1 Dorrance Hamilton Laboratories, Division of Nephrology, Department of Medicine, and 2 Department of Anatomy, Pathology, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107; and 3 Department of Clinical Pharmacology, University of Groningen, 9713 AV Groningen, The Netherlands
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ABSTRACT |
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Ca2+ influx has been
postulated to modulate the signaling pathway of transforming growth
factor- (TGF-
); however, the underlying mechanism and functional
significance of TGF-
-induced stimulation of Ca2+ influx
are unclear. We show here that TGF-
stimulates Ca2+
influx in mesangial cells without Ca2+ release. The influx
of Ca2+ is prevented by pharmacological inhibitors of
inositol 1,4,5-trisphosphate receptors (IP3R) as well as
specific antibodies to type III IP3R (IP3RIII)
but not to type I IP3R (IP3RI). TGF-
enhances plasma membrane localization of IP3RIII, whereas
the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA)
preferentially translocates to the nucleus. Untreated mesangial cells
exhibit actin filamentous protrusions on the cell surface, and
treatment with TGF-
dramatically reduces this pattern. The
alterations in the actin cytoskeleton by TGF-
are dependent on
TGF-
-induced Ca2+ influx. These studies identify a novel
pathway by which TGF-
regulates Ca2+ influx and induces
cytoskeletal alterations.
mesangial cells; signaling; filipodia; inositol
1,4,5-trisphosphate; transforming growth factor-
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INTRODUCTION |
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TRANSFORMING GROWTH
FACTOR- (TGF-
) has been implicated in
disease processes as diverse as cancer, inflammatory diseases, and
chronic fibrotic disorders. This is partly due to the observation that
TGF-
is a multifunctional cytokine that can inhibit cellular proliferation, enhance matrix accumulation, and suppress inflammation. The signaling pathway is initiated by binding of ligand to the TGF-
type II receptor (T
RII), and the complex then interacts with the
type I receptor (T
RI). Cross phosphorylation of T
RI by the
constitutive serine-threonine kinase of T
RII activates the
serine-threonine kinase of T
RI. The activated T
RI phosphorylates the receptor-regulated Smads (Smad2 and Smad3), thus enhancing association with Smad4 and migration to the nucleus (24).
Although the role of the Smad pathway is critical in mediating
inhibition of cell proliferation in many cell types, the role of Smads
in mediating other characteristics of TGF-
such as matrix production is less clear (38). Other pathways that have been
implicated in TGF-
signaling include the mitogen-activated protein
kinase pathway (10) and the protein kinase A pathway
(40), although their respective roles are less well defined.
An alternative pathway that may be involved in TGF- signaling is
regulation of intracellular Ca2+. Ca2+
regulation likely plays an important role in modulating TGF-
signaling as calmodulin, in the presence of Ca2+, binds
Smad2, Smad3, and Smad4 (43). Furthermore, overexpression of calmodulin reduced the effect of TGF-
on 3TP-Lux activity (43). The binding of calmodulin to Smad2 occurs at
specific sites in the Mad homology (MH)1 domain and inhibits
Smad2-dependent activity (33). Additionally, activated
Ca2+-calmodulin-dependent protein kinase II (CaM kinase II)
phosphorylates Smad2 and Smad4 and may inhibit their activity
(41). The basis for activation of calmodulin may be due to
an increase in intracellular cytosolic Ca2+ concentration
[Ca2+]c induced by TGF-
. Previously it was
established that TGF-
stimulates the
[Ca2+]c increase in fibroblasts (1,
29); however, the mechanisms underlying the TGF-
-induced
[Ca2+]c increase have not been described. In
addition, a direct functional role for the TGF-
-induced
[Ca2+]c increase independent of the Smad
pathway has not been identified.
The two sources of increasing [Ca2+]c are release of Ca2+ from internal stores and influx of Ca2+ from the extracellular space via channels located in the plasma membrane. In response to a variety of growth factors (e.g., platelet-derived, epidermal, and hepatocyte growth factors) and vasoactive agonists (e.g., angiotensin II and endothelin) there is an initial rise in [Ca2+]c via the inositol 1,4,5-trisphosphate (IP3)-gated intracellular Ca2+ channel (IP3R) (2). The generation of IP3 is mediated by the activation of phospholipase C isoforms by tyrosine kinase-linked receptors or G protein linked receptors. The COOH-terminal domain of the IP3R forms a channel in the membrane of intracellular Ca2+ storage sites and upon IP3 binding to the NH2-terminal region of the IP3R allows stored Ca2+ to enter the cytosol (30). Consequent to the release of stored intracellular Ca2+, there is a secondary influx of Ca2+ from the extracellular space. This influx of Ca2+ occurs via store-operated Ca2+ channels (SOC; Ref. 31) and appears to involve IP3Rs, possibly in association with another Ca2+ channel of the transient-receptor-potential channel family (TRP; Refs. 22, 32). The IP3Rs exist as three isoforms, and although they have different IP3 binding affinities and may exist in different spatial compartments (30), their distinct functional roles with respect to influx of Ca2+ are unclear.
The involvement of IP3Rs in TGF- signaling was suggested
by our prior study (36), wherein we found that short-term
TGF-
treatment (5-30 min) induced phosphorylation of the type I
IP3R (IP3RI) in mesangial cells. In the present
study, we evaluated the roles of the IP3RI and type III
IP3R (IP3RIII) in mediating TGF-
-induced
Ca2+ influx. Furthermore, we assessed the impact of
TGF-
-induced Ca2+ influx in relation to cytoskeletal
rearrangements in mesangial cells.
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EXPERIMENTAL PROCEDURES |
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Cell culture.
SV40 transformed murine mesangial cells (MMCs) were grown in DMEM with
10% serum and were rested overnight in serum-free DMEM before being
exposed to 10 ng/ml of TGF-1 (R&D Systems, Minneapolis, MN). All
reagents were obtained from Sigma (St. Louis, MO) unless otherwise noted.
Antibodies.
The following antibodies were used: rabbit polyclonal
anti-IP3RI (Affinity Bioreagents, Golden, CO), murine
monoclonal anti-NH2-terminal IP3RIII
(Transduction Laboratories, Lexington, KY), rabbit polyclonal anti-COOH-terminal IP3RIII (15), murine
monoclonal anti-sarcoplasmic-endoplasmic reticulum Ca2+
ATPase (SERCA; Affinity Bioreagents), and murine monoclonal
anti--actin (Sigma).
Ca2+ measurements.
To measure [Ca2+]c fluorescence, imaging
measurements of [Ca2+]c in MMCs were
performed as previously described (35). After cells were
grown on coverslips and loaded with fura 2-AM, TGF-1 was added in
the presence of 2 mM CaCl2 or in the absence of
Ca2+ in the extracellular medium. Time courses of
[Ca2+]c in individual cells were calculated
from fluorescence imaging pairs obtained using 340- and 380-nm
excitation (10-nm bandwidth) with a broadband emission filter passing
460-600 nm. To ensure that intracellular stores of
Ca2+ were not depleted in the low-Ca2+ media,
thapsigargin (2 µM; Alexis Biochemicals, San Diego, CA) was added to
cells in the presence of 2 mM CaCl2 or in the absence of
Ca2+ in the extracellular medium, and
[Ca2+]c was measured. Studies were carried
out using 3 different cell cultures during 3 parallel experiments on
each occasion, and 30-50 cells were monitored in each experiment.
Confocal microscopy.
MMCs grown on coverslips were exposed to TGF-1 for 5-30 min.
Immunostaining and confocal analysis for IP3RI and
IP3RIII were performed as previously described
(35). For SERCA, a double-antibody method with AlexaFluor
was utilized to amplify staining. Slides were visualized via confocal
microscopy, and representative regions were photographed. Confocal
analysis was performed with 1-µm sections and sequential
z-images to ascertain intracellular locations of proteins.
As a negative control, cells stained only with secondary antibody
showed minimal background fluorescence. The actin cytoskeleton was
visualized using rhodamine-phalloidin (Molecular Probes) staining on
control and TGF-
-treated cells according to the manufacturer's instructions.
Biotinylation and analysis of plasma membrane proteins.
Confluent MMCs grown on 100-mm dishes under control conditions or with
TGF- treatment were biotinylated with 10 ml of 0.5 mg/ml of the
membrane-impermeant biotinylating reagent sulfosuccinimidobiotin (sulfo-NHS-biotin; Pierce Chemical, Rockford, IL) for 30 min at 4°C.
After excess NHS-biotin was washed off, cells were lysed with 1 ml of
1% (wt/vol) Triton X-100 in 10 mM Tris · HCl, 150 mM NaCl,
1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µM
pepstatin A, and 10 µM leupeptin for 30 min at 4°C. The lysates were incubated with streptavidin-treated beads for 8-12 h at 4°C to recover biotinylated proteins. The recovered proteins were then run
on a 7% SDS-PAGE gel, transferred to nitrocellulose, and immunoblotted
with the IP3RI (1:2,000 dilution), IP3RIII
(1:5,000 dilution), or SERCA 2 (1:2,000 dilution) antibodies. To
control for an overall change in cellular protein levels of
IP3RI and IP3RIII, total cell lysates were
obtained from control and TGF-
-treated cells with lysis buffer
containing 1% Triton X-100. Protein (20 µg) was analyzed as above
and standardized by immunoblotting the membrane with anti-
-actin antibody.
Immunoelectron microscopy for IP3RIII. MMCs were fixed in wells by removing the medium and adding a mixture of freshly prepared 2% paraformaldehyde and 1% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) at room temperature. After washing and incubation for 2 h with the mouse monoclonal antibody for IP3RIII (1:2,000 dilution), the cells were incubated using the ABC method (Vectastain Elite ABC kit, Vector Laboratories). Subsequent to reacting with diaminobenzidine, a gold-substituted silver peroxidase enhancement reaction was performed to improve the visibility of the reaction product. After the incubation procedure, the cells were osmicated and dehydrated in a graded series of ethanol and propylene oxide and embedded in epon. The epon blocks were cut into ultrathin microsections of 60-75 nm and contrasted with uranyl acetate and lead citrate before being visualized.
Quantification of IP3RIII staining was performed by computerized quantification of staining of the peroxidase enhancement reaction product (ImagePro Plus 4.1, Media Cybernetics). For each group, at least six photographs taken from different cells were digitized at a resolution of 300 dpi (Agfa Snapscan 600 scanner). IP3RIII staining was quantified as the area of staining relative to the cell area investigated. Photographic material was quantified by an observer blinded to the experimental protocol. Computerized selection of stained areas was carefully checked by the observer to exclude any artifacts.Statistical analysis. Results are expressed as means ± SE. Significance was determined by Student's t-test or by ANOVA for cases with multiple comparisons. The variability within the groups was random. P < 0.05 was considered significant.
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RESULTS |
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TGF- increases Ca2+ influx without
Ca2+ release in mesangial cells.
To determine the time frame in which TGF-
has its maximal effect on
Ca2+ influx, [Ca2+]c transients
were measured in MMCs using fluorescence imaging with fura 2-AM
loading. After 1 min of TGF-
treatment, there was no observable
change in [Ca2+]c; however, after 25 min of
TGF-
treatment, the majority of cells exhibited an increase
in [Ca2+]c (Fig.
1A). The increase in
[Ca2+]c began gradually, 5 min after the
addition of TGF-
, and reached a plateau after 25 min (Fig.
1B). This pattern of a TGF-
-induced rise in
[Ca2+]c is unique among growth factors and
contrasts with the typical tyrosine kinase-linked or G protein-linked
receptors that induce an IP3-mediated rapid rise in
[Ca2+]c (2). In the absence of
extracellular Ca2+, there was no increase in
[Ca2+]c with TGF-
(Fig. 1B),
which suggests that Ca2+ was not being released from
intracellular stores. To further confirm that TGF-
induced
Ca2+ influx, MMCs were treated with TGF-
for increasing
amounts of time and the cells were exposed to
45Ca2+ for the last 30 s of treatment
(Fig. 2A).
There was a trend for TGF-
-induced 45Ca2+
influx after 5 and 15 min; however, this did not reach statistical significance. TGF-
-induced 45Ca2+ influx was
significant after 30 min.
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Ca2+ influx induced by TGF-
involves IP3RIII.
Plasma membrane-bound IP3Rs have been considered
to play a role in Ca2+ influx (17, 20);
therefore, we studied the effects of various inhibitors of
IP3Rs in mediating TGF-
-induced
45Ca2+ influx. Addition of heparin, which
interferes with IP3 binding and activation of the
IP3Rs (8, 19), inhibited TGF-
-induced Ca2+ influx (Fig. 2B). However, heparin may also
inhibit Ca2+ channels on the cell surface (27)
and may interact with TGF-
itself (21). Xestospongin
has been described to be a highly specific, cell-permeable inhibitor of
Ca2+ flux via the IP3R (7).
Addition of a low concentration of xestospongin (2 µM) also led to
attenuation of TGF-
-induced Ca2+ influx (Fig.
2B). Results of these studies suggest that IP3Rs play a critical role in TGF-
-induced Ca2+ influx.
TGF- enhances localization of IP3RIII to plasma
membrane.
To assess whether TGF-
alters the intracellular distribution pattern
of IP3R isoforms to promote Ca2+ influx, we
analyzed the same cell system by IP3R immunostaining. Under
control conditions, both IP3RI and IP3RIII are
primarily present in the cytoplasm (Fig.
3, A and B). After
TGF-
treatment, IP3RI takes on a more vesicular staining
pattern at or near the nucleus (Fig. 3A). In contrast,
IP3RIII exhibits increased intensity at the plasma membrane
after TGF-
treatment (Fig. 3B). Interestingly, both
IP3R isoforms appeared to have overall greater
intensity of immunostaining after TGF-
treatment. The divergence of
distribution of the two IP3R isoforms suggests that the
isoforms may be associated with different subcellular structures. We
therefore assessed the location of another endoplasmic reticulum
Ca2+ transport protein, SERCA, under control conditions and
with TGF-
treatment (Fig. 3C). In the basal state, SERCA
is in a vesicular perinuclear distribution, whereas after TGF-
treatment, SERCA moves into the nucleus.
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Immunoelectron microscopy for IP3RIII.
To better characterize the spatial intracellular distribution of
IP3RIII in mesangial cells under control and TGF-
treatment conditions, immunoelectron microscopy was performed with
antibody to the COOH-terminal region of IP3RIII (Fig.
6). Under control conditions,
IP3RIII is distributed diffusely in the cytosol with occasional intense immunostaining at the plasma membrane. On TGF-
treatment, there was a marked increase in IP3RIII at the
plasma membrane. Quantitation of IP3RIII staining revealed
a 10-fold increase of plasma membrane staining of IP3RIII
with TGF-
treatment (pixels/area ratio: control, 0.05 ± 0.02 vs. TGF-
treated, 0.48 ± 0.08; P = 0.0001).
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TGF--induced Ca2+ influx regulates
effect of TGF-
on actin cytoskeleton.
As Ca2+ has been closely associated with regulation of the
actin cytoskeleton (34), we questioned whether
TGF-
-induced Ca2+ influx was associated with
alterations in the actin cytoskeleton. Under control conditions,
the majority of rhodamine-phalloidin-stained mesangial cells
present exhibited numerous actin filament protrusions, or filopodia, on
the plasma membrane (Fig.
7). With
administration of TGF-
, there were a reduced number of filopodia
present on the cell surface, which was noted in the majority of cells
at 15 and 30 min (Fig. 7). To evaluate whether the TGF-
-induced effect on the actin cytoskeleton was related to Ca2+
influx, the studies were repeated in the presence and absence of
extracellular Ca2+. Under basal conditions, in the presence
of extracellular Ca2+, 92.3% of the cells exhibited
filipodia (Figs. 8 and
9). After 15 min of exposure to
TGF-
, the percentage of cells exhibiting actin filopodia were
markedly reduced (7.4%). This effect of TGF-
was dependent on
extracellular Ca2+, as cells incubated in
Ca2+-free media failed to elicit a TGF-
effect on
filopodia (91.8% untreated and 84.3% treated with TGF-
).
Furthermore, addition of the antibody to IP3RI did not
alter the TGF-
effect, whereas the presence of IP3RIII
antibody prevented the effect of TGF-
to reduce the number of
filopodia. Similar results were obtained when heparin was added
extracellularly to block TGF-
-induced Ca2+ influx (data
not shown).
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DISCUSSION |
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Based on the above evidence, we propose that TGF- stimulates
transmembrane Ca2+ influx in mesangial cells via the
involvement of cell membrane-associated IP3RIII.
Furthermore, Ca2+ influx is directly linked to acute
cytoskeletal changes induced by TGF-
. Localization of
IP3Rs to plasma membrane proteins has been recently
documented in several cell types and is not limited to the
IP3RIII isoform (39). However, our study
indicates for the first time that localization of IP3RIII
to the plasma membrane is enhanced by short-term TGF-
treatment.
Inhibition of IP3RIII by inhibitors or specific antibodies
demonstrates its critical role in facilitating Ca2+ influx.
The basis for IP3RIII to act as a Ca2+
channel at the plasma membrane is unclear, although several studies
have suggested that plasma-membrane-associated IP3Rs are
intimately associated with Ca2+ entry (3, 18).
It is conceivable that plasma membrane-bound IP3RIII
associates with other proteins to form an operative
Ca2+-entry pathway analogous to the association of
IP3R with plasma membrane-associated TRP in SOC entry
(22, 32). However, distinct from movement of
IP3Rs to the plasma membrane with SOC, our study suggests
that the IP3RIII isoform translocates to the plasma
membrane in response to TGF- without depletion of internal
Ca2+ stores. On reaching the plasma membrane,
IP3RIII may promote Ca2+ entry via TRP or
possibly other Ca2+ channels. Therefore, IP3Rs
associated at or near the plasma membrane are critical in mediating
Ca2+ influx with SOC as well as Ca2+ influx in
the absence of Ca2+ release from internal stores. A
specific role for the IP3RIII isoform in SOC has not yet
been determined. A role for IP3RIII has been implicated in
apoptosis of lymphocytes (3, 18), and this process
may be initiated by Ca2+ influx. It is tempting to
speculate that apoptosis induced by TGF-
may also involve
recruitment of IP3RIII to the plasma membrane.
The topology of the plasma membrane-bound
IP3RIII is unclear. Prior topology studies with
IP3RI (25) have found that intracellular IP3RI has its NH2- and COOH-terminal sites
facing the cytosolic aspect. By analogy, it would be predicted that
plasma membrane IP3RI and other isoforms of
IP3R would similarly have their NH2- and
COOH-terminal sites facing the intracellular aspect. However, there
have been no studies to specifically examine plasma membrane-bound IP3Rs. Based on our results with extracellular heparin and
extracellular antibodies to block TGF--induced Ca2+
influx, it would appear that the NH2- and COOH-terminal
sites of IP3RIII face the extracellular space. In addition,
we demonstrate that extracellular antibodies are able to recognize
IP3R isoforms in intact MMCs (see Fig. 5). However, it is
conceivable that heparin (4, 26, 27) and the antibodies
(23, 42) may enter the intracellular space and remain
active. Therefore, at the present time we cannot distinguish between
the possibilities that the IP3R inhibitors blocked the
function of IP3RIII by binding to intracellular or
extracellular NH2- and COOH-terminal sites. Studies are
under way to identify the topology of IP3RIII in mesangial cells.
Our finding that SERCA is translocated to the nucleus by TGF- was
unexpected. Of note, it has been recently observed that thapsigargin,
an inhibitor of SERCA, attenuates the activity of several
TGF-
-responsive promoters (41). The presence of SERCA in the nucleus after TGF-
treatment may be related to the activation of nuclear Ca2+-activated proteins (e.g., CaM kinase II)
with consequent effects on transcription factors regulating
TGF-
-responsive genes.
The overall conclusion of recent studies examining Ca2+,
calmodulin (33, 43), and CaM kinase II (41)
is that stimulation of Ca2+-regulated pathways leads to a
negative modulatory role in Smad-mediated pathways and cross talk with
the extracellular signal-regulated (ERK) pathway. However, these
studies have not identified a direct role for TGF--induced
Ca2+ influx that may be independent of the Smad and ERK
pathways. In our studies, we show that TGF-
-induced Ca2+
influx is critical for the effects of TGF-
on the actin
cytoskeleton. The role of the Smad pathway in mediating
Ca2+ influx by TGF-
cannot be excluded based on our
results. However, it would appear unlikely that Smads play a critical
role, as Smad-mediated processes generally require gene transcription
and are observed hours after TGF-
exposure, whereas Ca2+
influx and alteration of the actin filaments occur as early as 15-30 min after the addition of TGF-
.
The actin filamentous protrusions on the plasma membrane of MMCs
closely resemble filopodia. Filopodia have been described to be
present in neurites (14), ovarian granulosa cells
(9), and malignant cells (16) and appear to
be most closely associated with cell-cell communication and cell
spreading. Although [Ca2+]c has been
considered to regulate filopodia formation (34), there
have been no reports that TGF- inhibits filopodia formation. However, cyclosporin-induced membrane filament protrusions in adenocarcinoma cells have been found to be blocked by anti-TGF-
antibodies (11), which suggests that TGF-
may inhibit
filopodia in some cell types (the present study) and enhance it in
other cell types. The role of filopodia in mesangial cells is unclear but may be relevant to cell-cell communication with adjacent glomerular endothelial cells and matrix interaction. As membrane filopodia are
associated with cell proliferation, it is possible that the effect of
TGF-
on the actin cytoskeleton may be involved in limiting proliferation of cells and promoting cell hypertrophy. Of note, TGF-
has previously been found to inhibit proliferation (13) and induce cell hypertrophy (5) in mesangial cells.
Further studies are required to elucidate the downstream effects of
TGF-
-induced actin filament reorganization as well as the impact of
Ca2+ influx on cross talk with other well-described
pathways involved in TGF-
signaling.
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ACKNOWLEDGEMENTS |
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The authors thank Stephen R. Dunn for data presentation.
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FOOTNOTES |
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This study was funded by National Institutes of Health Grants R01 DK-53867 (to K. Sharma) and GM-58574 (to S. K. Joseph) and an American Diabetes Association research award (to K. Sharma).
Address for reprint requests and other correspondence: K. Sharma, Thomas Jefferson Univ., Suite 353, Jefferson Alumni Hall, 1020 Locust St., Philadelphia, PA 19107 (E-mail: Kumar.Sharma{at}mail.tju.edu).
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. Section 1734 solely to indicate this fact.
First published November 20, 2001;10.1152/ajprenal.00252.2001
Received 15 August 2001; accepted in final form 14 November 2001.
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