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Renal-Electrolyte and Hypertension Division of the Department of Medicine and the Penn Center for the Molecular Studies of Kidney Diseases, University of Pennsylvania, Philadelphia, Pennsylvania.
Correspondence to Dr. Fuad N. Ziyadeh, 700 Clinical Research Building, Renal-Electrolyte and Hypertension Division, University of Pennsylvania, 415 Curie Boulevard, Philadelphia, PA 19104-6144. Phone: 215-573-1837; Fax: 215-898-0189; E-mail: ziyadeh{at}mail.med.upenn.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1(I) collagen and fibronectin mRNA levels. Treatment with PD98059
(up to 25 µM) significantly inhibited these parameters. In contrast, 25
µM PD98059 had no significant effect on any of the parameters measured in
cells that were exposed to normal glucose. Overexpression of MAPK phosphatase
CL 100 prevented TGF-ß1 promoter activation by high glucose, confirming
the involvement of the MEK-ERK pathway in response to high glucose. The
conclusion is that activation of ERK in mesangial cells is responsible for
high-glucose-induced stimulation of TGF-ß1 and contributes to the
increased extracellular matrix expression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, three mitogen-activated protein kinase (MAPK) families have been identified and characterized (12); these are extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase. These kinases can be activated by various extracellular stimuli, including growth factors and environmental stresses, and they play an essential role in the signal transduction cascades that lead to alterations in cell growth and other key functions (12,13). With regard to the diabetic state, it has been reported that ERK is activated in glomeruli of diabetic rats as well as in mesangial cells cultured under high-glucose conditions (14,15). ERK activation in these cells is believed to occur through a PKC-dependent mechanism (14). A recent report also demonstrated that p38 MAP kinase was activated by relatively high concentrations of extracellular glucose in various type of cells, including mesangial cells (16). In contrast to these two kinases, JNK is not activated in the glomeruli of diabetic rats or stimulated in mesangial cells upon exposure to high glucose (17). Thus, the activation of the ERK pathway could be involved in the intracellular signaling cascade that leads to cellular dysfunction in diabetes mellitus. It is likely that the activation of ERK is involved in regulating cellular functions, including proliferation and protein synthesis. For example, ERK can activate Elk-1, a member of the ternary complex factor, resulting in c-fos transcription and the subsequent formation of the activator protein-1 (AP-1) transcription factor complex (18,19). However, the biologic consequences of ERK activation in mesangial cells by high ambient glucose have not been explored.
We postulated that increased renal TGF-ß bioactivity by high ambient
glucose is important in the pathogenesis of diabetic nephropathy
(4,20,21,22).
Studies that have used neutralizing anti—TGF-ß antibodies have
provided convincing evidence that the prosclerotic and hypertrophic effects of
high glucose are largely mediated by the autocrine production and activation
of TGF-ß in glomerular mesangial cells, proximal tubular cells, and
interstitial fibroblasts
(2,4,23,24).
We recently reported that high glucose or phorbol esters stimulate TGF-ß1
production in mouse mesangial cells through a transcriptional mechanism
(25). The TGF-ß1 promoter
has multiple AP-1—like consensus sites that respond to phorbol-ester/PKC
stimulation (26). From these
observations, it can be hypothesized that the activation of the PKC-ERK
pathway by high glucose could be responsible for the induction of TGF-ß1
and the production of extracellular matrix. To test this hypothesis, we
examined in the current study the effect of PD98059, a specific inhibitor of
MAPK/ERK kinase (MEK), on the expression of TGF-ß1, 1(I) collagen,
and fibronectin in mesangial cells that were cultured in high glucose. To
confirm the involvement of the MEK-ERK pathway, we also assessed TGF-ß1
promoter activity in response to high glucose after the overexpression of
CL100, a MAP kinase phosphatase.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ERK In Vitro Kinase Assay
Cells were lysed in a buffer containing 25 mM Tris-HCl (pH 7.4), 25 mM
NaCl, 80 mM ß-glycerophosphate, 10 mM sodium pyrophosphate, 1 mM
Na3VO4, 10 mM NaF, 4 mM ethyleneglycol-bis(ß
aminoethyl ether)-N,N'-tetraacetic acid, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 1% Triton X-100. The
cell lysates were centrifuged at 14,000 x g for 20 min, and
protein concentrations were measured by a protein assay kit (Bio Rad,
Richmond, CA). Cell lysates (400 µg) were incubated with 2 µg anti-ERK2
antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at 4°C. The
immunoprecipitates were recovered by incubating with protein G sepharose
(Amersham Pharmacia, Piscataway, NJ) for 1 h at 4°C. After centrifuging
and washing three times with cell lysis buffer and once with kinase buffer (20
mM Tris-HCl [pH 7.4], 10 mM MgCl2, 1 mM DTT, 1 mM
ethyleneglycol-bis(ß aminoethyl ether)-N,N'-tetraacetic
acid, and 1 µM protein kinase inhibitor), the immunoprecipitates were
incubated with 30 µl of a kinase buffer containing 20 µg of myelin basic
protein (MBP), 50 µM adenosine triphosphate (ATP) and 2 µCi
-[32P]ATP for 15 min at 25°C. The mixture (15 µl) was
spotted onto P-81 phosphocellulose paper (Whatman, Clifton, NJ), washed four
times in 0.5% phosphoric acid, and rinsed with 95% ethanol. Phosphorylation
activity was determined by a liquid scintillation counter.
Enzyme-Linked Immunosorbent Assay
Cell supernatants were frozen at —20°C until assayed by a
TGF-ß1 enzyme-linked immunosorbent assay (ELISA) kit according to the
manufacturer's specifications (R & D systems, Minneapolis, MN). In brief,
the supernatants were activated with 1 N HCl followed by neutralization with
1.2 N NaOH/0.5 M HEPES to measure total TGF-ß1. Samples were plated on
anti—TGF-ß type II receptor-coated microtiter plates and incubated
for 3 h at room temperature. After vigorous washing, wells were incubated with
anti—TGF-ß1 antibody conjugated to horseradish peroxidase for 1.5
h, and substrate solution was added. The reaction was stopped by adding stop
solution, and absorbance at 450 nm was measured. Total TGF-ß1 protein
production was reported per total cell protein content.
Immunoblot Analysis
Cell lysates were prepared in Laemmli sample buffer and boiled for 5 min.
Aliquots (30 µg) were subjected to sodium dodecyl sulfate (SDS)-10%
polyacrylamide gel and transferred to nitrocellulose membrane (Micron
Separations Inc., Westborough, MA). The membrane was blocked with 5% nonfat
dry milk in Tris-buffered saline-0.1% Tween 20 at 4°C overnight and then
probed with either anti-phospho-p44/42 antibody (New England Biolabs, Beverly,
MA) or anti-MAP kinase phosphatase (MKP)-1 antibody (Santa Cruz Biotechnology)
at room temperature for 3 h. A horseradish peroxidase-conjugated second
antibody was used to allow the detection of immunoreactive bands using the
enhanced chemiluminescence detection system (Amersham Pharmacia). For the
assessment of protein amount of ERK, the membrane was reprobed with anti-ERK2
antibody (Santa Cruz Biotechnology).
Northern Blot Analysis
Murine TGF-ß1, fibronectin, and 1(I) cDNA probes were
synthesized by PCR and cloned into the pCRII TA cloning vector (Invitrogen, La
Jolla, CA) as described previously
(28). Total RNA was isolated
using TRIzol reagent (Life Technologies BRL). For northern blots, 20 µg
total RNA was electrophoresed through a 1.2% agarose gel with 0.67 M
formaldehyde. The RNA was blotted onto nylon membrane (NEN Research Products,
Boston, MA) by the capillary method and UV cross-linked. Membranes were
prehybridized for 1 h at 65°C in a Church buffer containing 500 mM NaPO4
(pH 7.0), 5% SDS, 1% bovine serum albumin, and 1 mM
ethylenediaminetetraacetate. The cDNA was labeled with
[32P]deoxycytidine 5'-triphosphate (Amersham Pharmacia) using
a DNA labeling kit (Amersham Pharmacia). The membrane was hybridized in Church
buffer for 16 h at 65°C in a rotating oven and was washed twice in 2X SSC
(20X SSC: 3 M NaCl, 0.3 M sodium citrate, pH 7.0) for 10 min each at room
temperature, then in 2X SSC with 1% SDS for 15 min at 65°C, followed by
two 15-min high-stringency washes in 0.1% SSC, 0.1% SDS at 65°C, if
necessary. The membrane was autoradiographed with intensifying screens at
-70°C (Kodak, Wilmington, DE). The blots were stripped and subsequently
rehybridized with probes encoding mouse ribosomal protein L32 (mrpL32)
(29) to account for small
loading and transfer variations. Exposed films were scanned and analyzed with
the NIH Image 1.62 program, and RNA levels relative to those of mrpL32 were
calculated.
Plasmids, Transfection, and Luciferase Assay
Murine TGF-ß1 promoter-reporter chimeric constructs were kindly
provided by Dr. Andrew G. Geiser
(30). The construct pA835 used
contains -835 bp 5' from the A transcription start site of the murine
TGF-ß1 gene (30). The
luciferase reporter gene used was cloned into the
HindIII-KpnI site of the pXP2 vector
(31), which contains the
luciferase gene without regulatory elements. The MAP kinase phosphatase CL100
in the expression plasmid pSG5 (Stratagene, La Jolla, CA) was kindly provided
by Dr. Stephen M. Keyse
(32,33).
The ß-galactosidase-containing plasmid pCH110 (Amersham Pharmacia) was
used to control for transfection efficiency. MMC (50% confluent) were
transfected by the Fugene 6 transfection reagent (Roche Molecular
Biochemicals, Indianapolis, IN) following the manufacturer's instructions.
After 16 h of transfection, cells were incubated in either normal or high
glucose for an additional 24 h. Cells were harvested in reporter lysis buffer
(Promega, Madison, WI). Luciferase and ß-galactosidase assays were
performed with reagents from Promega. Luciferase activity was normalized to
ß-galactosidase activity.
Measurement of [3H]-Proline Incorporation
MMC were plated into 24-well plates (Nunclon, Roskilde, Denmark), and the
media were changed the next day so that cells could be exposed for another 72
h to either 5.5 or 25 mM glucose with or without PD98059. For the last 16 h,
cells were pulsed with 1 µCi [3H]-proline
(L-(2,3,4,5)-[3H]-proline, Amersham Pharmacia). Radiolabeled MMC
were washed twice in ice-cold phosphate-buffered saline and then precipitated
twice in ice-cold 10% TCA, redissolved in 0.5 ml of 0.5 N NaOH with 0.1%
Triton X-100. After neutralization with 0.5 N HCl, the incorporated
radioactivity was counted in a liquid scintillation counter. Proline
incorporation was corrected for the cellular protein content.
Statistical Analyses
Data are presented as mean ± SEM. ANOVA followed by Scheffe's test
was used for multiple comparisons. Two groups were compared by unpaired
t test. P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effect of PD98059 on High-Glucose—Induced TGF-ß1 Protein
Production
To assess the biologic significance of ERK activation, we first examined
whether PD98059 could inhibit TGF-ß1 production by high glucose. MMC were
cultured in media containing normal and high glucose with various
concentrations of PD98059 for 72 h. TGF-ß1 protein in the supernatant was
measured by ELISA. As we reported previously
(25), total TGF-ß1
production was significantly increased in supernatants of cells cultured in
high glucose compared with cells cultured in normal glucose. TGF-ß1
protein production was inhibited by exposure to PD98059 in a dose-dependent
manner. Twentyfive µM PD98059 completely prevented the increase in
TGF-ß1 by high glucose without changing TGF-ß1 production in cells
cultured in normal glucose (Figure
2).
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Effect of PD98059 on High-Glucose—Induced TGF-ß1 Gene
Expression
To confirm the inhibitory effect of PD98059 on TGF-ß1 production, we
examined the high-glucose—induced TGF-ß1 gene expression by
Northern blot analysis and promoter assay. TGF-ß1 mRNA was significantly
increased in cells cultured in high glucose, and PD98059 was able to inhibit
completely the increase in TGF-ß1 mRNA
(Figure 3). We next evaluated
the effect of PD98059 on TGF-ß1 promoter activity. Because we recently
demonstrated that only the pA835 promoter construct, which contains 835 bp
upstream from the transcription start site of the murine TGF-ß1,
exhibited significant activation by high glucose
(25), we used in this
experiment the pLA835 plasmid, which contains the same region of TGF-ß1
promoter fused to the luciferase gene. MMC were transiently contransfected
with pLA835 and the ß-galactosidase-containing plasmid pCH110, to control
for transfection efficiency. Cells were then cultured in high-glucose media
with or without PD98059 for an additional 24 h. Relative luciferase activity
to ß-galactosidase was calculated. As shown in
Figure 4, high glucose
significantly stimulated promoter activity of TGF-ß1, whereas PD98059
inhibited the induction of TGF-ß1 promoter activity. Treatment of
mesangial cells cultured in normal glucose media with 25 µM PD98059
affected neither baseline mRNA expression nor promoter activity.
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Effect of Overexpression of MAP Kinase Phosphatase CL100 on
TGF-ß1 Promoter Activity
To confirm further the involvement of ERK in TGF-ß1 induction under
high-glucose conditions, we cotransfected cells with a plasmid expressing MAP
kinase phosphatase CL100, which inactivates ERK
(32,33).
Contrasfection of MMC with pSG5-CL100 prevented the induction of TGF-ß1
promoter by high glucose; however, expression of the empty, control vector
produced the expected increment in TGF-ß1 promoter activity in
high-glucose media (Figure 5A).
The protein content of MAP kinase phosphatase and ERK were not changed in
cells transfected with CL100 (Figure
5B). We also confirmed that the overexpression of CL100 in the
cells prevented the activation of ERK by high glucose
(Figure 5C).
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Effect of PD98059 on High-Glucose—Induced Matrix
Production
We next assessed whether PD98059 could inhibit extracellular matrix
production induced by high glucose. We examined [3H]-proline
incorporation and performed Northern blot analysis for the expression of
1(I) collagen and fibronectin.
Figure 6 shows that
[3H]-proline incorporation into MMC was significantly increased in
cells grown in high-glucose media, as was expected
(24). The incubation with
PD98059 significantly inhibited the increment in [3H]-proline
incorporation into MMC. Northern blot analyses
(Figure 7) demonstrated that
the mRNA for 1(I) collagen and fibronectin were increased by
approximately 50% in high-glucose media and that PD98059 abolished these
increases without effecting mRNA expression in cells cultured in normal
glucose.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously reported that high glucose stimulates TGF-ß1 production in mouse mesangial cells through a transcriptional mechanism that involves a specific glucose-responsive DNA-binding element (25). We also recently reported that high glucose stimulates the TGF-ß type II receptor in mouse mesangial cells (22). In this study, we show that treatment of mesangial cells with PD98059 significantly inhibits the high-glucose—induced TGF-ß1 protein production, mRNA level, and promoter activity. Furthermore, and consistent with the requirement for ERK phosphorylation in the stimulation of TGF-ß1 by high glucose, we show that the overexpression of CL100, a MAPK phosphatase, prevents the activation of the TGF-ß1 promoter by high glucose.
ERK activation is able to activate Elk-1, a member of the ternary complex factors that enhance the expression of c-fos (18) and the subsequent DNA binding of the transcription factor AP-1 (19). The TGF-ß1 promoter has multiple AP-1—like consensus sites that respond to phorbol-ester/PKC stimulation (26). An increase in AP-1 binding has been reported to mediate the regulation of the TGF-ß1 gene (38). Thus, it is likely that the stimulation of ERK by high glucose transduces a signal to the nucleus, where it regulates the expression of certain target genes through AP-1 transactivation (18,19). In fact, it has been reported that high glucose stimulates the expression of c-fos and c-jun, components of the AP-1 complex (39), and the binding of AP-1 to DNA in cultured mesangial cells (40). Together, these observations suggest that the ERK pathway plays an important role in the transcriptional activation of TGF-ß1 in mesangial cells cultured under high-glucose conditions. Other studies have suggested that the ERK pathway is itself also involved in the signaling events downstream of the TGF-ß stimulus (41,42).
We also provide evidence that the activation of ERK by high glucose is
required for the production of extracellular matrix proteins, including
1(I) collagen and fibronectin. Treatment with PD98059 prevents the
high-glucose—induced uptake of proline, a major constituent of collagen
proteins, and the increased mRNA levels of 1(I) collagen and
fibronectin. There are two possible mechanisms for these inhibitory effects of
PD98059 on matrix expression. First, PD98059 may directly inhibit the
production of extracellular matrix components. Although several reports
incriminate PKC activation in renal matrix production in diabetes
(8,9,11),
little is known about the direct contribution of ERK activation on matrix
protein synthesis. Fibronectin gene expression can be regulated through the
binding of AP-1 to a cAMP response element in the promoter region
(43). A recent report has
demonstrated that PD98059 inhibits the mechanical stretch-induced fibronectin
production through the inhibition of the ERK—AP-1 pathway
(44). Furthermore, expression
of TGF-ß—induced type I collagen is mediated via the ERK pathway in
certain cell types, including mesangial cells
(41,42).
Together, these observations strongly suggest that ERK activation may be
directly involved in the regulation of extracellular matrix protein gene
expression, at least for type I collagen and fibronectin.
The second possibility is that the high-glucose—induced matrix production is inhibited by PD98059 through the inhibition of the profibrotic cytokine TGF-ß1. This possibility is supported by our previous report demonstrating that treatment of mesangial cells with neutralizing anti—TGF-ß antibodies attenuates high-glucose—induced proline uptake and the synthesis of types I and IV collagen (24). Furthermore, inhibition of TGF-ß bioactivity in diabetic mice using either neutralizing anti—TGF-ß antibodies (28,45) or antisense TGF-ß1 oligode-oxynucleotides (46,47) is capable of reversing the renal hypertrophy and upregulation of extracellular matrix expression. Together, these results suggest that inhibition of TGF-ß1 is one of the important approaches that can be considered for the prevention of the functional and structural consequences of diabetic renal disease.
In addition to hyperglycemia, many factors in the diabetic state, such as glomerular hypertension, oxidative stress, non-enzymatic glycation adducts, and growth factors, have been proposed to mediate diabetic nephropathy (5). Some of these factors, such as mechanical stretch (44), angiotensin II (48), endothelin (49), and advanced glycation end products (50), are able to activate ERK in cultured renal cells. These same factors are also activators of the TGF-ß system in the kidney (5). From all of these observations, we can conclude that the activation of ERK by high ambient glucose and other factors leads to increased TGF-ß1 production as well as extracellular matrix proteins. Renal overproduction of TGF-ß1, in turn, can further exaggerate the excess synthesis and accumulation of extracellular matrix proteins in the diabetic milieu (Figure 8). Thus, interventions at the level of ERK to intercept this intracellular signaling pathway may be a useful approach for inhibiting not only the TGF-ß system but also other pertinent downstream events that are characteristic of diabetic renal disease, such as increased matrix expression.
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Acknowledgments |
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