5-HT2A receptors stimulate mitogen-activated protein kinase via H2O2 generation in rat renal mesangial cells

Eddie L. Greene, Odette Houghton, Georgiann Collinsworth, Maria N. Garnovskaya, Toshio Nagai, Tahir Sajjad, Venugopala Bheemanathini, Jasjit S. Grewal, Richard V. Paul, and John R. Raymond

Nephrology Division, Department of Internal Medicine, Medical University of South Carolina, and Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29425


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Serotonin (5-HT) stimulates mitogenesis in rat renal mesangial cells through a G protein-coupled 5-HT2A receptor. We tested the hypothesis that oxidants might be involved in the signal transduction pathway linking the receptor to extracellular signal-regulated protein kinase (ERK). 5-HT rapidly increased the activity and phosphorylation of ERK. These effects were blocked by the 5-HT2A receptor antagonist ketanserin. The peak effect was noted at 5-10 min, and half-maximal stimulation was achieved at 10-30 nM 5-HT. Chemical inhibitor and activator studies supported the involvement of phospholipase C, protein kinase C (PKC), and reactive oxygen species (ROS, i.e., H2O2 and superoxide) generated by an NAD(P)H oxidase-like enzyme in the ERK activation cascade. Mapping studies supported a location for the NAD(P)H oxidase enzyme and the ROS downstream from PKC. Our studies are most consistent with an ERK activation pathway as follows: 5-HT2A receptor right-arrow Gq protein right-arrow phospholipase C right-arrow diacylglycerol right-arrow classical PKC right-arrow NAD(P)H oxidase right-arrow superoxide right-arrow superoxide dismutase right-arrow H2O2 right-arrow mitogen-activated extracellular signal-regulated kinase right-arrow ERK. These studies demonstrate a role for the 5-HT2A receptor in rapid, potent, and efficacious activation of ERK in rat renal mesangial cells. They support a role for oxidants in conveying the stimulatory signal from 5-HT, because 1) chemical antioxidants attenuate the 5-HT signal, 2) oxidants and 5-HT selectively activate ERK to a similar degree, 3) 5-HT produces superoxide and H2O2 in these cells, and 4) a specific enzyme [NAD(P)H oxidase] has been implicated as the source of the ROS, which react selectively downstream of classical PKC.

serotonin receptor; kidney; signal transduction; reactive oxygen species; NADP(H) oxidase


    INTRODUCTION
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INTRODUCTION
MATERIALS AND METHODS
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THE RENAL GLOMERULUS IS COMPOSED of several cell types, including endothelial cells, epithelial cells, and mesangial cells. Because they are located between the vascular and urine spaces, renal mesangial cells can be influenced by many vascular substances. Some of those substances, such as ANG II and arginine vasopressin (4, 7), glucose (5), thromboxane (37), interleukin-1 (43), and serotonin (5-HT) (1, 22, 40), can activate mesangial cell growth and may also participate in transcription cascades that lead to glomerulosclerosis.

One likely possibility for conveying the mitogenic signal is the group of kinases termed the mitogen-activated protein kinases (MAPK), particularly the subfamily termed the extracellular signal-regulated protein kinases (ERK). These kinases are activated by many mitogenic stimuli, including tyrosine kinase growth factor receptors and G protein-coupled receptors (42). Many of the intermediary signaling molecules that reside between the receptors and ERK have been identified, and they include a variety of lipid kinases, several protein tyrosine kinases, low-molecular-weight G proteins, and protein kinase C (PKC). Other accessory signaling molecules, including adapters such as Grb2, docking molecules such as Shc and IRS-1, and G protein-activating molecules such as Sos, are involved in conveying the growth signal to the ERK molecules (14). Yet, many important signaling intermediaries have not been identified or completely characterized. New evidence has implicated reactive oxygen species (ROS) as possible intermediates in activating ERK in many different cell types (3, 10, 14, 25, 32, 39). In cultured human glomerular mesangial cells, oxidants appear to be critical in mediating the interleukin-1 receptor-mediated activation of MAPKs (43). The interleukin-1 receptors are thought to convey their signals primarily through tyrosine phosphorylation reactions independent of heterotrimeric G proteins, but information on the role of oxidants in G protein-coupled receptor activation of ERK in mesangial cells is lacking. We hypothesized that G protein-coupled receptors might also stimulate mesangial cell ERK through pathways requiring the generation of ROS. For the present studies we examined the effects of 5-HT on ERK activity in mesangial cells. The 5-HT2A receptor expressed in mesangial cells is a prototypical receptor that couples to the Galpha q/11 family of heterotrimeric G proteins (16, 19, 27, 28). We recently showed that 5-HT increases the levels of transforming growth factor-beta mRNA and protein in mesangial cells and that those increases are mediated through a signal transduction pathway that requires mitogen-activated extracellular signal-regulated kinase (MEK, the kinase that phosphorylates and activates ERK) and the generation of ROS (19). Therefore, the present studies were performed to establish that 5-HT activates an ERK subtype in rat glomerular mesangial cells and, furthermore, to explore a potential role for ROS in transmitting the signal from 5-HT to the ERK molecules.


    MATERIALS AND METHODS
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Materials. Drugs and reagents were obtained from the following sources: A-23187, arsenite, buthionine sulfoximine (BSO), diamide, 5-HT, H2O2, alpha -lipoic acid (reduced form), 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF-109203X), myelin basic protein, N-acetylcysteine (NAC), pertussis toxin, phorbol 12-myristate 13-acetate (PMA), cytochrome c, and tert-butyl hydroperoxide from Sigma Chemical (St. Louis, MO); 1-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3), 1-(6-(17beta -3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl-1H pyrrole-2,5-dione (U-73122) and 1-(6-(17beta -3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl-2,5 pyrrilodine-dione (U-73343) from Biomol (Plymouth Meeting, PA); PD-98059 from Calbiochem (San Diego, CA); 2',7'-dichlorofluorescin diacetate (DCF-DA) from Molecular Probes (Eugene, OR); 1-(5-isoquinolinesulfonyl)-2-methylpeperazine and thapsigargin from LC Laboratories (Woburn, MA); and [alpha -32P]ATP from DuPont-NEN (Boston, MA). Cell culture media, serum, and antibiotics were obtained from GIBCO-BRL (Gaithersburg, MD) and culture flasks from Costar (Cambridge, MA). The phospho-MAPK kits were obtained from New England Biolabs (Beverly, MA).

Isolation and primary culture of rat glomerular mesangial cells. Mesangial cells were obtained from cortical sections of kidneys from young 100- to 150-g Sprague-Dawley rats by use of a standard sieving technique (30). Cells were incubated at 37°C in a humidified atmosphere of 95% air-5% CO2 and subcultured every 1-2 wk by trypsinization until pure cultures of mesangial cells were obtained. They were plated at a density of 2-5 × 104 cells/ml in RPMI medium supplemented with 20% FCS and antibiotics (100 U/ml of penicillin and 100 µg/ml of streptomycin). At 48 h before studies, cells were placed in serum-free RPMI medium supplemented with antibiotics. Cells from passages 5-16 were used.

ERK assays. ERK activity was measured in immune complexes with myelin basic protein as the substrate (17). For most experiments, ERK phosphorylation was used as a surrogate for kinase activity. ERK phosphorylation was assessed using a phosphorylation state-specific ERK antibody (New England Biolabs) that specifically recognizes tyrosine-204-phosphorylated (but not nonphosphorylated) ERK1 and ERK2 and does not react with closely related p38 MAPK or Jun kinases or stress-activated protein kinases (JNK/SAPKs). The phospho-ERK antibody was used at 1:1,000 dilution, whereas the control antibody, which recognizes equally well the phosphorylated and nonphosphorylated ERK, was used at 1:500 dilution per the manufacturer's recommendations. Blotting and visualization were carried out as previously described (17).

Measurement of superoxide anion production. Superoxide anion (O-2·) production was quantified by the cytochrome c reduction assay (31) with modifications. Briefly, cells were grown to 60-80% confluency in six-well culture plates and starved in serum- and phenol red-free medium for 48 h. The cells were further incubated in 1 ml of serum- and phenol red-free medium containing 200 µM cytochrome c and 1 µM 5-HT or 0.5 µM PMA in the presence or absence of 300 U/ml of superoxide dismutase (SOD) for 60 min at 37°C in a humidified incubator with 5% CO2. Cells were pretreated with inhibitors [50 µM diphenyleneiodonium (DPI) or 2 µM GF-109203X] for 30 min before application of 5-HT or PMA. Absorbance of the cell-free supernatant was measured spectrophotometrically at 550 nm. The following equation was used to determine O-2· produced in picomoles
O<SUP>−</SUP><SUB>2</SUB> ⋅ pmol/10<SUP>6</SUP> cells = 0.001 × [<IT>A</IT><SUB>550</SUB>(without SOD) − <IT>A</IT><SUB>550</SUB>(with SOD)] × 47.6

Measurement of intracellular H2O2 generation. The H2O2-sensitive fluorescent probe DCF-DA was used to assess the generation of intracellular H2O2 (8, 33). Nonfluorescent DCF-DA diffuses through the plasma membrane, where it is subsequently deacetylated enzymatically by cellular esterases to the polar compound 2',7'-dichlorofluorescein (DCF), which remains trapped in the cell and fluoresces in the presence of intracellular peroxides (H2O2 and lipid hydroperoxides). Cells in monolayer were incubated with Earle's balanced salt solution supplemented with 10 µM DCF-DA and 1% BSA (wt/vol) for 30 min at 37°C. The supernatant was removed and replaced with fresh unsupplemented Earle's solution before stimulation with 5-HT, which was added from a 1,000× stock directly to the Earle's solution before analysis. Relative fluorescence intensity and fluorescent images were obtained over time (0.5-20 min) by laser confocal scanning microscopy (LSMGB-200, Olympus Optical, Tokyo, Japan) at an excitation wavelength of 485 nm; emission was measured at a wavelength of 530 nm.


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5-HT induces phosphorylation and activation of ERK via the 5-HT2A receptor. Treatment of mesangial cells with 5-HT resulted in an increase in phosphorylation of ERK and in activation of ERK as determined by immunoprecipitation kinase assay in which myelin basic protein was the substrate (Fig. 1A). The phosphorylation and activation of ERK induced by 5-HT were inhibited by the specific 5-HT2A receptor antagonist ketanserin (10 µM) and mimicked by the specific 5-HT2A receptor agonist (R-[-]-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) (10 µM). Those findings verify that the signal is conveyed by the 5-HT2A receptor that is expressed in rat mesangial cells (16, 28). Figure 1B shows that the coupling of the 5-HT2A receptor to ERK phosphorylation was quite efficient, with an EC50 of 12 ± 7 nM. Notably, this coupling was about one order of magnitude more potent than that of this receptor for hydrolysis of inositol phosphates (~265 nM), a second messenger pathway that is almost universally linked to this receptor subtype (16). The time course (Fig. 1C) also is consistent with that expected for activation of ERK by G protein-coupled receptors, with phosphorylation first being apparent as early as 1 min, peaking at 5-15 min of exposure to 5-HT, and persisting for up to 60 min. Moreover, the signal was also blocked by the mitogen-activated extracellular signal-regulated kinase kinase (MEK1) inhibitor PD-98059, as expected for receptor-activated ERK (Fig. 1B).


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Fig. 1.   Serotonin (5-HT) activates extracellular signal-regulated kinase (ERK) in rat glomerular mesangial cells. A: cells were treated with test agents for 10 min. Cells treated with antagonist (ketanserin) were preexposed for 5 min before stimulation with 5-HT. ERK phosphorylation was assessed by immunoblot with a phosphorylation state-specific antibody (phospho-ERK blot), which was compared with results of blots probed with an ERK antibody that is not sensitive to ERK phosphorylation (ERK blot). Results of an activity assay that measures ability of immunoprecipitated ERK to phosphorylate myelin basic protein (MBP) are also shown (MBP substrate assay). There was excellent concordance between assays. DOI, (R-[-]-2,5-dimethoxy-4-iodoamphetamine hydrochloride. B: concentration-response curve for 5-HT-induced phosphorylation of ERK (). Ability of 1 µM 5-HT to phosphorylate ERK was nearly abolished by preincubation for 30 min with 50 µM (gray circle) and 100 µM (open circle ) PD-98059. C: time course of ERK phosphorylation after treatment with 1 µM 5-HT (0-60 min). Each experiment was performed >= 3 times in duplicate or triplicate. Error bars, SE. * P < 0.5 vs. vehicle control unless indicated by a spanning bar (A). Reverse Bonferroni correction was used to correct for multiple comparisons.

Involvement of phospholipase C and classical PKC and lack of involvement of pertussis toxin-sensitive G proteins in the phosphorylation of MAPK by 5-HT. 5-HT2A receptor classically signals by activating phospholipase C (PLC) and phorbol ester-sensitive classical PKC types. This typically occurs through non-pertussis toxin-sensitive G proteins, although we previously showed that in rat mesangial cells the 5-HT2A receptor inhibits cAMP and activates a proton efflux that are mainly sensitive to pertussis toxin (16). The potential involvement of pertussis toxin-sensitive Gi/o proteins was tested by preincubation of cells overnight with pertussis toxin (200 ng/ml). This treatment has previously been shown to greatly attenuate 5-HT2A receptor-inhibited cAMP and stimulation of proton efflux and to nearly completely eliminate the subsequent ADP-ribosylation of Gi/o proteins by pertussis toxin in these cells (16). In the present study, pertussis toxin had no effect on the ability of 5-HT to activate ERK (Fig. 2A), effectively ruling out a substantial role for Gi/o proteins in conveying the signal from the 5-HT2A receptor to ERK.


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Fig. 2.   Effects of treatment with pertussis toxin, phospholipase C (PLC) inhibitors, and protein kinase C (PKC) inhibitors on 1 µM 5-HT stimulation of ERK phosphorylation. A: ERK phosphorylation was not affected by preincubation overnight with pertussis toxin (200 ng/ml). 5-HT-induced ERK phosphorylation was blunted by 2 inhibitors of PLC (U-73122 and ET-OCH3) but not by an inactive analog of U-73122 (U-73343). B: 3 maneuvers that block PKC attenuated 5-HT-induced ERK phosphorylation. Treatments with 500 nM phorbol 12-myristate 13-acetate (PMA; PKC activator) for 10 min also resulted in ERK phosphorylation. Plots are representative of 3-5 separate experiments performed in triplicate. Error bars, SE. * P < 0.5 vs. 5-HT value in presence of vehicle control. Reverse Bonferroni correction was used to correct for multiple comparisons.

We previously showed that 5-HT activates PLC-mediated hydrolysis of inositol phosphates (16), whereas others have shown that 5-HT stimulates PKC in mesangial cells (27, 40). The involvement of PLC as a contributor to the activation of ERK was tested by incubating mesangial cells with two PLC inhibitors, ET-18-OCH3 and U-73122 (each at 20 µM), and U-73343, an inert analog of U-73122. Both PLC inhibitors significantly blocked the phosphorylation of ERK by 5-HT, but the inert analog U-73343 had no effect, confirming a role for PLC in this pathway (Fig. 2A).

PLC classically results in the generation of two second messengers, inositol trisphosphate (IP3) and diacylglycerol (DAG). DAG can directly activate classical types of PKC by interacting with its lipid-binding domain, and IP3 can indirectly activate PKC by increasing intracellular Ca2+, which interacts with the PKC Ca2+-binding domain. We therefore tested the potential involvement of classical PKC in the activation of ERK in two ways (Fig. 2B). First, cells were pretreated for 15 min with two PKC inhibitors, H-7 (50 µM) and GF-109203X. Second, cells were pretreated overnight with 1 µM PMA for 18 h to downregulate phorbol ester-sensitive PKC types. Those maneuvers inhibited the ability of 5-HT to phosphorylate ERK. Furthermore, in data not shown, another specific PKC inhibitor, chelerythrine, also attenuated the phosphorylation of ERK by 5-HT. Those results implicate phorbol ester-sensitive classical PKC as an intermediary between the 5-HT2A receptor and ERK. This hypothesis was supported further by the ability of PMA, a direct activator of PKC, to induce phosphorylation of ERK to a level equivalent to 5-HT. However, because the blockade of ERK activation was not total, it is likely that another signaling intermediate (such as a nonclassical PKC subtype or a tyrosine kinase) may mediate the remainder of the response.

Involvement of ROS in the phosphorylation of MAPK by 5-HT. To establish a potential role for ROS in conveying the stimulation of ERK by the 5-HT2A receptor, we established five criteria that would need to be fulfilled experimentally: 1) antioxidants should attenuate the effects of 5-HT, 2) the effects of 5-HT should be mimicked by direct application of molecules, which generate ROS, 3) a specific enzyme capable of generating ROS should be implicated, 4) there should be evidence of specificity in the actions of ROS on the ERK pathway, and 5) 5-HT should produce measurable amounts of ROS in a time scale similar to that of ERK activation.

To address the first criterion, we treated cells with two structurally distinct antioxidant molecules (the reduced form of alpha -lipoic acid and NAC) and then with 5-HT. NAC can serve as an antioxidant directly by protecting sulfhydryl groups from oxidation and indirectly by serving as a precursor for the synthesis of glutathione, an abundant endogenous cellular reducing antioxidant (35). alpha -Lipoic acid functions mainly as a scavenger of hydroxyl radicals, singlet oxygen, and hypochlorous acid. alpha -Lipoic acid may also exert antioxidant effects by chelation of transition metals. In addition, alpha -lipoic acid may have indirect antioxidant effects as well, by recycling other antioxidants or increasing cellular levels of glutathione (29, 35). Figure 3A demonstrates that overnight incubation with alpha -lipoic acid (500 µM) virtually eliminates the ability of 5-HT to activate ERK without affecting basal levels of phospho-ERK. The effect of alpha -lipoic acid could be reversed by coincubation with 400 µM racemic BSO, an inhibitor of gamma -glutamylcysteine synthase. Because gamma -glutamylcysteine synthase is the rate-limiting enzyme in glutathione synthesis, BSO treatment should deplete cells of the important antioxidant glutathione (20). Preincubation with 20 mM NAC for 30 min also markedly blunted the ability of 5-HT to increase the phosphorylation of ERK. Preincubation with 50 mM NAC blunted the response to a similar level, as did overnight treatment with alpha -lipoic acid (Fig. 3A). These experiments clearly implicate the redox state of the cell as critical in the ability of mesangial cells to respond to treatment with 5-HT. Moreover, the effect of NAC is not merely due to reduction of extracellular disulfide bonds contained within the 5-HT2A receptor, because the effect of PMA (which works at an intracellular site downstream of the receptor) is blunted by antioxidants as efficiently as is the effect of 5-HT stimulation (see Fig. 5).


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Fig. 3.   A: effect of modulators of cellular redox on ERK phosphorylation. Cells were exposed to 250 µM alpha -lipoic acid (alpha -LA) and/or 400 µM buthionine sulfoximine (BSO) overnight or to N-acetylcysteine (NAC) for 30 min before stimulation with 5-HT or vehicle. B: effects of oxidants on ERK phosphorylation. Cells were exposed to 500 µM H2O2, diamide, or tert-butyl hydroperoxide (t-butyl HP) or 400 µM sodium arsenite for 15 min. For some studies, cells were preincubated with BSO, alpha -lipoic acid, or NAC as described in A. C: concentration-response plot of ERK phosphorylation for 15-min treatments with H2O2. D: time course of ERK phosphorylation for 1 mM H2O2. Error bars, SE. * P < 0.5 vs. 5-HT value in presence of vehicle control. Reverse Bonferroni correction was used to correct for multiple comparisons.

If ROS participate in 5-HT2A receptor- and PKC-induced activation of ERK, it would be expected that oxidative stress might also activate ERK. Indeed, depletion of cellular glutathione stores with BSO treatment somewhat increased the basal phosphorylation of ERK in mesangial cells (Fig. 3A), although this effect was not statistically significant. To confirm the effects of oxidant stress on mesangial cell ERK, cells were treated with four substances that have been shown to induce oxidative stress: H2O2 (200 µM), sodium arsenite (400 µM), diamide (500 µM), and tert-butyl hydroperoxide (500 µM). H2O2 is produced in many cells in response to stress or specific hormones and may participate in the regulation of signaling pathways directly or through conversion to other oxidant molecules (35). Sodium arsenite is a sulfhydryl-reactive agent that has previously been shown to induce oxidative stress and activate ERK in PC-12 cells (25). Diamide penetrates cell membranes rapidly, where it efficiently oxidizes thiols (23). These three oxidant-generating molecules can interact efficiently with methionine and cysteine residues in proteins. Tert-butyl hydroperoxide is a molecule with oxidative capacity similar to H2O2. Unlike the three other oxidant-generating molecules, tert-butyl hydroperoxide selectively interacts with methionine residues of proteins, presumably because of its bulky butyl group (12). As shown in Fig. 3B, addition of H2O2, diamide, and sodium arsenite, but not tert-butyl hydroperoxide, increased the amount of phospho-ERK present in mesangial cell extracts. The stimulatory effect of H2O2 was markedly diminished in cells pretreated overnight with NAC or alpha -lipoic acid (Fig. 3B) but was not diminished in cells pretreated with BSO and alpha -lipoic acid (data not shown).

The stimulation was further characterized for H2O2 as being dependent on time and concentration, being apparent at 3 min, peaking at 15-30 min, and being maintained for >= 90 min. During a 10-min incubation, the stimulation was apparent at 200 µM H2O2, had peak effect at 1 mM, and had lesser effects at 10 and 25 mM (Fig. 3, C and D). Thus oxidant-generating substances mimic the effects of 5-HT on ERK, and this effect is specific for certain oxidant-generating molecules.

We hypothesized that mesangial cell NAD(P)H oxidase was the enzyme responsible for generating the ROS in response to 5-HT (19). Because NAD(P)H oxidase is a multicomponent enzyme (9), we were able to employ four types of pharmacological inhibitors of NAD(P)H oxidase to test its involvement in the activation of ERK by 5-HT. 4'-Hydroxy, 3'-methoxy acetophenone (HMAP) competes with NAD(P)H for a binding site on the oxidase but does not effectively complete an obligate two electron transfer to FAD (11, 34). This transfer to FAD is critical for enzyme activity, in that it is required to convert the obligate two electrons donated from NAD(P)H to a one-electron transfer required from the heme site of the NAD(P)H enzyme to O2 to yield O-2·. Another inhibitor, DPI, blocks FAD binding to the flavin site of the oxidase (11). Phenylarsine oxide (PAO) blocks distal electron transport in the NAD(P)H oxidase (24). Finally, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) is a serine protease inhibitor that also has been shown to block NAD(P)H oxidase by preventing assembly of the enzyme subunits (13).

Figure 4A shows that HMAP, DPI, and PAO markedly attenuated the activation of ERK by 5-HT. Because ROS could derive from mitochondrial sources and because it is possible that a flavin inhibitor such as DPI or a competitor of NAD(P)H such as HMAP could inhibit mitochondrial electron transport (thereby increasing mitochondrial production of ROS), we tested the effects of three inhibitors of mitochondrial electron transfer on phosphorylation of ERK induced by mitogens. Sodium azide, a blocker of complex IV, had no effect on ERK phosphorylation (Fig. 4A), making it unlikely that the ROS derive from the mitochondria. We also tested blockers of complex I (rotenone) and complex III (antimycin), neither of which had an effect on 5-HT-stimluated ERK phosphorylation (data not shown). The effects of PAO (100 µM) could be partially reversed by coincubation with British anti-Lewisite (BAL or 2,3-dimercaptopropanol, 20 µM), as has been shown to occur with purified NAD(P)H oxidase components (24). This feature is an important step in validating the interaction of PAO with the NAD(P)H oxidase and not some other signaling enzyme such as a tyrosine phosphatase. We did not expect that BAL would completely reverse the effects of PAO, because the two compounds are thought to compete for a single binding site within the NAD(P)H oxidase (24).


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Fig. 4.   Effect of NAD(P)H oxidase inhibitors on ERK phosphorylation. A: cells were exposed 100 µM 4'-hydroxy-3'-methoxyacetophenone (HMAP), 50 µM diphenyleneiodonium (DPI), 5 mM sodium azide, or 100 µM phenylarsine oxide (PAO) for 15-30 min before treatment with 5-HT or vehicle. Attenuation induced by PAO was reversed by coincubation with British anti-Lewisite (BAL), as would be expected for NAD(P)H oxidase. B: cells were exposed to 750 µM (each) 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) or other serine protease inhibitors with varying degrees of structural similarities to AEBSF for 15 min before treatment with 5-HT or vehicle. MAEBSF, 4-(2-N-methylaminoethyl)benzenesulfonyl fluoride; AEBSAc, aminoethylbenzenesulfonyl acetate; PMFS, phenylmethylsulfonyl fluoride; TLCK, Nalpha -p-tosyl-L-lysine chloromethylketone. Error bars, SE. * P < 0.5 vs. 5-HT value in presence of vehicle control. Reverse Bonferroni correction was used to correct for multiple comparisons.

AEBSF effectively blocked the activation of ERK by 5-HT (Fig. 4B). A series of protease inhibitors with varying degrees of structural similarities to AEBSF was also tested for the ability to inhibit the activation of ERK by 5-HT. As shown in Fig. 4B, 4-(2-N-methylaminoethyl)benzenesulfonyl fluoride, which shares the major structural features of AEBSF, was similarly able to inhibit ERK phosphorylation, as predicted from studies by Diatchuk et al. (13). In contrast, 4-(aminoethyl)benzenesulfonamide (in which the sulfonyl fluoride moiety is substituted with a sulfonamide), PMSF (which preserves only the sulfonyl fluoride moiety), or Nalpha -p-tosyl-L-lysine chloromethylketone (a structurally unrelated serine protease inhibitor) was unable to inhibit ERK phosphorylation. In fact, PMSF was consistently shown to slightly increase ERK phosphorylation. Thus these studies strongly implicate an NAD(P)H oxidase as a major source of the ROS required to activate mesangial cell ERK.

We performed pathway mapping studies (Figs. 5-7), which showed that the ROS and ROS-generating enzyme are localized to a specific region of the signal transduction cascade initiated by 5-HT. Figure 5A places the ROS and NAD(P)H oxidase downstream of PKC, in that ERK phosphorylation resulting from direct activation of PKC by PMA can be attenuated by ROS scavengers (NAC and alpha -lipoic acid) and by two NAD(P)H oxidase inhibitors (DPI and AEBSF). Figure 5B shows that the ROS are downstream of PKC and NAD(P)H oxidase, in that inhibition of both enzymes fails to attenuate the activation of ERK by direct exposure of mesangial cells to H2O2. Figure 5C uses a similar strategy to probe the relative locations of MEK (which is one step upstream from ERK) to PKC and the ROS. Figure 5C demonstrates that the ROS scavenger NAC and the NAD(P)H oxidase inhibitor DPI block phosphorylation of MEK by PMA. Those studies place PKC and NAD(P)H oxidase upstream of MEK. Furthermore, when H2O2 was directly applied to mesangial cells, the phosphorylation of MEK was enhanced; this effect was attenuated by NAC, but not by DPI.


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Fig. 5.   Studies designed to establish relative locations of PKC, NAD(P)H oxidase, reactive oxygen species (ROS), mitogen-activated extracellular signal-regulated kinase (MEK), and ERK in signal transduction pathway. A and B: phospho-ERK blots obtained after cells were treated with inhibitors before treatment with 1 µM PMA, 1 mM H2O2, or vehicle for 10 min. C: phospho-MEK blots obtained after cells were treated with inhibitors for 15 min and then with 1 µM 5-HT, 1 µM PMA, 1 mM H2O2, or vehicle for 15 min. Error bars, SE. * P < 0.5 vs. 5-HT value in presence of vehicle control. Reverse Bonferroni correction was used to correct for multiple comparisons.



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Fig. 6.   Measurement of intracellular H2O2 production by 2',7'-dichlorofluorescin diacetate (DCF-DA) confocal microscopic fluorescence detection in mesangial cells. Cells were treated with vehicle (top left) or 1 µM 5-HT (top right) after being loaded with H2O2-sensitive dye DCF-DA. Some cells were preincubated with NAC (bottom left) or DPI (bottom right) for 30 min before addition of 5-HT. Initial pictures in each set were taken as 5-HT (or vehicle) was being applied to cells. Subsequent pictures were taken at 5 and 30 min after exposure to 5-HT or vehicle.



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Fig. 7.   Measurement of intracellular superoxide production by cytochrome c reduction assay in mesangial cells. 5-HT (1 µM) and PMA (500 nM) stimulated production of superoxide. These effects were inhibited by DPI, GF-109203X, and superoxide dismutase (SOD). Error bars, SE. * P < 0.5 vs. 5-HT (or PMA) value in presence of vehicle control. Reverse Bonferroni correction was used to correct for multiple comparisons. Experiments were performed 3 separate times.

We assessed the intracellular production of H2O2 after administration of 5-HT. Figure 6 shows that application of 5-HT to quiescent, DCF-DA-loaded cells results in a time-dependent increase in fluorescence that was not present in vehicle-treated cells. These data demonstrate that 5-HT induces the rapid production of H2O2 in mesangial cells. Figure 6 shows that generation of H2O2 by 5-HT could be attenuated by DPI and NAC, confirming a role for NAD(P)H oxidase in the generation of H2O2 by 5-HT. Similar results (not shown) were obtained when PMA was used to increase production of H2O2 in mesangial cells, confirming a location for PKC that is upstream of the ROS. However, O-2·, and not H2O2, is the direct product of NAD(P)H oxidase. H2O2 could be produced from O-2· by the action of SOD, but we believed that our contention that 5-HT induces production of ROS (H2O2 and O-2·) through NAD(P)H oxidase needed to be confirmed using an assay that measures the production of O-2·.

We tested the ability of 5-HT to increase the production of O-2· by use of a cytochrome c reduction assay (Fig. 7). In their unstimulated state, rat mesangial cells produced ~200 pmol O-2· · min-1 · 106 cells-1, and this was increased threefold after treatment with 1 µM 5-HT or 500 nM PMA. These effects were blocked by DPI and suppressed below baseline by inclusion in the assay mixture of SOD, which converts O-2· to H2O2. They were also blocked by preincubation with GF-109203X, a specific inhibitor of PKC. Thus these studies are consistent with relative placements in the signaling cascade as follows: 5-HT2A receptor right-arrow G protein right-arrow PLC right-arrow DAG right-arrow classical PKC right-arrow NAD(P)H oxidase right-arrow O-2· right-arrow SOD right-arrow H2O2 right-arrow MEK right-arrow ERK.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Because the renal glomerulus could be exposed to 5-HT through local synthesis from the precursor molecule L-5-hydroxytryptophan (36, 38) or by release from platelets or other infiltrating cells, 5-HT from several sources could modulate the function of glomerular resident cells. Rat renal mesangial cells express a 5-HT2A receptor (28), the primary signaling pathway of which is thought to be activation of PLC (22, 40). However, in the glomerular mesangial cell, the signaling pathways linked to the 5-HT2A receptor are quite diverse. They include phosphoinositide metabolism (40), liberation of Ca2+ derived from intracellular pools (27, 40), activation of PKC (40), stimulation of vasodilator prostaglandin synthesis (22), Cl- conductance-related membrane depolarization, prolonged cytosolic alkalinization related to activation of electroneutral Na+/H+ exchange, enhanced Na+-independent Cl-/HCO-3 countertransport (27), inhibition of adenylyl cyclase (16), and activation of mitogenesis (40). The mitogenic response is of particular interest, in that it may play a key role in proliferative glomerulonephritis. Despite the diversity of signaling mechanisms available to the 5-HT2A receptor, little is known regarding the signal transduction pathways that mediate the mitogenic effect of 5-HT in mesangial cells.

The present studies support a role for PLC and classical forms of PKC (alpha , beta , or gamma ) in the stimulation of ERK initiated by the 5-HT2A receptor. That conclusion is based on the ability of overnight treatment of mesangial cells with phorbol esters or short-term treatment with PKC inhibitors to attenuate ERK phosphorylation. However, because those maneuvers are not specific for all types of PKC, we cannot comment on any potential roles for nonclassical PKC types in this process. Our results suggest that it is likely that classical forms of PKC mediate at least one-half of the stimulation by ERK, but they do not rule out an accessory role for other intermediates, such as nonclassical forms of PKC, or for tyrosine kinases.

Recent studies suggest that the ERKs are activated in response to mitogenic signals, hormones, cytokines, and growth factors in a variety of cell types (3). Excessive proliferation of resident glomerular cells can participate in the progression of chronic renal disease by altering glomerular architecture, synthesizing cytokines, or increasing the production of extracellular matrix in response to growth factors and other ligands. If unchecked, these processes can ultimately result in glomerular fibrosis. Oxidative stress and ROS production also could contribute to glomerular injury through several mechanisms. They may directly affect cellular function through lipid peroxidation of plasma membrane and subcellular membrane lipids and protein oxidation, thereby disrupting enzymatic functions. However, ROS are not necessarily always generated in quantities that are immediately cytotoxic and might function in some cases as second messengers (2). In the present study we have implicated H2O2 and O-2· as major participants in the activation of ERK by 5-HT in mesangial cells.

5-HT is among many potential growth factors released from activated platelets. In models of glomerulonephritis, including chronic glomerulonephritis, mitogenic ligands (5-HT, thromboxanes, sphingolipids, phospholipids) released from platelets are thought to play a possible role in proliferation and/or fibrosis. Moreover, the potential importance of the ERKs in renal glomerular disease has been recently highlighted in an animal model of acute proliferative glomerulonephritis. Our data complement nicely those recently reported by Bokemeyer et al. (6). They reported increased ERK activity in the renal cortex and glomeruli derived from an in vivo model of accelerated proliferative glomerulonephritis. Their studies suggested that macrophage activation resulted in increased ERK activation, since total body irradiation and resulting macrophage depletion reduced ERK activation in the model and protected the animals from progressive glomerulonephritis and proteinuria. Although they did not implicate oxidative stress, it is possible that ROS generated and released from macrophages or cytokine-induced ROS generation by neighboring resident glomerular cells was in part responsible for ERK activation.

Wilmer et al. (43) recently implicated ROS as critical intermediates in the activation of ERKs in human mesangial cells by interleukin-1beta . Interleukin-1beta signals through a receptor that activates tyrosine phosphorylation reactions, although the receptor itself does not possess intrinsic tyrosine kinase activities. This receptor is of a class that does not typically signal directly through G proteins. Our results demonstrate that a receptor (5-HT2A) that couples to cellular signaling cascades through Gq proteins also requires ROS as intermediate messengers for the activation of mesangial cell ERK. Moreover, this interaction fulfills five rigorous criteria implicating the ROS as second messengers in this system. First, antioxidants from two chemical classes attenuated the effects of 5-HT. Second, the effects of 5-HT were mimicked by direct application of three different oxidant molecules that interact with cellular thiols. Third, 5-HT was shown to produce measurable amounts of H2O2 and superoxide in a time scale similar to that of ERK activation. Fourth, a specific enzyme capable of generating ROS, NAD(P)H oxidase, was implicated by the use of four distinct types of inhibitors. Although NAD(P)H oxidase has classically been associated with neutrophils, two groups have used RT-PCR and immunoblot to document the presence of three or four subunits of the NAD(P)H oxidase in mesangial cells and glomerular podocytes (18, 21). Fifth, there was clear evidence of specificity in the actions of ROS on the ERK pathway. This last point is particularly important, in that ROS are short-lived, highly reactive molecules that are theoretically capable of eliciting some cellular effects through nonspecific toxicities. The effects on ERK are not likely to be nonspecific, because the ROS were localized to a specific region of the signal transduction pathway and because not every oxidant molecule applied to mesangial cells activated ERK. This second feature also has allowed us to generate the hypothesis that the ROS target a critical cysteine (rather than a methionine) residue.

Our studies are most consistent with an ERK activation pathway as follows: 5-HT2A receptor right-arrow G protein right-arrow PLC right-arrow DAG right-arrow PKC right-arrow NAD(P)H oxidase right-arrow O-2· right-arrow SOD right-arrow H2O2 right-arrow MEK right-arrow ERK. It is possible that MEK is the target of the ROS, although other signaling molecules could be targets as well. Ras and Raf are thought to be two steps and one step upstream, respectively, from MEK; although we did not specifically study their roles in this pathway, they are also likely potential targets for modification by ROS. It is also possible that an as yet unidentified protein could serve as the target in this transduction cascade.

Even though direct application of H2O2 results in ERK activation and 5-HT results in increased production of H2O2 in mesangial cells, other ROS might be involved. NAD(P)H oxidase does not directly produce H2O2, but O-2· is probably converted to H2O2 by SOD. H2O2, in turn, could yield hydroxyl radicals through Fenton chemistry (15). Any of those free radicals could potentially serve as the second messenger that leads to ERK activation.

Our results correlate well with those recently described by Lee et al. (25), who showed that 5-HT-generated superoxide mediates ERK activation and thymidine incorporation in CCL-39 hamster lung fibroblasts and in bovine pulmonary artery smooth muscle cells. Our results differ from theirs in two respects: 1) the effect of 5-HT in those cell types is exclusively or predominantly mediated by 5-HT transporters, rather than by receptors; and 2) they focused on superoxide as a key mediator of the effects of 5-HT, whereas we studied H2O2. Our results should also be contrasted to those of Ushio-Fukai et al. (41), who recently showed that ANG II activates ERK in vascular smooth muscle cells through a pathway that is independent of NAD(P)H oxidase or H2O2. Thus NAD(P)H oxidase and/or H2O2 does not appear to be universally linked to ERK activation in all cells or by all G protein-coupled receptors.

In conclusion, these studies present evidence that a prototypical Gq-coupled receptor (5-HT2A) can activate mesangial cell ERK through the generation of ROS. It is possible, although not yet proven, that this relationship is universal for all mesangial cell mitogens or mitogenic receptors. Because of the proposed roles of ERK and ROS in tissue damage and chronic renal disease processes, these studies underscore the need for a more detailed and mechanistic understanding of their interrelationships.


    ACKNOWLEDGEMENTS

We thank Pamela Wackym for excellent technical assistance.


    FOOTNOTES

This work was supported by the Department of Veterans Affairs (Merit Awards to J. R. Raymond and M. N. Garnovskaya), National Institutes of Health Grants DK-52448 and HL-03710 (to J. R. Raymond and E. L. Greene), a Robert Wood Johnson Faculty Development Award (to E. L. Greene), a laboratory endowment jointly supported by the Medical University of South Carolina Division of Nephrology and Dialysis Clinics, Inc. (to J. R. Raymond), an American Heart Association fellowship (to J. S. Grewal), and Medical University of South Carolina University Research Foundation awards (to M. N. Garnovskaya and E. L. Greene). M. N. Garnovskaya is a Research Scientist of the Department of Veterans Affairs.

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 and other correspondence: E. L. Greene, Rm. 829C CSB, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425 (E-mail: greeneel{at}musc.edu).

Received 27 August 1999; accepted in final form 3 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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