserum for 18 hours. Nuserum, a source of growth factors increased the percentage of cells that stained positive for BrdU, a marker of DNA synthesis, was doubled by sublytic C5b-9. a
b
Group 1 VEC, but the increase was less than 50% compared to controls. These results show that despite increased entry into the S phase (DNA synthesis) at 18 hours induced by C5b-9 attack, there was no corresponding increase in entry into (mitosis) or progression through (cytokinesis) the M phase of the cell cycle. Thus, sublytic C5b-9 attack either causes VEC to arrest at the G2/M phase or delays G2/M phase progression and entry into M phase, despite the presence of growth factors (Nuserum) to which they can respond. C5b-9-induced VEC proliferation is induced in part by a secreted mitogen
Fig. 1. Quantitation of cell number. Glomerular visceral epithelial cells (VEC) were divided into three groups and cells were counted when placed in media with Nuserum (0 hr) and at 24 and 48 hours. The cell number was increased in control VEC [groups 2 (e) and 3 (s)] compared to VEC exposed to sublytic C5b-9 (group 1; h) at 24 and 48 hours. There was no difference in cell number in control VEC at 24 and 48 hours. aP , 0.0010 vs. groups 2 and 3; bP , 0.0007 vs. groups 2 and 3; cP , 0.099 vs. group 3; dP , 0.24 vs. group 3.
Group 2 (anti-Fx1A without C5b-9) VEC. In contrast, there were only 0.52 6 0.73 mitotic figures/HPF in group 1 (sublytic C5b-9 attack) VEC (P , 0.001 vs. group 2) To determine the effect of sublytic C5b-9 on cytokinesis (cell division), cell number was measured at 24 and 48 hours following exposure to antibody with or without a complement source in VEC grown in the presence of Nuserum. Figure 1 shows that in control VEC (groups 2 and 3) grown in Nuserum, there was a progressive increase in cell number at 24 hours (P , 0.001 vs. 0 hr) and 48 hours (P , 0.001 vs. 24 hr). In contrast, cell number did not increase in VEC following sublytic C5b-9 attack (group 1) at 24 hours (P . 0.05 vs. groups 2 and 3). However, cell number did increase at 48 hours in
To determine if the increase in DNA synthesis (BrdU staining) induced by C5b-9 and growth factors was due to the release of mitogens from VEC, supernatants were collected after 18 hours from each group of VEC and transferred to normal VEC growing in Nuserum. DNA synthesis was measured by BrdU incorporation into DNA at 18 hours after the transfer. When VEC were incubated with supernatants from control VEC (groups 2 and 3), BrdU staining was not increased (19 6 1 vs. 18 6 0.7, P . 0.05). In contrast, incubating VEC with supernatants derived from VEC subjected to sublytic C5b-9 attack (group 1) increased BrdU staining 1.5-fold (28 6 0.1 vs. 18 6 0.7, P , 0.001). Taken together, these results suggest that the increase in DNA synthesis induced by C5b-9 attack was due in part to mitogen(s) “released” following exposure of VEC to sublytic C5b-9. Complement-induced VEC DNA synthesis is not due to bFGF The cytokine bFGF is increased in experimental forms of VEC injury in vivo and bFGF is mitogenic for VEC [31, 32]. To determine if C5b-9 induced DNA synthesis was due to the release of bFGF from injured cells, DNA synthesis was measured in the presence of a specific neutralizing anti-bFGF antibody. Anti-bFGF antibody did not reduce BrdU staining induced by C5b-9 stimulation compared to control IgG (67 6 2.5 vs. 62 6 1.5%,
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P . 0.05). The concentration of anti-bFGF antibody used was effective as judged by a reduction in DNA synthesis to baseline when bFGF was used as a mitogen in the presence of antibody (results not shown). Taken together, these results show that the increase in DNA synthesis induced by C5b-9 was not due to bFGF. Complement-induced VEC DNA synthesis is not inhibited by TGF-b1 Because the cytokine TGF-b1 has been shown to inhibit specific mitogen-induced VEC proliferation in vitro [31], we asked if TGF-b1 would inhibit complement-induced VEC proliferation. The concentration of TGF-b1 used in the current study reduced BrdU staining significantly in VEC exposed to growth factors to stimulate mitogenesis (26.5 6 0.5 vs. 9 6 1, P , 0.05). In contrast, DNA synthesis induced by sublytic C5b-9 was not reduced by TGF-b1 (52 6 1 vs. 49 6 0, P . 0.05). C5b-9 increases cell cycle proteins required for S phase To better understand the effects of sublytic C5b-9 on the VEC cell cycle, we measured the expression of the S phase cyclin A and its partner CDK2 in Group 1 (sublytic C5b-9 attack), and control group 2 (anti-Fx1A without C5b-9) and group 3 (no anti-Fx1A) VEC at 18 hours, the peak of DNA synthesis in this study when VEC were grown in Nuserum-containing media. These cell cycle regulatory proteins are required for DNA synthesis (S phase) in other cell types [21], including mesangial cells [30]. Less than 10% of control VEC stained positive for CDK2 (results not shown). There was a marked increase in the number of VEC staining positive for CDK2 in C5b-9 attacked VEC (38%, P , 0.001 vs. controls; not shown) at 18 hours. The increase in CDK2 protein levels was confirmed by Western blot analysis (Fig. 2). Figure 2 shows that cyclin A was also detected in low levels by Western blot analysis in control VEC (groups 2 and 3) grown in Nuserum for 18 hours. There was also an increase in cyclin A protein levels in C5b-9 attacked VEC (group 1) compared to controls at 18 hours (Fig. 1). These results demonstrate that expression of the S phase cell cycle proteins, CDK2 and cyclin A is increased in VEC exposed to sublytic C5b-9, data which demonstrate that these cells do exhibit the changes in cell cycle proteins required to proliferate. C5b-9 causes a differential expression of cyclin kinase inhibitors To further assess why DNA synthesis occurred in response to C5b-9 but proliferation did not, the levels of the cyclin kinase inhibitors (CKI), p21 and p27, were measured by Western blot analysis. These CKI have been shown to inhibit DNA synthesis by binding to cyclin A-CDK2 complexes. There was no change in p21 levels
Fig. 2. Western blot of S phase cell cycle regulatory proteins. Glomerular visceral epithelial cells (VEC) were sensitized to anti-Fx1A antibody and C5b-9 (group 1, lane 2), and grown in Nuserm-containing media for 18 hours with the antibody. Controls included VEC exposed to antibody without C5b-9 (group 2, lane 3) or no antibody (group 3, lane 1). Cyclin dependent kinase-2 (CDK2) and cyclin A levels increased in VEC subjected to sublytic C5b-9 injury compared to controls, and this coincided with the increase in DNA synthesis in these cells. C5b-9 caused a decrease in the levels of the cyclin kinase inhibitor p27, but did not alter p21 levels.
by Western blot analysis (Fig. 2) in VEC exposed to sublytic C5b-9 compared to control in the presence of Nuserum. In control VEC (groups 2 and 3) grown in Nuserumcontaining media where less than 20% of cells were undergoing DNA synthesis, p27 protein was readily detected by Western blot analysis (Fig. 2). In contrast, there was a marked decrease in p27 levels in VEC following exposure to sublytic C5b-9 (group 1) compared to controls (groups 2), and this coincided with the increase in DNA synthesis in these cells. These findings are also
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Fig. 3. Protein levels for cyclin B. The number of VEC staining positive for cyclin B (A) were higher in control VEC exposed to anti-Fx1A antibody without C5b-9 (group 2) compared to VEC attacked by anti-Fx1A with C5b-9 (group 1) (B). Western blot analysis for cyclin B, confirming a decrease in cyclin B levels in VEC exposed to sublytic C5b-9 (lane 2) compared to controls (lanes 1 and 3).
consistent with the cells exhibiting increased DNA synthesis in response to C5b-9. M phase cell cycle proteins are altered by C5b-9 Mitosis and cytokinesis are regulated by the M phase cell cycle proteins, cdc2 and its partner cyclin B [23]. Cyclin B protein was barely detected by immunostaining or Western blot analysis in VEC prior to sensitization with anti-Fx1A (results not shown). Cyclin B protein was detected by immunostaining and Western blot analysis in control VEC grown in serum for 24 hours after exposure to anti-Fx1A without C5b-9 (group 2) and control VEC not exposed to anti-Fx1A (group 3) (Fig. 3). In contrast, cyclin B was barely detected by immunostaining and Western blot analysis (Fig. 3) in VEC grown in Nuserum for 24 hours after exposure to sublytic C5b-9 (group 1), and the levels were lower in C5b-9 attacked VEC compared to controls.
cdc2 was barely detected by immunostaining and Western blot in VEC prior to exposure of Anti-Fx1A and C5b-9 (results not shown). Immunostaining (Fig. 4) and protein levels (Fig. 4) were readily detected in control VEC (groups 2 and 3) grown in Nuserum-containing media for 24 hours. In contrast, cdc2 immunostaining (Fig. 4) and protein levels by Western blot analysis (Fig. 3) were lower in C5b-9 attacked VEC (group 1) grown in Nuserum for 24 hours compared to controls. These results show that the failure of VEC to undergo mitosis and cytokinesis following sublytic C5b-9 attack was associated with decreased expression of the M phase cell cycle proteins, cyclin B and cdc2. DISCUSSION In the current study we have shown that VEC in vitro sensitized by incubation with low concentrations of anti-
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Fig. 4. Expression for cdc2. There was a decrease in immunostaining for cdc2 in VEC exposed to sublytic C5b-9 (group 1) (A) compared to control (group 2) (B). The Western blot analysis for cdc2 demonstrates that cdc2 levels decreased in C5b-9 stimulated VEC (lane 2) compared to control VEC that did (group 2, lane 3) or did not receive anti-VEC antibody (group 3, lane 1).
VEC IgG (anti-Fx1A) and then exposed to sublytic concentrations of fresh serum (C1) to activate complement and induce formation of membrane-inserted C5b-9 complexes, increase the expression of cyclin A and CDK2 and enter S phase in the presence of growth factors. However, C5b-9 decreases cyclin B and cdc2 expression, resulting in the delay in G2/M progression. In experimental membranous nephropathy induced by antibody to the visceral glomerular epithelial cell (VEC) or podocyte, glomerular injury that occurs as a consequence of C5b-9 attack on VEC results in cell activation and production of oxidants and proteases that damage underlying GBM [4, 33, 34]. Accompanying this process is a low-grade increase in DNA synthesis, which is complement-dependent [11]. However, there is no apparent increase in VEC number [11, 14]. This is in marked contrast to the mesangial cell [35], which also makes oxidants and proteases in response to antibody/complement attack in vivo [33], but exhibits a dramatic proliferative response as well, mediated by PDGF, with a substan-
tial increase in cell number [8]. To better understand the response of the VEC to sublytic C5b-9 and relate it to proliferation and cell cycle control, the present study was undertaken to examine the effects of sublytic C5b-9 on VEC proliferation in vitro where other variables can be better controlled. Earlier studies by our group [11] and others [31, 32, 36–38] have focused on the cytokines responsible for VEC proliferation. In the current study, VEC were briefly (30 min) exposed to an antibody directed to antigens on the VEC (anti-Fx1A) followed by sublytic concentrations of fresh, complement sufficient serum (C1) or control serum which lacked C6 (C2). The first finding in this study was that the sublytic C5b-9 attack (in the absence of a source of growth factors) was not sufficient by itself to increase DNA synthesis in vitro. However, prior sublytic injury to VEC by C5b-9 significantly augmented entry into the S phase of the cell cycle (DNA synthesis) induced by growth factors (Nuserum). In contrast, antibody binding to the VEC in the absence of
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C5b-9 (C2 serum) did not increase DNA synthesis compared to VEC grown in media containing Nuserum that had not been exposed to antibody. The results of this study differ from findings in other cell types. C5b-9 alone (in the absence of other growth factors) is sufficient to increase DNA synthesis in certain nonrenal cells, including 3T3 [39], B cells [40] and oligodendrocytes [41]. Thus, in contrast to these studies, we were unable to show that the sublytic C5b-9 attack by itself was sufficient to induce DNA synthesis in the absence of other mitogens. However, C5b-9 did increase DNA synthesis in the presence of growth factors. These results are the first, to our knowledge, to establish that sublytic C5b-9 can augment DNA synthesis in renal cells in vitro. To determine whether or not the sublytic C5b-9 effect on DNA synthesis after exposure to serum was a direct or indirect one, we exposed normal VEC to supernatants of C5b-9 stimulated VEC and found that these supernatants contained mitogenic activity. However, the increase in DNA synthesis was only 50% of that induced by direct C5b-9 stimulation. This discrepancy could reflect a loss of growth factors during culture or the transferring of supernatants. However, the results are also consistent with the possibility that C5b-9 has both a direct effect on proliferation independent of other mitogens as well as an indirect effect resulting from release of mitogens. The indirect effect is consistent with other studies that document the release of several potential mitogens from cells following C5b-9 attack, including reactive oxygen species, interleukin (IL)-1, IL-6 and tumor necrosis factor-a (TNF-a) [3, 42, 43]. Because bFGF is a mitogenic growth factor for VEC in vitro [32] and bFGF has been shown to be up-regulated following C5b-9 mediated VEC injury in experimental membranous nephropathy (PHN model) [12], we next examined if bFGF was the mitogen released following C5b-9 induced VEC stimulation in vitro. Our results show that neutralizing bFGF with a specific anti-bFGF antibody did not reduce C5b-9 induced DNA synthesis. The concentration of anti-bFGF antibody used reduced bFGF-stimulated DNA synthesis in VEC. However, we did not measure the concentration of bFGF in the supernatants. This indirect method used in this study showing that bFGF did not mediate C5b-9 induced DNA synthesis also differs from results reported in nonrenal cells [44]. These studies suggest that the mechanisms by which C5b-9 induces DNA synthesis may be cell type specific. PDGF has been shown to mediate C5b-9 induced mitogenesis in certain nonrenal cells [39]. Although we have previously shown an increase in PDGF synthesis by C5b-9 stimulated VEC [11], PDGF is not a likely mitogen for these cells since VEC lack PDGF receptors [11]. Further studies are needed to better define the nature of the mitogen released by VEC in response to C5b-9.
The postulate that C5b-9 may have a direct effect on DNA synthesis is consistent with what is known of the activation of cell signaling pathways by C5b-9, many of which participate in receptor mediated processes including the response to growth factors. Thus, sublytic C5b-9 can induce a transient increase in cytosolic calcium [40], activation of protein kinase C [45], phospholipase A2 [46], PGI2 formation [47] and the MAPK pathway [48, 49], and increased expression of certain transcription factors [41] and nuclear factor-kB (NF-kB) [50]. In this study we explored the possibility that the effect of C5b-9 on VEC proliferation occurs at the level of the cell cycle. Studies of C5b-9-induced effects on cell cycle in VEC in vitro are of particular interest if they help to understand events that occur in vivo. In both human and experimental membranous nephropathy the C5b-9 attack on VEC is an established (experimental) or likely (human) pathogenic mechanism, but mitotic arrest apparently occurs and no increase in VEC numbers is seen [14]. In other models of VEC injury, Rennke and Kritz have proposed that the inability of the VEC to proliferate in vivo may underlie the development of progressive glomerulosclerosis [14–17, 51]. In PHN, we have established that some increase in DNA synthesis assessed by PCNA staining occurs, mitotic figures are seen and S phase cyclins and the CDK-inhibitors p21 and p27 are increased [11, 52]. This increased expression of CKI may prevent progression through the S phase in vivo [52]. Our results in vitro are similar in some respects but differ in others. In vitro, C5b-9 also induced an increase in BrdU staining (in the presence of Nuserum, a source of growth factors) and an increase in S phase cyclin and CDK. However, the expression of the CKI’s p21 and p27 was reduced rather than increased. Despite this, progression through M phase and cell division did not occur, apparently due to reduced expression of M phase cyclins B and CDC2. Thus, the failure of VEC to proliferate in response to C5b-9 in vitro appears to reflect the effects on M phase cell cycle proteins rather than the effect of CKI. Another explanation may be that C5b-9 delays G2/M phase progression. The mechanisms were not determined in the current study. The reasons for this discrepancy in CKI expression in vivo and in vitro are not clear, but could reflect differences in the environment in which the VEC lives in vivo versus in vitro, differences in cell-matrix attachments, differences in the amount of C5b-9 to which they are exposed (there is likely more in vitro), and differences in the timing required for the anti-Fx1A antibody to be shed. Moreover, since M phase cyclins were not studied in vivo, it is quite possible that a similar block in the M phase occurs in this setting as well. In any event, together these studies show that effects of C5b-9 on cell cycle regulatory proteins may in large part explain the failure
Shankland et al: C5b-9 and podocyte proliferation
of VEC to divide and proliferate in response to antibodycomplement mediated injury. An alternative explanation for our results would be that the cell number did not increase in response to C5b-9 because cell death was increased. We believe this is unlikely for two reasons. First, in pilot studies we showed that although there was C5b-9 induced low-grade apoptosis in VEC, this occurred within the first three hours of attack, and not thereafter. Second, there was no decrease in cell number at 24 and 48 hours following the C5b-9 attack, which occurred after the increase in DNA synthesis (at 18 hr) in this study. Previous studies have shown that TGF-b limits DNA synthesis in many renal cell types, including mitogeninduced VEC proliferation in vitro [31]. Furthermore, we have shown that TGF-b is increased following complement-induced injury to the VEC in experimental membranous nephropathy [53]. Therefore, we examined whether or not TGF-b reduced C5b-9 induced DNA synthesis in VEC in vitro. We found that C5b-9 induced VEC DNA synthesis was not inhibited by TGF-b1. In contrast, TGF-b1 did reduce DNA synthesis in control VEC, indicating that the concentration of TGF-b1 used in this study could inhibit VEC proliferation. Of note, although we studied TGF-b1 here, our in vivo studies demonstrate up-regulation of the TGF-b2 and TGF-b3 isoforms in C5b-9 mediated VEC injury, while no changes are detected in TGF-b1 [53]. Although most studies suggest that all TGF-b isoforms have similar biological effects, we have not excluded the possibility that TGF-b2 or -b3 might inhibit C5b-9 induced VEC proliferation. In summary, our study provides the first evidence that sublytic C5b-9 augments growth factor induced DNA synthesis in VEC. However, C5b-9 alone is not sufficient to engage VEC in the S phase of the cell cycle. Furthermore, sublytic C5b-9 attack arrests VEC at G2/M phase, thereby preventing mitosis and cytokinesis in vitro. Thus, the ability of VEC to enter the S phase of the cell cycle, and to undergo low grade DNA synthesis but not divide and produce an increase in cell number following C5b-9 mediated injury is similar to C5b-9 mediated injury in experimental and human membranous nephropathy. ACKNOWLEDGMENTS This study was supported by Public Health Service grants DK34198, DK47659, DK52121, DK51096. We thank J.M. Roberts for the generous supply of antibodies to cyclin A. No reprints are available. Correspondence to Stuart J. Shankland, M.D., Division of Nephrology, University of Washington, P.O. Box 356521, Seattle, Washington 98195, USA. E-mail: [email protected]
APPENDIX Abbreviations used in this article are: bFGF, basic fibroblast growth factor; BrdU, 5-bromo-29-deoxyuridine; C1, complement sufficient
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PVG rats; C2, complement deficient PVG rats; CDK, cyclin dependent kinase; CKI, cyclin kinase inhibitors; GBM, gomerular basement membrane; IL, interleukin; LDH, lactate dehydrogenase; NADH, nicotinamide adenine dinucleotide; p21, p21WAF1,CIP1; p27, p27Kip1; TGF-b, transforming growth factor-b; TNF-a, tumor necrosis factor-alpha; VEC, glomerular visceral epithelial cell.
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