(Received for publication, November 30, 1994)
From the
Mesangial cells predominantly express platelet-derived growth factor (PDGF)-A chain mRNA and release PDGF. Mesangial cell PDGF-A chain mRNA abundance is regulated by several agents including phorbol esters. We have recently demonstrated that induction of PDGF-A chain mRNA abundance in response to phorbol 12-myristate 13-acetate is primarily due to gene transcription. We have now analyzed the 5`-flanking region of the PDGF-A chain promoter to identify DNA binding protein(s) which have the potential to regulate PDGF-A chain gene transcription in human mesangial cells. DNase I footprint analysis of the 5`-flanking region of the PDGF-A chain promoter identifies a DNase I protected region at the location -82 to -102 corresponding to the sequence 5`-GGCCCGGAATCCGGGGGAGGC-3`. Therefore, nuclear extracts from human mesangial cells contain a protein, PDGF-A-BP-1, that binds to a DNA sequence (-82 to -102) in the promoter region of the PDGF-A chain gene. Gel mobility shift analysis using labeled oligomer corresponding to the binding site for PDGF-A-BP-1 indicates that PDGF-A-BP-1 is induced by phorbol ester in mesangial cells as well as fat-storing cells (>20 fold). Egr-1 protein does not bind to labeled PDGF-A-BP-1 oligomer and does not compete with the binding of PDGF-A-BP-1. In addition, SP-1 binding sequence does not compete with the binding sequence of the mesangial cell protein. PDGF-A-BP-1 appears to represent a novel protein which is induced by phorbol ester and thus has the potential for an important role in the transcriptional regulation of the PDGF-A chain gene in mesangial cells and other vascular pericytes.
Platelet-derived growth factor (PDGF) ()is a
glycoprotein composed of two subunits referred to as A and B chains
that are encoded by two separate genes (reviewed in (1) ). The
dimeric forms of PDGF, PDGF-AA, -AB, and -BB, have been shown to be
biologically active and are involved in a receptor-mediated induction
of several of the biological effects of PDGF including cellular
proliferation, chemotaxis, and cell ruffling (reviewed in (1) ). PDGF has been implicated in the pathogenesis of diverse
processes including glomerulonephritis, atherosclerosis,
carcinogenesis, hypertension, wound healing, and tissue development
(reviewed in Ref 1). PDGF-A chain is a growth regulator in several cell
types(2, 3, 4) . PDGF-A chain mRNA is more
abundantly expressed than PDGF-B chain mRNA in human mesangial
cells(5) , vascular smooth muscle cells(6) , and
undifferentiated F9 embryonal carcinoma stem cells(7) .
Considerable interest has developed in recent years concerning the
regulation of PDGF-A chain gene expression due to its potential role in
growth and development as well as tissue morphogenesis and
differentiation(4, 7, 8) .
Glomerular mesangial cells, vascular pericytes in the renal microvasculature, are useful model to study PDGF-A chain gene regulation. A number of agents including growth factors have been shown to regulate steady state levels of PDGF-A chain mRNA abundance and gene transcription in these and other cells(5, 9, 10, 11, 12, 13) . Phorbol 12-myristate 13-acetate (PMA) is a potent inducer (10-15-fold) of PDGF-A chain mRNA (5, 9) . The increase in PDGF-A chain mRNA levels in cells treated with PMA ranges from 5- to 15-fold compared to control untreated cells (5, 9, 14) . We have also reported that PMA induces PDGF-A chain gene transcription in human mesangial cells(14) . We have now utilized DNase I footprint analysis to identify region(s) in the 5`-flanking sequence of PDGF-A chain promoter that binds to nuclear proteins from human mesangial cells. We found a unique protein binding domain in the PDGF-A chain promoter region that is regulated by phorbol esters. We also show by gel mobility shift and competition analyses that the binding of the identified nuclear protein PDGF-A-BP-1 is specific and is induced by PMA. This nuclear protein is likely to regulate PDGF-A chain function in mesangial cells.
Figure 1: PDGF-A chain gene promoter.
Figure 9: Gel mobility shift analysis using deleted sequences from wild type PDGF-A-BP-1 binding site. A, wild type (wt) or deletion mutant (mut) sequences are boxed in the diagram. Complementary pairs of single-stranded deoxynucleotides were synthesized and annealed (18) to form double-stranded oligomers. B, gel mobility shift analysis using wild type -82/-102 (lanes 1 and 2), mut -82/-96 (lanes 3 and 4), mut -88/-102 (lanes 5 and 6), and mut -82/-88, -95/-102 (lanes 7 and 8) as a labeled probe. Experimental details are as for Fig. 3. Nuclear extracts were prepared from human mesangial cells which were not treated (lanes 1, 3, 5, and 7) or treated with PMA (lanes 2, 4, 6, and 8) for 3 h. C, competition experiments show that unlabeled wild type -82/-102 or mut -82/-96 oligomer compete for the PDGF-A-BP-1 binding site in gel mobility shift analysis. Experimental details are as in B. With labeled wild type -82/-102 oligomer (lanes 1-5) and with labeled mut -82/-96 oligomer (lanes 6-10). Lanes (except 1 and 6) contained nuclear extracts treated with PMA (lanes 3-5 and 8-10) or without PMA treatment (lanes 2 and 7). For competition: 100-fold unlabeled wild type -82/-102 oligomer (lane 4) or mut -82/-96 oligomer (lane 9), and 100-fold nonspecific oligomer (lanes 5 or 10) was used for competition analysis.
Figure 3:
Induction of PDGF-A-BP-1 by phorbol ester
in gel mobility shift analysis in human mesangial cells. Nuclear
extracts were prepared from human mesangial cells made quiescent in
serum-free medium for 2 days and the same cells treated with
10M PMA for 3 h. Nuclear extracts were
incubated with 5`-end labeled specific oligomer probe
(GGCCCGGAATCCGGGGGAGGC) for 30 min on ice and then analyzed on 6.7%
polyacrylamide gel as described under ``Experimental
Procedures.'' The protein-DNA complexes are labeled as
A
, A
, and A
. 5 µg of nuclear
proteins were used in the gel mobility shift assays as follows: lane 1, labeled probe alone without nuclear extract; lane
2, nuclear extract from quiescent cells treated with PMA; lane
3, nuclear extract from cells maintained in serum-free
medium.
Figure 2: Identification of a region in PDGF promoter as a binding site for nuclear protein. Nuclear extracts were prepared from human mesangial cells maintained in complete medium. DNase I footprint analysis was performed as described under ``Experimental Procedures.'' Boundaries of the protected region from the DNase I digestion are marked. Lanes with (+) or without(-) nuclear extracts are designated.
Figure 4:
Induction of PDGF-A-BP-1 by phorbol ester
in gel mobility shift analysis in liver fat-storing cells. Nuclear
extracts were prepared from liver fat-storing cells made quiescent in
serum-free medium for 2 days and same cells treated with
10M PMA for 4 h. Nuclear extracts were
incubated with 5`-end labeled specific oligomer probe for 30 min on ice
and then analyzed on 6.7% polyacrylamide gel as described under
``Experimental Procedures.'' The protein-DNA complexes are
labeled as A
, A
, and A
. 5 µg of
nuclear proteins were used in the gel mobility shift assays as follows: lane 1, labeled probe alone without nuclear extract; lane
2, nuclear extract from quiescent cells; lane 3, nuclear
extract from cells maintained in serum-free medium and treated with
PMA.
Figure 5: Effect of specific and nonspecific competition on the PDGF-A-BP-1 in gel mobility shift analysis using nuclear extracts prepared from PMA-stimulated human mesangial cells. Experimental details are as for Fig. 3. Nuclear extracts were prepared from human mesangial cells which were treated with PMA for 3 h. Lane 1, labeled probe without nuclear extract; lanes 2-5 contained nuclear extracts with labeled probe as follows: lane 2, no poly(dI-dC); lane 3, no competitor; lane 4, 100-fold unlabeled specific oligomer; lane 5, 100-fold nonspecific oligomer.
Figure 6: Competition experiments demonstrating that unlabeled SP-1 oligomer does not compete for PDGF-A-BP-1 binding site in gel mobility shift analysis. Experimental details are as for Fig. 3. Lane 1, labeled probe without nuclear extract; lanes 2-5, contained nuclear extracts with labeled probe as follows: lane 2, no competitors; lane 3, 100-fold excess unlabeled specific oligomer; lane 4, 100-fold excess nonspecific oligomer; and lane 5, 100-fold excess SP-1 oligomer.
Figure 7: Competition studies using labeled SP-1 oligomer in gel mobility shift assays. Experimental details are as for Fig. 3except that labeled oligomer used in binding assays was SP-1 instead of oligomer corresponding to binding site for PDGF-A-BP-1. Lane 1, labeled SP-1 oligomer without nuclear extract; lanes 2-5 contained nuclear extracts and labeled SP-1 as follows: lane 2, no competitor; lane 3, 100-fold excess unlabeled SP-1 oligomer; lane 4, 100-fold excess nonspecific oligomer; lane 5; 100-fold excess unlabeled specific oligomer corresponding to binding site for PDGF-A-BP-1.
Figure 8: Effect of Egr-1 peptide and Egr-1 IgG on binding of labeled specific oligomer corresponding to the binding site for PDGF-A-BP-1 and nuclear extracts from human mesangial cells in gel mobility shift analysis. Experimental details are as for Fig. 3. Lanes 1, 3, and 4 are without nuclear extracts; lanes 2, 5, and 6, with nuclear extracts (5 µg). Egr-1 peptide, 1 µg (lanes 3, 4 and 7) was added without nuclear extracts. Egr-1 IgG was added to nuclear extracts and incubated for 90 min prior to addition of 5`-end labeled probe. Egr-1 IgG, 3 µg (lane 5) or 5 µg mg (lane 6).
Figure 10:
Functional analysis of PDGF-A-BP-1
sequences in response to PMA. A, PDGF-A promoter/CAT
constructs used in transient transfection assays. Location of the
PDGF-A-BP-1 binding site is also shown. B, CAT activity in
transiently transfected rat vascular smooth muscle cells. The figure
depicts an autoradiograph of a thin layer chromatogram used to separate
acetylated products from chloramphenicol. Cells were cotransfected with
2 µg of pSV2--galactosidase and 20 µg of either pACCATe36
or pACCATf11 constructs. After 24 h of transfection, cells were
incubated in serum-free media for 24 h followed by treatment with or
without PMA (10
M) for 24 h as indicated.
CAT activity was measured as described under ``Experimental
Procedures.'' C, relative CAT activity was estimated as
follows. Areas on the thin layer chromatogram corresponding to
acetylated products were cut from the thin layer chromatogram and
radioactivity was measured. The CAT activity was normalized with
-galactosidase activity for variation in transfection efficiency.
Relative CAT activity was calculated by normalizing to pACCATe36
(without PMA treatment). The data shown are the average of two
experiments.
The structure of the PDGF-A chain gene has been described in
detail by several investigators(23, 24) . The
expression of PDGF-A in mesangial and other cells has recently been
demonstrated to be regulated by a number of agents including phorbol
ester, epidermal growth factor, transforming growth factor and
, and
thrombin(5, 9, 10, 11, 12, 13) .
We have recently shown that PDGF-A chain gene transcription in human
mesangial cells is induced by phorbol ester(14) . To understand
the molecular mechanism(s) which regulate PDGF-A chain gene
transcription, we analyzed the 5`-flanking sequence of the human PDGF-A
chain promoter.
We have used DNase I footprinting and gel mobility shift analyses to identify protein(s) that bind to the 5`-flanking region of the PDGF-A chain promoter. Identification of potential regulatory elements will help understand PDGF-A chain gene regulation in mesangial and other cells that express abundant levels of PDGF-A chain mRNA. By DNase I footprint analysis, we deduced the boundaries of the nucleotide sequences protected from DNase I digestion which revealed a location at approximately -82 to -102 from transcription initiation site of the PDGF-A chain promoter. This suggests that nuclear extracts from human mesangial cells contain a nuclear protein (PDGF-A-BP-1) that binds to a DNA sequence (GGCCCGGAATCCGGGGGAGGC) in the 5`-flanking region of the PDGF-A chain promoter. It is noteworthy that by S1 nuclease analysis, S1 hypersensitive sites SHSI and SHSII have recently been identified in the 5`-flanking region of the PDGF-A chain promoter(25, 26) . The SHSI site is within the PDGF-A-BP-1 binding site, whereas the SHSII site is in the GC-rich region of the PDGF-A chain promoter(25, 26) . In addition, DNase I protection assays showed a protected region between -98 and -49 from the transcription start site and this region is implicated in the regulation of Wilms' tumor suppresser gene WTI(20) .
We have used gel mobility shift analyses to
further confirm the results obtained by DNase I protection analysis (Fig. 2). Data reported in this article demonstrate that the
binding site for PDGF-A-BP-1 in the 5`-flanking region of PDGF-A chain
promoter is specific. A comparison of identified sequences for binding
of PDGF-A-BP-1 to several known transcription factors shows no perfect
homology (Table 1). The binding site (-82 to -102)
for PDGF-A-BP-1 contains abundant G and C nucleotides similar to
transcription factors that are rich in GC nucleotides. The
transcription factors: Egr-1 (also called NGF-I-A, Krox,
Tis 8, Zif 268) and Egr-2 and Egr-3 (27, 28, 29, 30, 31) have
some homology at the 5`-end of the binding sequence for PDGF-A-BP-1 (Table 1). Egr-1 is encoded by an immediate early growth response
gene and has been implicated in cell growth and
differentiation(32) . Several other transcription factors
HIV-EP-1 (33) , MBP-1(34) , and PRDII-BF-1 (35) exhibit some homology with binding sequences for
PDGF-A-BP-1. MBP-1 has been implicated in cell proliferation
events(34) . The other transcription factors which exhibit some
homology to PDGF-A-BP-1 are: AGIE-BP-1(36) ,
H
TF
(37) , H
TF
(38, 39, 40) , KBF-1(41) ,
EBP-1(42) , and NF
B(39, 43) . In
addition, SP-1 has homology at the 3`-end of the PDGF-A-BP-1 binding
site sequence(44) . It is interesting to note that while
several transcription factors (Table 1) appear to show remarkable
sequence homology, they differ in their properties.
There are several binding sites for Egr-1 that are located in the GC-rich region of the 5`-flanking region of the PDGF-A chain promoter. However, Egr peptide does not form protein-DNA complexes when incubated with the labeled oligomer and preincubation of nuclear extracts with Egr-1 antibody does not interfere with binding of PDGF-A-BP-1 (Fig. 8). These data indicate that the putative factor is not Egr-1. In addition, there are a number of SP-1 binding sites located in the GC-rich region of PDGF-A chain promoter. Comparison of sequence homology for SP-1 binding site and that for PDGF-A-BP-1 reveal some homology at the 3`-end of the binding sequence for PDGF-A-BP-1. We therefore used SP-1 for competition analysis in gel mobility shift assays. By using labeled oligomer (-82 to -102) or SP-1 oligomer, we have shown that PDGF-A-BP-1 is not SP-1 (Fig. 6Fig. 7Fig. 8). This is due to the fact that the consensus sequence for SP-1 did not compete with the PDGF-A-BP-1 binding sequence ( Fig. 6and Fig. 7) and the PDGF-A-BP-1 binding sequence did not compete with SP-1 for binding (Fig. 7). Deletion of specific bases within the PDGF-A-BP-1 binding sequence demonstrates that sequences -82 to -96 of PDGF-A chain promoter are required for binding of the PDGF-A-BP-1 (Fig. 9). These data, together with functional analysis of PDGF-A-BP-1 sequences (Fig. 10), strongly suggest that PDGF-A-BP-1 is a novel nuclear protein.
In conclusion, we have identified by DNase I footprint analysis, a sequence (-82 to -102) in the 5`-flanking region of the PDGF-A chain promoter which is a site for binding of nuclear protein (PDGF-A-BP-1) in human mesangial cells. PMA caused severalfold induction of PDGF-A-BP-1 as demonstrated by gel mobility shift analysis. Based on gel mobility shift analysis with specific and nonspecific competitors, it appears that PDGF-A-BP-1 is a new nuclear protein and thus has the potential to play an important role in the regulation of PDGF-A chain gene transcription in mesangial cells and other vascular pericytes.