Nitric oxide modulates mechanical strain-induced activation of p38 MAPK in mesangial cells

A. J. Ingram1, L. James2, K. Thai2, H. Ly2, L. Cai2, and J. W. Scholey2

1 Department of Medicine, McMaster University, Hamilton, Ontario L8N 1Y2; and 2 University of Toronto, Toronto, Ontario, Canada M55 1A8


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mesangial cells (MC), grown on extracellular matrix (ECM) protein-coated plates and stretched, proliferate and produce ECM, recapitulating in vivo responses to increased glomerular capillary pressure (Pgc). Transduction of strain involves mitogen-activated protein kinases (MAPK), and we have shown that p38 MAPK is activated by strain in MC. Because in vivo studies show that nitric oxide (NO) in the remnant kidney limits glomerular injury without reducing Pgc, we studied whether NO attenuated stretch-induced p38 activation in MC. Increasing p38 activation occurred with increasing stretch, maximally at 10 min at -27-kPa vacuum. Cyclic strain increased nuclear translocation of phosphorylated p38 by immunofluorescent microscopy and nuclear protein binding to nuclear factor-kappa B (NF-kappa B) consensus sequences by mobility shift assay. Both events were largely abrogated by the p38 inhibitor SB-203580. The NO donors 3-morpholinosydnonimine, S-nitroso-N-acetylpenicillamine, and 8-bromoguanosine 3',5'-cyclic monophosphate, a stable cGMP analog, prevented p38 activation and nulcear translocation. Thus strain induces p38 activity and translocation to the nucleus and p38-dependent increases in nuclear protein binding to NF-kappa B. This pathway is attenuated by the NO donors or a cGMP analog.

nuclear factor-kappa B; kidney glomerulus; physical forces; mitogen-activated protein kinases; signaling


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN THE GLOMERULUS, mesangial cells (MC) serve as architectural support for capillary loops and consequently experience pulsatile stretch-relaxation (1). Little resident glomerular cell proliferation or sclerosis is demonstrable in normal animals (14). However, models of glomerular sclerosis can be generated by maneuvers increasing intraglomerular pressure (Pgc) by 10 mmHg (4, 9, 10). Moreover, in these models, maneuvers that decrease Pgc attenuate sclerosis, further implicating mechanical stress in glomerular injury (2, 10, 24). Our laboratory and others have demonstrated protection against sclerosis and MC proliferation in remnant glomeruli by oral L-arginine supplementation to increase nitric oxide (NO) production (18, 29). L-arginine increases NO production by the remnant kidney and reduces glomerular endothelin-1 expression but does not lower glomerular capillary pressure (18).

The effects of mechanical forces on MC in vitro have been studied by culturing cells in wells with deformable bottoms and applying vacuum to the well to generate alternating cycles of strain and relaxation. Initial experiments using this methodology showed increases in cellular calcium entry and total protein kinase C (PKC) activity within 5 min of the application of strain to MC (1), followed by induction of mRNA for the protooncogene and activator protein-1 transcription factor component c-fos at 30 min (1). This effect was blocked by PKC inhibition (1). Subsequently, increases in both MC proliferation (14) and collagenous and noncollagenous extracellular matrix (ECM) protein synthesis were observed by 48 h, the sine qua non of sclerotic injury (30, 37).

Our laboratory and others have studied the link between early events such as PKC activation and induction of transcription of c-fos in stressed MC (15, 19, 21). We demonstrated increases in p44/42 and p38 mitogen-activated protein kinases (MAPK) signaling in response to cyclic strain (19). Other investigators have found p38 MAPK activation in response to mechanical stress in cardiac myocytes (22) and in isolated hearts perfused at high pressure (7). Transfection of the specific upstream activators of p38 MAPK at the MAP kinase kinase level (MKK3 and MKK6) led to cardiomyocyte hypertrophy and protection from apoptosis (34). Although nuclear factor-kappa B (NF-kappa B) transactivational activity has not been studied in response to mechanical strain, it is seen in response to p38 MAPK activation by constitutively active MKK6 (39), and in response to the traditional p38 MAPK stimuli of hyperosmolarity (31) and cytokines such as tumor necrosis factor (32). NF-kappa B increases the expression of genes such as nitric oxide synthase (NOS) (3) and cyclooxygenase 2 (COX-2) (11) in MC, which are implicated in responses to intraglomerular hypertension. The effects of NO on mechanical strain-induced MC signaling have not been elucidated.

We hypothesized that NO would limit MC signaling in response to mechanical strain. Accordingly, we first sought to further characterize p38 MAPK activation in response to cyclic mechanical strain in MC and to link p38 MAPK activation to increased nuclear protein binding to NF-kappa B consensus sequences. We then studied the effect of NO on mechanical strain-induced p38 MAPK activation and nuclear translocation.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Sprague-Dawley rat MC were cultured in DMEM supplemented with 20% FCS (GIBCO-BRL), streptomycin (100 µg/ml), penicillin (100 U/ml), and 2 mM glutamine at 37°C in 95% air-5% CO2. Experiments were carried out in cells between passages 15 and 20.

Application of strain-relaxation. MC (2 × 106/well) were plated on to six well plates with flexible bottoms coated with bovine type I collagen (Flexcell International, McKeesport, PA). Cells were grown to confluence for 72 h, then rendered quiescent by incubation for 24 h in DMEM with 0.5% FCS. To characterize the time of maximum response, cells were initially exposed to cycles of strain-relaxation, generated by a cyclic vacuum produced by a computer-driven system (Flexercell Strain Unit 2000, Flexcell), for periods of 2, 5, 10, 30, and 60 min. Plates were exposed to continuous cycles of strain-relaxation, with each cycle comprising 0.5 s of strain and 0.5 s of relaxation, for a total of 60 cycles/min. Initially, vacuum pressures used were -10 to -27 kPa, inducing a 16-27% elongation in the diameter of the surface. Subsequent experiments were performed at the time and strain level of maximal response, 10 min and -27 kPa, respectively.

Inhibition of p38 kinase activity by SB-203580 was tested by culturing MC on coated plates and then exposing them to the osmotic stimulus of 400 mM sorbitol for 30 min with 10, 50 or 100 µM SB-203580.

For the experiments studying NO effects, either 70 µM S-nitroso-N-acetylpenicillamine (SNAP), 100 µM and 1 mM 3-morpholinosydnonimine (SIN-1), or 1 mM 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) was added 10 min before the initiation of stretch protocols.

Protein isolation. Cellular levels of p38 MAPK protein were determined in stretched and unstretched control cells at the indicated times and vacuum levels after the application of strain. Briefly, at the end of each strain protocol, media was removed and the cells were washed once with ice-cold PBS. PBS was then removed, and cells were harvested under nondenaturing conditions on ice by incubation for 5 min with 70 ml ice-cold cell lysis buffer/well [(in mM) 20 Tris (pH 7.4), 150 NaCl, 1 EDTA, 1 EGTA, 2.5 Na pyrophosphate, beta -glycerophosphate, and 1 Na orthovanadate, as well as 1% Triton X-100 and 1 µg/ml leupeptin] and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were then scraped into microcentrifuge tubes on ice and sonicated four times for 5 s each. After microcentrifugation at 14,000 rpm for 10 min at 4°C, the supernatant was transferred to a fresh microcentrifuge tube. Protein concentration was measured with the Bio-Rad assay kit (Bio-Rad, Mississauga, ON).

Western blotting for p38 MAPK. Forty micrograms of sample were then separated on a 10% SDS-PAGE gel. After electroblotting to a nitrocellulose membrane (Protran, Schleicher and Schuell, Keene, NH), membranes were incubated for 3 h at room temperature with 25 ml of blocking buffer [1× Tris-buffered saline (TBS), 0.1% Tween-20 with 5% wt/vol nonfat dry milk] and then overnight at 4°C with p38 MAPK polyclonal antibody (1:1,000, New England Biolaboratories, Beverly, MA) in 10 ml of antibody dilution buffer (1× TBS, 0.05% Tween-20 with 5% BSA) with gentle rocking. Membranes were then washed three times with 1× TBS with 0.1% Tween-20 (TTBS) and then incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2,000) in 10 ml of blocking buffer for 45 min at room temperature. After three further TBS washes, the membrane was incubated with LumiGlo reagent (KPL, Gaithersburg, MD) and then exposed to X-ray film (X-OMAT, Kodak, Rochester NY).

p38 MAPK kinase assay. After protein isolation from total cell lysate as above, 200 µg total protein were then incubated with phospho-p38 MAPK antibody (Thr180/Tyr182) immobilized on protein sepharose A beads (1:100; New England Biolaboratories) with gentle rocking overnight at 4°C. Lysate was then microcentrifuged for 30 s at 14,000 rpm to recover the beads, and the pellet was washed twice with 0.5 ml of 1× lysis buffer.

For the kinase assay, after immunoprecipitation, pellets were washed twice with 0.5 ml kinase buffer [(in mM) 25 Tris, 5 beta -glycerophosphate, 2 dithiothreitol (DTT), 0.1 Na orthovanadate, and 10 MgCl2], and the pellet was resuspended in 50 µl 1× kinase buffer supplemented with 200 µM ATP. p38 MAPK activity was then determined by adding 2 µg transcription factor ATF-2 fusion protein as substrate. After incubation for 30 min at 30°C, the reaction was terminated with 25 µM 3× SDS sample buffer [187.5 mM Tris · HCl (pH 6.8), 6% wt/vol SDS, 30% glycerol, 150 mM DTT, 0.3% wt/vol bromphenol blue], and the mixture was boiled for 5 min, vortexed, and then microcentrifuged for 2 min. Twenty microliters of sample were then run on an SDS-PAGE gel. After blotting to nitrocellulose, membranes were incubated for 3 h at room temperature with 25 ml of blocking buffer (1× TBS, 0.1% Tween-20 with 5% wt/vol nonfat dry milk) and then overnight at 4°C with phospho-specific anti-ATF-2 (Thr71) antibody 1:1,000 in 10 ml of antibody dilution buffer (1× TBS, 0.05% Tween-20 with 5% BSA). Gels were washed three times with TTBS and then incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2,000) for 1 h at room temperature. After three further TBS washes, the membrane was incubated with LumiGlo reagent (KPL) and then exposed to X-ray film (X-OMAT, Kodak).

Immunofluorescent microscopy of p38 MAPK translocation. After each strain protocol, cells were washed three times with PBS and fixed with 3.7% formaldehyde (300 µl/well) for 10 min at room temperature. Cells were washed three times with PBS and then permeabilized in 100% methanol for 5 min at -20°C, washed again with PBS, and incubated with anti-phospho-p38 MAPK (Thr180/Tyr182; New England Biolaboratories) 1:25 in PBS (after the primary antibody had been centrifuged at 12,000 rpm for 2 min and the supernatant was recovered) for 30 min at room temperature. Cells were washed three times in PBS and incubated with a secondary goat anti-rabbit green fluorescent-conjugated antibody (Alexis Biochemicals) 1:50 in PBS for 30 min at room temperature in the dark. Cells were washed and then mounted by removing the flexible base from each well and placing it directly on a glass slide, using one drop of antifade mount media (Slow Fade, Molecular Probes, Eugene, OR). A drop of mount media was then placed on top of the cells, which were then covered with a glass slip. Slides were stored at 4°C in the dark until confocal laser scanning microscopy was performed by using a Bio-Rad MRC-600 confocal microscope (Bio-Rad) within 10 days. To demonstrate phospho-p38 MAPK translocation in response to osmotic stress, MC were exposed to 400 mM sorbitol for 30 min and then were analyzed as above.

Nuclear protein binding to NF-kappa B consensus sequences. These experiments were performed according to published methods (23, 26). After washing in cold PBS, nuclear extracts of MC stretched at -27 kPa for 10 min were prepared by lysis in hypotonic buffer [(in mM) 20 HEPES, pH 7.9, 1 EDTA, 1 EGTA, 20 NaF, 1 Na3VO4, 1 Na4P2O7, 1 DTT, and 0.5 PMSF, as well as 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 0.6% Nonidet P-40], homogenized, and sedimented at 16,000 g for 20 min at 4°C. Pelleted nuclei were resuspended in hypertonic buffer with 0.42 mol/l of NaCl2 and 20% glycerol and rotated for 30 min at 4°C. After centrifugation for 20 min at 16,000 g, the supernatant was collected and protein concentration was measured with the Bio-Rad assay kit.

Double-stranded NF-kappa B consensus oligonucleotides were prepared by incubating 2 µl consensus oligonucleotide (sequences 5' AGT TGA GGG GAC TTT CCC AGG C 3' and 3' TCA ACT CCC CTG AAA GGG TCC G 5'; 1.75 pmol/ul; Promega, Madison, WI); 1 µl T4 polynucleotide kinase 10× buffer; 1 µl [gamma -32P]ATP (3,000 Ci/ml; DuPont, Boston, MA); and 5 µl nuclease-free water for 10 min at 37°C. The reaction was stopped by adding 1 µl of 0.5 M EDTA. Unlabeled [32P]ATP was removed from the oligonucleotide mixture with D-25 Sephadex columns (Clontech Laboratories, Palo Alto, CA).

The supernatants were used as nuclear proteins for the binding assay. Ten micrograms of nuclear proteins were incubated with 2 µg of poly(dI-dC) · poly(dI-dC) to block nonspecific protein-DNA binding (Pharmacia, Uppsala, Sweden) in binding buffer (20 mM HEPES, pH 7.9, 1.8 mM MgCl2, 2 mM DTT, 0.5 EDTA, 0.5 mg/ml BSA), incubated for 30 min, at room temperature, and then reacted with radiolabeled consensus oligonucleotides at room temperature for 20 min [50,000-100,000 counts/min (cpm)]. Unincorporated nucleotides were removed by using a Chroma Spin-10 column (Clontech Laboratories). Reaction mixtures were electrophoresed in a 6% polyacrylamide gel and autoradiographed. Competition experiments were performed by using 100× excess unlabeled NF-kappa B consensus oligonucleotides. To demonstrate the role of p38 MAPK activity in NF-kappa B induction, 50 µM SB-203580 was added 1 h before the strain protocols in one-half of the experiments.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Characterization of strain-induced p38 MAPK activity in MC. The first purpose of the present study was to determine the time and magnitude of strain dependence of p38 MAPK activation in MC. MC lysates were initially subjected to Western blot analysis of p38 MAPK expression, and subsequently analysis of p38 MAPK activity was performed by determination of the phosphorylation of an ATF-2 fusion protein by MC lysates. Cultures were exposed to strain for periods of 2, 5, 10 , 30, and 60 min at vacuum levels of -10, -14, -18, -22, and -27 kPa. Unstretched MC on identical plates were used as the control. Representative autoradiographs are shown in Figs. 1 and 2. In Fig. 1 the time course was explored as MC were stretched at -27 kPa for the times indicated above. No changes in the protein expression of p38 MAPK were observed (top). However, changes were seen in p38 MAPK activity at 2 min, reaching a maximum at 10 min and returning to baseline by 30 min (middle). The data from four consecutive separate experiments are graphically presented with SD bars (bottom). Activity at 5 and 10 min was different from unstretched baseline (P < 0.05, n = 4). In Fig. 2, dependence on the magnitude of strain was studied, as MC were exposed to the levels of vacuum outlined above for 10 min. Again, no changes in the protein expression of p38 MAPK were observed (top). In contrast, activity is increased at all strain levels and increases with increasing stretch (middle). The data from four consecutive experiments are graphically presented with SD bars, and activity at all levels of strain is different from baseline (P < 0.05, n = 4 experiments; bottom).


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Fig. 1.   Time course of p38 mitogen-activated protein kinases (MAPK) activation by mechanical strain. Mesangial cells were exposed to cyclic mechanical strain (-27 kPa, 60 Hz) for the times indicated. At the end of each time point, protein was isolated, electrophoresed, blotted to a nitrocellulose membrane, probed overnight with p38 MAPK polyclonal antibody, and visualized with a horseradish peroxidase-conjugated secondary antibody. Top: a representative Western blot. No change in amount of p38 MAPK protein present was observed at any time point. Middle: in contrast, p38 MAPK activity as measured by the phosphorylation of an ATF-2 target after immunoprecipitation of p38 MAPK was increased by cyclic strain, with maximal levels reached at 10 min. The positive control is 400 mM sorbitol for 30 min. Bottom: data from 4 separate experiments shown graphically. Error bars, SD. The 5- and 10-min time points are significantly different from control (P < 0.05, n = 4).



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Fig. 2.   Effect of strain magnitude on p38 MAPK activation. Mesangial cells were exposed to cyclic mechanical strain (60 Hz) for 10 min at the vacuum levels indicated. Protein was isolated, electrophoresed, blotted to a nitrocellulose membrane, probed overnight with p38 MAPK polyclonal antibody, and visualized with a horseradish peroxidase-conjugated secondary antibody. Top: a representative Western blot. No change in amount of p38 MAPK protein present was observed at any time point. Middle: in contrast, p38 MAPK activity as measured by the phosphorylation of an ATF-2 target after immunoprecipitation of p38 MAPK was increased by all levels of cyclic strain, with maximal levels reached at -27kPa. The positive control is 400 mM sorbitol for 30 min. Bottom: the data from 4 separate experiments shown graphically. Error bars, SD. All levels of strain are significantly different from control (P < 0.05, n = 4).

Inhibition by SB-203580. To determine the concentration of SB-203580 required to inhibit p38 MAPK activity, we used 400 mM sorbitol, an osmotic stimulus for p38 MAPK activation. Figure 3 shows markedly increased p38 MAPK activity after 10 min of 400 mM sorbitol was added to MC grown on flexible-bottom coated plates. Inhibition was seen with SB-203580 concentrations of 50 µM and above. Consequently, the concentration of 50 µM was used for subsequent experiments.


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Fig. 3.   Inhibition of p38 MAPK activity with SB-203580. Mesangial cells were exposed to 10 min of 400 mM sorbitol (Sorb) on flexible-bottom coated plates with varying concentrations of SB-203580 as shown. Protein was isolated, electrophoresed, blotted to a nitrocellulose membrane, probed overnight with p38 MAPK polyclonal antibody, and visualized with a horseradish peroxidase-conjugated secondary antibody. Inhibition was seen with SB-203580 concentrations of 50 µM and above. The densitometry shows the mean values from 3 separate experiments.

Immunofluorescent microscopy of p38 MAPK translocation. The nuclear translocation of active (phosphorylated) p38 MAPK induced by strain was studied by confocal microscopy, and representative photomicrographs are shown in Fig. 4. As shown in Fig. 4A, little phosphorylated p38 MAPK is identified by the primary antibody in unstretched MC, and the phospho-p38 MAPK present is primarily cytoplasmic in location. The addition of 400 mM sorbitol for 30 min to MC as an osmotic stress led to prompt nuclear translocation of phospho-p38 MAPK (B). Stretched MC (10 min, 60 Hz, -27 kPa) grown on type I collagen-coated plates but pretreated with 50 µM SB-203580 also show little phospho-p38 translocation (C). However, prompt induction and nuclear translocation of phospho-p38 MAPK are shown in response to 10 min of cyclic strain of -27kPa in the absence of inhibitor (D).


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Fig. 4.   Immunofluorescent microscopy of strain-induced p38 MAPK activation. Mesangial cells were grown on coated plates and exposed to sorbitol (400 mM) for 30 min or cyclic mechanical strain (-27 kPa, 60 Hz) for 10 min with and without SB-203580. After each protocol, cells were washed, permeabilized, and then incubated with anti-phospho-p38 (Thr180/Tyr182) for 30 min. Cells were then washed and incubated with a secondary goat anti-rabbit green fluorescent-conjugated antibody for 30 min. Cells were then washed, antifade mount media was added, and confocal laser scanning microscopy was performed by using a Bio-Rad MRC-600 confocal microscope. A: in unstretched mesangial cell. Little phosphorylated p38 MAPK is identified by the primary antibody, and it is primarily cytoplasmic in location. B: the addition of 400 mM sorbitol for 30 min to mesangial cells as an osmotic stress leads to prompt nuclear translocation of phospho-p38 MAPK. C: pretreatment of stretched meangical cells (10 min, 60 Hz, -27 kPa) with 50 µM SB-203580 results in little phospho-p38 translocation. D: prompt induction and nuclear translocation of phospho-p38 MAPK in response to 10 min of cyclic strain of -27 kPa in the absence of inhibitor.

Nuclear protein binding to NF-kappa B consensus sequences. We next sought to determine whether downstream intranuclear events were affected by strain and the consequent activation of p38 MAPK by assessing the binding of nuclear protein to NF-kappa B consensus sequences. Binding was studied from the time of maximal response (10 min) and at 30 and 60 min of strain to allow for downstream propagation of the signal. SB-203580 (50 µM) was added 10 min before strain protocols in one-half of the experiments.

Figure 5 shows a representative autoradiograph of four consecutive experiments of nuclear protein binding to NF-kappa B consensus sequence results. Retardation was maximal at 10 min, with decay seen through to 1 h, although binding remained increased over unstretched cells at all three time points. Addition of the p38 MAPK inhibitor SB-203580 essentially abrogated any retardation seen in response to strain, indicating that p38 MAPK activation is a necessary step in cellular signaling, leading to increased nuclear protein binding and consequent retardation of label seen in stretched MC. Specificity of the assay was confirmed by successful complete competition off of label by excess cold NF-kappa B sequences.


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Fig. 5.   Nuclear protein binding to nuclear factor-kappa B (NF-kappa B) consensus sequences. Mesangial cells were exposed to cyclic mechanical strain (-27 kPa, 60 Hz) for the times indicated. NF-kappa B gel shift assay (EMSA) was performed with isolated nuclear protein. Nuclear proteins were incubated with poly(dI-dC) · poly(dI-dC) and then reacted with radiolabeled consensus NF-kappa B oligonucleotides, electrophoresed, and autoradiographed. Retardation of the label, indicating binding of nuclear protein, was seen in stretched cells, maximally at 10 min. Addition of SB 203580 largely prevented retardation of the label, indicating p38 dependence of the mechanical signal. No retardation of radiolabel was seen in the absence of nuclear protein (neg). Specificity was ensured with competition experiments by using excess unlabeled NF-kappa B consensus oligonucleotide, which revealed no retardation of the label (extreme right lane). The autoradiograph is typical of 4 separate experiments. Two bands of retarded label are generally observed (26).

Effect of SNAP, SIN-1, and cGMP on strain-induced p38 MAPK signaling. To study the effect of NO on p38 MAPK activation in response to strain, either 70 µM SNAP, 100 µM and 1 mM SIN-1, or 1 mM 8-BrcGMP was added 10 min before the initiation of stretch protocols. Figure 6 shows that the increase in p38 MAPK activity seen with 10 min of cyclic strain could be completely inhibited with 70 µM SNAP (middle). The data from four consecutive separate experiments are graphically presented with SD bars (bottom). The inhibition at 10 min by SNAP was significant (P < 0.05, n = 4). Figure 7 shows that the increase in p38 MAPK activity seen with 10 min of cyclic strain could also be completely inhibited with another NO donor, SIN-1, and that the inhibition was independent of dose. Inhibition with 100 µM SIN-1 (top) and 1 mM SIN-1 (bottom) is shown. The data from four consecutive separate experiments at each dose are graphically presented with SD bars (bottom). The inhibition at 10 min by either 100 µM or 1 mM SIN-1 was significant (P < 0.05, n = 4). Figure 8 shows that the effect of the NO donors can be reproduced, in part, by a stable cGMP analog, as 8-BrcGMP also significantly inhibited the p38 MAPK activity seen at 10 min in response to strain (middle). Inhibition was not complete, however, as with SNAP or SIN-1. The data from four consecutive separate experiments are graphically presented with SD bars (bottom). The inhibition at 10 min by 8-BrcGMP was significant (P < 0.05, n = 4).


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Fig. 6.   Effect of S-nitroso-N-acetylpenicillamine (SNAP) on strain-induced p38 MAPK signaling. To study the effect of nitric oxide on p38 MAPK activation in response to strain, 70 µM SNAP was added 10 min before the initiation of stretch protocols. At the end of each time point, protein was isolated, electrophoresed, blotted to a nitrocellulose membrane, probed overnight with p38 MAPK polyclonal antibody, and visualized with a horseradish peroxidase- conjugated secondary antibody. A: a representative Western blot. No change in amount of p38 MAPK protein present was observed at any time point. B: p38 MAPK activity as measured by the phosphorylation of an ATF-2 target after immunoprecipitation of p38 MAPK was increased by cyclic strain at 10 min. Preincubation with SNAP completely prevented this increase. The positive control is 400 mM sorbitol for 30 min. C: the data from 4 separate experiments shown graphically. Error bars, SD. The 10-min time point with SNAP is significantly different from the 10-min control (P < 0.05, n = 4).



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Fig. 7.   Effect of 3-morpholinosydnonimine (SIN-1) on strain-induced p38MAPK signaling. To further study the effect of nitric oxide on p38 MAPK activation in response to strain, 100 µM or 1 mM SIN-1 was added 10 min before the initiation of stretch protocols. At the end of each time point, protein was isolated, electrophoresed, blotted to a nitrocellulose membrane, probed overnight with p38 MAPK polyclonal antibody, and visualized with a horseradish peroxidase-conjugated secondary antibody. Top: representative Western blots. No changes in the amount of p38 MAPK protein present were observed at any time point. Middle: p38 MAPK activity as measured by the phosphorylation of an ATF-2 target after immunoprecipitation of p38 MAPK was increased by cyclic strain at 10 min. Preincubation with SIN-1 at either 100 µM (top) or 1 mM (bottom) completely prevented this increase. The positive control is 400 mM sorbitol for 30 min. Bottom: the data from 4 separate experiments shown graphically for each SIN-1 dose. Error bars, SD. The 10-min time points with SIN-1 at either 100 µM or 1 mM are significantly different from the 10-min control (P < 0.05, n = 4).



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Fig. 8.   Effect of 8-bromo-cGMP (8-BrcGMP) on strain-induced p38 MAPK signaling. To determine whether the effect of nitric oxide on p38 MAPK activation in response to strain was dependent on the generation of cGMP, 1 mM 8-BrcGMP, a stable analog, was added 10 min before the initiation of stretch protocols. At the end of each time point, protein was isolated, electrophoresed, blotted to a nitrocellulose membrane, probed overnight with p38 MAPK polyclonal antibody, and visualized with a horseradish peroxidase-conjugated secondary antibody. A: a representative Western blot. No change in amount of p38 MAPK protein present was observed at any time point. B: p38 MAPK activity as measured by the phosphorylation of an ATF-2 target after immunoprecipitation of p38 MAPK was increased by cyclic strain at 10 min. Preincubation with 8-BrcGMP partly prevented this increase. The positive control is 400 mM sorbitol for 30 min. C: the data from 4 separate experiments shown graphically. Error bars, SD. The 10-min time point with 8-BrcGMP is significantly different from the 10-min control (P < 0.05, n = 4).

Concordant with these results, both SNAP and 8-BrcGMP prevented nuclear translocation of phospho-p38 MAPK in response to strain by immunofluorescent microscopy. Figure 9A shows unstretched MC grown on coated plates. After application of 10-min cyclic strain, nuclear translocation is seen (B). Both SNAP (C) and 8-BrcGMP (D) prevented this. Less staining was seen after 30-min stretch (E). There was no staining in the absence of primary antibody (F).


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Fig. 9.   Immunofluorescent microscopy of effect of SNAP and 8-BrcGMP on p38 MAPK translocation. Mesangial cells were grown on coated plates and exposed to cyclic mechanical strain (-27 kPa, 60 Hz) for 10 min with and without 70 µM SNAP or 1 mM 8-BrcGMP. After each protocol, cells were washed, permeabilized, and then incubated with anti- phospho-p38 (Thr180/Tyr182) for 30 min. Cells were then washed and incubated with a secondary goat anti-rabbit green fluorescent-conjugated antibody for 30 min. Cells were then washed, antifade mount media was added, and confocal laser scanning microscopy was performed by using a Bio-Rad MRC-600 confocal microscope. A: in unstretched mesangial cells, some phosphorylated p38 MAPK is identified by the primary antibody, which is primarily cytoplasmic in location. B: prompt nuclear translocation of phospho-p38 MAPK is seen in response to 10-min cyclic strain (60 Hz, -27 kPa). C: pretreatment of stretched mesangial cells with SNAP prevents phospho-p38 MAPK translocation. D: pretreatment of stretched mesangial cells with 8-BrcGMP prevents phospho-p38 MAPK translocation. E: much less nuclear phospho-p38 MAPK staining is seen after 30-min stretch. F: no staining is seen in the absence of primary antibody.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Considerable evidence supports the conclusion that increased physical force (which may be as little as a 20% increase in capillary pressure) triggers MC responses that ultimately contribute to the development of glomerulosclerosis (30, 37, 38) In vitro studies of the application of cyclic mechanical strain to MC have demonstrated that this stimulus results in the production of collagenous proteins (14) and fibronectin (15). MC proliferation has also been observed in response to strain (13, 14, 20).

Our laboratory (19) and others have studied how the mechanical signal is transduced in MC. The first site of transduction occurs at the cell membrane. Initial studies of mechanical strain in MC generally used increased expression of the protooncogene c-fos to study manipulations that might ultimately impact ECM protein synthesis and cell proliferation (1). Downregulation of PKC was shown to attenuate the c-fos expression induced by strain (13), as was calcium chelation by EGTA (13). Other membrane-associated events have also been implicated. For example, in MC, strain-induced transforming growth factor-beta expression is tyrosine kinase dependent (15). The proliferative effects of mechanical strain are to some extent matrix dependent. Cells adherent to fibronectin show the greatest strain-induced proliferative response, and this is inhibited by coincubation with RGD peptides (35). Integrin-focal adhesion complex interactions have been studied, and tyrosine phosphorylation of the focal adhesion associated kinase pp125FAK is seen in stretched MC (12).

Signaling of mechanical stimuli to the cell nucleus after membrane events involves the ubiquitous MAP kinase cascades. Each of the MAP kinase cascades consists of three protein kinases acting sequentially: a MKK, a MAP kinase activator, and a MAP kinase (8).

Our laboratory has reported that cyclic mechanical strain increases p38 MAPK activation and [3H]thymidine uptake in primary cultured MC adherent to collagen I (19). The first goal of the present study was to more fully characterize the time course of p38 MAPK activation and the dependence of p38 MAPK activation on the magnitude of stretch in MC exposed to cyclic strain. Cyclic strain, at a frequency of 60 Hz, led to an earlier increase in p38 MAPK activation than we originally reported. Basal p38 MAPK activity was low in static MC, but an increase in p38 MAPK activity was evident as early as 2 min after the application of strain, and peaked at 10 min. We also observed that p38 MAPK activity was dependent on the magnitude of stretch. Stretch-induced activation of p38 MAPK was accompanied by translocation of phospho-p38 MAPK into MC nuclei. Although the classic stimuli for p38 MAPK activation include hyperosmolality (19) and proinflammatory cytokines, such as interleukin-1beta (16), a link between a physical force and cellular p38 MAPK activation has also been reported in cardiac myocytes after aortic constriction (34). When cardiac myocytes are exposed to strain in vitro, both extracellular signal-regulated kinase (ERK) and p38 MAPK activation have also been observed (22). More recently, King and co-workers (17) have reported that high glucose concentrations also activate p38 MAPK in MC. Mechanical strain, therefore, appears to activate what has heretofore been considered an "inflammation" pathway in cardiac myocytes and MC.

Because p38 MAPK has been implicated in the signaling pathways of proinflammatory cytokines and the cytokine-induced nuclear factor NF-kappa B, the second goal of the present study was to determine whether cyclic mechanical strain led to a p38 MAPK- dependent increase in nuclear protein binding to NF-kappa B in MC. We observed that cyclic stretch increased nuclear protein binding to NF-kappa B in MC, and the increase was largely abrogated by inhibiting p38 MAPK activity with SB-203580. Although we did not link nuclear protein binding to NF-kappa B to transcriptional activation in our study, Akai and co-workers (1) have reported that cyclic stretch-relaxation induces COX-2 synthesis. COX-2 expression is regulated transcriptionally by NF-kappa B (3, 36), and it is tempting to speculate that mechanical strain induces COX-2 expression via p38 MAPK activation and NF-kappa B.

Our laboratory (18) and others (29) have shown that dietary supplementation with L-arginine, a NO donor, limits glomerular injury in the remnant kidney. L-arginine increases NO production by the remnant kidney but does not lower glomerular capillary pressure. Therefore, the third goal of the present study was to determine whether a NO donor would attenuate stretch-induced MC signaling, and specifically, whether NO would limit stretch-induced p38 MAPK activation. We first observed that the NO donor SNAP attenuated stretch-induced p38 MAPK activation in MC. Because NO can influence the activity of intracellular proteins by activating guanylate cyclase and increasing cGMP or by increasing the posttranslational modification of proteins via S-nitrosylation, we compared the effect of the NO donor SNAP to the effect of an active but stable cGMP analog, 8-BrcGMP, on stretch-induced p38 MAPK activation. Like SNAP, 8-BrcGMP also attenuated the activation of p38 MAPK by stretch in MC. Moreover, both SNAP and 8-BrcGMP prevented the nuclear translocation of phospho-p38 MAPK.

NO may be generated by constitutive enzymes [such as endothelial NOS (eNOS)] or by enzymes induced by inflammatory events [inducible NOS (iNOS)] (25). The concentration of NO donors that corresponds to levels of NO in vivo produced by each of these enzymes is uncertain. Chiu and co-workers (6) have found that SNAP at either 100 or 400 µM inhibits shear stress-induced p44/42 ERK activity in endothelia. A NOS inhibitor increased shear-induced p44/42 activity. As shear should not induce iNOS, it was postulated that eNOS events were being modeled (6). Studies of MC have generally used 1 mM concentrations of NO donors to mimic iNOS effects (5, 28). Accordingly, we used another NO donor, SIN-1, to ensure that p38 MAPK inhibition was not specific to SNAP or dose dependent. We observed that SIN-1 at either 100 µM or 1 mM also abrogated stretch-induced p38 MAPK activity.

We did not determine the mechanism responsible for the effect of NO on stretch-induced p38 MAPK activation, but interestingly, NO provided as SNAP inhibits the angiotensin II induction of ERK signaling by preventing the calcium-sensitive phosphorylation of proline-rich tyrosine kinase 2 (PYK2) (33). The activation of p38 MAPK by osmotic stress (sorbitol) has very recently been shown to be PYK2 dependent (27). Because PYK2 serves to phosphorylate pp125FAK in response to integrin-mediated signaling, it is quite plausible that p38 MAPK activation in response to mechanical strain and the inhibition by NO donors is mediated at this level. Further studies will be necessary to test this hypothesis.

In conclusion, these studies reveal cross-talk between mechanical strain-induced MC signaling and NO signaling and suggest parallels between mechanical strain and cytokine signaling. Cyclic strain leads to a rapid activation of p38 MAPK in MC. This activation is accompanied by translocation of phospho-p38 MAPK to the MC nucleus and a p38 MAPK-dependent increase in NF-kappa B binding, thus linking physical forces to the regulation of gene expression usually implicated in inflammation. This signaling pathway can be inhibited by NO donors and a stable cGMP analog.


    ACKNOWLEDGEMENTS

This work was supported by a grant from the Heart and Stroke Foundation of Canada and the Juvenile Diabetes Foundation/Medical Research Council of Canada (to J. W. Scholey) and from the Kidney Foundation of Canada (to A. J. Ingram). J. W. Scholey was also supported by a Scholarship Award from the Pharmaceutical Manufacturers' Association of Canada/Medical Research Council.


    FOOTNOTES

Address for reprint requests and other correspondence: A. J. Ingram, 500-25 Charlton Ave. East, Hamilton, Ontario, Canada L8N 1Y2 (E-mail: ingrama{at}fhs.mcmaster.ca).

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.

Received 13 September 1999; accepted in final form 29 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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