1 Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; 2 Department of Cell Biology, Peking Union Medical College, Beijing 100005, China; and 3 Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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We investigated the expression pattern and underlying mechanism that controls hepatocyte growth factor (HGF) receptor (c-met) expression in normal kidney and a variety of kidney cells. Immunohistochemical staining showed widespread expression of c-met in mouse kidney, a pattern closely correlated with renal expression of Sp1 and Sp3 transcription factors. In vitro, all types of kidney cells tested expressed different levels of c-met, which was tightly proportional to the cellular abundances of Sp1 and Sp3. Both Sp1 and Sp3 bound to the multiple GC boxes in the promoter region of the c-met gene. Coimmunoprecipitation suggested a physical interaction between Sp1 and Sp3. Functionally, Sp1 markedly stimulated c-met promoter activity. Although Sp3 only weakly activated the c-met promoter, its combination with Sp1 synergistically stimulated c-met transcription. Conversely, deprivation of Sp proteins by transfection of decoy Sp1 oligonucleotide or blockade of Sp1 binding with mithramycin A inhibited c-met expression. The c-met receptor in all types of kidney cells was functional and induced protein kinase B/Akt phosphorylation in a distinctly dynamic pattern after HGF stimulation. These results indicate that members of the Sp family of transcription factors play an important role in regulating constitutive expression of the c-met gene in all types of renal cells. Our findings suggest that HGF may have a broader spectrum of target cells and possess wider implications in kidney structure and function than originally thought.
hepatocyte growth factor; Sp1; Sp3; gene regulation; Akt kinase
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INTRODUCTION |
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HEPATOCYTE GROWTH FACTOR
(HGF) receptor is the product of the c-met
protooncogene, which is a membrane-spanning protein that belongs to the
receptor tyrosine kinase superfamily (3, 35). The
c-met gene was originally isolated from a human osteogenic sarcoma cell line that was treated in vitro with the chemical carcinogen
N-methyl-N'-nitro-N-nitrosoguanidine
(37). Mature c-met protein is a 190-kDa,
disulfide-linked heterodimer that consists of - and
-subunits
(37). The
-subunit is heavily glycosylated and is
completely extracellular. The
-subunit has an extracellular portion
that is involved in ligand binding and also has a transmembrane segment
and a cytoplasmic tyrosine kinase domain that contains multiple
phosphorylation sites. Both subunits are encoded within a single
open-reading frame and are produced from the proteolytic cleavage of a
170-kDa precursor (30). On binding to HGF, the c-met
receptor undergoes autophosphorylation of the tyrosine residues in its
cytoplasmic domain and initiates cascades of signal transduction events
that eventually lead to specific cellular responses (5,
31). It has been demonstrated that the HGF/c-met signaling
system plays a vital role in cell survival, proliferation,
migration, and differentiation in a wide spectrum of target
tissues including kidneys (21, 28, 33).
Because all biological activities of HGF are presumably mediated by a single c-met receptor, its expression is likely one of the crucial components that determine cell-type specificity and overall activity of HGF actions. Earlier studies indicated that the c-met gene is predominantly expressed in epithelial cells from different organs, whereas its ligand is primarily derived from the mesenchyme (46). This characteristic pattern of expression as well as the pleiotrophic nature of its actions makes HGF an important paracrine and/or endocrine mediator for mesenchymal/epithelial interactions, which are critical processes in organ development, tissue regeneration, and tumorigenesis under various physiological and pathological conditions. However, recent studies suggest that the c-met receptor is expressed at different levels in nonepithelial cells as well. For instance, c-met expression is observed in endothelial cells, various types of blood cells, and glomerular mesangial cells (4, 27, 52, 54). Because these cells also express HGF, these observations indicate that the autocrine pathway is another important mode of action for this paired receptor-ligand system, at least in certain types of cells.
The kidney is one of the organs in which the c-met receptor is abundantly expressed, although little is known about its function at normal physiological settings (21, 31). Earlier studies (26, 40) revealed that c-met protein is primarily expressed in renal tubular epithelial cells along the entire nephron in normal rat kidney. Little or no c-met protein was observed in other types of cells (such as renal interstitial fibroblasts) aside from renal tubules. However, it remains a question whether these cells truly do not express c-met or their expression level is instead below the detection limits by conventional approaches. Furthermore, the molecular mechanism that governs the constitutive expression of the c-met gene in various types of kidney cells remains largely unknown.
In this study, we examined the expression pattern of the c-met receptor in normal adult kidneys and in a wide variety of kidney cells in vitro. We found that c-met is ubiquitously expressed in normal kidney in a pattern that overlaps with that of the Sp family of transcription factors. Both Sp1 and Sp3 proteins bound to the promoter region of the c-met gene and functionally activated its transcription. All types of kidney cells tested in vitro expressed the functional c-met receptor and induced protein kinase B (PKB)/Akt phosphorylation after HGF stimulation.
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MATERIALS AND METHODS |
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Animals. Male CD-1 mice (body wt 20-24 g) were purchased from Harlan Sprague Dawley (Indianapolis, IN). The mice were housed in the animal facilities of the University of Pittsburgh Medical Center and had free access to food and water. Animals were treated humanely using approved procedures in accordance with the guidelines of the Institutional Animal Use and Care Committee of the National Institutes of Health at the University of Pittsburgh School of Medicine. The mice were killed by exsanguination while under general anesthesia. The kidneys were removed and immediately decapsulated. One part of the kidney was frozen in Tissue-Tek optimal cutting-temperature compound in preparation for cryosection. Another part was fixed in 10% neutral-buffered formalin and embedded in paraffin in preparation for histology and immunohistochemical staining.
Cell culture and treatment.
Mouse inner medullary collecting duct epithelial cell line 3 (mIMCD-3),
rat renal interstitial fibroblasts (NRK-49F), and Drosophila
Schneider line 2 (SL-2) cells were obtained from the American Type
Culture Collection (Rockville, MD). The human kidney proximal tubular
cell line (HKC) was provided by Dr. L. Racusen of Johns Hopkins
University. Rat glomerular mesangial cells were a gift of Dr. C. Wu of
the University of Pittsburgh. The conditionally immortalized mouse
podocyte cell line was established from the transgenic mouse that
carries a thermosensitive variant of the simian virus 40 (SV40)
promotor as described previously (34). mIMCD-3, HKC, and
NRK-49F cells were maintained in a 1:1 DMEM/Ham's F-12 medium (Life
Technologies, Grand Island, NY) mixture supplemented with 10% fetal
bovine serum (FBS). Mesangial cells were cultured in RPMI 1640 medium
supplemented with 20% FBS. To propagate podocytes, cells were cultured
on type I collagen at 33°C in the RPMI 1640 medium supplemented with
10% FBS and 10 U/ml mouse recombinant interferon (IFN)- (R & D
Systems, Minneapolis, MN) to enhance the expression of a
thermosensitive T antigen. To induce differentiation, podocytes were
grown at 37°C in the absence of IFN-
for 14 days under
nonpermissive conditions (34). SL-2 cells were grown in Schneider's medium (Life Technologies) supplemented with 10% FBS. For
chemical blockade of Sp binding, mIMCD-3 cells were treated with
mithramycin A (Sigma, St. Louis, MO) at different concentrations for
various periods of time.
Immunohistochemical staining. Kidney sections from paraffin-embedded tissues were prepared at 4-µm thickness using a routine procedure. Immunohistochemical localization was performed using the Vector MOM immunodetection kit (Vector Laboratories, Burlingame, CA) according to procedures described previously (7). The primary antibody against mouse c-met (sc-8057) was obtained from Santa Cruz Biochemical (Santa Cruz, CA). As a negative control, the primary antibody was replaced with nonimmune normal IgG, and no staining occurred.
Frozen section and immunofluorescence staining.
Cryosections were prepared at 5-µm thickness in a cryostat and were
fixed in a cold 1:1 methanol-acetone mixture for 10 min at 20°C.
Immunostaining was performed as described previously (51).
Briefly, cryosections were incubated with 20% normal donkey serum in
PBS for 30 min at room temperature to reduce background staining.
Sections were washed with PBS and incubated with primary antibodies in
PBS containing 1% BSA overnight at 4°C. The primary antibodies
against mouse c-met, Sp1 (sc-59), and Sp3 (sc-644) were obtained from
Santa Cruz Biochemical. Sections were then incubated for 1 h with
affinity-purified secondary antibodies (Jackson ImmunoResearch
Laboratories, West Grove, PA) at a 1:100 dilution in PBS that contained
1% BSA before being washed extensively with PBS. Slides were mounted
with antifade mounting media and examined on a Nikon Eclipse E600
epifluorescence microscope (Melville, NY) equipped with a digital camera.
Western blot analysis. Various types of kidney cells were lysed with SDS sample buffer (62.5 mM Tris · HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromophenol blue). Samples were heated at 100°C for 5-10 min and were then loaded and separated on precast 10% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA). The proteins were electrotransferred to a nitrocellulose membrane (Amersham, Arlington Heights, IL) in transfer buffer that contained 48 mM Tris · HCl, 39 mM glycine, 0.037% SDS, and 20% methanol at 4°C for 1 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). The membranes were then incubated for 16 h at 4°C with various primary antibodies in blocking buffer that contained 5% milk at the dilutions specified by the manufacturers. The phospho-specific Akt antibody (that detects Akt only when it is phosphorylated at specific sites) and the total Akt antibody (that detects Akt independently of phosphorylation state) were obtained from Cell Signaling (Beverly, MA). The antibodies against Sp1, Sp3, c-met, and actin were purchased from Santa Cruz Biochemical. The membranes were washed extensively in TBS buffer and were then incubated with horseradish peroxidase-conjugated secondary antibody (Sigma) at a dilution of 1:10,000 for 1 h at room temperature in 5% nonfat milk dissolved in TBS. Membranes were then washed with TBS buffer, and the signals were visualized using an ECL system (Amersham).
Preparation of nuclear protein extract.
For preparation of nuclear protein extracts, mIMCD-3 cells in an
exponential growth stage 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 methods described elsewhere
(24). Briefly, the pelleted cells were resuspended in 4 volumes of buffer A that contained protease inhibitors: 20 mM HEPES, pH 7.9, 0.5 M sucrose, 1.5 mM NaCl, 60 mM KCl, 0.15 mM
spermidine, 0.5 mM spermine, 0.5 mM EDTA, and 1 mM dithiothreitol plus
2 µg each of leupeptin, soybean trypsin inhibitor, antipain, and
chymostatin per milliliter. An equal volume of buffer A that contained 0.6% Nonidet P-40 was added with gentle mixing to lyse the
cells. Immediately after lysis, the solution was diluted with 8 volumes
of buffer A, and the nuclei were collected by centrifugation at 5,000 g for 30 min at 4°C. Nuclear protein was
extracted with 0.4 M KCl · TGM (10 mM
Tris · HCl, pH 7.6, 10% glycerol, 3 mM MgCl2, and 3 mM EGTA) buffer that contained protease
inhibitors as described. The lysate was centrifuged for 45 min at
50,000 g at 4°C, and the supernatant was then collected
and dialyzed against 60 mM KCl · TGM buffer using
a mini-dialysis system (Life Technologies). The insoluble material was
removed by centrifugation, and aliquots of protein extract were quickly
frozen and stored at 80°C after the protein concentration had been
determined using a bicinoninic acid (BCA) protein assay kit (Sigma).
Electrophoresis mobility shift assays.
A DNA fragment (F1) corresponding to 217 to
49 of the 5'-flanking
region of the human c-met gene was isolated via PCR
amplification of the c-met promoter as described previously
(23). The F1 fragment was labeled with 32P by
including
-[32P]dCTP (3,000 Ci/mmol; Amersham) in the
PCR reactions. The labeled probes were then gel purified and used in
the electrophoresis mobility shift assays (EMSAs) as described
previously (24). The nonspecific competitor was 4 µg of
Poly(dI-dC) · Poly(dI-dC) (Pharmacia, Piscataway,
NJ) added to 10 µl of reaction mixture. The binding reactions were
carried out at room temperature for 15 min before loading of 5%
nondenaturing polyacrylamide (19:1 acrylamide-bisacrylamide
ratio) gels. For competition experiments, a 100-fold molar excess
of unlabeled DNA fragments or double-stranded oligonucleotides (oligos)
was included in the reaction mixture. Oligos were chemically
synthesized by a commercial source (Life Technologies). Complementary
strands were annealed in a mixture of 10 mM
Tris · HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA by
heating the mixture to 95°C and cooling it to room temperature over a period of 3 h. The sequences of the oligos used in this
study are shown in Table 1. For
supershift experiments, specific antibodies against Sp1, Sp3, Egr-1,
and normal control IgG (Santa Cruz) were incubated with nuclear protein
extracts for 15 min at room temperature before reaction buffer was
added. Gels were run in 0.5× TBE (0.045 M
Tris · borate with 0.001 M EDTA) buffer at a
constant voltage of 190 V and were dried and autoradiographed with
intensifying screens.
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Immunoprecipitation. mIMCD-3 cells grown on 100-mm plates were lysed on ice in 1 ml of RIPA buffer that contained 1× PBS, 1% Nonidet P-40, 0.1% SDS, 10 µg/ml phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 1% protease inhibitor 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 fresh tubes. 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 each of anti-Sp1, anti-Sp3, and normal IgG, which was 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.
Plasmid construction, transfection, and reporter gene assay. The 0.2met-chloramphenicol acetyltransferase (CAT) and 0.1met-CAT chimeric plasmids, which contain 0.2 and 0.1 kb of the 5'-flanking region of the c-met gene, respectively, and the coding sequence for CAT, have been described elsewhere (23). The Drosophila SL-2 cells, which lack endogenous Sp transcription factors, were used for investigating the effects of Sp proteins on c-met promoter activity (6). At 24 h before transfection, the cells were seeded onto six-well plates at 2 × 105 cells/well. Cells were then transiently cotransfected with a constant amount of 0.2met-CAT or 0.1met-CAT chimeric plasmids and an increasing amount of either pPac-Sp1 or pPac-Sp3 expression vectors under the control of insect actin promoter. The DNA-calcium phosphate method was used according to the instructions of the CellPhect transfection kit (Pharmcia, Piscataway, NJ). Cells were incubated with DNA-calcium phosphate coprecipitation buffer for 16 h and washed twice with serum-free medium. Complete medium that contained 10% FBS was added, and the cells were incubated for an additional 24 h before harvest for CAT assays. After being washed in PBS, the cells were pelleted, resuspended in 150 µl of 0.25 M Tris · HCl at pH 7.5, and disrupted by three freeze-thaw cycles. The protein suspension was clarified by centrifugation at 15,000 g for 5 min at 4°C, and the supernatant was collected and assayed for CAT activity by a procedure described previously (23). Because Sp proteins are known to activate the transcription of many internal control reporter vectors driven under the SV40 early promoter and the thymidine kinase promoter (6, 16), the relative CAT activity in this study was reported after normalization for protein concentration. Protein concentration was determined using a BCA protein assay kit (Sigma). All experiments were repeated at least three times to ensure reproducibility. For deprivation of endogenous Sp proteins in renal epithelial cells with decoy oligos, mIMCD-3 cells were cotransfected with CAT reporter plasmids and either 20- or 50-fold molar excess of wild-type or mutant Sp1 oligos. At 36 h after transfection, cells were harvested, and CAT activities were determined.
Statistical analysis. Quantitation of the Western blots was performed by measuring the intensity of the hybridization signals with the use of NIH Image analysis software. Data were expressed as means ± SE. Statistical analyses of the data were carried out by t-test with the use of SigmaStat software (Jandel Scientific, San Rafael, CA). P < 0.05 was considered significant.
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RESULTS |
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Overlapping expression pattern of c-met and Sp family proteins in
normal kidney.
The expression of c-met protein in normal mouse kidney was examined by
immunohistochemical staining using a specific antibody against c-met.
As shown in Fig. 1, the c-met protein was
widely expressed in normal mouse kidney. All tubular epithelial cells along the entire nephron were positive for c-met protein, with high
levels observed in distal tubules and collecting duct epithelia. Weak
staining was also noticeable in the glomeruli, which was most likely
present in glomerular visceral epithelial cells (podocytes) and
mesangial cells. In contrast, c-met receptor staining in the renal interstitium of normal mouse kidney was extremely weak or nondetectable. When c-met antibody was replaced by normal IgG, no
staining occurred (Fig. 1B), suggesting the specificity of c-met staining. These results indicate that c-met protein is
constitutively expressed in normal adult kidney in a relatively
ubiquitous fashion with different levels in distinct types of kidney
cells.
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Correlation of c-met level with Sp family protein abundance in
various types of kidney cells in vitro.
We next examined c-met protein expression in a wide variety of kidney
cells in vitro by using Western blot analysis. As shown in Fig.
3, all types of kidney cells tested,
including glomerular mesangial cells, podocytes, proximal tubular
epithelial cells, collecting duct epithelial cells, and renal
interstitial fibroblasts, expressed different levels of c-met protein.
A single band of 145-kDa -subunit of c-met protein was observed in
all types of kidney cells in polyacrylamide gels under reducing
conditions. This observation is consistent with the in vivo data, which
demonstrates widespread expression of c-met protein in normal adult
kidney as described (see Fig. 1). Western blot analysis also exhibited that all of the kidney cells tested expressed distinctive levels of Sp1
and Sp3 transcription factors. A doublet of Sp1 protein that
represented a different phosphorylated status (15) at
~95 kDa was detected in all renal cells. Sp3 displayed two doublets at 124 and 84 kDa, respectively. Presumably, these different isoforms are derived from distinct internal translation initiations (18, 49). Intriguingly, a plot of the abundances of c-met, Sp1, and Sp3 proteins indicates that there is a remarkably tight correlation between the c-met and Sp protein levels in various types of kidney cells in vitro (Fig. 3C). These results establish that c-met
receptor levels in diverse types of kidney cells are in proportional
to, and are likely dictated by, the endogenous abundance of the Sp family of transcription factors.
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Binding of Sp1 and Sp3 to GC boxes of c-met promoter region.
To examine the possibility of Sp proteins participating in the
regulation of the c-met gene, we first determined whether Sp proteins interact with the c-met promoter by performing EMSA using a
DNA fragment that contained three cis-acting Sp1 sites (GC
boxes) from the c-met gene. When the DNA fragment was
incubated with nuclear protein extract derived from mIMCD-3 cells,
multiple DNA-protein complexes were formed that had retarded migration,
which resulted in three shifted bands in polyacrylamide gels under
nondenaturing conditions (C1-C3; see Fig.
5A). These binding complexes
were largely abolished by using an unlabeled F1 fragment itself as a
competitor in the incubation. Under the same conditions, the complexes
were also completely abrogated in the presence of a 100-fold molar
excess of the double-stranded oligo that corresponds to the Sp1 site of
the c-met gene. The binding complexes were intact when
incubated with a 100-fold molar excess of the mutated Sp1 oligo in
which two GGs were substituted with two TTs in the Sp1 binding region.
Other oligos with unrelated sequences such as activator protein 1 (AP-1), nuclear factor-B (NF-
B), and cAMP response element did
not interrupt the formation of the binding complexes (Fig.
5A), suggesting the specificity of these DNA-protein interactions. Supershift assay with specific antibodies revealed that
these binding complexes were contributed by Sp1 and Sp3 transcription factors (Fig. 5B). Incubation with Sp1 antibody caused a
further shift of the C2 complex and the formation of supershifted bands (SS1 and SS2), whereas the Sp3 antibody resulted in a supershift of the
C1 and C3 complexes and formation of SS3 and SS2 (Fig. 5B).
As expected, incubation with normal IgG or other unrelated antibodies
such as anti-Egr-1 transcription factor did not cause either a
supershift or inhibition of the complex formation.
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Functional cooperation among members of Sp family in activating
c-met transcription.
To determine the function of Sp1 and Sp3 proteins in regulating c-met
expression, we transfected the 0.2met-CAT reporter construct that
contains multiple Sp1 binding sites with expression vector for Sp1 and
Sp3 in Drosophila SL-2 cells. Because Drosophila
cells lack endogenous Sp activity, the cells provide a sensitive and reliable in vivo assay system for investigating the effects of Sp
proteins on gene transcription. As shown in Fig.
7A, Sp1 dramatically activated
c-met promoter activity in a dose-dependent manner. An ~18-fold
induction in reporter gene activity was observed after cotransfection
of SL-2 cells with 0.2met-CAT plasmid and 3 µg of Sp1 expression
vector pPac-Sp1. Sp3 alone also induced, to a much less extent, c-met
promoter activity. Cotransfection of 3 µg of Sp3 expression vector
pPac-Sp3 with 0.2met-CAT plasmid into SL-2 cells resulted in an
approximately eightfold induction of reporter activity (Fig.
7A).
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Deprivation of Sp family proteins by decoy oligos in renal
epithelial cells inhibits c-met promoter activity.
To further confirm the importance of the Sp family proteins in
regulating c-met transcription, we cotransfected the
0.2met-CAT reporter construct to mIMCD-3 cells with decoy oligo
corresponding to the Sp1 binding site, which competes with c-met
promoter Sp1 sites for binding with cellular trans-acting Sp
proteins. This strategy presumably leads to a decrease in the
availability of the cellular Sp proteins to the c-met promoter. As
shown in Fig. 8, introduction of the Sp1
decoy oligo markedly inhibited c-met gene transcription in a
dose-dependent fashion. Cotransfection of a 50-fold molar excess of Sp1
decoy oligo together with the 0.2met-CAT plasmid suppressed c-met
promoter activity by ~70%. In fact, the CAT reporter gene activity
was reduced by Sp1 decoy to a level similar to that elicited by the
0.1met-CAT plasmid in which three Sp1 binding sites were deleted.
However, transfection of the mutant Sp1 oligo that failed to bind Sp
proteins due to mutations in the Sp1 binding region (see Fig. 5) did
not significantly affect 0.2met-CAT reporter activity in renal
epithelial cells. Thus the abundance of endogenous cellular Sp family
proteins likely dictates the level of c-met expression in kidney cells.
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Blockade of Sp protein binding inhibits c-met expression in renal
epithelial cells.
We next investigated the effects of blockade of Sp binding via chemical
antagonist in renal epithelial cells on c-met receptor expression.
mIMCD-3 cells were treated with mithramycin A, a potent inhibitor of Sp
binding (39, 42), for various periods of time at different
concentrations. Because c-met expression is primarily regulated at the
transcriptional level in mIMCD-3 cells (25), the effect of
the Sp inhibitor on c-met expression was determined by measuring the
protein levels using Western blot analysis after various treatments. As
shown in Fig. 9, mithramycin A markedly inhibited c-met expression in mIMCD-3 cells in a dose-dependent manner.
At concentrations as low as 108 M, mithramycin A
repressed c-met expression by >80% after a 24-h incubation. The
kinetics of c-met inhibition by mithramycin A are presented in Fig.
9B. Blockade of Sp binding significantly inhibited c-met
expression as early as 12 h after incubation with the chemical
antagonist. Of note, the inhibitory effect of mithramycin A was
specific, because expression of other genes such as actin was not
blocked by this chemical inhibitor (Fig. 9). These results suggest that
the binding of Sp proteins to cognate cis-acting elements is
essential for constitutive expression of the c-met gene in
renal epithelial cells.
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Functionality of c-met receptor in different types of kidney cells
in vitro.
The ubiquitous expression pattern of c-met in the kidney prompted us to
investigate whether the c-met receptor is functional in all types of
kidney cells. To this end, we studied the phosphorylation and
activation of PKB/Akt kinase, which is a major signaling protein in the
pathway leading to cell survival (12, 36), in various types of kidney cells after HGF stimulation. Consistent with a previous
report (22), HGF induced marked Akt phosphorylation as
early as 5 min after stimulation in proximal tubular epithelial cells,
and this induction was sustained to at least 1.5 h after HGF
incubation (Fig. 10). In addition to
proximal tubular epithelial cells, all other types of kidney cells
tested, including glomerular mesangial cells, podocytes, collecting
duct epithelial cells, and renal interstitial fibroblasts, responded to
HGF stimulation and induced Akt phosphorylation and activation (Fig.
10). Therefore, the c-met receptor is indeed functional in all types of
kidney cells tested and responds to HGF stimulation to initiate signal transduction events that lead to cell survival.
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DISCUSSION |
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HGF and its specific c-met receptor are classically considered as a paired signaling system for mediating signal exchange between mesenchyme and epithelia via paracrine actions (46, 47). This assumption is largely based on observations that the c-met receptor is predominantly expressed in epithelial cells, whereas its ligand is mainly produced by mesenchyme-derived cells (46). In this study, we demonstrate that almost all kidney cells in normal adult animals express different levels of c-met receptor protein. In vitro, all types of kidney cells tested, including glomerular mesangial cells, podocytes, proximal tubular epithelial cells, collecting duct epithelial cells, and interstitial fibroblasts, expressed the functional c-met receptor and responded to HGF stimulation to induce PKB/Akt phosphorylation. Although the present study has not included renal glomerular and vascular endothelial cells, studies elsewhere indicate that endothelial cells also express the functional c-met receptor and respond to HGF stimulation (52). Altogether, these results suggest that c-met receptor expression in the kidney is widespread and ubiquitous. Because it is the receptor that determines the target specificity of HGF actions, our results suggest that HGF may have a broader spectrum of target cells in the kidney and thereby possess wider implications in kidney structure and function than previously envisioned.
Although the c-met receptor has been demonstrated to be expressed in renal tubular epithelial cells, its presence and function in other types of cells such as interstitial fibroblasts are uncertain. In contradiction to the present study, earlier observations often suggested an absence of c-met receptors in renal interstitial cells in normal rats (26). This discrepancy is probably attributable to the low sensitivity of the immunohistochemical staining approach that was employed previously. Consistent with this notion, extremely weak or no staining for the c-met receptor was also observed in the interstitium of mouse kidney in this study (see Fig. 1). The finding of ubiquitous expression of c-met in the kidney is supported by several lines of evidence. First, c-met receptor protein is detectable in all types of homogenous kidney cell populations via immunoblotting, a more sensitive detection approach (see Fig. 3). Second, c-met receptor expression in the kidney is clearly controlled by Sp1 and Sp3 transcription factors (see Figs. 5-8), whose expression in turn is ubiquitous. Third, all types of kidney cells tested in vitro retain the c-met receptor and undergo a cascade of signal transduction events leading to Akt kinase phosphorylation in response to HGF stimulation (see Fig. 10). It is of interest to note that despite the very low abundance of c-met receptors in NRK-49F cells and glomerular mesangial cells, the magnitude of the cellular responses, such as Akt activation after HGF stimulation, in these cells is compatible with that in other types of cells in which the c-met receptor is highly expressed (see Fig. 10). This obvious irrelevance of c-met abundance to cellular response insinuates that low levels of c-met receptors in these cells are not a limiting factor for optimal biological actions of HGF.
Because kidney cells with mesenchymal phenotypes such as interstitial fibroblasts and mesangial cells presumably express HGF (52), the presence of c-met receptors in these types of cells suggests an autocrine loop formation with simultaneous expression of both the receptor and its ligand in the same cell. These observations expand the modes of HGF action in normal kidney beyond the well-described paracrine and endocrine mechanisms. The physiological significance of HGF autocrine action is largely unknown. It is plausible to speculate that the autocrine signaling of HGF may be one of the pathways essential for the development and maintenance of normal kidney structure and function. In this regard, previous studies indicate that HGF and c-met are coexpressed in early metanephrogenic mesenchyme, which leads to the promotion of mesenchymal-to-epithelial cell transdifferentiation during nephrogenesis (1, 50). Similarly, both c-met and HGF are significantly induced in rat glomerular mesangial cells in response to interleukin-6 stimulation (27).
In light of the widespread expression of the c-met gene in
the kidney, it is not surprising to find that c-met is expressed in a
pattern that is overlapped with the ubiquitous Sp family of
transcription factors. The importance of Sp proteins in the constitutive expression of c-met in the kidney is established by
several lines of observations in this study. These include a tight
correlation between c-met receptor abundance and the levels of Sp
proteins in various types of kidney cells (see Fig. 3). This intrinsic
interconnection between c-met and Sp proteins is also evident during
podocyte differentiation (see Fig. 4). Hence, cellular endogenous Sp
protein level probably is a key molecular determinant for the
differential expression of c-met in diverse types of kidney cells. In
support of this, Sp proteins are found to bind to the promoter region
of the c-met gene and to functionally activate
c-met transcription. Conversely, deprivation of Sp proteins by a decoy strategy and blockade of Sp binding by chemical antagonist inhibits c-met expression in renal epithelial cells. Although the
present study uses the human c-met promoter, previous studies by Seol
and colleagues (44, 45) have demonstrated that the two Sp1
binding sites in the mouse c-met gene are also critical for
establishing basal c-met expression as well as for modulating induced
c-met transcription, suggesting that there is little
mechanistic difference in the regulation of c-met
transcription by Sp proteins in different species. Accordingly, a
recent report demonstrates that inhibition of c-met expression in human
primary hepatocytes by IFN- is mediated by decreased binding of Sp1
to the c-met promoter (41). In this context, it should
also be noted that Sp1-knockout embryos were severely
defective in development and all died around day 11 of
gestation (29), a time point precisely before the death of
c-met-knockout embryos at days 13 and
14 (2).
The Sp family of transcription factors consists of at least four
members with distinct expression patterns and diverse functions in the
different types of cells (49). All four proteins share similar structural features, including a highly conserved DNA binding
domain that consists of three zinc fingers close to the COOH-terminal
region (49). Sp1 is the prototype of this family and is
expressed ubiquitously. Sp1 functions as a transcriptional activator
for a large number of genes implicated in cell-cycle regulation,
hormonal activation, and embryogenic development. Sp2 exhibits a
significant structural difference from other members of the Sp family
and does not bind to the GC box but to a GT-rich element in the
promoter region of the T-cell receptor gene (19). Little
is known regarding Sp2 tissue distribution and its function. Although
Sp3 is also ubiquitously expressed, the expression of Sp4 is tissue
specific and largely restricted to the brain (48). Because
both Sp3 and Sp1 are often present in the same cell and are
indistinguishable in DNA binding specificity, Sp3 is generally considered to be an antagonist for Sp1 by functionally suppressing Sp1-mediated gene activation (10, 11, 17). However,
several reports also suggest a positive regulation of gene expression by Sp3 in different circumstances. For instance, Sp3 has been shown to
activate the promoter of the human 2(I) collagen gene and the mouse growth-hormone receptor gene (8, 13, 53). In
the present study, our data suggest that Sp3 alone activates c-met gene transcription, although to much less extent than
Sp1. In addition, Sp3 not only fails to inhibit Sp1-activated
c-met gene transcription, but also actually enhances
Sp1-induced c-met expression in a synergistic fashion (see Fig. 7).
These observations underline that members of the Sp family of
transcription factors interact with one another to produce either
positive or negative regulation of a particular gene, depending on the
context of specific promoter and cellular environments.
An interesting and novel finding in this study is the synergistic action of Sp1 and Sp3 in activating c-met gene transcription. Apart from the cotransfection data presented in Fig. 7B, Western blot analysis also reveals that the differences in c-met levels among diverse types of kidney cells are greater than in either Sp1 or Sp3 alone (see Fig. 3). This suggests a favorable interaction between cellular Sp1 and Sp3 proteins in establishing the constitutive expression of c-met in the kidney. In accordance with this, Sp1 and Sp3 are largely expressed in an identical pattern with comparable abundances in different types of normal kidney cells (see Fig. 2). Although the molecular mechanism that underlies the synergistic activation of the c-met gene by Sp1 and Sp3 remains unknown, physical interaction between two members of the Sp family may be of importance for such functional cooperation. Sp1 is known to be capable of forming homotypic interactions that lead to multimeric complexes (38), which mediate transcriptional synergism among multiple GC boxes. Moreover, many heterotypic interactions of Sp1 with diverse types of transcription factors, such as YY1, E2F, and Smad2/3, just to name a few, have been documented (9, 14, 20, 32, 43). Indeed, a direct interaction between Sp1 and Sp3 in renal epithelial cells has been demonstrated by coimmunoprecipitation with either Sp1 or Sp3 antibodies (see Fig. 6). In addition, supershift experiments using antibodies against either Sp1 or Sp3 (see Fig. 5) exhibit the presence of an additional complex (SS2) with identical size that presumably consists of Sp1, Sp3, and IgG. Therefore, heterotypic interaction between Sp1 and Sp3 occurs in renal epithelial cells and is probably critical for the synergistic activation of c-met gene transcription.
The finding of ubiquitous expression of c-met receptors in various types of renal cells suggests a broader spectrum of target cells for HGF in normal kidney than previously thought. Perhaps more surprisingly, the functional response to HGF by a particular type of cells appears to be unrelated to the cellular abundance of c-met receptors (see Fig. 10). These observations lead one to rethink the HGF biology in the kidney. It now appears certain that HGF signaling possesses important biological activities not only in tubular epithelial cells, where the c-met receptor is robustly expressed, but also in the cells with low abundance of c-met receptors, such as glomerular mesangial cells and interstitial fibroblasts. Undoubtedly, one of the great challenges in the future is to determine the exact function of HGF signaling in a particular type of renal cells in the context of whole kidney in vivo.
In summary, the c-met receptor is expressed in normal adult kidney and in diverse types of kidney cells in a ubiquitous fashion. The constitutive expression of the c-met gene is closely correlated with and primarily mediated by the synergistic actions of transcription factors Sp1 and Sp3. Physiologically, the c-met receptor in all types of kidney cells tested is functional and initiates a cascade of signal transduction events leading to Akt phosphorylation in response to HGF. In view of the fact that the receptor determines the target specificity of the ligand, these results suggest that HGF may have broader implications in kidney structure and function under various physiological and pathological conditions.
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ACKNOWLEDGEMENTS |
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The authors thank Drs. R. Tjian and G. Suske for providing the Sp1 and Sp3 expression plasmids.
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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 and by National Natural Science Foundation of China Grant 39825508. C. Dai and J. Yang were supported by postdoctoral fellowships from the American Heart Association Pennsylvania-Delaware Affiliate.
Address for reprint requests and other correspondence: Y. Liu, Dept. of Pathology, Univ. of Pittsburgh School of Medicine, 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.
10.1152/ajprenal.00200.2002
Received 22 May 2002; accepted in final form 4 August 2002.
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