Department of Pathology, University of Western Ontario, London, Ontario N6A 5C1, Canada
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ABSTRACT |
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Increased
extracellular matrix protein production leading to structural
abnormalities is a characteristic feature of chronic diabetic
complications. We previously showed that high glucose in endothelial
cell culture leads to the upregulation of basement membrane protein
fibronectin (FN) via an endothelin (ET)-dependent pathway involving
activation of NF-B and activating protein-1 (AP-1). To delineate the
mechanisms of basement membrane thickening, we used an animal model of
chronic diabetes and evaluated ET-dependent activation of NF-
B and
AP-1 and subsequent upregulation of FN in three target organs of
chronic diabetic complications. After 3 mo of diabetes, retina, renal
cortex, and myocardium demonstrated increased FN mRNA and increased
ET-1 mRNA expression. Increased FN expression was shown to be dependent
on ET receptor-mediated signaling, as the increase was prevented by the
dual ET receptor antagonist bosentan. NF-
B activation was most
pronounced in the retina, followed by kidney and heart. AP-1 activation
was also most pronounced in the retina but was similar in both kidney
and heart. Bosentan treatment prevented NF-
B activation in the
retina and heart and AP-1 activation in the retina and kidney. These data indicate that, although ETs are important in increased FN production due to diabetes, the mechanisms with respect to
transcription factor activation may vary depending on the
microenvironment of the organ.
activating protein-1; nuclear factor-B; endothelin; retina; kidney; heart; diabetes
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INTRODUCTION |
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CAPILLARY
BASEMENT MEMBRANE (BM) THICKENING is a hallmark of diabetic
microangiopathy and has been demonstrated in all target organs of
diabetic complications, including retina, kidney, and heart (4,
21). Although the mechanism is not clear, various biochemical
pathways have been associated with capillary BM thickening due to
diabetes (2, 4, 5, 21). At the molecular level, increased
extracellular matrix (ECM) protein synthesis is instrumental in BM
thickening (8, 15, 36). Fibronectin (FN), a glycoprotein of ~250 kDa, is a major component of the ECM. It is composed of two
similar, but not identical, polypeptides joined by disulfide bonds. FN
plays key roles in various cellular events, including cell adhesion,
motility, and tissue repair (3, 22, 41). However, its
overproduction may decrease the motility and replication of many cells,
including endothelial cells (28). Physiologically, upregulation of FN may lead to increased retinal vascular endothelial cell turnover due to diabetes (29). FN synthesis has been
demonstrated to increase in the retina of diabetic patients with
background retinopathy (36). Several factors may influence
augmented FN synthesis in diabetes. Vasoactive factors like endothelins
(ETs), by virtue of their extensive tissue distribution and widespread biological actions, are important mediators of pathogenic changes in
diabetic microangiopathy and may influence FN production (8, 9,
15). We have previously demonstrated (8, 12, 15) that ET-1 and ET-3 expressions are upregulated in the retina of diabetic rats and that ET receptor blockade prevents
hyperhexosemia-induced vasoconstriction and BM thickening in the retina
and glomeruli of diabetic rats. We have also shown (9)
that diabetes-induced myocardial focal scarring can be prevented by ET
antagonism. Several biochemical pathways, such as protein kinase C
(PKC) activation, nonenzymatic glycation, and activation of other
vasoactive factors, may promote increased ET synthesis and subsequent
FN increase (2, 4, 7, 21, 27). ETs have been shown to
produce fibrosis via activation of transcription factors NF-B and
activating protein (AP)-1. ET-1 activates NF-
B in the hepatic
stellate cells via the ETB receptor (16).
Angiotensin II-induced end organ damage in hypertension has been shown
to be mediated via ET receptor-dependent NF-
B and AP-1 activation
(31). Various pathways of tissue injury caused by
hyperglycemia in vivo or high glucose concentration in cell cultures
may activate transcription factors such as NF-
B and AP-1 and
subsequently alter the expression of several genes important in the
pathogenesis of diabetic complications (30, 32, 33).
NF-
B is present in several cell types, including endothelial cells
(39). Normally, inactive NF-
B exists in the cytoplasm,
bound to the inhibitory protein I
B. I
B is hydrolyzed following
stimulation, whereby the p50/p65 dimer translocates to the nucleus and
initiates transcription of various genes (1, 6, 17, 30,
32). Resynthesis of I
B, induced by NF-
B, allows
sequestration of NF-
B in the cytoplasm and termination of NF-
B
response (1, 6, 17). AP-1 consists of homodimers of Jun or
heterodimers of Fos and Jun (11, 38). It is also regulated by cellular stress. Coordinated participation of NF-
B and
another transcription factor such as AP-1 may activate the genes of the
effector molecules in various pathological conditions (11,
38). We have previously demonstrated (10) that
endothelial cells cultured in 25 mmol/l glucose upregulate the
expression of FN, thus mimicking the effects of diabetes. We have
further demonstrated that, in endothelial cells, such glucose-induced increased FN expression is mediated via ET through activation of both
NF-
B and AP-1 (10). The aim of the present study was to
determine the role of NF-
B and AP-1 in increased FN mRNA expression in the various clinically relevant target organs of diabetic
complications. We further investigated whether these changes are
dependent on ET alteration.
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MATERIALS AND METHODS |
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Reagents. All reagents were obtained from Sigma Chemical (St. Louis, MO) unless otherwise mentioned.
Animals. All animals were cared for in accordance with the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals. The University of Western Ontario Council on Animal Care Committee formally approved all experimental protocols. Male Sprague-Dawley rats of ~200 g were obtained from Charles River Canada (St. Constant, PQ, Canada). Diabetes was induced with a single intravenous injection of streptozotocin (65 mg/kg body wt, in citrate buffer). The presence of hyperglycemia was confirmed by blood glucose estimation (Surestep blood glucose meter; Lifescan, Burnaby, BC, Canada). Nondiabetic control animals received an equal-volume injection of citrate buffer. Diabetic rats were randomized into two groups, namely, 1) poorly controlled diabetics and 2) poorly controlled diabetics on bosentan treatment. Age- and sex-matched animals were used as nondiabetic controls.
Bosentan (courtesy of Dr. M. Clozel, Actelion, Basel, Switzerland) is a potent dual ETA and ETB receptor antagonist (35). Bosentan was administered by daily oral gavage at the dose of 100 mg · kg body wtPreparation of nuclear protein fractions.
Nuclear extracts of kidney, heart, and retina were prepared as
described elsewhere (13, 37). Rapid detection of
octamer-binding proteins with "miniextracts" was prepared from a
small amount of tissue (0.5-1 g) as previously described
(10, 37). Briefly, the tissues were homogenized, washed
with phosphate-buffered saline, and pelleted by centrifugation (1,500 g for 5 min). The pellet was resuspended in 0.4 ml of cold
buffer A (in mmol/l: 10 HEPES, pH 7.9, 10 KCl, 0.1 EDTA, 0.1 EGTA, 1 DTT, and 0.5 PMSF) by gentle pipetting. The cells were allowed
to swell on ice for 15 min. Twenty-five microliters of 10% IGEPAL
CA-630 were added, and cells were vortexed vigorously. The homogenate
was centrifuged (10,000 g for 30 s), and the nuclear
pellet was resuspended in 50 µl of ice-cold buffer C (in
mmol/l: 20 HEPES, pH 7.9, 0.4 NaCl, 1 EDTA, 1 EGTA, 1 DTT, and 1 PMSF).
The tube was vigorously rocked at 4°C for 15 min on a shaking
platform. The nuclear extract was centrifuged at 4°C (15,000 g for 5 min), and the supernatant was frozen at 70°C.
The protein concentrations were measured using the bicinchoninic acid
protein assay, with bovine serum albumin as a standard (Pierce,
Rockford, IL).
Electrophoretic mobility shift assay.
Electrophoretic mobility shift assays (EMSA) were performed following
established methodology and as described by us previously (10). Briefly, NF-B and AP-1 consensus oligonucleotide
(Promega, WI) DNA probes (Table 2) were
prepared by end labeling with
[
-32P]ATP (Amersham, QC, Canada) using T4
polynucleotide kinase. The probes were purified by ethanol
precipitation and resuspended in 10 mmol/l Tris and 1 mmol/l EDTA (pH
7.6). Five micrograms of nuclear proteins were incubated with 100,000 cpm of 32P-labeled consensus oligonucleotides for 30 min at
room temperature. The incubation was carried out in a buffer containing
10 mmol/l Tris (pH 7.5), 50 mmol/l NaCl, 1 mmol/l MgCl2,
5% glycerol, 0.05% IGEPAL CA-630, 0.5 mmol/l EDTA, 0.5 mmol/l DTT,
and 0.5 µg of poly(dI-dC). Protein-DNA complexes were resolved on a
standard 6% (NF-
B) and 4% (AP-1) nondenaturing polyacrylamide gel
in 0.5 × Tris-boric acid-EDTA running buffer. After 30 min of electrophoresis at 350 V, gels were dried under heated vacuum
onto Whatman paper and subjected to autoradiography from overnight to 3 days. Anti-NF-
B (p65) monoclonal antibody and anti-AP-1 (c-Jun)
polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were
used for the supershift assay. The specificity of binding was further
confirmed by incubation with 100-fold unlabeled oligonucleotides.
The blots were quantified with densitometry (10).
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RNA isolation. TRIzol reagent (Invitrogen, Burlington, ON, Canada) was used to isolate RNA. RNA was extracted with chloroform followed by centrifugation to separate into aqueous and organic phases. RNA was recovered from the aqueous phase by isopropyl alcohol precipitation and suspended in diethylpyrocarbonate-treated water (8, 15).
First-strand cDNA synthesis.
First-strand cDNA synthesis was performed using the Superscript-II
system (Invitrogen). RNA (3 µg) was added to oligo(dT) primers
(Invitrogen), denatured at 65°C, and quenched on ice for 10 min.
Reverse transcription was carried out by the addition of Moloney murine
leukemia virus reverse transcriptase and dNTP at 42°C for 50 min in a
total reaction volume of 20 µl. The reaction was terminated by
incubation at 70°C for 15 min. The resulting RT products were stored
at 20°C (8, 15).
Real-time RT-PCR. RT-PCR was carried out with the LightCycler (Roche Diagnostics Canada, Laval, PQ, Canada) using the SYBR Green I detection platform. This system allows amplification and detection of products in a single reaction tube. PCR reactions were performed in microcapillary tubes (Roche Diagnostics Canada), with a final volume of 20 µl. The reaction mixture consisted of 2.5 µl of 10 × PCR buffer (Invitrogen), 1.25 µl of 5 mM dNTP, 1.2 µl of 50 mM MgCl2 (1.6 µl for ET-1), 1 µl each of forward and reverse 10 µM primers, 0.5 µl of 5 U/µl Platinum Taq polymerase, 0.75 µl of 10 × SYBR Green I (Molecular Probes, Eugene, OR), 10.8 µl of H2O, and 1 µl of cDNA template.
The temperature profiles and primer sequences for PCR reactions are found in Table 1. To optimize the amplification of the genes, melting curve analysis (MCA) was used to determine the melting temperature (Tm) of specific products and primer dimers. According to the Tm value of specific products for respective genes, an additional step (signal acquisition step, 2-3°C below Tm) was added after the elongation phase of RT-PCR. This additional step in the PCR reactions allowed for signal acquisition from specific target products. The signal acquisition step was determined to be 83°C for
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Statistical analysis. The data are expressed as means ± SE and were analyzed by ANOVA followed by Student's t-test. Differences were considered significant at values of P < 0.05.
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RESULTS |
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Clinical monitoring. Poorly controlled diabetic animals demonstrated hyperglycemia (19.0 ± 2.5 vs. 4.6 ± 0.2 mmol/l in controls), reduced body weight gain (482 ± 18.2 vs. 627 ± 21.7 g), and increased glycated hemoglobin levels (17.4 ± 0.6 vs. 5.6 ± 0.4% in controls) compared with the age-matched nondiabetic control animals. These rats also demonstrated polyuria and glucosuria (data not shown). These data are indicative of diabetic dysmetabolism. Bosentan treatment had no effects on these parameters (20.7 ± 1.6 mmol/l, 489 ± 12.7 g, and 15.8 ± 0.5%, respectively). No alterations of blood pressure were seen in any of the groups.
Diabetes-induced ET-1 mRNA upregulation.
We have previously demonstrated (8, 9, 12, 15) that
chronic diabetes induces ET-1 mRNA upregulation in retina, kidney, and
heart. This study confirmed our previous finding that ET-1 mRNA
upregulation occurs in all target organs of diabetic complications (Fig. 1). We used a novel real-time
PCR-based assay to confirm our previous finding. A maximum (10-fold)
increase in ET-1 mRNA expression was seen in the retina in diabetes.
Diabetes also caused a significant (4-fold) increase in mRNA expression
in both kidney and heart. Interestingly, bosentan treatment lowered
ET-1 mRNA expression in all organs examined.
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Diabetes-induced increased FN synthesis is blocked by ET
antagonism.
We have previously demonstrated (8, 12, 15) that
hyperhexosemia-induced increased ET expression is an important mediator of increased ECM production in diabetes. One of the most important ECM
proteins overexpressed in these organs due to diabetes is FN (15,
36). In the present study, we used a real-time PCR method to
quantify FN mRNA expression. The primers used for FN RT-PCR were
designed to allow assessment of total FN, including all splice variants
of FN, in accordance with the guidelines for primer design for
LightCycler. In all three target organs of diabetic complications, we
found significantly increased FN mRNA expression in the poorly
controlled diabetic animals compared with the age- and sex- matched
nondiabetic rats (Fig.
2). Treatment of
poorly controlled diabetic animals with a dual ETA and
ETB antagonist completely prevented diabetes-induced
increased FN mRNA expression in both retina and kidney. A partial
prevention was noted in the heart, where bosentan-treated diabetic
animal mRNA levels were not significantly different from either poorly
controlled diabetic or nondiabetic rats (Fig. 2).
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NF-B activation in diabetes and modulation by ET antagonism.
As we have previously reported (10), high glucose and ETs
cause a time-dependent activation of NF-
B in endothelial cells. In
the present study, we have demonstrated that a similar activation of
NF-
B occurs in the target organs of diabetic complications (Fig.
3). The specificity of NF-
B activation
was established by the supershift assay and the incubation with
100-fold unlabeled oligonucleotides (Fig. 3). However, there were
variations in the level of activation. In parallel with ET-1
expression, the most pronounced activation was seen in the retina of
diabetic animals (4-fold compared with nondiabetic animals,
P < 0.01; Fig. 3). The arbitrary densitometric units,
although increased (2-fold, P < 0.05) in the kidney of
diabetic rats compared with the controls, were approximately one-half
the retinal levels in diabetics. The lowest level (one-tenth of the
retina in diabetes) of activation values were seen in the heart.
However, poorly controlled diabetic animals still showed a higher level
than the nondiabetic rats (P < 0.05). Diabetes-induced
NF-
B activation was prevented by bosentan treatment in both the
retina and heart but not the kidney (Fig. 3).
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AP-1 activation in diabetes and modulation by ET antagonism.
Transcription factor AP-1 also showed diabetes-induced activation. The
specificity of AP-1 activation was further established by incubation
with 100-fold unlabeled oligonucleotides (Fig.
4). However, there were variations in the
level of activation. The most pronounced activation was seen in the
retina of diabetic animals (6-fold compared with nondiabetic animals,
P < 0.01; Fig. 4). The arbitrary densitometric units,
although significantly increased (2-fold, P < 0.05) in
both the kidney and heart of diabetic rats compared with the controls,
were much lower compared with the retinal levels in diabetics.
Diabetes-induced AP-1 activation was prevented by bosentan treatment in
both the retina and kidney but not the heart (Fig. 4).
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DISCUSSION |
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The key findings of the present study are that diabetes causes FN
upregulation in the retina, kidney, and heart, the three organs
involved in chronic diabetic complications. Diabetes-induced, increased
tissue FN mRNA expression requires activation of both NF-B and AP-1,
at least in part, by an ET-mediated pathway, although the level of
activation varies from organ to organ. ET receptor antagonism prevents
FN mRNA expression in all three organs examined. However, the effects
on transcription factor activation were variable among organs. In the
retina, both NF-
B and AP-1 were important, whereas NF-
B
activation appeared to be the predominant mechanism of
diabetes-induced, ET-mediated FN synthesis in the heart. Similarly, AP-1 was the predominant mechanism in the kidney. Therefore, this study
has demonstrated that, in diabetes, activation of transcription factors
may be modulated by the tissue microenvironment.
Oxidative stress could be an important mechanism in the pathogenesis of
diabetic complications (2). NF-B and AP-1 can be
activated by oxidative stress (1, 11, 38). Previous studies have demonstrated NF-
B activation in retinal capillary pericytes exposed to high glucose levels as well as retinal tissues in
short-term diabetes (20, 34). Similarly, mesangial cells and renal tubular epithelial cells exposed to high glucose show NF-
B
activation (19, 23, 24). In the hearts of rats, short-term diabetes demonstrated both NF-
B and AP-1 activation
(32). This study has demonstrated that NF-
B and
AP-1 are differentially activated in various target organs of diabetic
complications after long-term diabetes. Furthermore, we have identified
the effect of such activation in the pathogenesis of a characteristic
lesion in diabetes. It is interesting to note that transcription factor activation exhibited variable patterns and levels in different tissues.
The strongest activation of NF-
B and AP-1 was seen in the retina in
diabetes, which also correlated with the highest ET-1 mRNA levels. The
exact reasons for differential transcription factor activation are not
clear. It appears that the tissue microenvironment may affect the
activation of transcription factors in diabetes. However, the findings
in this study may, in part, be helpful to explain the variation seen in
BM thickening in different target organs (14). Blockade of
NF-
B and AP-1 activation and augmented FN mRNA expression in
diabetes by bosentan indicate the important role ETs play with respect
to transcription factor activation leading to FN gene expression in diabetes.
We have previously demonstrated with endothelial cells how
glucose-induced, ET-mediated increased FN synthesis is dependent on
coordinated activation of both NF-B and AP-1 (10).
Several studies have demonstrated the effects of these transcription
factors on FN synthesis. In hepatoma cells, NF-
B was shown to play
both positive and negative regulator roles acting on the FN gene
(25). Although several potential NF-
B-binding sites
have been identified in the FN promoter, the functional significance of
only a few has been determined. The regulation of FN gene expression
has been shown to be offered by the
41 NF-
B-binding site
(25). The DNA sequence between +1 and +136 has been
determined for partial PKC-mediated activation of the FN gene in
hepatoma cells (25). A potential NF-
B p65 response
element present in the FN promoter may serve as a positive regulator of
FN gene expression (25). Identification and
characterization of an NF-
B-binding site in the promoter region of
the FN gene responsible for ET responsiveness in endothelial cells
require further investigation. An AP-1-binding site mediating
angiotensin II-induced transcriptional activation of the FN gene has
been shown in the promoter region (40). Whether ET-induced
activation of the FN gene also utilizes the same region of the gene
promoter remains to be investigated.
Furthermore, it is well known that hyperglycemia resulting from
diabetes activates PKC (2, 18, 21). Activation of PKC is
required for glucose-induced upregulation of ET in many cells, including endothelial cells (9, 27). Therefore, glucose
causing an upregulation of ET expression through PKC activation can
further activate transcription factors NF-B and AP-1 and thereby
increase FN expression. However, hyperhexosemia-induced ET upregulation can also be controlled by NF-
B and AP-1 (26, 27, 33).
Furthermore, because ET receptors are predominantly Gq
coupled, their activation may lead to activation of phospholipase C,
elevation of intracellular Ca2+ concentration, and
activation of PKC. Therefore, combining the results of our present
study with the previously published studies (7, 26, 27,
33), we can speculate that PKC, NF-
B, and AP-1 can control
both upstream and downstream effector molecules in the high
glucose-induced signaling pathway. However, the role of ET
receptor-mediated signaling in diabetes leading to NF-
B activation
may potentially be influenced by other factors (31), which
remain to be investigated.
In conclusion, we have demonstrated in three different organs that
diabetes-induced FN upregulation involves variable activation of
NF-B and AP-1 transcription factors via an ET-mediated pathway. The
observations made in the present study are likely to have relevance for
vasculopathy in diabetes.
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ACKNOWLEDGEMENTS |
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These studies were supported in part by grants from the Canadian Diabetes Association, in honor of Margaret Francis, and the Canadian Institute of Health Research (MOP 43841).
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. Chakrabarti, Dept. of Pathology, Dental Sciences Bldg, Univ. of Western Ontario, London, ON N6A 5C1, Canada (E-mail: schakrab{at}uwo.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 11, 2003;10.1152/ajpendo.00540.2002
Received 11 December 2002; accepted in final form 3 February 2003.
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