(Received for publication, November 16, 1994; and in revised form, December 15, 1994)
From the
We have recently identified a novel element (EFE 5/6) in the human elastin gene promoter that modulates the ability of insulin-like growth factor I (IGF-I) to up-regulate elastin gene transcription in aortic smooth muscle cells. In the present study, we have pursued the identification of those nuclear proteins binding to the EFE 5/6 element and affected by IGF-I treatment. Chelation inactivation and metal reactivation experiments together with supershift gel analyses demonstrated that Sp1 was one of the proteins affected by IGF-I. Southwestern and Western analyses showed that Sp1 was present in IGF-I nuclear extracts and capable of binding DNA after fractionation. Addition of retinoblastoma gene product (Rb) antibody mimicked the effect of IGF-I in gel shift analysis, suggesting that Sp1 binding may be regulated by an inhibitor normally associated with Rb. The fact that the phosphorylation state of Rb was affected by IGF-I was shown by Western blot analysis. The control smooth muscle cells transcribed the elastin gene at a high level without addition of IGF-I, so it is likely that disruption of Sp1 binding is the first step in allowing the binding of a more potent activating factor.
Elastin is an extracellular protein whose biological function is to provide elasticity to various connective tissues. The elastic properties imparted by elastin are essential to the structure of the aorta and large arteries of the cardiovascular system, where the proper maintenance of pressure gradients is critical to the continued functioning of the entire system. Reported studies suggest that elastogenesis is high during the development and growth of the aortic and arterial walls (Burnett et al., 1980; Shibahara et al., 1981; Foster et al., 1987, 1989), but very low in the mature animal (Lefevre and Rucker, 1983; Foster et al., 1990). Because the lack of renewed elastin repair has been implicated in the pathogenesis of cardiovascular disease (Davidson and Giro, 1986), much interest has centered on questions relating to how elastin gene expression is regulated.
Recent publications detailing the
complete nucleotide sequence of the human elastin gene and 5`-flanking
regions (Bashir et al., 1989; Yeh et al., 1989) have
provided a framework for investigating DNA-protein interactions
involved in modulating elastin gene transcription. Putative cis-acting
elements in the 5`-flanking region of the gene have been found by
computer homology search (Kahari et al., 1990), and broadly
defined functional areas of the gene have been identified through
transient transfections of various cell types with a series of deletion
reporter constructs (Fazio et al., 1990; Kahari et
al., 1990). Very little is currently known of specific sequences
within the elastin gene promoter that regulate transcription during any
phase of elastin expression. Our laboratory group has found that IGF-I ()increases transcription of the elastin gene in quiescent,
aortic SMC by disruption of several DNA-protein complexes associated
with -165 to -137 bp (EFE 5/6) in the human elastin gene
promoter (Wolfe et al., 1993). We have further shown by DNase
footprint analysis that deprotection of the EFE 5/6 element temporally
coincides with the major developmental burst of aortic elastogenesis
within the chick embryo (Rich et al., 1993). The objective of
the present communication was to gain some insight into the mechanism
of IGF-I action by first identifying those trans-acting factors whose
binding to the elastin promoter was disrupted by IGF-I and whose
removal coincided with a higher level of transcription.
The P-labeled DNA fragments (0.1-2.5 ng, 40-200
fmol, 20,000-100,000 cpm), nuclear extract (5-30 µg of
protein), and 1.0-10 µg of poly(dI-dC) were brought to a
volume of 35 µl with 7 µl of 5
binding buffer (50
mM HEPES (pH 7.9), 5 mM dithiothreitol, 0.5% Triton,
and 2.5% glycerol), 7 µl of extraction buffer (counting the volume
of nuclear extract), and the appropriate volume of H
O. The
final concentration of the binding mixture was 6 mM HEPES, 70
mM NaCl, 0.3 mM MgCl
, 0.04 mM EDTA, 1.2 mM dithiothreitol, 0.1% Triton, and 5.5%
glycerol. Reactions were incubated at 25 °C for 30 min, and the
products were resolved by electrophoresis through a 4% native
polyacrylamide gel in 90 mM Tris borate, 2.0 mM EDTA
buffer, pH 8.3. Electrophoresis was performed at 25 °C at 100 V (10
V/cm). The gels were dried and analyzed by autoradiography. In
competition experiments, 50-100-fold molar excess of unlabeled,
competitor DNA was pre-incubated with nuclear extracts for 30 min at 25
°C before the addition of
P-labeled DNA. The
supershift gel mobility assays were performed with these minor changes.
The nuclear extract was preincubated with specified antibody
preparations for 1 h at room temperature before incubation with labeled
oligonucleotide as described above, followed by loading onto the gel.
For chelation inactivation experiments, specified amounts of EDTA or
1,10-phenanthroline were added to nuclear extracts with binding buffer
and 1.0 µg of poly(dI-dC) and incubated at room temperature for 30
min. Radiolabeled DNA was then added, and the mixture was incubated for
another 30 min at the same temperature. For metal ion reactivation,
specified amounts of either MgCl or ZnCl
were
added to the nuclear extract after the incubation with
1,10-phenanthroline, and the mixture was allowed to sit at room
temperature for an additional 20 min before final incubation with
radiolabeled DNA.
For Southwestern analysis, the blot was first washed twice (10
min each, room temperature) in 20 mM HEPES (pH 7.9), 3 mM MgCl, 40 mM KCL, and 10 mM
-mercaptoethanol, blocked for 60 min in 20 mM HEPES
(pH 7.9) containing 4% milk, and then washed once in binding buffer
consisting of 10 mM HEPES (pH 7.9), 70 mM NaCl, 1
mM dithiothreitol, 0.3 mM MgCl
, and 0.1%
Triton X-100. Hybridization of the blot with 2.5 ng/ml radiolabeled EFE
5/6 (2
10
cpm) was performed for 3 h at room
temperature in binding buffer containing BSA (60 µg/ml) and
poly(dI-dC) (37.5 µg/ml). Prior to exposure of the gel to x-ray
film, it was washed twice (5 min at room temperature) in binding buffer
containing 0.01% Triton X-100. For competition of binding, a 2-fold
excess of Sp1 consensus oligomer (5 ng/ml) was incubated with the blot
for 1 h prior to the addition of radiolabeled probe.
Figure 1:
Gel retardation analysis of the EFE 5/6
probe with nuclear proteins extracted from control and IGF-I-treated
SMC. Quiescent cultures of SMC were treated with 50 ng/ml of IGF-I for
24 h. Control SMC were prepared in parallel but without the addition of
IGF-I. Proteins were extracted from isolated nuclei, incubated with the P-labeled EFE 5/6, and subjected to mobility shift
analysis as described in the text using 10 µg poly(dI-dC) in the
reaction mix. The left panel compares the binding of the EFE
5/6 probe (1 ng) to nuclear extracts (20 µg) isolated from control
and IGF-I-treated SMC. In the right panel, nuclear extract (25
µg) isolated from control SMC was incubated with 1 ng of
P-labeled EFE 5/6 element or a
P-labeled
28-bp nonspecific sequence as specified. The competition of binding
complexes was performed by adding either 50- or 100-fold molar excess
of unlabeled EFE 5/6 element or a 28-bp nonspecific sequence to the
binding reaction for 30 min before adding the radiolabeled DNA. Control
and IGF-I refer to the treatment of SMC used for isolation of nuclear
proteins, and NS 28MER refers to a nonspecific 28-bp oligomer
containing the same base composition of EFE 5/6 but a random sequence.
Bands denoted I, II, and III represent the three major complexes formed
with nuclear proteins isolated from control SMC. Complex I is composed
of two closely migrating bands designated
Ia,b.
Figure 2:
Sensitivity of EFE 5/6 complex formation
to metal chelation and reactivation. Nuclear proteins (10 µg)
isolated from control SMC were preincubated with 1 µg of
poly(dI-dC) in the presence of chelator prior to the addition of 1 ng
of P-labeled EFE 5/6 probe as detailed in the text. Panel A shows gel shift analysis performed in the presence of
specified concentrations of EDTA. Panel B presents a
comparable study using 1,10-phenanthroline and also includes the
addition of either MgCl
or ZnCl
to the binding
mixture after incubation with
1,10-phenanthroline.
Figure 3:
Supershift gel mobility assay using
antibody to Sp1. A gel mobility shift assay was performed using 1
µg of poly(dI-dC), 1 ng of P-labeled EFE 5/6 probe,
and 10 µg of control SMC nuclear extract. Sp1 antibody (2 µg)
was added to the gel shift reaction mixture for 1 h before addition of
the radiolabeled EFE 5/6 probe. As controls, Sp1 antibody (2 µg)
was added to the free probe, 2 µl of rabbit antiserum raised
against soluble elastin peptides was used to replace the Sp1 antibody,
and a synthetic peptide (2.5 µg) that was used to elicit the Sp1
antibody was used to block the Sp1 antibody (0.5
µg).
Figure 4:
Binding of purified Sp1 to the EFE 5/6
probe. Human recombinant Sp1 (0.4 footprinting units) was incubated
with 1 ng of P-labeled EFE 5/6 probe independently or
together with either 5 µg of nuclear extract isolated from control
SMC or 5 µg of BSA in the presence of 1 µg of poly(dI-dC). Gel
shift analysis was performed as described in the text. As controls, the
radiolabeled probe was incubated separately with 5 µg of nuclear
extract or 5 µg of BSA.
To further explore the role of Sp1 in complex formation, a human recombinant form of Sp1 was used in gel shift assays to compare its binding properties to that of the endogenous rat nuclear protein(s). Fig. 4shows a gel shift assay where Sp1 was incubated with labeled EFE 5/6 alone, in the presence of subsaturating amounts of nuclear extract, or a general protein carrier, i.e. BSA. The results showed that Sp1 by itself did not form a detectable complex, however, when Sp1 was added in the presence of nuclear extract or BSA, a low mobility complex formed that was comparable in electrophoretic mobility to complex I. It is interesting to note that other studies involving the binding of purified Sp1 to putative Sp1 elements have found that Sp1 does not bind unless cell nuclear extract is present (Alemany et al., 1992; Kutoh and Schwander, 1993). The proposed explanation for this phenomenon is that the nuclear proteins added modify or participate in specific protein-protein interactions that allow Sp1 to bind. In our system, there is clearly no specific interaction between nuclear proteins and the exogenous Sp1 since the addition of a carrier protein (BSA) results in the same degree of Sp1 complex formation as does the nuclear extract. Also evident in Fig. 4is that the addition of Sp1 together with nuclear extract results in the elimination of complex II. Although this result showed that the purified Sp1 preparation can effectively compete with the protein(s) involved in complex II, it does not differentiate whether the competed protein is a unique zinc-dependent protein or a modified form of Sp1. In experiments not shown, titration of the DNA probe with increasing amounts of recombinant Sp1 resulted in the formation of only one complex even at the lowest amount of protein.
Competition gel shift assays were performed to further confirm the presence of Sp1. Radiolabeled EFE 5/6 binding to protein in the control nuclear extract was competed with a consensus Sp1 site and a putative Sp1 site (EFE 3) located downstream from EFE 5/6 at -119 to -97 bp. As shown in Fig. 5A, the Sp1 consensus sequence competed as effectively as the EFE 5/6 element for proteins involved in complexes I and II as well as complex III, whereas the EFE 3 sequence caused some diminution of complexes I and II and had no effect on complex III formation. Since the Sp1 consensus sequence and to some extent the EFE 3 sequence competed with proteins involved in binding the EFE 5/6 element, these oligomers were radiolabeled and subjected to gel shift analysis using either nuclear extract isolated from control SMC or purified Sp1 in the presence of BSA (Fig. 5B). The consensus Sp1 oligomer formed two major complexes with the nuclear proteins that possessed electrophoretic mobilities comparable with complexes I and II. The EFE 3 oligomer formed a minor complex that comigrated with complex I. Both the EFE 5/6 and consensus Sp1 oligomers bound recombinant Sp1 very strongly, whereas the EFE 3 oligomer formed a weak complex, illustrating that its affinity was much lower.
Figure 5:
Comparison of the binding affinity of EFE
5/6 with a Sp1 consensus sequence and another putative Sp1 binding
sequence (EFE 3) located within the elastin promoter. Panel A,
10 µg of control SMC nuclear extract with 1 µg of poly(dI-dC)
was preincubated with 50 molar excesses of unlabeled EFE 5/6, a
Sp1 consensus sequence, EFE 3, or a nonspecific 28-bp oligomer (NS
28MER, see legend to Fig. 1) prior to the addition of 1 ng of
P-labeled EFE 5/6 probe. The reaction mixtures were
subjected to gel shift analysis as described. Panel B, 10
µg of control SMC nuclear extract with 1 µg of poly(dI-dC) was
incubated with 1 ng of
P-labeled EFE 5/6, EFE 3, or Sp1
consensus sequence. Direct binding of human recombinant Sp1 (0.4
footprinting units) was also examined with 1 ng of
P-labeled EFE 5/6, EFE 3, or the Sp1 consensus sequence in
the presence of 5 µg of BSA and 10 µg of poly(dI-dC). Gel shift
analysis was performed as described in the
text.
In a
seminal study of the functional DNA binding domain of Sp1, Kadonaga et al.(1987) found that purified human Sp1 was insensitive to
1,10-phenanthroline chelation at concentrations as high as 1.0
mM. In the present study, we have shown that complexes I and
II are eliminated by as little as 80 µM 1,10-phenanthroline (see Fig. 2B). Since the
experimental conditions that we used were different than those reported
by Kadonaga et al.(1987), we tested whether under our
experimental protocol Sp1 binding is susceptible to chelation by
1,10-phenanthroline. Human recombinant Sp1 was incubated with
radiolabeled EFE 5/6 and Sp1 consensus oligomers in the presence of
increasing amounts of 1,10-phenanthroline (Fig. 6). A
dose-dependent loss of Sp1 binding was noted with complete abrogation
of binding achieved at 40 µM for the consensus sequence
and 20 µM for the EFE 5/6 sequence. Further, Sp1 binding
was restored to both sequences by the addition of 80 µM ZnCl.
Figure 6:
Sensitivity of recombinant Sp1 binding to
chelation by 1,10-phenanthroline. Recombinant Sp1 (0.4 footprinting
units) was preincubated in binding buffer containing 5 µg of BSA
and 10 µg of poly(dI-dC) in the absence or presence of specified
concentrations of 1,10-phenanthroline prior to the incubation with 1 ng
of P-labeled EFE 5/6 or Sp1 consensus probes, and gel
shift analysis was performed as described. Postchelation addition of
specified amounts of ZnCl
and MgCl
was
performed as described in Fig. 2B.
Figure 7:
Gel retardation analysis of the Sp1
consensus and EFE 3 probes with nuclear proteins extracted from control
and IGF-I-treated SMC. 20 µg of nuclear proteins extracted from
control and IGF-I-treated SMC were incubated with 1 ng of P-labeled EFE 3 or Sp1 consensus probes and subjected to
mobility shift analysis as described in the text using 1 µg
poly(dI-dC) in the binding mix.
To gain some insight
into the mechanism by which IGF-I abrogates Sp1 binding in the SMC,
Western and Southwestern analyses were performed to determine 1) if Sp1
were present in nuclear extracts isolated from IGF-I-treated SMC and 2)
if immunologically detectable forms of Sp1 were capable of binding the
EFE 5/6 element. The Western blot given in Fig. 8A showed that nuclear extracts isolated from both control and
IGF-I-treated SMC contained Sp1 and further demonstrated that the ratio
of the two major species detected, i.e. 106 and 95 kDa, did
not differ between the two samples. The human recombinant Sp1 sample
used for this study is reported by the supplier (Promega) to be
comprised of two major species representing a
phosphorylated/glycosylated form (106 kDa) and an unmodified form (95
kDa). Although our analysis of the human recombinant Sp1 and rat SMC
nuclear protein samples showed that both animal species possessed
immunologically reactive polypeptides of similar size, we have not
determined the phosphorylation or glycosylation states of either
polypeptide. Southwestern analysis of the nuclear extracts run on the
same gel using radiolabeled EFE 5/6 as a probe showed that the binding
of Sp1 within extracts isolated from control and IGF-I-treated SMC is
comparable (Fig. 8B). Interestingly, both extracts
exhibited binding with only the 106-kDa species. The specificity of the
reaction is illustrated in the rightpanel where
binding of the radiolabeled EFE 5/6 oligomer to the 106-kDa band was
lost when competed with the unlabeled Sp1 consensus oligomer. These
data show that Sp1 is present in the nuclear extracts of IGF-I-treated
cells and is capable of binding its cognate sequence after
fractionation by SDS-polyacrylamide gel electrophoresis. The results
also suggest that IGF-I is not causing a major post-translational
modification in the Sp1 protein since the migration patterns of the
control and IGF-I samples are virtually identical. The two other
proteins visualized by hybridization with the EFE 5/6 probe possessed
apparent molecular masses of 60 and 42 kDa. The possible role of these
proteins in contributing to complex III formation will be presented
elsewhere. ()
Figure 8:
Comparison of the steady-state levels and
DNA binding ability of Sp1 in nuclear extracts obtained from control
and IGF-I-treated SMC. Panel A, 5 footprinting units of
recombinant Sp1 and either 50 µg of control or IGF-I-treated
nuclear proteins were subjected to Western blot analysis as detailed in
the text. Calculation of molecular weights was based on the migration
of phosphorylase B (97, 400), BSA (66, 200), ovalbumin (45,000), and
carbonic anhydrase (31,000) using linear regression analysis at a
confidence level of 95% and resulting in a r value
of 0.999. Panel B, duplicate samples examined in the Western
blot were probed with radiolabeled EFE 5/6 in a Southwestern analysis
as specified (details given in the text). The farrightlane represents 50 µg of control nuclear extract that
was competed with unlabeled Sp1 consensus oligonucleotide (2:1) before
addition of the
P-labeled EFE
5/6.
Figure 9:
The
effect of Rb antibody on the formation of complexes I and II. Gel
mobility shift analysis was performed with 10 µg of SMC nuclear
extracts, 10 µg of poly(dI-dC), 1 ng of P-labeled EFE
5/6, and 2 µg of the indicated polyclonal antibody preparation as
specified.
Since these results suggested that Rb may be mediating the effect of IGF-I on Sp1 binding, we examined whether IGF-I had any direct effect on the steady-state levels or phosphorylation state of Rb. The Southwestern blot shown in Fig. 8B was washed and probed with antibody to Rb. The results given in Fig. 10showed that within the control nuclear extract, Rb exists primarily in the underphosphorylated form, whereas after IGF-I treatment the hyperphosphorylated form predominated. This result establishes a link between IGF-I treatment of SMC and the phosphorylation state of Rb, which is particularly interesting since Chen et al.(1994) have postulated that the nonphosphorylated form of Rb binds the Sp1 inhibitor. The fact that the phosphorylated form of Rb is increased by IGF-I would be consistent with the loss of Sp1 binding.
Figure 10: The effect of IGF-I on the steady-state level and phosphorylation of Rb. The nitrocellulose blot pictured in Fig. 8was washed three times with TTBS, blocked as described in the text, and then incubated with Rb monoclonal antibody (1.0 µg/ml) for 2 h at room temperature. Detection of immunoreactive bands was performed as described in the text. The assignment of the phosphorylated and non-phosphorylated forms of Rb was established by using the electrophoretic mobility of Rb found in nuclear extracts isolated from C2C12 mouse myoblast and myotubes, respectively.
The overall goal of this study was to gain insight into the mechanism by which IGF-I increases the transcriptional level of the elastin gene in aortic SMC. The data presented do provide insight into at least one component of IGF-I action on the elastin gene and additionally supply important information relative to a general effect of IGF-I on SMC gene expression and on a specific factor potentially controlling the SMC cell cycle. The finding that Rb may regulate Sp1 binding in the SMC is significant and has implications independent of IGF-I action. Other investigators have shown that elastin synthesis in rat SMC is inversely related to cell proliferation, and the major burst of elastogenesis occurs after confluency (Toselli et al., 1992). These observations, together with the data reported here, suggest that Rb plays a key role in transcriptional regulation of the elastin gene and perhaps other matrix genes by allowing Sp1 binding when the cells are prohibited from entering S phase, thereby establishing a boundary between cell growth and matrix formation.
The addition of IGF-I to cultures of quiescent, neonatal SMC does not induce elastin gene transcription but rather increases the transcriptional level above an already existent high level. It is therefore possible that the EFE 5/6 sequence may be a positive element within a specified time frame of SMC elastogenesis by complexing with Sp1 to allow increased frequency of Pol II initiation in confluent, non-proliferating SMC. In this light, our initial reference to the IGF-I effect on elastin gene transcription as one of derepression should be reconsidered. Our previous study had shown that the removal of the EFE 5/6 binding site by deletion of sequences spanning -195 to -137 bp from the -195 to +1-bp chloramphenicol acetyltransferase reporter construct resulted in an increased activity of a chloramphenicol acetyltransferase reporter gene driven by -136 to +1 of the elastin promoter (Wolfe et al., 1993). Although the term derepression adequately describes the results obtained, the terminology used may be inappropriate since it implies that EFE 5/6 sequence is a repressor element within the elastin gene proximal promoter. It may be that the removal of this sequence allows the binding of Sp1 to a lower binding site in the truncated promoter, i.e. the EFE 3 sequence, whose position is closer to the CAAT box.
It is interesting to note that Alemany et al.(1990) have shown that IGF-I and insulin induce Sp1
binding to an Sp1-binding sequence adjacent to the CAAT box in the
proximal promoter of the -crystallin gene. These investigators
have also found that the binding of Sp1 to this sequence is
developmentally regulated in embryonic chick lens (Alemany et
al., 1992). Similar to these studies, we report here that Sp1 is
involved in the IGF-I up-regulation of elastin gene transcription, and
we have previously shown that the proteins interacting with the EFE 5/6
sequence are developmentally regulated in embryonic chick aorta (Rich et al., 1993). However, the major difference in the role of
Sp1 as dictated by IGF-I treatment is activation of transcription by
binding in the case of the
-crystallin gene and up-regulation of
transcription by disruption of binding within the elastin gene. It thus
appears as if IGF-I can activate or increase transcription by at least
two separate pathways involving Sp1 as a focal point. The cells used
for these studies differed significantly in that the cells used to
examine the
-crystallin gene involved chick embryonic lens
epithelial cells that are stimulated to differentiate by IGF-I and
insulin. The cells we have used in this study are differentiated aortic
SMC that are already transcribing the elastin gene. A key to the
differences in IGF-I responses may be related to events controlled by
the cell cycle.
Our initial goal was to investigate the mechanism by
which IGF-I up-regulates the elastin gene. Our studies have shown that
Sp1 binding to the elastin promoter is lost as a consequence of IGF-I
treatment; however, this does not explain why elastin gene
transcription is increased. In fact, one would predict that the loss of
Sp1 binding would decrease transcription. It is interesting to note
that Terpstra (1994) found that in vivo competition
experiments involving cotransfection of the EFE 5/6 sequence with a
chloramphenicol acetyltransferase reporter construct containing
-195 to +2 bp of the human elastin promoter resulted in
decreased chloramphenicol acetyltransferase activity, whereas a
nonspecific oligomer possessing the same base composition had no
effect. These results confirm the hypothesis that the loss of Sp1 from
the EFE 5/6 sequence in itself does not result in increased
transcriptional activity. Since the up-regulation of the elastin gene
transcription by IGF-I requires protein synthesis (Wolfe et
al., 1993), it is possible that IGF-I induces the synthesis of
another activating protein that may bind to a sequence overlapping a
portion of the EFE 5/6 sequence. Alternatively, the protein synthesis
required by the IGF-I may involve a target protein controlling Rb
either transcriptionally and/or post-transcriptionally. We previously
have shown that when the -195 to +2-bp region of the elastin
promoter was incubated with nuclear extracts obtained from control and
IGF-I-treated SMC and analyzed by gel shift analysis, two complexes
were lost by IGF-I treatment, and a third complex was increased.
Although this was not a consistent finding in all of the primary
cultures examined at that time, we have performed a number of other
experiments that suggest that removal of Sp1 by IGF-I treatment results
in the binding of an AP-2-like protein to a sequence overlapping the
3`-end of EFE 5/6. ()
In conclusion, based primarily on the data obtained within this study and preliminary data obtained from parallel studies, our current working hypothesis is that IGF-I up-regulates elastin gene transcription through two steps. One of these involves the disruption of Sp1 binding, perhaps through alteration of the level or phosphorylation state of Rb, and the second involves binding of an activator that is capable of bringing the trans-activation complex to a higher level of activity. We are currently pursuing which of the steps require protein synthesis and are also expanding our investigations of the role of Rb in controlling elastogenesis within SMC.