Institute of Medical Science and Department of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8
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ABSTRACT |
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In high glucose (HG), mesangial
cells (MCs) lose their contractile response to endothelin-1 (ET-1)
coincidently with filamentous (F)-actin disassembly. We postulated that
these MC phenotypic changes are mediated by altered protein kinase C
(PKC) isozyme activity, myosin light chain (MLC20)
phosphorylation, or Ca2+ signaling. MCs were growth
arrested for 24 h in 0.5% fetal bovine serum (FBS)-DMEM in 5.6 (normal glucose; NG) or 30 mM glucose (high glucose; HG). In HG, the
planar area was reduced [2,608 ± 135 vs. 3,952 ± 225 (SE)
µm2 in NG, P < 0.01, n = 31] with no contractile response to 0.1 µM ET-1. Mannitol did not
affect cell size or ET-1 response. Confocal imaging of fluo 3- loaded
cells revealed that the peak intensity of ET-1-induced Ca2+
signaling was not altered in HG vs. NG. Immunoblotting of
phosphorylated MLC20 showed that HG increased mono- and
decreased unphosphorylated MLC20 (42 ± 16 and 49 ± 15 vs. 13 ± 3 and 80 ± 4% of total in NG,
P < 0.05, n = 3), but the peak
phosphorylation responses to ET-1 were identical in NG and HG. ET-1
stimulated translocation of PKC- and -
from cytosolic to membrane
and particulate fractions identically in NG and HG but did not cause
PKC-
translocation. In HG, membrane accumulation of PKC-
was
observed. Membrane PKC-
activity measured by immunoprecipitation and
32P phosphorylation of PKC-
pseudosubstrate
peptide was 190 ± 18% of NG (P < 0.01, n = 4), which was completely inhibited by pretreatment with a myristoylated peptide inhibitor (ZI). In HG, pretreatment with
ZI for 24 h restored normal MC size and contractile and F-actin disassembly responses to ET-1. In conclusion, in HG, decreased MC size
is due to decreased F-actin assembly, and loss of contractile response
to ET-1 occurs in the presence of normal Ca2+ signaling and
normal MLC20 phosphorylation. In HG, altered F-actin and
contractile functions in MCs are mediated by PKC-
.
endothelin-1; calcium signaling; myosin light chain
phosphorylation; protein kinase C-
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INTRODUCTION |
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ENDOTHELIN-1 (ET-1) is a potent vasoconstrictor that is synthesized by glomerular endothelial cells (33), epithelial cells (45), and mesangial cells (40). ET-1-stimulated mesangial cell contraction can regulate glomerular capillary surface area and filtration rate (3). However, mesangial cells cultured in high glucose (8, 17) and glomeruli isolated from streptozotocin (STZ)-diabetic rats fail to contract in response to ANG II, (24), ET-1, or raised intracellular Ca2+ (17). In diabetes, high glucose-induced loss of preglomerular afferent arteriolar contractile response to vasoconstrictors leads to elevated intraglomerular pressure, accelerating the progression of glomerulosclerosis (58).
The precise mechanisms by which high glucose alters the phenotype of mesangial cell and vascular smooth muscle cell (VSMC) responsiveness are not known. Reduced Ca2+ signaling mediated by either high glucose-induced protein kinase C (PKC) inhibition of inositol 1,4,5-trisphosphate (IP3) release (34) or via high glucose-induced inhibition of receptor-operated channels (35) have been proposed. Work from our laboratory has demonstrated that isolated glomeruli and mesangial cells cultured in 25 mM (high) glucose display normal Ca2+ signaling to ET-1, ANG II, and arginine vasopressin (AVP) as measured by 45Ca2+ efflux and fura 2 spectrofluorimetry, respectively (53). The normal IP3 response observed in high glucose is attributed to upregulation of myo-inositol transport, which prevents mesangial cell myo-inositol depletion (4).
The initial Ca2+ transient induced by vasoconstrictor peptides in VSMCs and mesangial cells is accompanied by Ca2+/calmodulin (CaM)-dependent activation of myosin light chain kinase (MLCK) and the phosphorylation of the regulatory 20-kDa myosin light chain (MLC20) at serine-19 and threonine-18. This leads to the activation of the actin-activated myosin ATPase, interaction of filamentous (F)-actin and myosin, and cellular contraction (42, 46). PKC, a ubiquitous serine-threonine protein kinase, is also implicated in the regulation of contraction in both smooth muscle and mesangial cells (8, 9, 50, 52). Activation of mesangial cell PKC isozymes by hyperglycemia in diabetes represents an important pathway potentially contributing to altered cytoskeletal responsiveness (2, 8, 30). PKC can directly phosphorylate MLC20 at serine-1, serine-2, and threonine-9 without activating contraction (52) and indirectly stimulates MLC20 phosphorylation at serine-19 and threonine-18 by inhibiting myosin light chain phosphatase (MLC-PP). PKC can also stimulate contraction, independent of MLC20 phosphorylation, by phosphorylating the actin regulatory proteins calponin and caldesmon (15, 16).
Although we have previously shown that ET-1-stimulated mesangial cell
contraction involves the activation of PKC-, -
, and, to a lesser
extent, -
in normal glucose (9), little is known about
PKC isozyme regulation of mesangial cell MLC20
phosphorylation. In high glucose, mesangial cell PKC isozyme expression
patterns, subcellular distribution, and activity are altered (1,
2, 20, 21, 23, 55). In mesangial cells, Kikkawa et al.
(23) and, recently, Amiri and Garcia (1)
demonstrated membrane translocation of PKC-
and -
after 72 and
120 h of high glucose. Therefore, high glucose-induced activation
of selective PKC isozymes that mediate cytoskeletal restructuring may
be implicated in the mechanism of reduced contractility. We have
observed that mesangial cells in high glucose for 48 h demonstrate
F-actin disassembly, which is reversed by inhibition of the polyol
pathway and appears to be mediated through a PKC-dependent mechanism
(61). Loss of mesangial cell contractility to ET-1 is also
restored with aldose reductase inhibition (8).
In this study we postulated that high glucose may alter mesangial cell
Ca2+ signaling, MLC20 phosphorylation, or PKC
isozyme activity, mediating cytoskeletal dysfunction and
hypocontractility. We examined the effects of high glucose on
ET-1-stimulated mesangial cell planar area reduction (contraction),
Ca2+ signaling, MLC20
phosphorylation, PKC isozyme distribution, and F-actin disassembly. The
use of a myristoylated PKC- peptide inhibitor
(myr-RRGARRWRK; ZI) was used to determine the role of the PKC-
isozyme in the regulation of contraction and F-actin assembly in high glucose.
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MATERIALS AND METHODS |
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Materials
DMEM, penicillin, streptomycin, and trypsin were purchased from GIBCO BRL Life Technologies (Burlington, ON). Fetal bovine serum (FBS) was purchased from Wisent (St. Bruno, PQ). 12 Phorbol-13 myristate (PMA), ET-1, leupeptin, pepstatin A, aprotinin, benzamidine, Tween 20, sodium orthovanadate, dithiothreitol (DTT), and polyclonal anti-PKC-Mesangial Cell Planar Area Measurements
Mesangial cells (passages T5-T10) were cultured as previously described (18) in 20% FBS-DMEM in 5.6 mM (normal) glucose, pH 7.4, supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 mM HEPES.Mesangial cells (100,000 cells/dish) were directly growth arrested for
24 h on 35-mm plastic dishes (Falcon, Becton-Dickinson Laboratories, Lincoln Park, NJ) in either 2 ml 0.5% FBS-DMEM with 5.6 or 30 mM (high) glucose. The dishes were transferred to the heated
stage of an inverted phase-contrast light microscope (Bausch and Lomb,
New York, NY) and maintained in 5% CO2 in air at 30°C. The images were captured by a Hitachi KP-113 solid-state television camera (Hitachi Denshi, Tokyo, Japan). After visualization, the image
was digitized to represent time 0. To downregulate PKC in normal glucose, cells were pretreated for 24 h with 0.1 µM PMA. To inhibit PKC-, cells were pretreated for 24 h with 10 µM of ZI. This peptide has been shown to inhibit PKC-
activity in
Xenopus laevis oocytes (10) and in pancreatic
islet
-cells (47). ET-1 was added directly to the
medium to achieve a final concentration of 0.1 µM for cells in normal
or high glucose in the absence or presence of ZI. To control for
osmolarity, cells were growth arrested in 0.5% FBS in normal glucose
supplemented with 24.4 mM mannitol. The same group of cells
(n = 10-20/group) was digitized serially for the
next 60 min. Images were captured on a 486 DX PC and digitized with
Sigma Scan morphometric software (Jandel Scientific, San Rafael, CA).
Only those cells with a clearly defined border perimeter were used for
planar surface area measurement. Changes in individual cell surface
planar area were calculated for all of the cells at each time point.
Confocal Imaging of Intracellular Ca2+
Cells were sparsely plated on glass coverslips to minimize overlap of individual cells. With the use of our previously published methods (54), growth-arrested cells were loaded with 2.5 µM fluo 3 in DMEM (containing CaCl2) with 0.02% Pluronic F-127 and 1 mg/ml BSA for 30 min at 37°C. The cells were washed once in DMEM without fluo 3 and then incubated in the dark in DMEM for 30 min at room temperature (RT). The coverslip was mounted in a chamber on the stage of a Zeiss confocal microscope (LSM 410, Düsseldorf, Germany), and the cells were imaged at 30°C in response to 0.1 µM ET-1. An argon laser was focused with an inverted objective lens (Axiovert 100, ×10), and the pixel resolution was set at 512 × 512 with a gray-scale level of 0 (minimum) to 255 (maximum). A section through the maximum diameter of each cell was digitized at consecutive time points after the introduction of ET-1. To standardize the fluorescence intensity measurement between experiments, the image contrast and brightness levels were adjusted optimally and then kept constant for the experiments in the study. The pinhole (size = 25), scanning time (0.546 s), zoom (1), and magnification of the confocal scanning system were identical for each experiment. The digitized confocal images were analyzed using National Institutes of Health (NIH) Image software (version 1.62) for the Macintosh (NIH, Bethesda, MD), without enhancement.Analysis of MLC20 Phosphorylation
Phosphorylation of the 20-kDa regulatory MLC20 was measured using a modified method from Persechini et al. (36). The separation of unphosphorylated, monophosphorylated, and diphosphorylated forms of MLC20 was accomplished with glycerol PAGE followed by electrophoretic transfer of protein to nitrocellulose membranes and immunoblot analysis.Mesangial cells were growth arrested for 24 h on 100-mm plastic dishes in 0.5% FBS-DMEM containing 5.6 or 30 mM glucose. Cells were treated with either 0.1 µM ET-1, PMA, or ionomycin for 0, 2, 5, 10, 20, and 40 min at 30°C. To inhibit PKC-dependent MLC20 phosphorylation, cells were exposed to 0.1 µM PMA for 24 h to downregulate PKC. Trypsin-detached cells served as a negative control. After treatment, cells were rinsed with PBS and cellular proteins were precipitated in 600 µl of 10% trichloroacetic acid containing 2 mM DTT at 4°C. Precipitates were scraped from the plates and centrifuged at 15,000 g for 15 min. The supernatant fraction was decanted, and the pellet was extracted three times in 6 ml of diethyl ether containing 2 mM DTT at 4°C for 2 min, followed by air drying at room temperature (RT) for 30 min to remove residual ether. The pellet was then dissolved in 100 µl of sample buffer containing 8 M urea, 20 mM Tris base, 23 mM glycine, and 2 mM DTT, pH 8.6. After centrifugation and protein determination, 100 µg of the urea-solubilized samples were electrophoresed at 400 V for 1.5 h at 10°C in 1.0-mm minigels containing 10% polyacrylamide, 0.5% bisacrylamide, 40% glycerol, 20 mM Tris base, and 23 mM glycine. The reservoir buffer contained (in mM) 20 Tris, 23 glycine, 1 sodium thioglycolate, and 1 DTT. Separated proteins were then electroblotted to 0.22-µm nitrocellulose (Bio-Rad) membranes at 24 V for 3 h, employing a 20 mM Tris-23 mM glycine-20% methanol transfer buffer, pH 7.6.
After transfer, membranes were blocked in 5% skim milk in Tris-buffered saline plus Tween 20 (TTBS) and then incubated for 1 h at RT in a rabbit polyclonal anti-MLC20 antibody (1:1,000) in 5% skim milk in TTBS. For chemiluminescent detection, the membranes were incubated with an HRP-conjugated anti-rabbit secondary antibody, dipped into luminol substrate solution for 1 min, and developed on film, as described above. Densitometry was performed using NIH Image 1.62 analysis software, and MLC20 phosphorylation was expressed as the percentage of MLC20 in un-, mono-, or diphosphorylated form.
Immunoblotting of Ser19-MLC20 and the ETA Receptor
In a separate series of experiments, phosphorylation of Ser19-MLC20 and ETA receptor expression were analyzed in total mesangial cell lysates. Cells were growth arrested in six-well plates for 24 h in 0.5% FBS-DMEM in normal or high glucose. To determine if high glucose altered ETA receptor expression, cells were growth arrested in 0.5% FBS-DMEM in normal or high glucose for 1, 2, 3, 5, and 7 days and then lysed in 150 µl of 2× SDS sample buffer at 100°C. After protein determination, 15 µg of cellular protein were subjected to SDS-PAGE using 15 and 10% minigels for Ser19-MLC20 and the ETA receptor, respectively. The proteins were transferred to polyvinylidene difluoride (PVDF; Millipore, Bedford, MA) membranes, blocked as described above, and probed for 1 h at RT (1:1,000 in 5% skim milk in TTBS) with either a monoclonal anti-Ser19-MLC20 antibody or a sheep polyclonal anti-ETA receptor antibody (US Biologicals, Swampscott, MA). Chemiluminescent detection and densitometry were performed as described above.Cell Fractionation and PKC Immunoblots
PKC isozymes were probed in cytosolic, membrane, and particulate fractions of 24-h-growth-arrested mesangial cells in normal and high glucose in the absence and presence of 0.1 µM ET for 10 min at 30°C. As described previously (9, 21), cellular fractions were obtained using sequential ultracentrifugation in the absence (cytosolic) or presence of the detergents 1% Triton X-100 (membrane) and 10% SDS (particulate). Total PKC isozyme expression was determined in cells lysed in 2× SDS sample buffer at 100°C. Protein content was measured using a Bio-Rad Dc Lowry protein assay (Bio-Rad). Fifteen microliters of protein were subjected to SDS-PAGE on 10% gels followed by overnight transfer onto PVDF membranes. Membranes were then exposed to polyclonal anti PKC-Confocal Fluorescence Imaging
Mesangial cells were cultured on glass coverslips under conditions identical to those described above. The cells were fixed with 3.7% formaldehyde for 15 min at RT followed by plasma membrane and nuclear membrane permeabilization with 100% methanol atMesangial cell F- and G-actin were simultaneously labeled with the fluorescent probes rhodamine-phalloidin (F-actin) and FITC-DNase 1 (G-actin) according to our previously published methods (60). Cells were cultured on glass coverslips under experimental conditions described above. After being washed in PBS (4°C), the cells were fixed in 3.7% formaldehyde in PBS for 15 min at RT and permeabilized with 0.1% Triton X-100 for 10 min. After additional washes in PBS, the cells were incubated simultaneously in the dark with 200 µl of 0.165 µM rhodamine-phalloidin and 0.3 µM FITC-DNase 1 for 20 min. Coverslips were mounted on glass slides and imaged with a dual-channel Zeiss LSM 410 confocal laser-scanning microscope, as described above (Confocal Imaging of Intracellular Ca2+).
Measurement of PKC- and PKC-
Activity
PKC- activity was determined in total cell lysates using
immunoprecipitation and 32P phosphorylation of
Ser25-PKC-
pseudosubstrate peptide. To determine the
specificity of the PKC-
inhibitor, cells were pretreated with 10 µM ZI for 24 h. Cells were lysed in 500 µl of lysis buffer
containing (in mM) 150 NaCl, 25 HEPES, 1 EGTA, 2 EDTA, 10 NaF, 50
-glycerophosphate, 1 Na3VO4, 2.5 bensamidine, and 1 phenylmethylsulfonyl fluoride, as well as 10 µg/ml
leupeptin, pepstatin, and aprotinin, 10 nM microcystin, and 1%
Triton-100, pH 7.5. Lysates were triturated five times through a
25-gauge needle, placed on ice for 30 min, and centrifuged at 15,000 g for 5 min to remove cellular debris. Three hundred
micrograms of total cellular protein were immunoprecipitated with 2 µg of rabbit polyclonal anti-PKC-
(Santa Cruz
Biotechnology) for 3 h, followed by the addition of 40 µl protein G-Sepharose for 1 h. The immunocomplexes were
centrifuged, washed in lysis buffer, followed by three washes in kinase
buffer. Immunoprecipitated PKC-
was assayed in 40 µl of kinase
buffer that contained 5 µg of Ser25-PKC-19-31-
pseudosubstrate peptide (GIBCO BRL Life Technologies) but was otherwise
identical to that used for determining PKC-
activity. The reaction
was terminated by the addition of quenching buffer, followed by
spotting on P81 filter disks, subsequent washing in 75 mM phosphoric
acid, and scintillation counting, as described above. Data for PKC-
and PKC-
activities are expressed as the percentage of those
observed in normal glucose.
Statistical Analyses
All results are expressed as means± SE. Statistical analysis was performed using InStat 2.01 statistics software (Graph Pad, Sacramento, CA). The means of two groups were compared using an unpaired Student's t-test. The means of three or more groups were compared by one-way ANOVA with Bonferroni's multiple comparison. To compare multiple groups with basal normal glucose, Dunnett's multiple comparison was utilized. Differences described as significant are P < 0.05 unless stated otherwise. ![]() |
RESULTS |
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Effects of High Glucose and Chronic Phorbol Ester on Mesangial Cell Planar Area
Figure 1 demonstrates the change in planar surface area of mesangial cells growth arrested for 24 h in 5.6 (normal) and 30 mM (high) glucose and stimulated with 0.1 µM ET-1 at 30°C. Basal planar surface area in cells cultured in 5.6 mM glucose was 3,952 ± 225 µm2 (n = 53 cells). Cells incubated in 30 mM glucose displayed a smaller basal planar area of 2,608 ± 135 µm2 (n = 31 cells, P < 0.01 vs. normal glucose at time 0). This was not due to osmolarity as cells cultured in normal glucose supplemented with 24.4 mM mannitol were no different from cells in normal glucose alone (3,424 ± 238 µm2, n = 28 cells).
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In normal glucose, administration of ET-1 for 60 min decreased planar area to 77 ± 3% of the original surface area (P < 0.05 vs. normal glucose at time 0) (Fig. 1B). Mannitol in normal glucose did not alter mesangial cell responsiveness to ET-1 at 60 min (81 ± 2% of basal area).
Effects of High Glucose on ETA Receptor Expression
To determine whether the loss of ET-1 responsiveness in high glucose was due to reduced ETA receptor expression, mesangial cells were cultured in 0.5% FBS-DMEM in 5.6 or 30 mM glucose for up to 7 days, and total cell lysates were examined by immunoblot for the presence of the ETA receptor. In the representative immunoblot shown in Fig. 2, the antibody generated against a 39-kDa cytosolic fragment of the ETA receptor reacted with cellular proteins of 64-, 50-, and 39-kDa molecular mass. The intensities of the three dominant bands were not altered by high glucose. Equal protein loading was confirmed by staining the PVDF membrane with Ponceau S.
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Confocal Fluorescence Imaging of ET-1-Stimulated Intracellular Ca2+ Signaling
In normal glucose, fluo 3-loaded cells displayed perinuclear and cytosolic Ca2+ fluorescence staining at time 0 (Fig. 3A). The administration of 0.1 µM ET-1 stimulated an increase in cytosolic and nuclear Ca2+ beginning at 20 s and peaking at 40-60 s. Fluorescence intensity returned to basal levels at 80-100 s. This rapid Ca2+ response to ET-1 was seen in >90% of cells examined (n = 44 cells in 3 independent experiments).
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In Fig. 3B at time 0, cells in high glucose displayed a basal perinuclear and cytosolic staining pattern that was no different from that seen in normal glucose. However, an increase in cytosolic and nuclear Ca2+ did not occur until 40 s after the addition of 0.1 µM ET-1 and reached a peak at 70-80 s. Fluorescence intensity returned to basal levels at 120-200 s. Although the Ca2+ response was delayed compared with that for normal glucose, peak fluorescence pixel intensity (130 ± 13 in normal glucose vs. 117 ± 12 in high glucose) and the number of cells responding to ET-1 (>90%, n = 44 cells) were not different.
Phosphorylation of MLC20
Effects of ET-1, phorbol ester, and ionomycin on MLC20
phosphorylation.
The change in ET-1-induced mesangial cell planar surface area was used
as an indirect measurement of contraction. Glycerol-urea PAGE was used
to measure MLC20 phosphorylation, the key regulatory step
preceding smooth muscle cell contraction. As shown in Fig. 4, at time 0 the majority of
MLC20 was unphosphorylated [81 ± 5 (SE)%,
n = 3], with 13 ± 4 and 6 ± 2% mono- and
diphosphorylated, respectively. In comparison, trypsinized suspended
cells contained 97 ± 5 un- and 3 ± 1% monophosphorylated
MLC20. ET-1 at 2 min significantly stimulated
MLC20 phosphorylation. Mono- and diphosphorylated MLC20 increased to 60 ± 9 and 24 ± 11% of
total, respectively (P < 0.05 vs. time 0,
n = 3), whereas unphosphorylated MLC20
decreased to 16 ± 7% of total (P < 0.05 vs.
time 0, n = 3). The effect of ET-1 was
sustained at 40 min, with 29 ± 7, 40 ± 4, and 31 ± 5% un-, mono-, and diphosphorylated, respectively (P < 0.05 vs. time 0). Downregulation of PKC with 24-h PMA did
not alter time 0 MLC20 phosphorylation (72 ± 4, 22 ± 10, 6 ± 2% un-, mono-, and diphosphorylated, respectively) and did not affect ET-1-stimulated phosphorylation at 10 min (22 ± 6, 40 ± 10, and 38 ± 13% un-, mono-,
diphosphorylated, respectively, vs. 23 ± 8, 45 ± 2, and
32 ± 8% with no PMA).
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Effects of high glucose on ET-1-stimulated MLC20
phosphorylation.
Densitometric analysis of ET-1-stimulated MLC20
phosphorylation in high glucose is shown in Fig.
5. At time 0, high glucose significantly increased monophosphorylated MLC20, whereas
it decreased unphosphorylated MLC20 (42 ± 16 and
49 ± 15% of total, respectively, vs. 13 ± 3 and 80 ± 4% of total in normal glucose at time 0, P < 0.05, n = 3). ET-1 at 2 min significantly stimulated
diphosphorylation of MLC20. Mono- and diphosphorylated
MLC20 increased to 62 ± 20 and 28 ± 11% of
total, respectively (P < 0.05 vs. time 0,
n = 3), whereas unphosphorylated MLC20
decreased to 10 ± 1% of total (P < 0.05 vs.
time 0, n = 3). The effect of ET-1 on
diphosphorylated MLC20 was sustained at 40 min, with
34 ± 20% of total MLC20 diphosphorylated (P < 0.05 vs. time 0). Downregulation of
PKC with 24-h PMA did not alter basal MLC20 phosphorylation
in high glucose (54 ± 20, 37 ± 19, and 9 ± 4% un-,
mono-, di-phosphorylated, respectively) and did not affect
ET-1-stimulated phosphorylation at 10 min (20 ± 10, 39 ± 13, and 41 ± 21% un-, mono-, diphosphorylated, respectively, vs.
24 ± 11, 40 ± 6, and 36 ± 15% with no PMA).
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Effects of high glucose on phosphorylated
Ser19-MLC20.
The use of glycerol-urea PAGE effectively separated the un-, mono-, and
diphosphorylated MLC20. However, this method cannot distinguish which serine or threonine is phosphorylated. To determine whether Ser19-MLC20, the dominant site
phosphorylated by MLCK and Rho kinase, was the monophosphorylated
MLC20, we immunoblotted total cell lysates with a
monoclonal phospho-specific Ser19-MLC20
antibody. As shown in Fig. 6A,
basal levels of phospho-Ser19-MLC20 were not
altered by high glucose (92 ± 8% of normal glucose, n = 8).
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Confocal imaging of ET-1-stimulated phosphorylated Ser19-MLC20. Basal phospho-Ser19-MLC20 cellular distribution in mesangial cells in normal glucose is shown in Fig. 6B. Fluorescence was localized primarily to the cell membrane along with a fibrillar, cytosolic staining pattern. ET-1 administration failed to significantly alter the staining pattern. In high glucose, the intensity and staining pattern were not different from that in normal glucose. In high glucose, ET-1 also failed to change the distribution and fluorescence intensity of the membrane staining.
Mesangial Cell PKC Isozymes
Effects of high glucose on basal and ET-1-stimulated PKC isozyme
distribution.
In normal glucose, ET-1 stimulates the translocation of PKC-, -
,
and -
isozymes, but not PKC-
, from cytosolic to membrane and
particulate fractions (9). To determine whether high
glucose alters ET-1-stimulated PKC isozyme translocation, we
immunoblotted cytosolic, membrane, and particulate cellular fractions
with PKC isozyme-specific antibodies. As shown in Fig.
7, PKC-
, -
, and -
were present
in all three fractions in both normal and high glucose and translocated
in response to ET-1 from cytosolic to membrane and particulate
fractions to a similar extent. In response to ET-1 in normal vs. high
glucose, PKC-
distribution in membrane and particulate fractions,
respectively, was 87 ± 17 and 133 ± 10% of that in basal
normal glucose (P < 0.05) vs. in high glucose 92 ± 20 and 142 ± 6% of basal normal glucose levels
(P < 0.05, n = 4). PKC-
distribution in membrane and particulate fractions, respectively, was
300 ± 76 and 134 ± 3% of that in basal normal glucose
(P < 0.01 and P < 0.05, respectively)
vs. in high glucose 287 ± 74 and 137 ± 24% of that in
basal normal glucose (P < 0.01 and P < 0.05, respectively). PKC-
distribution in membrane and particulate fractions, respectively, was 205 ± 42 and 133 ± 16% of that in basal normal glucose (P < 0.01 and
P < 0.05, respectively) vs. in high glucose 184 ± 52 and 190 ± 56% of that in basal normal glucose
(P < 0.01 and P < 0.05, respectively,
n = 4). Cells treated with 0.1 µM PMA for 10 min
served as a positive control and demonstrated the disappearance of
PKC-
, -
, and -
from the cytosolic fraction (12 ± 9, 2 ± 1, and 4 ± 1% of distribution in basal normal glucose, respectively, P < 0.01, n = 4). This
was accompanied by enhanced recovery of isozymes in membrane (289 ± 97, 513 ± 90, and 267 ± 43% of basal normal glucose,
respectively, P < 0.01, n = 4) and
particulate fractions (376 ± 46, 205 ± 33, and 244 ± 70% of basal normal glucose, respectively, P < 0.01, n = 4).
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Effects of high glucose on total PKC isozyme content.
In high glucose, it was postulated that the enhanced recovery of
PKC- in the membrane might be attributed to enhanced total PKC
expression. Therefore, we determined the effects of 24-h high glucose
on total PKC expression by immunoblotting total cell lysates (Fig.
8). PKC-
, -
, -
, -
, and
-
I were expressed as a single band in mesangial cells,
but PKC-
was not expressed. High glucose stimulated a significant
2.7-fold increase in PKC-
(272 ± 75%, P < 0.05, vs. normal glucose, n = 6), whereas PKC-
(125 ± 12%, n = 6), -
(90 ± 10%,
n = 6), -
(102 ± 2%, n = 6),
and -
I (90 ± 12%, n = 6) remained
unchanged.
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PKC- and High Glucose-Induced Loss of Mesangial Cell Contraction
to ET-1
Effects of high glucose on immunoprecipitated PKC- and PKC-
activity.
Figure 9 shows the effects of high
glucose on membrane PKC-
activity in the absence and presence of the
ZI. High glucose increased PKC-
activity to 190 ± 18% of
normal glucose (P < 0.01, n = 4).
Twenty-four-hour pretreatment with 10 µM ZI in either normal or high
glucose significantly inhibited activity to 45 ± 10 and 73 ± 4% of normal glucose, respectively (P < 0.01, n = 4). Mannitol did not significantly affect PKC-
activity (99 ± 11% of normal glucose, n = 4).
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Effects of ZI on mesangial cell planar area and ET-1-stimulated
contraction.
Basal planar surface area in cells cultured in normal glucose was
3,952 ± 225 µm2 (n = 53 cells)
(Fig. 10A). In comparison,
cells incubated in 30 mM glucose displayed a smaller basal planar area
of 2,608 ± 135 µm2 (n = 31 cells,
P < 0.01 vs. normal glucose at time 0).
This was not due to osmolarity as cells cultured in normal glucose
supplemented with 24.4 mM mannitol were no different from cells in
normal glucose (3,624 ± 238 µm2, n = 28 cells). Pretreatment of cells in high glucose with 10 µM ZI for
24 h restored the area to values seen in normal glucose (3,875 ± 213 µm2, n = 40 cells). In
normal glucose, pretreatment with ZI did not alter basal planar area
(3,871 ± 277 µm2, n = 23 cells). ZI
pretreatment in normal glucose did not alter MC responsiveness to ET-1
at 60 min (69 ± 5 vs. 77 ± 3% of basal area in normal
glucose) as shown in Fig. 10B. Cells in high glucose did not
respond to ET-1. In high glucose, pretreatment with ZI restored the
ET-1 response, decreasing planar area to 80 ± 2% of basal area,
similar to the change in normal glucose.
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Effects of ZI on mesangial cell F-/G-actin in high glucose.
In Table 1, the average pixel
intensity/cell values for F-actin and G-actin are presented. F-/G-actin
ratio was calculated for each cell and the mean ± SE is reported
for each group. In high glucose, the F-/G-actin ratio was significantly
reduced to 1.5 ± 0.2 (P < 0.05 vs. normal
glucose, n = 44 cells) from a value of 1.9 ± 0.1 in normal glucose. The addition of 0.1 µM ET-1 in normal glucose
resulted in an F-/G-actin ratio that was not different from that seen
in the basal high glucose state (1.5 ± 0.1, n = 44). In high glucose, ET-1 did not alter the mesangial cell F-/G-actin ratio (1.4 ± 0.1, n = 36). Mannitol (normal
glucose+24.4 mM mannitol for 24 h) had no effect on the F- or
G-actin content or F-/G-actin ratio (2.0 ± 0.2, n = 26 cells).
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DISCUSSION |
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In this study, we examined the signaling events related to
mesangial cell contractile and cytoskeletal dysfunction in high glucose. PKC has been implicated as an underlying mechanism of high
glucose-induced VSMC dysfunction (25). In high glucose, lack of mesangial cell contractility in response to ET-1 and F-actin disassembly is not due to altered Ca2+ signaling or
phosphorylation of MLC20 but is PKC- mediated.
The observations that after exposure to 24-h high glucose mesangial
cells are smaller in size, unresponsive to ET-1 (8), and
display PKC-mediated F-actin disassembly (61) resemble our earlier studies in higher passage cells. We reported previously that
mesangial cell reduced planar area, responsiveness to ET-1, and F-actin
disassembly were reversed by tolrestat, an aldose reductase inhibitor
(21). Aldose reductase inhibition of the polyol
pathway prevented the de novo synthesis of diacylglycerol (DAG)
(8, 29, 49) and thus activation of DAG-sensitive PKC
isozymes at 48 h (21). At 48 h, high glucose
stimulated the membrane translocation of PKC- and -
(21). In the present study, total PKC-
but not PKC-
was increased by 24-h high glucose, whereas PKC-
was increased in
the membrane fraction only. Although the exact mechanisms of PKC-
activation are not known, phosphatidic acid (PA), a precursor of DAG,
is reported to activate PKC-
(32). In our earlier
studies (21), tolrestat, through inhibition of de novo
PA synthesis, may have reduced PKC-
activity concomitantly with
either PKC-
and/or -
.
It is possible that the diminished responsiveness of mesangial cells to ET-1 in high glucose may be mediated by reduced endothelin-receptor A (ETA) expression (19). Hargrove et al. (14) demonstrated in cultured rat mesangial cells that high glucose for 24 h upregulates the production of ET-1 and ET-1 mRNA twofold. Increased ET-1 production may cause downregulation and decreased expression of mesangial cell ETA receptors (19). By contrast, glomerular ETA receptor expression was upregulated in glomeruli from alloxan-diabetic rabbits (22), whereas hyperglycemia did not alter kidney ETA receptor expression in humans or in STZ-diabetic rats (41). In our study, 1-7 days of high glucose did not alter mesangial cell ETA receptor expression. This suggests that reduced mesangial cell ETA receptor expression does not likely account for the high glucose-induced contractile dysfunction in response to ET-1.
As observed in Fig. 3, A and B, in high glucose
ET-1 stimulated intracellular Ca2+ to similar levels
observed in normal glucose. Although peak intensity of fluo 3-labeled
release was not different, a time delay in the measured response in
high glucose was consistently observed. We have previously shown that
under the identical culture conditions used in this study
(9), acute PMA does not elicit a Ca2+ signal.
This suggests that PKC activation does not directly stimulate Ca2+ signaling in mesangial cells. However, others have
shown that prior PKC activation may inhibit signaling. Interaction may
be at the level of G protein coupling (57) or the
cytoskeleton (37). Disruption of F-actin with cytochalasin
D in platelets inhibits Ca2+ entry (39).
Treatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), also inhibits Ca2+ entry
(39). PI 3-kinase is a potent activator of atypical
PKC- (59). In our hands, cytochalasin D pretreatment of
mesangial cells did not affect either timing or intensity of
ET-1-stimulated Ca2+ release (data not shown). This
suggests that the time delay observed in Ca2+ signaling in
response to ET-1 in high glucose is not attributable to mesangial cell
F-actin disassembly. The potential role of PI 3-kinase in high
glucose-induced activation of mesangial cell PKC-
and stress fiber
disassembly was not explored.
Stress fibers are composed not only of actin but also contain myosin,
tropomyosin, and -actinin and require the small GTP-binding protein
Rho for their formation (38). Stress fiber assembly is
usually preceded by MLC20 phosphorylation, whereas stress
fiber disassembly is a consequence of MLC20
dephosphorylation (5). In mesangial cells, Kreisberg et
al. (28) demonstrated that cAMP-mediated stress fiber
disassembly preceded MLC20 dephosphorylation. In our study,
basal MLC20 phosphorylation was markedly increased in high
glucose, whereas F-actin was disassembled, resembling the effects of
agonist stimulation. Fukuda et al. (11) reported increased
basal MLC20 phosphorylation in platelets from type 2 diabetic patients, but the organization of actin was not studied. Because MLC20 phosphorylation is a balance between kinase
and phosphatase activities (42), it is possible that high
glucose may enhance MLCK activity or increase PKC- or Rho-mediated
inhibition of phosphatase activity (31, 43). In this
study, neither acute nor chronic PMA, in either normal or high glucose,
affected basal MLC20 phosphorylation. The lack of
MLC20 phosphorylation in response to acute PMA suggests
that the PMA-induced mesangial cell contraction observed in our earlier
studies (8, 9) must be MLC20 phosphorylation independent. In our earlier study, chronic PMA inhibited contraction by
downregulating PKC-
, -
, and -
in the cytosolic, membrane, and
particulate fractions (9), thus reducing DAG-sensitive PKC
isozyme content. PKC isozymes may stimulate contraction by initiating a
kinase cascade involving the phosphorylation of the actin regulatory
proteins calponin, caldesmon (16), and/or myristoylated alanine-rich C-kinase substrate (48).
In high glucose, ET-1 stimulated phosphorylation of MLC20 to levels seen in normal glucose, which was not accompanied by cell contraction. This might be explained by the preferential phosphorylation of PKC-specific sites serine-1, serine-2, or threonine-9 of the MLC20 by ET-1 in high glucose. Phosphorylation of MLC20 at these sites does not result in myosin-ATPase activation and contraction (52). If high glucose enhanced PKC-mediated inhibition of MLC-PP, phosphorylation of Ser19-MLC20 would increase. This was not detected by either immunoblotting or confocal imaging with a Ser19-MLC20 antibody. The effects of high glucose on either Rho expression or activity are not known. Therefore, in high glucose, MLC20 phosphorylation does not appear to correlate with either F-actin disassembly or lack of contraction in response to ET-1. High glucose must be exerting its actions at another cytoskeletal target.
In this study, high glucose for 24-h enhanced membrane accumulation of
PKC-. This was much earlier than the 5-day time point reported by
others (1, 24). This was not accompanied by a concomitant
decrease in cytosolic and/or particulate fraction and may be explained
by the fact that the relative abundance of PKC-
in these fractions
may hinder the detection of small compartmental changes in
distribution. Immunocomplexed PKC-
membrane activity was
significantly increased by high glucose and was normalized by
pretreatment with the myristoylated ZI. ZI was specific for the PKC-
isozyme, as pretreatment with ZI did not affect immunoprecipitated PKC-
activity in either normal or high glucose. Although total protein expression and activity of PKC-
were increased by high glucose, this was not accompanied by changes in fractional
distribution. PKC-
may be activated by PKC-
-mediated
Ser660 phosphorylation (62). Furthermore,
overexpression of constitutively active PKC-
results in
phosphorylation of PKC-
coexpressed in HEK-293 cells
(62). In our study, high-glucose-induced
PKC-
-mediated activation of PKC-
is unlikely, as pretreatment of
cells in high glucose with the ZI, which inhibited PKC-
activity,
did not inhibit PKC-
activity. In COS-7 cells,
H2O2 causes phosphorylation of Tyr502 and Tyr523, which results in prolonged
activation of PKC-
(26). Recent studies by Konishi et
al. (27) using phosphorylation-site specific antibodies
and mass spectrometric analyses have shown that
H2O2-induced PKC-
activation requires
phosphorylation of Tyr311 and is not accompanied by
membrane translocation. Mesangial cells cultured in 30 mM glucose for
as little as 1 h generate H2O2, as
detected by dichlorofluorescein (13), but it is not known whether this is associated with PKC isozyme tyrosine phosphorylation and activation.
The observations that 24-h high glucose-stimulated, PKC--dependent
F-actin disassembly was reversed by ZI, which also reversed cell size
and response to ET-1, suggest that the smaller mesangial cell planar
area is the result of F-actin disassembly. The mechanism underlying
PKC-
-mediated disassembly is not known. PKC-
can directly
associate with the actin cytoskeleton in fibroblasts (12).
In NIH-3T3 fibroblasts, atypical PKC-
and -
are reported to
mediate Cdc42-mediated stress fiber disassembly (6) and have also been implicated in regulating Ras-mediated stress fiber disassembly (51). In mesangial cells, high glucose for
48 h does not affect the expression of Ras but increases membrane
associated Ras activity (56). It is not known whether high
glucose-induced Ras activation results in increased PKC-
activity
and F-actin disassembly. The recent findings of Cortes et al.
(7) that perfused glomeruli from 9-mo-old STZ-diabetic
rats display F-actin disassembly and loss of stress fibers strongly
suggest that the F-actin disassembly we have observed in cultured
mesangial cells in high glucose mimic the in vivo findings.
In summary, this study identifies the importance of PKC- in the
mediation of high glucose-induced loss of mesangial cell responsiveness
to ET-1. Loss of the contractile response to ET-1 is not due to reduced
Ca2+ signaling, phosphorylation of MLC20, or
altered DAG-sensitive PKC isozyme activation but may be related to
PKC-
-dependent F-actin disassembly. This cytoskeletal
disorganization alters the mesangial cell phenotype and may account for
the early effects of high glucose-induced cellular dysfunction.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors thank Hong Hua for technical assistance with confocal microscopy. We also thank Dr. C. S. Packer (Indiana University, Indianapolis, IN) and Dr. Yasuharu Sasaki (Asahi Chemical, Ltd., Shizuoka, Japan) for MLC20 and anti-phospho-Ser19-MLC20 antibodies, respectively.
![]() |
FOOTNOTES |
---|
This work was jointly funded by the Juvenile Diabetes Foundation International and Canadian Institutes of Health Research.
Address for reprint requests and other correspondence: C. I. Whiteside, Medical Sciences Bldg., Rm. 7302, 1 King's College Cir., University of Toronto, Toronto, Ontario, Canada M5S 1A8 (E-mail: catharine.whiteside{at}utoronto.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published August 15, 2001; 10.1152/ajprenal.00055.2001
Received 23 February 2001; accepted in final form 13 August 2001.
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