Department of Medicine, Nephrology Division, Medical University of South Carolina and the Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29425
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ABSTRACT |
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We examined the links between fibrotic and
proliferative pathways for the
5-HT2A receptor in rat mesangial
cells. Serotonin (5-hydroxytryptamine, 5-HT) induced transforming
growth factor-1 (TGF-
1) mRNA in a concentration-dependent (peak
at 30 nM 5-HT) and time-dependent fashion. For 10 nM 5-HT, the effect
was noticeable at 1 h and maximal by 6 h. Inhibition of
1) protein kinase C (PKC), 2) mitogen- and extracellular
signal-regulated kinase kinase (MEK1) with
2'-amino-3'-methoxyflavone (PD-90859), and
3) extracellular signal-regulated
kinase (ERK) with apigenin attenuated this effect. The effect was
blocked by antioxidants,
N-acetyl-L-cysteine
(NAC) and
-lipoic acid, and mimicked by direct
application of
H2O2. TGF-
1 mRNA induction was also blocked by diphenyleneiodonium and
4-(2-aminoethyl)-benzenesulfonyl fluoride, which inhibit NAD(P)H oxidase, a source of oxidants. 5-HT increased the amount of TGF-
1 protein, validating the mRNA studies and demonstrating that 5-HT potently activates ERK and induces TGF-
1 mRNA and protein in mesangial cells. Mapping studies strongly supported relative positions of the components of the signaling cascade as follow:
5-HT2A receptor
PKC
NAD(P)H oxidase/reactive oxygen species
MEK
ERK
TGF-
1 mRNA. These studies demonstrate that mitogenic
signaling components (PKC, MEK, and oxidants) are directly linked to
the regulation of TGF-
1, a key mediator of fibrosis. Thus a single stimulus can direct both proliferative and fibrotic signals in renal
mesangial cells.
proliferation; fibrosis; mesangial cell; serotonin; transforming growth factor
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INTRODUCTION |
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GLOMERULAR MESANGIAL CELLS have critical roles in the structural integrity and ultrafiltration functions of the kidney. Their morphological localization juxtaposed to the vascular compartment renders them susceptible to a number of vasoactive substances (1, 10), including constrictors like angiotensin II, arginine vasopressin, thromboxane, and serotonin (5-hydroxytryptamine, 5-HT) (16, 22, 37) and relaxants such as dopamine and PGE2 (23). These substances may play key roles in regulating glomerular functions through their actions on mesangial cells. Mesangial cells are normally quiescent, but when activated can proliferate excessively and/or lead to alterations in the amount and composition of extracellular matrix (ECM) in the renal glomerulus. Because early proliferation and subsequent fibrosis are hallmarks of nearly all forms of progressive glomerular diseases (1, 10), an understanding of how various vasoactive substances affect glomerular cell proliferation and ECM homeostasis might yield valuable insight into the common pathways that lead to irreversible renal disease.
Recent studies have implicated mitogen-activated protein kinases (MAPK) of the ERK ("extracellular signal-regulated kinase") family (7) in the pathogenesis of proliferative glomerulonephritis, in experimental antiglomerular basement membrane glomerulonephritis (3) and in human renal cell carcinoma (26). The ERKs are activated by dual-specificity (threonine and tyrosine) protein kinases called MAPKK/MEK. When phosphorylated, the ERKs are capable in turn of phosphorylating a variety of diverse targets such as effector kinases and transcription factors, thereby regulating the expression of different genes associated with mitogenesis (40). Thus there is ample rationale for implicating mesangial cell ERKs as participants in the early proliferative phase of glomerular injury.
In later phases of glomerular disease, abnormal ECM metabolism results
in an increased accumulation of ECM and in alteration of matrix
composition. Although many mitogens can stimulate production and/or
decrease degradation of ECM, the signaling pathways involved in these
processes remain obscure. A key signaling molecule prototypically associated with fibrotic processes in many tissues including the renal
glomerulus is transforming growth factor- (TGF-
). TGF-
is an
important member of a large superfamily of pleiotropic cytokines that
play crucial roles during embryonic development, ECM protein synthesis,
immune system turnover, cell proliferation, and apoptosis (15, 21, 36).
Of several distinct isoforms of TGF-
, TGF-
1 is the most abundant.
Increased TGF-
1 mRNA and/or protein levels have been consistently
demonstrated in various animal models of glomerulosclerosis (17, 45)
and in progressive renal disease in humans (4, 35). Although the cell
type responsible for TGF-
1 production in the glomerulus has not yet
been identified, mesangial cells may be one of the major sources. In
addition to autoinduction of its own production (14, 42), TGF-
1
regulates the expression of cellular reactive oxygen species (ROS),
which have been implicated in fibrotic processes (31).
Despite their recognized importance in the development of progressive
chronic renal diseases, the precise links between the mechanisms of
glomerular mesangial cell proliferation and glomerular fibrosis are
poorly understood. In the current study, we treated rat mesangial cells
with 5-HT to examine its effects on TGF-1 metabolism and to test the
hypothesis that ERKs regulate the expression of TGF-
1. Mesangial
cells possess a 5-HT2A receptor
(12, 25), which was recently linked to the production of TGF-
1 and
collagen through a protein kinase C (PKC)-dependent mechanism (20).
Because 5-HT, which is mitogenic, has also been linked to fibrosis in other cell types, it served as an ideal test substance with which to
study the potential links between proliferative and fibrotic signals in
mesangial cells.
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MATERIALS AND METHODS |
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Chemicals. MaxiScript in vitro
transcription kit and RPA II Kit was obtained from Ambion (Austin, TX).
2'-Amino-3'-methoxyflavone (PD-98059),
4-4-fluorophenyl-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB-203580), apigenin, and bisindolylmaleimide (GF-109203) were from
Calbiochem (San Diego, CA).
[-32P]UTP (800 Ci/mmol) was from NEN Life Science Products (Boston, MA).
The bioactive form of recombinant human TGF-
1, anti-TGF-
1 antibody and Quantikine human TGF-
1 immunoassay kit were from R&D
Systems (Minneapolis, ME). Phospho-MAPK (p44/p42) antibody, phospho-SEK1/MKK4 (Thr223)
antibody, alkaline phosphatase (AP)-linked rabbit IgG
secondary antibody, and CDP Star substrate were purchased from New
England Biolabs (Beverly, MA). AP-conjugated donkey
anti-chicken IgY(H+L) secondary antibody was obtained from Jackson
ImmunoResearch Laboratory (West Grove, PA). Precast 4-20%
Tris-glycine gradient minigels were bought from Novex (San Diego, CA).
All other chemical and reagents were obtained from Sigma (St. Louis, MO).
Cell culture. Primary mesangial cell cultures were raised from glomeruli isolated by sieving the kidneys of 75- to 150-g male Rattus norvegicus Sprague-Dawley rat as described previously (12, 37). Cells were cultured in RPMI-1640 [GIBCO-BRL (Life Technologies), Gaithersburg, MD] supplemented with 20% FBS and 100 U/ml penicillin and 100 µg/ml streptomycin, then incubated at 37°C in a humidified atmosphere of 95% air-5% CO2. Cells were identified as mesangial cells by their characteristic morphology and contractile properties. Cells from passages 6-16 were used in all experiments. Quiescence was induced by transferring the 60-70% confluent cell cultures to RPMI-1640 with 0.5% BSA, 100 U/ml of penicillin, and 100 µg/ml of streptomycin for 48 h prior to the treatments with various chemicals.
TGF- Western immunoblotting
assay. Equal numbers of quiescent cells (~2 × 105 cells) in 12-well culture
plates were treated with various agonists for 10 min, whereas
antagonist treatments were carried out for 30 min prior to agonist
treatments. Cells were kept at 37°C in an incubator under 5%
CO2 atmosphere during all of the
treatments. After aspirating the medium, cells were lysed and scraped
in 100 µl of Laemmli buffer. Cells were sonicated for 30 s to shear
DNA and to reduce viscosity. Samples were heated to 95-100°C
in boiling water for 5 min and then shifted to ice. After briefly
centrifuging, 20 µl of each sample was loaded on to 4-20%
Tris-glycine gradient mini-precast gels for electrophoresis. Proteins
were then electrotransferred onto Immobilon-P membranes. Membranes were
blocked with blocking buffer (5% nonfat milk in PBS and 0.1%
Tween-20) for 1 h before incubating overnight at 4°C with primary
antibody (1:1,000 dilution). The membranes were then washed three times
with blocking buffer and incubated further for 1 h with alkaline
phosphatase-linked anti-rabbit IgG secondary antibody (1:1,000
dilution), except for the TGF-
1 protein assay in which case an
AP-conjugated donkey anti-chicken IgY(H+L) secondary
antibody was used. After being washed three times with blocking buffer
and three times with wash buffer (PBS, 0.1% Tween-20), membranes were
incubated for 5 min at room temperature with CDP Star substrate (1:500
dilution). Membranes were then soaked dry between the folds of filter
papers and exposed to Kodak X-Omat AR film for 10 s to 15 min and
quantified using a GS-670 densitometer and Molecular Analyst software
(Bio-Rad, Hercules, CA).
In vitro transcription. The wild-type
TGF-1 sequence in pBlueScript II KS+ plasmid, pRTGF
1 (American
Type Culture Collection, catalog no. 63197; DNA sequence
accession no. X52498) was linearized with
BamH I. To get a 245-nt riboprobe, in
vitro transcription was performed using T3 DNA-directed RNA polymerase.
In brief, 32P-labeled antisense
mRNA was transcribed using
[
-32P]UTP (800 Ci/mmol), RNasin, nucleotides, and buffer conditions as
described in the Ambion MaxiScript manual. Internal standards of
-actin or GAPDH riboprobe of low specific activities were transcribed using pTRIPLEScript and higher concentrations (0.4 mM) of
cold UTP. On completion of the transcription reaction, the template DNA
was removed by adding RNase-free DNase 1. The labeled transcripts were
then purified by phenol/chloroform extractions and ethanol
precipitation. Sizes of the transcripts were verified by
electrophoresing samples through denaturing 1.2% agarose/formaldehyde gels.
RNA isolation. Total RNA was isolated
by a single-step method (5) using acid guanidinium
thiocyanate-phenol-chloroform extraction from 80% confluent cells from
at least one 100-mm dish. The aqueous phase containing RNA was
transferred to a new Eppendorf tube, and RNA was precipitated with 0.7 vol of isopropanol. The RNA pellets were then washed with 70% ethanol
to remove salts and isopropanol. Purified RNA was air dried and
reconstituted in RNase-free distilled water and stored at
80°C until used.
RNase protection assay. RNase
protection assay (RPA) was carried out following the manufacturer's
instructions provided with the Ambion RPA II kit. In brief, 20 µg of
sample RNA was hybridized with 50 × 104 cpm of antisense riboprobe in
hybridization buffer [80% deionized formamide/100 mM sodium
citrate (pH 6.4), 300 mM sodium acetate (pH 6.4), and 1 mM EDTA]
for 16-20 h at 48°C. After hybridization, excess single-strand
riboprobe and the unhybridized portion of sample RNA was digested away
with RNases A and T1 cocktail (Solution Bx, Ambion) supplemented with
1% blue coprecipitant solution (Ambion) to visualize the pellet. The
reaction mixture was incubated at 37°C for 30 min. The RNases were
inactivated, and protected double-stranded RNA was precipitated in a
single step by adding Solution Dx (Ambion) and incubating at
20°C for 20 min. Samples were then centrifuged for 15 min,
and the pellets were air dried and reconstituted in 8 µl of gel
loading buffer (8% sucrose, 0.025% bromphenol blue, 0.025% xylene
cyanol). The protected segments were isolated on a 5% native
polyacrylamide gel. Gels were dried and exposed to Kodak X-Omat AR
films for 4-16 h.
-Actin or GAPDH was used to normalize the
mRNA loading on the gel. Dried gels were also exposed to a
phosphorimager intensifying screen for the same period for further
analysis by the Storm 860 imaging system (Molecular Dynamics, Sunnyvale, CA). Densitometric analysis was performed using ImageQuant supplied by the same manufacturer.
Potentiometric measurement of cellular reduction
rate. We used a modified Cytosensor microphysiometer to
probe the redox state of mesangial cells by an electrochemical
potentiometric means. We previously used the microphysiometer fitted
with silicon sensors to measure proton efflux in mesangial cells (12).
In the current studies, we used custom modified gold electrodes
(Molecular Devices, Sunnyvale, CA) and an extracellular
solution containing ferricyanide/ferrocyanide. Menadione/menadiol was
added to the perfusate as a shuttle for carrying electrons across the
plasma membrane so that the ability to reduce iron could be measured.
The procedure has been validated for several cell types and described
in detail recently (29). Cells were grown and maintained as previously
described (12). Cells (2.5 × 106) were seeded onto
polycarbonate inserts (Costar, Cambridge, MA) the night prior to
experimentation. After attachment, they were incubated in serum-free
Ham's F-12 medium supplemented with 0.5% BSA and antibiotics until
the following day, when they were placed into microphysiometer chambers
(12, 29). Prior to each experiment, the chambers and electrodes were
washed with distilled water and equilibrated with redox medium
consisting of PBS (145 mM Na+, 4 mM KCl, 1 mM Mg2+, 1 mM
Ca2+, 143 mM
Cl, and 10 mM phosphate, pH
7.4) supplemented with 10 mM glucose, 20 mM HEPES, 1 mg/ml of
endotoxin-free BSA, and 100 µM each of ferricyanide and ferrocyanide.
Once loaded, the chambers were maintained at 37°C and perfused with
redox medium. The microphysiometer was calibrated to 0 V during
perfusion in the absence of menadione/menadiol. Following calibration,
measurements were made by switching the perfusate to redox media
containing 10 µM menadione. Cells were perfused with
menadione-containing medium, then switched to identical medium (to
control for valve-switching artifacts) or medium containing test
compounds. The chambers were perfused for 58 s, then flow was stopped
for 32 s during each 90-s pump cycles. The rate of change in the
potential was measured during seconds
20-30 of the stop-flow period. The
long delay between stopping perfusion and the reduction rate
measurement was needed, because the gold electrodes do not equilibrate
with the extracellular ferri(o)cyanide redox pair as rapidly as the
standard pH-sensing electrodes equilibrate with protons (29). Data are
presented as percentage change from basal values, which were calculated
as the mean values of five consecutive readings taken prior to the
readings taken with the test compounds.
Quantitation of activated TGF-1
concentrations. The TGF-
1 quantitation assay was
carried out following the manufacturer's instructions (R&D Systems).
In brief, 80% and 50% confluent quiescent mesangial cells in 6-well
plates were treated with 5-HT and/or the MEK inhibitor PD-98059 for 6 and 24 h. Experiments were also performed in which cells were
pretreated with PD-98059 for 1 h, followed by stimulation with 5-HT for
6 and 24 h. To activate latent TGF-
1, the cell culture supernatants
were treated with 0.2 vol of 1 N HCl for 10 min at room temperature.
Neutralization (to pH 7.2-7.6) of the acidified sample was
achieved by adding 0.2 vol of 1.2 N NaOH/0.5 M HEPES solution. Volumes
of 200 µl of standard or activated samples were added per well to a
96-well microplate coated with soluble TGF-
receptor type II. The
microplate was then incubated at room temperature for 3 h. After three
washings with wash buffer (0.05% Tween-20 in 1× PBS), 200 µl
of horseradish peroxidase-conjugated polyclonal anti-TGF-
1 antibody
(1:1,000 dilution) was added to each well and incubated at room
temperature for further 90 min. A volume of 200 µl of substrate
solution (1:1 mixture of
H2O2
and the chromogen tetramethylbenzidine solution) was added to each well
after three washings with wash buffer. The reaction was stopped by
adding 50 µl of 2 N sulfuric acid to each well. The optical density
of each well was determined within 30 min using a microplate reader
(Titertek MultiScan MCC/340; ICN, Costa Mesa, CA) set to take dual
endpoint readings at 450 nm and a reference wavelength of 540 nm. The
standard curve was generated using four parameter logistic curve fit,
and from this curve concentrations of activated TGF-
1 in various
samples were determined.
Phospho-ERK immunoblots. ERK phosphorylation was assessed using a phosphorylation state-specific ERK antibody (New England Biolabs) which specifically recognizes Tyr204-phosphorylated (but not nonphosphorylated) ERK-1 and ERK-2. The phospho-MAPK antibody was used at 1:1,000 dilution, whereas the control antibody, which recognizes equally well the phosphorylated and nonphosphorylated MAPK, was used at 1:500 dilution per the manufacturer's recommendations. After treatment, cells were scraped into Laemmli buffer, boiled for 3 min, and subjected to SDS-PAGE under reducing conditions with 4-20% precast gels (Novex). After semi-dry transfer to polyvinylidine difluoride membranes, the membranes were blocked with a BLOTTO buffer (5% defatted dried milk in 10 mM Tris, 150 mM NaCl, and 1% Tween-20, pH 8.0). The membranes were incubated overnight with the BLOTTO containing the phospho-MAPK antibody (at 1:1,000 dilution). The membranes were washed, then exposed to goat anti-rabbit alkaline phosphatase-conjugated IgG (1:1,000) in BLOTTO for 1 h, then washed again. Immunoreactive bands were visualized by a chemiluminescent method (CDP Star, New England Biolabs) using preflashed Kodak XAR film.
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RESULTS |
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TGF-1 induction. We
first tested for the induction of TGF-
1 mRNA by 5-HT by ribonuclease
protection assay. Cells treated with 5-HT showed both
concentration- and time-dependent increases in TGF-
1 mRNA levels.
5-HT induced detectable increases in TGF-
1 mRNA at 2 h, which were
maximal after 6 h of treatment (Fig.
1A). At 30 nM, 5-HT induced nearly peak stimulation (Fig.
1B), which coincides closely with
the concentration of 5-HT required for maximal stimulation of the ERKs
(data not shown). The EC50
concentrations for 5-HT activation of ERK and increased TGF-
1 mRNA
were both ~10 nM, suggesting a possible close linkage between their
signal transduction pathways. Although Nebigil et al. (25)
have identified and sequenced a
5-HT2A receptor in mesangial
cells, we verified that the 5-HT2A
receptor was responsible for TGF-
1 mRNA induction. Ketanserin
tartrate, a selective
5-HT2A/5-HT2C
receptor inhibitor, blocked the induction of TGF-
1.
2,5-Dimethoxy-4-iodoamphetamine hydrochloride (DOI HCl), a potent and
selective
5-HT2A/5-HT2C receptor agonist, increased the induction of TGF-
1 (Fig.
1C). Because the
5-HT2C receptor is not present in
these cells (12, 25), the results are consistent with activation of the
5-HT2A receptor.
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Lack of involvement of pertussis toxin-sensitive G
protein and involvement of PKC. Pertussis toxin was
able to block the induction of TGF-1 mRNA by 5-HT only slightly
(Fig.
2A)
under conditions previously shown to cause ADP-ribosylation of nearly
all mesangial cell Gi/o proteins
(12). As the 5-HT2A receptor is
nearly always coupled to activation of PKC, we tested the role for PKC
in the increase of TGF-
1 mRNA in mesangial cells. Phorbol
12-myristate, 13-acetate (PMA), a direct PKC activator, was used to
establish that TGF-
1 mRNA increases can be mediated via PKC
activation. PMA treatment (1 µM for 6 h) increased TGF-
1 mRNA.
Prolonged treatment with PMA for 24 h, which exhausts cells of PKC by
accelerating its degradation, blocked the induction of TGF-
1 mRNA by
5-HT (Fig. 2B). TGF-
1 mRNA
induction by 5-HT as well as by PMA was blocked by 500 nM GF-109203, a
highly selective cell-permeable PKC inhibitor (Fig.
2B). Similarly, 100 µM
(±)-1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride
(H-7), a broad-based cell-permeable serine/threonine kinase inhibitor,
also blocked TGF-
1 mRNA induction by 5-HT (Fig. 2B).
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Involvement of ERKs and MEK1. One hour
of pretreatment with the MEK inhibitor PD-98059, which binds to the
inactive form of mitogen- and extracellular signal-regulated kinase
kinase (MEK1) and prevents its activation, blocked the
stimulation of TGF-1 mRNA nearly to the basal level (Fig.
2C). Similarly, apigenin (4,5,7-trihydroxyflavone) known to inhibit MAPK, also blocked TGF-
1
mRNA induction (Fig. 2C). To test
the specificity of the inhibition of TGF-
1 mRNA by the MEK inhibitor
PD-98059, and by apigenin, we studied the effects of SB-203580, a
highly specific inhibitor of the MAPK homolog p38. This compound had no
effect on TGF-
mRNA induction by 5-HT, whereas it blocked
phosphorylation of p38 by the
5-HT2A receptor (data not shown).
The lack of involvement of
stress-activated/jun-activated
protein kinases (SAPK/JUNK) was demonstrated by an immunoblot assay
showing that 5-HT does not lead to phosphorylation of stress-activated
protein kinase kinase (SEK1/MKK4), which is the functional equivalent
of MEK1 in the SAPK/JUNK pathway (data not shown).
Involvement of ROS in the increase in
TGF- mRNA. ROS derived from NAD(P)H
oxidase have previously been shown to be important for the activation
of MAPKs in mesangial and vascular smooth muscle cells (32, 41, 44).
Because inhibition of MEK and ERK blocked induction of TGF-
1 mRNA by
5-HT, we tested the effects of two antioxidants on the ability of 5-HT
to increase TGF-
1 mRNA. A thiol-based antioxidant
(N-acetyl-L-cysteine,
NAC) blocked the stimulation of TGF-
1 mRNA nearly to the basal level
(Fig.
3A), suggesting that the generation of ROS is critical in the signaling cascade that upregulates TGF-
1 mRNA.
-Lipoic acid
(DL-thioctic acid) (33), a broad
range antioxidant, also significantly reduced the TGF-
1 mRNA
induction by 5-HT compared with the basal level (Fig.
3A). Thus two chemically distinct
antioxidants blocked the upregulation of TGF-
1 mRNA by 5-HT. If 5-HT
induces TGF-
1 mRNA through the generation of oxidant molecules, then
one would expect that application of exogenous ROS should mimic that
effect. Accordingly, various concentrations (300 pM to 1 µM) of
H2O2
were applied to mesangial cells, and after 6 h, the cells were
harvested for analysis by ribonuclease protection assay.
Because
H2O2
is a labile and reactive molecule, the
H2O2
was replenished every hour. The treatment of cells with
H2O2
resulted in upregulation of TGF-
1 mRNA at a threshold level of 1 µM
H2O2
(Fig. 3B).
H2O2
at 1 µM increased TGF-
1 mRNA to a level that was equivalent
to that induced by 5-HT. These studies support a role for ROS as second
messengers in the pathway from the
5-HT2A receptor in mesangial
cells, which results in increased expression of TGF-
1 mRNA.
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NAD(P)H oxidase enzymes have been increasingly implicated as
participants in the generation of ROS in various cell types including the mesangium (32, 41). Thus we tested the effects of two chemical
inhibitors of NAD(P)H oxidase on the induction of TGF-1 mRNA by
5-HT. Diphenyleneiodonium chloride (DPI), which interferes with the
flavin binding site of NAD(P)H oxidase, and
4-(2-aminoethyl)-benzenesulfonyl fluoride HCl (AEBSF), an irreversible
serine protease inhibitor that prevents the activation of NADPH oxidase
by preventing assembly of its subunits (8), were also able to block
TGF-
1 message induction by 5-HT (Fig.
3C).
If 5-HT increases TGF-1 mRNA through the generation of ROS, then we
should be able to detect rapid changes in cellular redox potential
induced by 5-HT. To address this issue, we used a redox shuttle system
consisting of ferrous and ferric iron, and the shuttle compound,
menadione, to facilitate extracellular detection of changes in
intracellular redox states. Figure
4A shows
representative tracings of cell monolayers exposed for five pump cycles
to 1 µM 5-HT in the presence or the absence of cells. In chambers
with cells, 1 µM 5-HT increased the reduction rate by 13 ± 2%
(n = 3), and 5 µM 5-HT increased the
reduction rate by 17 ± 2% (n = 9). In empty chambers, 5-HT elicited no changes in the reducing rate,
ruling out simple chemical interactions of 5-HT with the redox shuttle
system components. In chambers exposed to perfusate not containing the
electron shuttle, menadione, 5-HT did not elicit an increase in
reduction rate, confirming that the shuttle is necessary for reducing
equivalents to be transferred from the intracellular environment to the
extracellular space (Fig. 4B, solid
bar). The selective 5-HT2A
receptor partial agonist DOI increased the reduction rate by 11 ± 2% (Fig. 4A,
n = 6). Application of sodium azide,
which increases cellular oxidative stress by inhibiting the
mitochondrial electron transport chain, also increased the reduction
rate by 11 ± 3% (n = 3),
demonstrating that a receptor-independent oxidative stimulus also
increases the reduction rate. To demonstrate that the increase in
reduction rate is not a peculiarity of the 5-HT2A receptor, we treated cells
with 10 µM ATP to stimulate another endogenous
Gq-coupled purinergic receptor.
ATP increased the reduction rate by 8 ± 1%
(n = 6). The effect of ATP is not simply due to increased energy supplies, as the intracellular concentration of ATP in most cells (1-5 mM) is in vast excess to
the amount that we applied to the mesangial cells.
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Mapping of the relative positions of PKC, NAD(P)H
oxidase, ROS, and ERK in the TGF-1
cascade. Figure 5 shows the
results of mapping studies in which TGF-
1 production was stimulated
by PMA. PMA increased TGF-
1 mRNA by ~1.5-fold, and this increase could be attenuated by at least 60% by PD-98059, DPI, or
NAC. Those studies suggest the placement of MEK and NAD(P)H oxidase downstream of PKC in the TGF-
1 cascade. To confirm this placement, we examined the effects of PD-98059, DPI, or NAC to inhibit the phosphorylation of ERK induced by treatment for 10 min with 1 µM PMA.
PMA increased the phosphorylation of ERK to >500% of control levels,
and this effect was almost completely inhibited by PD-98059, DPI, or
NAC.
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5-HT induced TGF-1 protein
quantitation. Upregulation of most mRNA species is only
important if there is a concomitant increase in the quantities and/or
activities of the proteins that they encode. To determine whether
TGF-
1 mRNA induction is accompanied by a subsequent increase in
TGF-
1 protein, we performed a quantitative sandwich ELISA. Cells
treated with 5-HT showed activated TGF-
1 protein more than six
times the basal level. The increase in the active form of
TGF-
1 protein was blocked by the MEK inhibitor, PD-98059 (Fig.
6A). The
results were similar in cells that were 50% and 80% confluent at 6- and 24-h incubation with 5-HT. Similar results were seen with an
immunoblot of the active form of TGF-
(Fig.
6B).
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TGF-1 autoinduction of
TGF-
1 mRNA. In some cells, TGF-
1 has
been shown to support its own induction. We tested the effects of
TGF-
1 on TGF-
1 mRNA in mesangial cells and found that there was a
concentration-dependent increase in TGF-
1 mRNA (Fig.
7). Thus, in mesangial cells, TGF-
1 has
the potential to autoinduce its own expression.
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DISCUSSION |
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The molecular mechanisms underlying the abnormal proliferation and
fibrosis in chronic renal diseases are only now being unraveled. It is
very likely that dysregulation of ERKs is involved in the early
proliferative phase of chronic renal diseases, whereas TGF- is
likely involved in the later fibrotic phase. Nevertheless, the
relationship between those two phases or their molecular mediators has
remained elusive. What is new about the current work is that we have
described a clear relationship between ERK activity and TGF-
1 mRNA
levels and protein in mesangial cells. In addition, our data also
suggest that ROS play a role in the regulation of TGF-
1 expression
by 5-HT. These findings are particularly interesting in light of prior
evidence that has implicated ROS in the activation of ERKs in mesangial
cells (44).
Our work was facilitated by the use of a single substance, 5-HT, which
has been linked both to abnormal cellular proliferation and to fibrotic
diseases. Serotonin (i.e., 5-HT) is known as a potent mitogen in many
cells, activating cell growth through both pertussis toxin-sensitive
(2, 34) and -insensitive G-proteins (13). It has also been implicated
in fibrotic disorders, like the retroperitoneal fibrosis associated
with methysergide (18), carcinoid heart disease (43), and pulmonary
hypertension/aortic valve disease associated with fenfluramine and
phentermine (6, 19). 5-HT was also recently shown to
increase the expression of collagen in mesangial cells through the
actions of PKC and TGF-1, with TGF-
1 residing downstream of PKC
(20). The potential significance of these observations is enhanced by
an extensive older literature in which 5-HT has been implicated in
various forms of nephropathy (9, 24).
Previous evidence has supported the possibility that various effects of
5-HT are mediated by the cytokine, TGF-1. Zhang et al. (46)
demonstrated in the isolated Aplysia
ganglion that TGF-
1 mediates the effects of 5-HT on long-term
facilitation. Similarly, Pousset et al. (28) showed in rat primary
hippocampal astrocytes that 100 pM 5-HT induced TGF-
1 mRNA in 4 h in
vitro. While this report was under review, Masaya et al. (20) linked 5-HT to production TGF-
1 and type IV collagen in mesangial cells. In
the present studies, we have demonstrated that rat mesangial cells
respond to 5-HT treatment (5-HT2A
receptor subtype) in a concentration- and treatment time-dependent
manner by inducing TGF-
1 mRNA. We further demonstrated that an
increase in TGF-
1 mRNA levels is accompanied by increased TGF-
1
protein levels. 5-HT-treated cells showed TGF-
1 protein levels that
were as high as six times the basal values, as detected by Western blot
and ELISA. This is an essential finding that validates the significance of the increased mRNA levels.
Several groups previously showed that thromboxane (38, 39) increases
TGF-1 through pathways that involve PKC and ROS in mesangial cells,
whereas 5-HT increases TGF-
1 through PKC (20). Moreover, it is
already known that PKC is a critical intermediate in the activation of
proliferation in mesangial cells by the
5-HT2A receptor (11, 30).
The increase in TGF-1 mRNA by PMA, a direct activator of PKC, and
the blockade of 5-HT-induced increases in TGF-
1 mRNA by PKC
inhibitors, supported the involvement of PKC in the regulatory pathway
initiated by 5-HT. The main data linking ERK with TGF-
1 induction
are provided by the blockade induced by PD-98059, a specific inhibitor
of MEK1, and by apigenin, an inhibitor of ERKs. The specificity of the
involvement of the ERK type of MAPK was demonstrated by lack of
involvement of SAPK/JUNK or of p38 MAPK. These studies link a specific
mitogenic cascade (MEK1
ERK) with a fibrogenic cascade
(TGF-
1) in rat mesangial cells.
We tested a potential role for ROS in the induction of TGF-1 mRNA by
5-HT. Four lines of evidence supported a role for ROS in this process.
First, the induction of TGF-
1 mRNA was attenuated by treating cells
with two structurally distinct thiol antioxidants (NAC; and the reduced
form of
-lipoic acid) prior to stimulation with 5-HT. NAC serves as
an antioxidant by directly protecting sulfhydryl groups from oxidation
or indirectly by serving as a precursor for the synthesis of
glutathione, an abundant endogenous cellular reducing antioxidant, and
by recycling other antioxidants (27, 33).
-Lipoic acid is a
scavenger of hydroxyl radicals, singlet oxygen, and hypochlorous acid
and may exert antioxidant effects by chelation of transition metals
(33).
The second line of evidence supporting the involvement of ROS is that
direct application of the weak oxidant,
H2O2,
also induced TGF-1 mRNA to a degree similar to that demonstrated for
5-HT. This effect was seen at a low threshold
concentration of 1 µM, suggesting that this effect is not secondary
to nonspecific cellular toxicity. The third line of evidence is
provided by experiments demonstrating that the induction of TGF-
1
mRNA could be blocked by two structurally distinct chemical inhibitors
of NAD(P)H oxidase, which is a major enzymatic generator of cellular
oxygen free radicals. Both DPI (a flavin site blocker)
and AEBSF [which prevents assembly of the functional NAD(P)H
oxidase enzyme] also decreased the TGF-
1 mRNA induction by
5-HT. The fourth line of evidence demonstrated that 5-HT rapidly alters
mesangial cell redox as assessed by microphysiometry. Mapping studies
strongly supported a relative position of the NAD(P)H oxidase and ROS
in the signaling cascade as follows:
5-HT2A receptor
PKC
NAD(P)H oxidase/ROS
MEK
ERK
TGF-
1 mRNA. However, because PD-98059, a specific inhibitor of MEK1,
completely blocked the phosphorylation of ERK induced by PMA, but only
attenuated the increase in TGF-
1 mRNA induced by PMA by ~60%
(Fig. 5), it is possible that a PKC-driven,
MEK1-independent pathway also exists. Based on our studies, and those
of others (14, 16, 20, 22, 30, 39), we propose a signal transduction
model as depicted in Fig. 8.
|
A final point of interest is the ability of TGF-1 to induce its own
mRNA in mesangial cells. This finding supports the possibility that an
initial mitogenic exposure to a substance such as 5-HT could lead to
proliferation via direct activation of ERKs and secondarily to generate
a sustained fibrotic stimulus by inducing TGF-
1 expression which in
turn is able to sustain its own induction. Overall, these studies
provide a potential link between the mechanisms that induce
proliferation and those that induce and sustain fibrosis in renal
mesangial cells.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Georgiann Collinsworth and Pamela Wackym for excellent technical assistance and Dr. Baby G. Tholanikunnel for helpful suggestions.
![]() |
FOOTNOTES |
---|
This work was supported by Dept. 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 laboratory endowment jointly supported by the MUSC Division of Nephrology and Dialysis Clinics, Inc. (to J. R. Raymond), fellowships from the American Heart Association (to Y. V. Mukhin and J. S. Grewal), and by MUSC Univ. Research Foundation awards (to E. L. Greene and M. N. Garnovskaya).
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 Univ. of South Carolina, 171 Ashley Ave., Charleston, SC 29425 (E-mail: greeneel{at}musc.edu).
Received 7 October 1998; accepted in final form 17 March 1999.
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