Mesangial cell-reduced Ca2+ signaling in high glucose is due to inactivation of phospholipase C-3 by protein kinase C
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《美国生理学杂志》
Institute of Medical Science, University Health Network, Department of Medicine, University of Toronto, Toronto, Canada
ABSTRACT
In high glucose, glomerular mesangial cells (MCs) demonstrate impaired Ca2+ signaling in response to seven-transmembrane receptor stimulation. To identify the mechanism, we first postulated decreased release from intracellular stores. Intracellular Ca2+ was measured in fluo-3-loaded primary cultured rat MCs using confocal fluorescence microscopy. In high glucose (HG) 30 mM for 48 h, the 25 nM ionomycin-stimulated intracellular Ca2+ response was reduced to 82% of that observed in normal glucose (NG). In NG 5.6 mM, Ca2+ responses to endothelin (ET)-1 and platelet-derived growth factor (PDGF) were unchanged in cells cultured in 50 nM Ca2+ vs. 1.8 mM Ca2+. Depletion of intracellular Ca2+ stores with thapsigargin eliminated ET-1-stimulated Ca2+ responses. Incubation in 30 mM glucose (HG) for 48 h or stimulation with phorbol myristate acetate (PMA) for 10 min eliminated the Ca2+ response to ET-1 but had no effect on the PDGF response. Downregulation of protein kinase C (PKC) with 24-h PMA or inhibition with G6976 in HG normalized the Ca2+ response to ET-1. Because ET-1 and PDGF stimulate Ca2+ signaling through different phospholipase C pathways, we hypothesized that, in HG, PKC selectively phosphorylates and inhibits PLC-3. Using confocal immunofluorescence imaging, in NG, a 1.6- to 1.7-fold increase in PLC-3 Ser1105 phosphorylation was observed following PMA or ET-1 stimulation for 10 min. In HG, immunofluorescent imaging and immunoblotting showed increased PLC-3 phosphorylation, without change in total PLC-3, which was reversed with 24-h PMA or G6976. We conclude that reduced Ca2+ signaling in HG cannot be explained by reduced Ca2+ stores but is due to conventional PKC-dependent phosphorylation and inactivation of PLC-3.
endothelin-1; platelet-derived growth factor; thapsigargin; ionomycin; fluo-3
WHEN EXPOSED TO HIGH glucose, glomerular mesangial cells transform into dedifferentiated myofibroblasts. This phenotype demonstrates significant changes in cell signaling including activation of PKC-, -1, -, -, and -, reduced Ca2+ responsiveness to signal transduction via seven-transmembrane receptors, and enhanced activation of mitogen-activated protein kinases (1, 4, 8, 10, 27). In high glucose, mesangial cell dysfunctional characteristics include F-actin disassembly, loss of normal contraction to peptide vasoconstrictors, and enhanced expression of transforming growth factor-1 and collagen IV, contributing to progressive diabetic glomerulopathy (5, 31). A mechanistic link between activation of PKC in high glucose and reduced Ca2+ signaling is reported by our lab (10) and others (18), although the exact mechanism(s) are not fully understood. PKC may inhibit capacitative Ca2+ influx through store-operated channels (SOC) in mesangial cells as postulated by Mené et al. (18). SOCs are highly expressed in mesangial cells, account for significant capacitative influx on depletion of intracellular stores, and have interactions with PKC (16, 17). Another possibility for the mechanism is that PKC may cause impaired Ca2+ release from endoplasmic reticulum stores.
In this study, using confocal laser-scanning microscopy of fluo-3 AM-loaded mesangial cells, we compared the effects of high glucose on the Ca2+ signaling responses to endothelin (ET)-1 and platelet-derived growth factor (PDGF). These autocoid growth factors are produced by mesangial cells (23, 28) and stimulate them through two different classes of receptors and signal transduction pathways. Our purpose was to identify divergence in the signaling mechanisms with respect to the effects of high glucose. ET-1 binds to its seven-transmembrane G protein-coupled receptor and, through activation of heterotrimeric G proteins, stimulates phospholipase C-3 (PLC-3) to break down phosphoinositide bisphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol (DAG) (11, 24). IP3 binds to its receptor on the endoplasmic reticulum causing Ca2+ release into the cytosol and nucleus. In mesangial cells, DAG-sensitive PKC-, -, and - are activated by ET-1 (7). PLC-3 activity is downregulated following phosphorylation of its Ser1105 residue via PKC, protein kinase A, or G (26, 29, 30). PDGF binds to its tyrosine kinase receptor, stimulating dimerization and autophosphorylation of the receptor. This is followed by association of PLC-1 to phosphorylated tyrosine residues on the receptor via its src homology-2 (SH2) domain (12). The subsequent activation of PLC-1 causes IP3 and DAG production (21). Deactivation of PLC-1 occurs following disassociation from the tyrosine kinase receptor and appears to be independent of PKC (13). We postulated that the different phospholipase C molecules in the signal transduction pathways for ET-1 and PDGF could be a point of divergence in the effects of high glucose on signal transduction.
In high glucose, mesangial cell DAG-sensitive PKC isozymes are activated in the absence of peptide hormone receptor occupancy and signal transduction (1, 4). We postulated that in mesangial cells exposed to high glucose, decreased Ca2+ signaling in response to ET-1 may be a result of DAG-sensitive PKC deactivation of PLC-3. Furthermore, we hypothesized that PDGF-stimulated Ca2+ signaling would be unaffected by high glucose.
To test these hypotheses, first we determined whether the reduced Ca2+ signaling in response to ET-1 in high glucose was due to reduced release from intracellular stores. Then, the mesangial cell Ca2+ signaling responses to ET-1 and PDGF were compared in normal and high glucose. To identify the role of DAG-sensitive PKC isozymes in modulating Ca2+ signaling in response to these two peptide hormones, phorbol myristate acetate (PMA) was used to either prestimulate PKC for 10 min or to downregulate DAG-sensitive PKC isozymes over 24 h. Finding no inhibition of the PDGF response in either high glucose or following the activation of DAG-sensitive PKC isozymes, we examined the role of PKC-dependent phosphorylation of PLC-3 using confocal immunofluorescence imaging. We found enhanced conventional PKC-dependent phosphorylation of PLC-3 in high glucose and that inhibition of PLC-3 phosphorylation normalized Ca2+ signaling in response to ET-1 in high glucose.
MATERIALS AND METHODS
Materials. DMEM was purchased from Invitrogen (Burlington, ON). FBS was purchased from Wisent (St. Bruno, PQ). Trypsin was purchased from GIBCO BRL Life Technologies (Burlington, ON). Fluo-3,5-(and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA; Fluo-3) was purchased from Molecular Probes (Eugene, OR). Pluronic acid was purchased from Sigma (St. Louis, MO). PMA, ET-1, and ionomycin were purchased from BD Biosciences (Mississauga, ON). PDGF was purchased from Boehringer Mannheim (Mannheim, Germany). Anti-IP3 receptor type I and III antibody was purchased from Cedarlane Laboratories (Hornby, ON). G6976 was purchased from Kymiya Biomedical (Seattle, WA). Anti-PLC-3 and anti-phospho-Ser1105-PLC-3 antibodies were purchased from Cell Signaling Technologies (Beverly, MA). Fluorescein (FITC)-conjugated AffiniPure goat anti-rabbit IgG was purchased from Jackson Immunoresearch Laboratories (West Grove, PA).
Cell culture. In this study, we used primary mesangial cells that were originally isolated and cultured from collagenase-treated glomeruli by sieving kidney cortex of 150- to 200-g male Sprague-Dawley rats (6). Mesangial cells (passages T8-T18) were grown for 2–5 days to confluence in DMEM containing 20% FBS, 5.6 mM normal glucose, pH 7.4, supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. Before all experiments, the mesangial cells were growth-arrested in 0.5% FBS-DMEM containing either 5.6 mM D-glucose, 30 mM D-glucose, or 24.4 mM L-glucose + 5.6 mM D-glucose (as an osmotic control) for 48 h.
Immunoblotting of PKC isozymes and PLC-3. Expression levels of PKC-, -1, -, -, and - and of total PLC3 were analyzed in total cell lysates. Cells were grown on 10-cm plates and growth-arrested in 5.6 or 30 mM glucose 0.5% FBS-DMEM for 48 h. To determine the effects of PKC downregulation, cells were stimulated with PMA for 24 h. Cells were then lysed in 2x SDS sample buffer at 100°C and disrupted by passage through a 26-gauge needle. For isolation of nuclear-enriched fractions, cells were lysed with buffer A without Triton and nuclei were freed with a Dounce homogenizer using five strokes of the "loose" pestle and five strokes of the "tight" pestle. The nuclear-enriched fractions were then isolated through centrifugation, mixed with 2x SDS sample buffer at 100°C, and disrupted by passage through a 26-gauge needle. The protein content was measured using the Bio-Rad Dc Lowry protein assay (Bio-Rad Laboratories, Hercules, CA). Ten microliters of protein were then subjected to SDS-PAGE on 10% gels, followed by transfer of the protein onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked in 5% skim milk in TTBS for 1 h and then rabbit polyclonal antibodies against specific PKC isozymes and PLC-3 described above were added to their respective membranes at a concentration of 1:1,000 and 1:500, respectively, in 5% skim milk in TTBS for 1 h. After being washed three times in TTBS, FITC-conjugated goat anti-rabbit secondary antibody at a concentration of 1:3,000 in 5% skim milk in TTBS was added to the membranes. The secondary antibody was detected by chemiluminescence and the membranes were developed on Kodak X-Omat Blue film (PerkinElmer Life Sciences, Boston, MA).
Confocal fluorescence imaging of intracellular Ca2+. Cells were plated and grown to subconfluence on glass coverslips under the above conditions. Growth-arrested cells were loaded with 2.5 μM fluo-3 in DMEM (containing CaCl2) plus 0.02% Pluronic F-127 for 60 min at 37°C and then washed with fluo-3-free 0% FBS-DMEM. The coverslips were then mounted in the chamber of a Zeiss confocal microscope (LSM 410; Düsseldorf, Germany), and the cells were imaged at room temperature (RT) before and during stimulation with 100 nM ET-1, 50 ng/ml PDGF, or 100 nM ionomycin for 10 min with or without pretreatment with 100 nM PMA or 10 μM thapsigargin. During stimulation, cells were immersed in either 1.8 mM [Ca2+] or 50 nM [Ca2+] extracellular medium. Confocal laser-scanning microscopy was performed as previously described (5) using an excitation wavelength of 488 nm and a scanning time of 1 s per image. The images were taken through the center of the cell nuclei and the digitized images were captured every 15–30 s for 120–240 s. The digitized images for each time point following stimulation were analyzed to determine total cell pixel intensity using Scion Image Analysis (Scion, Frederick, MD). For each condition, 30 cells from different coverslips of three separate experiments were analyzed. Intracellular Ca2+ concentration ([Ca2+]i) was calculated from pixel intensity as follows:
where Kd for fluo-3 was 390 nM (9). The Rmin and Rmax represent the fluorescence intensities measured with stimulation under minimum free Ca2+ (bound with EGTA) and maximum Ca2+ (with 100 nM ionomycin stimulation plus 10 mM CaCl2).
Confocal immunofluorescence imaging. Mesangial cells were grown for 2–5 days to confluence in DMEM containing 20% FBS, 5.6 mM normal glucose, pH 7.4, supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were growth-arrested in 0.5% FBS-DMEM containing either 5.6 mM D-glucose, 30 mM D-glucose, or 24.4 mM L-glucose + 5.6 mM D-glucose (as an osmotic control) for 48 h. After stimulation with either 10 min PMA, ET-1, or PDGF, the cells were then washed three times with PBS and fixed in 3.7% formaldehyde for 10 min at RT. The cells were then washed three times with PBS and then permeabilized with 100% methanol at –20°C for 10 min. After another three washes with PBS, cells were blocked with 1% goat serum plus 0.1% BSA in water for 60 min at RT. Rabbit polyclonal anti-IP3R type I or III, anti-PLC-3, and anti-phospho-Ser1105-PLC-3 were diluted to 1:50 in blocking solution and added to their respective coverslips for 60 min at 37°C. After three washes with PBS, polyclonal secondary goat anti-rabbit antibody of dilution 1:100 was added to each coverslip for 60 min at 37°C. The coverslips were then washed three times with PBS and mounted on glass slides. The cells were analyzed by confocal fluorescence microscopy using an excitation wavelength of 488 nm as the fluorescein (FITC)-conjugated AffiniPure goat anti-rabbit secondary IgG antibody has absorption and emission wavelengths of 492 and 520 nm, respectively. Total cell pixel intensity per cell was measured using Scion Image as above, where the pixel intensity ranged from 1 (low) to 256 (high) on a standard gray scale.
Statistical analysis. All results are expressed as means ± SE. Total cell number for each test group is pooled from three to four independent experiments. Statistical analysis of pixel intensity/cell was performed using InStat 2.01 statistics software (Graph Pad, Sacramento, CA). The means of two groups were compared using a Student's t-test. The means of three or more groups were compared by one-way ANOVA with a Tukey posttest.
RESULTS
Mesangial cell Ca2+ signaling in response to ET-1 and PDGF. Stimulation with ET-1 or PDGF caused a Ca2+ signaling response in 80–90% of mesangial cells. All responsive cells were selected for analysis. Stimulation of mesangial cells with ET-1 or PDGF caused a total cell Ca2+ response of 144 ± 4 pixel intensity/cell (n = 56) over a basal level of 65 ± 3 (n = 56; Fig. 1), corresponding to [Ca2+] ranges of 403–469 nM (peak) and 84–101 nM (basal), respectively.
Contribution to Ca2+ signaling from intracellular stores. To identify the component of ET-1- and PDGF-induced Ca2+ signaling in mesangial cells primarily arising from release of intracellular stored Ca2+, we studied Ca2+ responses to ET-1 and PDGF in 1.8 mM [Ca2+] and 50 nM [Ca2+] extracellular medium. The Ca2+ response to ET-1 was unchanged ranging from 144 ± 4 pixel intensity/cell in 1.8 mM [Ca2+] to 140 ± 3 pixel intensity/cell in 50 nM [Ca2+] (Fig. 1). In normal and low [Ca2+] medium, the Ca2+ response to PDGF was unchanged at 150 ± 4 and 143 ± 4 pixel intensity/cell (n = 40–54).
To deplete intracellular Ca2+ stores, mesangial cells were pretreated with 10 μM thapsigargin for 10 min and then stimulated with ionomycin or ET-1. After thapsigargin pretreatment, stimulation with ionomycin caused no change in Ca2+ levels above a basal value of 55 ± 3 pixel intensity/cell, confirming that thapsigargin successfully depleted Ca2+ stores (data not shown). Following thapsigargin pretreatment for 10 min, the peak response to ET-1 fell from 144 ± 4 pixel intensity/cell to a basal level of 56 ± 2 pixel intensity/cell (P < 0.001, n = 40).
Effect of high glucose on intracellular Ca2+ stores. To test whether 48-h incubation in high glucose altered intracellular Ca2+ stores, we tested the Ca2+ response to ionomycin in 50 nM [Ca2+] extracellular medium in 5.6 mM (normal) and 30 mM glucose (high). The Ca2+ response to ionomycin fell from 157 ± 5 pixel intensity/cell in normal glucose to 129 ± 6 pixel intensity/cell in high glucose (P < 0.001 vs. normal glucose, n = 41–47; Fig. 2), suggesting a reduction in intracellular Ca2+-dependent Ca2+ release or decreased availability of stored Ca2+ in high glucose.
DAG-sensitive PKC inhibition by PMA downregulation. To determine the effects of PMA incubation for 24 h on the expression of specific PKC isozymes in normal glucose, total cell lysates were examined by Western immunoblotting for the presence of PKC-, -1, -, -, and -. The DAG-sensitive PKC isozymes were completely downregulated, whereas no effect was observed on the DAG-insensitive PKC- (Fig. 3).
Effect of high glucose and PKC on the ET-1 and PDGF Ca2+ responses. To test the effects of 48-h high glucose on ET-1- and PDGF-induced Ca2+ signaling, mesangial cells were growth-arrested for 48 h in either 5.6 or 30 mM glucose and then stimulated with ET-1 or PDGF for 10 min. The Ca2+ response to ET-1 was reduced from 123 ± 4 pixel intensity/cell in 5.6 mM glucose to 44 ± 2 pixel intensity/cell in 30 mM glucose (P < 0.001 vs. normal glucose, n = 88–96; Fig. 4, A and B). To test the effect of PKC activation on Ca2+ signaling, PKC was stimulated by incubation with PMA for 10 min in normal glucose before exposure to ET-1 or PDGF. Following preactivation of PKC, the Ca2+ response to ET-1 fell from 123 ± 4 to a basal level of 59 ± 2 pixel intensity/cell (P < 0.001 vs. normal glucose without PKC activation, n = 52–96; Fig. 4, A and B). To determine the effects of downregulation of PKC in normal and high glucose Ca2+ signaling, mesangial cells were incubated in 5.6 and 30 mM glucose with PMA for 24 h and then stimulated with ET-1. Downregulation of PKC normalized the ET-1-stimulated Ca2+ response in high glucose to 144 ± 4 pixel intensity/cell (P < 0.001 vs. high glucose without PKC downregulation n = 48–88). Inhibition of the conventional PKC isozymes (- and -) with G6976 similarly normalized the ET-1-stimulated Ca2+ response in high glucose to 121 ± 7 pixel intensity/cell, while having no significant effect in normal glucose (P < 0.001 vs. high glucose without G6976 n = 56–88). The Ca2+ response to ET-1 was unaffected by 48-h incubation in 5.6 mM glucose ± 24.4 mM L-glucose (Fig. 4C).
The Ca2+ response to PDGF decreased modestly from 148 ± 4 pixel intensity/cell in 5.6 mM glucose to 133 ± 5 pixel intensity/cell in 30 mM glucose (P < 0.05 vs. normal glucose, n = 38–54). The Ca2+ response to PDGF was unchanged from 148 ± 4 to 141 ± 3 pixel intensity/cell with PKC preactivation (n = 53–54; Fig. 5).
Effect of high glucose on expression of the IP3 type I receptor. To determine the effects of high glucose conditions on expression of the IP3 type I receptor, the intracellular IP3 type I receptor was observed by confocal immunofluorescence imaging and total cell lysates were examined by Western immunoblotting. In high glucose, using both immunofluorescence imaging (n = 127–166) and immunoblotting (n = 5), quantitative analysis showed no significant change compared with normal glucose in the expression levels of the IP3 type I receptor (Fig. 6, A and B). The IP3 type III receptor was not detected.
Expression and phosphorylation of PLC-3. To determine the effects of high glucose on expression of PLC-3, cells were examined by confocal immunofluorescence microscopy for the presence of PLC-3. The expression of PLC-3 was unchanged from 71 ± 1 pixel intensity/cell in normal glucose to 70 ± 1 pixel intensity/cell in high glucose (n = 95–101; Fig. 7, A and B). PLC-3 was localized throughout the cytoplasm and was found to be elevated in the nucleus of the cell. Preboiling of the primary antibody to PLC-3 resulted in no immunufluorescence labeling of the mesangial cell cytosol or nucleus (not shown). Immunoblotting of total PLC-3 showed no difference in PLC-3 expression in normal and high glucose and with PKC downregulation (Fig. 7, C and D).
To determine the effects of high glucose on PLC-3 phosphorylation at the inhibitory Ser1105 site, mesangial cells were examined by confocal immunofluorescence microscopy and nuclear-enriched cellular preparations were immunoblotted. Using immunofluorescence imaging, phosphorylated PLC-3 increased from 57 ± 1 pixel intensity/cell in normal glucose to 117 ± 2 pixel intensity/cell in high glucose (P < 0.01, n = 162–174; Fig. 8). In normal glucose, phosphorylated PLC-3 was found in elevated levels in the nucleus, whereas, in high glucose, it was found more evenly distributed throughout the cell as well as in the nucleus. Phosphorylated PLC-3 was unchanged from 57 ± 1 pixel intensity/cell in normal glucose to 56 ± 1 pixel intensity/cell in high L-glucose (n = 162–191).
After pretreatment with 10-min PMA, the amount of phosphorylated PLC-3 increased from 57 ± 1 to 91 ± 3 pixel intensity/cell (P < 0.01 vs. normal glucose without PKC activation, n = 86–162) and demonstrated a nuclear distribution. After ET-1 stimulation in 5.6 mM glucose, the amount of phosphorylated PLC-3 increased from 57 ± 1 to 94 ± 2 pixel intensity/cell (P < 0.01 vs. normal glucose without ET-1 stimulation, n = 162–191), and phosphorylated PLC-3 was also localized to the nucleus similar to that observed with acute stimulation with PMA. By contrast, stimulation with PDGF in 5.6 mM glucose caused no visible change in PLC-3 phosphorylation (Fig. 9C). In high glucose for 48 h, pretreatment with PMA for the final 24 h resulted in a decrease in phosphorylated PLC-3 from 117 ± 2 to 54 ± 1 pixel intensity/cell (P < 0.01 vs. high glucose without PKC downregulation, n = 157–174). Similarly, the conventional PKC isozyme inhibitor G6976 abrogated high glucose-induced phosphorylation of PLC-3. In high glucose, acute stimulation with PMA or ET-1 for 10 min caused no additional increase in immunofluorescence or cellular distribution of phosphorylated PLC-3 (data not shown).
Western immunoblotting under the same conditions demonstrated corollary findings of enhanced phosphorylation of PLC-3 in the nucleus in high glucose and with 10-min PMA pretreatment (Fig. 9). Downregulation of DAG-sensitive PKC with PMA for 24 h prevented high glucose-induced phosphorylation of PLC-3 as did addition of G6976. These results were seen both in the total cell (Fig. 9, A and B) and in the nuclear-enriched fraction (Fig. 9, C and D).
DISCUSSION
In this study, we showed that, in high glucose, ET-1-stimulated Ca2+ signaling in mesangial cells is inhibited by the phosphorylation at the inactivation site of PLC-3 by a DAG-sensitive PKC mechanism. The fluo-3 confocal imaging method detected predominantly intracellular Ca2+ signaling in response to ET-1 and PDGF that remained unchanged when the cells were switched from 1.8 mM [Ca2+] extracellular medium to 50 nM [Ca2+] medium and disappeared with depletion of intracellular Ca2+ stores with thapsigargin. High glucose may have caused a small, but significant, depletion in intracellular Ca2+ stores as seen by a decreased Ca2+ signal on stimulation with ionomycin or PDGF. The loss of Ca2+ response to ET-1 in high glucose was mimicked in normal glucose by a 10-min PMA pretreatment. Correspondingly, downregulation of DAG-sensitive PKCs with 24-h PMA or inhibition of the conventional PKC isozymes (- and -) with G6976 normalized the Ca2+ response in high glucose, confirming that this inhibition is due to activation of DAG-sensitive PKC. By contrast, the Ca2+ response to PDGF in high glucose and following 10-min PMA pretreatment was reduced by <10% in keeping with the finding of modest reduction in intracellular stored Ca2+ availability. To explain this difference between the Ca2+ response to ET-1 and PDGF in high glucose, we tested whether PKC specifically inhibits the ET-1 response by phosphorylating PLC-3. We found that both high glucose and 10-min PMA pretreatment caused phosphorylation of PLC-3, with no change in total PLC-3 expression, and that downregulation of PKC with 24-h PMA or inhibition of the conventional PKC isozymes (- and -) with G6976 prevented PLC-3 phosphorylation in high glucose. These data conclusively demonstrate a DAG-sensitive conventional PKC-dependent mechanism of high glucose inhibition of ET-1-induced Ca2+ signaling in mesangial cells.
Our data reflect current understanding of high glucose inhibition of Ca2+ signaling in mesangial cells, although the specifics of the origin of the Ca2+ signal are variable. Mené et al. (18) reported that high glucose causes inhibition of capacitative influx through store-operated channels in mesangial cells through a PKC-dependent mechanism. Nutt and O'Neil (20) showed that high glucose inhibits Ca2+ signaling not from capacitative influx nor from intracellular stores, but from a separate source that they postulate to be receptor-operated Ca2+ influx. Differences between these studies and ours reflect methodological variances and the focus on different aspects of signaling. First, the measurement of Ca2+ signaling in the previously reported studies was achieved by loading cells with the UV-light-excitable Ca2+ fluorophore fura-2 and measuring Ca2+ fluorescence changes by obtaining the excitation-emission ratio using a dual-excitation fluorometer (18, 20). Our study made use of the visible light-excitable Ca2+ fluorophore fluo-3 and measured Ca2+ fluorescence changes using a confocal laser-scanning microcope of wavelength 488 nm. It is likely that the methods of detecting fluorescence using these fluorophores vary in their sensitivity to different elements of Ca2+ signaling. Fura-2 appears to detect both Ca2+ influx and intracellular store release, whereas fluo-3 identifies primarily intracellular store release. Previous results from our lab indicate that, in mesangial cells, fura-2 detects 30–40% of Ca2+ signaling as influx into the cells (25). The fluo-3 fluorescence measurement of Ca2+ signaling in our current study identifies primarily intracellular Ca2+ release. In high glucose, we observed less Ca2+ release from intracellular stores by ionomycin or PDGF stimulation that may be due to reduced refilling of intracellular Ca2+ stores via store-operated channels, as previously described (18).
Our results suggest that, in mesangial cells, DAG-sensitive PKC isozymes act as a negative feedback mechanism to control the strength and duration of the seven-transmembrane receptor-stimulated Ca2+ signal. In high glucose, this negative feedback loop appears to be permanently activated due to de novo synthesis of DAG and subsequent PKC activation. In other in vitro studies, high glucose has been found to increase total PKC-, -2, and - as well as plasma and nuclear membrane-associated PKC-, -, and - (14). In addition, an increase in membrane-associated PKC-, -, and - was found in isolated diabetic rat glomeruli, which was reversed by near normalization of blood glucose by treatment with insulin (2). Our lab (8) reported that, in high glucose, stimulation of mesangial cell ERK1/2 by ET-1 is enhanced via a DAG-sensitive PKC mechanism. Recent work from our lab identified a role for reactive oxygen species (ROS) in PKC-dependent inhibition of Ca2+ signaling in high glucose (10). In this study, ROS production in high glucose was located to NADPH oxidase as antisense to NADPH oxidase reversed high glucose inhibition of Ca2+ signaling. H2O2 was found to cause membrane translocation of PKC- and -. These results suggested an interaction between ROS and PKC in the high glucose inhibition of Ca2+ signaling. In high glucose, decreased Ca2+ signaling occurs with many changes in mesangial cells that contribute to their transformation into a myofibroblast phenotype.
A point of interest in our study was the change in the intracellular pattern of phosphorylated PLC-3 following acute stimulation with ET-1 or 10-min PMA in normal glucose vs. that observed following 48-h exposure to high glucose. On activation of DAG-sensitive PKCs and stimulation with ET-1, we observed a very strong localization of phosphorylated PLC-3 in the nucleus, whereas, in high glucose, a more evenly distributed pattern of PLC-3 throughout the cytoplasm was observed. On immunofluorescence analysis, total PLC-3, in normal glucose, demonstrated both a cytosolic and nuclear distribution. In high glucose, we found no change in total PLC-3 immunofluorescence intensity or pattern on acute stimulation with PMA or ET-1. This suggests that the phosphorylated PLC-3 seen in large quantities in the nucleus with PKC activation or ET-1 stimulation may reflect in situ nuclear activation and phosphorylation. This is in agreement with studies recognizing the localization of PLC in the nucleus (19, 22). PLC-1 has been shown to reside in large quantities the nucleus and to possess a COOH-terminal basic amino acid sequence that is responsible for its nuclear localization (3, 15). It has been shown that when DAG-sensitive PKCs are activated, they translocate to the nucleus as well as to the plasma membrane and also that this nuclear translocation is attributable to DAG produced by nuclear PLC (19).
By contrast, the cytosolic distribution of phosphorylated PLC-3 throughout the cytoplasm in high glucose may simply reflect a steady-state pattern of deactivated enzyme. We found that downregulation of PKCs for 24 h with PMA was sufficient to reverse phosphorylation of PLC-3 that occurred with 48-h high glucose exposure. This suggests a turnover of PLC-3 phosphorylation and the potential for restoration of PLC-3 function following normalization of glucose.
In summary, the loss of mesangial cell Ca2+ signaling responses to ET-1 in high glucose is due to a DAG-sensitive conventional PKC mechanism that includes deactivation of PLC-3 at Ser1105. Future directions will explore the specific PKC isozymes involved in the inhibition of Ca2+ signaling and the interactive role of ROS.
GRANTS
This research was supported by The Canadian Institutes of Health Research and The Canadian Diabetes Association.
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. Section 1734 solely to indicate this fact.
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ABSTRACT
In high glucose, glomerular mesangial cells (MCs) demonstrate impaired Ca2+ signaling in response to seven-transmembrane receptor stimulation. To identify the mechanism, we first postulated decreased release from intracellular stores. Intracellular Ca2+ was measured in fluo-3-loaded primary cultured rat MCs using confocal fluorescence microscopy. In high glucose (HG) 30 mM for 48 h, the 25 nM ionomycin-stimulated intracellular Ca2+ response was reduced to 82% of that observed in normal glucose (NG). In NG 5.6 mM, Ca2+ responses to endothelin (ET)-1 and platelet-derived growth factor (PDGF) were unchanged in cells cultured in 50 nM Ca2+ vs. 1.8 mM Ca2+. Depletion of intracellular Ca2+ stores with thapsigargin eliminated ET-1-stimulated Ca2+ responses. Incubation in 30 mM glucose (HG) for 48 h or stimulation with phorbol myristate acetate (PMA) for 10 min eliminated the Ca2+ response to ET-1 but had no effect on the PDGF response. Downregulation of protein kinase C (PKC) with 24-h PMA or inhibition with G6976 in HG normalized the Ca2+ response to ET-1. Because ET-1 and PDGF stimulate Ca2+ signaling through different phospholipase C pathways, we hypothesized that, in HG, PKC selectively phosphorylates and inhibits PLC-3. Using confocal immunofluorescence imaging, in NG, a 1.6- to 1.7-fold increase in PLC-3 Ser1105 phosphorylation was observed following PMA or ET-1 stimulation for 10 min. In HG, immunofluorescent imaging and immunoblotting showed increased PLC-3 phosphorylation, without change in total PLC-3, which was reversed with 24-h PMA or G6976. We conclude that reduced Ca2+ signaling in HG cannot be explained by reduced Ca2+ stores but is due to conventional PKC-dependent phosphorylation and inactivation of PLC-3.
endothelin-1; platelet-derived growth factor; thapsigargin; ionomycin; fluo-3
WHEN EXPOSED TO HIGH glucose, glomerular mesangial cells transform into dedifferentiated myofibroblasts. This phenotype demonstrates significant changes in cell signaling including activation of PKC-, -1, -, -, and -, reduced Ca2+ responsiveness to signal transduction via seven-transmembrane receptors, and enhanced activation of mitogen-activated protein kinases (1, 4, 8, 10, 27). In high glucose, mesangial cell dysfunctional characteristics include F-actin disassembly, loss of normal contraction to peptide vasoconstrictors, and enhanced expression of transforming growth factor-1 and collagen IV, contributing to progressive diabetic glomerulopathy (5, 31). A mechanistic link between activation of PKC in high glucose and reduced Ca2+ signaling is reported by our lab (10) and others (18), although the exact mechanism(s) are not fully understood. PKC may inhibit capacitative Ca2+ influx through store-operated channels (SOC) in mesangial cells as postulated by Mené et al. (18). SOCs are highly expressed in mesangial cells, account for significant capacitative influx on depletion of intracellular stores, and have interactions with PKC (16, 17). Another possibility for the mechanism is that PKC may cause impaired Ca2+ release from endoplasmic reticulum stores.
In this study, using confocal laser-scanning microscopy of fluo-3 AM-loaded mesangial cells, we compared the effects of high glucose on the Ca2+ signaling responses to endothelin (ET)-1 and platelet-derived growth factor (PDGF). These autocoid growth factors are produced by mesangial cells (23, 28) and stimulate them through two different classes of receptors and signal transduction pathways. Our purpose was to identify divergence in the signaling mechanisms with respect to the effects of high glucose. ET-1 binds to its seven-transmembrane G protein-coupled receptor and, through activation of heterotrimeric G proteins, stimulates phospholipase C-3 (PLC-3) to break down phosphoinositide bisphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol (DAG) (11, 24). IP3 binds to its receptor on the endoplasmic reticulum causing Ca2+ release into the cytosol and nucleus. In mesangial cells, DAG-sensitive PKC-, -, and - are activated by ET-1 (7). PLC-3 activity is downregulated following phosphorylation of its Ser1105 residue via PKC, protein kinase A, or G (26, 29, 30). PDGF binds to its tyrosine kinase receptor, stimulating dimerization and autophosphorylation of the receptor. This is followed by association of PLC-1 to phosphorylated tyrosine residues on the receptor via its src homology-2 (SH2) domain (12). The subsequent activation of PLC-1 causes IP3 and DAG production (21). Deactivation of PLC-1 occurs following disassociation from the tyrosine kinase receptor and appears to be independent of PKC (13). We postulated that the different phospholipase C molecules in the signal transduction pathways for ET-1 and PDGF could be a point of divergence in the effects of high glucose on signal transduction.
In high glucose, mesangial cell DAG-sensitive PKC isozymes are activated in the absence of peptide hormone receptor occupancy and signal transduction (1, 4). We postulated that in mesangial cells exposed to high glucose, decreased Ca2+ signaling in response to ET-1 may be a result of DAG-sensitive PKC deactivation of PLC-3. Furthermore, we hypothesized that PDGF-stimulated Ca2+ signaling would be unaffected by high glucose.
To test these hypotheses, first we determined whether the reduced Ca2+ signaling in response to ET-1 in high glucose was due to reduced release from intracellular stores. Then, the mesangial cell Ca2+ signaling responses to ET-1 and PDGF were compared in normal and high glucose. To identify the role of DAG-sensitive PKC isozymes in modulating Ca2+ signaling in response to these two peptide hormones, phorbol myristate acetate (PMA) was used to either prestimulate PKC for 10 min or to downregulate DAG-sensitive PKC isozymes over 24 h. Finding no inhibition of the PDGF response in either high glucose or following the activation of DAG-sensitive PKC isozymes, we examined the role of PKC-dependent phosphorylation of PLC-3 using confocal immunofluorescence imaging. We found enhanced conventional PKC-dependent phosphorylation of PLC-3 in high glucose and that inhibition of PLC-3 phosphorylation normalized Ca2+ signaling in response to ET-1 in high glucose.
MATERIALS AND METHODS
Materials. DMEM was purchased from Invitrogen (Burlington, ON). FBS was purchased from Wisent (St. Bruno, PQ). Trypsin was purchased from GIBCO BRL Life Technologies (Burlington, ON). Fluo-3,5-(and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA; Fluo-3) was purchased from Molecular Probes (Eugene, OR). Pluronic acid was purchased from Sigma (St. Louis, MO). PMA, ET-1, and ionomycin were purchased from BD Biosciences (Mississauga, ON). PDGF was purchased from Boehringer Mannheim (Mannheim, Germany). Anti-IP3 receptor type I and III antibody was purchased from Cedarlane Laboratories (Hornby, ON). G6976 was purchased from Kymiya Biomedical (Seattle, WA). Anti-PLC-3 and anti-phospho-Ser1105-PLC-3 antibodies were purchased from Cell Signaling Technologies (Beverly, MA). Fluorescein (FITC)-conjugated AffiniPure goat anti-rabbit IgG was purchased from Jackson Immunoresearch Laboratories (West Grove, PA).
Cell culture. In this study, we used primary mesangial cells that were originally isolated and cultured from collagenase-treated glomeruli by sieving kidney cortex of 150- to 200-g male Sprague-Dawley rats (6). Mesangial cells (passages T8-T18) were grown for 2–5 days to confluence in DMEM containing 20% FBS, 5.6 mM normal glucose, pH 7.4, supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. Before all experiments, the mesangial cells were growth-arrested in 0.5% FBS-DMEM containing either 5.6 mM D-glucose, 30 mM D-glucose, or 24.4 mM L-glucose + 5.6 mM D-glucose (as an osmotic control) for 48 h.
Immunoblotting of PKC isozymes and PLC-3. Expression levels of PKC-, -1, -, -, and - and of total PLC3 were analyzed in total cell lysates. Cells were grown on 10-cm plates and growth-arrested in 5.6 or 30 mM glucose 0.5% FBS-DMEM for 48 h. To determine the effects of PKC downregulation, cells were stimulated with PMA for 24 h. Cells were then lysed in 2x SDS sample buffer at 100°C and disrupted by passage through a 26-gauge needle. For isolation of nuclear-enriched fractions, cells were lysed with buffer A without Triton and nuclei were freed with a Dounce homogenizer using five strokes of the "loose" pestle and five strokes of the "tight" pestle. The nuclear-enriched fractions were then isolated through centrifugation, mixed with 2x SDS sample buffer at 100°C, and disrupted by passage through a 26-gauge needle. The protein content was measured using the Bio-Rad Dc Lowry protein assay (Bio-Rad Laboratories, Hercules, CA). Ten microliters of protein were then subjected to SDS-PAGE on 10% gels, followed by transfer of the protein onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked in 5% skim milk in TTBS for 1 h and then rabbit polyclonal antibodies against specific PKC isozymes and PLC-3 described above were added to their respective membranes at a concentration of 1:1,000 and 1:500, respectively, in 5% skim milk in TTBS for 1 h. After being washed three times in TTBS, FITC-conjugated goat anti-rabbit secondary antibody at a concentration of 1:3,000 in 5% skim milk in TTBS was added to the membranes. The secondary antibody was detected by chemiluminescence and the membranes were developed on Kodak X-Omat Blue film (PerkinElmer Life Sciences, Boston, MA).
Confocal fluorescence imaging of intracellular Ca2+. Cells were plated and grown to subconfluence on glass coverslips under the above conditions. Growth-arrested cells were loaded with 2.5 μM fluo-3 in DMEM (containing CaCl2) plus 0.02% Pluronic F-127 for 60 min at 37°C and then washed with fluo-3-free 0% FBS-DMEM. The coverslips were then mounted in the chamber of a Zeiss confocal microscope (LSM 410; Düsseldorf, Germany), and the cells were imaged at room temperature (RT) before and during stimulation with 100 nM ET-1, 50 ng/ml PDGF, or 100 nM ionomycin for 10 min with or without pretreatment with 100 nM PMA or 10 μM thapsigargin. During stimulation, cells were immersed in either 1.8 mM [Ca2+] or 50 nM [Ca2+] extracellular medium. Confocal laser-scanning microscopy was performed as previously described (5) using an excitation wavelength of 488 nm and a scanning time of 1 s per image. The images were taken through the center of the cell nuclei and the digitized images were captured every 15–30 s for 120–240 s. The digitized images for each time point following stimulation were analyzed to determine total cell pixel intensity using Scion Image Analysis (Scion, Frederick, MD). For each condition, 30 cells from different coverslips of three separate experiments were analyzed. Intracellular Ca2+ concentration ([Ca2+]i) was calculated from pixel intensity as follows:
where Kd for fluo-3 was 390 nM (9). The Rmin and Rmax represent the fluorescence intensities measured with stimulation under minimum free Ca2+ (bound with EGTA) and maximum Ca2+ (with 100 nM ionomycin stimulation plus 10 mM CaCl2).
Confocal immunofluorescence imaging. Mesangial cells were grown for 2–5 days to confluence in DMEM containing 20% FBS, 5.6 mM normal glucose, pH 7.4, supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were growth-arrested in 0.5% FBS-DMEM containing either 5.6 mM D-glucose, 30 mM D-glucose, or 24.4 mM L-glucose + 5.6 mM D-glucose (as an osmotic control) for 48 h. After stimulation with either 10 min PMA, ET-1, or PDGF, the cells were then washed three times with PBS and fixed in 3.7% formaldehyde for 10 min at RT. The cells were then washed three times with PBS and then permeabilized with 100% methanol at –20°C for 10 min. After another three washes with PBS, cells were blocked with 1% goat serum plus 0.1% BSA in water for 60 min at RT. Rabbit polyclonal anti-IP3R type I or III, anti-PLC-3, and anti-phospho-Ser1105-PLC-3 were diluted to 1:50 in blocking solution and added to their respective coverslips for 60 min at 37°C. After three washes with PBS, polyclonal secondary goat anti-rabbit antibody of dilution 1:100 was added to each coverslip for 60 min at 37°C. The coverslips were then washed three times with PBS and mounted on glass slides. The cells were analyzed by confocal fluorescence microscopy using an excitation wavelength of 488 nm as the fluorescein (FITC)-conjugated AffiniPure goat anti-rabbit secondary IgG antibody has absorption and emission wavelengths of 492 and 520 nm, respectively. Total cell pixel intensity per cell was measured using Scion Image as above, where the pixel intensity ranged from 1 (low) to 256 (high) on a standard gray scale.
Statistical analysis. All results are expressed as means ± SE. Total cell number for each test group is pooled from three to four independent experiments. Statistical analysis of pixel intensity/cell was performed using InStat 2.01 statistics software (Graph Pad, Sacramento, CA). The means of two groups were compared using a Student's t-test. The means of three or more groups were compared by one-way ANOVA with a Tukey posttest.
RESULTS
Mesangial cell Ca2+ signaling in response to ET-1 and PDGF. Stimulation with ET-1 or PDGF caused a Ca2+ signaling response in 80–90% of mesangial cells. All responsive cells were selected for analysis. Stimulation of mesangial cells with ET-1 or PDGF caused a total cell Ca2+ response of 144 ± 4 pixel intensity/cell (n = 56) over a basal level of 65 ± 3 (n = 56; Fig. 1), corresponding to [Ca2+] ranges of 403–469 nM (peak) and 84–101 nM (basal), respectively.
Contribution to Ca2+ signaling from intracellular stores. To identify the component of ET-1- and PDGF-induced Ca2+ signaling in mesangial cells primarily arising from release of intracellular stored Ca2+, we studied Ca2+ responses to ET-1 and PDGF in 1.8 mM [Ca2+] and 50 nM [Ca2+] extracellular medium. The Ca2+ response to ET-1 was unchanged ranging from 144 ± 4 pixel intensity/cell in 1.8 mM [Ca2+] to 140 ± 3 pixel intensity/cell in 50 nM [Ca2+] (Fig. 1). In normal and low [Ca2+] medium, the Ca2+ response to PDGF was unchanged at 150 ± 4 and 143 ± 4 pixel intensity/cell (n = 40–54).
To deplete intracellular Ca2+ stores, mesangial cells were pretreated with 10 μM thapsigargin for 10 min and then stimulated with ionomycin or ET-1. After thapsigargin pretreatment, stimulation with ionomycin caused no change in Ca2+ levels above a basal value of 55 ± 3 pixel intensity/cell, confirming that thapsigargin successfully depleted Ca2+ stores (data not shown). Following thapsigargin pretreatment for 10 min, the peak response to ET-1 fell from 144 ± 4 pixel intensity/cell to a basal level of 56 ± 2 pixel intensity/cell (P < 0.001, n = 40).
Effect of high glucose on intracellular Ca2+ stores. To test whether 48-h incubation in high glucose altered intracellular Ca2+ stores, we tested the Ca2+ response to ionomycin in 50 nM [Ca2+] extracellular medium in 5.6 mM (normal) and 30 mM glucose (high). The Ca2+ response to ionomycin fell from 157 ± 5 pixel intensity/cell in normal glucose to 129 ± 6 pixel intensity/cell in high glucose (P < 0.001 vs. normal glucose, n = 41–47; Fig. 2), suggesting a reduction in intracellular Ca2+-dependent Ca2+ release or decreased availability of stored Ca2+ in high glucose.
DAG-sensitive PKC inhibition by PMA downregulation. To determine the effects of PMA incubation for 24 h on the expression of specific PKC isozymes in normal glucose, total cell lysates were examined by Western immunoblotting for the presence of PKC-, -1, -, -, and -. The DAG-sensitive PKC isozymes were completely downregulated, whereas no effect was observed on the DAG-insensitive PKC- (Fig. 3).
Effect of high glucose and PKC on the ET-1 and PDGF Ca2+ responses. To test the effects of 48-h high glucose on ET-1- and PDGF-induced Ca2+ signaling, mesangial cells were growth-arrested for 48 h in either 5.6 or 30 mM glucose and then stimulated with ET-1 or PDGF for 10 min. The Ca2+ response to ET-1 was reduced from 123 ± 4 pixel intensity/cell in 5.6 mM glucose to 44 ± 2 pixel intensity/cell in 30 mM glucose (P < 0.001 vs. normal glucose, n = 88–96; Fig. 4, A and B). To test the effect of PKC activation on Ca2+ signaling, PKC was stimulated by incubation with PMA for 10 min in normal glucose before exposure to ET-1 or PDGF. Following preactivation of PKC, the Ca2+ response to ET-1 fell from 123 ± 4 to a basal level of 59 ± 2 pixel intensity/cell (P < 0.001 vs. normal glucose without PKC activation, n = 52–96; Fig. 4, A and B). To determine the effects of downregulation of PKC in normal and high glucose Ca2+ signaling, mesangial cells were incubated in 5.6 and 30 mM glucose with PMA for 24 h and then stimulated with ET-1. Downregulation of PKC normalized the ET-1-stimulated Ca2+ response in high glucose to 144 ± 4 pixel intensity/cell (P < 0.001 vs. high glucose without PKC downregulation n = 48–88). Inhibition of the conventional PKC isozymes (- and -) with G6976 similarly normalized the ET-1-stimulated Ca2+ response in high glucose to 121 ± 7 pixel intensity/cell, while having no significant effect in normal glucose (P < 0.001 vs. high glucose without G6976 n = 56–88). The Ca2+ response to ET-1 was unaffected by 48-h incubation in 5.6 mM glucose ± 24.4 mM L-glucose (Fig. 4C).
The Ca2+ response to PDGF decreased modestly from 148 ± 4 pixel intensity/cell in 5.6 mM glucose to 133 ± 5 pixel intensity/cell in 30 mM glucose (P < 0.05 vs. normal glucose, n = 38–54). The Ca2+ response to PDGF was unchanged from 148 ± 4 to 141 ± 3 pixel intensity/cell with PKC preactivation (n = 53–54; Fig. 5).
Effect of high glucose on expression of the IP3 type I receptor. To determine the effects of high glucose conditions on expression of the IP3 type I receptor, the intracellular IP3 type I receptor was observed by confocal immunofluorescence imaging and total cell lysates were examined by Western immunoblotting. In high glucose, using both immunofluorescence imaging (n = 127–166) and immunoblotting (n = 5), quantitative analysis showed no significant change compared with normal glucose in the expression levels of the IP3 type I receptor (Fig. 6, A and B). The IP3 type III receptor was not detected.
Expression and phosphorylation of PLC-3. To determine the effects of high glucose on expression of PLC-3, cells were examined by confocal immunofluorescence microscopy for the presence of PLC-3. The expression of PLC-3 was unchanged from 71 ± 1 pixel intensity/cell in normal glucose to 70 ± 1 pixel intensity/cell in high glucose (n = 95–101; Fig. 7, A and B). PLC-3 was localized throughout the cytoplasm and was found to be elevated in the nucleus of the cell. Preboiling of the primary antibody to PLC-3 resulted in no immunufluorescence labeling of the mesangial cell cytosol or nucleus (not shown). Immunoblotting of total PLC-3 showed no difference in PLC-3 expression in normal and high glucose and with PKC downregulation (Fig. 7, C and D).
To determine the effects of high glucose on PLC-3 phosphorylation at the inhibitory Ser1105 site, mesangial cells were examined by confocal immunofluorescence microscopy and nuclear-enriched cellular preparations were immunoblotted. Using immunofluorescence imaging, phosphorylated PLC-3 increased from 57 ± 1 pixel intensity/cell in normal glucose to 117 ± 2 pixel intensity/cell in high glucose (P < 0.01, n = 162–174; Fig. 8). In normal glucose, phosphorylated PLC-3 was found in elevated levels in the nucleus, whereas, in high glucose, it was found more evenly distributed throughout the cell as well as in the nucleus. Phosphorylated PLC-3 was unchanged from 57 ± 1 pixel intensity/cell in normal glucose to 56 ± 1 pixel intensity/cell in high L-glucose (n = 162–191).
After pretreatment with 10-min PMA, the amount of phosphorylated PLC-3 increased from 57 ± 1 to 91 ± 3 pixel intensity/cell (P < 0.01 vs. normal glucose without PKC activation, n = 86–162) and demonstrated a nuclear distribution. After ET-1 stimulation in 5.6 mM glucose, the amount of phosphorylated PLC-3 increased from 57 ± 1 to 94 ± 2 pixel intensity/cell (P < 0.01 vs. normal glucose without ET-1 stimulation, n = 162–191), and phosphorylated PLC-3 was also localized to the nucleus similar to that observed with acute stimulation with PMA. By contrast, stimulation with PDGF in 5.6 mM glucose caused no visible change in PLC-3 phosphorylation (Fig. 9C). In high glucose for 48 h, pretreatment with PMA for the final 24 h resulted in a decrease in phosphorylated PLC-3 from 117 ± 2 to 54 ± 1 pixel intensity/cell (P < 0.01 vs. high glucose without PKC downregulation, n = 157–174). Similarly, the conventional PKC isozyme inhibitor G6976 abrogated high glucose-induced phosphorylation of PLC-3. In high glucose, acute stimulation with PMA or ET-1 for 10 min caused no additional increase in immunofluorescence or cellular distribution of phosphorylated PLC-3 (data not shown).
Western immunoblotting under the same conditions demonstrated corollary findings of enhanced phosphorylation of PLC-3 in the nucleus in high glucose and with 10-min PMA pretreatment (Fig. 9). Downregulation of DAG-sensitive PKC with PMA for 24 h prevented high glucose-induced phosphorylation of PLC-3 as did addition of G6976. These results were seen both in the total cell (Fig. 9, A and B) and in the nuclear-enriched fraction (Fig. 9, C and D).
DISCUSSION
In this study, we showed that, in high glucose, ET-1-stimulated Ca2+ signaling in mesangial cells is inhibited by the phosphorylation at the inactivation site of PLC-3 by a DAG-sensitive PKC mechanism. The fluo-3 confocal imaging method detected predominantly intracellular Ca2+ signaling in response to ET-1 and PDGF that remained unchanged when the cells were switched from 1.8 mM [Ca2+] extracellular medium to 50 nM [Ca2+] medium and disappeared with depletion of intracellular Ca2+ stores with thapsigargin. High glucose may have caused a small, but significant, depletion in intracellular Ca2+ stores as seen by a decreased Ca2+ signal on stimulation with ionomycin or PDGF. The loss of Ca2+ response to ET-1 in high glucose was mimicked in normal glucose by a 10-min PMA pretreatment. Correspondingly, downregulation of DAG-sensitive PKCs with 24-h PMA or inhibition of the conventional PKC isozymes (- and -) with G6976 normalized the Ca2+ response in high glucose, confirming that this inhibition is due to activation of DAG-sensitive PKC. By contrast, the Ca2+ response to PDGF in high glucose and following 10-min PMA pretreatment was reduced by <10% in keeping with the finding of modest reduction in intracellular stored Ca2+ availability. To explain this difference between the Ca2+ response to ET-1 and PDGF in high glucose, we tested whether PKC specifically inhibits the ET-1 response by phosphorylating PLC-3. We found that both high glucose and 10-min PMA pretreatment caused phosphorylation of PLC-3, with no change in total PLC-3 expression, and that downregulation of PKC with 24-h PMA or inhibition of the conventional PKC isozymes (- and -) with G6976 prevented PLC-3 phosphorylation in high glucose. These data conclusively demonstrate a DAG-sensitive conventional PKC-dependent mechanism of high glucose inhibition of ET-1-induced Ca2+ signaling in mesangial cells.
Our data reflect current understanding of high glucose inhibition of Ca2+ signaling in mesangial cells, although the specifics of the origin of the Ca2+ signal are variable. Mené et al. (18) reported that high glucose causes inhibition of capacitative influx through store-operated channels in mesangial cells through a PKC-dependent mechanism. Nutt and O'Neil (20) showed that high glucose inhibits Ca2+ signaling not from capacitative influx nor from intracellular stores, but from a separate source that they postulate to be receptor-operated Ca2+ influx. Differences between these studies and ours reflect methodological variances and the focus on different aspects of signaling. First, the measurement of Ca2+ signaling in the previously reported studies was achieved by loading cells with the UV-light-excitable Ca2+ fluorophore fura-2 and measuring Ca2+ fluorescence changes by obtaining the excitation-emission ratio using a dual-excitation fluorometer (18, 20). Our study made use of the visible light-excitable Ca2+ fluorophore fluo-3 and measured Ca2+ fluorescence changes using a confocal laser-scanning microcope of wavelength 488 nm. It is likely that the methods of detecting fluorescence using these fluorophores vary in their sensitivity to different elements of Ca2+ signaling. Fura-2 appears to detect both Ca2+ influx and intracellular store release, whereas fluo-3 identifies primarily intracellular store release. Previous results from our lab indicate that, in mesangial cells, fura-2 detects 30–40% of Ca2+ signaling as influx into the cells (25). The fluo-3 fluorescence measurement of Ca2+ signaling in our current study identifies primarily intracellular Ca2+ release. In high glucose, we observed less Ca2+ release from intracellular stores by ionomycin or PDGF stimulation that may be due to reduced refilling of intracellular Ca2+ stores via store-operated channels, as previously described (18).
Our results suggest that, in mesangial cells, DAG-sensitive PKC isozymes act as a negative feedback mechanism to control the strength and duration of the seven-transmembrane receptor-stimulated Ca2+ signal. In high glucose, this negative feedback loop appears to be permanently activated due to de novo synthesis of DAG and subsequent PKC activation. In other in vitro studies, high glucose has been found to increase total PKC-, -2, and - as well as plasma and nuclear membrane-associated PKC-, -, and - (14). In addition, an increase in membrane-associated PKC-, -, and - was found in isolated diabetic rat glomeruli, which was reversed by near normalization of blood glucose by treatment with insulin (2). Our lab (8) reported that, in high glucose, stimulation of mesangial cell ERK1/2 by ET-1 is enhanced via a DAG-sensitive PKC mechanism. Recent work from our lab identified a role for reactive oxygen species (ROS) in PKC-dependent inhibition of Ca2+ signaling in high glucose (10). In this study, ROS production in high glucose was located to NADPH oxidase as antisense to NADPH oxidase reversed high glucose inhibition of Ca2+ signaling. H2O2 was found to cause membrane translocation of PKC- and -. These results suggested an interaction between ROS and PKC in the high glucose inhibition of Ca2+ signaling. In high glucose, decreased Ca2+ signaling occurs with many changes in mesangial cells that contribute to their transformation into a myofibroblast phenotype.
A point of interest in our study was the change in the intracellular pattern of phosphorylated PLC-3 following acute stimulation with ET-1 or 10-min PMA in normal glucose vs. that observed following 48-h exposure to high glucose. On activation of DAG-sensitive PKCs and stimulation with ET-1, we observed a very strong localization of phosphorylated PLC-3 in the nucleus, whereas, in high glucose, a more evenly distributed pattern of PLC-3 throughout the cytoplasm was observed. On immunofluorescence analysis, total PLC-3, in normal glucose, demonstrated both a cytosolic and nuclear distribution. In high glucose, we found no change in total PLC-3 immunofluorescence intensity or pattern on acute stimulation with PMA or ET-1. This suggests that the phosphorylated PLC-3 seen in large quantities in the nucleus with PKC activation or ET-1 stimulation may reflect in situ nuclear activation and phosphorylation. This is in agreement with studies recognizing the localization of PLC in the nucleus (19, 22). PLC-1 has been shown to reside in large quantities the nucleus and to possess a COOH-terminal basic amino acid sequence that is responsible for its nuclear localization (3, 15). It has been shown that when DAG-sensitive PKCs are activated, they translocate to the nucleus as well as to the plasma membrane and also that this nuclear translocation is attributable to DAG produced by nuclear PLC (19).
By contrast, the cytosolic distribution of phosphorylated PLC-3 throughout the cytoplasm in high glucose may simply reflect a steady-state pattern of deactivated enzyme. We found that downregulation of PKCs for 24 h with PMA was sufficient to reverse phosphorylation of PLC-3 that occurred with 48-h high glucose exposure. This suggests a turnover of PLC-3 phosphorylation and the potential for restoration of PLC-3 function following normalization of glucose.
In summary, the loss of mesangial cell Ca2+ signaling responses to ET-1 in high glucose is due to a DAG-sensitive conventional PKC mechanism that includes deactivation of PLC-3 at Ser1105. Future directions will explore the specific PKC isozymes involved in the inhibition of Ca2+ signaling and the interactive role of ROS.
GRANTS
This research was supported by The Canadian Institutes of Health Research and The Canadian Diabetes Association.
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. Section 1734 solely to indicate this fact.
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