Inhibition of type I and III IP3Rs by TGF-beta is associated with impaired calcium release in mesangial cells

Kumar Sharma1, Tracy A. Mc Gowan1, Lewei Wang1, Muniswamy Madesh2, Vince Kaspar1, Gabor Szalai2, Andrew P. Thomas2, and György Hajnóczky2

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-beta 1 (TGF-beta 1; 10 ng/ml, 24 h) leads to downregulation of both type I and III IP3Rs as measured by immunoblot and confocal analysis. TGF-beta 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-beta 1. Functional effects of TGF-beta 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-beta 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-beta 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-beta 1; inositol 1,4,5-trisphosphate receptors; calcium mobilization; mesangial cells


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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-beta (PLC-beta )-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 gamma -isoform of PLC (PLC-gamma ), 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-beta (TGF-beta 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-beta 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-beta 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-beta 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-beta 1 may be inhibiting IP3R function. Recently we demonstrated that TGF-beta 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-beta 1 to mesangial cells leads to decreased expression of both IP3R isoforms. Mesangial cells pretreated with TGF-beta 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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Materials. [32P] Inorganic phosphate, [gamma -32P]ATP, and [gamma -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-beta 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-beta 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-beta 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-beta 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 beta -actin (Sigma Chemical). MMC cells were also grown on coverslips and treated with TGF-beta 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-beta 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-beta 1 treatment. The coverslips with control MMCs or TGF-beta 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-beta 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-beta 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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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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Fig. 1.   Subcellular location of type I and III 1,4,5-trisphosphate receptors (IP3Rs) in murine mesangial cells (MMC) and effects of transforming growth factor-beta 1 (TGF-beta 1) treatment. MMCs under control conditions (A and C) or with TGF-beta 1 treatment (10 ng/ml, 24 h: B and D) were fixed on coverslips and immunofluorescence was performed with anti-type I IP3R antibody (A, B) and anti-type III IP3R antibody. Slides were observed by confocal microscopy and representative regions were photographed.



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Fig. 2.   Immunoblot analysis of cytosolic and membrane fractions with type I IP3R and type III IP3R antibody. Cytosolic (20 µg of protein) and membrane (20 µg of protein) fractions of MMCs were resolved on a 7% SDS-PAGE, transferred to nitrocellulose, and probed with anti-type I IP3R antibody (top) and with anti-type III IP3R antibody (bottom).

TGF-beta 1 treatment decreases both type I and III IP3R isoforms after 24 h. MMC were treated with TGF-beta 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-beta 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 beta -actin (bottom). With 24 h of TGF-beta 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-beta 1 treatment (Figure 1D).


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Fig. 3.   TGF-beta treatment reduces type I IP3R and type III IP3R immunoreactivity. Whole cell lysates (20 µg of protein) of control MMCs or MMCs treated with TGF-beta 1 (10 ng/ml) for indicated time periods were probed with anti-type I IP3R antibody (top), anti-type III IP3R antibody (middle), and anti-beta -actin antibody (bottom).

The protein half-life of type I IP3R is not reduced by TGF-beta treatment. We had previously found that TGF-beta 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-beta 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-beta 1 treatment. In addition, we measured whether TGF-beta 1 would affect IP3 levels. TGF-beta 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-beta 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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Fig. 4.   Turnover rate of type I IP3R protein is not affected by TGF-beta 1 treatment in MMCs. Confluent MMCs were pulse-labeled with Trans 35S-labeled methionine (100 µCi/ml) for 60 min in methionine-free DMEM and then chased for indicated times with 25 mM methionine. Cells were then treated with TGF-beta 1 (10 ng/ml) for indicated time periods. Detergent extracts were immunoprecipitated with anti-type I IP3R antibody, electrophoresed, transferred to nitrocellulose, and then autoradiographed. The experiment was repeated 3 times with similar results.

EGF-induced [Ca2+]c responses is attenuated in mesangial cells pretreated with TGF-beta 1. To demonstrate that reduction of the IP3Rs by TGF-beta 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-beta (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-beta 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-beta 1 responded to EGF. A similar degree of inhibition of Ca2+ release was noted in cells pretreated with TGF-beta 1 when PDGF was substituted for EGF (data not shown).


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Fig. 5.   Effect of TGF-beta 1 on EGF-stimulated cytosolic free calcium concentration ([Ca2+]c) responses in intact MMCs. [Ca2+]c was monitored in fura 2-loaded MMCs by using fluorescence imaging. Epidermal growth factor (EGF; 200 ng/ml) was added as indicated by arrow. A: representative mean [Ca2+]c traces recorded in both naive (thick line) and TGF-beta -pretreated (10 ng/ml, 24 h, thin line) MMCs. The mean trace represents values obtained from 40 cells in control condition and 40 cells with TGF-beta 1 pretreatment. B: peak [Ca2+]c rise calculated as percent of prestimulation [Ca2+]c. Values are means ± SE from 3 separate experiments. The difference between EGF-induced [Ca2+]c increases in naive and TGF-beta 1-pretreated MMCs was statistically significant (P < 0.03).

Sensitivity of IP3Rs to IP3 in permeabilized mesangial cell. To determine whether the reduction in Ca2+ mobilization after 24 h of TGF-beta 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-beta 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-beta 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-beta 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-beta 1 treatment. Thus the size of the total IP3-sensitive and IP3-insensitive Ca2+ stores is unchanged with TGF-beta 1 treatment. However, the decreased protein expression of both type I and III IP3R by TGF-beta 1 treatment is associated with a specific reduction in submaximal IP3-mediated calcium release.



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Fig. 6.   Effect of TGF-beta 1 on IP3-induced Ca2+ release. IP3-induced Ca2+ release was monitored fluorometrically by using fura 2/FA in incubation medium of permeabilized mesangial cells maintained in suspension. Cells were permeabilized with digitonin in the presence of 2 mM MgATP and an ATP regenerating system, and Ca2+ uptake was allowed to reach a steady state during 390-s incubation. Sequential additions of 1 µM CaCl2 (Ca2+), 400 nM, and 10.5 µM IP3 were then made as indicated, followed by 2 µM thapsigargin (Tg) and 10 µM ionomycin (Iono). A: representative time courses for Ca2+ responses by cells treated overnight with 10 ng/ml TGF-beta 1 (thin line) or solvent (thick line). B: combined IP3 dose-response data calculated from measurements of type shown in A. Values are means ± SE of data from 8 pairs. * P < 0.05 TGF-beta vs. control sample treated with 400 nM IP3.


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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-beta 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-beta 1 treatment, and we have previously demonstrated reduction of type I IP3R mRNA by TGF-beta 1 treatment (28). The functional consequence of the effect of TGF-beta 1 on IP3Rs is evidenced by the demonstration that mesangial cells pretreated with TGF-beta 1 have reduced Ca2+ mobilization in response to agonists that normally raise intracellular IP3 levels. Furthermore, reduction of IP3R isoforms by TGF-beta 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-beta 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-beta 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-beta 1-pretreated cells, demonstrating that the Ca2+ pools themselves are unaltered. In addition, ionomycin-induced calcium release is unchanged in TGF-beta 1 treated cells, demonstrating that total intracellular amounts of calcium are not altered.


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Fig. 7.   A model to describe how reduction of IP3Rs would lead to reduced calcium release with a submaximal dose of IP3 but a normal amount of calcium release with maximal dose of IP3. Top left: normal distribution of IP3Rs (open circle ) in membrane of a calcium storage pool (large filled ovals). A submaximal dose of IP3 (low IP3, top middle) would be expected to bind to a few IP3Rs (activated IP3R, ), and thus only part of entire calcium store will be released. With maximal dose of IP3 (high IP3, top right), all IP3Rs will be bound by IP3 and all IP3-sensitive sites will release their store of calcium. With TGF-ß1 treatment, there is an ~70% reduction in number of IP3Rs (bottom left). It would be expected that a submaximal dose of IP3 would lead to reduced extent of calcium release (bottom middle) compared with control condition (top middle). Assuming that residual IP3Rs show unchanged sensitivity to IP3 and that each calcium storage site still retains at least 1 IP3R, a maximal dose of IP3 (bottom right) will activate all remaining IP3Rs, and this is sufficient to release all stored calcium.

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-beta 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-beta 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-beta 1 treatment. Further studies are required to evaluate these short-term effects of TGF-beta 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-beta 1 affects intracellular calcium in a variety of cell types. In Sertoli cells, TGF-beta 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-beta 1 (>60 min) in Rat-1 cells (22) and NIH3T3 cells (1). In vascular smooth muscle cells, short-term TGF-beta 1 (30 s) treatment enhanced angiotensin II-induced [Ca2+]c but longer durations of TGF-beta 1 treatment (>30 min) reduced angiotensin II-induced [Ca2+]c (39). In our prior study with MMC, TGF-beta 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-beta 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-beta 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-beta 1. Thus dysregulation of intracellular Ca2+ by chronic upregulation of TGF-beta 1 in glomerular diseases would limit mitogenic and vasoconstrictive effects and may channel the mesangial cell to a fate of cell hypertrophy and vasodilation.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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