Bone Morphogenetic Protein 2 Inhibits Platelet-derived Growth
Factor-induced c-fos Gene Transcription and DNA
Synthesis in Mesangial Cells
INVOLVEMENT OF MITOGEN-ACTIVATED PROTEIN KINASE*
Goutam
Ghosh Choundhury
§,
Yong-soo
Kim,
Matthias
Simon¶,
John
Wozney
,
Stephen
Harris,
Nandini
Ghosh-Choundhury**
, and
Hanna E.
Abboud
From the Departments of ** Medicine and Pathology, The University of
Texas Health Science Center and
Audie L. Murphy Memorial
Veterans Affairs Medical Center, San Antonio, Texas 78284-7882 and the
Genetics Institute, Cambridge, Massachusetts 01810
 |
ABSTRACT |
Bone morphogenetic proteins (BMPs) play an
important role in nephrogenesis. The biologic effect and mechanism of
action of these proteins in the adult kidney has not yet been studied.
We investigated the effect of BMP2, a member of these growth and differentiation factors, on mitogenic signal transduction pathways induced by platelet-derived growth factor (PDGF) in glomerular mesangial cells. PDGF is a growth and survival factor for these cells
in vitro and in vivo. Incubation of mesangial
cells with increasing concentrations of BMP2 inhibited PDGF-induced DNA
synthesis in a dose-dependent manner with maximum
inhibition at 250 ng/ml. Immune complex tyrosine kinase assay of PDGF
receptor
immunoprecipitates from lysates of mesangial cells treated
with PDGF showed no inhibitory effect of BMP2 on PDGF receptor tyrosine
phosphorylation. This indicates that the inhibition of DNA synthesis is
likely due to postreceptor events. However, BMP2 significantly
inhibited PDGF-stimulated mitogen-activated protein kinase (MAPK)
activity that phosphorylates the Elk-1 transcription factor, a
component of the ternary complex factor. Using a fusion protein-based
reporter assay, we also show that BMP2 blocks PDGF-induced
Elk-1-mediated transcription. Furthermore, we demonstrate that BMP2
inhibits PDGF-induced transcription of c-fos gene, a
natural target of Elk-1 that normally forms a ternary complex that
activates the serum response element of the c-fos gene.
These data provide the first evidence that in mesangial cells, BMP2
signaling cross-talks with MAPK-based transcriptional events to inhibit
PDGF-induced DNA synthesis. One target for this inhibition is the early
response gene c-fos.
 |
INTRODUCTION |
Bone morphogenetic proteins are a group of growth and
differentiation factors that were originally identified by their
ability to form ectopic bone (1, 2). Recently several members of this
family have been cloned. These proteins contain seven highly conserved
cysteines in the C terminus that are characteristic of the
TGF-
1 family of proteins
and thus fall into that broad superfamily (3-6). BMPs play an
important role in the development of many organs, including lung,
heart, teeth, gut, skin, and particularly the kidney (4). BMP7, a
member of this large family, has been shown to be abundantly expressed
in the ureteric bud epithelium before condensation of mesenchyme during
nephrogenesis. BMP7 is also expressed during glomerulogenesis (7). BMP7
null mice die after birth because of early cessation of kidney
development (8, 9). The rudimentary kidneys at birth lack glomeruli, the filtering units, likely due to lack of differentiation of metanephric mesenchymal cells (8).
High affinity binding of BMP2, another member of this
family, has been identified in a large variety of tissues and cells,
including the kidney (10). Similar to TGF-
, BMPs exert their effect
via type I and type II transmembrane serine-threonine kinase receptors
(4-6). Binding of BMPs to the receptor induces phosphorylation of the
type I receptor in the intracellular GS domain (6). Smad1 and Smad5
have been identified recently as downstream targets of the BMP
receptor. Upon BMP receptor activation, these cytoplasmic proteins
undergo serine phosphorylation that leads to its translocation to the
nucleus (11, 12). Furthermore the C-terminal region of Smad1 contains a
transcriptional activation domain (11, 13).
BMPs initiate signaling that involves synergistic and antagonistic
interplay of different pathways. It has been reported recently that
activation of epidermal growth factor receptor, a tyrosine kinase,
leads to phosphorylation of Smad1 in serine residues in a
MAPK-dependent manner. This phosphorylation inhibits
localization of Smad1 in the nucleus thus inhibiting its
transcriptional function (14). It is known that activators of tyrosine
kinase receptors, e.g. fibroblast growth factor receptor,
oppose the effect of BMP2 during limb bud outgrowth (15). Similarly,
epidermal growth factor receptor can block expression of osteogenic
differentiation markers induced by BMP2 (16). On the contrary,
fibroblast growth factor-induced genes essential for tooth development
can be blocked by BMP2 (17). These data indicate that there is
cross-talk between BMP receptor serine threonine kinase and its
components and the receptor tyrosine kinase cascades.
Platelet-derived growth factor (PDGF) is a potent mitogen for many
mesenchymal cells, including mesangial cells, vascular pericytes that
occupy the glomerular microvascular bed (18, 19). During embryogenesis
activation of PDGF receptor
(PDGFR) in multipotent mesenchymal
cells is necessary for development, growth, and survival of glomerular
mesangial cells (20, 21). In the adult kidney, overexpression and
activation of PDGFR is associated with proliferative and fibrotic
responses (22). One of the major signal transduction pathways initiated
by PDGF in target cells is the activation of MAPK (23, 24). Tyrosine phosphorylation of PDGFR creates binding sites for a number of proteins
characterized by the presence of about 100-amino acid residue sequence
motifs known as Src homology 2 domain (19, 25). Binding of one such Src
homology 2 domain-containing protein, SHP-2, to the
tyrosine-phosphorylated receptor stimulates the guanine nucleotide
exchange factor Son of sevenless to activate Ras, an upstream regulator
of MAPK cascade (19, 24, 26). We have shown recently that PDGF
stimulates PI 3-kinase which in turn regulates MAPK activity in
mesangial cells. Thus there is cross-talk between these two pathways
that lead to MAPK activation and cell growth (27). Inhibition of either
pathway blocks PDGF-induced mesangial cell proliferation, an important
biological response of these cells during glomerular pathology
(22).
Here we show that recombinant BMP2 inhibits PDGF-induced DNA synthesis
in mesangial cells in a dose-dependent manner. The mechanism involves BMP2 inhibition of PDGF-induced MAPK activity resulting in inhibition of Elk-1-dependent transcription of
an appropriate reporter gene. In support of this mechanism, we show that BMP2 inhibits PDGF-mediated c-fos gene transcription in
mesangial cells.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Tissue culture materials and LipofectAMINE were
obtained from Life Technologies, Inc. Nonidet P-40,
phenylmethylsulfonyl fluoride and Na3VO4 were
purchased from Sigma. GAL4-Elk-1 fusion plasmid and the
GAL-4-luciferase reporter construct were obtained from Stratagene. The
dual luciferase kit was purchased from Promega. Recombinant PDGF BB was
obtained from Amgen. TGF-
1 was purchased from R & D Systems. BMP2
was from the Genetics Institute. PDGFR
antibody was purchased from
Upstate Biotechnology Inc. MAPK antibody was from Santa Cruz.
[3H]Thymidine and [
-32P]ATP were from
Amersham Pharmacia Biotech.
Cell Culture--
Rat glomerular mesangial cells (kindly
provided by Dr. Jeff Kreisberg) or human mesangial cells were isolated
and characterized as described (27, 28). The human and rat cells were
used between 6th and 10th passages and 10th and 20th passages,
respectively. Cells were maintained in RPMI 1640 tissue culture medium
supplemented with antibiotic/antifungal solution and 17% fetal bovine
serum. Cells were serum-deprived for 48 h and treated with BMP2
for 30 min before addition of PDGF. For ease of transfection of rat
mesangial cells, biochemical studies and transfections were carried out in these cells.
Measurement of DNA Synthesis--
DNA synthesis in human
mesangial cells was measured as incorporation of
[3H]thymidine into trichloroacetic acid-insoluble
material as described previously (27, 29).
Immunoprecipitation and Tyrosine Kinase Assay--
Cells were
lysed in radioimmune precipitation buffer (20 mM Tris-HCl,
pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 0.1% aprotinin, and 1% Nonidet P-40)
at 4 °C for 30 min. The lysates were collected and centrifuged at
10,000 × g for 30 min at 4 °C. Protein was
determined in the cleared supernatant using the Bio-Rad reagent. For
PDGFR immunoprecipitation, equal amounts of protein were incubated with
the PDGFR
antibody on ice for 30 min. Protein A-Sepharose beads were
added and incubated at 4 °C for 3 h on a rotating device. The
beads were washed three times with radioimmune precipitation buffer and
twice with buffer A (50 mM Tris-HCl, pH 7.4, 0.5 mM Na3VO4). Tyrosine kinase
activity of PDGFR was measured directly on the Immunobeads as described (30). Briefly, the immunobeads were resuspended in kinase assay buffer
(50 mM HEPES, pH 7.4, and 10 mM
MnCl2). 20 µCi of [
-32P]ATP was added,
and the reaction was incubated at 30 °C for 15 min. At the end of
the reaction, 2 × sample buffer was added, and the labeled
proteins were separated on 7.5% SDS gel and visualized by autoradiography.
MAPK Assay--
MAPK assay was performed as described earlier
(27). Briefly, cleared cell lysates were immunoprecipitated with
MAPK-specific antibody. The immunobeads were resuspended in MAPK assay
buffer (10 mM HEPES, pH 7.4, 10 mM
MgCl2, 0.5 mM dithiothreitol, and 0.5 mM Na3VO4) in the presence of 0.5 mg/ml myelin basic protein (MBP), 0.5 µM cAMP-dependent
protein kinase inhibitor, and 25 µM cold ATP plus 1 µCi
of [
-32P]ATP. This reaction was incubated at 30 °C
for 30 min. The mixture was then incubated on ice for 10 min followed
by 15% SDS gel analysis. Phosphorylated MBP was visualized by
autoradiography. The bands were quantitated by densitometry.
Immunoblotting--
Immunoblotting of MAPK was performed as
described previously (29, 31).
Transient Transfections and Luciferase Assay--
Transient
transfection of mesangial cells was performed with LipofectAMINE with a
modification of vendor's protocol. Briefly, 1 µg of firefly
luciferase reporter plasmid was used. In experiments with GAL4 DNA
binding domain-Elk-1 transactivation domain fusion protein, 50 ng of
the fusion construct was used. To standardize for transfection
efficiency, 25 ng of cytomegalovirus-Renilla luciferase reporter
plasmid was included. These plasmids were mixed with 0.2 ml of Opti-MEM
medium and 4 µl of LipofectAMINE. The mixture was incubated at room
temperature for 45 min. To this mixture, 0.8 ml of Opti-MEM medium was
added. Mesangial cell monolayers in 35-mm culture dishes were washed
twice with 2.5 ml of serum-free RPMI 1640 and washed once with 2 ml of
Opti-MEM. To this monolayer the DNA/LipofectAMINE mix was added. The
cells were incubated at 37 °C at 5% CO2 for 5 h,
after which 1 ml of fresh medium with twice the concentration of serum
was added. The cells were grown for another 24 h. The cells were
then made quiescent by incubation in serum free RPMI 1640 for 48 h. BMP2 at a concentration of 250 ng/ml was added for 30 min before the
addition of 10 ng/ml PDGF for 12 h. The cells were lysed in
extraction buffer, and the luciferase activity was determined using the
dual luciferase assay kit according to the method provided by the manufacturer.
Northern Analysis--
Total RNA was isolated from mesangial
cells by guanidinium isothiocyanate lysis and centrifugation on cesium
chloride gradients (32). Total RNA was separated on
formaldehyde-agarose gels and probed with fibronectin cDNA
essentially as described (33). Fibronectin cDNA was a kind gift of
Dr. Jeffrey Barnes.
Data Analysis--
Significance of the data was determined by
unpaired Student's t test.
 |
RESULTS |
BMP2 Modulates PDGF-induced DNA Synthesis--
We and others (18,
19, 34) have shown previously that PDGF is a potent mitogen for
mesangial cells in culture and that TGF-
inhibits PDGF-induced DNA
synthesis. BMP2 falls in the TGF-
superfamily of proteins (3, 6). To
test the effect of BMP2 in mesangial cells, we first identified the
receptor in these cells with total RNA as template in reverse
transcriptase polymerase chain reaction using specific primers for BMP
receptor type IA (data not shown). We then studied the effect of
different concentrations of BMP2 on PDGF-induced DNA synthesis. As
shown in Fig. 1, PDGF stimulates DNA
synthesis in mesangial cells. Significant inhibition of DNA synthesis
by BMP2 was observed at doses higher than 50 ng/ml. 50% attenuation of
PDGF-mediated DNA synthesis was obtained with 250 ng/ml BMP2. No
further inhibition was obtained even at a dose as high as 500 ng/ml of
BMP2.

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Fig. 1.
Effect of BMP2 on PDGF-induced DNA synthesis
in human mesangial cells. Serum-deprived mesangial cells were
incubated with different concentration of BMP2 for 30 min before
addition of 10 ng/ml PDGF BB. [3H]Thymidine incorporation
was measured as an index of DNA synthesis. Results are mean ± S.E. of three independent experiments, each done in triplicate. *,
p < 0.05 versus PDGF-untreated cells. @,
p < 0.05 versus PDGF-treated, but
BMP2-untreated, cells.
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Effect of BMP2 on PDGFR and MAPK Activation--
PDGF exerts its
biological effects by stimulating the tyrosine kinase activity of its
receptor. We have shown recently that inhibition of PDGFR tyrosine
kinase activity blocks PDGF-induced DNA synthesis in mesangial cells
(31). Thus the inhibitory effect of BMP2 on PDGF-induced DNA synthesis
(Fig. 1) may result from inhibition of tyrosine kinase activity of
PDGFR. To test the effect of BMP2 on PDGFR tyrosine kinase activity, we
treated mesangial cells with BMP2 followed by stimulation with PDGF.
The cell lysate was immunoprecipitated with PDGFR antibody and the
receptor-associated tyrosine kinase activity was measured by
immune-complex kinase assay. The results show that PDGF stimulates
PDGFR-associated tyrosine kinase activity (Fig.
2, compare lane 2 with
lane 1), and incubation of mesangial cells with BMP2 and
PDGF does not inhibit PDGF-induced intrinsic tyrosine kinase activity
of the PDGFR (Fig. 2, compare lane 4 with lane
2). These data indicate that BMP2 does not modulate PDGF function
at the level of receptor autophosphorylation but may alter downstream
signaling molecule(s) of the PDGFR kinase cascade.

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Fig. 2.
Effect of BMP2 on PDGFR activation.
Serum-deprived mesangial cells were treated with 250 ng/ml BMP2 for 30 min before addition of 10 ng/ml PDGF for 5 min. Equal amounts of
lysates were immunoprecipitated with PDGFR -specific antibody, and
the immunoprecipitates were used in immune complex kinase assay as
described under "Experimental Procedures." The numbers
in the left margin indicate molecular mass markers in
kilodaltons.
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We have shown recently that PDGF increases MAPK activity in mesangial
cells. Activation of this kinase cascade is essential for many
mitogens, including PDGF (23, 27). To test if the inhibitory effect of
BMP2 on PDGF-induced DNA synthesis is via modulation of MAPK activity,
we measured the activity of MAPK in MAPK immunoprecipitates after BMP2
and PDGF treatment of mesangial cells. BMP2 significantly inhibited
MAPK activity stimulated by PDGF (Fig.
3A). Quantitation of these
data revealed that BMP2 caused approximately 60% inhibition of
PDGF-induced MAPK activity (Fig. 3B). These data suggest
that BMP2-mediated inhibition of PDGF-induced DNA synthesis may also
result from attenuation of MAPK activity.

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Fig. 3.
Effect of BMP2 on PDGF-induced MAPK
activity. A, serum-deprived mesangial cells were
incubated with BMP2 for 30 min. The cells were treated with PDGF for 5 min. 100 µg of cell lysate was immunoprecipitated with MAPK-specific
antibody, and the immunoprecipitates were used in an in
vitro MAPK assay in the presence of MBP as substrate and
[ -32P]ATP. Phosphorylated MBP was separated on a 15%
SDS gel. Molecular mass markers are shown in kilodaltons in the
left margin. The bottom panel shows the
immunoblot analysis of cell lysates with MAPK antibody. B,
scanning densitometry of A. Phosphorylated MBP bands were
scanned in two dimensions, and the density was corrected for the
background present in the lane. Data represent corrected densities and
are expressed as arbitrary units.
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TGF-
, another member of this family, is known to inhibit DNA
synthesis in a variety of cells, including mesangial cells (34). Since
we demonstrated in Fig. 3 that one of the mechanism of BMP2-induced inhibition of DNA synthesis may involve inhibition of MAPK, we studied
the effect of TGF-
on PDGF-induced MAPK activity. Mesangial cells
were treated with TGF-
and PDGF. MAPK was immunoprecipitated from
the cell lysate, and its activity was assayed. As shown in Fig.
4, similar to BMP2, TGF-
also
inhibited PDGF-induced MAPK activity in mesangial cells. Therefore,
TGF-
intercepts an early signal transduction pathway to elicit its
inhibitory effect on DNA synthesis.

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Fig. 4.
Effect of TGF- on
PDGF-induced MAPK activity. Serum-deprived mesangial cells were
incubated with 2 ng/ml TGF- 1 for 30 min followed by stimulation with
10 ng/ml PDGF for 5 min. MAPK was immunoprecipitated and assayed by
immune complex kinase assay using MBP as substrate as described in the
legend to Fig. 3. Molecular mass markers are shown in kilodaltons in
the left margin. The bottom panel shows the
immunoblot analysis of cell lysates with MAPK antibody.
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BMP2 Inhibits MAPK-mediated Transcription--
Activation of MAPK
stimulates transcription of a variety of target genes that are
necessary for the mitogenic effect of growth factors (23, 24). Part of
this transcriptional activation is mediated by direct phosphorylation
of certain substrate transcription factor(s) by MAPK. One target
protein of MAPK is the ternary complex factor Elk-1. Elk-1 belongs to a
subgroup of ETS domain family of transcription factors (35). Addition
of mitogens to cells stimulates serine phosphorylation of the
C-terminal domain of Elk-1 by MAPK. This phosphorylation of Elk-1 is
necessary for its ability to transactivate target genes (36, 37). To
test whether the regulation of MAPK by BMP2 in mesangial cells results in Elk-1-mediated transcriptional activation, we performed
cotransfection assays with an expression vector encoding the Elk-1
C-terminal transactivation domain fused to the GAL4 DNA binding domain
and a reporter plasmid under the control of GAL4 DNA element. This is a
sensitive assay for Elk-1 phosphorylation and activation. As expected,
PDGF increases GAL4-dependent reporter gene expression in
mesangial cells transfected with GAL4-ElK-1 fusion plasmid, suggesting
activation of Elk-1 transcription factor (Fig.
5A). Prior exposure of
mesangial cells to BMP2 inhibited PDGF-induced reporter gene expression
in the Elk-1-dependent promoter. Many mitogens, including
PDGF, are potent activators of c-fos mRNA expression
(38, 39). Elk-1 forms a ternary complex with serum response factor and
the serum response element (SRE) that is present in many genes (35).
Thus SRE present in the c-fos gene promoter is a target of
Elk-1 in mitogen-stimulated cells (39). Regulation of Elk-1 activity
modulates the transcription of the c-fos gene. We performed
transient transfection assays in mesangial cells with a reporter gene
under the control of c-fos promoter that includes SRE. PDGF
stimulates c-fos gene transcription (Fig. 5B). Exposure of mesangial cells to BMP2 inhibits both basal as well as
PDGF-induced c-fos transcription (Fig. 5B).
Collectively these data indicate that BMP2 blocks transcriptional
events that result from activation of MAPK by PDGF.

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Fig. 5.
BMP2 inhibits PDGF-induced
transcription. A, effect of BMP2 on PDGF-induced
Elk-1-mediated transcription of reporter gene. Mesangial cells were
transfected with 1 µg of GAL4-luciferase reporter plasmid, 50 ng of
GAL4-Elk-1 fusion plasmid, and 25 ng of cytomegalovirus-Renilla plasmid
as described under "Experimental Procedures." Serum-starved
transfected cells were treated with BMP2 for 30 min followed by PDGF,
and the luciferase activity was measured in the cell lysate as
described under "Experimental Procedures." B, effect of
BMP2 on PDGF-induced c-fos transcription. A reporter
construct in which the luciferase cDNA is driven by a 550-bp
fragment of c-fos promoter was transiently transfected with
cytomegalovirus-Renilla plasmid into mesangial cells. Serum-starved
transfected cells were treated with BMP2 and PDGF, and the luciferase
activity was determined in the cell lysate as described in
A.
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Effects of BMP2 and TGF-
on Fibronectin mRNA Expression in
Mesangial Cells--
Increased extracellular matrix production is one
of the feature of mesangial proliferative glomerulonephritis that
includes fibrosis (40). One major biological activity of TGF-
,
besides inhibition of DNA synthesis, is stimulation of matrix
production in many cells, including mesangial cells (41). In these
cells, TGF-
increases the formation of collagen type I and
fibronectin (42). The increase in fibronectin production in response to TGF-
is mediated at least partially at the transcriptional level (43). However, the effect of BMP2, a member of the TGF-
family, on
matrix gene expression has not been studied in mesangial cells. Mesangial cells were treated with either TGF-
or BMP2 for different periods of time. Total RNAs were analyzed by Northern blotting using a
cellular fibronectin cDNA probe. The data show that after both 8 and 16 h of stimulation, TGF-
increases fibronectin mRNA expression (Fig. 6, lanes 3 and 6). Unlike TGF-
, BMP2 does not increase fibronectin
mRNA expression in mesangial cells. This finding may have broader
implication to the potential use of BMP2 as antimitotic agent in
proliferative and fibrotic disorders.

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Fig. 6.
Effect of TGF- and
BMP2 on fibronectin gene expression. Mesangial cells were
incubated with either TGF- 1 or BMP2 for the indicated time periods.
10 µg of total RNA was separated electrophoretically in
formaldehyde-agarose gel and transferred to gene screen membrane.
Upper panel, transferred RNAs were hybridized with
fibronectin cDNA. The lower panel shows the ethidium
bromide staining of the same blot.
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 |
DISCUSSION |
These studies demonstrate that BMP2 inhibits PDGF-induced DNA
synthesis in mesangial cells by blocking PDGF-stimulated MAPK activity
that leads to inhibition of Elk-1-dependent transcriptional events and c-fos gene expression. BMP2 had no inhibitory
effect on PDGFR activation. These data provide the first evidence that BMP2 blocks DNA synthesis in mammalian cells by blocking MAPK-mediated signal transduction.
We have shown previously that PDGF-mediated signal transduction in
mesangial cells is initiated in the plasma membrane by tyrosine
phosphorylation of the dimerized PDGF receptor (29, 31, 32). Three
major signal transduction pathways are activated by PDGFR. These are
phospholipase C
, phosphatidylinositol 3-kinase and MAPK (19, 24,
25). Present evidence indicates that each of these signaling molecules
contributes to mitogenic effect of PDGF. There is evidence of
cross-talk among these signaling pathways. For example, we have
recently shown that inhibition of PI 3-kinase blocks PDGF-induced DNA
synthesis and that PI 3-kinase is an upstream regulator of MAPK in
mesangial cells (27).
BMP2 is a member of the TGF-
superfamily of proteins, although they
differ from the TGF-
family in several features. They contain seven
conserved cysteines instead of nine present in TGF-
. Unlike TGF-
,
each BMP family member is glycosylated (3). Both TGF-
and BMPs
function through distinct type I and type II receptors (5, 6). TGF-
binds to type II receptor and then recruits the type I protein to
undergo oligomerization (5). In the absence of type II receptor TGF-
cannot interact with type I receptor alone. BMPs can interact with both
receptors independently with low affinity; however, in the presence of
both receptors, BMPs bind with a very high affinity and can signal to
induce appropriate genes. We have shown previously that TGF-
inhibits PDGF-induced mesangial cell proliferation (34). It is known
that TGF-
exerts its inhibitory effect by increasing the expression
of p27, leading to the inhibition of cyclin-dependent
kinases, a point of convergence of many mitogenic signaling pathways
(44). Thus inhibition of cyclin-dependent kinases causes
cells to arrest in G1 and prevents their entry into S
phase. Whether BMP2 regulates any cyclin-dependent kinase
inhibitors is not known. It should be emphasized that the concentration
of PDGF that was used in our studies elicits maximum stimulation of DNA
synthesis in mesangial cells similar to that observed with serum (data
not shown). In this report, we show that 100 ng/ml BMP2, which is
rather a high concentration, significantly inhibits PDGF-induced DNA
synthesis in mesangial cells (Fig. 1). There is a wide variability
among various cell types in threshold doses of BMP2 that result in
inhibition of DNA synthesis. In a recent report, Ide et al.
(45) demonstrated that BMP2 at a dose of 100 ng/ml significantly
inhibited the growth of human prostate cancer cells stimulated by serum
and androgen. It remains to be determined if such requirement of high
concentration of BMP2 reflects number or affinity of BMP2 receptors on
the target cells. Moreover, the pathophysiologic relevance of these
concentrations of BMP2 to concentrations achieved locally in cell or
tissue microenvironment remains to be determined.
Receptor tyrosine kinases are known to be phosphorylated at serine and
threonine residues by downstream signaling kinases, and this
phosphorylation inactivates receptor function (46, 47). PDGFR may also
be negatively modulated by serine phosphorylation directly via BMP
receptor signaling upon exposure of mesangial cells to BMP2. However,
our results argue against this possibility, since BMP2 does not inhibit
ligand-induced PDGFR-associated tyrosine kinase activity (Fig. 2). As
discussed earlier, attenuation of PI 3-kinase and MAPK results in
inhibition of PDGF-induced DNA synthesis (27). However the inhibitory
effect of BMP2 on PDGF-mediated DNA synthesis was only 50% (Fig. 1),
suggesting that BMP2 does not completely block all the parallel early
mitogenic signal transduction pathways. Indeed little inhibitory effect
of BMP2 was observed on PDGF-induced PI 3-kinase activity (data not
shown). Although BMP2 inhibits PDGF-stimulated MAPK activity
significantly (Fig. 3), it also was not sufficient to completely block
PDGF-induced DNA synthesis. In fact, BMP2 alone stimulates MAPK
activity very weakly (e.g. Fig. 3A,
compare lane 3 with lane 1 and Fig. 3B). These data indicate that MAPK is regulated in a
positive manner by BMP2 alone, while in the presence of PDGF
stimulation, it exerts a negative regulatory signal. The signal
initiated by BMP2 in the presence of PDGF is not sufficient to
completely inhibit MAPK activity induced by PDGF (Fig. 3). It should be
emphasized that TGF-
also inhibits PDGF-induced MAPK activity in
mesangial cells (Fig. 4). This action of TGF-
demonstrates that
along with its late effects on cell cycle proteins, it also blocks
early mitogenic signal transduction pathways. MAPK is primarily
regulated by activation of Ras, Raf1, and MEK (23, 24). Also protein kinase C
can stimulate MAPK activity in cells via direct
phosphorylation of Raf1 (48). PDGF is known to activate all these
enzymes (27, 29, 32). Which of these proteins is targeted by BMP2 or
TGF-
to exert their inhibitory effect is not yet known.
Early PDGF growth signals are amplified and eventually integrate into
the nucleus to initiate de novo transcription of genes and
synthesis of proteins that are necessary for their biological function.
There is evidence that MAPK can positively regulate activities of
several transcription factors. MAPK activity is also involved in the
post-transcriptional control of gene expression (23). In
Drosophila, the ETS domain containing transcription factor,
PointedP2, is phosphorylated by Rolled/ERKA at a single conserved
threonine-proline residue that is essential for R7 photoreceptor cell
differentiation (49, 50). In mammalian cells, MAPK directly phosphorylates the ETS domain transcription factors Elk-1 in at least
three serine residues in the C-terminal C-box (23, 36). Mutation of the
MAPK phosphorylation sites in Elk-1 reduced serum-induced transcriptional activation by a GAL4-Elk-1 fusion protein-GAL4 reporter
assay (35, 51). We now provide evidence that inhibition of PDGF-induced
MAPK activity by BMP2 blocks GAL4-Elk-1-mediated transcription (Fig.
5A). These data indicate that Elk-1-mediated transcription
is a necessary component of the mitogenic effect of PDGF.
c-fos expression is regulated by SRE present in its
promoter. The SRE has been shown to be sufficient for rapid
transcriptional induction of this gene in response to many growth
factors (38, 39). The serum response factor is a 64-kDa protein with
DNA binding and transactivation properties. After binding to its
cognate core sequence in the SRE, it recruits Elk-1 to the 5' end of
the core sequence. The recruitment of Elk-1 in the ternary complex involving SRF and DNA depends upon the phosphorylation of its C-terminal domain by MAPK (23, 35, 36). Mutation of these phosphorylation sites inhibits c-fos gene expression (51).
Our data show a linear relation of MAPK activation and c-fos
transcription by PDGF (Fig. 5B). Furthermore, inhibition of
PDGF-induced MAPK by BMP2 inhibits c-fos gene expression
(Fig. 5B). We should emphasize that treatment of mesangial
cells with BMP2 also inhibits the basal transcription of the
c-fos gene (Fig. 5B). These data indicate that
apart from the effect of BMP2 on MAPK that leads to inhibition of
PDGF-induced c-fos expression, BMP2 modulates other pathways that regulate basal transcription of this gene.
The role of BMP2 in the biology of mesangial and other kidney cells
remains to be determined. TGF-
plays a major role in fibrotic
disorders, including glomerulosclerosis (41). This deleterious action
of TGF-
is due to its effect on matrix accumulation in glomerular
cells, including mesangial cells (41). However, unlike TGF-
, BMP2 at
doses that inhibit DNA synthesis does not stimulate fibronectin gene
expression in mesangial cells (Fig. 6). Nakaoka et al. (52)
have demonstrated recently that in vascular smooth muscle cells,
TGF-
, but not BMP2, stimulates collagen synthesis. These findings
may have important therapeutic implication in proliferative and
fibrotic disorders. Mesangial cell proliferation and matrix synthesis
are prominent features of inflammatory glomerular diseases. BMP2 may
prove to be an effective antiproliferative agent for
mesangioproliferative disorders without matrix expansion.
 |
ACKNOWLEDGEMENT |
We thank Sergio Garcia for help with the cell culture.
 |
FOOTNOTES |
*
This work was supported in part by a Veterans Administration
Medical Research Service grant and National Institutes of Health Grants
DK 50190 (both to G. G. C.), DK43988 (to H. E. A.),
and AR 44728 (to S. H.).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.
§
To whom all correspondence should be addressed: Dept. of Medicine,
The University of Texas Health Science Center, 7703 Floyd Curl Dr., San
Antonio, TX 78284-7882. E-mail: choundhuryg{at}uthscsa.edu.
¶
Supported by a Research Fellowship from the German Research
Foundation and by a fellowship award from the National Kidney Foundation.

Supported by an institutional American Cancer Society grant.
 |
ABBREVIATIONS |
The abbreviations used are:
TGF-
, transforming growth factor-
;
BMP, bone morphogenetic protein;
MAPK, mitogen-activated protein kinase;
PDGF, platelet-derived growth factor;
PDGFR, PDGF receptor;
PI, phosphatidylinositol;
MBP, myelin basic
protein;
SRE, serum response element.
 |
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