Departments of 1 Diabetes and 2 Molecular Biology, Beckman Research Institute of the City of Hope, Duarte 91010; 4 Division of Nephrology, Harbor-UCLA Research and Education Institute, Torrance, California 90509; and 3 Department of Endocrinology, University of Virginia Medical School, Charlottesville, Virginia 22908
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ABSTRACT |
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The lipoxygenase (LO) pathway of
arachidonate metabolism and mitogen-activated protein kinases (MAPKs)
can mediate cellular growth and ANG II effects in vascular smooth
muscle cells. However, their role in renal mesangial cells (MC) is not
very clear. ANG II treatment of rat MC significantly increased 12-LO
mRNA expression and formation of the 12-LO product
12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE;
P < 0.03]. ANG II-induced [3H]leucine
incorporation was blocked by an LO inhibitor,
cinnamyl-3,4-dihydroxy--cyanocinnamate (P < 0.02).
12(S)-HETE and ANG II directly induced cellular hypertrophy and fibronectin (FN) expression (P < 0.01) to a
similar extent. ANG II and 12(S)-HETE led to activation of
p38MAPK and its target transcription factor cAMP-responsive
element-binding protein (CREB). ANG II- and
12(S)-HETE-induced CREB activation and
[3H]leucine incorporation were blocked by the
p38MAPK inhibitor SB-202190. A specific molecular inhibitor
of rat 12-LO mRNA, namely, a novel ribozyme, could attenuate ANG
II-induced FN mRNA. Thus p38MAPK-dependent CREB activation
may mediate ANG II- and LO product-induced FN expression and cellular
growth in rat MC. ANG II effects may be mediated by the LO pathway.
These results suggest a novel interaction between LO and
p38MAPK activation in MC matrix synthesis associated with
renal complications.
angiotensin II; 12(S)-hydroxyeicosatetraenoic acid; hypertrophy; p38MAPK; cAMP-responsive element-binding protein; fibronectin; rat
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INTRODUCTION |
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INCREASED MESANGIAL
CELL (MC) hypertrophy and matrix deposition is a hallmark of
renal diseases such as diabetic nephropathy. This can be mediated by
the action of hyperglycemia and several growth factors such as
angiotensin II (ANG II), platelet-derived growth factor (PDGF), and
transforming growth factor- (57). However, the specific
molecular and signaling transduction mechanisms involved are not clear.
These growth factors can bind to their specific receptors and activate
intracellular phospholipases, leading to the formation of arachidonic
acid, which can be further metabolized by cyclooxygenases, cytochrome
P-450 oxygenases, and lipoxygenases (LOs)
(46). LOs are a family of nonheme
iron-containing enzymes that insert molecular oxygen into
polyunsaturated fatty acids such as arachidonic and linoleic acids
(59). LOs are classified as 5-, 8-, 12-, and 15-LOs on the
basis of their ability to insert molecular oxygen at the corresponding
carbon atom of arachidonic acid (11, 59). 12-LO action can
lead to the formation of oxidized lipids such as
12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] (59).
Three major isoforms of 12-LO have been cloned: platelet-, leukocyte-, and epidermal-type 12-LO (11, 59). Leukocyte-type 12-LO has been cloned from porcine and mouse leukocytes (5, 62). The presence of leukocyte-type 12-LO (also termed 12/15-LO) has also been demonstrated in various cell types and tissues, including vascular smooth muscle cells (VSMC), adrenal cells, and rat brain and kidney (14, 17, 23, 24 35, 52). Leukocyte-type 12/15-LO has been implicated in the pathogenesis of atherosclerosis and restenosis (7, 10, 15, 32-34, 40, 61). In VSMC, high glucose, ANG II, and PDGF, all factors implicated in the pathogenesis of diabetic nephropathy, significantly increased 12-LO activity and expression (32, 35). Furthermore, the downstream functional effects of these factors in VSMC, including hypertrophy and migration, were attenuated by LO blockade (15, 32, 34, 40), indicating the key role of LO activation in the vascular effects of these factors. However, the biochemical and functional role of the 12-LO pathway and its products in glomerular MC is not clear.
In the kidney, the pathogenic importance of the 5-LO pathway has been demonstrated in experimental immune-mediated glomerular disease (27, 58), but it has not been evaluated in diabetic nephropathy. Evidence suggests that, in glomeruli, 12- and 15-LO activities predominate, leading to the synthesis of 12(S)- and 15(S)-HETE (3, 20). Rat tissues express mainly leukocyte-type 12/15-LO (17, 52), and Katoh et al. (24) identified and partially sequenced a leukocyte-type 12/15-LO from rat glomeruli. Moreover, high glucose concentrations could induce arachidonic acid release in MC, along with cyclooxygenase product synthesis and protein kinase C (PKC) activation (56), but the LO pathway was not assessed. An anti-inflammatory role for 15-LO has been suggested during immune-mediated tissue injury (29).
We recently showed for the first time that 12/15-LO mRNA and protein are increased in glucose-stimulated MC and in experimental diabetic nephropathy (23). Furthermore, fibronectin (FN) expression in vivo in the diabetic rat renal glomeruli was associated with increased glomerular 12-LO expression (23), implicating the 12-LO pathway in the pathogenesis of diabetic nephropathy. However, it is not known whether the 12-LO product 12(S)-HETE has direct effects on MC hypertrophy or matrix synthesis. Furthermore, the molecular mechanisms of 12(S)-HETE actions in MCs have also not been examined. Studies from our laboratory as well as others have also implicated members of the mitogen-activated protein kinase (MAPK) pathway, such as extracellular signal-regulated kinase (ERK1/2) and p38MAPK, in the pathogenesis of diabetic nephropathy (19, 21, 22, 50). In the present study, our objective was to determine 1) whether the 12/15-LO pathway is induced by ANG II in rat MC, 2) whether the LO pathway mediates ANG II effects on hypertrophy and FN expression in rat MC, and 3) whether 12(S)-HETE can activate p38MAPK and its target transcription factors, such as cAMP-responsive element-binding protein (CREB), which are related to matrix gene expression. Our results show for the first time that oxidized lipids, such as 12(S)-HETE, can directly induce MC hypertrophy and matrix production with potency similar to that of ANG II. Furthermore, LO products can act as direct signaling molecules and also mediate the effects of ANG II by activating key members of the MAPK pathway. Thus 12-LO activation and the product 12(S)-HETE may be involved in multiple events related to the development of diabetic nephropathy.
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MATERIALS AND METHODS |
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Materials.
12(S)-Hydroxyeicosa-5Z,8Z,10E,14Z-tetraenoic
acid [12(S)-HETE] and its stereoisomer
12(R)-HETE were purchased from Biomol (Plymouth Meeting,
PA), inhibitor for p38MAPK (SB-202190) from Calbiochem (La
Jolla, CA), and the ERK pathway inhibitor U-1026 from Upstate
Biotechnology (Lake Placid, NY). The LO inhibitor
cinnamyl-3,4-dihydroxy--cyanocinnamate (CDC; Biomol, Plymouth
Meeting, PA) was added from 1,000-fold concentrates in ethanol.
Protease inhibitor cocktail (Complete) was obtained from Roche
Biochemicals (Indianapolis, IN); gel shift oligonucleotides for CREB
and SP-1 transcription factors from Santa Cruz Biotechnology (Santa
Cruz, CA); antibodies for phosphospecific and
nonphospho-p38MAPK, -ERK1/2, and -CREB proteins and
horseradish peroxidase-conjugated secondary antibodies from Cell
Signaling (Beverly, MA); antibody to FN (monoclonal antibody 1940 against cellular FN), used for immunoblotting, from Chemicon
International (Temecula, CA); the chemiluminescence reagent Supersignal
from Pierce (Rockford, IL); and primers for Quantum RNA 18S internal
standards from Ambion (Austin, TX).
Quantitation of 12-LO mRNA levels. Rat leukocyte-type 12/15-LO mRNA levels were quantitated by a specific nested RT-PCR method using gene-specific primers, as recently described (23).
12(S)-HETE assay.
Rat MC were serum depleted and treated with ANG II for 10 or 30 min. At
the end of the incubation period, the culture dishes were placed on
ice, and the supernates were aspirated and saved at 70°C.
12(S)-HETE in these cell supernates was extracted on C-18
columns and quantitated by a specific radioimmunoassay, as described
earlier by us (30, 35).
Cell culture and treatment with 12(S)-HETE. Primary cultures of rat MC were obtained and characterized as described earlier (23). They were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, L-glutamine (2 mM), HEPES (7 mM), streptomycin (100 µg/ml), and penicillin (100 U/ml). Quiescent rat MC in 100-mm dishes were preincubated for 60 min in RPMI 1640 medium containing 0.2% BSA and then treated with vehicle (0.1% ethanol) or 12(S)-HETE or ANG II for the indicated time intervals. At the end of the incubation periods, cells were washed with PBS and processed for whole cell lysates, nuclear protein extraction, or RNA extraction as described below. In some experiments, rat MC were pretreated with inhibitors or corresponding vehicle.
Cell lysis and immunoblotting. After various incubations, washed cells were lysed and used for immunoblotting as described earlier (23, 42). Protein bands on Western blots were visualized using Supersignal chemiluminescence reagent. Immunoblots were scanned using a GS-800 densitometer, and protein bands were quantified with Quantitation One software (Bio-Rad Laboratories, Hercules, CA). The following antibodies were used: pERK1/2 (1:3,000), ERK1/2MAPKs (1:5,000), pp38 and p38MAPKs (1:2,000), pCREB and CREB (1:1,000), and FN (1:1,000).
Incorporation of [3H]leucine. Serum-depleted rat MC were incubated overnight with [3H]leucine and 12(S)-HETE or ANG II at the indicated concentrations, and radioactivity in trichloroacetic acid-insoluble protein precipitates was determined by scintillation counting.
FN ELISA.
Serum-starved rat MC in 24-well dishes were treated with ANG II or
12(S)-HETE for 24 h. The conditioned medium and cell
lysates were collected and stored at 70°C for FN measurement by a
specific sandwich ELISA (34).
FN mRNA quantitation by RT-PCR. After stimulation with agonists, RNA was extracted using RNA-STAT60 as described by the manufacturer, and expression of FN RNA was determined with a relative multiplex RT-PCR. cDNA was generated with 1 µg of RNA using random hexamers and Maloney murine leukemia virus RT. Then cDNA corresponding to 0.05 µg of RNA was used in a multiplex PCR containing rat FN-specific primers (23) paired with Quantum RNA 18S internal standards. The PCR was performed for 25 cycles at 94°C for 30 s, 62°C for 30 s, and 72°C for 30 s in GeneAmp 9700 (Applied Biosystems, Foster City, CA). The PCR products were fractionated on agarose gels and photographed using Alpha Imager 2000 (Alpha Inotech, San Leandro, CA). DNA bands corresponding to FN and 18S RNA were quantified with Quantitation One software. Results are expressed as fold stimulation over control after normalization for 18S RNA levels.
Designing a modified ribozyme targeting rat leukocyte-type 12-LO.
A hammerhead ribozyme (Rz) was designed to cleave the rat
leukocyte-type 12-LO mRNA. The sequence of rat leukocyte-type 12-LO cDNA has been previously reported (17, 52). Our Rz was
designed to cleave 12-LO mRNA at the 3' end of a GUC triplet located
nine bases downstream of the AUG start site of translation (Fig.
1). The Rz contained a chimeric DNA-RNA
sequence, in which the RNA bases of the substrate complementary arms
were replaced with deoxyribonucleotides, and ribonucleotides were
present in the core center area. Such substitutions would prevent
exoribonuclease activity and reduce the number of endoribonuclease
targets within the Rz (49). To further improve stability,
the Rz contained two phosphorothioate linkages at 5' and 3' ends (Fig.
1). As a control, a catalytically inactive mutant Rz (MRz) was
generated by a single base substitution at the core center position 5 (G-to-A). Importantly, this Rz in Fig. 1 is a new-generation Rz, which
is a modification of the long Rz we have tested in VSMC in vitro and in
vivo (15). The key modification is the replacement of the
stem loop of the original "long" rat 12-LO Rz (15)
with four propane diols (Fig. 1). This type of modified Rz has been
suggested to greatly improve catalytic efficiency, because the propane
diols ease rigidity, improve flexibility, and, hence, increase access
to the substrate. The added advantage of this modified short Rz is its
relative cost effectiveness. All the oligonucleotides were chemically
synthesized and purified at the DNA Synthesis Core at City of Hope.
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Transfection of Rz into cells.
Rat MC were plated in 60-mm dishes, and on the next day (80%
confluent) they were transfected with 12-LO Rz or 12-LO MRz
oligonucleotides using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in
RPMI 1640 medium containing 1% serum. After 6 h, cells were
washed with PBS, and fresh medium containing 1% FBS was added. On the
next day, cells were serum depleted for 6 h in RPMI 1640 medium
containing 0.2% BSA, and 106 M ANG II was added. After
overnight incubation, RNA was extracted, and FN mRNA levels were
determined by RT-PCR using 18S RNA as an internal standard.
Nuclear extract preparations and gel shift assays. Nuclear extracts were prepared and gel shift assays were performed to evaluate DNA binding of nuclear proteins (42, 60). Synthetic oligonucleotides containing consensus-binding sequences for transcription factors CREB or SP-1 were used as probes for electrophoretic mobility shift assay. Gels were dried, protein-DNA complexes were visualized on a PhosphorImager, and radioactivity in each complex was quantified using Imagequant software (Molecular Dynamics, Sunnyvale, CA).
Data analysis. Values are means ± SE of multiple experiments. Paired Student's t-tests were used to compare two groups, or ANOVA with Dunnett's or Tukey-Kramer's post tests was used for multiple groups using PRISM software (Graph Pad, San Diego, CA). Statistical significance was detected at the 0.05 level.
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RESULTS |
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ANG II increases activity and expression of 12-LO in rat MC.
To determine whether ANG II could lead to the activation of the 12-LO
pathway in rat MC, we treated serum-depleted rat MC in 100-mm dishes
with 107 or 10
8 M ANG II for 10 or 30 min.
12(S)-HETE in the supernates was extracted and quantitated
by a validated radioimmunoassay that is specific for
12(S)-HETE and does not cross-react with
12(R)-HETE (30). Figure
2A shows that ANG II led to a
dose-dependent increase in the levels of released 12(S)-HETE
at 10 and 30 min.
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Involvement of the 12-LO pathway in ANG II-induced cellular
hypertrophy.
To evaluate the functional significance of the ANG II stimulatory
effects on 12-LO activity and expression, we hypothesized that 12-LO
activation may mediate the hypertrophic effects of ANG II in rat MC. We
therefore examined the effects of a specific pharmacological 12-LO
inhibitor, CDC. Figure 3 shows that ANG II significantly increased [3H]leucine incorporation (an
index of cellular hypertrophy) into the rat MC, and this was
significantly attenuated by pretreatment with CDC. There was no effect
with CDC alone (not shown). Thus 12-LO activation appears to be
necessary for ANG II-induced rat MC hypertrophy.
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Growth-promoting effects of 12-HETE in rat MC.
Next, we asked whether 12(S)-HETE has direct
growth-promoting effects in MC. Figure
4A shows that
12(S)-HETE could indeed significantly increase
[3H]leucine incorporation in rat MC, with a peak response
at 107 M. Figure 4B shows that ANG II and
12(S)-HETE led to dose-dependent increases in total cell
protein content in the rat MC as another index of cellular hypertrophy.
Furthermore, the effects of 12(S)-HETE were comparable to
the effect of ANG II. These new results demonstrate the hypertrophic
effects of oxidized lipids in MC.
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Effect of 12-HETE on FN expression in rat MC.
Several lines of evidence indicate that increased deposition of
extracellular matrix proteins such as FN plays a key role in the
pathogenesis of kidney disease such as diabetic nephropathy. ANG II has
been shown to increase FN expression (18, 31) in MC. We
examined whether 12(S)-HETE could also lead to FN expression at the mRNA, as well as the protein, level. Rat MC were stimulated with
12(S)-HETE or ANG II (107 M each) for 4, 6, and 24 h, and expression of FN mRNA was determined by relative
multiplex RT-PCR (see MATERIALS AND METHODS), with 18S RNA
used as an internal control. Figure 5,
A and B, shows that, in
12(S)-HETE-treated cells, FN mRNA expression increased in
4 h and remained elevated up to 24 h after stimulation. In the case of ANG II, FN mRNA showed an increase by 6 h and remained elevated 24 h after stimulation (Fig. 5, A and
C). Thus ANG II and 12(S)-HETE increased FN mRNA
expression in rat MC. The effects of 12(S)-HETE appeared to
be more potent than the effects of ANG II.
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Effect of the 12-LO Rz on ANG II-induced FN mRNA expression.
Because pharmacological inhibitors may be associated with potential
nonspecific effects, we designed a novel short new-generation Rz as a
molecular inhibitor to specifically inhibit 12-LO mRNA expression. The
Rz and the control mutant MRz are depicted in Fig. 1. Rat MC were
transfected with the Rz or the MRz (see MATERIALS AND
METHODS) and then treated with 106 M ANG II for
6 h. Total RNA was then subjected to competitive RT-PCR to
quantitate FN mRNA levels. Results shown in Fig.
7 indicate that the Rz could reduce ANG
II-induced FN mRNA expression by >50%. However, the control MRz had
no effect under these conditions (Fig. 7). These results suggest that
ANG II-induced FN is mediated, at least in part, by the LO pathway.
Furthermore, they illustrate the utility of Rz to reduce the expression
of genes related to the progression of diabetic nephropathy.
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Activation of MAPKs and CREB by 12(S)-HETE and ANG II.
We next wanted to examine the specific signal transduction mechanisms
by which 12(S)-HETE exerts its growth-promoting effects in
rat MC and compare them with those of ANG II. We therefore examined the
activation of key growth- and stress-related MAPKs. Serum-depleted VSMC
were treated with 12(S)-HETE or ANG II (107 M
each) for 5, 10, 30, and 60 min, and MAPK activation was determined by
immunoblotting with phosphospecific antibodies that recognize only the
activated kinases. As shown in Fig.
8A, a marked increase in
p38MAPK activation could be seen within 5 min after
stimulation with 12(S)-HETE and remained elevated until 60 min (top) as detected by increase in the levels of the
phospho-p38MAPK band. When the same blot was stripped and
probed with antibody to nonphospho-p38MAPK, equal amounts
of p38MAPK were present in all the lanes
(middle). Similar results were obtained with ANG II (Fig.
8B). Immunoblotting with phospho-ERK or phospho-c-Jun
NH2-terminal kinase (JNK) antibodies showed that 12(S)-HETE did not significantly activate ERK1/2 or JNK
(results not shown). These results demonstrate that
12(S)-HETE is a potent activator of p38MAPK but
not ERK1/2 or JNK in rat MC.
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Activation of p38MAPK and CREB by 12(S)-HETE, but not
by 12(R)-HETE.
To test the specificity of 12(S)-HETE signaling, rat MC were
stimulated with 12(S)-HETE or its stereoisomer
12(R)-HETE (which is not an LO metabolite) for 5 min. Then
the cell lysates were analyzed for p38MAPK and CREB
activation. As shown in Fig. 9,
12(S)-HETE stimulated the activation of p38MAPK
and CREB compared with control cells. In contrast,
12(R)-HETE had no effect on the activation of
p38MAPK or CREB. When the blot in Fig. 9A was
stripped and probed with a pan-specific p38MAPK antibody,
the results showed that 12(S)-HETE does not alter total p38
levels and that equal amounts of protein were loaded in all the lanes.
Thus 12(S)-HETE, but not 12(R)-HETE, was able to
stimulate p38MAPK and CREB in rat MC, and, hence, the
effects are specific to the LO product.
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Involvement of p38MAPK in 12(S)-HETE-induced CREB
phosphorylation.
To investigate the upstream kinase(s) involved in
12(S)-HETE-mediated phosphorylation of CREB and to determine
whether CREB is a target of 12(S)-HETE-induced
p38MAPK activation, we tested the effect of MAPK inhibitors
on CREB phosphorylation. Serum-starved rat MC were pretreated with the
p38MAPK inhibitor SB-202190 (106 M) or the
specific ERK pathway inhibitor U-0126 (10
5 M) for 30 min
before stimulation with 12(S)-HETE for 5 min. The cell
lysates were then immunoblotted with the antibody specific to
phospho-CREB, and the results are shown in Fig.
10. Phosphorylation of CREB was clearly
inhibited by SB-202190 but not by U-0126. The blot was then stripped
and probed with phospho-p38MAPK antibody. As shown in Fig.
10 (middle), SB-202190 completely blocked p38MAPK activation; in contrast, U-0126 had no effect.
There was no change in total p38 levels under these conditions (Fig.
10, bottom). These results demonstrate for the first time
that p38MAPK mediates 12(S)-HETE-induced
phosphorylation of CREB in rat MC.
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Stimulation of CREB DNA binding activity by 12(S)-HETE.
Because CREB phosphorylation can lead to increased binding to specific
cAMP-responsive elements in the promoter of key genes involved in MC
hypertrophy, we evaluated the effects of 12(S)-HETE on CREB
DNA binding. Serum-depleted rat MC were stimulated with 12(S)-HETE for 15-120 min. Nuclear extracts were then
prepared and analyzed for CREB DNA binding activity by gel shift assay using 32P-labeled oligonucleotides containing consensus
binding sites for the transcription factor CREB (Fig.
11A) or a constitutively active transcription factor SP-1 (Fig. 11B). DNA binding
activity of CREB was increased by 12(S)-HETE within 15 min,
peaking between 15 and 60 min and continuing until 120 min (Fig.
11A). However, in the same nuclear extracts,
12(S)-HETE had no effect on SP-1 DNA binding activity,
indicating the specificity of 12(S)-HETE for the activation
of CREB DNA binding. These results demonstrate that
12(S)-HETE can induce DNA binding activity of CREB in rat MC.
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Role of p38 MAPK activation in mediating growth-promoting effects
of ANG II and 12(S)-HETE in rat MC.
We next examined whether p38MAPK activation plays a
functional role in the growth effects of 12(S)-HETE or ANG
II. Serum-depleted VSMC were pretreated with the p38MAPK
inhibitor SB-202190 (106 M) for 15 min and then
stimulated with 12(S)-HETE or ANG II (10
7 M
each) for 24 h. ANG II- and 12(S)-HETE-induced
[3H]leucine incorporation into rat MC was significantly
blocked by the p38MAPK inhibitor (Fig.
12). SB-202190 alone had no effect.
Thus p38MAPK appears to mediate the growth-promoting
effects of 12(S)-HETE and ANG II in rat MC.
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DISCUSSION |
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Although it is known that oxidized lipids can have multiple adverse effects on renal functions, much less is known about their cellular effects or the specific signaling and molecular mechanisms by which they lead to the expression of key genes associated with diseases such as diabetic nephropathy. In the present study, we report for the first time in rat MC that the LO product 12(S)-HETE can lead to cellular growth, FN protein and mRNA expression, and p38MAPK and CREB transcription factor activation with potency similar to that of ANG II. Furthermore, the LO pathway could mediate the effects of ANG II. These studies demonstrate that bioactive lipids such as these LO products can directly initiate intracellular signaling and gene expression in renal cells. We recently showed that oxidized lipid products of arachidonic acid and linoleic acid metabolism via the LO pathway had growth and inflammatory effects in VSMC (34, 38, 42). Furthermore, the LO pathway could mediate the growth-promoting effects of ANG II as well as the chemotactic effects of PDGF (15, 32, 34, 40) and also plays a role in neointimal responses to vascular injury (10, 37). Thus bioactive lipids of the LO pathway may play a role in microvascular and macrovascular complications, particularly those related to diabetes.
We observed that 12(S)-HETE could directly lead to the activation of p38MAPK but not ERK1/2 or JNK MAPKs. In contrast, the isomer 12(R)-HETE, which is not an LO product, did not have these stimulatory effects, indicating specificity for the LO pathway. 12(S)-HETE, but not 12(R)-HETE, also induced the phosphorylation of CREB transcription factor. The p38MAPK inhibitor SB-202190 blocked these effects. 12(S)-HETE also increased DNA binding activity of CREB but not SP-1, as assessed by electrophoretic mobility shift assay. ANG II could also induce p38MAPK activation, confirming a recent report (50), as well as CREB phosphorylation at Ser133. CREB is a key target of p38MAPK (48) and plays a role in FN transcription (8, 9, 31, 25). Furthermore, earlier studies have implicated CREB activation in glucose- and ANG II-induced FN synthesis in MC and other cells (25, 31, 42, 45). Our present studies suggest the involvement of the p38MAPK-CREB pathway in 12(S)-HETE- and ANG II-induced FN transcription. Furthermore, ANG II effects on MC hypertrophy and FN expression may be mediated, at least in part, by the LO pathway. This is supported by our observations that the LO inhibitor blocks ANG II-induced leucine incorporation and that the 12-LO Rz blocks ANG II-induced FN mRNA expression.
Additional functional evidence was obtained from our results showing that 12(S)-HETE could directly lead to cellular hypertrophy and that a p38MAPK inhibitor blocked the hypertrophic effects of ANG II and 12(S)-HETE. Earlier reports indicated that oxidant stress and activation of the ERK and p38MAPK pathways might mediate the hypertrophic effects of ANG II in VSMC (42, 51). Taken together, our results demonstrate that 12-LO activation plays a key role in MAPK activation and cellular growth in rat MC, properties that implicate it in the pathology of glomerulosclerosis. We recently showed that a VSMC cell line overexpressing 12-LO could consume nitric oxide at greater rates than control mock-transfected VSMC, suggesting that LOs may contribute to renal dysfunction not only by the bioactivity of their lipid products, but by also serving as a catalytic sink for nitric oxide (6). These studies provide additional support for the important role of 12-LO in vascular and renal disorders.
12(S)-HETE could lead to cellular effects and gene regulation by various mechanisms. HETEs can activate PKC directly or indirectly by incorporating into membrane phospholipids, which then generate HETE-containing diacylglycerol species to activate PKC (26, 36). In the present studies, PKC activation upstream of p38MAPK may also be a key operative mechanism. Earlier studies have indicated that 12(S)-HETE can activate members of the MAPK pathway in fibroblasts and VSMC (41, 42, 54, 55). Cardiac fibroblasts overexpressing 12-LO had increased p38MAPK, which seemed to mediate the increased growth in these cells (53). LO products can modulate calcium currents in VSMC (47) and also induce oxidant stress as well as inflammatory gene expression (38, 44). In cancer cells, 12(S)-HETE has been shown to induce multiple signaling events through a potential receptor-mediated mechanism (28). However, it is not known whether renal cells have 12(S)-HETE receptors. Hence, the specific upstream mechanisms by which oxidized lipids such as 12(S)-HETE initiate signaling and MAPK activation in rat MC are not clear.
The regulation of pathological gene expression by lipids such as 12(S)-HETE could be relevant to several renal complications, including diabetic nephropathy. Increased levels of 12(S)-HETE have been observed in hypertensive and diabetic patients (2, 13). LO has been implicated in the oxidation of low-density lipoproteins and in the pathogenesis of atherosclerosis (33, 39, 61). We recently demonstrated increased 12-LO expression in a swine model of diabetes-induced atherosclerosis under hyperlipemic and hyperglycemic states (33). 12-LO expression was also increased in a rat carotid artery model of balloon injury and restenosis (15, 37). In this model, neointimal thickening in vivo could be significantly attenuated by a conventional long Rz directed to the rat leukocyte-type 12-LO, which was also effective in reducing PDGF-induced migration in rat VSMC (15, 40). In the present study, we showed that specific blockade of rat 12/15-LO with a modified "short" Rz could inhibit ANG II-induced FN mRNA expression in rat MC. This further supports the involvement of LO activation in ANG II-induced hypertrophy and FN expression.
Rzs are RNA enzymes that catalytically cleave specific RNA sequences, resulting in irreversible inactivation of the target RNAs (4, 43). Rzs have a decided advantage over antisense oligonucleotides because of their cleavage and catalytic activity, which lower the concentrations required for reducing gene expression (1, 16). We recently showed that a Rz directed to porcine leukocyte-type 12-LO is effective in vitro and ex vivo in porcine VSMC (40, 16). We also designed and demonstrated the ex vivo and in vivo efficiency of a rat 12-LO Rz in VSMC and a restenosis model (15). Rzs are therefore powerful tools to evaluate the mechanistic and functional consequences of reduced 12-LO activity and expression. Our results illustrate the utility of Rz, especially newer-generation Rz, to reduce expression of genes related to the pathogenesis of diabetic nephropathy.
Our recent studies have shown that 12-LO expression is stimulated by high-glucose culture of rat MC and also increased in glomeruli from diabetic rats in parallel with FN expression (23). Furthermore, the expression and activity of p38MAPK, as well as its upstream kinase activator MKK3/6, were also increased in this model (22). Because high-glucose culture of rat MC can increase p38MAPK activation, our present studies suggest a novel interaction between the p38MAPK and 12-LO pathways in mediating the effects of ANG II and high glucose on matrix deposition and glomerulosclerosis. Hence, therapeutic modalities and novel Rz technology to block 12-LO may be beneficial in the treatment of diabetic renal diseases.
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ACKNOWLEDGEMENTS |
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The authors thank Piotr Swiderski for help with the synthesis of the ribozyme and mutant ribozyme oligonucleotides.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1 DK-58191 (to R. Natarajan) and RO1 DK-397921 (to J. L. Nadler) and by the Juvenile Diabetes Research Foundation Grants (to R. Natarajan and S. G. Aadler).
Address for reprint requests and other correspondence: R. Natarajan, Dept. of Diabetes, Beckman Research Institute of the City of Hope, 1500 East Duarte Rd., Duarte, CA 91010 (E-mail: rnatarajan{at}coh.org).
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.
June 11, 2002;10.1152/ajprenal.00181.2002
Received 9 May 2002; accepted in final form 3 June 2002.
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