Sp1 and Sp3 transcription factors synergistically
regulate HGF receptor gene expression in kidney
XIANGHONG ZHANG,1,2YINGJIAN LI,1CHUNSUN DAI,1
JUNWEI YANG,1PETER MUNDEL,3AND YOUHUA LIU1
1Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261;
2Department of Cell Biology, Peking Union Medical College, Beijing 100005, China; and
3Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461
Submitted 22 May 2002; accepted in final form 4 August 2002
Zhang, Xianghong, Yingjian Li, Chunsun Dai, Jun-
wei 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 investi-
gated 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 expres-
sion 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 be-
tween 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 block-
ade 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 transcrip-
tion 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 struc-
ture and function than originally thought.
hepatocyte growth factor; Sp1; Sp3; gene regulation; Akt
HEPATOCYTE GROWTH FACTOR (HGF) receptor is the prod-
uct 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 origi-
nally isolated from a human osteogenic sarcoma cell
line that was treated in vitro with the chemical carcin-
ogen N-methyl-N?-nitro-N-nitrosoguanidine (37). Ma-
ture c-met protein is a 190-kDa, disulfide-linked het-
erodimer that consists of ?- and ?-subunits (37). The
?-subunit is heavily glycosylated and is completely
extracellular. The ?-subunit has an extracellular por-
tion 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 autophosphoryla-
tion 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 presum-
ably 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 pre-
dominantly 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 inter-
actions, which are critical processes in organ develop-
ment, tissue regeneration, and tumorigenesis under
various physiological and pathological conditions. How-
ever, 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 glo-
merular 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
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.
Am J Physiol Renal Physiol 284: F82–F94, 2003;
0363-6127/03 $5.00 Copyright © 2003 the American Physiological Societyhttp://www.ajprenal.orgF82
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 set-
tings (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 ques-
tion 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 function-
ally activated its transcription. All types of kidney cells
tested in vitro expressed the functional c-met receptor
and induced protein kinase B (PKB)/Akt phosphoryla-
tion after HGF stimulation.
MATERIALS AND METHODS
Animals. Male CD-1 mice (body wt 20–24 g) were pur-
chased 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 pro-
cedures 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 de-
capsulated. 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 histol-
ogy and immunohistochemical staining.
Cell culture and treatment. Mouse inner medullary collect-
ing duct epithelial cell line 3 (mIMCD-3), rat renal intersti-
tial 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 tu-
bular 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 es-
tablished from the transgenic mouse that carries a thermo-
sensitive 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 differ-
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 par-
affin-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 proce-
dures described previously (7). The primary antibody against
mouse c-met (sc-8057) was obtained from Santa Cruz Bio-
chemical (Santa Cruz, CA). As a negative control, the pri-
mary antibody was replaced with nonimmune normal IgG,
and no staining occurred.
Frozen section and immunofluorescence staining. Cryosec-
tions 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 previ-
ously (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 contain-
ing 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
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% bromo-
phenol 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 (Amer-
sham, Arlington Heights, IL) in transfer buffer that con-
tained 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 sec-
ondary antibody (Sigma) at a dilution of 1:10,000 for 1 h at
room temperature in 5% nonfat milk dissolved in TBS. Mem-
branes 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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