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High Ambient Glucose Enhances Sensitivity to TGF-1 via Extracellular Signal—Regulated Kinase and Protein Kinase C Activities in Human Mesangial Cells

Department of Pediatrics, Feinberg School of Medicine, Northwestern University; and Children’s Memorial Institute for Education and Research, Chicago Illinois

Correspondence to Dr. Tomoko Hayashida, Pediatrics W-140, 303 E Chicago Avenue, Ward 12-112, Chicago, IL 60611-3008; Phone: 312-503-2918; Fax: 312-503-1181; E-mail: hayashida{at}northwestern.edu

	Abstract

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ABSTRACT. High ambient glucose activates intracellular signaling pathways to induce cytokines such as TGF-1 in the extracellular matrix accumulation of diabetic nephropathy. These same pathways also may directly modulate TGF-1 signaling. R-Smad phosphorylation, association with Smad4, and nuclear accumulation after TGF-1 treatment (1.0 ng/ml) were significantly higher in mesangial cells that were conditioned to 20 mM glucose for 72 h than mesangial cells in 6.5 mM glucose, suggesting that high glucose enhanced responsiveness to TGF-1. Neither TGF-1 bioactivity nor TGF- receptor binding was significantly different between in 6.5 and 20 mM glucose-conditioned cultures. Furthermore, adding a neutralizing anti–TGF-1 antibody during glucose conditioning did not affect the enhanced Smad responsiveness, indicating that enhancement likely did not result from increased TGF- expression. In contrast, a mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK inhibitor, PD98059, completely abrogated the effect of high glucose. Glucose stimulation of ERK was inhibited by the general protein kinase C (PKC) inhibitor calphostin C and by the PKC-specific inhibitor rottlerin, whereas Gö6976, an inhibitor of conventional PKC, had no effect on ERK activity. Specificity of the PKC inhibitors was further verified by PKC and kinase assay. High glucose increased expression of several PKC isozymes, but only PKC showed proportionally increased membrane translocation and kinase activity in cells that were conditioned to 20 mM glucose. Finally, both ERK and PKC inhibition during glucose conditioning abrogated enhanced 1(I) collagen mRNA and promoter induction by TGF-1. Taken together, these data strongly suggest that heightened ERK and PKC activity in high ambient glucose conditions interact with the Smad pathway, leading to enhanced responsiveness to TGF-1 and increased extracellular matrix production in mesangial cells.

	Introduction

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Diabetic nephropathy is a leading cause of end-stage renal disease (ESRD). A variety of parameters influence the development of diabetic kidney disease, including the clinical factors of hypertension or hyperlipidemia (1), physical factors such as hyperfiltration and/or increased intraglomerular pressure (2), or hyperinsulinemia (3). An important predictor of diabetic renal complications is high blood glucose levels (4), which clearly have deleterious effects on kidney cell biology (5).

Diabetic glomerulosclerosis includes mesangial expansion and accumulated extracellular matrix (ECM) (6). ECM accumulation in diabetes can result from decreased degradation as a result of either reduced expression of matrix-degrading enzymes or resistance of glycated matrix to those enzymes (7). High glucose concentration in culture medium stimulates increased expression of ECM constituents such as collagen, fibronectin, and laminin (8). However, the mechanisms by which high ambient glucose levels lead to ECM accumulation are not well understood.

One potential mechanism that has been defined for glucose is the stimulation of fibrogenic cytokine production (6). In particular, TGF- is a potent stimulus of ECM expression (9) that has been correlated clinically and in experimental models with diabetic nephropathy (reviewed in reference 10). TGF-1 gene polymorphisms have been implicated in diabetic complications (11). In vitro, high-glucose culture induces TGF-1 mRNA or protein expression (12) at the transcriptional level (13). Moreover, intraperitoneal administration of neutralizing antibody to TGF-1 attenuates the increase in mRNA for collagen and fibronectin in the kidneys of streptozotocin (STZ)-induced diabetic mice (14) and preserves glomerular function in type 2 diabetic db/db mice (15). Furthermore, TGF-1 inhibition may ameliorate even established changes (16). These data strongly suggest that TGF- is a major mediator of sclerosis in diabetic nephropathy.

Data from our laboratory and others (17) have characterized a central role for the Smad signal transduction pathway in TGF-1–stimulated mesangial cell collagen expression. TGF- exist in a latent form that is activated by release from a stabilizing protein. TGF- receptor (TR) binding stimulates phosphorylation of the R-Smads Smad2 and Smad3, which form hetero-oligomeric complexes with Smad4. This complex translocates to the nucleus and acts as a transcriptional regulator of gene expression (18). Recent findings indicate that this classical paradigm for TGF- signaling may be more complex (19). In mesangial cells, TGF-1 activates both extracellular signal–regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinase pathways (20). Preincubation with an ERK inhibitor reduces TGF-1–stimulated Smad phosphorylation and association, as well as collagen production and promoter activities, suggesting that ERK activity is necessary for an optimal response to TGF-1 (21). Furthermore, protein kinase C (PKC) is specifically activated by TGF-1 in mesangial cells to modulate collagen expression via interaction with Smads (22). Importantly, whereas these other pathways modulate Smad activity and collagen expression, their activation in the absence of Smad activity does not lead to collagen production (21). Nonetheless, our data indicate that modification of Smad signaling by other cascades is important in regulating TGF-–stimulated collagen production.

This complexity led us to speculate that the hyperglycemic microenvironment could directly enhance Smad signaling as well as induce TGF- expression. Excessive ambient glucose may activate intracellular signals in multiple ways. First, high osmolarity can itself affect cell function. Second, aberrant metabolism of high ambient glucose such as de novo synthesis of diacylglycerol (DAG) or activation of the polyol pathway can initiate signals such as PKC activity (23). Third, high ambient glucose impairs the intracellular energy balance (NADH/NAD), possibly leading to increased mitochondrial superoxide production (24). Fourth, generation of nonenzymatically glycosylated proteins may activate PKC (25) or ERK (26). Any of these events could have an impact on Smad signaling. For example, elevated ERK activity, which can enhance Smad activity (19,21), has been reported in glomeruli from diabetic rats or in mesangial cells cultured with high-glucose medium (27–29).

In the present series of experiments, we examined whether high ambient glucose enhances TGF-1–stimulated Smad signaling to promote collagen expression in mesangial cells. One potential mechanism is through PKC activity (28). The PKC family of serine/threonine kinases consists of at least 12 isozymes that are classified into (1) conventional PKC, -_I and _II, - that depend on Ca²⁺ and DAG or phorbol ester for activation; (2) novel PKC, -, -, and - that are Ca²⁺ independent but DAG or phorbol ester sensitive; and (3) atypical PKC, -, and - that require neither Ca²⁺ nor phospholipids. The most intensively studied PKC isozyme in diabetes is the isozyme, and its specific inhibition by LY333531 prevents diabetic nephropathy both in STZ-induced Sprague-Dawley (SD) rat diabetes (30) and in db/db mice (31). Roles for other PKC isozymes in diabetes, particularly and isozymes, also have become of interest (32). However, information regarding how a particular isozyme affects other intracellular signals is limited. Here, we report a potential mechanism by which high glucose modulates TGF-1/Smad signaling via activation of ERK and/or PKC, leading to ECM accumulation in human mesangial cells.

	Materials and Methods

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Active, recombinant human TGF-1 (R & D Systems, Minneapolis, MN) was reconstituted as a 4-µg/ml stock solution in 4 mM HCl with 1 mg/ml BSA. PD98059, calphostin C, rottlerin, Gö6976 (Calbiochem, San Diego, CA), and PMA (Sigma, St. Louis, MO) were dissolved in DMSO as stock solutions. Antibodies were purchased from the following vendors: anti-Smad2/3 (N-19); anti-Smad4 (B-8); anti-ERK1 and 2; anti-PKC (C-20), _II (C-18), (C-19), (C-20), (C-15), (C-15), (C-18), (C-20), and (N-20) from Santa Cruz Biotechnology (Santa Cruz, CA); phospho Smad2 from Upstate (Lake Placid, NY); phospho ERK1/2 from Promega (Madison, WI); neutralizing anti–TGF-1 from R & D; and Oregon Green 514 anti-rabbit IgG from Molecular Probes (Eugene, OR). PKC substrates and reagents for kinase assays were purchased from Upstate.

Cell Culture and Glucose Conditioning
Human mesangial cells were isolated by differential sieving of minced human renal cortex as described previously and maintained with DMEM/Ham’s F12 (glucose 16.5 mM) supplemented with 20% heat-inactivated FBS, glutamine, penicillin/streptomycin, sodium pyruvate, HEPES buffer, and 8 µg/ml insulin (20). After 3 d of preconditioning to 6.5 mM glucose at the seventh passage, the cells were then passaged and cultured in DME containing 6.5 or 20 mM glucose for up to 72 h. Mannitol was used as an osmotic control. If mentioned, neutralizing antibody or inhibitors were present throughout the conditioning period, and the media were replaced every 24 h.

Cell Lysis, Immunoprecipitation, and Immunoblotting
The cells were lysed on ice in RIPA buffer (50 mM Tris/HCl [pH 7.5], 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) that contained protease and phosphatase inhibitors (1 mM PMSF; 1 mM EDTA; 1 µg/ml leupeptin, aprotinin, and pepstatin A; 1 mM sodium orthovanadate; 50 mM sodium fluoride; 40 mM -glycerophosphate) and then centrifuged at 18,000 x g for 10 min at 4°C. The immunoprecipitates from 500 µg of lysate protein were absorbed with 20 µl of protein G–Sepharose and then eluted by boiling for 5 min in 50 µl of 2x Laemmli reducing buffer. The resulting immunoprecipitates or 25 µg of protein from whole cell lysates were electrophoresed through a 6 to 10% SDS-PAGE gel, then transferred onto Immobilon-P (PVDF) membranes (Millipore, Bedford, MA). After immunoblotting, immunoreactive bands were visualized by chemiluminescence reagent according to the manufacturer’s protocol (Santa Cruz Biotechnology). The resulting bands were densitometrically analyzed using NIH Image 1.61 program for Macintosh.

Nuclear Protein Extraction
Nuclear proteins were extracted by addition of buffer B (HEPES 20 mM [pH 7.9], NaCl 0.4 M) to the nuclear particles isolated from cells incubated with a hypotonic buffer A (HEPES 20 mM [pH 7.9], KCl 10 mM) followed by membrane disruption with 0.5% NP-40 (33).

Cytosol/Membrane Separation
For cytosol/membrane protein separation, cells were scraped into a detergent-free buffer (20 mM Tris/HCl [pH 7.5], 0.5 mM EDTA, 0.5 mM EGTA, 10 mM -mercaptoethanol) that contained protease and phosphatase inhibitors and disrupted using a Dounce homogenizer, and the cytosolic fraction (supernatant) was separated by centrifugation at 100,000 x g. The pellets were resuspended in the buffer that contained 0.5% Triton-X and incubated for 30 min on ice, and the detergent extract was collected as the detergent-soluble particulated (membrane) fraction (22).

RNA Isolation and Northern Analysis
Total RNA was isolated with Trizol (Invitrogen, Carlsbad, CA) and then electrophoresed through a 1.2% agarose–1.1% formaldehyde gel, transferred onto a nylon membrane (MagnaGraph; MSI, Westborough, MA), and immobilized by UV cross-linking. Membranes were probed with ³²P-labeled cDNA probes as described previously (20).

Luciferase Assay
Glucose-conditioned cells were replated on six-well plates at 5.0 x 10⁴/well. After 24 h, cells were transfected with specific plasmids along with CMV-SPORT -galactosidase (Invitrogen) as a control for transfection efficiency. A total of 0.5 µg/well of each DNA along with a reporter gene (p3TP-Lux (34) or –0.42(I) collagen promoter (33)) were transfected in serum-free medium using Fugene6 (2 µl/1 µg DNA; Roche Molecular Biochemical, Indianapolis, IN) according to the manufacturer’s instructions. Three hours later, 1.0 ng/ml TGF-1 or vehicle was added to cultures. The cells were harvested in reporter lysis buffer (Promega) after a 24-h incubation. Luciferase and -galactosidase activities were measured as described previously (20). Each condition was tested in triplicate, and experiments were repeated at least three times for statistical analysis.

Immunocytochemistry
Cells that were conditioned to glucose for 72 h were replated on coverslips coated with 1 mg/ml gelatin and then fixed with 3.7% formaldehyde followed by permeabilization with 1% Triton X. The cells were stained for specific PKC isozymes or control rabbit IgG and visualized with Oregon Green 514-conjugated secondary antibodies. Images were evaluated under a confocal microscope (LSM510 META, Zeiss).

PKC Kinase Assay
PKC activity was determined as described previously (22). Briefly, after conditioning, cells that were treated with PMA for indicated periods were harvested simultaneously in complete RIPA buffer. PKC isozyme precipitated with an antibody (2 µg/sample) was then incubated with a substrate peptide and with [³²P]-ATP in 20 mM MOPS (pH 7.2), 25 mM -glycerophosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol in the presence of 500 µM cold ATP, and 75 mM MgCl₂ at 30°C for 30 min. The incorporated radioactivity was spotted onto p81 phosphocellulose paper and measured by scintillation counting after washing with 1% phosphoric acid and then acetone.

TGF- Binding Assay
Cells that were grown on 24-well plates with either 6.5 or 20 mM glucose media for 72 h were analyzed for TGF- binding as described previously (35). Briefly, after removal of endogenous TGF- with glycine/NaCl buffer (20 mM glycine, 135 mM NaCl [pH 3.0]), cells were incubated with ¹²⁵I-labeled TGF-1 (final concentration, 1 to 40 pM in binding buffer; 0.2% BSA, 128 mM NaCl, 5 mM KCl, 5 mM MgSO₄, 1.2 mM CaCl₂, 50 mM HEPES [pH 7.4]) for 2 h at 22°C. Nonspecific binding was determined in wells that contained 100-fold or greater excess unlabeled TGF-1. The supernatant was removed for free radioactivity determination, and cell lysates that were solubilized with 1% TritonX-100 that contained 10% glycerol and 20 mM HEPES (pH 7.4) were counted for radioactivity. Binding data were analyzed with Prism3 software (GraphPad Software).

Bioassay for TGF-1
A mink lung epithelial cell line (MLEC; clone 32) that was stably transfected with an 800-bp fragment of the 5' end of plasminogen activator inhibitor-1 (PAI-1) gene fused to the luciferase reporter gene in a p2-LUC–based vector (36) (D. Rifkin, New York University Medical Center, New York, NY) was maintained with selection medium (DME containing 200 µg/ml G418; Geneticin; Invitrogen). Levels of bioactive TGF- in the media were evaluated by induction of PAI-1 promoter activity 24 h after application of samples to the MLEC plated on a 24-well plate. Total secreted TGF- was also determined by heat activating the samples at 80°C for 5 min. The luciferase activity was converted to pM by using standard curves, obtained with dilutions of recombinant TGF-1, that were linear with R² > 0.95 up to 124 pM.

	Results

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Enhanced Smad Activity in High-Glucose Conditions
To determine whether high ambient glucose affects Smad signaling, we first examined the mesangial cell Smad response to a single TGF-1 stimulation. Cells were conditioned to 20 mM glucose for up to 72 h and then treated with 1.0 ng/ml TGF-1 in fresh medium for 30 min. Smad2 phosphorylation detected by immunoblotting with an anti–phospho-Smad2 antibody, which recognizes a specific C-terminal serine-phosphorylation site that is necessary for Smad activation by TGF- (18), was slightly but significantly induced without exogenous TGF-1 treatment after exposure to high glucose (Figure 1, odd lanes). In addition, the cells that were conditioned to 20 mM glucose demonstrated increased TGF-1 stimulation of Smad2 phosphorylation (Figure 1, even lanes). R-Smad total serine phosphorylation (Figure 2A, top) and association with Smad4 (Figure 2A, fourth panel) also were increased, by 2.1- and 2.7-fold, respectively. We also detected some Smad4 association with Smad2/3 in cells in 20 mM glucose even without TGF-1 stimulation (Figure 2A, fourth panel, second and third lanes) but not in 6.5 mM glucose medium. 20 mM mannitol as an osmotic control had no effects on Smad activities (Fig 2A, right). Exposure to 20 mM glucose or mannitol had little effect on Smad expression (Fig 2A, second and fifth panels). R-Smad translocates to the nucleus upon activation. Significantly higher amounts of phospho-Smad2 were detected in the nucleus of cells cultured in 20 mM glucose medium (Figure 2B, top, and solid bars in the graph), whereas Smad2/3 (Figure 2B, second panel) and Smad4 (Figure 2B, third panel) levels detected in the nuclear fraction after TGF-1 stimulation were equivalent in the 6.5- and 20-mM cultures. A similar pattern was observed by immunocytochemistry (data not shown). Furthermore, TGF-1–responsive p3TP-Lux reporter (34) activity was induced more strongly in 20 mM glucose–conditioned cells than those in 6.5 mM glucose (16.5- versus 6.7-fold; Figure 3). Taken together, these data indicate that mesangial cells exposed to high ambient glucose show enhanced Smad responsiveness to TGF-1.

View larger version (36K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Effect of high ambient glucose on TGF-1 stimulation of Smad2 C-terminal phosphorylation. Human mesangial cells that were preconditioned to 6.5 mM glucose, followed by exposure to 20 mM glucose up to for 72 h, were treated with a single dose of recombinant TGF-1 (1.0 ng/ml, 30 min). Cells were then lysed, and C-terminal–specific serine phosphorylation was determined by immunoblotting with an antibody that recognizes p465/467 Smad2 (top). Smad2/3 levels were also determined with aliquots of the same sample run in parallel (bottom). Results from densitometric analysis of three independent experiments are shown as a graph. , without TGF-1 treatment; , with TGF-1 treatment. *P < 0.05 versus day 0, with TGF-1; P < 0.05 versus day 0, without TGF-1.

View larger version (42K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Effect of glucose conditioning on TGF-1 stimulation of Smad activity. (A) R-Smads, stimulated with TGF-1 (1.0 ng/ml, 30 min) after 24- or 72-h conditioning to either 6.5 or 20 mM glucose, were immunoprecipitated with anti-Smad2/3 antibody, and serine phosphorylation (top) and Smad4 association (fourth panel) were evaluated by immunoblotting. Smad2/3 or Smad4 expression levels and immunoprecipitated Smad2/3 levels are also shown (second, fifth, and third panels, respectively). Results from TGF-1–treated cells cultured for 72 h in the presence mannitol (3) or control (0) are shown in the right panel. (B) Nuclear fractions prepared from cells that were glucose conditioned for 72 h and then treated with TGF-1 for the indicated times were subjected to electrophoresis, and nuclear pSmad2 (top), Smad2/3 (middle), and Smad4 (bottom) were evaluated by immunoblotting. The graph in the bottom panel shows results from densitometric analysis of nuclear pSmad2 levels from three separate experiments. , 6.5 mM glucose; , 20 mM glucose. Two-way ANOVA revealed significant enhancement of TGF-1–induced nuclear pSmad2 levels by 20 mM glucose (P < 0.05).

View larger version (21K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Effect of high ambient glucose on TGF-1 induction of p3TP-Lux activity. Mesangial cells that were conditioned for 72 h in the presence of PD98059 (10 µM) or DMSO as a vehicle were transfected with the p3TP-Lux reporter construct, and the activity induced by 24-h stimulation with 1.0 ng/ml TGF-1 was evaluated. The graph shows mean ± SEM of luciferase activities corrected for -galactosidase expression from a representative experiment performed in triplicate. Similar results were obtained from three individual experiments, and the difference in fold induction between 6.5 and 20 mM glucose–conditioned cells was statistically significant (P < 0.05, n = 3).

Extracellular Factors Potentially Related to Smad Activity: TGF- Production and Receptor Binding Capacity

View larger version (33K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Role of ambient TGF-1 in glucose-enhanced Smad responsiveness. (A) Cells that were conditioned to 6.5 or 20 mM glucose in the presence of neutralizing anti–TGF-1 antibody (5 µg/ml) or control IgG were then stimulated with TGF-1 (1.0 ng/ml, 30 min). R-Smad phosphorylation (top) and Smad4 association (third panel) were determined in Smad2/3-immunoprecipitated samples by immunoblotting. Levels of immunoprecipitated Smad2/3 were evaluated with the same membrane stripped for 20 min at 55°C (second panel). Smad4 levels were analyzed in whole cell lysates run in parallel (fourth panel). (B) TGF- bioactivity in conditioned media collected during the first (1) or last (3) 24 h of glucose conditioning and those accumulated over the entire 72 h (3*) were assayed in plasminogen activator inhibitor-1 promoter–expressing mink lung epithelial cell line (left half and the inset). Total TGF- secretion also was evaluated after heat activation of the rest of the conditioned media (right half). The box plot shows TGF- activities (pM, mean ± SEM) from a representative experiment in which six samples were measured in duplicate. Similar results were obtained in three separate experiments. *P < 0.05 versus values in day 1; P < 0.01 versus values in corresponding 6.5 mM glucose–conditioned media. All values for TGF- in heat-activated conditioned media were significantly higher than those for untreated media (P < 0.001).

View larger version (20K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Effect of ambient glucose levels on TGF- receptor capacity. Representative saturation binding curves for specific binding to 125I-TGF-1 and Scatchard plots (insets) in cells conditioned to 6.5 (left) or 20 (right) mM glucose for 72 h are shown. Each condition was tested in triplicate. Similar results were obtained in three experiments performed independently.

View larger version (40K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Role for extracellular signal–regulated kinase (ERK) activity in glucose-enhanced Smad activity. R-Smad phosphorylation (top panel and bottom left graph) and Smad4 association (fourth panel and bottom right graph) were evaluated in cells conditioned to 6.5 or 20 mM glucose in the presence of PD98059 (10 µM) or control vehicle as described in Figure 4. The bottom graphs show relative changes in TGF-1 responses determined by the intensities of immunoreactive bands (mean ± SEM, n = 5) compared with that in cells in 6.5 mM glucose in the absence of PD98059. Values are corrected for loading with immunoprecipitated Smad2/3 (third panel and left graph) or total Smad4 (right graph). In both cases, significant action of the inhibitor on the effects of glucose concentrations was detected by two-way ANOVA (P < 0.05).

Mechanism of ERK Activation in High Glucose: Role for PKC

View larger version (46K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Glucose activation of ERK and role of protein kinase C (PKC) isozymes. (A) Glucose-conditioned mesangial cells in the presence (third lane) or absence (left two lanes) of neutralizing anti–TGF-1 antibody (5 µg/ml) were lysed, and ERK activity was evaluated by immunoblots. Osmotic control for 20 mM glucose is shown in the fourth lane depicted as 20. (B) Cells were conditioned to glucose in the presence of calphostin C (CalC; 0.1 µM), Gö6976 (Gö; 10 nM), rottlerin (Rot; 5 µM), or DMSO (DM) as a vehicle, and ERK activity in the whole-cell lysates was evaluated by immunoblotting with phospho-ERK1/2 antibody (top). Aliquots of the same sample were run in parallel and blotted for ERK1/2 expression (bottom). Intensities of the resulting immunoreactive bands after standardization for ERK1/2 levels are shown as fold increase (mean ± SEM, n = 3) over the control (6.5 mM, DMSO) in the bottom graph. *P < 0.05 versus values in 6.5 mM, DMSO.

View larger version (55K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Effect of ambient glucose levels on PKC isozyme expression and activity. (A) Whole-cell lysates from 6.5 or 20 mM glucose–conditioned cells were electrophoresed in multiple pairs. The membranes with each pair were immunoblotted with isozyme-specific antibodies, and immunoreactive bands were visualized by chemiluminescent reagent, followed by simultaneous exposure. (B) Cytosolic and detergent-soluble particulated (membrane) fractions prepared from the glucose-conditioned cells were subjected to immunoblotting with isozyme-specific PKC antibodies. The graphs show relative PKC isozyme expression differences between 6.5 and 20 mM glucose culture in cytosolic or membrane fractions. Values (mean ± SEM, n = 6) are expressed as intensity ratios of immunoreactive bands comparing cells in 20 mM glucose with those in 6.5 mM glucose. As an internal control, signal intensities were standardized for PKCII signal intensities, because these did not differ with different glucose concentrations (fourth panel). (C) Cells that were plated on gelatin-coated cover slips and conditioned for 72 h were fixed and stained for PKC (top) or control rabbit IgG (bottom).

View larger version (16K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 9. PKC activation in 20 mM glucose and specificity of PKC inhibitors. (A) PKC activity was determined by in vitro kinase assay with a PKC-specific substrate with PKC immunoprecipitates from the cells that were conditioned to 6.5 or 20 mM glucose. Data are presented as fold increase over the value in control cells conditioned to 6.5 mM glucose. *P < 0.05 (mean ± SEM, n = 3). (B) Cells were treated with PMA (100 nM, 15 min) in the presence of inhibitors (CalC, 0.1 µM; Gö, 10 nM; Rot, 5 µM) or control vehicle (DMSO), and PKCII or - immunoprecipitated were subjected to in vitro kinase assay performed with a specific PKC substrate (QKRPSQRSKYL for PKCII, in the presence of PKA inhibitor and excess phosphatidyl serine and diacylglycerol; ERMRPPKRQGSVRRRV for PKC). Results from a representative experiment measured in duplicate are shown as kinase activity relative to the controls. Similar results were obtained in three separate experiments.

Contribution of ERK and PKC to TGF-1–Stimulated Collagen Gene Activation in High-Glucose Conditions

View larger version (53K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 10. (A) Effect of high ambient glucose on TGF-1 induction of 2(I) collagen promoter activity. Cells that were conditioned to glucose in the presence of PKC inhibitors (CalC, 0.1 µM; Gö, 10 nM; Rot, 5 µM) were then transfected with a –0.42(I) collagen promoter, and promoter activity was determined after a 24-h incubation with TGF-1. The graph shows fold induction of the activity over control (mean ± SEM, n = 3) in each condition, and the numbers above the bars represent ratios of fold induction of 20 mM glucose culture over 6.5 mM glucose culture. *P < 0.05 versus values in 20 mM, DMSO; P < 0.05 versus values in 6.5 mM, DMSO; P < 0.05 versus values in 6.5 mM, Rot; P < 0.05 versus ratios of values in 20 mM/6.5 mM in DMSO. (B) Effect of ERK or PKC inhibition on heightened 1(I) collagen expression by TGF-1 in high-glucose culture. Glucose-conditioned cells in the presence of inhibitors as described in A were treated with 1.0 ng/ml TGF-1 during the last 24 h of a 72-h conditioning. Mannitol was used as an osmotic control for 20 mM glucose (right, 20). TGF-1 induction of 1(I) collagen mRNA expression (top) was calculated after correction for loading determined by 28S (bottom) and demonstrated under the blot. Similar results were obtained from three independent experiments, and representative blots are shown.

	Discussion

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These data support a role for Smads in diabetic nephropathy. However, the sclerosing process in diabetic kidneys is chronic and changes become apparent approximately a decade after the onset of diabetes, whereas maximal Smad activity in response to a single TGF-1 exposure is observed as early as 30 min after the treatment (33). Therefore, although the enhanced Smad activity in the diabetic milieu could be attributed partly to increased TGF-1 expression, the long-term effects of high ambient glucose related to relatively transient Smad activity are less clear. To begin to address this issue, we examined Smad responses to a single stimulation with TGF-1 after high-glucose conditioning and found that mesangial cells so treated showed enhanced responsiveness to TGF-1.

In the present study, we found that mesangial cells conditioned to 20 mM glucose showed increased Smad phosphorylation, multimerization, and nuclear translocation of phospho Smad. These events seem to be mediated by the activation of ERK MAP kinase and PKC. In contrast to previous studies indicating a role for conventional PKC in enhancing glucose-stimulated TGF- expression, our studies show that Smad activation is enhanced independent of either production of active TGF- or TR binding capacity. Instead, our results suggest that enhanced sensitivity to TGF-1 induced by high-glucose conditioning is most likely due to events downstream of receptor binding. A similar phenomenon was previously reported in which mesangial cells seemed to be sensitized to another cytokine, endothelin, through novel PKC activity induced by high glucose (47). Our experiments focused on the effects of TGF-1, because this is the major isoform that has been associated with ECM accumulation and is upregulated in diabetic kidneys as demonstrated by immunohistochemistry or by mRNA expression (reviewed in reference 10). Regardless of the isoform involved, our data do not necessarily reflect local TGF- bioactivity, which we found was not significantly different between 6.5 and 20 mM glucose culture, because the majority of TGF- exists in latent form.

Our understanding of regulation of intracellular signaling networks has become increasingly complex. The current paradigm for Smad regulation implicates multiple signaling pathways modulating Smad activity (18,19), as would likely be the case in the diabetic milieu. We previously reported that ERK enhances Smad activity in TGF-1 induction of collagen production (20,21). The observations in the present report indicate that, in addition to stimulating TGF-1 expression as has previously been reported (48), glucose-stimulated ERK could directly interact with the Smad pathway to enhance responses to TGF-1. Similarly, our laboratory recently reported that PKC modulates TGF-1/Smad activity (22). In the present study, we show that the PKC isozyme contributes to glucose activation of the ERK pathway to enhance collagen transcription but does not contribute to the release of TGF-1. Thus, the mechanism of action of high glucose described here is distinct from that previously reported for classical PKC such as PKC (30,31). Although our study has, on the basis of our previous work (22), focused on the isozyme as a potential intracellular agent mediating enhanced TGF- signaling in high glucose, our results do not obviate the possible involvement of other PKC isozymes either via interaction with (49,50) or independent of (32) Smad activity. In the current study, inhibition of MEK/ERK or PKC alone did not completely reverse high glucose–enhanced promoter activity. Thus, ERK and/or PKC may have additional effects. However, we have found that stimulation of ERK or PKC without Smad activation does not increase collagen expression (21), suggesting a central role for Smads. In view of this complexity, progress in identifying novel therapies for renal fibrosis will require further characterization of critical signaling pathway interactions that could be disrupted to ameliorate diabetic nephropathy.

	Acknowledgments

 
This work was supported in part by grant R01 DK49362 from the NIDDK. The data were presented in part at the 35th meeting of the American Society of Nephrology (2002).

We thank Susan Hubchak for isolating and characterizing the human mesangial cells, D. Rifkin for MLEC clone 32, J. Massague for the p3TP-Lux construct, Y. Yamada for cDNA for human 1(I) collagen, and H. Saga for 28S rRNA. We also thank other members of the Schnaper laboratory for helpful discussions.

	References

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