1 Department of Medicine and 2 Department of Anatomy, Cell Biology, and Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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Inositol
1,4,5-trisphosphate receptors (IP3Rs) mediate cytosolic
free calcium concentration
([Ca2+]c) signals in response to a
variety of agonists that stimulate mesangial cell contraction and
proliferation. In the present study, we demonstrate that
mesangial cells express both type I and III IP3Rs and that
these receptors occupy different cellular locations. Chronic treatment
with transforming growth factor-1 (TGF-
1; 10 ng/ml, 24 h) leads
to downregulation of both type I and III IP3Rs as measured
by immunoblot and confocal analysis. TGF-
1 treatment does not affect
IP3 levels, and downregulation of type I IP3R
is not due to enhanced degradation of the protein, as the half-life of
type I IP3R is unchanged in the presence or absence of
TGF-
1. Functional effects of TGF-
1-induced downregulation of the
IP3Rs were evaluated by measuring
[Ca2+]c changes in response to
epidermal growth factor (EGF) in intact cells and sensitivity of
[Ca2+]c release to IP3
in permeabilized cells. TGF-
1 pretreatment led to a significant
decrease of [Ca2+]c release
induced by EGF in intact cells and by submaximal
IP3 (400 nm) in permeabilized cells. Total
IP3-sensitive [Ca2+]c
stores were not changed, as assessed by stimulation with maximal doses
of IP3 (10.5 µm) and thapsigargin-mediated calcium
release in permeabilized cells. We conclude that prolonged exposure to TGF-
1 leads to downregulation of both type I and III
IP3Rs in mesangial cells and this is associated with
impaired sensitivity to IP3.
transforming growth factor-1; inositol 1,4,5-trisphosphate
receptors; calcium mobilization; mesangial cells
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INTRODUCTION |
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THE MESANGIAL CELL IS A PIVOTAL cell in the glomerulus
in both normal and diseased states. It forms the stalk of the
glomerular capillary loop and decreases filtration surface area by
contracting in response to vasoconstrictors, such as angiotensin II and
endothelin. These vasoconstrictors stimulate phospholipase C-
(PLC-
)-coupled receptors leading to the formation of inositol
1,4,5-trisphosphate (IP3) and subsequent
IP3-mediated Ca2+ release (4). Proliferation of
mesangial cells is a characteristic finding in many glomerular diseases
and is largely mediated by mitogenic growth factors such as
platelet-derived growth factor (PDGF), epidermal growth factor (EGF),
and fibroblast growth factor (FGF). The mitogenic growth factors also
stimulate formation of IP3, via the
-isoform of PLC
(PLC-
), and consequent IP3-mediated cytosolic free
calcium concentration [Ca2+]c
release (34). IP3 specifically binds to IP3
receptors (IP3Rs), which are located primarily in the
endoplasmic reticulum (4) and possibly in or adjacent to the plasma
membrane (5, 17). A given cell type may contain more than one of the
three major isoforms of the IP3Rs (type I, II, or III) and
moreover, various IP3R isoforms may mediate different
cellular functions. Thus knowing the specific identity, cellular
location, and modulators of the IP3R isoforms is imperative
to understanding their role in both the physiology and pathophysiology
of mesangial cell function.
Transforming growth factor- (TGF-
1) is stimulated in a variety of
kidney diseases characterized by altered mesangial cell function,
including diabetic nephropathy in both its early and later stages (29).
The inhibition of TGF-
1 by neutralizing antibodies prevents
glomerular hypertrophy in the murine model of diabetic kidney disease
(27). Interestingly, despite the fact that diabetes mellitus also
stimulates a variety of vasoconstrictors and mitogenic growth factors
(9, 23), the diabetic glomerulus appears to be in a vasodilated state
(13, 14) with only a mild degree of mesangial cell proliferation (24,
25). Assuming that TGF-
1 overexpression is acting to counteract the
effects of vasoconstrictive and mitogenic growth factors, a possible
mechanism for this action is via inhibition of IP3-mediated
calcium increase in mesangial cells. Previous investigations, from our
laboratory and others, have demonstrated that short-term TGF-
1
treatment (<60 min) inhibits PDGF-induced
[Ca2+]c in mesangial cells (2) and
angiotensin II-induced [Ca2+]c in
vascular smooth muscle cells (39). As the common pathway for the
release of [Ca2+]c by both PDGF and
angiotensin II is at the binding site of IP3 to the
IP3R, we postulated that TGF-
1 may be inhibiting
IP3R function. Recently we demonstrated that TGF-
1
stimulates phosphorylation of the type I IP3R (28) and that
short-term exposure (2-8 h) leads to downregulation of type I
IP3R expression. In the present study, we demonstrate that
mesangial cells contain both type I and type III IP3R
isoforms, each in a different cellular location, and that long-term
exposure (24 h) of TGF-
1 to mesangial cells leads to decreased
expression of both IP3R isoforms. Mesangial cells
pretreated with TGF-
1 were also found to have decreased Ca2+
mobilization in response to EGF in intact cells and to IP3
in permeabilized cells, yet these cells maintained normal
[Ca2+]c stores.
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EXPERIMENTAL PROCEDURES |
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Materials.
[32P] Inorganic phosphate,
[-32P]ATP, and
[
-32P]CTP were from Du Pont de Nemours-New
England Nuclear, Boston, MA. An enhanced chemiluminescence kit was
purchased from Amersham, Arlington Heights, IL. TGF-
1 was purchased
from R&D Systems, Minneapolis, MN. IP3 and thapsigargin
were purchased from Alexis Biochemicals, San Diego, CA. All other
reagents were from Sigma Chemical, St. Louis, MO, unless otherwise noted.
Cell culture. An SV40 transformed murine glomerular mesangial cell line (MMC), which has been previously described (37), was used in these studies. These cells retain many of the differentiated characteristics of mesangial cells in primary culture (37).
Confocal analysis of IP3R isoforms.
MMCs were grown on no. 1 coverslips pretreated with
poly-D-lysine (0.1 mg/ml for 5 min), in DMEM (GIBCO
Laboratories, Grand Island, NY) with 10% FCS. After cells were
adherent, they were rested in DMEM-0% FCS overnight and then exposed
to TGF-1 (10 ng/ml) for 24 h. Cells were then washed with PBS three
times and fixed with 3.7% formaldehyde for 10 min at room temperature.
After repeated washing, cells were permeabilized with 0.05%
Triton-X-100 in PBS (PBS/TX) for 10 min, blocked with 4% normal goat
serum (NGS) in PBS/TX for 10 min, and then incubated with a rabbit
polyclonal antibody to the type I IP3R (Affinity
Bioreagents, Boulder, CO) or with a mouse monoclonal type III
IP3R antibody (Transduction Laboratories, Lexington, KY)
(1:200) for 30 min at 37°C. Cells were washed with PBS/TX, blocked
again with 4% NGS, and the appropriate secondary antibody applied
(fluoroscein-conjugated-goat anti-rabbit or anti-mouse antibody, 1:150
dilution, Rockland, Gilbertsville, PA) for 30 min at 37°C. After
repeated washing, cells were postfixed with 3.7% formaldehyde for 10 min at room temperature, washed, and the coverslips were placed on
glass slides and mounted with SlowFade (Molecular Probe, Eugene, OR).
Slides were visualized with confocal microscopy (courtesy of Dr. James
Keen, Thomas Jefferson University), and representative regions were
photographed. Control cells, stained only with secondary antibody,
showed minimal background fluorescence. To confirm localization of
specific IP3R isoforms an additional rabbit polyclonal
antibody specific to type I IP3R (15) and monoclonal
antibodies specific to type I and III IP3R were also
utilized (31).
Immunoblot analysis of cytosol and membrane fractions for IP3R isoforms. To determine whether the IP3R isoforms were present in membrane or cytosol fractions, MMCs were lysed by freeze-thaw method in a buffer containing 50 mM Tris · HCl (pH 7.2), 150 mM NaCl, 1 mM EDTA, 1 mM polymethylsulfonyl fluoride (PMSF), and 5 µg/ml each of aprotonin and leupeptin three times and centrifuged at 14,000 g for 15 min. The supernatant was designated as the cytosolic fraction. The pellet was then treated with the same buffer plus the addition of 1% (wt/vol) Triton X-100 for 60 min at 4°C. The Triton-X-treated fraction was then centrifuged for 15 min at 4°C, and the supernatant was designated as the membrane fraction. Protein concentration of cytosol and membrane fractions were quantified by the Bio-Rad DC assay (Bio-Rad Labs, Hercules, CA), and 20 µg of protein were resolved on a 7% SDS-PAGE gel, transferred to nitrocellulose, and immunoblotted with a rabbit polyclonal antibody raised to the COOH-terminus of type I IP3R from brain, kindly provided by Dr. Suresh Joseph (15), or mouse monoclonal antibody to type III IP3R (Transduction Laboratories, Lexington, KY). A monoclonal antibody specific to type II IP3R was kindly provided by Dr. Katsuhiko Mikoshiba (31). The primary antibody was then removed, and the membrane was incubated with horseradish peroxidase-conjugated secondary antibody. Immunoreactive bands were detected by using enhanced chemiluminescence.
TGF- regulation of IP3R isoforms.
MMCs grown in DMEM with 10% FCS were harvested and plated onto 100-mm
dishes with growth media. After reaching 80% confluence, cells were
incubated in serum-free DMEM overnight. During the subsequent 24 h of
incubation, cells were treated with TGF-
1 (10 ng/ml) for the last 6 and 24 h, washed with PBS three times, and harvested in lysis buffer
that contained 50 mM Tris · HCl (pH 7.2), 150 mM
NaCl, 1% (wt/vol) Triton X-100, 1 mM EDTA, 1 mM PMSF, and 5 µg/ml
each of aprotonin and leupeptin. All samples, including the control
samples, were harvested after the same overall duration of incubation.
Protein concentration of samples were quantified and equal amounts of
protein were run on a 7% SDS-PAGE gel, transferred to nitrocellulose,
and immunoblotted with the antibodies to the type I (15) or type III
IP3R, as above. To verify equal loading of proteins the
membranes were separately immunoblotted with a monoclonal antibody for
-actin (Sigma Chemical). MMC cells were also grown on coverslips and
treated with TGF-
1 (10 ng/ml, 24 h) prior to confocal analysis with
antibodies to type I (Affinity Bioreagents) and III IP3R,
as described above.
Metabolic labeling and immunoprecipitation of MMC cell extracts. MMCs were grown to confluence in 100-mm dishes with DMEM/10% FCS then rested in DMEM-0% FCS for 24 h. For pulse-chase experiments, the medium was replaced with methionine-free DMEM for 15 min and then 100 µCi/ml Tran 35S-labeled methionine (ICN Biochemicals, Costa Mesa, CA) were added and cells were incubated for 60 min at 37°C. At the end of the labeling period, the medium was removed, the dishes washed twice with ice-cold PBS solution, and chase medium (DMEM with 50 mM cold methionine) was added at the indicated times. The cells were then harvested by washing twice with ice-cold PBS and then scraping cells into 1 ml of lysis buffer, as above. The cells were solubilized on ice for 30 min, and insoluble material was removed by centrifugation for 10 min at 25,000 g. All the extracts were precleared for 2 h by using 25 µl of a 50% (vol/vol) slurry of Staphylococcus aureus cell wall (Calbiochem, La Jolla, CA). Type I IP3R was immunoprecipitated from the labeled extracts by incubation for 4 h with 100 µl of protein A-Sepharose beads, and 100 µg of antibody were raised to the COOH-terminus of type I IP3R from brain (15). The immunoprecipitates were washed once in lysis buffer, containing an additional 0.5 M NaCl and 0.1% SDS, and twice in lysis buffer alone. The labeled proteins were resolved on 7% SDS-PAGE, transferred to nitrocellulose, autoradiographed, and finally immunoblotted with type I IP3R antibody to locate the receptor.
IP3 measurements.
IP3 was measured by using a Biotrak kit from Amersham.
Briefly, MMCs were treated with TGF-1 (10 ng/ml) for various time periods in 100-mm dishes, washed with PBS twice, 500 µl of ice-cold perchloric acid were added to the cell monolayer, and cells were incubated on ice for 20 min. Cells were then scraped into siliconized tubes and centrifuged at 2,000 g for 15 min at 4°C. The
supernatant was neutralized with ice-cold 10 M KOH to pH 7.5 and
centrifuged again at 2,000 g for 15 min at 4°C. The
supernatant was then assayed for IP3, by using the
manufacturer's guidelines.
Fluorescence imaging measurements of
[Ca2+]c
in intact MMCs.
MMCs were grown on coverslips as noted above, and the growth medium was
changed to serum-free DMEM overnight prior to TGF-1 treatment. The
coverslips with control MMCs or TGF-
1 (10 ng/ml, 24 h)-treated MMCs
were then placed in an extracellular medium [2% BSA/extracellular
matrix (ECM)] consisting of 121 mM NaCl, 5 mM NaHCO3, 10 mM Na-HEPES, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, 10 mM glucose, and 2%
bovine serum albumin (BSA), pH 7.4. To monitor
[Ca2+]c cells were loaded with 5 µM fura 2-AM for 30 min in the presence of 100 µM sulfinpyrazone at
room temperature. Sulfinpyrazone was also present during the imaging
measurements to minimize dye loss. Dye-loaded cells were washed
2-3 times with 2% BSA/ECM. Imaging measurements were performed in
ECM containing 0.25% BSA (0.25% BSA/ECM) at 35°C. Fluorescence
images were acquired by using an Olympus IX70 inverted microscope
fitted with a ×40 (UApo, NA 1.35) oil immersion objective and a
cooled CCD camera (PXL, Photometrics) under computer control. The
computer also controlled a scanning monochromator (DeltaRam, PTI) to
select the excitation wavelength (7). Time courses of
[Ca2+]c in individual cells were
calculated from fluorescence image pairs obtained by using 340- and
380-nm excitation (10 nm bandwidth) with a broad-band emission filter
passing 460-600 nm. EGF (Sigma Chemical) and thapsigargin were
added, and direct visualization of cells was performed to monitor
[Ca2+]c. Experiments were carried
out with three different experiments by using 2-3 parallel dishes
per experiment, and 30-50 cells were monitored in each dish.
Permeabilization of MMCs and IP3-mediated calcium
release.
Control MMC or TGF-1 (10 ng/ml, 24 h) treated MMC were harvested by
cell scraping with a rubber policeman and washed three times in a
Ca2+-free buffer (in mM: 120 NaCl, 5 KCl, 1 KH2PO4 and 20 HEPES/Tris, as well as 100 EGTA
µM, pH 7.4). The cells were then resuspended in 1.8 ml of
intracellular medium (ICM), composed of (in mM) 120 KCl, 1 KH2PO4, 10 NaCl, and 20 HEPES/Tris, (pH 7.2),
as well as 1 µg/ml antipain, 1 µg/ml leupeptin, 1 µg/ml
pepstatin, 1 µM Ruthenium Red, ATP regenerating system (5 mM
phosphocreatine, 5 U/ml creatinine kinase, and 2 mM Mg-ATP) and
permeabilized with 25 µg/ml digitonin. The ATP regenerating system
was added to ensure adequate calcium uptake by the ER. The ICM was
pretreated with Chelex to remove contaminating calcium, as previously
reported (12). Fura 2 (1.5 µM), in the free acid form, was then added
to permeabilized cells to monitor [Ca2+]c.
Ca2+ measurements were performed by using a similar
protocol as described previously (11). IP3 was added at two
doses (400 nM and 10.5 µM) to evaluate if TGF-
1 pretreatment
affects sensitivity to IP3R. Thapsigargin was added after
IP3 to measure total calcium stored in ER. Ionomycin was
added to release any other cellular stores of calcium.
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RESULTS |
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MMCs express both type I and III IP3R isoforms.
By using specific antibodies for type I and type III IP3R
in confocal analysis, a clear distinction of the intracellular
distribution of type I and type III IP3R isoforms can be
demonstrated (Figure 1A and
C). Type I IP3R is localized to discrete structures
primarily in a perinuclear distribution (Figure 1A). Type III
IP3R is in a diffuse homogeneous distribution, apparently
throughout the cytoplasm (Figure 1C). As the cells were fixed
prior to permeabilization, intracellular sites were accessible to the
antibodies. Similar immunostaining patterns were obtained with a
separate rabbit polyclonal antibody specific to type I IP3R
(15) and monoclonal antibodies to type I and III IP3Rs (31)
(data not shown). Cell fractionation studies were performed and crude
membrane fractions and cell lysates were analyzed by Western
immunoblotting (Figure 2). Both the type I
and III IP3R isoforms were present exclusively in the
membrane fraction. Of note, MMC did not express type II
IP3R by immunoblotting (data not shown). Thus the combined
analyses from confocal and cell fractionation/immunoblotting show that
the type I IP3R was present in the membrane fraction of
perinuclear structures and apparently in a different subcellular site
than type III IP3R.
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TGF-1 treatment decreases both type I and III
IP3R isoforms after 24 h.
MMC were treated with TGF-
1 for various time points and the protein
expression of IP3R isoforms was assessed by immunoblot (Figure 3) and confocal analysis (Figure
1B and D). After 6 h of TGF-
1 treatment (Figure 3,
top), there was a reduction of type I IP3R
protein, confirming our previous observations (28); however, type III
IP3R was not reduced at this time point (Figure 3,
middle). Equivalent amounts of total protein lysates were
loaded in each lane as evidenced by immunoblotting with an antibody
against
-actin (bottom). With 24 h of TGF-
1 treatment,
both type I and type III IP3R protein levels were reduced
compared with control values. By confocal analysis (Figure 1B),
the distribution of type I IP3R remains in discrete
perinuclear structures but with decreased intensity of staining. Type
III IP3R remained in a diffuse distribution throughout the
cell, yet also showed decreased staining intensity after 24 h of
TGF-
1 treatment (Figure 1D).
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The protein half-life of type I IP3R is not reduced by
TGF- treatment.
We had previously found that TGF-
reduces steady-state expression of
type I IP3R mRNA levels within the first 4 h of treatment (28), suggesting that an important effect of TGF-
1 was to reduce synthesis of type I IP3R. To establish whether degradation
of type I IP3R protein was involved as well, we performed
35S-methionine pulse-chase experiments to establish the
half-life of type I IP3R in MMC (Figure
4). The half-life under control conditions
was 2 h and was unchanged with TGF-
1 treatment. In addition, we
measured whether TGF-
1 would affect IP3 levels. TGF-
1
treatment had no effect on IP3 levels in MMCs at early time
points (10-60 s) or at greater durations (2-6 h) (data not shown). Thus the mechanism of TGF-
1-induced reduction of type I
IP3R in mesangial cells was not due to enhanced degradation of the protein and was unrelated to increases in IP3
levels.
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EGF-induced
[Ca2+]c
responses is attenuated in mesangial cells pretreated with
TGF-1.
To demonstrate that reduction of the IP3Rs by TGF-
1
would lead to a reduction in [Ca2+]c
signals evoked by an agonist that stimulates IP3
production we measured [Ca2+]c in
MMCs in response to EGF. Basal levels of
[Ca2+]c prior to addition of EGF
were not significantly different in control cells and cells pretreated
with TGF-
(97 ± 9 vs. 105 ± 6 nM, respectively). As noted in
Figure 5, EGF increases
[Ca2+]c under control conditions;
however, the degree of EGF-stimulated [Ca2+]c rise was reduced by 73% in
MMCs pretreated with TGF-
1. Analyzing individual cells, by using a
threshold of calcium mobilization of 10% of the thapsigargin-induced
[Ca2+]c rise, we found that 82%
(334/408) of cells responded to EGF under control conditions whereas
only 26% (98/371) of cells pretreated with TGF-
1 responded to EGF.
A similar degree of inhibition of Ca2+ release was noted in
cells pretreated with TGF-
1 when PDGF was substituted for EGF (data
not shown).
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Sensitivity of IP3Rs to IP3 in permeabilized
mesangial cell.
To determine whether the reduction in Ca2+ mobilization
after 24 h of TGF-1 treatment is due to reduction of
IP3R expression, we permeabilized cells with digitonin and
exposed them to varying concentrations of IP3 in cell
suspensions. Ca2+ release was measured with fura 2 added to
the intracellular medium. As shown in Figure
6, pretreatment of MMCs with TGF-
1 led
to a 50% reduction in IP3-mediated calcium release at a
dose of 400 nM of IP3. Maximal dose of IP3
(10.5 µM) resulted in a similar release of calcium in both control
and TGF-
1 treated cells. Addition of the sarcoplasmic or endoplasmic
reticulum Ca2+-ATPase (SERCA) Ca2+ pump
inhibitor, thapsigargin (to deplete the entire endoplasmic reticulum
calcium store), also led to an equivalent release of calcium in both
control and TGF-
1-treated cells by blocking the pathway of calcium
reuptake into the endoplasmic reticulum. Addition of the calcium
ionophore, ionomycin, further released additional sites of sequestered
intracellular calcium and was again unchanged by TGF-
1
treatment. Thus the size of the total IP3-sensitive and IP3-insensitive Ca2+ stores is unchanged
with TGF-
1 treatment. However, the decreased protein expression of
both type I and III IP3R by TGF-
1 treatment is
associated with a specific reduction in submaximal
IP3-mediated calcium release.
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DISCUSSION |
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In this report, we have demonstrated that mesangial cells express type
I and III IP3Rs in different subcellular locations and that
both are reduced by chronic TGF-1 treatment. The mechanism of
reduction of type I IP3R appears to be solely at
the level of synthesis, as the half-life of the protein was unchanged
by TGF-
1 treatment, and we have previously demonstrated reduction of
type I IP3R mRNA by TGF-
1 treatment (28). The functional consequence of the effect of TGF-
1 on IP3Rs is evidenced
by the demonstration that mesangial cells pretreated with TGF-
1 have reduced Ca2+ mobilization in response to agonists that
normally raise intracellular IP3 levels. Furthermore,
reduction of IP3R isoforms by TGF-
1 was associated with
a reduced sensitivity of IP3-mediated Ca2+
release in permeabilized mesangial cells. Total stores of IP3
and non-IP3-mediated calcium stores, however, were
not affected by TGF-
1 treatment.
Regulation of [Ca2+]c via the
IP3R is an important initial step in transducing the
signals of a variety of factors that lead to a coordinated response.
Recently, it has been demonstrated that both the absolute magnitude of
the initial calcium transient and the frequency of oscillations of
calcium are important determinants of the effects of calcium-mediated
actions on cell function (8, 18, 32). It is believed that most
IP3Rs reside in the membrane of the endoplasmic reticulum
and likely control discrete functional calcium pools (3, 21). In a
model (see Figure 7) where there is an
excess of IP3Rs per calcium pool, a submaximal increase in
IP3 levels may be sufficient to interact with at least one of these receptors and thus discharge all the calcium present in that
pool. However, if the protein level of IP3Rs is also a rate-limiting step, then it would follow that submaximal levels of
IP3 would be insufficient to reach the few
IP3Rs present and that many more calcium pools would be
left untouched. Such a model fits well with our data. We find that a
submaximal dose of IP3 mobilizes a fixed amount of calcium
in untreated, permeabilized cells but that IP3 sensitivity
is reduced in TGF-1 pretreated, permeabilized cells. As cells are
permeabilized, we have bypassed all steps from the cell membrane to
generation of IP3 in this system. The lack of
difference of calcium rise noted with maximal IP3 dose
suggests that each calcium pool contains at least one IP3R
and that the remaining IP3Rs are functioning appropriately. Similar data were reported by Bokkala et al. (6) in hepatocytes. In
their study angiotensin II reduced type I and type III IP3R protein levels by 80%, calcium release to submaximal doses of IP3 was reduced by 50%, but calcium release to maximal
dose of IP3 was not altered, compared with control (6). In
the present study, we also found that the calcium pools controlled by
the SERCA pumps are unchanged between control and TGF-
1-pretreated cells, demonstrating that the Ca2+ pools themselves are
unaltered. In addition, ionomycin-induced calcium release is unchanged
in TGF-
1 treated cells, demonstrating that total intracellular
amounts of calcium are not altered.
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Several studies have demonstrated that IP3R expression is
reduced by agonists such as carbachol in neuroblastoma cells (35, 36),
angiotensin II in hepatic cells (6), and vasopressin in a vascular
smooth muscle cell line (30). It is likely that the mechanisms
underlying the effects of these agonists are similar, as they all raise
intracellular IP3 and promote degradation of IP3R protein. In the latter two studies (6, 30),
proteasomal degradation of type I IP3R was demonstrated
with the use of proteasomal inhibitors. Although in a prior study with
Rat-1 fibroblasts, TGF-1 treatment induced an increase in
IP3 levels (22), we find that IP3 levels are
not elevated in mesangial cells implying a different mechanism for
TGF-
1 induced downregulation of the IP3R. However, the
use of 35S-methionine pulse-chase experiments may not
clearly demonstrate regulation of protein within the first hour after
labeling, as intracellular labeled 35S-methionine continues
to be incorporated into protein even with cold methionine in the
medium. Thus it is conceivable that there may be both enhanced
degradation of existing type I IP3R protein and enhanced
synthesis of new IP3R protein with short-term TGF-
1 treatment. Further studies are required to evaluate these short-term effects of TGF-
1. It is interesting to note, that although protein degradation was enhanced by both carbachol and vasopressin, these agents also inhibited mRNA expression of type I IP3R (35)
and/or inhibited the promoter activity of type I IP3R (30).
In addition, the half-life of type I IP3R is substantially
shorter (2 h) in MMCs, compared with the half-life reported in
hepatocytes and smooth muscle cells (8-12 h) (16, 30). As the MMC
is an immortalized cell line, this may affect type I IP3R
protein turnover (33).
Recent analysis of rat kidney tissue for IP3R isoforms has demonstrated that the glomerulus contains only type I and type III IP3R and not type II IP3R (20, 38). This fits well with our data in MMCs as we found expression of both type I and III IP3R but were unable to demonstrate type II IP3R by immunoblotting. Several studies have suggested that the various IP3R isoforms are located at different intracellular sites and thus may control different cellular functions. For example, in B cells, type III IP3R is primarily at the cell membrane and may be important in controlling calcium influx and mediating apoptosis (17). Further data to support the concept that there are distinct IP3R isoforms at different sites are supplied by the demonstration that submaximal doses of IP3 stimulate calcium release from intracellular storage sites, whereas high doses of IP3 mediate calcium influx across the plasma membrane (26). It has also been found that plasma membrane-derived IP3R differ from intracellular IP3R with relation to Ca2+ and ATP sensitivity (19). Additional studies are required to establish the precise intracellular sites of the IP3R isoforms in mesangial cells and to determine their specific functional roles.
Several studies have demonstrated that TGF-1 affects intracellular
calcium in a variety of cell types. In Sertoli cells, TGF-
1 enhances
calcium influx after several hours of treatment (1-4 h) but
inhibits follicle-stimulating hormone-induced increase of
[Ca2+]c (10). Calcium influx was
also stimulated by TGF-
1 (>60 min) in Rat-1 cells (22) and NIH3T3
cells (1). In vascular smooth muscle cells, short-term TGF-
1 (30 s)
treatment enhanced angiotensin II-induced
[Ca2+]c but longer durations of
TGF-
1 treatment (>30 min) reduced angiotensin II-induced
[Ca2+]c (39). In our prior study
with MMC, TGF-
1 treatment for 15-30 min markedly reduced
PDGF-induced [Ca2+]c increase (2).
Although, the above studies found that both calcium influx and release
were modulated by TGF-
1, the study designs did not exclusively test
one component vs. the other. In the present study, by using a
permeabilized cell system where only calcium release is being measured,
we demonstrate that chronic TGF-
1 treatment reduces the sensitivity
of IP3-regulated Ca2+ stores.
The major implication of our data is that agonists that stimulate
IP3 would not lead to maximal Ca2+ release in
mesangial cells exposed to a high local concentration of TGF-1. Thus
dysregulation of intracellular Ca2+ by chronic upregulation
of TGF-
1 in glomerular diseases would limit mitogenic and
vasoconstrictive effects and may channel the mesangial cell to a fate
of cell hypertrophy and vasodilation.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-02308 and American Diabetes Association Research Award (to K. Sharma), and (NIDDK) Grant DK-51526 (to G. Hajnóczky). The work was performed at the Dorrance H. Hamilton Laboratories of Thomas Jefferson University.
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FOOTNOTES |
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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: K. Sharma, Div. of Nephrology, Dept. of Medicine, Thomas Jefferson Univ., Suite 353, JAH 1020 Locust St., Philadelphia, PA 19107 (E-mail: Kumar.Sharma{at}mail.tju.edu).
Received 14 May 1999; accepted in final form 18 January 2000.
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