TGF-beta -induced Ca2+ influx involves the type III IP3 receptor and regulates actin cytoskeleton

Tracy A. McGowan1, Muniswamy Madesh2, Yanqing Zhu1, Lewei Wang1, Mark Russo1, Leo Deelman3, Rob Henning3, Suresh Joseph2, Gyorgy Hajnoczky2, and Kumar Sharma1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ca2+ influx has been postulated to modulate the signaling pathway of transforming growth factor-beta (TGF-beta ); however, the underlying mechanism and functional significance of TGF-beta -induced stimulation of Ca2+ influx are unclear. We show here that TGF-beta 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-beta 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-beta dramatically reduces this pattern. The alterations in the actin cytoskeleton by TGF-beta are dependent on TGF-beta -induced Ca2+ influx. These studies identify a novel pathway by which TGF-beta regulates Ca2+ influx and induces cytoskeletal alterations.

mesangial cells; signaling; filipodia; inositol 1,4,5-trisphosphate; transforming growth factor-beta


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

TRANSFORMING GROWTH FACTOR-beta (TGF-beta ) 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-beta 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-beta type II receptor (Tbeta RII), and the complex then interacts with the type I receptor (Tbeta RI). Cross phosphorylation of Tbeta RI by the constitutive serine-threonine kinase of Tbeta RII activates the serine-threonine kinase of Tbeta RI. The activated Tbeta 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-beta such as matrix production is less clear (38). Other pathways that have been implicated in TGF-beta 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-beta signaling is regulation of intracellular Ca2+. Ca2+ regulation likely plays an important role in modulating TGF-beta signaling as calmodulin, in the presence of Ca2+, binds Smad2, Smad3, and Smad4 (43). Furthermore, overexpression of calmodulin reduced the effect of TGF-beta 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-beta . Previously it was established that TGF-beta stimulates the [Ca2+]c increase in fibroblasts (1, 29); however, the mechanisms underlying the TGF-beta -induced [Ca2+]c increase have not been described. In addition, a direct functional role for the TGF-beta -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-beta signaling was suggested by our prior study (36), wherein we found that short-term TGF-beta 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-beta -induced Ca2+ influx. Furthermore, we assessed the impact of TGF-beta -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-beta 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-beta -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-beta 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.

To measure Ca2+ influx, cells were grown on six-well plates, washed with Ca2+-free physiological saline solution (PSS) buffer that contained (in mM) 145 NaCl, 5 KCl, 1 MgCl2, 10 glucose, and 5 HEPES, pH 7.4, and were then placed in PSS with 0.12 mM CaCl2 as previously described (1). Cells were treated with TGF-beta 1 for various amounts of time. For experiments to study the effects of heparin, xestospongin, or antibodies, the cells were pretreated with TGF-beta 1 for 30 min before addition of IP3R inhibitors or antibodies for the last 5 min. Cells were then labeled with 1 µCi of 45Ca2+/well for the final 30 s of incubation before being washed and harvested in lysis buffer. 45Ca2+ was counted using a liquid scintillation counter. Experiments were performed three times, and samples were analyzed in triplicate.

Confocal microscopy. MMCs grown on coverslips were exposed to TGF-beta 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-beta -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-beta 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-beta -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-beta -actin antibody.

To determine whether extracellular IP3R antibodies would recognize their respective antigens, MMCs were plated on 100-mm dishes in DMEM that contained 10% fetal calf serum. After reaching 90% confluence, cells were rested overnight in serum-free DMEM. The rabbit polyclonal anti-IP3RI (Affinity Bioreagents) or murine monoclonal anti-NH2-terminal IP3RIII (Transduction Laboratories) antibodies were added to cells at a 1:500 dilution in 5 ml of serum-free DMEM for 30 min at 37°C. Unbound antibodies were washed off three times with ice-cold PBS, and 1 ml of 0.25 M Tris · HCl (pH 7.8) was added before cells were harvested with a cell scraper into 1.5-ml Eppendorf tubes. Subsequently cells were subjected to three cycles of freeze-thaw lysis. Protein lysate (600 µg) was then added to 70 µl of protein A-agarose and rotated overnight at 4°C. After centrifugation at 3,000 rpm for 5 min at 4°C, the pellet was washed 4× with wash buffer that contained 0.1% (wt/vol) Triton X-100, 50 mM Tris · HCl, pH 7.4, 300 mM NaCl, 5 mM EDTA, and 0.02% (wt/vol) sodium azide. The pellet was then resuspended in sample buffer, boiled for 5 min, and loaded onto a 7% SDS-PAGE gel. Immunoblotting was performed as described.

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.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

TGF-beta increases Ca2+ influx without Ca2+ release in mesangial cells. To determine the time frame in which TGF-beta 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-beta treatment, there was no observable change in [Ca2+]c; however, after 25 min of TGF-beta 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-beta , and reached a plateau after 25 min (Fig. 1B). This pattern of a TGF-beta -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-beta (Fig. 1B), which suggests that Ca2+ was not being released from intracellular stores. To further confirm that TGF-beta induced Ca2+ influx, MMCs were treated with TGF-beta 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-beta -induced 45Ca2+ influx after 5 and 15 min; however, this did not reach statistical significance. TGF-beta -induced 45Ca2+ influx was significant after 30 min.


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Fig. 1.   Transforming growth factor (TGF)-beta stimulates Ca2+ entry in mesangial cells. A: a representative group of fura 2-AM-loaded murine mesangial cells under control conditions and with TGF-beta treatment (10 ng/ml) showing increased intracellular cytosolic Ca2+ concentration ([Ca2+]c) after TGF-beta treatment. Elevation in [Ca2+]c appears as green-to-red shift in the green/red overlay images (380:340 nm excitation; width of frame corresponds to 121 µm). B: time course of TGF-beta -induced rise in [Ca2+]c was evaluated from 3 sets of experiments with fura 2-AM loaded cells. Top: a rise in [Ca2+]c was noted after 5 min of TGF-beta treatment and continued for 30 min. Bottom: in the absence of extracellular Ca2+, TGF-beta treatment failed to evoke an increase in [Ca2+]c. [Ca2+]ec, extracellular Ca2+ concentration; Rfura2, 340/380 ratio.



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Fig. 2.   TGF-beta induced Ca2+ influx is inhibited by inositol 1,4,5-trisphosphate receptors (IP3R) inhibitors. A: murine mesangial cells (MMCs) were treated with TGF-beta for various amounts of time and exposed to 45Ca2+ for the final 30 s of incubation before harvest. There is a progressive increase in uptake of 45Ca2+ between 5 and 30 min of TGF-beta treatment. B: MMC were treated with TGF-beta for 30 min with the addition of extracellular heparin (1 or 10 ng/ml) or xestospongin (2 µM) for the last 5 min and then exposed to 45Ca2+ for the final 30 s before harvest. Experiments were repeated three times with similar results. C: influx of 45Ca2+ was assessed in the presence or absence of extracellular COOH-terminal antibodies against type I IP3R (IP3RI; 1:500 dilution) or COOH- and NH2-terminal antibodies against type III IP3R (IP3RIII; 1:500 dilution) for the last 5 min before harvest. Experiments were repeated three times with similar results. Results are expressed as 45Ca2+ influx by counts/min (cpm) or as percent control. Values are means ± SE of data from three separate experiments; n = 3-6 per condition. *P < 0.05 vs. control.

The roles of SOC and voltage-gated Ca2+ channels were evaluated after TGF-beta exposure. TGF-beta did not stimulate IP3-mediated Ca2+ release, as an increase in [Ca2+]c did not occur in the absence of extracellular Ca2+ (Fig. 1B). To ensure that internal stores of Ca2+ were not depleted in the Ca2+-free condition, thapsigargin-induced Ca2+ release was monitored and found not to be significantly affected under either medium (340:380 ratio of 0.64 ± 0.06 at baseline in 2 mM Ca2+ bath increasing to 1.18 ± 0.30 with thapsigargin, and 340:380 ratio of 0.49 ± 0.04 at baseline in Ca2+-free bath increasing to 1.02 ± 0.25 with thapsigargin). In addition, we previously found that IP3 levels did not increase after TGF-beta treatment in MMCs (35). The role of voltage-gated Ca2+ channels has been implicated in mediating TGF-beta -induced Ca2+ influx (12); however, [Ca2+]c was not increased in MMCs with exposure to 20 or 40 mM KCl as measured by 45Ca2+ or by fura 2 imaging (data not shown), which suggests that classic voltage-gated channels were not operating in our cells.

Ca2+ influx induced by TGF-beta 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-beta -induced 45Ca2+ influx. Addition of heparin, which interferes with IP3 binding and activation of the IP3Rs (8, 19), inhibited TGF-beta -induced Ca2+ influx (Fig. 2B). However, heparin may also inhibit Ca2+ channels on the cell surface (27) and may interact with TGF-beta 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-beta -induced Ca2+ influx (Fig. 2B). Results of these studies suggest that IP3Rs play a critical role in TGF-beta -induced Ca2+ influx.

Mesangial cells have a predominance of IP3RI and IP3RIII isoforms (28, 35, 37), and both of these isoforms have been described to be located at or near the plasma membrane in various cell types (6, 17, 39). Therefore, we focused on IP3RI and IP3RIII as candidate proteins that may facilitate Ca2+ influx on stimulation with TGF-beta . To determine whether an isoform of IP3R at the cell surface is required for the effects of TGF-beta on Ca2+ influx, the 45Ca2+-influx studies were repeated in the presence or absence of extracellular antibodies specific for IP3RI or IP3RIII (Fig. 2C). As seen in Fig. 2C, TGF-beta -mediated Ca2+ influx is prevented by an antibody directed against either the COOH or NH2 terminus of IP3RIII but not with an antibody to IP3RI.

TGF-beta enhances localization of IP3RIII to plasma membrane. To assess whether TGF-beta 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-beta 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-beta treatment (Fig. 3B). Interestingly, both IP3R isoforms appeared to have overall greater intensity of immunostaining after TGF-beta 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-beta treatment (Fig. 3C). In the basal state, SERCA is in a vesicular perinuclear distribution, whereas after TGF-beta treatment, SERCA moves into the nucleus.


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Fig. 3.   TGF-beta treatment promotes IP3RIII localization to plasma membrane. MMCs under control conditions or in the presence of TGF-beta (10 ng/ml, 5 min) were incubated with antibody to either IP3RI (A), IP3RIII (B), or sarcoplasmic-endoplasmic reticulum Ca2+- ATPase (SERCA; C) and analyzed with confocal microscopy (width of frame corresponds to 105 µm). After treatment with TGF-beta , IP3RI and SERCA took on a vesicular and/or nuclear distribution (A and C) whereas a major fraction of IP3RIII was close to the cell surface (arrowheads; B). Experiments were performed three times with similar results.

To confirm that there is an increased amount of IP3RIII at the plasma membrane after exposure to TGF-beta , plasma-membrane proteins were isolated with sulfo-NHS-biotin to biotinylate the cell-surface proteins (6, 39). Plasma membrane proteins were assessed for the presence of IP3RI, IP3RIII, and SERCA. At baseline, both IP3RI and IP3RIII are present in plasma membrane proteins and are further increased after TGF-beta treatment (Fig. 4, A and B). However, SERCA was not found in plasma membrane proteins (Fig. 4C), which suggests that pools of IP3Rs exist that are spatially distinct from SERCA. Overall levels of IP3RI and IP3RIII did not vary in total cell lysates with TGF-beta treatment (Fig. 4, D-F).


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Fig. 4.   Immunoblot analysis of biotinylated proteins after TGF-beta treatment. Biotinylated proteins from externally biotinylated MMCs under control conditions or after 5 min of TGF-beta treatment were assessed for IP3RI (A), IP3RIII (B), and SERCA (C). TGF-beta treatment enhanced IP3RI and IP3RIII localization in the biotinylated fraction compared with control, whereas SERCA was absent from the biotinylated pool regardless of TGF-beta treatment. To assess whether overall levels of IP3R isoforms were regulated by short-term TGF-beta treatment, total cell lysates were immunoblotted with antibodies for IP3RI (D) and IP3RIII (E). Membranes were also immunoblotted with antibody to beta -actin (F) to ensure equivalent loading. Experiments were repeated three times with similar results.

As previously noted in Fig. 2C, addition of IP3RIII antibodies to intact cells inhibited TGF-beta -induced Ca2+ influx. Therefore, it would be presumed that extracellular antibodies are able to recognize the IP3Rs located on the cell surface. To directly test this concept, MMCs were exposed to anti-IP3RI or anti-IP3RIII antibodies and vigorously washed with PBS before lysing of the cells. The cell lysates were then incubated with protein A-agarose beads, and the immunoprecipitate was analyzed by Western blot analysis. As indicated in Fig. 5, only the lysates from MMCs exposed to extracellular antibodies exhibited a band for the respective IP3R. This result suggests that the extracellular antibodies do indeed recognize their respective epitopes in intact MMCs.


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Fig. 5.   Extracellular IP3R antibodies recognize IP3R isoforms in intact MMCs. To determine whether extracellular antibodies recognize IP3Rs in intact cells, anti-IP3RI antibody (1:500 dilution) was added to MMCs before lysis of cells and immunoblot analysis for IP3RI (left). Similarly, anti-IP3RIII antibody (1:500 dilution) was added to cells before lysis and subsequent immunoblot analysis for IP3RIII (right). A band for IP3RI or IP3RIII is noted only under the condition of addition of extracellular antibodies. Experiment was repeated three times with similar results.

Immunoelectron microscopy for IP3RIII. To better characterize the spatial intracellular distribution of IP3RIII in mesangial cells under control and TGF-beta 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-beta 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-beta treatment (pixels/area ratio: control, 0.05 ± 0.02 vs. TGF-beta treated, 0.48 ± 0.08; P = 0.0001).


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Fig. 6.   Increased plasma membrane localization of IP3RIII after TGF-beta treatment by immunogold electron microscopic analysis. Electron microscopy (×20,000 magnification) was performed for IP3RIII localization by immunogold staining. Under control conditions, IP3RIII was located in the cytosol and occasionally on the plasma membrane (A). With TGF-beta treatment (10 ng/ml, 15 min), there was an accentuation of IP3RIII on plasma membrane (B). Bottom-right field is further magnified ×3 for the control sample (C) and with TGF-beta treatment (D). Experiment was performed twice with similar results.

TGF-beta -induced Ca2+ influx regulates effect of TGF-beta on actin cytoskeleton. As Ca2+ has been closely associated with regulation of the actin cytoskeleton (34), we questioned whether TGF-beta -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-beta , 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-beta -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-beta , the percentage of cells exhibiting actin filopodia were markedly reduced (7.4%). This effect of TGF-beta was dependent on extracellular Ca2+, as cells incubated in Ca2+-free media failed to elicit a TGF-beta effect on filopodia (91.8% untreated and 84.3% treated with TGF-beta ). Furthermore, addition of the antibody to IP3RI did not alter the TGF-beta effect, whereas the presence of IP3RIII antibody prevented the effect of TGF-beta to reduce the number of filopodia. Similar results were obtained when heparin was added extracellularly to block TGF-beta -induced Ca2+ influx (data not shown).


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Fig. 7.   TGF-beta -induced reorganization of actin filaments: time course. MMCs cultured on coverslips in serum-free growth medium for 24 h were treated with TGF-beta 1 (10 ng/ml) at the times indicated (B-D). After fixation, cells were stained with rhodamine-phalloidin and evaluated by confocal microscopy. Under control conditions (A), cells exhibited long, thin filamentous protrusions from the cell surface (filopodia); however, after 15 (C) and 30 min (D) of TGF-beta treatment, filopodia were largely absent. Experiments were repeated three times with similar results.



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Fig. 8.   TGF-beta -induced reorganization of actin filaments: role of Ca2+ influx and IP3RIII. MMCs cultured on coverslips in serum-free growth medium for 24 h were treated with 10 ng/ml of TGF-beta 1 for 15 min (B, D, F, H) or solvent (A, C, E, G). After fixation, cells were stained with rhodamine-phalloidin and evaluated by confocal microscopy. To test the role of Ca2+ influx, cells were washed and incubated in Ca2+-free medium (C) before addition of TGF-beta (D). To evaluate the role of IP3Rs, MMCs were preincubated with antibodies to IP3RI (IP3RIAb, 1:500 dilution) for 5 min (E and F) or antibodies to IP3RIII (IP3RIIIAb, 1:500 dilution; G and H) before addition of TGF-beta . Experiments were repeated three times with similar results.



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Fig. 9.   Quantitative analysis of the presence of filopodia on cell surfaces under control conditions and with TGF-beta treatment. Under basal conditions, 92% of cells expressed filopodia from the cell surface, whereas with TGF-beta 1 treatment (10 ng/ml, 15 min), only 8% of cells exhibited filopodia. Absence of extracellular Ca2+ or anti-IP3RIII antibodies blocked the effect of TGF-beta (84 and 82% of cells, respectively, exhibited filopodia with TGF-beta ), whereas anti-IP3RI antibody failed to block the effect of TGF-beta . Between 73 and 115 cells were counted from three separate experiments under each condition. Values are expressed as means ± SE. *P < 0.05 vs. corresponding control group.

The effect of TGF-beta on the actin cytoskeleton appears to be specifically dependent on TGF-beta -induced Ca2+ influx, because the addition of thapsigargin to raise intracellular Ca2+ had no effect on the actin cytoskeleton (Fig. 10). Addition of ionomycin led to cell detachment and was therefore not interpretable (data not shown).


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Fig. 10.   Thapsigargin has no effect on actin cytoskeleton. MMCs cultured on coverslips in serum-free growth medium for 24 h were treated with solvent (A) or 2 µM thapsigargin for 15 min (B). After fixation, cells were stained with rhodamine-phalloidin and evaluated by confocal microscopy. In contrast to the effects of TGF-beta , thapsigargin failed to decrease the number of the actin filamentous protrusions noted on the plasma membrane. Experiments were repeated three times with similar results.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Based on the above evidence, we propose that TGF-beta 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-beta . 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-beta 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-beta 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-beta 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-beta -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-beta was unexpected. Of note, it has been recently observed that thapsigargin, an inhibitor of SERCA, attenuates the activity of several TGF-beta -responsive promoters (41). The presence of SERCA in the nucleus after TGF-beta 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-beta -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-beta -induced Ca2+ influx that may be independent of the Smad and ERK pathways. In our studies, we show that TGF-beta -induced Ca2+ influx is critical for the effects of TGF-beta on the actin cytoskeleton. The role of the Smad pathway in mediating Ca2+ influx by TGF-beta 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-beta exposure, whereas Ca2+ influx and alteration of the actin filaments occur as early as 15-30 min after the addition of TGF-beta .

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-beta inhibits filopodia formation. However, cyclosporin-induced membrane filament protrusions in adenocarcinoma cells have been found to be blocked by anti-TGF-beta antibodies (11), which suggests that TGF-beta 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-beta on the actin cytoskeleton may be involved in limiting proliferation of cells and promoting cell hypertrophy. Of note, TGF-beta 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-beta -induced actin filament reorganization as well as the impact of Ca2+ influx on cross talk with other well-described pathways involved in TGF-beta signaling.


    ACKNOWLEDGEMENTS

The authors thank Stephen R. Dunn for data presentation.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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