Modulation of renal tubular cell function by RGS3
W.
Grüning1,
T.
Arnould1,
F.
Jochimsen1,
L.
Sellin1,
S.
Ananth1,
E.
Kim2, and
G.
Walz1
1 Renal Division, Department of
Medicine, Beth Israel Deaconess Medical Center, Harvard Medical
School, Boston 02215; and
2 Laboratory of Molecular and
Developmental Neuroscience, Massachusetts General Hospital, Harvard
Medical School, Boston, Massachusetts
02114
 |
ABSTRACT |
The recently
discovered family of regulators of G protein signaling (RGS)
accelerates the intrinsic GTPase activity of certain G
subunits,
thereby terminating G protein signaling. Particularly high mRNA levels
of one family member, RGS3, are found in the adult kidney. To establish
the temporal and spatial renal expression pattern of RGS3, a polyclonal
antiserum was raised against the COOH terminus of RGS3. Staining of
mouse renal tissue at different gestational stages revealed high levels
of RGS3 within the developing and mature tubular epithelial cells. We
tested whether RGS3 can modulate tubular migration, an important aspect
of tubular development, in response to G protein-mediated signaling.
Several mouse intermedullary collecting duct (mIMCD-3) cell lines were
generated that expressed RGS3 under the control of an inducible
promoter. Lysophosphatidic acid (LPA) is a potent chemoattractant that
mediates its effects through heterotrimeric G proteins. We found that
induction of RGS3 significantly reduced LPA-mediated cell migration in
RGS3-expressing mIMCD-3 clones, whereas chemotaxis induced by
hepatocyte growth factor remained unaffected by RGS3. Our findings
suggest that RGS3 modulates tubular functions during renal development
and in the adult kidney.
renal tubular epithelial cells; kidney development; chemotaxis; G
protein; regulators of G protein signaling
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INTRODUCTION |
RGS PROTEINS belong to a gene family with structural
and functional homologies sharing a region of ~125 amino acids, the
regulator of G protein signaling (RGS) domain, which accelerates the
intrinsic GTPase activity of certain G
subunits (reviewed in Refs.
3, 19, 20, 32, 33). Members of this protein family have been shown to
regulate pheromone signaling in yeast and a variety of G protein-linked
signaling pathways in mammalian cells. RGS proteins stabilize activated
G
subunits in a conformation that favors the release of bound GTP
(4, 57). In addition, RGS proteins appear to antagonize G protein
signaling through a direct interaction with G
and G
subunits (30,
54, 61). Currently, ~20 different RGS proteins are known (reviewed in
Ref. 3). RGS proteins are widely expressed and can be found in
virtually all organs (6, 15, 16, 22, 29, 34, 51, 52). Only a few family
members display a restricted expression pattern; for example, RGS7 is found predominantly in the brain and lung (28, 34), RGS8 is found
almost exclusively in the brain (28, 49), and RGS9 is expressed only in
the retina and in the brain (28, 29). Although RGS3 is expressed in
several organs, including heart, skeletal muscle, lung, liver, and
brain (7, 22, 28, 62), RGS3 exceeds the expression of other RGS family
members in the kidney (22, 34, 62). In contrast, RGS2, RGS4, RGS5,
RGS7, RGS9, RET-RGS1, RGS12, RGS14, and RGS16 are absent or only
expressed at low levels in the kidney in comparison with other organs
(10, 22, 25, 29, 34, 51, 53, 62).
RGS3 encodes a protein of 519 amino acids with a relative molecular
mass of 57 kDa (22) and has been implicated in the regulation of
several signaling cascades. Expression of RGS3 impairs interleukin-8 receptor and lysophosphatidic acid (LPA)-mediated ERK1/ERK2 activation (9, 22), modulates the G protein-gated inwardly rectifying potassium
channel (21), and inhibits the
Gq
-coupled
production of inositol phosphate (9, 41) as well as the
Gs
-coupled production of cAMP (9), although the mechanism by which RGS3 impairs
Gs
-coupled
signaling remains unclear. Intrigued by the high expression levels of
RGS3 in the kidney, we decided to analyze the spatial and temporal
expression of RGS3 during renal development. A polyclonal antiserum
raised against the COOH-terminal 100 amino acids of RGS3 revealed a
predominantly tubular expression pattern during renal embryogenesis. To
examine the effect of RGS3 on tubular epithelial cell motility, a mouse
intermedullary collecting duct (mIMCD-3) cell line was generated that
expressed RGS3 under the control of an inducible promoter. With the use
of this model system, we demonstrated that RGS3 modulates tubular cell
migration in response to heterotrimeric G protein signaling.
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MATERIALS AND METHODS |
Plasmids and reagents. The cDNA of the
full-length human RGS3 (kindly provided by K. M. Druey) was fused to a
Flag-His tag sequence MLDYKDDDDKHHHHHHHHH and subcloned
into CDM8 (F.RGS3). A fragment containing the COOH-terminal 300 bp was
generated by PCR and cloned into pMAL-c3 (New England Biolabs, Beverly,
MA) to produce the maltose-binding protein (MBP) fusion protein
MBP-RGS3421-520. For the
generation of an inducible, stable cell line, RGS3 followed by the
encephalomyocarditis virus (EMCV) internal ribosome entry site
(EMCV-IRES) (kindly provided by P. Sarnow) and the green fluorescent
protein (gfp) (kindly provided
by B. Seed) were subcloned into the expression vector pIND (Invitrogen,
San Diego, CA). A control pIND vector was generated containing the
EMCV-IRES followed by gfp, but lacking
RGS3. LPA, epidermal growth factor (EGF), angiotensin II, bradykinin,
and wortmannin were obtained from Sigma (St. Louis, MO). Pertussis
toxin (PTX) was obtained from GIBCO, Life Technologies (Gaithersburg,
MD), and L. G. Cantley kindly provided hepatocyte growth factor (HGF).
LPA was dissolved in chloroform:methanol:acetic acid
(90:5:5) and further diluted in DMEM. HGF was dissolved in
DMSO. Solvents alone were used in control experiments at the
appropriate final concentrations.
Generation and affinity purification of an antiserum
specific for RGS3. Gel-purified
MBP-RGS3421-520 was used to
immunize rabbits (Cocalico Biologicals, Reamstown, PA) with a standard
immunization protocol. For affinity purification of the rabbit
antiserum, recombinant
MBP-RGS3421-520 was coupled
to AminoLink Plus (Pierce, Rockford, IL), and RGS3-specific antiserum
was eluted from the AminoLink column with 0.1 M glycine-HCl (pH 3.0)
and dialyzed against PBS. The concentration of the affinity-purified RGS3 antiserum was determined by ELISA.
Western blot analysis. Western blot
analysis was performed to confirm the specificity of the RGS3
antiserum. Human embryonic kidney cells (HEK 293T) were transiently
transfected with plasmids encoding Flag-tagged hRGS3, hRGS7, or vector
(pCDM8) with the calcium phosphate method. After 24 h of incubation,
cells were harvested and lysed in sample buffer. The samples were
fractionated by SDS-PAGE and transferred to a polyvinylidene difluoride
membrane (NEN Biological Products, Boston, MA). The membrane was
incubated with affinity-purified anti-hRGS3 antiserum, followed by
horseradish perioxidase-coupled anti-rabbit IgG (Amersham, Life
Science, Arlington Heights, IN) and ECL (SuperSignal, Pierce). Blocking
experiments were performed with recombinant
MBP-RGS3421-520. The
expression of hRGS3 in the inducible mIMCD-3 cell lines was determined
at 24 and 56 h after induction with 1 µM muristerone A.
Immunohistochemistry. Mouse (Swiss
Webster) fetal kidneys (days
E14-E18,
newborn and adult) were fixed in 4% paraformaldehyde (Sigma) in
phosphate buffer (pH 7.4) for 30-90 min and incubated in 30%
sucrose (Acros Organics, NJ) in phosphate buffer (pH 7.4) overnight at
4°C. The tissue was embedded in OCT compound (VWR Scientific, Bridgeport, NJ), and 8-µm sections were prepared at
24°C. The sections were initially incubated in TBS, pH 7.6, and endogenous peroxidase was blocked with 1%
H2O2
(Sigma) in methanol (Fisher Scientific, Pittsburgh, PA) for 30 min,
rinsed in deionized H2O, followed by two
washes in TBS for 5 min. For antigen retrieval, sections were heated to
90°C in ACCU-TUF (Accurate Chemicals, Westbury, NY) for 10 min,
cooled for 10 min, and washed again. Heat-inactivated normal goat serum
(Sigma) was used at 5% in TBS, pH 7.6, for 30 min to block unspecific
bindings. Tissue sections were incubated with anti-RGS3 or preimmune
rabbit IgG at a concentration of 10 or 20 µg/ml in TBS, pH 7.6, containing 1% normal goat serum for 60 min at room temperature or
overnight at 4°C. After two rinses in TBS on an orbital shaker for
8 min, the sections were incubated with an anti-rabbit IgG-biotinylated
secondary antibody (Vector Laboratories, Burlingame, CA) at a 1/200
dilution in 50 mM Tris, pH 7.6, for 30 min. Regular ABC Reagent (Vector
Laboratories) was added to slides for 30 min. Staining was developed
with diaminobenzidine (Research Genetics, Huntsville, AL),
and counterstaining was performed with 1× hematoxylin (Harris
Formula, Surgipath, Richmond, IL). Sections were dehydrated in
75-100% ethanol, saturated with xylene (Fisher Scientific), and
mounted with xylene-based Cytoseal (Stephen's Scientific, Riverdale,
NJ). The same procedure was used to detect RGS3 in the inducible stable
cell lines at 24 or 48 h after induction.
Generation of stable mIMCD-3 cell
lines. The mIMCD-3 (48) was cultured in DMEM
(Bio-Whittaker, Walkersville, MD) supplemented with 8% calf serum
(Sigma). To avoid adverse effects by RGS3 during cell propagation and
phenotypic changes during selection, an inducible gene expression
system for mammalian cells was utilized (Ecdysone System, Invitrogen,
San Diego, CA). The pVgRXR vector directing the expression of the
heterodimeric ecdysone-retinoid receptor (VgEcR/RXR) was transfected
into mIMCD3 cells, followed by a selection in zeocin at 25 µg/ml.
Positive clones were identified with a pIND reporter plasmid that
mediates the expression of gfp.
Expression of GFP was induced by muristerone A (1 µM), a steroid
analog that binds to the heterodimeric nuclear VgEcR receptor and
mediates the recognition of a modified ecdysone-responsive element. A
second transfection introduced the pIND vector directing the expression of RGS3-EMCV-IRES-gfp, or the control
pIND plasmid expressing EMCV-IRES-gfp,
and was followed by a double selection with zeocin and geneticin (400 µg/ml; GIBCO, Life Technologies). Positive clones were identified by
green fluorescence after induction with 1 µM muristerone A.
Chemotaxis assays. The chemotaxis
assay was essentially performed as described (17). A modified Boyden
chamber (Neuro Probe, Cabin John, MD) with 48 single wells was used to
test for gradient-directed cell movement activated by different
chemoattractants or control medium (DMEM). Polycarbonate membranes
(8-µm pore size; Poretics, Livermore, CA) were coated with rat tail
collagen type I (Upstate Biotechnology) and washed in PBS. DMEM
containing HGF, LPA, FBS, angiotensin II, bradykinin, EGF, or solvent
controls was added to the bottom chamber and overlaid with the
membrane. Cells (2 × 104)
were added to the upper chamber. Incubation was carried out at 37°C
for 4 h. The membranes were then removed, fixed, and stained with
Diff-Quik (Baxter Healthcare, Miami, FL). Cells that had passed through
the pores of the membrane were counted by light microscopy. The mean
value ± SD of each condition (n = 6-12) was determined per square millimeter. PTX or wortmannin was
added to the cell culture 1 h before assay and was added at the same concentration to both the upper and the lower chamber during the assay.
Proliferation assays. The mIMCD-3 cell
lines were seeded at a density of 12,500 cell/well in 12-well plates
and incubated overnight in DMEM containing LPA, EGF, angiotensin II,
bradykinin, or HGF or serum at concentrations as indicated. Cells were
pulsed with 1 µCi of
[3H]thymidine (NEN)
per each well. After 24 h, cells were washed with cold PBS, air-dried,
and lysed with 200 µl of 1.5 N sodium hydroxide for 30-60 min at
37°C. Samples of 100 µl were added to 5 ml ScintiVerse (Fisher,
Fair Lawn, NJ), and counts per minute were measured with a Tri-Carb
Liquid Scintillation Analyzer 2200CA (Packard, Meridawn, CT). PTX or
wortmannin was added for 18 h before cell harvesting as indicated. In
selected experiments, [3H]thymidine
incorporation was followed for up to 5 days.
Cell viability. Cell viability was
assessed with trypan blue exclusion. Whereas higher pertussis
concentrations exerted a toxic effect, >95% of cells remained viable
at PTX concentrations <150 ng/ml. No toxic effects were observed for
wortmannin at 10 nM.
Statistical analysis. Data are
presented as means ± SE and represent at least three independent
experiments; statistical significance was determined by the Student's
t test.
 |
RESULTS |
RGS3 is expressed in the embryonal and adult
kidney. Because RGS3 mRNA is expressed at significant
levels in the adult kidney (22), we hypothesized that a more precise
spatial and temporal definition of the renal RGS3 expression would
provide the first clues for its putative function in the kidney. A
COOH-terminal fragment of RGS3 fused to the MBP was used to generate an
antiserum in rabbits. The specificity of the affinity-purified
antiserum was confirmed by Western blot analysis of HEK 293T cells,
transiently transfected with constructs expressing RGS3, RGS7, or pCDM8
vector. As demonstrated in Fig.
1A, the
anti-MBP-RGS3 antiserum specifically recognized RGS3, but not, for
example, RGS7. RGS3 displays an electrophoretic mobility of ~83 kDa,
significantly higher than its predicted molecular mass,
which is potentially due to a high percentage of charged amino acids
(>30%) and/or extensive posttranslational modification. Pretreatment
of the antiserum with MBP-RGS3 completely abolished the detection of
RGS3 (Fig. 1B). With the use of this affinity-purified antiserum, embryonal mouse kidneys at embryonal days
E14 to
E18, newborn and adult mouse kidney,
were analyzed by immunohistochemistry. RGS3 was detectable in the mouse
embryonal kidney as early as E14. Staining was distinct and initially
limited to the apical plasma membrane and the brush border of
developing tubules and S-shape bodies (Fig.
2A).
At day
E16 (Fig.
2B), RGS3 expression intensified and
a more lateral and basolateral staining of tubular structures became
apparent. This staining pattern, involving all tubular structures at
later gestational stages, was detectable at
day
E18 (Fig.
2C), in newborn tissue (Fig.
2D), and in the adult kidney (Fig.
2E). No significant mesangial
staining was detectable at later stages of embryonal development or in the adult kidney.

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Fig. 1.
Specificity of
anti-MBP-RGS3421-520
antiserum. Western blot analysis of human embryonic kidney (HEK 293T)
cells transiently transfected with Flag-tagged RGS3- or RGS7-expressing
constructs or pCDM8 vector. A: RGS3
antiserum recognized RGS3 but did not detect RGS7.
B: detection of RGS3 was inhibited
after incubation of antiserum with recombinant
MBP-RGS3421-520 for 10 and 30 min and completely abrogated after 120 min.
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Fig. 2.
Immunohistochemical pattern of RGS3 expression in mouse fetal and adult
kidney. A: at
day
E14, RGS3 antiserum revealed a
predominant staining of apical tubular structures.
B: at
day
E16, a more basolateral staining of
tubules and a faint staining of mesangial cells was detectable.
Day
E18
(C) and postnatal
day 1 (D) revealed an increasing staining
of proximal tubules and collecting ducts. Uniform staining of virtually
all proximal tubular structures was maintained in adult mouse kidney
(E). No significant mesangial
staining was detectable at this time point.
F: pretreatment of antiserum with
recombinant MBP-RGS3421-520
abrogated any specific staining.
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Generation of an RGS3-expressing mIMCD-3 cell
line. The predominantly tubular expression pattern of
RGS3 prompted us to generate a tubular epithelial cell line that
expresses RGS3. To avoid effects that a negative regulator of G protein
signaling, such as RGS3, might exhibit on cell growth and cellular
phenotype, the inducible ecdysone system was utilized to express RGS3
in a mIMCD-3 cell line in response to muristerone A. Control cell lines
lacking RGS3 were generated in a similar fashion. Screening for
positive clones was facilitated by inserting the
gfp downstream of EMCV-IRES in tandem
with the RGS3 cDNA. After induction, ~95% of cells of an individual
RGS3 (Fig. 3,
A and
B) or control clone (Fig. 3, C and
D) became positive for GFP and
~100% of cells stained positive for RGS3 ~48 h after induction
(Fig. 3, E and
F). There was virtually no staining
in clones before induction (Fig. 3E)
or in control clones (data not shown). Western blot analysis confirmed
the expression of RGS3 protein in the inducible mIMCD-3 cell clones. A
specific band for RGS3 was detectable at 24 and 56 h after induction
(Fig. 4). RGS3 was not detectable in
uninduced cell lines or in cell lines lacking the RGS3 expression
cassette.

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Fig. 3.
Generation and selection of RGS3-expressing mIMCD-3 cell lines.
RGS3-expressing mIMCD-3 clones and control mIMCD-3 clones were
identified by their ability to express green fluorescent protein (GFP).
A and
B: phase-contrast and fluorescent
images, respectively, of an mIMCD-3 cell clone expressing RGS3.
C and
D: phase-contrast and fluorescent
images, respectively, of a control cell clone lacking RGS3. More than
90% of cells were positive for GFP 36 h after induction with 1 µM
muristerone A. Induction of RGS3 and/or GFP was not accompanied by a
detectable change of phenotype of selected mIMCD-3 cell clones.
E and
F: expression of RGS3 before and after
48 h of induction with 1 µM muristerone A, respectively.
Nearly 100% of cells became positive by immunohistochemistry with
anti-MBP-RGS3421-520
antiserum. Control clones were negative for RGS3 before or after
induction (data not shown).
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Fig. 4.
Expression of RGS3 protein in RGS3-mIMCD-3 cell clones. RGS3 was
detectable by Western blot analysis with RGS3-specific antiserum in 2 independent RGS3-expressing mIMCD-3 clones (VI7, VI21) but not in
control clone c16 after 48 h of induction with 1 µM muristerone
A.
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RGS3 inhibits LPA-activated cell movement in mIMCD-3
cells. An important aspect of tubulogenesis requires
the coordinated cellular movement of developing tubular epithelial
cells, and a number of hormones and growth factors have been implicated
to control this process. We analyzed the effect of RGS3 on the
chemotactic behavior of wild-type, control, and RGS3-expressing mIMCD-3
cells exposed to angiotensin II, bradykinin, LPA, EGF, HGF, and serum. All cell lines exhibited a pronounced chemotactic response to LPA, EGF,
HGF, and serum but not to different concentrations of angiotensin II or
bradykinin (Fig.
5A). The
LPA-mediated chemotaxis was concentration dependent, and a maximal
response was found at normal LPA serum concentration ranging from 5 to
20 µM (Fig. 5B); levels over 60 µM inhibited chemotaxis because of toxicity. The migratory responses
of two different control mIMCD-3 cell lines (c16 and c18) lacking the
RGS3 transgene are shown in Fig. 5C.
Induction of these clones with muristerone A for 48 h had no effect on
the chemotaxis in response to serum or LPA. However, the induction of
RGS3 expression in the RGS3-expressing mIMCD-3 cell lines (VI7 and
VI21) by muristerone A resulted in a significant inhibition of
migration in response to LPA (Fig.
5D)
(P < 0.001); in a total of 16 experiments, RGS3 reduced the LPA-mediated migration by 44-58%.
In addition, a less pronounced but reproducible reduction in migration
was also observed in response to the serum; RGS3 expression reduced the
number of migrated mIMCD-3 cells by 12-25% (P < 0.05). In comparison, PTX at
100 ng/ml, a commonly used dose (10, 11) that did not affect the
viability of the migrating mIMCD-3 cells as assessed by trypan blue
exclusion, inhibited both serum and LPA-dependent migration by 83 and
80%, respectively, but had no significant effect on the HGF-mediated
chemotaxis (Fig. 5E). Because PTX
impaired LPA-mediated chemotaxis in a dose-dependent manner (Fig.
5F), it is likely that the effects
of LPA on mIMCD-3 cell movement are
Gi
linked. Interestingly, at an
intermediate concentration of 40 ng/ml PTX that typically exerts
~50% inhibition of LPA-dependent migration (Fig.
5F), PTX did not augment the inhibitory effects of RGS3 on LPA- and serum-dependent chemotaxis (Fig.
5G), supporting the notion that both
PTX and RGS3 inhibit identical,
Gi
-dependent signaling.

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Fig. 5.
RGS3 inhibits lysophosphatidic acid (LPA)-dependent migration of
mIMCD-3 cells. A: LPA at a
concentration of 10 µM (4.4 µg/ml) triggered a vigorous chemotactic
response of mIMCD-3 cells comparable with effects of 40 ng/ml
hepatocyte growth factor (HGF) or 10% FBS. No significant differences
were detected comparing wild-type mIMCD-3 cells (solid bars), uninduced
control (hatched bars), or uninduced RGS3-mIMCD3 cell lines (open
bars). B: LPA-mediated mIMCD-3 cell
migration was concentration dependent. Active concentration range of
LPA matched physiological serum concentration of 5-20 µM. At
higher levels, cell motility decreased because of solvent toxicity.
C: 2 control clones (c16, c18) lacking
RGS3 exhibited no change in serum or LPA-dependent cell movement before
(solid bars) and after (hatched columns) induction with 1 µM
muristerone A. D: expression of RGS3
inhibits LPA-mediated chemotaxis. Induction of RGS3 (hatched bars) in 2 independent RGS3 mIMCD-3 clones (VI7, VI21) resulted in significant
inhibition of migration in response to LPA compared with migratory
response before induction (solid bars,
P < 0.001). Expression of RGS3 also
inhibited migration in response to serum; although this effect was less
dramatic, it was highly reproducible
(P < 0.05).
E: pertussis toxin (PTX) at 100 ng/ml
blocked LPA- and serum-dependent chemotaxis of mIMCD-3 cells (hatched
bars), whereas effects of HGF remained virtually unaffected. Solid
bars, control. F: PTX inhibited
(hatched bars) chemotaxis in response to LPA in a
concentration-dependent fashion. Assessment of cell viability by trypan
blue revealed that >96% of cells remained viable at a PTX
concentration <150 ng/ml. Solid bars, control.
G: PTX at 40 ng/ml failed to augment
inhibitory effects of RGS3 on LPA- or FBS-stimulated mIMCD-3 cells.
Chemotaxis in response to HGF remained unaffected by either PTX or RGS3
(solid and open bars, RGS3-mIMCD-3 cells before and after induction,
respectively; shaded and hatched bars, mIMCD-3 cells before and after
induction in presence of PTX, respectively).
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Effect of RGS3 on DNA synthesis in mIMCD-3
cells. Although RGS3 clearly reduced the migratory
responses of mIMCD-3 cells after activation of G protein-linked
signaling, we were able to demonstrate that this effect was not due to
an unspecific cytopathic effect. We analyzed the proliferative
responses of RGS3-expressing cell lines triggered by serum, LPA, or
HGF. Exponential incorporation of
[3H]thymidine was
observed by wild-type mIMCD-3 cells (data not shown), the control
mIMCD-3 cells, and RGS3-expressing mIMCD-3 cells over a period of
2-5 days at normal serum concentrations (Fig.
6A) or
reduced serum concentrations (Fig.
6B). Whereas the proliferation of mIMCD-3 cells was not affected by LPA at
concentrations between 5 and 40 µM (Fig.
6C), HGF mediated a dose-dependent
proliferative response of mIMCD-3 cells (Fig.
6C). Consistent with previous reports (6), this proliferative response was inhibited by wortmannin, but not by PTX (Fig. 6D) or the
induction of RGS3 expression (Fig. 6E).

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Fig. 6.
RGS3 does not affect DNA synthesis and proliferation of mIMCD-3 cells.
A: RGS3 does not inhibit
serum-mediated proliferation of mIMCD-3 cells.
[3H]thymidine
incorporation was monitored in RGS3-expressing mIMCD-3
cells over a 5-day period. Induction of RGS3 did not significantly
affect proliferative behavior of mIMCD-3 cells (solid and open bars,
control mIMCD-3 cells before and after induction with muristerone A,
respectively; shaded and hatched bars, RGS3-mIMCD-3 cells before and
after induction, respectively). B:
after 48 h, RGS3 does not affect proliferative responses at reduced
serum concentrations (72 h after induction),
[3H]thymidine
incorporation was virtually the same in all 4 groups: control mIMCD-3
cell clone before (solid bars) and after (open bars) induction;
RGS3-mIMCD-3 cell clone before (shaded bars) and after (hatched bars)
induction. C: LPA had no effect on
wild-type mIMCD-3 cells at a concentration from 5 to 40 µM (hatched
bars), whereas HGF increased
[3H]thymidine
incorporation (solid bars). D:
wortmannin (WM; 10 nM) but not PTX (100 ng/ml) blocked
HGF (40 ng/ml)-activated DNA synthesis (filled bars); neither WM nor
PTX affected LPA-mediated (hatched bars) or serum-mediated
[3H]thymidine uptake
(open bars). E: HGF-activated
[3H]thymidine uptake
was not impaired by RGS3 in mIMCD-3 cell lines
(left, control mIMCD-3 cells;
right, RGS3-mIMCD-3
cells) before (solid bars) or after (hatched bars)
induction.
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 |
DISCUSSION |
Most RGS proteins belong to a family of GTPase-activating proteins that
limit signaling through seven membrane-spanning receptors by binding
and inactivating Gi
subunits
(reviewed in Refs. 3, 19, 20, 32, 33). The expression of RGS3 in the
fetal kidney suggests that this protein may regulate important
signaling events during renal development. Several signaling cascades,
including the prostaglandin, endothelin, and renin-angiotensin system,
are linked to seven membrane receptors and their heterotrimeric G proteins and have been implicated in certain aspects of renal development. For example, tubular abnormalities have been demonstrated after inhibition of the renin-angiotensin system by
angiotensin-converting enzyme inhibitors (37), and the temporal and
spatial expression of kallikrein and bradykinin
B2 receptors in the
differentiating epithelium of the distal nephron, the site of kinin
formation, supports the hypothesis that kinins modulate the maturation
of certain nephron segments (23, 24). The importance of prostaglandin signaling during renal development is underlined by the severe renal
dysgenesis observed in cyclooxygenase-2-deficient mice (18, 40).
To test whether RGS3 can modulate a complex tubular function, we
examined the effects of RGS3 on LPA-mediated chemotaxis of tubular
epithelial cells. The phospholipid LPA (1-acyl-glycerol-3-phosphate) activates a specific, seven transmembrane-spanning G protein-coupled receptor leading to remarkably divergent biological responses (reviewed
in Refs. 26, 42). LPA triggers cytoskeletal changes through activation
of Rho (35, 55, 60) that are accompanied by phosphorylation of FAK (12)
and the recruitment of the cytoskeletal proteins talin, vinculin, and
paxillin to the newly formed focal adhesions (1). LPA mediates
Ras-dependent phosphorylation and activation of the mitogen-activated
protein kinases ERK1/ERK2 and is mitogenic for several cells and
tissues (8, 13, 14, 46, 59, 60). LPA functions as a growth and survival
factor for tubular epithelial cells and glomerular mesangial cells and has been shown to regulate the function of Madin-Darby canine kidney
and mesangial cells (2, 27, 31, 36). Finally, LPA induces migration of
fibroblasts and epithelial cells that is, at least in part, mediated by
a pertussis-sensitive G protein subunit (47, 50, 58). LPA is an
intermediate product of the membrane phospholipid metabolism, but can
also be produced by activated platelets and other tissues (reviewed in
Ref. 36), and is present at a concentration of 5-20 µM in normal
serum (38, 39, 42). LPA triggered the chemotaxis of mIMCD-3 cells in a
dose-dependent fashion. We found that induction of RGS3 expression in
this cell line resulted in a ~50% inhibition of the LPA-mediated migration, consistent with the extent of other RGS3-mediated inhibitory events (9, 41). The RGS3-dependent effect on mIMCD-3 cell migration was
highly specific for the G protein-mediated chemotaxis; other potent
chemotactic agents such as HGF and EGF were not affected by the
induction of RGS3 expression. Furthermore, RGS3 failed to affect DNA
synthesis and proliferation of mIMCD3 cells, indicating that the effect
of RGS3 on tubular cell migration was highly selective and not due to
an unspecific cytopathic effect. Our results demonstrate that RGS
proteins, and RGS3 in particular, may regulate G protein-dependent signaling during renal tubular development. Although abrogation of G
protein signaling by deletion of certain G
subunits has not revealed
selective renal abnormalities (43-45), their ubiquitous distribution and participation in a broad range of cellular functions predicts an involvement of G proteins and their regulatory molecules during embryogenesis. Indeed, studies by Stow et al. (56) have demonstrated that Gi
family
members, susceptible to the activity of RGS3, are present in all
tubular segments. Our study provides the first clues that RGS3 may
exert a dual role, regulating complex cellular functions such as
tubular cell migration during embryogenesis, whereas the regulation of
more specialized functions, for example, the modulation of G
protein-regulated ion channels, may predominate in the differentiated,
mature tubular cell.
Bowman et al. (5) have recently demonstrated that RGS1, RGS3, and RGS4,
but not RGS2, can reduce chemotaxis in response to activation of G
protein-coupled receptors. Because most RGS family members bind and
accelerate the intrinsic GTPase activity of
Gi
and
Gq
subunits, it is not
surprising that RGS proteins display overlapping functions. Although it
is currently unknown how the specificity and activity of these proteins
are controlled, several mechanisms have been proposed (reviewed in
Refs. 3, 32). For example, Sst2p, the prototypic yeast RGS, is
regulated at the transcriptional level (reviewed in Ref. 19), whereas a
restricted temporal and spatial expression appears to curtail the
function of other RGS proteins (11, 29). RGS3 is expressed in tubular
epithelial cells during renal embryogenesis and in the adult kidney. It
is therefore conceivable that this family member modulates the
threshold of multiple
Gi
-dependent signaling events
in tubular epithelial cells. Work remains to be done to determine
whether other RGS proteins are present in tubular epithelial cells and
to delineate their the specific functions in the kidney.
 |
ACKNOWLEDGEMENTS |
We are grateful to Kate Spokes and Lloyd G. Cantley for
experimental advice.
 |
FOOTNOTES |
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant RO1-DK-52897 (G. Walz) and a grant
by the Polycystic Kidney Research Foundation (G. Walz). W. Grüning and F. Jochimsen were supported by the Deutsche
Forschungsgemeinschaft (Gr 1612/1-1 and Jo 270/1-1). E. Kim
was supported by National Institute of Mental Health Grant MH-01147.
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: G. Walz, Renal
Div., Dept. of Medicine, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215 (E-mail:
gwalz{at}bidmc.harvard.edu).
Received 27 August 1998; accepted in final form 23 December 1998.
 |
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