Nephrology Division, Department of Internal Medicine, Medical University of South Carolina, and Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29425
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ABSTRACT |
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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 Gq protein
phospholipase C
diacylglycerol
classical PKC
NAD(P)H oxidase
superoxide
superoxide dismutase
H2O2
mitogen-activated extracellular
signal-regulated kinase
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
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INTRODUCTION |
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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 Gq/11 family
of heterotrimeric G proteins (16, 19, 27, 28). We recently showed that
5-HT increases the levels of transforming growth factor-
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.
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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, -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-(17
-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl-1H pyrrole-2,5-dione (U-73122) and
1-(6-(17
-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
[
-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 (O2·) 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
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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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RESULTS |
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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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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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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
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DISCUSSION |
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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
(,
, or
) 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
O2· 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-1.
Interleukin-1
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 G protein
PLC
DAG
PKC
NAD(P)H oxidase
O
2·
SOD
H2O2
MEK
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
O2· 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.
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ACKNOWLEDGEMENTS |
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We thank Pamela Wackym for excellent technical assistance.
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FOOTNOTES |
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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.
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