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Diabetes 50:2376-2383, 2001
© 2001 by the American Diabetes Association, Inc.

High Glucose–Enhanced Mesangial Cell Extracellular Signal–Regulated Protein Kinase Activation and {alpha}1(IV) Collagen Expression in Response to Endothelin-1

Role of Specific Protein Kinase C Isozymes

Hong Hua1,2, Howard J. Goldberg2, I.G. Fantus1,2, and Catharine I. Whiteside1,2

1 Institute of Medical Science, the University Health Network
2 Department of Medicine, University of Toronto, Toronto, Canada


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
High glucose (HG) stimulates glomerular mesangial cell (MC) expression of extracellular matrix, a process involving protein kinase C (PKC) isozymes and enhanced signaling by autocrine peptides such as endothelin-1 (ET-1). The purpose of this study was to identify the specific PKC isozymes mediating the effects of HG on MC extracellular signal–regulated protein kinase (ERK1/2) signaling and {alpha}1(IV) collagen expression in response to ET-1. HG (30 mmol/l for 72 h) enhanced ET-1–stimulated {alpha}1(IV) collagen mRNA expression from 1.2 ± 0.1–fold to 1.9 ± 0.2–fold (P < 0.05 vs. normal glucose [NG] + ET-1), and the effect was significantly reduced by Calphostin C or the MEK (mitogen-activated protein kinase kinase) inhibitor PD98059. In transiently transfected MCs, dominant-negative (DN)–PKC-{delta}, -{epsilon}, or -{zeta} inhibited ET-1 activation of ERK1/2. Likewise, downstream of ERK1/2, ET-1 stimulated Elk-1–driven GAL4 luciferase activity to 11 ± 1–fold (P < 0.002 vs. NG + ET-1) in HG, and DN-PKC–{delta}, –{epsilon}, or –{zeta} attenuated this response to NG levels. HG enhanced ET-1–stimulated intracellular {alpha}1(IV) collagen protein expression, assessed by confocal immunofluorescence imaging, showed that individual DN–PKC-{delta}, -{epsilon}, -{zeta}, as well as DN–PKC-{alpha} and -ß, attenuated the response. Thus, HG-enhanced ET-1 stimulation of {alpha}1(IV) collagen expression requires PKC-{delta}, -{epsilon}, and -{zeta} to act through an ERK1/2-dependent pathway and via PKC-{alpha} and -ß, which are independent of ERK1/2.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Diabetic nephropathy is characterized by glomerular mesangial expansion through increased synthesis and decreased degradation of extracellular matrix (ECM) proteins, particularly {alpha}1(IV) collagen, fibronectin, and laminin ß1 (1,2,3,4,5,6,7). Accumulation of ECM proteins progressively obliterates the glomerular capillaries, ultimately resulting in renal failure. Sustained hyperglycemia is the major cause of diabetic nephropathy (8,9). The response of mesangial cells (MCs) to high glucose (HG) includes activation of protein kinase C (PKC) and aberrant synthesis of growth factors, including vasomodulatory agents, such as endothelin-1 (ET-1), which contribute to ECM expression (10,11,12,13,14,15,16).

PKC isozymes are a family of serine/threonine protein kinases encoded by at least 12 different genes (17,18,19,20,21,22). The conventional PKCs ({alpha}, ß, and {gamma}) are stimulated by diacylglycerol (DAG) and phosphatidylserine and require Ca2+. The novel PKCs ({delta}, {epsilon}, {eta}, and {theta}) are activated by DAG and phosphatidylserine but are insensitive to Ca2+. The atypical PKCs ({zeta}, {iota}, and {lambda}) and PKC-µ/protein kinase D are sensitive only to phosphatidylserine. MCs express PKC-{alpha}, -ß, -{delta}, -{epsilon}, and -{zeta} in culture (23,24) and PKC-{alpha}, -ßII, -{delta}, and -{epsilon} in vivo, as identified through immunogold labeling of rat glomerular cells in situ (25). HG alters the subcellular distribution of PKC isozymes in cultured MCs (26,27) and increases PKC activity in glomeruli of streptozotocin-induced diabetic rats (25,28,29) through de novo synthesis of DAG (5,30). To date, the exact role of specific PKC isozymes mediating HG-enhanced ECM expression by MCs is unknown. We and others (31,32) have reported that in HG, MC extracellular signal–regulated protein kinase (ERK1/2) signaling responses to autocoid growth factors, e.g., ET-1, are enhanced and PKC-dependent. Recently, the importance of mitogen-activated protein kinases (MAPKs) in diabetic complications has been reviewed (33,34,35).

Endothelins are involved in the pathogenesis of glomerular disease (36,37,38,39). A direct role for ET-1 in the progression of diabetic nephropathy is suggested by enhanced endothelin mRNA expression in cultured cells (40) and diabetic animals (41,42) and by stimulation of MC mitogenesis, a known response to ET-1 (36,37). ET-1 binding to its G-protein–coupled receptors stimulates phospholipase C hydrolysis of phosphatidylinosital bisphosphate to generate two second messengers, inositol trisphosphate and DAG, which stimulate release of Ca2+ and PKC activation, respectively (38). ET-1 activates PKC-{alpha}, -{delta}, and -{epsilon} in primary-cultured rat MCs (43). ET-1 signal transduction also involves MAPKs, including ERK1/2, with the subsequent regulation of immediate-early genes (44,45).

The purpose of this study was to identify the specific PKC isozymes that mediate the effects of HG on MC ERK1/2 signaling and {alpha}1(IV) collagen expression in response to ET-1. The role of the individual PKC isozymes that cause activation of ERK1/2 MAPK and the downstream stimulation of the transcription factor Elk-1 were identified by cotransfection of individual dominant-negative (DN)-PKC cDNA, with an Elk-1 activation domain fused to the yeast GAL4 DNA binding domain and a GAL4-driven luciferase reporter construct. This system reports on the activation of Elk-1 by upstream ERK1/2. We also used dual-channel confocal imaging to simultaneously observe in individual cells the effects of hemagglutinin (HA)-tagged DN-PKC isozymes on ET-1 activation of ERK1/2, identified by immunofluoresence labeling of phospho-ERK1/2. We found that PKC-{delta}, -{epsilon}, and -{zeta} target ERK1/2, leading to {alpha}1(IV) collagen expression, and that PKC-{alpha} and -ß also regulate ET-1–induced {alpha}1(IV) collagen expression in HG but likely function independently of the ERK1/2 and Elk-1 pathways.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
MC culture.
Primary MCs were isolated from Sprague-Dawley rat kidney glomeruli as previously described (46). Primary MC passages 11–20 were used for all studies.

Transient transfection.
Primary rat MCs were grown in 24-well plates to 80% confluence in Dulbecco’s modified Eagle’s medium (DMEM) containing 20% fetal bovine serum (FBS). The media was changed to 0.5% FBS DMEM, and the cells were transfected in triplicates per experiment using FuGENE6 (Roche Diagnostics, Laval, Canada) with the following plasmids: 10% pFA2-Elk1 composed of the yeast GAL4 DNA binding domain (1–147) and fused to the transactivation domain of Elk-1 (307–428) (Stratagene, La Jolla, CA), 45% pFR-luc (Stratagene), which contains multiple binding sites of GAL4 fused to luciferase, and 45% DN PKC isozymes with a lysine to arginine point mutation at the ATP binding site (gifts from Dr. J. Soh and Dr. I.B. Weinstein, Columbia University, New York, NY) (47) or empty vector pcDNA3 (Invitrogen, Carlsbad, CA). Cells were transfected in normal glucose (NG) 5.6 mmol/l or HG 30 mmol/l for 72 h and stimulated with ET-1 (100 nmol/l) (Sigma, St. Louis, MO) during the last 16 h (predetermined optimal time point). Cell extracts were collected from each well, and luciferase activity was measured by injecting luciferin (Sigma) into the lysate. Light emission was measured for 10 s with a plate luminometer. Elk-GAL4 luciferase activity (the average of three wells per condition in each experiment) was expressed as fold stimulation over basal (cells transfected with pFA2-Elk1, pFR-luc, and pcDNA3 in the absence of ET-1 stimulation). Where indicated, MCs were pretreated with Calphostin C (1 µmol/l for 1 h) (Calbiochem, San Diego, CA) or PD98059 (100 µmol/l for 1 h) (Calbiochem) and stimulated with ET-1 (100 nmol/l for 16 h). To determine the specificity of the DN-PKC constructs, MCs were transfected with AP-1 luciferase (Stratagene) in the presence of constitutively active (CA)-PKC and DN-PKC or empty vector pcDNA3 for 72 h. CA-PKC activation of AP-1 luciferase was expressed as fold stimulation over AP-1 luciferase in the presence of empty vector pcDNA3. Expression vectors for DN–PKC-ßI and -ßII, and CA–PKC-{epsilon} and -{zeta}, which contain mutations in their pseudosubstrate regions, were provided by Dr. R.V. Farese (University of South Florida, Tampa, FL).

Western blot analysis.
MCs were grown in DMEM containing 20% FBS in 6-well plates and were growth-arrested in NG or HG for 72 h. Cellular protein was extracted with 2x sample buffer (0.13 mol/l Tris-base, pH 6.8, 20% glycerol, and 4% SDS). Aliquots were taken for protein assay using Bradford Protein Assay (BioRad, Hercules, CA). The remaining cell extracts were denatured in 4x sample buffer (0.13 mol/l Tris, 40% glycerol, 8% SDS, 4% ß-mercaptoethanol, and 0.02% bromophenol blue). Equal amounts of protein were separated by SDS-PAGE at 120 V for 1–2 h. The protein was transferred to Immobilon polyvinylidine fluoride membranes (Millipore, Bedford, MA) overnight at 4°C in transfer buffer (25 mmol/l Tris-base, 192 mmol/l glycine, pH 8.3, and 20% methanol). The membranes were blocked in 5% skim milk powder in Tris buffer (pH 8.0) containing 0.05% Tween-20 and then probed with the indicated antibody. The immunoblots were visualized with enhanced chemiluminescence (KPL, Gaithersburg, MD). The following antibodies and dilutions were used: anti–phospho-ERK1/2 (New England Biolabs, Beverly, MA) at 1:3,000, anti–total-ERK1/2 (New England Biolabs) at 1:2,000, and anti-HA (Babco) at 1:200. HRP-labeled goat anti–rabbit IgG (BioRad) and HRP-labeled goat anti–mouse IgG (Jackson ImmnoResearch) were used at 1:5,000.

Confocal immunofluorescence.
MCs were cultured on glass coverslips and transfected as described above. Cells were fixed in 3.7% formaldehyde and permeabilized with Triton X-100. Nonspecific binding was blocked with 1% goat serum containing 0.1% bovine serum albumin and then incubated with anti–PKC-{zeta} at 1:100 (Sigma), anti–phospho-ERK1/2 at 1:50, anti–collagen IV at 1:100 (Biodesign International, Saco, Maine), or anti-HA at 1:1,000. Fluorescein isothiocyanate–conjugated goat anti–mouse IgG at 1:100 (Jackson ImmunoResearch) or Rhodamine-conjugated goat anti–rabbit IgG at 1:100 (Jackson ImmunoResearch) were used as secondary antibodies. Immunofluorescence was imaged using a Zeiss confocal laser-scanning microscope (LSM 410; Zeiss), with excitation and emission wavelength of 488 and 520 nm, respectively.

Northern blot analysis.
MCs were grown in 10-cm plates and growth-arrested in 0.5% FBS DMEM containing NG or HG for 72 h. Total RNA was extracted using the Qiagen (Santa Clarita, CA) Rneasy kit according to the manufacturer’s instructions and was then quantitated by spectrophotometry at 260 nm. Equal amounts of RNA (20 µg) were run on a 1% agarose gel (8 V/cm for 3 h) containing 20 mmol/l guanidine isothiocyanate. RNA was transferred onto Gene Screen Plus membranes (New England Nuclear, Boston, MA), prehybridized in 200 mmol/l Na2PO4, 300 mmol/l NaH2PO4, pH 7, 7% SDS, 1 mmol/l EDTA, 1% BSA, 1 mmol/l Na4P2O7, and 125 µg/ml salmon sperm DNA and hybridized overnight at 55°C in the same buffer and 1 x 106 cpm/ml of radiolabeled murine {alpha}1(IV) collagen cDNA probe (a gift from Dr. D. Templeton, University of Toronto) that was labeled by random priming with an Amersham Pharmacia Biotech (Piscataway, NJ) T7QuickPrime labeling kit. The membrane was washed in 2 x sodium chloride–sodium citrate (SSC) (1 x SSC is 150 mmol/l sodium chloride and 15 mmol/l sodium citrate, pH 7.0) containing 0.1% SDS and exposed to Kodak Blue XB-1 film at -70°C for 24–72 h. The blots were reprobed with an 18S ribosomal RNA probe to control for loading.

Statistical analysis.
All values are expressed as means ± SE. Significance of results was determined with Instat 2.1 (GraphPad, Sacramento, CA). Comparisons were performed using an unpaired Student’s t test, and P < 0.05 was considered to indicate a significant difference. Where applicable, the means of three or more groups were compared by one-way analysis of variance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HG-enhanced ET-1–induced expression of {alpha}1(IV) collagen mRNA requires PKC and ERK1/2.
Given the importance of ECM accumulation to the pathogenesis of diabetic nephropathy, we selected {alpha}1(IV) collagen gene expression as an end point for characterizing the effects of HG and ET-1. ET-1 (100 nmol/l for 16 h) stimulated primary rat MC {alpha}1(IV) collagen mRNA expression to 1.2 ± 0.1–fold (n = 4, above control) in NG (5.6 mmol/l for 72 h), and the effect was enhanced in HG (30 mmol/l for 72 h) to 1.9 ± 0.2–fold (n = 4, P < 0.05 vs. NG + ET-1) (Fig. 1A). HG alone increased {alpha}1(IV) collagen mRNA expression to 1.3 ± 0.1–fold. Pretreatment with Calphostin C (1 µmol/l for 1 h) prevented ET-1 stimulation of {alpha}1(IV) collagen mRNA in both NG to 0.7 ± 0.03–fold (n = 4, P < 0.01 vs. NG + ET-1) and HG to 0.9 ± 0.1–fold (n = 4, P < 0.01 vs. HG + ET-1). By contrast, when cells were pretreated with PD98059 (100 µmol/l for 1 h), {alpha}1(IV) collagen mRNA expression was significantly decreased in HG (1.2 ± 0.1–fold, n = 4, P < 0.05 vs. HG + ET-1 in the absence of PD98059) but not in NG.



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  		FIG. 1. Effects of Calphostin C and PD98059 on ET-1–increased expression of {alpha}1(IV) collagen mRNA in NG and HG. MCs were growth-arrested in NG or HG for 72 h, pretreated with Calphostin C (CC, 1 µmol/l for 1 h) or PD98059 (PD, 100 µmol/l for 1 h), and then stimulated with ET-1 (100 nmol/l for 16 h). {alpha}1(IV) collagen mRNA was detected by Northern blotting, as described in RESEARCH DESIGN AND METHODS. A: Northern blot of {alpha}1(IV) collagen mRNA. B: The densitometric analysis of four experiments. The results are expressed as fold stimulation of {alpha}1(IV) collagen mRNA/18S ratio over control. *P < 0.01 vs. NG + ET-1; **P < 0.05 vs. NG + ET-1; #P < 0.01 vs. HG + ET-1; ##P < 0.05 vs. HG + ET-1.

 
Transient expression and specificity of HA-tagged DN-PKCs.
To address the role of specific PKC isozyme signaling by ET-1 upstream of {alpha}1(IV) collagen mRNA expression, primary rat MCs were transiently transfected with DN-PKC constructs. All transfected DN-PKC isozymes were detected by immunoblotting total cell lysates using a primary anti-HA antibody (Fig. 2A). Expression of HA-tagged DN-PKCs was also detected by immunofluorescence (Fig. 2B). The percent of transfected cells was calculated in three separate experiments for each DN-PKC to determine the transfection efficiency. Figure 2C illustrates DN–PKC-{zeta} transfected cells in a phase contrast field. The transfection efficiency for each DN-PKC expressed as a percentage of total number of cells counted was as follows: DN–PKC-{alpha} 19 ± 3%, n = 156 cells; DN–PKC-{delta} 30 ± 6%, n = 126 cells; DN–PKC-{epsilon} 24 ± 3%, n = 374 cells; DN–PKC-{zeta} 17 ± 3%, n = 122 cells. Figures 2D and E illustrate that in cells transfected with DN–PKC-{zeta}, the amount of PKC-{zeta} was increased over the endogenous PKC-{zeta}. Confocal imaging of other DN-PKCs also showed increased expression of PKCs in transfected cells similar to DN–PKC-{zeta} (data not shown).



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  		FIG. 2. Expression of HA-tagged DN-PKCs. MCs were tranfected as described in RESEARCH DESIGN AND METHODS. A: Western blot of DN-PKC expression using anti-HA antibody. B: Confocal immunofluorescence of HA-tagged DN–PKC-{zeta}. C: Confocal immunofluorescence of HA-tagged DN–PKC-{zeta} superimposed onto the corresponding phase contrast image. D and E: Confocal immunofluorescence of HA-tagged DN–PKC-{zeta} and a polyclonal antibody to PKC-{zeta}, respectively.

 
The specificity of the DN-PKCs was determined by cotransfecting MCs with CA-PKCs in the presence of a reporter gene containing multiple AP-1 sites linked to luciferase (AP-1 luciferase). CA–PKC-{epsilon} and -{zeta} increased AP-1 luciferase activity by 3- and 2.5-fold, respectively (Fig. 3). DN–PKC-{epsilon} but not DN–PKC-{alpha} inhibited CA–PKC-{epsilon} activation of AP-1 luciferase (Fig. 3). Likewise, DN–PKC-{zeta} but not DN–PKC-{alpha} completely inhibited CA–PKC-{zeta} activation of AP-1 luciferase.



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  		FIG. 3. Selective inhibition of CA-PKC stimulation of AP-1 by DN-PKCs. MCs were transfected with CA-PKC alone or cotransfected with DN-PKC, as described in RESEARCH DESIGN AND METHODS. Luciferase activity was measured in total cell lysate. *P < 0.05 vs. CA-{epsilon}; **P < 0.05 vs. CA-{zeta}.

 
HG-enhanced ET-1 stimulation of phospho-ERK1/2 requires PKC-{delta}, -{epsilon}, and -{zeta}.
Figure 4A is a representative immunoblot illustrating ET-1 stimulation of MC ERK1/2 phosphorylation. ET-1 (100 nmol/l for 10 min) caused a rapid increase of phospho-ERK1/2 in NG that was enhanced in HG (30 mmol/l for 72 h). Inhibition of PKCs with chronic phorbol myristic acid (PMA) (100 nmol/l for 24 h) or Calphostin C (1 µmol/l for 4 h) partially inhibited ET-1 activation of ERK1/2 in both NG and HG (Fig. 4A). Chronic PMA downregulated phorbol ester-sensitive PKC-{alpha}, -{delta}, -{epsilon}, and -ßI but not atypical PKC-{zeta} (Fig. 4B).



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  		FIG. 4. ET-1–induced increase of phospho-ERK1/2 is PKC-dependent. A: MCs were growth-arrested in 5.6 mmol/l (NG) or 30 mmol/l (HG) glucose for 72 h with or without chronic exposure to PMA (cP, 100 nmol/l for 24 h) or Calphostin C (CC, 1 µmol/l for 4 h) and then stimulated with ET-1 (100 nmol/l for 10 min). Representative blot using phospho-specific ERK1/2 and total ERK1/2 antibodies. B: MCs were growth-arrested in NG, pretreated with PMA (cP, 100 nmol/l for 24 h), and probed for total PKCs.

 
At the single-cell level, in the absence of ET-1 stimulation, a small amount of phospho-ERK1/2 was detected in the nucleus (data not shown). ET-1 increased the intensity of phospho-ERK1/2 in the nucleus (Figs. 5A–F). To determine the effect of specific PKC isozymes on ET-1 activation of ERK1/2, MCs were growth-arrested on glass coverslips, transfected with DN-PKCs, and stimulated with ET-1 (100 nmol/l for 10 min). In cells expressing HA-tagged DN–PKC-{delta}, -{epsilon}, or -{zeta} (Figs. 5A and C), ET-1–increased staining of phospho-ERK1/2 (Figs. 5B and D) in HG was partially or completely inhibited, whereas cells expressing HA-tagged DN–PKC-ßI (Fig. 5E) did not alter ET-1–increased phosphorylation of ERK1/2 (Fig. 5F). In NG, DN–PKC-{delta}, -{epsilon}, and -{zeta} also diminished the ET-1 increase of phospho-ERK1/2 immunoreactivity (data not shown).



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  		FIG. 5. Role of PKC isozymes in ET-1–induced increase of phospho-ERK1/2. MCs were grown on glass coverslips and transfected with DN-PKCs for 72 h, acutely stimulated with ET-1 (100 nmol/l for 10 min), and imaged for phospho-ERK1/2 and HA-tagged DN-PKC using dual-channel confocal immunofluorescence.

 
ET-1 stimulation of Elk-GAL4 luciferase activity in HG requires PKC-{delta}, -{epsilon}, and -{zeta}.
Next, we used a functional assay to study activation of the transcription factor, Elk-1, by HG and ET-1. Phosphorylation of ERK1/2 drives Elk-1–dependent luciferase expression (Elk-GAL4 luciferase activity). HG (30 mmol/l for 72 h) alone increased Elk-GAL4 luciferase activity to 1.6-fold above the value in NG alone. ET-1 stimulated Elk-GAL4 luciferase activity in NG (5.6 mmol/l) to 6.0 ± 1.0–fold (n = 10) and in HG (30 mmol/l for 72 h) to 11 ± 1.0–fold (n = 10, P < 0.002 vs. NG + ET-1) (Fig. 6). To control for osmotic effects, MCs were exposed to 25 mmol/l mannitol instead of HG. In the presence of mannitol, ET-1 activated Elk-GAL4 only to fivefold (n = 2). In NG, most DN-PKCs did not significantly alter ET-1 stimulation of Elk-GAL4 luciferase (Fig. 6), with the exception of DN–PKC-ßII, which independently increased Elk-GAL4 luciferase to 10 ± 2.0–fold (n = 4, P < 0.02 vs. NG + ET-1). In HG, DN–PKC-{delta}, -{epsilon}, and -{zeta} reversed the enhancement to 7.0 ± 1.0–fold, 7.0 ± 1.0–fold, and 6.0 ± 2.0–fold (n = 7 for each DN-PKC, P < 0.03 vs. HG + ET-1 in the absence of DN-PKCs), respectively. Neither DN–PKC-ßI (9.0 ± 2.0–fold, n = 4) nor -ßII (11 ± 2.0–fold, n = 4) altered HG-enhanced ET-1 activation of Elk-1. Thus, in HG, ET-1 stimulation of Elk-1 requires PKC -{delta}, -{epsilon}, and -{zeta}.



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  		FIG. 6. The effects of DN-PKCs on ET-1 stimulation of Elk-GAL4 luciferase activity. MCs were transfected with pFA2–Elk-1 (encoding the GAL4 DNA binding domain fused to the Elk-1 transcriptional activation domain), pFr-luc (containing GAL4 binding sites fused to a luciferase reporter gene), and DN-PKCs or an empty pcDNA3 vector in NG or HG as described in RESEARCH DESIGN AND METHODS. {square}, NG + ET-1; {blacksquare}, HG + ET-1. *P < 0.002 vs. NG + ET-1; **P < 0.03 vs. HG + ET-1 in the absence of DN-PKCs.

 
PKCs and ERK1/2 are required for HG-enhanced ET-1 increase of {alpha}1(IV) collagen protein expression.
In NG, ET-1 (100 nmol/l for 24 h) increased immunoreactivity of {alpha}1(IV) collagen protein compared with control, and the effect was enhanced in HG (Figs. 7A–C). Pretreatment with Calphostin C, chronic PMA, or PD98059 in HG decreased ET-1 stimulation of {alpha}1(IV) collagen protein expression (Figs. 7D–F). To determine the role of specific PKC isozymes, MCs were growth-arrested in HG, transiently transfected with DN-PKCs, and stimulated with ET-1. Figure 8 is a representative montage of two separate experiments that show MCs expressing DN–PKC-{alpha}, -{delta}, -{epsilon}, -{zeta}, -ßI, or -ßII attenuated HG-enhanced ET-1–increased staining of {alpha}1(IV) collagen. Thus, ET-1–increased expression of {alpha}1(IV) collagen mRNA correlated with increased protein levels, and several PKC isozymes contributed to ET-1–induced {alpha}1(IV) collagen expression in HG.



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  		FIG. 7. ET-1 increase of {alpha}1(IV) collagen protein is enhanced in HG. Confocal immunofluorescence imaging of anti–{alpha}1(IV) collagen expressed by MCs in NG or HG and stimulated with ET-1 (100 nmol/l for 24 h). A: Absence of ET-1 stimulation. B: ET-1 stimulation in NG. C: ET-1 stimulation in HG. MCs in HG were pretreated with Calphostin C (1 µmol/l for 4 h) (D), chronic PMA (100 nmol/l for 24 h) (E), or PD98059 (100 µmol/l for 1 h) (F) and then stimulated with ET-1. Bar denotes 25 µm.

 


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  		FIG. 8. Role of specific PKC isozymes in HG-enhanced ET-1 activation of {alpha}1(IV) collagen protein. Dual-channel confocal immunofluorescence images of MCs transiently transfected with DN-PKCs in HG and stimulated with ET-1 (100 nmol/l for 24 h). A– L: Double labeling of HA-tagged DN-PKCs and anti–{alpha}1(IV) collagen. Arrows indicate the cells transfected with DN-PKC and reduced {alpha}1(IV) collagen expression.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This work further elucidates the role of specific PKC isozymes in MCs exposed to HG that may contribute to the pathogenesis of diabetic nephropathy. We report that expression of MC {alpha}1(IV) collagen mRNA and protein stimulated by ET-1 was enhanced in HG and required PKC-{delta}, -{epsilon}, and -{zeta} to mediate their effect through an ERK1/2 pathway and for PKC-{alpha} and -ß to do so through an ERK1/2-independent pathway. To identify the action of specific PKC isozymes, we transfected primary rat MCs with DN-PKC mutant constructs. The isozyme specificity of the DN-PKC constructs was ascertained by the ability of DN-PKC mutants to selectively inhibit CA-PKC isozyme activation of a cotransfected AP-1 luciferase construct. Identical DN-PKC constructs are reported to be isozyme specific by Majumder et al. (48), who identified specificity by measuring individual PKC isozyme activity. Similarly, other groups have successfully used DN- and CA-PKC constructs in an isozyme-selective manner (47,49,50,51,52). Confocal imaging demonstrated that ET-1 stimulation of ERK1/2 required PKC-{delta}, -{epsilon}, and -{zeta}. Using Elk-GAL4 luciferase as a read-out, DN–PKC-{delta}, -{epsilon}, and -{zeta} also reversed HG-enhanced ET-1 stimulation of the transcription factor Elk-1. Calphostin C or chronic exposure to PMA partially inhibited the ERK1/2 response to ET-1, indicating that PKC-independent pathways are also involved. Nevertheless, in HG the enhanced activation of ERK1/2 in response to ET-1 was completely abolished by PKC inhibition. Finally, HG-enhanced ET-1 stimulation of {alpha}1(IV) collagen mRNA correlated with increased protein expression determined by confocal immunofluorescence imaging. All DN-PKC isozymes tested were able to attenuate ET-1 stimulation of {alpha}1(IV) collagen protein expression observed in individual cells. This finding parallels the observation that {alpha}1(IV) collagen mRNA expression in response to HG and ET-1 was abolished by PKC inhibition. We conclude that these PKC isozymes function sequentially or in alternative pathways to regulate the enhanced expression of MC {alpha}1(IV) collagen in HG and in response to ET-1.

Although several studies have examined the activation of PKC isozymes in HG (25,28,29,53,54), the downstream consequences of PKC activation by HG are not as well documented. Previously, we (31) have shown a partial PKC dependence of HG-enhanced ET-1 activation of MC ERK1/2, suggesting that this MAPK is one possible downstream target of HG activation of PKC. In the present study, inhibition of PKC by chronic PMA or Calphostin C partially attenuated ET-1 stimulation of ERK1/2. We showed that exposure to PMA for 24 h downregulated DAG-sensitive PKCs but not atypical PKC-{zeta}. Although Calphostin C competitively inhibits binding at the DAG and the phorbol ester–binding site, several studies have shown Calphostin C can also inhibit the function of PKC-{zeta} (55,56,57,58). Previous reports suggest that MC PKC-{zeta} may be activated in HG (26) and that activation of ERK1/2 in HG (without agonist) is inhibited by Calphostin C (32,59). These observations in combination with our findings support the postulate that in HG the activation of PKC-{zeta} may contribute to enhanced ERK1/2 activation and that Calphostin C may inhibit this pathway. Nonetheless, at the single-cell level, we have isolated the specific PKC isozymes required for ET-1 activation of ERK1/2. We showed that DN–PKC-{delta}, -{epsilon}, or -{zeta} partially or completely prevented ET-1 stimulation of phospho-ERK1/2. Downstream of ERK1/2, we found that DN–PKC-{delta}, -{epsilon}, and -{zeta} reversed HG-enhanced ET-1 activation of Elk-GAL4 luciferase.

HG causes increased expression of MC matrix mRNA and proteins, including {alpha}1(IV) collagen (5,7,60). Growth factors such as ET-1 also influence matrix expression. In cultured rat MCs, ET-1 stimulates the expression of fibronectin and {alpha}1(IV) collagen mRNA (61). Our report is the first to identify the role of specific PKC isozymes in HG-enhanced {alpha}1(IV) collagen expression in response to an autocoid stimulus. We found that several PKC isozymes were necessary for HG-enhanced ET-1 activation of {alpha}1(IV) collagen protein expression (Fig. 8). Because all DN-PKCs tested abrogated HG-enhanced ET-1 increase of {alpha}1(IV) collagen immunoreactivity, whereas only DN–PKC-{delta}, -{epsilon}, and -{zeta} attenuated ET-1 stimulation of ERK1/2, we further identified that {alpha}1(IV) collagen protein synthesis also requires PKC-{alpha} and -ß independent of the ERK1/2 pathway. Certainly, several signaling pathways lead to expression of matrix proteins in the diabetic milieu. Because transforming growth factor-ß1 (TGF-ß1) is strongly implicated in HG-induced matrix expression (60,62,63,64,65), MC-specific PKC isozyme action may also require the effect of this autocoid growth factor (66,67,68).

Inhibition of individual PKC isozymes may be sufficient to prevent excess {alpha}1(IV) collagen expression from contributing to progressive diabetic nephropathy. Because PKC isozymes are important for normal cellular function, future therapeutic intervention should target inhibition of those PKC isozymes involved in the pathogenesis of disease.


   ACKNOWLEDGMENTS
 
This study was supported by the Juvenile Diabetes Foundation, the Medical Research Council of Canada, and Ontario Graduate Scholarship (H.H.).

The authors acknowledge Dr. J.A. Dlugosz and S. Munk for their technical assistance and Dr. S.C. Hubchak for suggesting the source of the collagen antibody.


   FOOTNOTES
 
Address correspondence and reprint requests to Catharine Whiteside, MD, PhD, FRCP(C), Medical Sciences Building, Rm. 7302, 1 King’s College Circle, University of Toronto, Toronto, Canada M5S 1A8. E-mail: catharine.whiteside{at}utoronto.ca.

Received for publication 18 December 2000 and accepted in revised form 18 July 2001.

CA, constitutively active; DAG, diacylglycerol; DMEM, Dulbecco’s modified Eagle’s medium; DN, dominant-negative; ECM, extracellular matrix; ERK1/2, extracellular signal–related protein kinase; ET-1, endothelin-1; FBS, fetal bovine serum; HG, high glucose; MAPK, mitogen-activated protein kinase; MC, mesangial cell; NG, normal glucose; PKC, protein kinase C; PMA, phorbol myristic acid; SSC, sodium chloride–sodium citrate.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
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