Background

High glucose causes glomerular mesangial growth and increased matrix synthesis contributing to diabetic glomerulopathy. Our purpose was to determine if high glucose alters endothelin-1 (ET-1) or platelet-derived growth factor-B activation of mesangial cell diacylglycerol-sensitive protein kinase C (PKC) isoforms and subsequent stimulation of mitogen-activated protein kinase (MAPK; p42, p44).

Methods

Rat mesangial cells in primary culture were growth arrested for 48 hours in glucose 5.6 mM (NG) or 30 mM (HG). PKC-, PKC-, and PKC- translocation from the cytosol-to-membrane and cytosol-to-particulate (cytoskeleton, nucleus) cellular fractions were measured by immunoblot using isoform-specific monoclonal antibodies. PKC isoforms were visualized also by confocal immunofluorescence microscopy. MAPK activation was measured by immunoblot using phospho-MAPK antibody and by detection of Elk-1 fusion protein phosphorylation following phospho-MAPK immunoprecipitation.

Results

In NG, ET-1 stimulated cytosol-to-membrane translocation of PKC- and PKC- but not PKC-. In HG, the pattern of ET-1–stimulated PKC- and PKC- changed to a cytosol-to-particulate distribution, which was confirmed by confocal immunofluorescence imaging. Platelet-derived growth factor-B did not cause translocation of PKC-, PKC-, or PKC- in either NG or HG. In HG, both basal and ET-1–stimulated MAPK activities were increased significantly. In HG, down-regulation of PKC isoforms with phorbol ester prevented the increased stimulation of MAPK by ET-1.

Conclusion

In HG, the enhanced activation of mesangial cell MAPK by ET-1 is PKC dependent and associated with altered translocation of PKC- and PKC-. Enhanced mesangial cell signaling responsiveness to vasoactive peptides in HG may constitute an important mechanism contributing to diabetic nephropathy.

METHODS

Primary culture of rat glomerular mesangial cells

Mesangial cells were obtained from glomeruli of 200 g male Spague-Dawley rats as previously described³². Briefly, glomeruli were isolated by selective sieving of chopped renal cortex and initially plated in minimal essential medium containing D-valine (GIBCO BRL, Life Technologies, Mississauga, Ontario, Canada), 5 units/ml insulin (GIBCO BRL), 100 units/ml penicillin-streptomycin (GIBCO BRL), and 20% fetal bovine serum (FBS; Wisent Inc., St.-Bruno, Quebec, Canada). Primary mesangial cells were identified by positive immunofluorescence for the intermediate filaments desmin and vimentin and were negative for cytokeratin and factor VIII. The cells demonstrated a positive contractile response to ET-1 (Sigma Aldrich, Oakville, Ontario, Canada). Mesangial cells from passages 8 to 10 were used for all experiments and were cultured to 90% confluence in 100 20 mm dishes in Dulbecco's modified Eagle's medium (DMEM) containing 5.6 mM glucose, L-glutamine, 110 mg/liter sodium pyruvate, prepared by the addition of 3.7 g/liter NaHCO₃, 20 mM HEPES acid, and 20% FBS.

Experimental protocol

Prior to experimentation, the cells were growth-arrested in DMEM containing 0.5% FBS and 5.6 or 30 mM glucose for 48 hours, in the presence or absence of 1 M phorbol 13-myristate 12-acetate (PMA), and were then serum deprived for two hours. To determine the effects of high glucose on mesangial cell PKC isoform translocation following agonist stimulation, a set of basal (unstimulated), PMA-stimulated and agonist-stimulated cytosol, membrane or particulate fractions were analyzed together on the same gel. The responses were compared with the basal, unstimulated condition in 5.6 mM glucose in each experiment. Prior to harvest, serum-deprived cells in 5.6 mM or 30 mM glucose were stimulated with 10 or 100 nM ET-1, 3 ng/ml PDGF-B (Boehringer Mannheim, Laval, Quebec, Canada) or 100 nM PMA for 10 minutes at 37°C. The agonists were dissolved in DMEM (not less than 20 l) and pipetted directly into the medium to achieve the final concentration.

Cytosol, membrane and particulate fractionation

At the end of the 10 minute stimulation, the cells were washed three times with phosphate buffered saline (PBS) at 4°C and were scraped in 80 l lysis buffer containing 1 mM NaHCO₃, 5 mM MgCl₂, 50 mM Tris-HCl, 2 mM ethylene glycol tetraacetic acid, 10 mM ethylenediaminetetraacetic acid (EDTA), 0.86 mM phenylmethylsulfonylfluoride (PMSF), 25 g/ml leupeptin, 10 mM benzamidine, and 100 mM dithiothreitol (DTT; New England Biolabs, Mississauga, Ontario, Canada). Fresh protease inhibitors and DTT were prepared and added on the day of the experiment. The cell lysate (two to three plates) was passed six times through a 26.5 inch gauge needle (Becton Dickinson, Mississauga, Ontario, Canada). After 30 minutes at 4°C in the lysis buffer, the lysate was centrifuged at 100,000 g at 4°C to separate cytosol (supernatant) from the pellet. For the membrane preparation, the pellets were washed with 30 l lysis buffer and solubilized in 100 to 170 l lysis buffer at 4°C containing 10 l/ml Triton-X-100 (BDH, Toronto, Ontario, Canada) by passing through a 26.5 inch gauge needle. After a second centrifugation at 100,000 g at 4°C, the membrane (supernatant) fractions were placed on ice, and the remaining fractions (Triton-X insoluble) were discarded. For the isolation of the particulate fraction, following the initial centrifugation, the pellet was solubilized in 250 to 300 l lysis buffer at room temperature containing 10 l/ml Triton-X-100 and 2% sodium dodecyl sulfate (SDS) and passed first through a six inch gauge then a 26.5 inch gauge needle followed by filtration through disposable, large-pore filters (Wheaton, Millville, NJ, USA) to remove the insoluble DNA. All cytosol, membrane and particulate fraction samples were assayed for protein in quadruplicate using the modified micro Lowry protein assay (Bio Rad D_c Assay Kit; Bio-Rad, Mississauga, Ontario, Canada) with bovine serum albumin (BSA; ICN Pharmaceuticals, Montreal, Quebec, Canada) as the standard. Each sample was added to one-third volume of 4 Laemmli sample buffer (0.260 M Tris-base, pH 6.8 with HCl, 4% -mercaptoethanol, 8% SDS, 40% glycerol, 0.04% bromophenol blue), boiled for two minutes, and stored at -20°C.

Protein kinase C isoform immunoblotting

Equal amounts of protein (15 g) from each sample and rat brain PKC-positive control were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 10% polyacrylamide) at 100 V for two hours. The gel was equilibrated in transfer buffer at room temperature, and the proteins were transferred to PVDF Immobilon hydrophobic membranes (Millipore, Bedford, MA, USA) overnight at 4°C in transfer buffer (25 mM Tris-base, 192 mM glycine, pH 8.3, plus 20% methanol).

After transfer, the membrane was rinsed with H₂O and blocked with 5% skim milk powder in Tris-buffered saline, pH 8.0, containing 0.05% Tween-20 (TTBS), for four hours with slow shaking at room temperature. The blocking solution for analysis of the particulate samples also contained 5% BSA, fraction V (ICN Pharmaceuticals). After rinsing in H₂O, the membrane was incubated in a 1:500 dilution (0.5 g/ml) of primary monoclonal antibody (Transduction Labs, Mississauga, Ontario, Canada) for PKC-, PKC-, or PKC-, or polyclonal antibody for PKC- (Sigma Chemical Co., St. Louis, MO, USA) in TTBS + 5% skim milk for one hour at room temperature. The membrane was washed three times (five min) with TTBS and was then incubated for 20 minutes at room temperature with slow shaking in a 1:5000 dilution (TTBS + 5% skim milk) of horseradish peroxidase (HRP)-labeled secondary goat antimouse IgG (BioCan Scientific, Mississauga, Ontario, Canada) to detect primary monoclonal antibodies or goat antirabbit IgG (Bio-Rad) to detect polyclonal antibodies. The blots were washed three times (seven min) with TTBS and once (10 min) with TBS.

Uniform loading of protein in each lane was confirmed by staining the membrane with Ponceau S (Sigma). The secondary antibodies were visualized using enhanced chemiluminescence (Kirgegaard & Perry Laboratories Inc., Gaithersburg, MD, USA). The blot was exposed to Kodak X-omat film (Kodak, Toronto, Ontario, Canada) for one minute. Densitometry was performed with a Macintosh gel photodocumentation system (ImageStore 5000; Ultraviolet Products, San Gabriel, CA, USA) using a white-light transilluminator. The area under each peak of the intensity curve was analyzed (arbitrary units) using NIH Image software (version 1.62; National Institutes of Health, Bethesda, MD, USA) for the Macintosh. Data from five separate experiments were pooled to analyze each PKC isoform. All results were expressed as a ratio compared with 5.6 mM glucose. Immunoblot densities from the same gel were compared, and the percentage of normal glucose basal values was pooled for statistical analysis.

Immunofluorescence labeling of PKC isoforms and confocal microscopy

Mesangial cells were cultured on glass cover slips and were treated as described earlier here. The cells were fixed with 3.7% formaldehyde for 15 minutes at room temperature followed by plasma membrane and nuclear membrane permeabilization with 100% methanol at -20°C for 10 minutes. After washing three times with PBS, the cell proteins were blocked with 1% goat serum plus 0.1% BSA in PBS for 60 minutes at room temperature. Mouse monoclonal anti-PKC- or PKC- antibodies were diluted to 1:100 in blocking solution and added to each cover slip for 60 minutes at 37°C. After washing three times with PBS, the FITC-conjugated secondary antibody (Molecular Probes Inc., Eugene, OR, USA) diluted 1:160 in blocking solution was added for 60 minutes at 37°C in the dark, and was then washed and mounted on glass slides with Aqua-Poly mount (Polysciences, Inc., Warrington, PA, USA) and Slowfade reagent (Molecular Probes). The FITC-labeled PKC isoforms were imaged using a confocal laser scanning image system (LSM 410; Zeiss, Düsseldorf, Germany) with FITC excitation and emission wavelengths of 488 nm and 520 nm, respectively. The cells were visualized with an oil-immersion inverted objective lens (Axiovert 100, 63), and the image pixel resolution was 512 512 with a gray level scale of 0 (minimum) to 255 (maximum) intensity. To standardize the fluorescence intensity for all of the experimental preparations, the confocal image contrast and brightness levels were adjusted optimally for each PKC isoform and were then kept constant. The pinhole (size = 20), scanning time (0.546 seconds), and magnification (63 1.4) of the confocal scanning system were constant for all image analysis. Incubation with FITC-conjugated secondary antibody alone demonstrated no significant labeling.

Analysis of MAPK activation

Phosphorylation of MAPK analyzed by immunoblot

The effect of high glucose exposure for 2.5, 6, 12, 24, and 48 hours on MAPK phosphorylation was analyzed. After 48 hours in normal and high glucose, stimulation with ET-1 10 nM or PDGF-B 3 ng/ml for 10 minutes was performed as described earlier here for the PKC isoform translocation studies.

Following agonist stimulation, the cells were washed three times in PBS at 4°C and scraped on ice with a rubber spatula in 80 l lysis buffer [50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1% Nonidet P-40 (BDH), 0.25% sodium deoxycholate (Sigma), 1 mM PMSF, 1 g/ml aprotinin (Sigma), 1 g/ml leupeptin, 1 g/ml pepstatin (Sigma), 1 mM orthovanadate, 1 mM NaF]. The lysate was then passed through a 26.5 inch needle four times and incubated at 4°C for 20 minutes. The remaining particulate content was removed with a 10-minute centrifugation at 10,000 g, 4°C. The supernatant was removed and assayed for protein using the modified micro-Lowry method. One-third volume of 4 Laemmli sample buffer was added, and the sample was boiled for two minutes followed by storage at -20°C until immunoblotting.

The SDS-PAGE, transfer and blocking were performed identically as described for the PKC isoform immunoblot (earlier here). A molecular weight standard sample (New England Biolabs) was also loaded on each gel to verify the molecular size of each band. After washing, the membrane was exposed to 1:3000 dilution of affinity-purified rabbit polyclonal IgG phospho-specific MAPK antibody (New England Biolabs) in TTBS + 5% BSA overnight at 4°C with slow shaking. This antibody detects p42 and p44 MAPK when catalytically activated by phosphorylation at both Thr 202 and Tyr 204. It does not cross-react with phosphorylated stress-activated protein kinase or p38 MAPK homologues. To test the specificity of the phospho-specific antibody, negative and positive phospho-MAPK control samples were loaded on each gel. The negative control was a kinase-inactive p42 protein (New England Biolabs), and the positive control was a fully phosphorylated (by MAPK kinase) p42 protein purified free from nonphosphorylated MAPK (New England Biolabs).

After washing briefly in H₂O, the membranes were washed three times (five minutes) at room temperature in TTBS with vigorous shaking, followed by exposure to a 1:5000 dilution of HRP-conjugated antirabbit secondary antibody (Bio-Rad) for 20 minutes at room temperature with gentle shaking. The blots were washed and detected as described earlier here.

The same membranes were used to detect total MAPK protein. To extinguish the chemiluminescence reaction without stripping the protein, the membrane was exposed to 15% peroxide for 15 minutes at room temperature, with gentle shaking. After a five-minute wash, the membrane was reblocked, and the immunoblot was repeated using anti-MAPK IgG (mouse) monoclonal antibody (Transduction Labs), which reacts with both p42 and p44, detecting both nonphosphorylated and phosphorylated MAPK. This was followed by HRP-conjugated secondary antibody (antimouse), which does not cross-react with the original antiphosphorylated MAPK IgG (rabbit). Densitometry was performed on both sets of blots, and the results were expressed as a ratio of phospho-MAPK to total MAPK, to control for differences in the amounts of cellular MAPK. Data from four separate experiments were pooled for statistical analysis.

Elk-1 fusion protein phosphorylation following phospho-MAPK immunoprecipitation

Following agonist stimulation as described earlier here, the cells were rinsed once with ice-cold PBS. Then lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM -glycerolphosphate, 1 mM Na₃VO₄, 1 g/ml leupeptin, and 1 mM PMSF was added, and the plates were left on ice for five minutes. The cells were scraped off the plate, sonicated (four times for five seconds), and microcentrifuged for 10 minutes at 4°C, and the supernatant was collected and assayed for protein using the modified micro-Lowry protein method. To immunoprecipitate active MAPK, phospho-specific MAPK p42, p44 monoclonal antibody (1:400 dilution, New England Biolabs) was added to each 200 g sample protein. After overnight incubation at 4°C, protein A sepharose beads were added and incubated for three hours at 4°C. The immunoprecipitates were then microcentrifuged for 30 seconds at 4°C. The pellets were washed twice with lysis buffer and twice with kinase buffer containing 25 mM Tris (pH 7.5), 5 mM -glycerolphosphate, 2 mM DTT, 0.1 mM Na₃VO₄, 10 mM MgCl₂, suspended in 50 l kinase buffer supplemented with 200 M adenosine triphosphate and 2 g Elk-1 fusion protein followed by incubation for 30 minutes at 30°C. The reaction was terminated with the addition of 25 l of 3 SDS sample buffer. The samples were boiled for five minutes, vortexed, and microcentrifuged. Immunoblotting was performed as described earlier here using 12% SDS-PAGE. After blocking with TTBS containing 5% skim milk for three hours at room temperature, the membrane was incubated with phospho-specific Elk-1 antibody (1:2000 dilution) overnight at 4°C. Active MAPK (New England Biolabs) was used as a positive control. Elk-1 fusion protein phosphorylation was detected with the enhanced chemiluminescence method, and densitometry was performed as described earlier here.

Statistical analysis

All final densitometric ratio data were expressed as mean standard error (SE). Statistical analysis was performed using InStat software (version 2.01; Graph Pad Software, Sacramento, CA, USA) for Macintosh. To compare the translocation from cytosol-to-membrane and cytosol-to-particulate results, one-way analysis of variance for unpaired samples was performed. To compare all conditions with basal NG, the Dunnett multiple comparisons was used. To compare other selected data, an unpaired, two-tailed Student t-test was used.

RESULTS

Effect of high glucose on PKC isoform translocation in response to PMA, ET-1, and PDGF

The recovery of total mesangial cellular protein from the 100 mm culture plates was no different in 30 mM glucose compared with 5 mM glucose, 2.48 0.17 mg/ml and 2.53 0.18 mg/ml, respectively.

PKC-

In the cytosol and membrane fractions, a single band of 82 kDa was observed Figure 1a, which comigrated with the rat brain positive control. No difference in the basal (unstimulated) cytosol or membrane fractions was observed in high glucose compared with the low glucose condition Figure 1 a, b. PMA stimulated translocation of PKC- from the cytosol to membrane fraction in both normal and high glucose. No significant translocation was observed when the cells were stimulated with ET-1 or PDGF in either normal or high glucose. In a separate experiment, during 48 hours of exposure to 1 M PMA, PKC- disappeared from both the cytosol and membrane fractions, indicating down-regulation Figure 1c.

Figure 1.

(A) Representative paired immunoblots of mesangial cell PKC- in the cytosol and membrane fractions following 48 hours in normal glucose (NG; 5.6 mM) or high glucose (HG; 30 mM) and 10-minute stimulation with phorbol 13-myristate 12-acetate (PMA; 100 nM), platelet-derived growth factor (PDGF-B; 3 ng/ml), or endothelin-1 (ET-1; 100 nM). The rat brain control PKC- (+) was identified as a single 82 kDa band identical to the mesangial cell samples. (B) PKC- immunoblot densitometry data (mean SEM, N = 5 experiments) from mesangial cell cytosol and membrane fractions are presented as percentage above normal glucose basal (100%). Symbols are: () normal glucose; () high glucose 30 mM; *P < 0.05 vs. NG glucose basal; **P < 0.01 vs. NG basal. (C) In mesangial cells, cytosol and membrane-associated PKC- was down-regulated with phorbol 13-myristate 12-acetate (PMA; 1 M) for 48 hours in normal (5.6 mM) or high (30 mM) glucose followed by stimulation with acute PMA 100 nM or endothelin-1 (ET-1; 100 nM). Rat brain was used as positive control (+).

Full figure and legend (14K)

In the particulate fractions Figure 2, the same 82 kDa band was observed, and PMA stimulated translocation from the cytosol to the particulate fractions. No translocation from the cytosol-to-particulate fraction was stimulated by ET-1 Figure 2 or PDGF-B (data not shown).

Figure 2.

(Upper panel) Representative paired immunoblots of mesangial cell PKC- in the cytosol and particulate fractions following 48 hours in normal glucose (NG; 5.6 mM) or high glucose (HG; 30 mM) and 10-minute stimulation with phorbol 13-myristate 12-acetate (PMA; 100 nM) or endothelin-1 (ET-1; 100 nM). Rat brain control (+) was used as a positive control. (Lower panel) PKC- immunoblot densitometry data (mean SEM, N = 5 experiments) from mesangial cell cytosol () and particulate fractions () are presented as percentage above NG basal (100%). *P < 0.05 vs. NG basal.

Full figure and legend (28K)

PKC-

In the cytosol and membrane fractions, a single band of 78 kDa was observed Figure 3a, which comigrated with the positive control. In basal (unstimulated) high glucose, a significant increase in the membrane fraction (P < 0.01 vs. normal glucose basal), but no change in the cytosol fraction, was observed Figure 3 a, b. PMA and ET-1 stimulated a significant decrease in the cytosol in both normal and high glucose. In normal glucose, PMA and ET-1 stimulated a significant increase in the membrane fraction. By contrast, in high glucose, the membrane-associated PKC- declined below the basal level. PDGF-B stimulation caused no translocation of PKC-. Chronic exposure to PMA down-regulated PKC- in both the cytosol and membrane fractions Figure 3c. PKC- was observed in the particulate fraction at 78 kDa. In normal glucose, PMA and ET-1 caused a significant decline in cytosol but no change in the particulate fraction Figure 4. In high glucose, translocation to the particulate fraction stimulated by ET-1 was increased significantly (P < 0.05 vs. 5.6 mM glucose plus ET-1). PDGF-B caused no translocation to the particulate fraction (data not shown).

Figure 3.

(A) Representative paired immunoblots of mesangial cell PKC- in the cytosol and membrane fractions following 48 hours in normal glucose (NG; 5.6 mM) or high glucose (HG; 30 mM) and 10-minute stimulation with phorbol 13-myristate 12-acetate (PMA; 100 nM), platelet-derived growth factor-B (PDGF-B; 3 ng/ml), or endothelin-1 (ET-1; 100 nM) are presented. The rat brain control PKC- (+) was identified as a single 78 kDa band identical to the mesangial cell samples. (B) PKC- immunoblot densitometry data (mean SE, N = 5 experiments) from mesangial cell cytosol and membrane fractions are presented as percentage above NG basal (100%). Symbols are: () normal glucose (NG); () high glucose 30 mM; *P < 0.05 vs. NG basal; **P < 0.01 vs. NG basal; §P < 0.05 vs. HG basal. (C) In mesangial cells, cytosol and membrane-associated PKC- was down-regulated with phorbol 13-myristate 12-acetate (PMA; 1 M) for 48 hours in normal glucose (NG; 5.6 mM) or high glucose (HG; 30 mM) followed by stimulation with acute phorbol 13-myristate 12-acetate (PMA; 100 nM) or endothelin-1 (ET-1; 100 nM). Rat brain was used as a positive control (+).

Full figure and legend (15K)

Figure 4.

(Upper panel) Representative paired immunoblots of mesangial cell PKC- in the cytosol and particulate fractions following 48 hours in normal glucose (NG; 5.6 mM) or high glucose (HG; 30 mM) and 10-minute stimulation with phorbol 13-myristate 12-acetate (PMA; 100 nM) or endothelin-1 (ET-1; 100 nM). Rat brain was used as a positive control. (Lower panel) PKC- immunoblot densitometry data (mean SE, N = 5 experiments) from mesangial cell cytosol () and particulate () fractions presented as percentage above NG basal (100%). *P < 0.05 vs. NG basal; **P < 0.05 vs. NG + ET-1.

Full figure and legend (31K)

Representative confocal immunofluorescence images cut through the mesangial cell nucleus labeled with primary monoclonal antibody to PKC- are shown in Figure 5. In normal glucose, PKC- was distributed throughout the cytosol, and in the presence of ET-1, increased PKC- label was observed particularly at plasma membrane locations. In high glucose, increased fluorescence and plasma membrane highlights were observed in the basal state, and a further change in intensity and nuclear localization was observed in high glucose plus ET-1.

Figure 5.

PKC- immunofluorescence. Mesangial cells growth-arrested on glass cover slips were cultured in normal (5.6 mM) glucose or high (30 mM) glucose for 48 hours, followed by 10 minutes of stimulation with endothelin-1 (ET-1; 100 nM). PKC- was detected in fixed, permeabilized cells by confocal scanning immunofluorescence microscopy using monoclonal anti-PKC– antibody and FITC-conjugated secondary antibody (magnification 240).

Full figure and legend (73K)

PKC-

In the cytosol and membrane fractions, PKC- was observed as a single band of 90 kDa that comigrated with the positive control, as demonstrated in Figure 6. In high glucose, a significant increase in membrane-associated PKC- was observed (P < 0.05 vs. 5.6 mM glucose basal). No change in cytosolic PKC- was apparent in high glucose. PMA and ET-1 stimulated a very significant translocation of PKC- from the cytosol to the membrane fractions in both normal and high glucose Figure 6 a, b. In high glucose, after ET-1 stimulation, there was a proportionately smaller increase in the membrane-associated PKC- despite a decrease in cytosol content similar to that observed in normal glucose Figure 6 a, b. PDGF-B did not cause translocation of PKC-. Chronic exposure to PMA down-regulated PKC- in both the cytosol and membrane fractions Figure 6c.

Figure 6.

(A) Representative paired immunoblots of mesangial cell PKC- in the cytosol and membrane fractions following 48 hours in normal glucose (NG; 5.6 mM) or high glucose (HG; 30 mM) and 10 minutes of stimulation with phorbol 13-myristate 12-acetate (PMA; 100 nM), platelet-derived growth factor (PDGF-B; 3 ng/ml), or endothelin-1 (ET-1; 100 nM). The rat brain control PKC- (+) was identified as a single 90 kDa band identical to the mesangial cell samples. (B) PKC- immunoblot densitometry data (mean SE, N = 5 experiments) from mesangial cell cytosol and membrane fractions were compared with basal (unstimulated) normal glucose (5.6 mM; NG) and are presented as percentage above NG basal (100%). Symbols are: () normal glucose; () high glucose 30 mM; **P < 0.01 vs. NG basal; §NS vs. HG basal. (C) In mesangial cells, cytosol and membrane-associated PKC- was down-regulated with phorbol 13-myristate 12-acetate (PMA; 1 M) for 48 hours in normal (5.6 mM; NG) or high (30 mM; HG) glucose, followed by stimulation with acute PMA 100 nM or endothelin-1 (ET-1; 100 nM). Rat brain was used as a positive control (+).

Full figure and legend (15K)

Protein kinase C- was observed in the particulate fraction as a single 90 kDa band Figure 7. In normal glucose, PMA and ET-1 stimulated a significant decrease in the cytosol content, with no change in the particulate fraction. In high-glucose, ET-1 stimulation was associated with a significant increase of PKC- in the particulate fraction (P < 0.05 vs. 5.6 mM glucose plus ET-1).

Figure 7.

(Upper panel) Representative paired immunoblots of mesangial cell PKC- in the cytosol and particulate fractions following 48 hours in normal glucose (NG; 5.6 mM) or high glucose (HG; 30 mM) and 10-minute stimulation with phorbol 13-myristate 12-acetate (PMA; 100 nM) or endothelin-1 (ET-1; 100 nM) are illustrated. (Lower panel) PKC- immunoblot densitometry data (mean SE, N = 5 experiments) from mesangial cell cytosol () and particulate () fractions are presented as percentage above NG basal (100%). *P < 0.05 vs. NG basal; **P < 0.05 vs. NG + ET-1.

Full figure and legend (33K)

Representative confocal immunofluorescence images cut through the mesangial cell nucleus labeled with primary monoclonal antibody to PKC- are shown in Figure 8. In the normal glucose basal state, PKC- was distributed throughout the cytoplasm. In the presence of ET-1, increased intensity and translocation to the nucleus were observed. In high glucose, increased intensity within the cytoplasm, including a cytoskeletal distribution and nuclear labeling of PKC-, was apparent. In high glucose, the addition of ET-1 caused redistribution of PKD- into a filamentous pattern.

Figure 8.

PKC- immunofluorescence. Mesangial cells growth arrested on glass cover slips were cultured in normal (5.6 mM) glucose or high (30 mM) glucose for 48 hours, followed by a 10-minute stimulation with endothelin-1 (ET-1; 100 nM). PKC- was detected in fixed, permeabilized cells by confocal scanning immunofluorescence microscopy using monoclonal anti–PKC- antibody and FITC-conjugated secondary antibody (magnification 240).

Full figure and legend (90K)

MAPK activation in high glucose and in response to PMA, ET-1, and PDGF-B

There was a time-dependent phosphorylation of MAPK on exposure of the mesangial cells to high glucose over 48 hours, as assessed by immunoblotting of phospho-MAPK. The increase in phosphorylation was observed between 24 and 48 hours. The total cellular content of MAPK was unchanged, whereas the phospho-MAPK/total MAPK (p42 plus p44) increased to 1.5- 0.3-fold above the 5.6 mM glucose value (P < 0.05, N = 5).

In response to acute PMA, PDGF or ET-1 increase in phospho-MAPK occurred as early as five minutes and was no different at 10, 15, or 30 minutes (data not shown). Figure 9 illustrates that acute exposure to PMA, or PDGF-B augmented the amount of phospho-MAPK in mesangial cells similarly in normal and high glucose. Chronic exposure to PMA, which down-regulated PKC, abolished the response to acute re-exposure to PMA but did not alter the response to PDGF-B (N = 5).

Figure 9.

Mitogen-activated protein kinase (MAPK) activation by platelet-derived growth factor (PDGF)-B. Growth-arrested cells were cultured in normal glucose (NG; 5.6 mM) or high glucose (HG; 30 mM) in the presence or absence of phorbol 13-myristate 12-acetate (PMA; 1 M) for 48 hours followed by two hours of serum-deprivation. Cells in NG, NG + PMA (chronic), and HG were stimulated with acute PMA 100 nM for 10 minutes. Cells in NG, NG + PMA (48 hr), HG, and HG + PMA (48 hr) were stimulated with PDGF-B 3 ng/ml for 10 minutes. In the same mesangial cell total lysates, anti-phospho-MAPK antibody (P, upper blot) or anti-MAPK antibody (T, lower blot) were used to detect phosphorylated and total MAPK, respectively. A negative (-) phospho-MAPK and positive (+) phospho-MAPK are included in the upper blot. *P < 0.05 vs. NG basal, N = 5 experiments.

Full figure and legend (45K)

Figure 10 illustrates that acute exposure to ET-1 10 nM also stimulated an increase in phospho-MAPK. In contrast to PDGF-B, the response to ET-1 in high glucose was significantly greater than that in normal glucose (P < 0.05, N = 5). Furthermore, chronic exposure to PMA diminished this enhanced activation such that the responses to ET-1 in normal and high glucose were no longer different.

Figure 10.

Mitogen-activated protein kinase (MAPK) p42, p44 phosphorylation following endothelin-1 (ET-1) stimulation. Growth-arrested cells were cultured in normal glucose (NG; 5.6 mM) or high glucose (HG; 30 mM) in the presence or absence of phorbol 13-myristate 12-acetate (PMA; 1 M) for 48 hours followed by two hours of serum deprivation. Cells in NG, NG + PMA (chronic), and HG were stimulated for 10 minutes with acute PMA 100 nM for 10 minutes. Cells in NG, NG + PMA (48 hr), HG, and HG + PMA (48 hr) were stimulated with ET-1 (10 nM) for 10 minutes. In the same mesangial cell total lysates, anti-phospho-MAPK antibody (P, upper blot) or anti-MAPK antibody (T, lower blot) were used to detect phosphorylated and total MAPK, respectively. A negative (-) phospho-MAPK and positive (+) phospho-MAPK, as described in the Methods section, are included in the upper blot. A total MAPK positive control is shown in the lower immunoblot panel. *P < 0.05 vs. NG basal; **P < 0.05 vs. NG basal; §P < 0.05 vs. NG + ET-1; §§P < 0.05 vs. HG + ET-1; N = 5 experiments.

Full figure and legend (46K)

To further identify the activation of MAPK, following immunoprecipitation of activated MAPK with phospho-MAPK antibody from total cell lysate, phosphorylation of Elk-1 fusion protein was examined. As illustrated in Figure 11, in high-glucose, ET-1 10 nM stimulated MAPK activity significantly above that observed in normal glucose. Preincubation with PMA diminished this enhanced activation, returning the level to that observed during mesangial cell stimulation by ET-1 in normal glucose.

Figure 11.

Elk-1 fusion protein phosphorylation. Growth-arrested mesangial cells were cultured in normal glucose (NG; 5.6 mM) or high glucose (HG; 30 mM) in the presence or absence of phorbol 13-myristate 12-acetate (PMA; 1 M) for 48 hours and were then serum deprived for two hours. Cells were then stimulated for 10 minutes with ET-1 10 nM, and total cell lysate was immunoprecipitated with anti-phospho-MAPK antibody. The activity of MAPK p42, p44 was observed using Elk-1 fusion protein as the substrate for phosphorylation that was detected with antiphospho-Elk-1 antibody. A representative immunoblot is demonstrated (N = 3 experiments).

Full figure and legend (37K)

DISCUSSION

Mesangial cells express the DAG-sensitive PKC isoforms -, -, and -. In normal glucose, ET-1 stimulation caused cytosol-to-membrane translocation of PKC- and PKC- but no translocation to the particulate fraction. PDGF-B caused no translocation of these PKC isoforms. Confocal immunofluorescence imaging revealed that ET-1 caused translocation of PKC- to peripheral membrane sites Figure 5 and redistribution of PKC- to the nucleus and perinuclear area Figure 8. Increased intensity of PKC isoform immunofluorescence is associated with activation, as previously reported^19,33. In the high-glucose basal condition, immunoblot analysis revealed an increased proportion of PKC- and PKC- in the membrane fraction. The immunofluorescence patterns of mesangial cell PKC- and PKC- after 48 hours of high glucose resembled the patterns in normal glucose following ET-1 stimulation. In high glucose, following ET-1 stimulation, immunoblotting demonstrated a change in the translocation pattern of both PKC- and PKC- to a cytosol-to-particulate distribution. Immunofluorescence imaging indicated that in high glucose, ET-1 stimulated translocation of PKC- to the nucleus, and PKC- became localized to the nucleus and cytoskeleton compartment. Taken together, the immunoblot and immunofluorescence data suggest that high glucose causes an increase in membrane-associated PKC- and PKC-. ET-1 stimulates both PKC- and PKC-, and in high glucose, the pattern of translocation is altered from a predominantly cytosol-to-membrane distribution to a cytosol-to-particulate (cytoskeleton/nucleus) pattern.

We found that basal MAPK activity is increased in high glucose as reported previously in cultured mesangial cells and glomeruli isolated from diabetic rats¹⁷. A rise in activity was observed between 24 and 48 hours of exposure to high glucose, without an alteration in total MAPK. PMA, ET-1, and PDGF all activated MAPK p42, p44, as demonstrated by the appearance of increased phospho-MAPK and enhanced phosphorylation of Elk-1 fusion protein. In high glucose, phosphorylation of MAPK p42, p44 following PDGF-B was unchanged, whereas ET-1 10 nM stimulation caused a significant increase in phospho-MAPK. When the DAG-sensitive PKC isoforms were down-regulated during chronic exposure to PMA, ET-1–dependent MAPK activation was no different in high glucose compared with the normal glucose condition Figure 11. These data suggest that in high glucose, the enhanced MAPK activation is mediated by a DAG-sensitive PKC pathway. The altered pattern of ET-1–stimulated PKC- and PKC- translocation may contribute to enhanced MAPK activation.

Our finding of increased membrane association and altered immunofluorescence patterns of PKC- and PKC- in the presence of high glucose is in keeping with the report of Haller et al³³, who demonstrated in vascular smooth muscle cells, by immunoblot and confocal microscopy, the translocation of PKC-, PKC-, PKC-, and PKC- within 6 to 12 hours of exposure to high glucose. However, in cultured rat mesangial cells after three to five days of high glucose, Kikkawa et al reported enhanced recovery of membrane-associated PKC- and PKC-, but not PKC- and PKC-³⁴. The differences between the latter study and our own may be due to differences in cellular fraction isolation or to the timing of analysis following exposure to high glucose or to both. We have previously observed that in high glucose, the increase in mesangial cell total and membrane-associated PKC- and PKC- occurs between 24 and 48 hours^29,30. Our immunoblot finding of increased mesangial cell membrane-associated PKC- and PKC- following 48 hours of high glucose is consistent with increased activity³¹, although a coincident decrease in cytosol content was not observed. This may be explained by an increase in total amount of cellular PKC- and PKC- in high glucose. Then, during a small amount of translocation, no apparent decrease in basal cytosolic content would be observed compared with normal glucose. Although the confocal images illustrate a definite change caused by high glucose of immunofluorescence intensity and probable translocation of PKC- and PKC- in the basal state, these data cannot distinguish between increased amount of PKC isoform in association with a pattern of activation/translocation in high glucose¹⁹. A more detailed study examining the time-dependent effects of high glucose on total cellular content of each PKC isoform is required to address this important question adequately.

The finding that high glucose alters the translocation pattern of PKC- and PKC- in response to ET-1 is novel. The work of Mochly-Rosen et al indicates that individual PKC isoforms are anchored to different subcellular structures, for example, cytoskeleton, via specific receptors for activated C kinase (RACKs)^35,36. RACKs are not PKC substrates, but enable PKC isoforms to colocalize with their substrates, thus facilitating phosphorylation. PKC-, PKC-, and PKC- may bind to cytoskeletal structures, as demonstrated in a wide variety of mammalian cell types, including rat pituitary cells³⁷ and neurons³⁸. One explanation for our finding of increased translocation of PKC- and PKC- to the particulate fraction in high glucose following ET-1 stimulation may be an alteration in the expression or function of RACKs specific for these isoforms. Perhaps the increased membrane association of PKC- and PKC- in high glucose in the basal state precludes translocation from the cytosol to the membrane sites following ET-1 stimulation.

In growth-arrested mesangial cells, activation of DAG-sensitive PKC isoforms with PMA caused a dramatic increase in MAPK phosphorylation, equivalent to PDGF-B stimulation of MAPK phosphorylation, which was PKC independent. PDGF-B may signal, in part, through DAG-insensitive PKC-, which is expressed in rat mesangial cells²², and possibly altered in high glucose³⁴. The lack of effect of high glucose on PDGF-B activation of MAPK suggests that any contribution of PKC- is either minimal or unaltered under these conditions.

In normal glucose, ET-1 stimulates MAPK principally through a PKC-independent pathway, including p21^ras39. In high glucose, the increased MAPK phosphorylation caused by ET-1 appeared to be entirely PKC dependent. Thus, our data indicate that in high glucose, the PKC-dependent pathway stimulated by ET-1, involving PKC- and PKC-, is altered. The increased membrane content of PKC- and PKC- in high glucose alone and the altered translocation pattern by ET-1 in the presence of high glucose are both associated with enhanced MAPK activation. The link between PKC and the MAPK pathway may be at the level of Raf (a MAPK kinase kinase), which may be directly phosphorylated and activated by DAG-sensitive PKC^40,41. Raf is known to bind not only to membrane but also to cytoskeletal elements to become activated⁴². We speculate that in high glucose, the increased membrane recovery and, after ET-1 stimulation, particulate localization of PKC- and PKC- enhance the activity of Raf and, in turn, MAPK kinase. Alternatively, PKC may influence MAPK-specific phosphatases^43,44, resulting in increased MAPK activity. Phorbol esters increase the activity of some protein tyrosine phosphatases by phosphorylation at specific serine residues^45,46. Further analysis of the intermediate signaling steps is required to define the molecular mechanisms of high glucose on PKC-activation of MAPK.

These data demonstrate that normal mesangial cell signaling pathways, for example, ET-1, may be altered in the presence of a high-glucose environment. The notion that normal physiological stimuli lead to abnormal responses in the diabetic milieu provides a potentially unifying mechanism linking many factors that appear to contribute to progressive diabetic glomerulosclerosis. Our work has focused on the early effects of high glucose. Prolonged exposure to elevated glucose or to recurrent changes in plasma glucose levels in the diabetic state may lead to altered acute responses and gene expression stimulated by vasoactive peptides, growth factors, and cytokines. MAPK is a final common pathway for many extracellular signals in mammalian cells^47,48,49. Specific MAPK substrates include p90^rsk, phospholipase A₂, caldesmon, c-jun, and c-myc^50,51. AP-1 is activated by c-fos expression due to increased phosphorylation by MAPK of the ternary complex factor Elk-1 and serum response factor⁵². In the pathogenesis of diabetic nephropathy, increased mesangial cell growth^1,2,8,13, enhanced expression of transforming growth factor-₁^1,53,54, and extracellular matrix protein synthesis^54,55 may be downstream events that respond to activated MAPK^16,17. Thus, selective inhibition of this key signaling pathway in organs targeted for diabetic complications may provide an important new direction for the development of therapeutic interventions.

In summary, ET-1 stimulates mesangial cell PKC- and PKC-, whereas PDGF-B has no effect on PKC-, PKC-, or PKC-. In normal glucose, PKC- and PKC- were found in the cytosol, and ET-1 caused translocation from the cytosol to membrane compartment. In high glucose, more membrane recovery of PKC- and PKC- was observed by immunoblotting. By immunofluorescence imaging, in high glucose the pattern of PKC- appeared to be more membrane associated and PKC- localized in a cytoskeletal and nuclear pattern. ET-1 stimulation in high glucose caused translocation of PKC- and PKC- from the cytosol to a cytoskeletal/nuclear compartment. Also, in high glucose, mesangial cell MAPK activity was increased, and ET-1–stimulated MAPK activity was enhanced compared with that observed in normal glucose and was PKC dependent. Therefore, in high-glucose, enhanced activation of mesangial cell MAPK by ET-1 may be due to altered translocation and activation of either, or both, PKC- and PKC-.

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Acknowledgments

This research was supported jointly by the Juvenile Diabetes Foundation International and the Medical Research Council of Canada. Dr. Tsiani was supported by the Banting and Best Diabetes Center, University of Toronto.