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Am J Physiol Endocrinol Metab 293: E1280-E1288, 2007. First published August 21, 2007; doi:10.1152/ajpendo.00223.2007
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Reactive oxygen species, PKC-beta1, and PKC-{zeta} mediate high-glucose-induced vascular endothelial growth factor expression in mesangial cells

Ling Xia,1,2 Hong Wang,1,2 Snezana Munk,1,2 Helena Frecker,2 Howard J. Goldberg,2,3 I. George Fantus,2,3 and Catharine I. Whiteside1,2

1University Health Network, 3Mt. Sinai Hospital and 2Department of Medicine, University of Toronto, Toronto, Ontario, Canada

Submitted 10 April 2007 ; accepted in final form 4 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vascular endothelial growth factor (VEGF) is implicated in the development of proteinuria in diabetic nephropathy. High ambient glucose present in diabetes stimulates VEGF expression in several cell types, but the molecular mechanisms are incompletely understood. Here primary cultured rat mesangial cells served as a model to investigate the signal transduction pathways involved in high-glucose-induced VEGF expression. Exposure to high glucose (25 mM) significantly increased VEGF mRNA evaluated by real-time PCR by 3 h, VEGF cellular protein content assessed by immunoblotting or immunofluorescence within 24 h, and VEGF secretion by 24 h. High-glucose-induced VEGF expression was blocked by an antioxidant, Tempol, and antisense oligonucleotides directed against p22phox, a NADPH oxidase subunit. Inhibition of protein kinase C (PKC)-beta1 with the specific pharmacological inhibitor LY-333531 or inhibition of PKC-{zeta} with a cell permeable specific pseudosubstrate peptide also prevented enhanced VEGF expression in high glucose. Enhanced VEGF secretion in high glucose was prevented by Tempol, PKC-beta1, or PKC-{zeta} inhibition. In normal glucose (5.6 mM), overexpression of p22phox or constitutively active PKC-{zeta} enhanced VEGF expression. Hypoxia inducible factor-1{alpha} protein was significantly increased in high glucose only by 24 h, suggesting a possible contribution to high-glucose-stimulated VEGF expression at later time points. Thus reactive oxygen species generated by NADPH oxidase, and both PKC-beta1 and -{zeta}, play important roles in high-glucose-stimulated VEGF expression and secretion by mesangial cells.

NADPH oxidase; vascular endothelial growth factor; p22phox; protein kinase C-beta1; protein kinase C-{zeta}

DIABETIC NEPHROPATHY, the leading cause of end-stage renal disease in North America, is characterized by proteinuria, diminished glomerular filtration rate, podocyte injury, mesangial matrix accumulation, progressive glomerulosclerosis, and tubulointerstitial disease (41, 52). Increased local expression of peptide growth factors, such as ANG II, transforming growth factor-beta (TGF-beta), and vascular endothelial growth factor (VEGF), is implicated in these pathological abnormalities (41, 52). Long-term clinical trials of individuals with type I and type II diabetes have demonstrated that hyperglycemia is a causal factor for the development of diabetic nephropathy (13, 48). The biochemical actions of intracellular high glucose may be initiated by the generation of reactive oxygen species (ROS) in the mitochondria and the ensuing increased flux through side pathways in glycolysis (7). This leads to activation of protein kinase C (PKC), advanced glycation end-product (AGE) formation, and other effects. However, the molecular mechanisms linking these earliest effects of high glucose to the excess expression and secretion of peptide growth factors remain incompletely understood.

VEGF (also called VEGF-A) is a member of a family of secreted 34–42 kDa dimeric glycoproteins related to the platelet-derived growth factor family (8, 43). VEGF potently stimulates vasculogenesis, angiogenesis, microvascular permeability, and nitric oxide release by binding to one of two tyrosine kinase receptors, VEGFR1 (also called Flt-1) and VEGFR2 (Flk-1), on endothelial cells (15). Alternate splicing produces a number of different isoforms, of which VEGF165 and VEGF164 are the most abundant in humans (15) and rats (38), respectively. High glucose causes upregulation of VEGF in mesangial cells and podocytes in vivo (10, 12, 44, 51), whereas VEGFR2 receptors are expressed mainly on glomerular endothelial cells (12).

Mesangial cell PKC-{alpha}, -beta, -{delta}, and –{epsilon} are activated in high glucose, presumably through de novo diacylglycerol synthesis (2, 21, 25). Indeed, PKC-beta is necessary for the development of experimental diabetic nephropathy in rodents, as shown by studies with a specific PKC inhibitor, ruboxistaurin (LY-333531), and more recently with PKC-beta knockout mice (24, 32). Although diacylglycerol does not modulate PKC-{zeta} directly, PKC-{zeta} activity is elevated in the presence of high glucose, as assessed by membrane translocation and an in vitro kinase activity of PKC-{zeta} immunoprecipitated from mesangial cell membranes (2, 14, 27). In turn, PKC-{zeta} contributes to high-glucose-induced filamentous actin disassembly, the generation of ROS from NADPH oxidase, and is required for enhanced collagen IV expression (14, 27).

Mesangial cell NADPH oxidase is activated by both classical PKC isozymes (PKC-{alpha} or -beta) and atypical PKC-{zeta} (23, 53). We and others have shown that mesangial cells or podocytes incubated in high glucose generate ROS dependent on NADPH oxidase and this is sustained by high-glucose-stimulated increases in the expression of the p22phox and p47phox (16, 20, 45, 53) subunits of NADPH oxidase. The significance of NADPH oxidase in the pathogenesis of diabetic nephropathy is underscored by the finding that apocynin, a pharmacological inhibitor of NADPH oxidase, suppresses proteinuria and mesangial matrix expansion in streptozotocin-induced diabetic rats and in db/db mice (4, 45).

In diabetic nephropathy, mesangial cells transform into a prosclerotic phenotype that can be mimicked by exposing mesangial cells in vitro to high glucose (19, 25, 41, 52). Mesangial cells secrete VEGF in vitro in response to several factors relevant to diabetes, including TGF-beta, AGEs, and ANG II (39, 50, 55). High glucose or overexpression of GLUT1, a glucose transporter, also upregulates VEGF expression in mesangial cells, podocytes, and retinal pigment epithelial cells in vitro (10, 18, 22, 33, 37, 54, 56), and this effect is blocked by general PKC inhibitors (10, 18, 22). The exact pathways involved in high glucose regulation of VEGF remain unknown.

VEGF is of particular interest because of its possible autocrine and paracine action in altering glomerular permeability and causing proteinuria in diabetic nephropathy. The present study was designed to further elucidate the mechanisms responsible for the enhanced expression and secretion of VEGF in high ambient glucose using mesangial cells as a model. We postulated that, in mesangial cells exposed to high glucose, upregulation of VEGF expression was related to the earliest glucose-induced signaling mechanisms involving ROS and PKC isozymes. Here we demonstrate a requirement for NADPH oxidase-induced ROS and for two PKC isoforms (beta1 and {zeta}), in high-glucose-stimulated VEGF synthesis and secretion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials. Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl) was obtained from Sigma (St. Louis, MO). Gö-6976 was purchased from EMD Biosciences (San Diego, CA). LY-333351 was from Axxora (San Diego, CA). The myristoylated PKC-{zeta} inhibitor (myr-RRGARRWRK), the p22phox phosphorothioate antisense oligonucleotides (5'-GAT CTG CCC CAT GGT GAG GAC C-3'), and all the primers for real-time PCR were synthesized by The Center for Applied Genomics at The Hospital for Sick Children (Toronto, ON, Canada).

Cell culture. Rat mesangial cells were isolated and grown as we have previously described (20, 53). The cells were routinely growth-arrested in 0.5% FBS for 48 h before the start of experiments.

Plasmids and mesangial cell transfection. A plasmid expression vector for constitutively active PKC-{zeta}, which contains an amino terminal p60 c-Src myristoylation sequence (MGSNKSKPK) fused to rat PKC-{zeta} cDNA and a carboxy terminal Flag epitope, was a gift of Dr. Alex Toker (Harvard Medical School, Boston, MA; see Ref. 11). A plasmid containing the cDNA for the NADPH oxidase subunit, p22phox, was generous gift from Dr. Kathy Griendling (Emory University, Atlanta, GA; see Ref. 49). A p22phox expression vector was generated by excising a 731-bp EcoR1/BamH1 fragment from this plasmid and subcloning it into the cytomegalovirus-based expression plasmid, pcDNA3.1MycHis (Invitrogen, Carlsbad, CA). Antisense oligonucleotides against p22phox were transfected as we previously reported. The transfection efficiency exceeded 90% as demonstrated by confocal immunofluorescence imaging of p22phox in the transfected cultured mesangial cells (not shown) previously reported (20, 53). Mesangial cells were transfected with plasmid constructs using Lipofectamine-2000 (Invitrogen) or Fugene 6 (Roche, Indianapolis, IN), as specified by the manufacturers.

Immunofluoresence staining. VEGF was detected by staining mesangial cells with VEGF antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) followed by confocal immunofluorescence microscopy as described (20, 27, 53).

Western immunoblotting and cellular fractionation. Western blots were performed with primary antibodies against VEGF (EMD Bioscience), p22phox, PKC-beta1, PKC-{zeta} (Santa Cruz Biotechnology), protein kinase B (Akt) phosphoserine-473 and Akt (Cell Signaling Technology, Danvers, MA), beta-actin (Sigma-Aldrich, St. Louis, MO), and hypoxia-inducible factor-1{alpha} (HIF-1{alpha}; Novus Biologicals, Littleton, CO) as we previously described (20, 27, 53).

Total cell lysates and cellular membrane fractions were prepared as previously reported (14, 53). A mesangial cell crude nuclear fraction was obtained by lysing cells in 50 mM Tris·HCl, pH 7.5, 10 mM EGTA, 2 mM EDTA, 1 mM benzamidine, 1 mM NaF, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 25 µg/ml leupeptin. With the use of a chilled 1-ml Dounce homogenizer, the lysates were processed with five strokes using a "loose" pestle and then five strokes with a "tight" pestle to separate the nuclei. The lysate was then centrifuged at 4°C at 500 g for 10 min three times with resuspension in the above buffer. Next, the nuclear-enriched pellet was suspended in the above buffer with the addition of 1% Triton X-100, passed five times through a 26-gauge needle, and centrifuged at 15,000 g for 5 min. The supernatant was used as the nuclear-enriched fraction.

VEGF mRNA measured by real-time PCR. RNA was isolated from mesangial cells using an RNeasy kit (Qiagen, Valencia, CA). The RNA was reverse transcribed and subject to real-time PCR as we have described (53). The primers for VEGF were VEGF sense, 5'-GATGAGATAGAGTATATCTTCAAGCCGT-3' and VEGF antisense, 5'-TCTATCTTTCTTTGGTCTGCATTCAC-3' (GenBank: NM_031836 [GenBank] ). To measure VEGF mRNA stability, mesangial cells were treated with 5 µg/ml actinomycin D, and RNA was harvested and analyzed for VEGF mRNA levels by real-time PCR as we previously described (53).

PKC-beta1 kinase assay. PKC-beta1 activity was measured in total mesangial cell lysates, cellular membrane fraction, and the crude nuclear fraction as prepared above. The solublized samples were immunoprecipitated in the following buffer: 25 mM HEPES, 150 mM NaCl, 1 mM EGTA, 2 mM EDTA, 10 mM NaF, 50 mM beta-glycerophosphate, 1 mM sodium orthovanadate, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 25 µg/ml leupeptin. PKC-beta1 was immunoprecipitated with 2 µg anti-PKC-beta1 antibody (Santa Cruz) and 65 µl protein G Plus Agarose. The purified PKC-beta1 samples were used for an in vitro kinase assay as we previously described for PKC-{zeta}, using a MARCKS peptide substrate (Biomol, Plymouth Meeting, PA; see Ref. 27).

Enzyme-linked immunosorbent assay for VEGF. The concentration of VEGF164 was measured in the conditioned media of cultured mesangial cells using a kit from R&D Systems (Minneapolis, Minnesota) according to the manufacturer's instructions.

Statistical analyses. Results are expressed as means ± SE. Statistical analyses were performed with Instat 2.01 (Graph Pad, Sacramento, CA). Unpaired Student's t-tests were used to compare the means of two groups, and ANOVA was applied to compare the means of three or more groups followed by the Tukey-Kramer post hoc test. P < 0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
High glucose stimulates mesangial cell VEGF expression. To investigate the regulation of VEGF, rat mesangial cells were exposed to either 5.6 mM (normal glucose) or 25 mM (high glucose) glucose for up to 48 h. As displayed in Fig. 1A, cellular VEGF protein levels, determined by Western blotting, were increased significantly (1.75-fold) by 3 h. This was not due to an osmotic effect, since 25 mM D-glucose but not 19.4 mM L-glucose plus 5.6 mM D-glucose increased VEGF immunostaining in mesangial cells (Fig. 1B). Figure 1C shows that VEGF protein levels were increased by D-glucose concentrations as low as 15 mM. Secreted VEGF164 detected by enzyme-linked immunosorbent assay (ELISA) was increased significantly in response to high glucose in mesangial cell media after 24 h and was 2.5-fold higher by 48 h (Fig. 1D).


Figure 1
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  		Fig. 1. Mesangial cell vascular endothelial growth factor (VEGF) mRNA and protein levels increase in high glucose. Mesangial cells were exposed to normal glucose (NG, 5.6 mM) or high glucose (HG, 25 mM) for the indicated times before measurement of VEGF expression. A: VEGF immunoblot (top) and VEGF protein levels quantified relative to 5.6 mM glucose, n = 7 (bottom). B: VEGF immunofluorescence in mesangial cells cultured for 48 h in NG, 5.6 mM D-glucose + 19.4 mM L-glucose (LG), or HG. C: VEGF Western blot following mesangial cell exposure to increasing concentration of D-glucose for 48 h. D: VEGF secretion in conditioned media relative to NG measured by ELISA, n = 4. E: VEGF mRNA levels determined by real-time PCR in four experiments. F: analysis of VEGF mRNA stability. VEGF mRNA levels were measured by real-time PCR at the indicated times after mesangial cells were incubated with 5.6 or 25 mM glucose for 6 h and then treated with 5 µg/ml actinomycin D. Results are expressed relative to NG at the time of actinomycin addition, n = 5. *P < 0.05 vs. NG. **P < 0.01 vs. NG. ***P < 0.001 vs. NG.

 
VEGF mRNA levels, assessed by real-time quantitative PCR, were increased significantly at 3 h in high glucose and were 2.5-fold higher by 48 h (Fig. 1E). VEGF gene expression is regulated by transcriptional activation and through changes in mRNA stability, as observed in AMP-activated protein kinase-stimulated myotubes (34). Therefore, mesangial cells were incubated with 5.6 or 25 mM glucose for 6 h and treated with actinomycin D to stop transcription, and VEGF mRNA levels were monitored by PCR. Figure 1F shows that high glucose did not reduce the decay rate of VEGF mRNA following the addition of actinomycin-D, indicating that high glucose does not increase VEGF mRNA stability.

High-glucose-induced VEGF expression is dependent on ROS generated by NADPH oxidase. Previously, we found that high-glucose-induced increases in collagen IV expression and suppression of calcium signaling by high glucose required ROS produced by NADPH oxidase (20, 53). This motivated us to ask whether ROS produced by NADPH oxidase are necessary for high-glucose-stimulated VEGF expression. Figure 2, A and B, demonstrates that the superoxide dismutase mimetic Tempol abolished high-glucose-induced increases in VEGF protein levels detected by Western blotting or immunofluorescence, respectively. To establish a specific role for NADPH oxidase in mediating the action of high glucose on VEGF, mesangial cells were transfected with antisense or scrambled oligonucleotides against the p22phox NADPH oxidase subunit and incubated in 5.6 or 25 mM glucose, and cell extracts were analyzed by Western blotting. Consistent with our previous report (53), antisense oligonucleotides directed against p22phox downregulated p22phox protein levels under normal and high-glucose conditions (Fig. 3A). Interestingly, knocking down p22phox expression blocked the increase in VEGF protein in high glucose detected by both Western blotting (Fig. 3A) and immunostaining (Fig. 3B). Further supporting a role for NADPH oxidase, transiently overexpressing p22phox was sufficient to upregulate VEGF expression in normal glucose (Fig. 3C).


Figure 2
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  		Fig. 2. Inhibition of high-glucose-induced VEGF expression by the antioxidant Tempol. Mesangial cells were pretreated with 100 nM Tempol or its vehicle, DMSO, for 1 h and then exposed to 5.6 mM glucose (NG) or 25 mM glucose (HG). A: VEGF immunoblot in mesangial cells exposed to HG for 6 h (top) and VEGF expression quantified relative to NG, n = 4 (bottom). B: VEGF immunofluorescence in mesangial cells exposed to HG for the indicated times (top) and VEGF expression quantified as pixel intensity per cell (n = 100–155 cells) in three experiments (bottom). *P < 0.01 vs. NG. **P < 0.001 vs. NG. ***P < 0.001 vs. HG.

 

Figure 3
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  		Fig. 3. Antisense oligonucleotides directed against the NADPH oxidase subunit p22phox prevent high-glucose-stimulated VEGF expression. Mesangial cells were transiently transfected with antisense oligonucleotides against p22phox or scrambled oligonucleotides (scr) and then exposed to 5.6 mM (NG) or 25 mM (HG) glucose. A: VEGF or p22phox immunoblot in mesangial cells incubated for 48 h in NG or HG. beta-Actin was used as a loading control. VEGF or p22phox expression relative to NG control was quantified in three experiments (bottom). B: VEGF immunofluorescence in mesangial cells exposed to NG or HG for the indicated times (left) and VEGF expression (pixel intensity/cell; n = 183–330 cells) from three experiments (right). C: VEGF and p22phox immunoblots (left) in mesangial cells transfected for 48 h with a p22phox expression vector or an empty vector (pcDNA3), or untransfected (labeled C). Left: VEGF and p22phox expression relative to NG, n = 5. *P < 0.001 vs. NG control, scr, or empty vector. **P < 0.001 vs. HG control or scr.

 
Role of PKC-beta1 and -{zeta} in high-glucose-stimulated VEGF expression. One of the fundamental consequences of exposure to high glucose is increased diacylglycerol synthesis and activation of conventional and novel PKC isozymes (see above). Indeed, certain PKC isozymes are necessary for NADPH oxidase activity and collagen IV expression in mesangial cells (19, 27, 53). Accordingly, we explored the relationship between PKC and high-glucose-induced VEGF expression, focusing on PKC-beta1 and -{zeta}, which are involved in the regulation of NADPH oxidase. As depicted in Fig. 4, A and B, Gö-6976, an inhibitor of the conventional PKC isoforms, PKC-{alpha} and -beta, prevented high-glucose-stimulated VEGF protein expression detected by Western blotting or immunofluorescence.


Figure 4
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  		Fig. 4. Protein kinase C inhibitor Gö-6976 interferes with high-glucose-induced VEGF expression. Mesangial cells were pretreated with 300 nM Gö-6976 or vehicle (DMSO) for 24 h and then cultured in 5.6 (NG) or 25 (HG) mM glucose for the indicated times. A: VEGF immunoblot (top) and VEGF expression quantified relative to NG, n = 4 (bottom). B: VEGF immunofluorescence (top) and VEGF expression (pixel intensity/cell; n = 165–248 cells) quantified in three experiments (bottom). *P < 0.001 vs. NG. **P < 0.01 vs. HG. ***P < 0.001 vs. HG.

 
To better define the role of PKC-beta1, we sought evidence that PKC-beta1 is activated by high glucose under the present experimental conditions. In mesangial cells incubated in 25 mM glucose for 3 h, the amount of PKC-beta1 was increased in whole cell extracts and slightly increased in crude nuclear fractions, which include some associated endoplasmic reticulum membrane fractions, relative to cells maintained in 5.6 mM glucose (Fig. 5A). In preliminary experiments, PKC-beta1 in vitro kinase activity was assayed in immunoprecipitates and found to be modestly increased in total cell lysates and cellular membrane fractions in high-glucose compared with normal glucose conditions (not shown). As displayed in Fig. 5B, the in vitro kinase activity of PKC-beta1 immunoprecipitated from crude nuclear fractions was 3.8-fold higher in cells exposed to 25 mM glucose compared with 5.6 mM glucose. The stimulation of mesangial cells in normal glucose with phorbol myristate acetate was used as a positive control.


Figure 5
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  		Fig. 5. Role of protein kinase C (PKC)-beta1 in high-glucose-induced VEGF expression. A: PKC-beta1 immunoblot of whole cell extracts (top) or crude nuclear fractions (bottom) in mesangial cells exposed to 5.6 mM (NG) or 25 mM (HG) glucose for the indicated times. B: PKC-beta1 in vitro kinase activity in nuclear fractions relative to NG in mesangial cells exposed to NG or HG for 48 h in the presence or absence of the specific PKC-beta inhibitor LY-333531 (200 nM), n = 4. C: VEGF mRNA levels detected by real-time PCR in mesangial cells pretreated with 200 nM LY-333531, n = 6. D and E: VEGF protein levels detected by Western blot or immunofluorescence in mesangial cells pretreated with 200 nM LY-333531 (top) and VEGF expression quantified relative to NG in each experiment, n = 3 [bottom (D) and right (E)]. *P < 0.05 vs. NG. **P < 0.01 vs. NG. ***P < 0.01 vs. HG1h or HG3h. #P < 0.001 vs. NG or NG + LY-333531. ##P < 0.001 vs. HG3h or HG48h.

 
These results prompted us to ascertain a functional role for PKC-beta in high-glucose-stimulated VEGF expression. To this end, mesangial cells were incubated with the PKC-beta-specific inhibitor LY-333531 (25). The results obtained by real-time RT-PCR (Fig. 5C), Western blotting (Fig. 5D), or immunofluorescence (Fig. 5E) demonstrate that inhibition of PKC-beta1 suppressed the increase in VEGF expression induced by high glucose, with little effect on basal levels.

Given that mesangial cell PKC-{zeta} is also activated by high glucose, as we have previously reported (27), and is a mediator of high-glucose-stimulated ROS formation (see above), we evaluated the participation of PKC-{zeta} in VEGF synthesis triggered by high glucose. Mesangial cells were treated with a cell-permeant, myristoylated peptide PKC-{zeta} pseudosubstrate inhibitor (ZI), which binds to the PKC-{zeta} substrate binding domain and blocks access to the PKC-{zeta} catalytic domain (27). Pretreatment of mesangial cells with ZI eliminated the increase in VEGF in cells incubated in 25 mM glucose relative to 5.6 mM glucose as detected by real-time PCR (Fig. 6A), Western blotting (Fig. 6B), or confocal immunofluorescence imaging (Fig. 6C). VEGF secretion in the mesangial cell media was inhibited by ZI, Tempol, and Gö-6976 (Fig. 6D). Congruent with these results, transient overexpression of constitutively active PKC-{zeta} elevated VEGF expression (Fig. 6E). ZI did not act by increasing Akt phosphorylation, as recently reported in endothelial cells (26 and Fig. 6F).


Figure 6
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  		Fig. 6. PKC-{zeta} pseudosubstrate peptide inhibitor blocks increased VEGF expression in response to high glucose. A: VEGF mRNA levels in mesangial cells pretreated with 10 µM PKC-{zeta} pseudosubstrate peptide inhibitor (ZI) or vehicle for 24 h and then incubated in 5.6 mM (NG) or 25 mM (HG) glucose for an additional 3 h, n = 4. B: VEGF immunoblot (top) in mesangial cells pretreated with 10 µM ZI or vehicle for 24 h and then incubated in 5.6 mM (NG) or 25 mM (HG) glucose for an additional 6 h. Bottom: VEGF expression quantified relative to NG, n = 3. C: VEGF immunofluorescence (top) in mesangial cells pretreated with ZI or vehicle and exposed to NG or HG for the indicated times. Bottom: VEGF expression was quantified in three experiments. D: VEGF secretion by mesangial cells in conditioned media relative to NG was measured by ELISA. Mesangial cells were pretreated with 10 mM ZI, 100 nM Tempol, 300 nM Gö-6976, or vehicle (C), and then incubated in NG or HG for an additional 48 h, n = 4. E: VEGF, PKC-{zeta}, and beta-actin (loading control) immunoblots (top) in mesangial cells transiently transfected with a constitutively active PKC-{zeta} expression vector or empty vector (C). VEGF and PKC-{zeta} expression were quantified in four experiments (bottom). F: phospho- and total protein kinase B (Akt) immunoblot in mesangial cells treated with ZI or vehicle. *P < 0.05 vs. NG. **P < 0.01 vs. empty vector. ***P < 0.001 vs. NG. #P < 0.01 vs. HG6h. ##P < 0.001 vs. HG, ###P < 0.01 vs. HG, no Z1.

 
HIF-1{alpha} expression in high glucose. HIF-1{alpha} is a basic helix loop helix transcription that is a critical regulator of hypoxia-induced VEGF gene expression (17). In addition to hypoxia, various growth factors increase HIF-1{alpha} expression, leading us to postulate that HIF-1{alpha} might be upregulated by high glucose (17). Therefore, mesangial cells were incubated with 5.6 or 25 mM glucose, and HIF-1{alpha} expression was monitored by Western blotting. Incubation of the cells for 24 h with cobalt chloride (100 µM) served as a positive control. As shown in Fig. 7, there were pronounced increases in HIF-1{alpha} after 24 to 48 h of high glucose but not at earlier time points, such as 3 h, when we have observed significant upregulation of VEGF mRNA. This suggests a possible contribution of HIF-1{alpha} to high-glucose-enhanced VEGF expression at later time points.


Figure 7
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  		Fig. 7. Hypoxia inducible factor-1{alpha} (HIF-1{alpha}) expression in HG. HIF-1{alpha} immunoblot (top) in mesangial cells incubated in 5.6 mM glucose (NG) or 25 mM glucose (HG) for the indicated times. HIF-1{alpha} expression relative to NG was quantified in five experiments (bottom). Treatment with 100 µM cobalt chloride for 24 h served as a positive control. *P < 0.05 vs. NG. **P < 0.001 vs. NG.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It is well established that high ambient glucose concentrations lead to enhanced VEGF expression, but only limited information is available on the mechanisms involved. Using mesangial cells as a model, we observed that high glucose enhanced VEGF expression both at the protein level, detected by Western blotting, immunofluorescence, and ELISA, and at the mRNA level, detected by PCR. This latter effect was not the result of increased RNA stability.

The present results demonstrate for the first time that ROS generated by NADPH oxidase are necessary for high-glucose-induced VEGF expression. Previous reports support the role of NADPH oxidase in influencing VEGF expression in other settings. For example, knocking down NOX3 expression prevented stimulation of VEGF expression by insulin in Hep G2 cells (9). Hypoxia-induced VEGF expression in retinal endothelial cells and VEGF expression in an in vivo model of ischemic retinopathy were mitigated by specifically inhibiting NADPH oxidase (1). Although Brownlee (7) has clearly shown that ROS generated by glucose metabolism in the mitochondria are necessary for a variety of high-glucose effects, interfering with NADPH oxidase activity also eliminates high-glucose-induced ROS in mesangial cells or podocytes (16, 20, 45, 53). These seemingly disparate results may be explained by mutual interactions between mitochondria-generated ROS and NADPH oxidase, causing both to be necessary for high-glucose-induced ROS formation. Thus, in glomerular podocytes exposed to high glucose, inhibitors of both NADPH and mitochondrial metabolism abrogate ROS formation (45). Serum withdrawal from 293T cells results in the generation of ROS in mitochondria that subsequently activate NADPH oxidase (28).

The finding of increased HIF-1{alpha} levels could partly account for high-glucose-induced VEGF expression at 48 h. These high-glucose-enhanced HIF-1{alpha} levels may be attributable to the secondary release of growth factors or to increased ROS levels, given the recent finding that ROS are generated by NADPH oxidase in response to insulin-augmented HIF-1{alpha} levels (5). Enhanced HIF-1{alpha} protein levels have previously been noted in high-glucose-treated mesangial cells (54) and in GLUT1-overexpressing mesangial cells, where they were partly responsible for elevated VEGF expression (37). At earlier time points, high-glucose-stimulated increases in VEGF mRNA may be the result of activation of other transcription factors. Beside HIF-1{alpha}, the transcription factor surfactant protein-1 (Sp1) is a key regulator of the VEGF promoter and a target of the signaling pathways implicated in high-glucose-regulated VEGF expression in the present study. For example, oxidative stress and PGE2 elevate VEGF levels and promoter activity by increasing Sp1 DNA binding rather than by acting on HIF-1{alpha} (6, 40). Interestingly, PKC-{zeta} stimulates VEGF transcription through Sp1, possibly by phosphorylating Sp1 (31, 36). PKC-beta is indirectly linked to Sp1 in that PKC-beta can activate extracellular-regulated kinase (ERK), as demonstrated in studies of neointimal expansion in a mouse model of atherosclerosis (3). In turn, ERK can phosphorylate Sp1 and increase its DNA binding (30, 47). Other ROS-sensitive transcription factors, such as activator protein-1, that bind to the VEGF promoter (35, 42) could also mediate the effects of high-glucose-induced ROS on VEGF transcription, as suggested by studies in mesangial cells overexpressing GLUT1 (37).

Our experiments further revealed that two PKC isoforms, PKC-beta1 and PKC-{zeta}, are required for the upregulation of VEGF in high glucose, whereas previous studies did not address the role of specific PKC isoforms. These conclusions were based on the use a specific pharmacological inhibitor of PKC-beta1 and a pseudosubstrate peptide inhibitor for PKC-{zeta}. In agreement, it was recently reported that VEGF expression is not increased in PKC-beta null mice with diabetes (32). These findings are further reinforced by reports that VEGF expression is upregulated by PKC-beta1 or -{zeta} in other contexts. For instance, AGEs stimulate VEGF through PKC-beta in endothelial cells (29), and stretch-induced VEGF secretion is dependent on PKC-{zeta} in retinal capillary pericytes (46). Notably, Sp1-PKC-{zeta} interactions were shown to activate VEGF transcription in renal carcinoma cells (36). The exact relationship between PKC-beta1 and -{zeta} is unclear at present. These PKC isoforms may operate in parallel, with PKC-beta1 stimulating NADPH oxidase (23) and PKC-{zeta} increasing both NADPH oxidase and Sp1 activities. Theoretically, they may also act sequentially, with PKC-beta1 increasing the generation of phosphatidic acid, a PKC-{zeta} activator, through phospholipase D. Krotova et al. (26) reported that ZI activates eNOS through Akt phosphorylation in endothelial cells. However, in our hands, ZI did not cause Akt phosphorylation in mesangial cells, suggesting that Akt phosphorylation in high glucose may be cell specific.

In summary, as depicted in Fig. 8, we have provided evidence that inputs from at least three high-glucose-induced signaling pathways, PKC-beta1 and -{zeta}, and NADPH oxidase, are required for high glucose to enhance VEGF mRNA and protein levels, and secretion. These results highlight PKC-beta1 and NADPH oxidase as being attractive therapeutic targets for reducing VEGF-dependent proteinuria and other manifestations of diabetic nephropathy.


Figure 8
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  		Fig. 8. Summary of pathways required for high-glucose-induced VEGF expression. NADPH oxidase, PKC-beta1, and PKC-{zeta} act to increase VEGF expression. ROS, reactive oxygen species.

 


   ACKNOWLEDGMENTS
 
This research was supported by a grant from the Canadian Diabetes Association. L. Xia is a postdoctoral fellow supported by the Canadian Diabetes Association.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Whiteside, Medical Sciences Bldg., Rm. 7302, 1 King's College Circle, Univ. of Toronto, Toronto, ON, 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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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