Am J Physiol Renal Physiol 284: F82–F94, 2003;
10.1152/ajprenal.00200.2002.
Sp1 and Sp3 transcription factors synergistically
regulate HGF receptor gene expression in kidney
XIANGHONG ZHANG,1,2 YINGJIAN LI,1 CHUNSUN DAI,1
JUNWEI YANG,1 PETER MUNDEL,3 AND YOUHUA LIU1
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
Submitted 22 May 2002; accepted in final form 4 August 2002
HEPATOCYTE GROWTH FACTOR (HGF) receptor is the product of the c-met protooncogene, which is a membranespanning 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). Ma-
ture 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 openreading 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.
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: [email protected]).
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.
hepatocyte growth factor; Sp1; Sp3; gene regulation; Akt
kinase
F82
0363-6127/03 $5.00 Copyright © 2003 the American Physiological Society
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Zhang, Xianghong, Yingjian Li, Chunsun Dai, Junwei Yang, Peter Mundel, and Youhua Liu. Sp1 and Sp3
transcription factors synergistically regulate HGF receptor
gene expression in kidney. Am J Physiol Renal Physiol 284:
F82–F94, 2003; 10.1152/ajprenal.00200.2002.—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.
REGULATION OF C-MET GENE BY SP PROTEINS
MATERIALS AND METHODS
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 differAJP-Renal Physiol • VOL
entiation, 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-␮m 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-␮m 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 䡠 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% SDSpolyacrylamide 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 䡠 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 䡠 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
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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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REGULATION OF C-MET GENE BY SP PROTEINS
1⫻ PBS, 1% Nonidet P-40, 0.1% SDS, 10 ␮g/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 ␮g of normal rabbit IgG and 20 ␮l 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 ␮g each of anti-Sp1, anti-Sp3, and normal IgG, which
was followed by precipitation with 20 ␮l 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 ␮l of 0.25 M Tris 䡠 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
Table 1. Oligonucleotide sequences used in this study
Oligo
Sp1
Mutant Sp1
Activator protein 1
Nuclear factor-␬B
CRE
Sequence
Location at c-met Promoter
5⬘-gatcGTCGTGGGCGGGGCAGAG
CAGCACCCGCCCCGTCTCctag-5⬘
5⬘-gatcGTCGTGTTCGGGGCAGAG
CAGCACAAGCCCCGTCTCctag-5⬘
5⬘-gatcCCGGCTGAGTCACTGGCA
GGCCGACTCAGTGACCGTctag-5⬘
5⬘-gatcAATGGGAACTTTCCTTTT
TTACCCTTGAAAGGAAAActag-5⬘
5⬘-gatcCAGAATGACATCACATGG
GTCTTACTGTAGTGTACCctag-5⬘
⫺149 to ⫺132
CRE, cAMP response element.
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⫺149 to ⫺132
⫹119 to ⫹137
⫺1152 to ⫺1135
⫺2341 to ⫺2324
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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 ␮g 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 䡠 TGM (10 mM Tris 䡠 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 䡠 TGM buffer using a minidialysis 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 ␮g of Poly(dI-dC) 䡠 Poly(dIdC) (Pharmacia, Piscataway, NJ) added to 10 ␮l 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 䡠 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 䡠 borate with 0.001 M EDTA) buffer at a constant voltage
of 190 V and were dried and autoradiographed with intensifying screens.
Immunoprecipitation. mIMCD-3 cells grown on 100-mm
plates were lysed on ice in 1 ml of RIPA buffer that contained
REGULATION OF C-MET GENE BY SP PROTEINS
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.
RESULTS
Fig. 1. Immunohistochemical localization of c-met protein in normal
mouse kidney. A: ubiquitous presence of c-met protein in normal
kidney is shown. Specific staining for c-met protein was visible in
renal tubules along the entire nephron. Weak staining was also
observed in renal glomeruli (arrow). B: as a negative control, no
staining was observed in the kidney when c-met primary antibody
was replaced with normal IgG. Scale bar, 20 ␮m.
AJP-Renal Physiol • VOL
trast, 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.
The widespread expression pattern of c-met in normal kidney led us to speculate that there might
be ubiquitous trans-acting factor(s) responsible for
its transcriptional activation. Earlier studies (23) revealed the presence of multiple GC boxes (Sp1 binding
sites) in the promoter region of the c-met gene, which
potentially implicates the Sp family of transcription
factors in regulating c-met expression in the kidney.
Therefore, we examined the expression patterns of Sp1
and Sp3 transcription factors and the relationships
with c-met in normal kidney via double immunofluorescence staining for simultaneous detection of both
c-met and Sp proteins. As shown in Fig. 2, there was
close correlation between the expression patterns of
c-met protein and the Sp family of transcription factors
in normal adult kidney. In kidney sections, c-met protein expression completely overlapped with the presence of nuclear Sp1 protein (Fig. 2). Similarly, Sp1 and
Sp3 proteins were also concomitantly expressed in the
nuclei of various kidney cells, suggesting a possibly
functional coupling and cooperation with one another.
Except on rare occasions in which some cells expressed
more Sp1 than Sp3 or vice versa, Sp1 and Sp3 were
largely expressed in comparable amounts in many cells
of normal adult kidney (Fig. 2).
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
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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 con-
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REGULATION OF C-MET GENE BY SP PROTEINS
in proportional to, and are likely dictated by, the endogenous abundance of the Sp family of transcription
factors.
Comparison of the c-met receptor levels from podocytes of the differentiated and nondifferentiated states
uncovered that c-met receptor expression was repressed in differentiated podocytes (Fig. 3B). This inhibition of c-met expression during podocyte differentiation was accompanied by decreased Sp1 and Sp3
protein levels (Fig. 3B). To further explore the correlation between c-met expression and Sp protein abundances, we examined the kinetics of c-met and Sp
protein expression during podocyte differentiation induced by switching podocyte culture to 37°C in the
absence of INF-␥ (34). As shown in Fig. 4, c-met inhibition occurred in the first 4 days of podocyte differentiation, and levels were sustained beyond this time
point. Again, there was a strong association between
c-met and Sp protein levels during this period of podocyte differentiation (r2 ⫽ 0.94).
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 comAJP-Renal Physiol • VOL
plexes 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 antiEgr-1 transcription factor did not cause either a supershift or inhibition of the complex formation.
Careful examination of the supershift data revealed
that band SS2 was formed in the reactions involving
both anti-Sp1 and anti-Sp3 antibodies (Fig. 5B, lanes 3
and 4). These results implied that SS2 could be attributable to an interacting complex that potentially consisted of Sp1, Sp3, and IgG, which suggests that two
members of Sp family proteins (Sp1 and Sp3) may
physically interact with each other. To test this hypothesis, we performed additional coimmunoprecipitation experiments to demonstrate a direct interaction
between Sp1 and Sp3 in renal epithelial cells. As
shown in Fig. 6A, when cell lysate derived from
mIMCD-3 cells was immunoprecipitated with Sp1 an-
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Fig. 2. Colocalization of c-met protein with Sp family of transcription factors in normal mouse kidney. Representative micrographs demonstrate a close association of c-met protein with Sp1 and Sp3 transcription factors.
Immunofluorescence staining for both Sp1 (red) and c-met (green) in cryosections of normal mouse kidney (top).
Merging of two micrographs (top right) illustrates the overlapping pattern of c-met and Sp1 localization.
Immunofluorescence staining for Sp1 (red) and Sp3 (green) in normal kidney (bottom). Merging of two micrographs
(bottom right) demonstrates a largely overlapping pattern of Sp1 and Sp3. Cell nuclei with approximately equal
amounts of both Sp1 and Sp3 (yellow) are indicated (arrowheads). Scale bar, 20 ␮m.
REGULATION OF C-MET GENE BY SP PROTEINS
tibody, Sp3 protein was detected in the precipitated
complexes. Similarly, Sp1 protein was also found in the
complexes immunoprecipitated by Sp3 antibody (Fig.
6B). Thus there is a physical interaction between Sp1
and Sp3 proteins that could potentially lead to a functional cooperation in regulating c-met transcription in
renal epithelial cells.
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
AJP-Renal Physiol • VOL
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 ␮g of Sp1
expression vector pPac-Sp1. Sp3 alone also induced, to
a much less extent, c-met promoter activity. Cotransfection of 3 ␮g 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).
To investigate the potentially functional interaction
between Sp1 and Sp3 proteins in activating c-met transcription, we cotransfected SL-2 cells with the 0.2metCAT construct plus both Sp1 and Sp3 expression vectors. As presented in Fig. 7B, the combination of Sp1
and Sp3 resulted in a dramatically synergistic induction of c-met gene transcription. The magnitude of the
CAT reporter induction that was elicited by the combined Sp1 and Sp3 was greater than the additive effect
obtained by Sp1 and Sp3 individually. Hence, both Sp1
and Sp3 activate c-met transcription in a functionally
cooperative manner.
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.
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
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Fig. 3. Close correlation of c-met expression with the protein levels
of Sp1 and Sp3 transcription factors in various types of kidney cells.
Cell lysates from major types of kidney cells, including human
proximal tubular cells (HKC), mouse inner medullary collecting duct
epithelial cells (mIMCD-3), rat renal interstitial fibroblasts (NRK49F), rat mesangial cells (RMC), and mouse glomerular visceral
epithelial cells (podocyte), were immunoblotted with specific antibodies against c-met, Sp1, Sp3, and actin. A: representative Western blot
analysis of five different types of kidney cells. B: Western blot
analysis of c-met and Sp protein expression of nondifferentiated
(ND) and differentiated (D) podocytes. Positive signals with each
antibody are indicated (right) as are molecular sizes (in kDa; left). C:
linear regression of the abundance of c-met protein vs. the combined
levels of Sp1 and Sp3 in various types of kidney cells. Quantitative
data from densitometric analysis of Western blots were plotted after
correction with actin to ensure equal loading. Regression-line equation and correlation coefficient are shown.
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REGULATION OF C-MET GENE BY SP PROTEINS
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 10⫺8 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 inFig. 5. Electrophoresis mobility shift assay
(EMSA) demonstrates that both Sp1 and Sp3
proteins bind to the GC boxes in the c-met promoter region. A: 32P-labeled DNA fragment (F1)
corresponding to the nucleotide sequence ⫺217 to
⫺50 of the c-met promoter region was incubated
with nuclear protein extract (NPE) from
mIMCD-3 cells in EMSA reaction mixture. Three
major DNA-protein complexes (designated C1,
C2, and C3) were formed between renal epithelial
cell nuclear proteins and c-met promoter. Competition for binding was performed by including a
100-fold molar excess of unlabeled F1 itself or
oligonucleotides corresponding to Sp1, mutant
Sp1 (Sp1m), activator protein 1 (AP-1), nuclear
factor-␬B (NF-␬B), and cAMP response element
(CRE) as indicated. Sequences of these oligonucleotides are presented in Table 1. B: identification of these DNA-protein complexes (C1–C3)
was characterized by supershift analysis with
specific antibodies and normal rabbit IgG. Supershifted protein-DNA complexes with Sp1 antibody are designated as SS1 and SS2, and those
with Sp3 antibody are denoted as SS3 and SS2
(arrows).
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Fig. 4. Concomitant alterations of c-met expression with Sp1 and
Sp3 transcription factors during podocyte differentiation. Mouse
podocyte differentiation was initiated by switching the culture from
33 to 37°C in the absence of interferon-␥ under nonpermissive
conditions. At various time points (in days) as indicated, podocytes
were harvested and the cell lysate was immunoblotted with specific
antibodies against c-met, Sp1, Sp3, and actin. Identification (right)
and molecular sizes (in kDa; left) of the proteins are indicated.
hibitory 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 cisacting elements is essential for constitutive expression
of the c-met gene in renal epithelial cells.
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.
We found that the Akt phosphorylation and activation induced by HGF in different types of kidney cells
displayed distinctly dynamic patterns (Fig. 10). The
activation of Akt kinase by HGF took place in glomerular mesangial cells (RMC) and renal interstitial fibroblasts (NRK-49F) in a similarly dynamic fashion (Fig.
10). The Akt kinase was activated markedly and rapidly (as early as 5 min), but this induction was transient, and the phosphorylated Akt abundance quickly
returned toward baseline level after 1 h (Fig. 10). The
dynamics of Akt activation in mIMCD-3 cells and dif-
REGULATION OF C-MET GENE BY SP PROTEINS
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gested 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 popula-
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Fig. 6. Physical interaction between Sp1 and Sp3 proteins in renal
epithelial cells was demonstrated by coimmunoprecipitation.
mIMCD-3 epithelial cells were immunoprecipitated (IP) with Sp1
and Sp3 antibodies or normal rabbit IgG. Resulting complexes were
separated on polyacrylamide gels and immunoblotted (IB). A: immunoprecipitation with Sp1 antibody and blotting with Sp3 and Sp1
antibodies. B: immunoprecipitation with Sp3 antibody and blotting
with Sp1 and Sp3 antibodies. Coimmunoprecipitated Sp1 and Sp3
were evident in both experiments.
ferentiated podocytes were comparable to delayed responses starting at 30 and 60 min, respectively, after
HGF stimulation. Conversely, HGF induced rapid and
sustained Akt phosphorylation in HKC cells and nondifferentiated podocytes.
DISCUSSION
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 sugAJP-Renal Physiol • VOL
Fig. 7. Sp1 and Sp3 synergistically activate c-met promoter activity.
Reporter 0.2met-chloramphenicol acetyltransferase (CAT) plasmid
that contains the c-met promoter sequence corresponding to ⫺223 to
⫹60 linked to the coding region of the CAT gene was transiently
cotransfected with Sp1 and Sp3 expression vectors into Drosophila
Schneider cells. A: cell homogenates were prepared 48 h after cotransfection with a fixed amount (3 ␮g) of 0.2met-CAT reporter
plasmid and increasing amounts of individual pPac-Sp1 and pPacSp3 expression vectors as indicated. B: cells were cotransfected with
3 ␮g of 0.2met-CAT reporter construct and various amounts of
individual pPac-Sp1 and pPac-Sp3 plasmids or in combination as
indicated. Relative CAT activities (fold induction, with transfection
of 0.2met-CAT plasmid alone taken as 1.0) are presented as means ⫾
SE from 3 independent experiments. *P ⬍ 0.05; **P ⬍ 0.01.
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REGULATION OF C-MET GENE BY SP PROTEINS
tions 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 reAJP-Renal Physiol • VOL
Fig. 9. Chemical blockade of Sp1 binding with mithramycin A inhibits c-met expression in renal epithelial cells in dose- and timedependent manners. A: mIMCD-3 cells were incubated with various
concentrations of mithramycin A for 24 h. B: Cells were incubated
with 10⫺7 M of mithramycin A for different periods of time. Cell
lysates were separated on polyacrylamide gels and immunoblotted
with c-met antibody. The same blots were reprobed with actin to
ensure equal loading of the samples.
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Fig. 8. Deprivation of Sp proteins by cotransfection of decoy oligonulceotides inhibits c-met promoter activity. A: schematic representation of chimeric reporter plasmids 0.2met-CAT and 0.1met-CAT.
Three Sp1 sites were located at the c-met promoter region between
nucleotides ⫺68 and ⫺223 (ovals). B: mIMCD-3 cells were cotransfected with 0.2met-CAT plasmid and a 20- or 50-fold molar excess of
Sp1 or mutant Sp1 decoy oligos (Sp1mut oligo), respectively, as
indicated. Cells were also transfected with 0.1met-CAT construct as
a control in which the three Sp1 sites of the c-met promoter region
were deleted. Relative CAT activities (with 0.2met-CAT plasmid
alone taken as 100) are presented as means ⫾ SE from 3 independent experiments. **P ⬍ 0.01.
ceptors 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 cmet 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 mod-
REGULATION OF C-MET GENE BY SP PROTEINS
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Fig. 10. Activation of protein kinase B/Akt in various types of kidney cells in response to HGF stimulation. Major
types of kidney cells, including RMC cells, NRK-49F cells, mIMCD-3 cells, HKC cells, mouse differentiated
glomerular podocytes (D), and nondifferentiated podocytes (ND) were incubated with 20 ng/ml human recombinant
HGF for various periods of time as indicated. A: cell lysates were immunoblotted with antibodies against
phospho-specific Akt or total Akt, respectively. Molecular size markers (in kDa) are shown. B: dynamic patterns of
Akt phosphorylation and activation in various types of kidney cells after HGF stimulation. Quantitative data from
densitometric analysis of Western blots were plotted after correction with total Akt.
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REGULATION OF C-MET GENE BY SP PROTEINS
AJP-Renal Physiol • VOL
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.
The authors thank Drs. R. Tjian and G. Suske for providing the
Sp1 and Sp3 expression plasmids.
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.
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ulating 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
REGULATION OF C-MET GENE BY SP PROTEINS
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