ROS stimulate reorganization of mesangial cell-collagen gels by tyrosine kinase signaling

Roy Zent1, Menachem Ailenberg1, Gregory P. Downey2, and Melvin Silverman1

1 Membrane Biology Group and 2 Division of Respiratory Medicine, Department of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8


    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Reactive oxygen species (ROS) initiate multiple pathological and physiological cellular responses, including tyrosine phosphorylation of proteins. In this study, we investigated the effects of ROS on cell-extracellular matrix interactions utilizing the floating three-dimensional collagen gel assay. Exposure of mesangial cells grown in three-dimensional culture to H2O2, 3-amino-1,2,4-triazole (a catalase inhibitor), or puromycin is associated with gel reorganization accompanied by tyrosine phosphorylation of multiple proteins, including focal adhesion kinase (FAK). Neutrophils cocultured with mesangial cells in three-dimensional culture also induce mesangial cell-collagen gel reorganization and initiate tyrosine phosphorylation of a similar set of proteins. Collectively, these results show that ROS of either endogenous or exogenous origin can modulate mesangial cell-extracellular matrix interactions through initiation of a phosphotyrosine kinase signaling cascade. Consequently, ROS may play a role as signaling molecules that regulate mesangial cell-extracellular matrix interactions in both physiological and pathological conditions.

reactive oxygen species; protein-tyrosine kinase; neutrophils; glomerular mesangium; puromycin


    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

CONSTITUTIVE PRODUCTION of reactive oxygen species (ROS) by mesangial cells originates from an intrinsic NADPH oxidase (21) that normally functions at a low level but increases in response to inflammatory stimuli such as cytokines (20). Neutrophils, monocytes, and macrophages also possess an NADPH oxidase, but their production of ROS is considerably greater than that of mesangial cells. ROS are implicated as causative agents in glomerular disease, and the source of ROS may be either the mesangial cell itself or "passenger" leukocytes such as neutrophils, monocytes, or macrophages (22). ROS also play a role in the pathogenesis of puromycin aminonucleoside nephrosis (7, 22, 23). However, in this case, the generation of ROS is as a result of an anthracycline compound undergoing a single-electron reduction to a free radical semiquinone species, which is subsequently reoxidized in the presence of molecular oxygen to form reactive oxygen metabolites (22).

Although ROS are highly reactive molecules with the potential to cause cell injury, they also initiate a number of physiological responses, including tyrosine phosphorylation of growth factor receptors (15) and p42/p44MAPK in vascular smooth muscle cells and neutrophils (3, 5). Furthermore, both proliferative and migratory effects of platelet-derived growth factor (PDGF) on vascular smooth muscle cells are, at least in part, dependent on the production of ROS (26). In cultured mesangial cells, ROS induce tyrosine phosphorylation of the PDGF receptor pp60c-src and a 125-kDa protein thought to be focal adhesion kinase (FAK) (9). In addition, high concentrations of ROS act as phosphatase inhibitors, and in the presence of sodium vanadate, ROS induce focal adhesion formation (6). The chemical basis of these various effects of ROS is unknown but may be due to direct oxidation of critical protein sulfhydryl groups or through formation of transition metal complexes (14).

Normal function of the mesangium, which includes controlling glomerular filtration rate, is dependent on complex mesangial cell-extracellular matrix interactions. These cell-extracellular matrix interactions can be studied in a realistic in vitro system by growing mesangial cells in three-dimensional collagen gels in which the cells exhibit a phenotype resembling the in vivo state (16, 17, 29). When cells are placed in collagen, they attach to it via integrins within 30 min of being plated (10). By 4 h, they begin to spread and develop lamellipodia (11). As cells spread, the actin cytoskeleton generates force that is transmitted to the extracellular matrix via integrins, resulting in extracellular matrix remodeling (8, 18, 25). Once the extracellular matrix is remodeled, cells migrate and form new cell-substratum attachments. If the collagen gel in which the cells are embedded is not fixed to the culture dish, then the extracellular matrix remodeling results in the diameter of the gel becoming smaller. This decrease in size of the gel is referred to as gel contraction.

Remodeling of collagen gels is induced by many agonists, including fetal bovine serum (FBS) and PDGF (10, 11, 29). The cell attachment, spreading, and migration essential for gel reorganization is dependent on tyrosine phosphorylation of a number of proteins, including FAK (29). Although remodeling of the gels takes at least 6 h, the tyrosine phosphorylation of the necessary signaling molecules is maximal 45 min after addition of the agonist (29).

Because ROS increase tyrosine phosphorylation of a number of proteins, including a 125-kDa protein thought to be FAK (9), and tyrosine kinase phosphorylation is implicated in mesangial cell-collagen gel reorganization (29), we investigated the effects of ROS on mesangial cells embedded in floating three-dimensional collagen I gels. For logistical reasons, we used collagen I rather than collagen IV, which is the normal extracellular matrix found in the mesangium. This remains a physiologically relevant model (17, 28) because mesangial cells express alpha 1beta 1, alpha 2beta 1, and alpha 3beta 1 integrins that bind both collagen I and collagen IV (19). We show that low concentrations of H2O2 induce mesangial cells to reorganize collagen I gels. This process is dependent on tyrosine phosphorylation of several proteins, among which FAK is prominent. Similarly, when ROS are produced by the mesangial cells themselves in response to 3-amino-1,2,4-triazole (an endogenous catalase inhibitor) or puromycin or, alternatively, when ROS are generated exogenously by neutrophils, mesangial cells reorganize collagen gels. Because collagen gel reorganization is dependent on cell attachment, spreading, and migration, these results imply that ROS are signaling molecules that may influence cell-extracellular matrix interactions in the mesangium under both physiological and pathological conditions.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Materials. MEM, Hanks' balanced salt solution (HBSS), and FBS were supplied by GIBCO (Grand Island, NY). Collagen type I, collagenase type Ia, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), protein G-sepharose 4B, puromycin aminonucleoside, 3-amino-1,2,4-triazole, N-formyl-Met-Leu-Phe (fMLP), and N-t-BOC-Met-Leu-Phe (t-BOC) were obtained from Sigma (St. Louis, MO). Diisopropyl fluorophosphate was purchased from Calbiochem (La Jolla, CA). Monoclonal anti-phosphotyrosine antibodies and monoclonal anti-FAK antibodies were purchased from Upstate Biotechnologies (Lake Placid, NY). Polyclonal anti-FAK antibodies were purchased from Santa Cruz (La Jolla, CA). Catalase was obtained from Worthington Laboratories. Diphenyleneiodonium (DPI) was a gift from Dr. Sergio Grinstein (Hospital for Sick Children, Toronto, Ontario, Canada).

Tissue culture. Rat glomeruli were isolated from kidneys of 3-wk-old male Sprague-Dawley rats by the graded-sieve technique (12). Mesangial cells isolated from these glomeruli were grown in 100-mm plates in MEM containing 15% FBS as previously described (2). Experiments were performed on cells between passages 5 and 15.

Neutrophil preparation. Human neutrophils (>98% pure) were isolated from citrated whole blood obtained by venipuncture using dextran sedimentation and discontinuous plasma-Percoll gradients as previously described (13). The cells were used for the gel contraction assays immediately after isolation. To minimize proteolysis of mesangial cell proteins after extraction in detergent, we pretreated the neutrophils for 30 min with 5 mM diisopropyl fluorophosphate and washed them three times in PBS before use.

Gel contraction assay. The gel contraction assay is based on previously described methods (2). Mesangial cells were placed in a solution of MEM and type I collagen and allowed to gelate for 3 h. The substances tested were dissolved in MEM and added to the gels. In a variety of cell types for which the gel contraction assay has been utilized, FBS empirically causes the greatest amount of gel contraction (10, 11). We elected to quantitate the effects of ROS on gel contraction relative to those induced by FBS. The diameter of the gel was measured at the specified time points using a dissecting microscope (Wild Leitz) at ×16 magnification, and the percentage of maximal contraction was calculated using the following formula: %gel contraction = 100 × [(AMEM - Atest)/(AMEM - AFBS)], where AMEM is the area of the MEM-treated gel, Atest is the area of the test substance-treated gel, and AFBS is the area of the FBS-treated gel.

Cell-viability studies. Cell viability was assessed by the tetrazolium salt (MTT) method, as well as by trypan dye exclusion, as described previously (28).

Immunoblotting. To ensure that protein loads were comparable in the various immunoblotting protocols, we added an equal number of cells to each collagen gel. Cells embedded in collagen gels were isolated by dissolving the collagen with collagenase type I (20 µg/ml) mixed with the phosphatase inhibitors (1 mM Na3VO4, 30 mM sodium pyrophosphate, and 50 mM NaF). Cells were then lysed for 20 min at 4°C in a lysis buffer [20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.15 U/ml aprotonin, and 1 mM Na3VO4]. The lysates were cleared by centrifugation at 10,000 g for 10 min at 4°C, after which the supernatants were collected and equal protein loading was verified by the Bradford protein assay. The proteins were dissolved in a 5× SDS-PAGE sample buffer and electrophoresed on a 10% polyacrylamide slab gel. Proteins were transferred electrophoretically to nitrocellulose and blocked overnight (0.25% gelatin, 10% ethanolamine, and 0.1 M Tris). The nitrocellulose was probed with a monoclonal anti-phosphotyrosine (4G10) antibody at a concentration of 1:5,000 for 2 h, followed by a 1-h incubation with a 1:5,000 dilution of a horseradish peroxidase-conjugated sheep anti-mouse antibody. Immunoreactive bands were visualized by enhanced chemiluminescence. On each blot, there were lysates of mesangial cells extracted from collagen gel cells incubated in either MEM (negative control) or FBS (positive control) or treated with test ROS agonists. Molecular-weight standards were run in parallel. We prepared Figs. 1, 2, 5, 6, and 9 by making composites comparing selected individual lanes from the same slab gels.

Immunoprecipitation of FAK. Cells were prepared in a similar fashion to that used for immunoblotting, except that the lysis and solubilization was carried out in a modified RIPA buffer (Tris · HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1 µg/ml aprotonin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM Na3VO4, and 1 mM NaF) for 20 min at 4°C. The lysates were cleared by centrifugation at 10,000 g for 10 min at 4°C, and the supernatants were collected and equalized for protein loading by the Bradford protein assay. Equal volumes of lysate were immunoprecipitated with an anti-FAK polyclonal antibody for at least 1 h at 4°C, after which they were incubated with protein G-sepharose beads (precleared by 0.25% ovalbumin) for an additional 2 h. Immunoprecipitates were washed three times in the lysis buffer and divided into fractions, each of which was electrophoresed separately on a 10% polyacrylamide slab gel and subjected to immunoblotting using either a monoclonal anti-phosphotyrosine or a monoclonal anti-FAK antibody, as described in Immunoblotting. In this way, it was possible to monitor changes in phosphorylation and verify the equivalency of the amount of immunoprecipitated FAK.

Statistics. All gel contraction assays were performed in duplicate on at least three different occasions. The results of the gel contraction assays were normalized as shown in METHODS. Means ± SE of the pooled data of the contraction assays were calculated and are shown graphically. When single points were measured, the means ± SE of the assays were calculated, and the significance of the differences of their means was compared using the Student's t-test. The immunoblot and immunoprecipitation experiments were done at least three times each, and representative examples are shown.


    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Short-term exposure of mesangial cells in three-dimensional culture to high concentrations of H2O2 induces anchorage-dependent tyrosine phosphorylation. To determine if exposure of mesangial cells to H2O2 in three-dimensional culture resulted in tyrosine phosphorylation of cellular proteins, we prepared collagen gels as described in METHODS and left them in MEM overnight. The following day, the gels were exposed to increasing concentrations of H2O2 (2.5-10 mM) for 45 min, after which the cells were lysed, solubilized, and subjected to immunoblotting with an anti-phosphotyrosine antibody. As shown in Fig. 1A, exposure of mesangial cells in three-dimensional culture to H2O2 induced tyrosine phosphorylation of several proteins in a dose-dependent manner (lanes 2-4). In a parallel set of experiments (Fig. 1B), cells were grown on tissue culture plates, after which they were serum starved for 48 h, trypsinized, suspended in MEM for 1 h, and then exposed to H2O2. These cells showed little evidence of increased protein tyrosine phosphorylation relative to cells in three-dimensional culture (Fig. 1B). Lysates from the unattached cells in suspension, as well as from the three-dimensional culture systems, were immunoprecipitated with a polyclonal anti-FAK antibody. The immunoprecipitate was divided into equal fractions; one-half was immunoblotted with a monoclonal anti-phosphotyrosine antibody, and the other half was immunoblotted with a monoclonal anti-FAK antibody. As shown in Fig. 1C, an equivalent amount of FAK was immunoprecipitated from the lysates, but the degree of phosphorylation after exposure to 10 mM H2O2 increased for cells grown in three-dimensional culture (compare lanes 1 and 2), but not for unattached mesangial cells in suspension (compare lanes 3 and 4).


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Fig. 1.   H2O2 induces protein tyrosine phosphorylation in mesangial cells grown in 3-dimensional collagen culture. A: mesangial cells were embedded in collagen and incubated in MEM overnight. The following morning, cells were either left in MEM or exposed to increasing concentrations of H2O2 for 45 min, after which they were extracted from gels, lysed, and subjected to immunoblotting with monoclonal anti-phosphotyrosine antibody. Lane 1, basal tyrosine phosphorylation in presence of MEM; lanes 2-4; protein tyrosine phosphorylation 45 min after addition of 2.5, 5, and 10 mM of H2O2, respectively. B: mesangial cells were grown to confluence on plastic culture dishes, serum starved for 48 h, and trypsinized; suspended cells were then treated with varying concentrations of H2O2 for 45 min. Lane 1, basal tyrosine phosphorylation in MEM; lanes 2-4, tyrosine phosphorylation after addition of 2.5, 5, and 10 mM of H2O2, respectively. C: cells prepared in a fashion similar to protocols described in A and B were either left in MEM or exposed to 10 mM of H2O2 for 45 min. Lysates were immunoprecipitated with a polyclonal focal adhesion kinase (FAK) antibody and then immunoblotted with either a monoclonal anti-phosphotyrosine antibody (bottom) or a monoclonal FAK antibody (top). Lanes 1 and 3, basal level of FAK phosphorylation of cells grown in 3-dimensional collagen gels or in suspension, respectively; lanes 2 and 4, immunoprecipitates corresponding to lanes 1 and 3, respectively, after exposure to 10 mM H2O2.

ROS induce gel contraction and tyrosine kinase phosphorylation. Mesangial cells grown in three-dimensional culture and exposed to H2O2 demonstrated a pattern of protein tyrosine phosphorylation similar to that which occurs during FBS- and PDGF-induced mesangial cell-collagen gel contraction (29). It was therefore of interest to examine whether exposure of mesangial cells grown in three-dimensional culture to ROS might also induce gel contraction. Because mesangial cells require several hours to significantly remodel collagen gels, an experimental protocol that allowed cells to be exposed to ROS for a sufficient length of time was designed. Concentrations of H2O2 (2.5-10 mM) that resulted in protein tyrosine phosphorylation of mesangial cells within 45 min (see Fig. 1) were cytotoxic to cells after longer exposure, as determined by the MTT assay (data not shown). Therefore, three-dimensional mesangial cell-collagen gels were exposed for 6 h to much lower concentrations of H2O2 (25-800 µM) augmented by a low concentration of the catalase inhibitor 3-amino-1,2,4-triazole (0.625 mM). This concentration of 3-amino-1,2,4-triazole did not change cell viability as monitored by the MTT assay. Under these conditions, H2O2 stimulated gel contraction that exhibited a dose-response curve with maximal gel contraction that occurred at 200 µM H2O2 and returned to basal levels at 800 µM H2O2 (Fig. 2A). The percentage of gel contraction was measured at 6 h and is expressed as a percentage of maximal gel contraction induced by 3% FBS (see METHODS).


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Fig. 2.   H2O2 induces mesangial cell-collagen gel contraction and tyrosine kinase phosphorylation. A: mesangial cells (2.5 × 105) were embedded in collagen and incubated in MEM overnight. Gels were incubated in increasing concentrations of H2O2 in presence of 0.625 µM 3-amino-1,2,4-triazole (ATZ) for 6 h, and percentage of gel contraction was calculated as a percentage of maximal fetal bovine serum (FBS)-induced gel contraction. B: mesangial cells embedded in collagen gels and incubated overnight in MEM were exposed to H2O2 (200 uM) and ATZ (0.625 µM) in the presence of genistein (1-5 µg/ml), daidzein (1-5 µg/ml), lavendustin A (2.5-40 µM), or lavendustin B (2.5-40 µM) for 6 h. Percentage of gel contraction is shown as a percentage of maximal FBS-induced gel contraction. C: gels prepared in same way as in Fig. 1A. Lane 1, basal tyrosine phosphorylation; lane 2, gels exposed to H2O2 (200 µM) and ATZ (0.625 mM) for 45 min.

To determine whether mesangial cell-induced collagen gel remodeling in response to ROS was dependent on tyrosine kinase phosphorylation, we performed gel contraction experiments similar to those described in Fig. 2A in the presence of tyrosine kinase inhibitors. As shown in Fig. 2B, genistein (1-5 µg/ml) and lavendustin B (2.5-40 µM) inhibited gel contraction induced by H2O2 (200 µM) and 3-amino-1,2,4-triazole (0.625 mM), whereas their inactive isomers, daidzein (1-5 µg/ml) and lavendustin A (2.5-40 µM), did not.

Experiments were attempted to assess whether low concentrations of H2O2 (200 µM) would induce tyrosine phosphorylation of mesangial cells grown in three-dimensional collagen gels. H2O2 (200 µM) without the addition of 3-amino-1,2,4-triazole did not result in tyrosine phosphorylation relative to controls. However, if H2O2 (200 µM) and 3-amino-1,2,4-triazole (0.625 mM) were added together and the cells were lysed 45 min later, there was increased tyrosine phosphorylation of multiple protein bands (Fig. 2C), similar to that observed when mesangial cells were exposed to high concentrations of H2O2 (Fig. 1).

These results demonstrate that ROS exert a concentration-dependent differential effect on mesangial cell-induced gel contraction. As the ROS concentration increased, gel contraction increased, reached a peak, and declined to control levels. The fact that tyrosine kinase inhibitors inhibited gel contraction, and that gel contraction was associated with increased tyrosine phosphorylation, implies that gel contraction is dependent on tyrosine phosphorylation of specific proteins.

3-Amino-1,2,4-triazole and puromycin induce protein tyrosine phosphorylation along with stimulation of gel contraction. In earlier work (28), we demonstrated that mesangial cells produce ROS when exposed to either puromycin or 3-amino-1,2,4-triazole. Given that mesangial cells remodel collagen gels when exposed to low concentrations of H2O2, we decided to evaluate the effects of low concentrations of both these substances on mesangial cell-collagen gel contraction. Gels were prepared and incubated overnight in MEM, after which they were exposed to either 3-amino-1,2,4-triazole (0.625-10 mM) or puromycin (2.5-20 µg/ml). The percentage of gel contraction was measured at 6 h and is expressed as a percentage of maximal gel contraction induced by 3% FBS. 3-Amino-1,2,4-triazole (Fig. 3) and puromycin (Fig. 4) both induced gel contraction in a dose-dependent fashion, yielding similar biphasic curves (i.e., as the concentration was increased, gel contraction also increased, peaked, and then decreased back to control levels). Moreover, inspection of Figs. 3 and 4 reveals that, in each case, gel contraction was inhibited by addition of either catalase (12.5 µg/ml; Figs. 3A and 4A), which degrades H2O2, or the flavoprotein inhibitor DPI (0.625 µM; Figs. 3B and 4B), which inhibits NADPH oxidase (24).


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Fig. 3.   ATZ induces mesangial cell-collagen gel contraction. A: mesangial cells (2.5 × 105) embedded in collagen gels and incubated overnight in MEM were exposed to increasing concentrations of ATZ or ATZ plus catalase (CAT) (12.5 µg/ml). Gel diameter was measured 6 h after exposure. Percentage of gel contraction is expressed as percentage of maximal FBS-induced gel contraction. B: gels were exposed to ATZ or ATZ plus 0.625 µM diphenyleneiodonium (DPI).


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Fig. 4.   Puromycin (PA) induces mesangial cell-collagen gel contraction. A: gels (2.5 × 105 mesangial cells) were exposed to increasing concentrations of PA or PA plus CAT (12.5 µg/ml). Percentage of gel contraction induced by PA is expressed as a percentage of maximal FBS-induced gel contraction. B: gels were exposed to PA alone or PA + DPI (0.625 µM).

We next determined whether 3-amino-1,2,4-triazole- and puromycin-mediated mesangial cell-collagen gel contraction resulted in protein tyrosine phosphorylation. To do this, we exposed mesangial cells in collagen gels to 3-amino-1,2,4-triazole (2.5 mM) or puromycin (5 µg/ml) for 45 min, after which the cells were lysed and cellular proteins were separated by SDS-PAGE and immunoblotted with an anti-phosphotyrosine antibody. As shown in Fig. 5, A and B, exposure to 3-amino-1,2,4-triazole and puromycin resulted in increased tyrosine phosphorylation of multiple protein bands, similar to that observed when mesangial cells were exposed to H2O2. In each case, addition of ROS inhibitors DPI (0.625 µM) and catalase (12.5 µg/ml) reversed the 3-amino-1,2,4-triazole- and puromycin-induced tyrosine phosphorylation (lanes 3 and 4, respectively, in Fig. 5, A and B), confirming that it is indeed the production of ROS that results in initiation of the tyrosine phosphorylation. Immunoprecipitation of FAK from cells treated with either 3-amino-1,2,4-triazole (2.5 mM) or puromycin (5 µg/ml) revealed increased phosphorylation of FAK after exposure to these compounds (Fig. 5C).


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Fig. 5.   Low concentrations of ATZ and PA induce tyrosine phosphorylation. A: mesangial cells (2.5 × 105) were prepared in a fashion similar to that described in Fig. 1. Lane 1, basal tyrosine phosphorylation; lane 2, extent of tyrosine phosphorylation induced by incubation with ATZ (2.5 mM) for 45 min. ATZ-induced protein tyrosine phosphorylation is reversed by addition of DPI (0.625 µM; lane 3) or CAT (12.5 µg/ml; lane 4). B: lane 1, basal tyrosine phosphorylation; lane 2, tyrosine phosphorylation induced by PA (5 µg/ml); lanes 3 and 4, reversal of phosphorylation by DPI (0.625 µM) and CAT (12.5 µg/ml), respectively. C: mesangial cell lysates were immunoprecipitated with a polyclonal anti-FAK antibody and immunoblotted with an anti-phosphotyrosine antibody (bottom) or an anti-FAK monoclonal antibody (top). Lane 1, FAK phosphorylation under basal conditions; lane 2, increased phosphorylation of FAK after 45-min incubation with ATZ (2.5 mM); lane 3, phosphorylation of FAK after 45-min incubation with PA (5 µg/ml).

These experiments demonstrate that low concentrations of ROS produced by mesangial cells in response to 3-amino-1,2,4-triazole and puromycin induce gel contraction that is accompanied by tyrosine phosphorylation of several proteins, including FAK. However, with increasing concentrations of ROS production by mesangial cells, gel contraction is inhibited.

FMLP induces gel contraction and protein tyrosine phosphorylation. FMLP stimulates NADPH oxidase via a specific formylpeptide receptor of the seven transmembrane type, resulting in ROS production and activation of downstream effector pathways including kinases and phosphatases (4). To test if FMLP had any effect on three-dimensional mesangial cell cultures, we prepared gels as described in METHODS, incubated them overnight in MEM, and then exposed them to increasing concentrations of FMLP. Figure 6A shows that the maximal effect of FMLP (0.1 µM) induced 40% of the maximal gel contraction caused by FBS over a 6-h period. No gel contraction relative to FMLP was induced in the presence of the inactive receptor competitor t-BOC FMLP (0.1 µM) (P < 0.01), confirming the specificity of the response. Catalase and DPI, respectively, decreased FMLP-induced contraction to 17% and 16% of maximal contraction induced by FBS, which was significantly less than the 40% of maximal gel contraction induced by FMLP alone (P < 0.01). Although gel contraction by FMLP in the presence of catalase and DPI was significantly less than by FMLP alone, the inhibition was only ~60% of maximal FMLP-induced contraction. This suggests that FMLP-induced gel contraction is only partially due to stimulation of mesangial cell ROS by FMLP.


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Fig. 6.   N-formyl-Met-Leu-Phe (FMLP) induces gel contraction and tyrosine phosphorylation. A: mesangial cells (2.5 × 105) embedded in collagen gels were incubated overnight in MEM and treated with either FMLP (0.1 µM), N-t-BOC-Met-Leu-Phe (t-BOC) FMLP (0.1 µM), CAT alone (12.5 µg/ml), FMLP (0.1 µM) + CAT, DPI alone (0.625 µM), or FMLP (0.1 µM) + DPI. Percentage of gel contraction was measured after 6 h and is shown as percentage of maximal FBS-induced gel contraction. Data points represent means ± SE of 3 experiments performed in duplicate. * Significant difference (P < 0.01) in amount of contraction vs. stimulation of gel by FMLP alone. B: collagen gels prepared in a fashion similar to those in Fig. 1. Lane 1, basal tyrosine phosphorylation; lanes 2-4, induction of tyrosine phosphorylation after 45-min exposure to FMLP alone (0.1 µM), FMLP (0.1 µM) + CAT (12.5 µg/ml), or FMLP (0.1 µM) + DPI (0.625 µM), respectively.

As shown in Fig. 6B, FMLP-induced contraction was accompanied by tyrosine phosphorylation of a number of proteins and the FMLP-induced tyrosine phosphorylation was partially reversed by catalase and DPI. Comparison of Figs. 5B and 6B reveals that the degree of reversal of FMLP-induced tyrosine phosphorylation by catalase and DPI was less than that of 3-amino-1,2,4-triazole- and puromycin-induced protein tyrosine phosphorylation, consistent with the finding that catalase and DPI only partially inhibit FMLP-induced gel contraction (see Fig. 6A).

In summary, our data suggest that mesangial cells possess FMLP receptors and that activation of these receptors stimulates mesangial cell spreading, attachment, and migration by at least two different tyrosine phosphorylation pathways, one of which is due to an FMLP-stimulated increase in ROS production.

Neutrophils induce mesangial cell-collagen gel contraction that is accompanied by protein tyrosine phosphorylation. Neutrophils produce a basal concentration of ROS, as demonstrated by the fact that 106 cells produce 0.5 nM H2O2/min when incubated in PBS (27). To assess whether this neutrophil-derived ROS could induce gel contraction through cell-cell interactions, we created a coculture system in which 2.5 × 105 mesangial cells embedded in collagen gels were prepared and left in MEM overnight. The following day, neutrophils (2 × 106/ml) were added to the gels and the amount of gel contraction was measured after 6 h and compared with maximal gel contraction induced by FBS.

As shown in Fig. 7, addition of neutrophils stimulated gel contraction to ~70% of the maximal gel contraction caused by FBS after 6 h. Inspection of Fig. 7 shows that "coculture" of neutrophils in the presence of catalase and DPI resulted in only 8% and 5% of maximal gel contraction, respectively, which is significantly less than the untreated gels (P < 0.01). In contrast to FMLP-induced contraction, this means that stimulation of mesangial cell spreading and migration by exposure to neutrophils is predominantly due to production of ROS. In control experiments, gel remodeling did not occur when neutrophils were added to gels that did not contain mesangial cells (data not shown).


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Fig. 7.   Neutrophils (Neut) induce gel contraction. Collagen gels (2.5 × 105 mesangial cells) were prepared and allowed to incubate in MEM overnight. The following morning, freshly prepared human neutrophils (2 × 106/ml) were added to gels either alone or in presence of CAT (12.5 µg/ml) or DPI (0.625 µM). Effect of neutrophils was compared with FBS-induced contraction 6 h later. Data points represent means ± SE of 3 experiments performed in duplicate. * Significant inhibition (P < 0.01) of neutrophil-induced contraction by CAT and DPI.

To determine whether the effect of neutrophils on gel contraction was due to secretion of factors other than ROS, we carried out experiments with conditioned medium from neutrophils. Neutrophils were kept in MEM for 24 h, after which the conditioned medium was added to mesangial cell-collagen gels in increasing concentrations. In contrast to gel contraction induced by neutrophils, no gel contraction resulted from addition of the conditioned medium (data not shown).

To determine whether increasing the concentration of neutrophils affected the mesangial cell-induced gel contraction assay, collagen gels were incubated in MEM overnight and exposed to increasing numbers of neutrophils in the presence or absence of DPI or catalase. Gel contraction was measured at 6 h. As shown in Fig. 8, neutrophil-induced gel contraction produced a biphasic dose-dependency curve as a function of increasing neutrophil density. Maximal contraction was observed at ~2 × 106 neutrophils/ml (based on an estimate of 8 neutrophils per mesangial cell). When neutrophil density reached 8 × 106/ml (32 neutrophils per mesangial cell) or more, gel contraction was markedly impaired. Catalase and DPI partially blocked the neutrophil-induced gel contraction at low ratios of neutrophils to mesangial cells. This is to be expected if the gel contraction were stimulated by ROS. But as the ratio of neutrophils to mesangial cells increased, presumably accompanied by an ever-increasing production of ROS, inhibition of ROS production appeared to be protective of the mesangial cells in the gels, and they maintained some degree of gel contraction. Thus catalase and DPI have a dual effect. At low concentrations of neutrophils, they reverse ROS-stimulated gel contraction; however, at high neutrophil concentrations, when ROS production may become cytotoxic, catalase and DPI are protective.


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Fig. 8.   Neutrophils induce a biphasic response in mesangial cell-collagen gel contraction. Gels (2.5 × 105 mesangial cells) were prepared and incubated overnight, after which they were exposed to neutrophils in increasing concentrations, either alone or in presence of CAT (12.5 µg/ml) or DPI (0.625 µM). Percentage of gel contraction was measured 6 h after addition of neutrophils and is expressed as a percentage of maximal FBS-induced gel contraction.

Next, mesangial cells embedded in collagen gels were exposed to neutrophils (2 × 106/ml), after which the collagen gels were washed, the mesangial cells were extracted from the gels and lysed, and the protein was solubilized, electrophoresed, and immunoblotted with an anti-phosphotyrosine antibody. Inspection of Fig. 9A shows that, relative to controls, exposure to neutrophils resulted in tyrosine phosphorylation of a number of proteins (compare lanes 1 and 2). This protein tyrosine phosphorylation was reversed by the addition of catalase and DPI (lanes 3 and 4, respectively). Immunoprecipitation with anti-FAK antibody (Fig. 9B) revealed increased phosphorylation of FAK in mesangial cells exposed to FMLP (lane 2) or neutrophils (lane 3), compared with control (lane 1).


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Fig. 9.   Neutrophils induce protein tyrosine phosphorylation in 3-dimensional collagen gels. A: lysates from collagen gels were prepared in a fashion similar to those described in Fig. 1. To ensure that only mesangial cells were lysed, gels were washed 3 times in Hanks' balanced salt solution to remove neutrophils from surface of gels before lysis. Lane 1, basal tyrosine phosphorylation activity; lane 2, tyrosine phosphorylation 45 min after addition of neutrophils (2 × 106/ml). Lanes 3 and 4 show that tyrosine phosphorylation induced by neutrophils can be reversed by addition of CAT (12.5 µg/ml) and DPI (0.625 µM), respectively. B: collagen gels prepared in a fashion similar to those in Fig. 1C and immunoprecipitated with anti-FAK antibody. Lane 1, basal phosphorylation of FAK; lanes 2 and 3, increasing phosphorylation of FAK after 45-min incubation with FMLP (0.1 µM) or neutrophils (2 × 106/ml), respectively. Bottom: FAK immunoprecipitates immunoblotted with an anti-phosphotyrosine antibody; top: FAK immunoprecipitates immunoblotted with an anti-FAK antibody.

Collectively, these results imply that neutrophils produce ROS, which in low concentrations induce gel contraction accompanied by tyrosine phosphorylation of several proteins, including FAK. However, when large concentrations of neutrophils are added, they produce substances (including ROS) that inhibit gel contraction. Thus ROS produced by neutrophils appear to modulate mesangial cell function in a concentration-dependent manner, similar to ROS generated endogenously by mesangial cells.


    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Until recently, the effects of ROS on biological tissue have been evaluated principally in terms of potential to cause cell damage because of their high reactivity. It is becoming increasingly evident, however, that ROS may also play a physiological role as signaling molecules influencing a variety of cellular functions. The present study supports a regulatory role for ROS in mesangial cell function. We found that, at low concentrations, ROS induced mesangial cell attachment, spreading, and migration, which, in a three-dimensional collagen culture system, leads to matrix remodeling and gel contraction. Moreover, the ROS-induced gel contraction is dependent on tyrosine phosphorylation of a number of proteins, including FAK. The data demonstrate that ROS, produced either endogenously by mesangial cells or exogenously from other cellular sources such as neutrophils, modulate the behavior of mesangial cells in a concentration-dependent manner. Regardless of whether the source of ROS was H2O2, 3-amino-1,2,4-triazole, puromycin, or neutrophils, the same biphasic dose dependency was observed. At low concentrations, ROS increasingly stimulated gel contraction until a threshold (peak) was reached. Above that threshold, the ROS effect waned and gel contraction reduced to background levels.

It was recently shown that exposure of mesangial cells grown in two-dimensional culture to high concentrations of H2O2 increases tyrosine phosphorylation of the PDGF receptor, pp60c-src, and a 125-kDa protein presumed to be FAK (9). We confirmed that short-term exposure of mesangial cells grown in two-dimensional culture results in increased tyrosine phosphorylation of a number of proteins (data not shown), and we also demonstrated that similar exposure of mesangial cells to high concentrations of H2O2 in three-dimensional culture leads to stimulation of tyrosine phosphorylation of multiple proteins, including FAK.

The present study demonstrates that low concentrations of H2O2, as well as generation of ROS by mesangial cells after exposure to 3-amino-1,2,4-triazole, puromycin, or FMLP, or by neutrophils, each separately induce collagen gel remodeling, presumably by increasing attachment, spreading, and migration of mesangial cells. Although FBS is the most potent stimulator of gel contraction (10, 11), the mechanism whereby it induces gel contraction is unknown. In the present investigation, we found that ROS produce ~50-70% of maximal gel contraction caused by FBS. This effect is as powerful as, or more powerful than, any mediator other than FBS that has been used to induce collagen gel contraction (29). Although this is an entirely in vitro empirical finding, it suggests that ROS can be quite potent modulators of mesangial cell remodeling of collagen gels.

The present study was carried out using a three-dimensional collagen gel culture system because mesangial cells behave differently in this environment than in the usual two-dimensional culture. In three-dimensional culture systems, mesangial cells adopt a nonproliferative phenotype similar to that found in vivo rather than the proliferative phenotype found in two-dimensional culture systems (16, 17, 29). Cells in three-dimensional culture also respond differently to growth factors like PDGF compared with cells in the two-dimensional state. Mesenchymal cells, including mesangial cells, express fewer PDGF receptors with decreased autophosphorylation when grown under these conditions (17, 29). This resembles the situation in the adult kidney where little PDGF and few PDGF receptors are found (1).

There are several possible mechanisms that would explain how low concentrations of ROS might induce gel contraction. H2O2 is a broad-spectrum phosphatase inhibitor (14); this phosphatase inhibition may result in tyrosine phosphorylation of FAK, paxilline, pp60c-src, tensin, or other tyrosine kinases that may, in turn, lead to focal adhesion formation, subsequent cell attachment, and migration. This would be consistent with the finding that exposure of cells in a two-dimensional culture system to high concentrations of H2O2, albeit in the presence of vanadate (another phosphatase inhibitor), results in formation of focal adhesions (6). On the other hand, when mesangial cells were trypsinized from plastic and then exposed to H2O2, tyrosine phosphorylation was not markedly increased. This latter observation implies that cellular signaling responses to H2O2 are anchorage dependent. Therefore, it is possible that H2O2 has a direct effect on integrins, causing a conformational change in either the extracellular or intracellular domain, leading to integrin-mediated cell attachment to ligand and subsequent focal adhesion formation with resultant tyrosine phosphorylation. Finally, there is the possibility that ROS activate some other intracellular signaling molecule(s) functionally linked to phosphorylation of cytoskeletal proteins, with secondary recruitment of FAK to focal adhesion complexes.

Our findings related to neutrophils cocultured with mesangial cells may be relevant to those situations in which there is a significant inflammatory presence in the glomerulus. The release of ROS from "passenger" neutrophils could affect the behavior of constituent mesangial cells, depending on the extent of the neutrophil infiltrate. Small numbers of inflammatory cells might lead to functional activation (i.e., through stimulation of intracellular signaling), whereas, in the presence of a large cellular infiltrate, the large amount of ROS might simply cause cytotoxicity and loss of function.

Further experiments are necessary to determine if it is possible to extrapolate a possible physiological role in the intact glomerulus from the modulatory role of ROS in mesangial cell spreading and migratory behavior that we have observed in vitro.


    ACKNOWLEDGEMENTS

We thank Gary Song for help in preparing the manuscript and Martin Schwartz for helpful comments.


    FOOTNOTES

R. Zent is the recipient of a Fellowship from The Kidney Foundation of Canada. The Kidney Foundation of Canada and the Medical Research Council of Canada funded this work.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: M. Silverman, Room 7207, Medical Sciences Bldg., Univ. of Toronto, Toronto, Ontario, Canada M5S 1A8.

Received 27 April 1998; accepted in final form 22 October 1998.


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Am J Physiol Renal Physiol 276(2):F278-F287
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