1 Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; and 2 Department of Cell Biology, Peking Union Medical College, Beijing 100005, China
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ABSTRACT |
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Hepatocyte
growth factor (HGF) receptor, the product of the c-met
protooncogene, is transcriptionally regulated by a wide variety of
cytokines as well as extracellular environmental cues. In this report,
we demonstrate that c-met expression was significantly suppressed by
oxidative stress. Treatment of mouse renal inner medullary collecting
duct epithelial cells with 0.5 mM H2O2
inhibited c-met mRNA and protein expression, which was concomitant with induction of Egr-1 transcription factor. Ectopic expression of Egr-1 in renal epithelial cells markedly inhibited endogenous c-met
expression in a dose-dependent fashion, suggesting a causative effect
of Egr-1 in mediating c-met suppression. The cis-acting element responsible for H2O2-induced c-met
inhibition was localized at nucleotide position 223 to
68 of c-met
promoter, in which reside an imperfect Egr-1 and three Sp1-binding
sites. Egr-1 markedly suppressed c-met promoter activity but did not
directly bind to its cis-acting element in the
c-met gene. Induction of Egr-1 by oxidative stress
attenuated the binding of Sp1 to its cognate sites, but it did not
affect Sp1 abundance in renal epithelial cells. Immunoprecipitation
uncovered that Egr-1 physically interacted with Sp1 by forming the
Sp1/Egr-1 complex, which presumably resulted in a decreased
availability of unbound Sp1 as a transcriptional activator for the
c-met gene. Thus it appears that inhibition of c-met
expression by oxidative stress is mediated by the interplay between Sp1
and Egr-1 transcription factors. Our findings reveal a novel
transcriptional regulatory mechanism by which Egr-1 sequesters Sp1 as a
transcriptional activator of c-met via physical interaction.
hepatocyte growth factor; c-met receptor; gene transcription; tubular epithelial cells; H2O2
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INTRODUCTION |
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HEPATOCYTE GROWTH
FACTOR (HGF) receptor is a member of the receptor tyrosine kinase
superfamily and is encoded by the c-met protooncogene
(3, 29). Mature c-met receptor is a heterodimeric protein
consisting of an extracellular -subunit and a
-subunit harboring
an extracellular portion, a transmembrane segment and the cytoplasmic
tyrosine kinase domain (32). On binding to its specific
ligand, c-met receptor undergoes autophosphorylation of multiple
tyrosine residues in its cytoplasmic region and triggers cascades of
signal transduction events that lead to such diverse cellular responses
as cell survival, proliferation, migration, and differentiation
(5, 6, 17, 24-26). Because all biological activities
of HGF are mediated by a single receptor, the expression and levels of
c-met protein not only determine the cell-type specificity of HGF
actions but also dictate the overall activities of this paired
signaling system. Consistent with this view, earlier studies indicate
that it is the abundance and activity of c-met receptor, rather than
HGF itself, that best correlate with the biological function of HGF in
diverse organs under physiological and pathological conditions
(8, 28, 39).
The expression of the c-met receptor is primarily regulated at the transcriptional level (21). Unlike its ligand, which is expressed mainly in mesenchyme-derived cells in normal tissues (19, 26), c-met expression is relatively ubiquitous with a widespread pattern. For instance, in normal adult kidney and cultured kidney cells, c-met protein is detected in all types of renal cells including glomerular mesangial cells, podocytes, proximal tubular cells, collecting duct epithelial cells, and interstitial fibroblasts, although high level of c-met is only observed in distal tubules and collecting duct epithelia under normal circumstances (46). This prevalent pattern of c-met constitutive expression is closely overlapped with, and predominantly dictated by, the ubiquitous specificity protein (Sp) family of transcription factors (18, 37, 46). Both Sp1 and Sp3 avidly bind to the GC boxes (Sp1-binding sites) in the c-met promoter region and transcriptionally activate its expression; and together, they interact with each other, forming heterodimeric complexes and elicit synergistic actions on c-met gene transcription (46). The broad expression pattern of the c-met gene likely highlights a wide implication of HGF in normal cell physiology in diverse organs.
A wide variety of cytokines, growth factors, and hormones has been reported to induce c-met expression in renal epithelial cells and other types of cells (21, 23, 27, 36). Expression of the c-met gene is also induced rapidly in the injured organs after diverse types of tissue-injurious insults (20, 21). Contrary to HGF whose expression is often stimulated in both injured and distal intact organs, c-met induction exclusively occurs at the site of injury and is principally correlated with the injury and subsequent repair process in a site-specific manner (14, 21, 34). In addition, c-met expression is induced in renal epithelial cells by altered extracellular environmental cues such as a high concentration of glucose in vitro and in diabetic states in vivo (22). Hence, in response to a vast diversity of stimuli, the c-met gene is commonly activated to induce its expression, a process that may be necessary and important for injury repair and regeneration. However, little is documented as to c-met transcriptional suppression in response to certain conditions, and it also remains largely unknown about the molecular mechanism governing c-met inhibition.
In this report, we demonstrate that oxidative stress inhibits c-met gene expression at both mRNA and protein levels. The suppression of c-met expression is accompanied by induction of Egr-1 transcription factor in renal epithelial cells after H2O2 treatment. Our results suggest that Egr-1 sequesters Sp1 by physical interaction, which leads to a decreased availability of free Sp1 for c-met transcriptional activation.
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MATERIALS AND METHODS |
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Cell culture and treatment. Mouse inner medullary collecting duct epithelial cell line 3 (mIMCD-3) was obtained from American Type Culture Collection (ATCC, Manassas, VA). Human kidney proximal tubular cells (HKC) were provided by Dr. L. Racusen of Johns Hopkins University (Baltimore, MD). Cells were maintained in DMEM and Ham's F-12 medium (1:1 of DMEM/F12; Life Technologies, Grand Island, NY) supplemented with 10% FBS. Cells were seeded in plastic petri dishes (100 mm; Falcon) at 60-70% confluence. After being cultivated for 16 h in the complete medium containing 10% FBS, cells were serum-starved for 24 h and incubated for various periods of time as indicated in the absence (control) or presence of 0.5 mM H2O2 (Sigma-Aldrich, St. Louis, MO), except when otherwise indicated. The cells were then collected for analysis of gene expression at both mRNA and protein levels by Northern and Western blot analyses, respectively.
RNA preparation and Northern blot analysis.
Total RNA was extracted from the cells using Ultraspec RNA solution
(Biotecx, Houston, TX) according to the instructions specified by the
manufacturer. Samples of 20 µg total RNA were electrophoresed on
1.0% formadehyde-agarose gels and then transferred to GeneScreen plus
nylon membrane (DuPont, Boston, MA) by capillary blotting followed by
ultraviolet cross-linking. Membranes were prehybridized for 4 h at
65°C in a buffer containing 6× SSC, 5× Denhardt's solution, 1%
SDS, 10% dextran sulfate, and 100 µg/ml denatured salmon sperm DNA.
32P-labeled DNA probes were prepared by the random primer
labeling kit (Stratagene, La Jolla, CA) using
[-32P]dCTP. The rat c-met cDNA probe was
generated in our laboratory as described previously (23).
The human egr-1 cDNA was obtained from the ATCC. Denatured probes were
added to the same hybridization buffer at a concentration of
1-2 × 106 cpm/ml, and hybridization was allowed
to proceed at 65°C for 16 h. Membranes were washed and exposed
to X-ray film (Eastman Kodak, Rochester, NY) at
80°C with the aid
of an intensifying screen as described elsewhere (21, 44).
Quantitation was performed by determination of the intensity of the
hybridization signals using the National Institutes of Health Image program.
Western blot analysis. The mIMCD-3 cells were solubilized with RIPA lysis buffer (1% Nonidet P-40, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 2 µg/ml aprotin, 2 µg/ml leupeptin in PBS) at 4°C for 20 min, and the supernatants were collected after centrifugation at 13,000 g at 4°C for 10 min. Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit with bovine serum albumin as a standard (Sigma), and cell lysates were mixed with an equal amount of 2× loading buffer (100 mM Tris · HCl, 4% SDS, 20% glycerol, and 0.2% bromophenol blue). Samples were heated at 100°C for 10 min before loading and separated on precasted 10% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA) under nonreducing conditions. The proteins were transferred to a nitrocellulose membrane (Amersham, Arlington Heights, IL) in transfer buffer containing 48 mM Tris · HCl, 39 mM glycine, 0.037% SDS, and 20% methanol at 4°C for 2 h. Nonspecific binding to the membrane was blocked for 1 h at room temperature with 5% nonfat milk in TBS buffer (20 mM Tris · HCl, 150 mM NaCl, and 0.1% Tween 20), and then the membrane was incubated for 1 h with various primary antibodies followed by incubation for 1 h with a secondary horseradish peroxidase-conjugated IgG in 5% nonfat milk. The specific antibodies against c-met, Egr-1, Sp1, Sp3, and actin, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The signals were visualized by the enhanced chemiluminescence system (ECL, Amersham) as described elsewhere (43).
Construction of plasmid, DNA transfection, and luciferase assay. The various chimeric plasmids containing different lengths of the 5'-flanking region of the human c-met gene linked to the coding sequence for chloramphenicol acetyltransferase (CAT) had been described previously (18). In this study, the highly sensitive firefly luciferase reporter system was used. For that purpose, various DNA fragments containing different lengths of the c-met promoter region were excised with SstI and BglII from different c-met-CAT reporter plasmid vectors. The DNA fragments were gel purified and then subcloned into the SstI/BglII site of the promoterless pGL3-Basic luciferase expression vector (Promega, Madison, WI). The resultant chimeric reporter plasmids (designated as pGL3-0.7met, pGL3-0.2met, or pGL3-0.1met, etc.) were verified by detailed restriction mapping. For transient transfection, the mIMCD-3 cells were seeded in six-well plates at 2 × 105 cells/well. The cells were then transfected with various luciferase reporter plasmids using the Lipofectamine 2000 reagent according to the instructions specified by the manufacturer (Life Technologies). A fixed amount of internal control reporter Renilla reniformis luciferase driven under thymidine kinase (TK) promoter (pRL-TK, Promega) was cotransfected for normalizing the transfection efficiency and correcting firefly luciferase activity. After transfection, the cells were incubated for an additional 24 h in the absence or presence of 0.5 mM H2O2 before being harvested for luciferase assay. Luciferase assay was performed using the Dual Luciferase Assay System kit essentially according to the protocol specified by the manufacturer (Promega). Relative luciferase activity of each construct (arbitrary unit) was reported as fold-induction over pGL3-0.1met after normalizing for transfection efficiency. All experiments were repeated in a minimum of three separate experiments (triple wells per experiment) to assume reproducibility. The values from these experiments were combined and subjected to analysis of variance using SigmaStat statistical software (Jandel Scientific, San Rafael, CA). P < 0.05 was considered significant.
Preparation of nuclear protein extract.
The control and H2O2-treated mIMCD-3 cells were
cultured in the conditions described above. For preparation of nuclear
protein extracts, cells were washed twice with cold PBS and scraped off the plate with a rubber policeman. Cells were collected and the nuclei
were isolated according to the method described elsewhere (9,
46). Briefly, the pelleted cells were resuspended in 4 vol of
buffer A containing protease inhibitors (10 mM HEPES, pH
7.9, 1.5 mM MgCl2, 0.5% Nonidet P-40, 10 mM KCl, 0.2 mM
PMSF, and 0.5 mM dithiothreitol plus 1% protease inhibitor cocktail from Sigma-Aldrich). The cell suspension was transferred into a Dounce
homogenizer immediately after incubation on ice for 10 min to allow the
cells to swell. The cells were then lysed by 10 strokes. Cell nuclei
were collected by centrifugation at 3,500 g for 15 min at
4°C and suspended in the same volume of buffer A without
Nonidet P-40. The nuclei were resuspended in 150 µl of buffer
B (20 mM HEPES, pH 7.9, 10% glycerol, 1.5 mM MgCl2, 10 mM KCl, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM dithiothreitol plus
protease inhibitor cocktail). Fifty-microliters of buffer B
containing 1.6 M KCl were added into the nuclei suspension in a
dropwise fashion, followed by incubation on ice for 1 h. The nuclear extracts were collected by centrifugation at 4°C at 25,000 g for 30 min. Aliquots of protein extract were quickly
frozen and stored at 80°C after the protein concentration had been
determined using a BCA protein assay kit (Sigma-Aldrich).
EMSA.
The double-strand Sp1/Egr-1 oligonucleotide (oligo) corresponding to
the nucleotide sequence of 86~
61 in the c-met promoter which
contains a perfect Sp1 site overlapped with an imperfect Egr-1 site was
labeled with [
-32P]ATP using T4 kinase
(Life Technologies). An oligo containing two Egr-1-binding sites was
purchased from Santa Cruz Biotechnology and used as a positive control
for Egr-1 binding. The labeled probes were then gel purified and used
in EMSA as described previously (46). Four micrograms of
Poly(dI-dC)-Poly(dI-dC) (Pharmacia, Piscataway, NJ) were used as the
nonspecific competitor in 10 µl reaction mixture. The binding
reactions were carried out at 37°C for 30 min before loading of 5%
nondenaturing polyacrylamide (19:1, acrylamide:bisacrylamide) gels. For
competition experiments, 100-fold molar excess of unlabeled
double-strand oligo was included in the reaction mixture except where
indicated otherwise. For supershift experiments, specific antibodies
against Sp1, Sp3, Egr-1, and normal control IgG (Santa Cruz) were
incubated with nuclear protein extracts for 30 min at 37°C before
addition of reaction buffer. Gels were run in 0.5× TBE buffer (0.045 M
Tris · borate, 0.001 M EDTA) at a constant
voltage of 190 V, dried, and autoradiographed with intensifying screens.
Immunoprecipitation. Renal epithelial mIMCD-3 cells grown on 100-mm plate were lysed on ice in 1 ml RIPA buffer containing 1× PBS, 1% Nonidet P-40, 0.1% SDS, 10 µg/ml phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 1% protease inhibitors cocktail (Sigma). Whole cell lysates were clarified by centrifugation at 12,000 g for 10 min at 4°C, and the supernatants were transferred into a fresh tube. To preclear cell lysates, 0.25 µg of normal rabbit IgG and 20 µl of protein A/G Plus-Agarose (Santa Cruz) were added into 1 ml of whole cell lysates. After incubation for 1 h at 4°C, supernatants were collected by centrifugation at 1,000 g for 5 min at 4°C. Lysates were immunoprecipitated overnight at 4°C with 1 µg of anti-Sp1 and anti-Egr-1, respectively, followed by precipitation with 20 µl of protein A/G Plus-Agarose for 3 h at 4°C. After four washes with RIPA buffer, the immunoprecipitates were boiled for 5 min in SDS sample buffer. The resulting precipitated complexes were separated on SDS-polyacrylamide gels and blotted with various antibodies as described above.
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RESULTS |
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Oxidative stress inhibits c-met expression while concomitantly
inducing egr-1.
We investigated the effects of oxidative stress on c-met
gene expression in renal epithelial cells in vitro. As shown in Fig. 1, incubation of renal collecting duct
epithelial cells (mIMCD-3) with 0.5 mM H2O2
markedly suppressed the steady-state levels of c-met mRNA in a
time-dependent manner. At 6 h after H2O2
treatment, ~70% inhibition of c-met mRNA was observed in mIMCD-3
cells, as determined by quantitatively measuring the intensity of the
hybridization signal. This inhibition of c-met expression by
H2O2 was also dose dependent, and significant
inhibition of c-met mRNA was observed at a concentration as low as 0.05 mM (data not shown). Of note, at the concentration of 0.5 mM as used in
this study, H2O2 did not significantly cause
cell death, as the lactase dehydrogenase activity in the supernatant of
control and H2O2-treated mIMCD-3 cell culture
is essentially identical (data not shown). H2O2
also had no appreciable effects on housekeeping gene GAPDH expression.
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Ectopic expression of egr-1 suppresses endogenous c-met expression
in renal epithelial cells.
To test a direct relevance of Egr-1 induction and c-met suppression, we
investigated the effects of ectopic expression of exogenous Egr-1 on
c-met expression in renal tubular epithelial cells by transfecting the
Egr-1 expression vector. As shown in Fig.
3, forced expression of exogenous Egr-1
suppressed endogenous c-met expression in mIMCD-3 cells in a
dose-dependent fashion. Quantitative determination of the relative
abundances exhibited a closely inverse relationship between c-met and
Egr-1 proteins in mIMCD-3 cells following transfection of different
amounts of Egr-1 expression vector (Fig. 3C). Similarly,
expression of exogenous Egr-1 also inhibited endogenous c-met
expression in HKC cells (Fig. 3, B and D). Thus
these results establish that Egr-1 may directly mediate the suppression
of c-met expression by oxidative stress in renal tubular epithelial
cells.
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Promoter region responsible for c-met inhibition by oxidative
stress contains both Egr-1- and Sp1-binding sites.
To further investigate how Egr-1 mediates c-met inhibition by oxidative
stress in renal epithelial cells, we defined the cis-acting element(s) responsible for c-met inhibition in the promoter region of
the c-met gene. Various lengths of the c-met promoter region coupled with the firefly luciferase reporter gene were transfected into
renal epithelial mIMCD-3 cells. The luciferase activities were
determined in the transfected cells after incubation in the absence or
presence of 0.5 mM H2O2 for 24 h. As shown
in Fig. 4, H2O2
significantly suppressed the reporter activities of pGL3-0.7met and pGL3-0.2met constructs that contain 0.7- and 0.2-kb
5'-flanking regions of the c-met gene, respectively.
However, no significant inhibition of reporter activity was observed
after transfection with pGL3-0.1met construct in response to
H2O2 treatment (1.0 ± 0.03 in control vs.
0.58 ± 0.06 in H2O2 group,
P = 0.692, n = 3). Hence, the
cis-acting element responsible for c-met inhibition was
largely localized at a 156-bp region corresponding to nucleotide position 223 to
68 of c-met gene. Sequence analysis
revealed that there are an imperfect Egr-1- and three Sp1-binding sites within this region (Fig. 4C) (18), which led us
to speculate that alterations in cellular abundance or activity of
Egr-1 and/or Sp proteins might be involved in mediating c-met
inhibition in response to oxidative stress.
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Egr-1 inhibits c-met promoter activity but does not directly bind
to the cis-acting element.
To establish the functional significance of Egr-1 in controlling
c-met gene transcription, we examined the effects of
overexpression of Egr-1 on c-met promoter activity by cotransfecting
the Egr-1 expression vector and c-met promoter reporter construct into
renal epithelial cells. As shown in Fig.
5, transfection of increasing amounts of
Egr-1 expression vector and pGL3-0.2met reporter construct that
contains an imperfect Egr-1-binding site suppressed c-met promoter
activity in a dose-dependent manner, suggesting that Egr-1 is critical
for c-met transcriptional suppression.
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Induction of Egr-1 by oxidative stress attenuates the binding of
Sp1 to its cognate site but does not affect the cellular abundance of
Sp proteins.
Given that Egr-1 does not bind to the c-met promoter, we next compared
the binding ability of Sp1 protein to the Sp1/Egr-1 site under basal
and H2O2-treated conditions. As shown in Fig. 7, A and C,
induction of Egr-1 by oxidative stress significantly suppressed the
binding of Sp proteins to the c-met promoter. Weak Sp1 and Sp3 binding
was observed when nuclear extract rich in Egr-1 from
H2O2-treated mIMCD-3 cells was used, suggesting
that a high level of Egr-1 may interfere with Sp protein binding. Of interest, preabsorption of the nuclear extract from
H2O2-treated mIMCD-3 cells with anti-Egr-1
antibody tended to restore the binding ability of Sp proteins (Fig. 7,
B and D). This implies that a high level of Egr-1
induced by oxidative stress is likely responsible for the attenuation
of Sp proteins binding to c-met promoter in renal epithelial cells.
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Physical interaction of Egr-1 with Sp1.
The observation that Egr-1 does not directly bind to the c-met promoter
(Fig. 6) raises an intriguing possibility that Egr-1 may affect the
binding of Sp proteins to the c-met promoter through protein-protein
interaction. To test this hypothesis, we examined the interaction
between Egr-1 and Sp1 by a coimmunoprecipitation approach using
specific antibodies against Egr-1 and Sp1. As shown in Fig.
8, when cell lysates derived from either
control or H2O2-treated renal epithelial
mIMCD-3 cells were immunoprecipitated with anti-Sp1 antibody, Egr-1 was
detected in the precipitated complexes (Fig. 8A). The
abundance of Egr-1 present in the precipitated complexes was largely
proportional to the cellular Egr-1 level after
H2O2 treatment in a time-dependent manner.
Similarly, Sp1 protein was also detected in the reciprocal experiments
when cell lysates were immunoprecipitated with Egr-1 antibody (Fig.
8B), suggesting that Egr-1 binds to Sp1 through a
protein-protein interaction. However, in control mIMCD-3 cells that are
poor in Egr-1, Sp1 was not found in the Egr-1 immunoprecipates (Fig.
8B). Therefore, it is conceivable that after oxidative
stress, increased Egr-1 tends to bind Sp1 protein via protein-protein
interaction, leading to sequestration of Sp1 for c-met gene
transcriptional activation.
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DISCUSSION |
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HGF receptor signaling plays a critical role in controlling diverse types of cellular responses such as cell survival, proliferation, migration, and differentiation (17, 25, 26, 42). Given the importance of the c-met receptor in cell physiology, it is easy to realize that its expression is tightly controlled under normal physiological conditions. Aberrant regulation of the c-met expression has been implicated in developing various types of tumors in transgenic animal models and in patients (2, 4, 7, 12, 40). Although many cytokines, growth factors, and hormones have been demonstrated to upregulate c-met expression in a wide variety of cells including renal epithelial cells (21, 27), little is known as to the extracellular cues that suppress its expression (35). In this report, we demonstrated that oxidative stress suppresses c-met expression in renal epithelial cells in a time- and dose-dependent fashion. This inhibition of c-met expression by oxidant stress is likely mediated by the interplay between Sp1 and Egr-1 transcription factors, in which induced Egr-1 sequesters Sp1's function as a transcriptional activator of the c-met gene through physical interaction.
One of the intriguing features in renal epithelial cells after oxidative stress is the concomitant and reciprocal expression of c-met and Egr-1 transcription factors (Figs. 1 and 2). This inverse association between c-met and Egr-1 was later confirmed by ectopic expression of Egr-1 via transfection (Fig. 3). Egr-1 is the prototypic member of the Egr family of transcription factors whose expression is rapidly induced in response to various stimuli (31, 47). Egr-1 contains a DNA binding domain that is homologous to that found in Sp1 and recognizes and binds to the consensus GC-rich sequence 5'-GCGGGGGCG-3' of its target genes in a zinc-dependent manner (11, 15). Although Egr-1 binding usually results in activation of gene transcription, it also suppresses gene transcription depending on specific context of the promoters (1, 33). As to the c-met gene, we found that Egr-1 was unable to bind to the c-met promoter (Fig. 6) but markedly suppressed its transcription (Fig. 5) and protein expression (Fig. 3), despite robust induction of Egr-1 transcript (Fig. 1), protein (Fig. 2), and its DNA-binding activity (Fig. 6C) in renal epithelial cells after H2O2 treatment. The inability of Egr-1 to bind to the cis-element is likely attributable to the existence of an imperfect match (8 nucleotides of 9) in the putative Egr-1 site of the c-met promoter. This underscores that a single mutation in the core sequence of the Egr-1-binding site can cripple the interaction between the trans-acting factor and cis-acting element.
The involvement of Sp1 in mediating oxidative stress-triggered c-met suppression is initially hinted by the identification of a cis-acting H2O2-responsive region, which harbors three Sp1-binding sites and an imperfect Egr-1 site (Fig. 4). Moreover, DNA-protein interaction studies reveal an attenuated binding of Sp proteins (Sp1 and Sp3) to their cognate sites when using nuclear extract from H2O2-treated renal epithelial cells (Fig. 7). It should be noted that there is an additional perfect Sp1-binding site located at the nucleotide position of +29 to +34 relative to the c-met transcriptional initiation site (18), which could explain the marginal reduction in luciferase activity in the pGL3-0.1met construct after H2O2 treatment (Fig. 4). These observations are consistent with earlier studies demonstrating that the Sp family of transcription factors is essential for establishing the constitutive expression of c-met gene in diverse types of cells (18, 36, 37, 46). For instance, c-met expression in normal adult kidney is largely overlapped with Sp1 and Sp3, and the abundance of c-met is tightly proportional to endogenous cellular levels of Sp proteins (46). Of importance, both Sp1 and Sp3 proteins trans-activate the c-met promoter in a highly synergistic way. However, treatment of renal epithelial cells with H2O2 induced no alteration in cellular levels of both Sp1 and Sp3 (Fig. 7E). Such observation establishes that oxidant stress suppresses c-met expression by a mechanism involving a possible interplay between Sp proteins and induced Egr-1 rather than any alteration in cellular level of Sp proteins.
Interplay between Egr-1 and Sp1 transcription factors has been
described to mediate the transcriptional regulation of many genes
(1, 10, 16, 30, 41). Extensive studies indicate that the
molecular mechanism governing the interaction between Egr-1 and Sp1 is
overwhelmingly controlled through directly competing for binding to the
overlapping cis-acting element. In many promoters containing
GC-rich elements, Sp1- and Egr-1-binding sites often are partially
overlapped by sharing several nucleotides in their respective core
binding sequence. Thus overexpression or induction of one factor,
leading to an increased cellular concentration, will result in
"displacement" of another factor for binding to the overlapping
sites. Such displacement of Sp1 by Egr-1, or vice versa, could lead to
either transcriptional activation or repression, depending on a
particular promoter and cellular content. For example, Egr-1 competes
with Sp1 protein for an overlapping region in the promoter of
platelet-derived growth factor A and functions as a positive activator
(16). Thus the Egr-1/Sp1 displacement mechanism may be an
important regulatory pathway in the control of inducible gene
expression (16). Contrary to this report, Egr-1 has been shown to act as a negative regulator and represses Sp1-mediated activation of many genes including protein-tyrosine phosphatase 1B
(10), Epstein-Barr virus C (30), and
1-adrenergic receptor (1). These studies
suggest that Egr-1 and Sp1 often elicit opposite regulation of
particular genes by competing for binding to an overlapping site and
thereby displacing with each other. Hence, competition for DNA binding
between inducible Egr-1 and constitutive Sp1 may provide a defined
means of transcriptional regulation. Such a mechanism, however, could
not account for the regulation of c-met expression in renal epithelial
cells after oxidative stress, because Egr-1 does not directly bind to
the c-met promoter (Fig. 6).
The action of Egr-1 on gene transcription is also regulated through its phosphorylation by an inducible casein kinase II (13, 38, 45). This represents an alternative mechanism implicating the interplay between Egr-1 and Sp1 in modulating gene transcription, besides the displacement of DNA binding. Previous studies show that estrogen blocks macrophage colony-stimulating factor (M-CSF) gene expression by decreasing casein kinase II-dependent phosphorylation of Egr-1 (38). Phosphorylated Egr-1, possibly due to conformational changes of specific domains that modulate the affinity of Egr-1 for Sp1, binds less ardently to Sp1, which leads to higher levels of free Sp1 for stimulating activation of the M-CSF gene (38). Therefore, posttranslational modification of Egr-1 also plays a critical role in regulating gene transcription by modulating the affinity of Egr-1 for Sp1. However, because it has not been tested whether oxidative stress affects the phosphorylation of Egr-1 in this study, it remains elusive whether this mode of regulation is related to c-met suppression by oxidative stress in renal epithelial cells.
Our findings in the present study unravel a distinctive mode of interaction between Egr-1 and Sp1 transcription factors, in which Egr-1 sequesters Sp1 for its transcriptional activation of the c-met gene. The sequestration of Sp1 by Egr-1 is most likely mediated through protein-protein interaction. This conclusion is consistent with, and supported by, several lines of observations. First, despite increased amounts of Egr-1 expression after oxidative stress in renal epithelial cells, Egr-1 did not directly bind to the c-met promoter. This excludes the possibility of a direct "displacement" of Sp1 by increasing Egr-1 after H2O2 treatment via competition for binding. Second, physical interaction between Egr-1 and Sp1 is clearly evident, as illustrated by coimmunoprecipitation (Fig. 8). It is unlikely that formation of the Egr-1/Sp1 complex requires the binding of both proteins to the cis-acting DNA element in the c-met promoter, because such a complex was absent in the EMSA studies (Fig. 6). Third, ectopic expression of Egr-1 functionally suppresses c-met promoter and protein expression in renal epithelial cells, a scenario resembling that in renal epithelial cells where Egr-1 is abundant following oxidative stress. Because Egr-1 suppression of c-met expression does not require its binding to DNA element, our findings on Egr-1 sequestration of Sp1 may have broad implications in the regulation of any genes that are transcriptionally controlled by ubiquitous Sp1 and potentially provide a generalized mechanism elucidating how inducible Egr-1 protein controls the expression of a wide variety of genes, regardless of whether they have an Egr-1-binding site in their promoter regions.
In summary, we showed herein that oxidative stress suppresses c-met expression in renal epithelial cells in a time- and dose-dependent fashion. We demonstrate that Egr-1, the product of an inducible immediate-early responsive gene, is a key mediator of c-met inhibition, primarily by its sequestration of Sp1 as a transcriptional activator for the c-met gene via physical interaction. The results of this study provide the mechanistic insights into the transcriptional repression of the c-met gene and will form the basis for further analyses of the regulation of this important signaling molecule under normal and pathological settings.
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ACKNOWLEDGEMENTS |
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We thank Drs. R. Tjian and F. Rauscher III for generously providing plasmid vectors.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants DK-02611, DK-54922, and DK-61408 (Y. Liu).
Address for reprint requests and other correspondence: Y. Liu, Dept. of Pathology, Univ. of Pittsburgh, S-405 Biomedical Science Tower, 200 Lothrop St., Pittsburgh, PA 15261 (E-mail: liuy{at}msx.upmc.edu).
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 4, 2003;10.1152/ajprenal.00426.2002
Received 16 December 2002; accepted in final form 31 January 2003.
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