©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcriptional Regulation of the Elastin Gene by Insulin-like Growth Factor-I Involves Disruption of Sp1 Binding
EVIDENCE FOR THE ROLE OF Rb IN MEDIATING Sp1 BINDING IN AORTIC SMOOTH MUSCLE CELLS (*)

(Received for publication, November 16, 1994; and in revised form, December 15, 1994)

Donna E. Jensen (§) Celeste B. Rich Anita J. Terpstra Stephen R. Farmer Judith Ann Foster (¶)

From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 (^1)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.


MATERIALS AND METHODS

Reagents

Human recombinant Sp1 and a double-stranded oligodeoxynucleotide corresponding to the Sp1 consensus sequence ATTCGATCGGGGCGGGGCGACC were purchased from Promega. Rabbit polyclonal IgG preparations directed against amino acid residues 520-538 of human Sp1, the carboxyl-terminal 15 amino acids of the human Rb p110 sequence (Rb C-15), the carboxyl-terminal 14 amino acids of human egr-1 (egr-1 588 X), and the carboxyl-terminal 24 amino acids of human egr-3 (egr-3 C-24) were purchased from Santa Cruz Biotechnology. The peptide antigen used to elicit antiserum to Sp1 was also purchased from Santa Cruz Biotechnology. A mouse monclonal antibody directed against Rb was obtained from Transduction Laboratories. Three single-stranded DNA oligomers and their complementary strands were purchased from Oligos Etc. These included CGTAATTGTCCCCTCCCCGCGGCCCCCT (EFE 5/6, -165 to -137 bp), TCTCGCACGCTCGCACGCTCCTCCGCCT (a scrambled sequence of EFE 5/6 designated NS 28MER), and TCTCCCGCCCTCCCGCCCGCCCT (EFE 3, -119 to -97 bp).

Preparation of Nuclear Extracts

Neonatal rat smooth muscle cells were isolated from the aortae of 2-3 day Sprague-Dawley rats, cultured, rendered quiescent, and treated with IGF-I at 50 ng/ml medium (6.5 nM) as previously described (Wolfe et al., 1993). Nuclei from control and IGF-I-treated cell cultures were isolated by the procedure of Dean et al. (1986), and nuclear proteins were extracted essentially according to the method of Dignam et al.(1983) with the modifications introduced by Ritzenthaler et al.(1991). Total protein within each sample was determined by the BCA protein assay (Pierce). The extracts were stored at -80 °C in extraction buffer consisting of 20 mM HEPES (pH 7.9), 0.35 M NaCl, 1.5 mM MgCl(2), 0.2 mM EDTA, 25% glycerol, 1.0 µM diisopropyl fluorophosphate, 0.5 µg/ml leupeptin, 2.0 µg/ml aprotinin, and 0.7 µg/ml pepstatin.

Gel Mobility Shift Assay

Duplex oligomers were prepared by heating complementary strands in 10 mM Tris (pH 8.0) and 10 mM MgCl(2) to 95 °C for 5 min and then slowly cooling to room temperature over a 2-h period. The homogeneity of annealed oligomers was examined by gel electrophoresis. Deoxyribonucleic acid fragments were radiolabeled with T4 polynucleotide kinase and separated from free [P]dATP by the Sephadex G-50 spun-column procedure (Sambrook et al., 1989).

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 times 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(2)O. The final concentration of the binding mixture was 6 mM HEPES, 70 mM NaCl, 0.3 mM MgCl(2), 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(2) or ZnCl(2) 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.

Western and Southwestern Blot Analysis

Triplicate 50-µg samples of control or IGF-I-treated SMC nuclear protein were analyzed by 8.0% SDS-polyacrylamide gel electrophoresis according to Laemmli(1970). One lane of the gel included molecular weight standards. The gel was electrophoretically transferred to nitrocellulose as described by Towbin et al.(1979). The nitrocellulose was cut to allow each set of samples to be probed with either antibody or radiolabeled probe. For Western analysis, the blot was washed twice (10 min each, room temperature) in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween-20 (referred to as TTBS buffer) and then blocked for 60 min at room temperature in TTBS containing 5% milk. The blot was incubated with Sp1 or Rb antibody (1 µg/ml) in TTBS, 5% milk for 2 h at room temperature followed by incubation for 1 h with horseradish peroxidase conjugated with protein A or goat anti-mouse IgG diluted 1:3000 in TTBS containing 5% milk. The blot was washed twice with TTBS (10 min each, room temperature) followed by an additional 10-min wash with 10 mM Tris-HCl (pH 8.0) and 150 mM NaCl and then incubated with Amersham ECL reagents according to the manufacturer's directions for 1 min and then exposed to x-ray film.

For Southwestern analysis, the blot was first washed twice (10 min each, room temperature) in 20 mM HEPES (pH 7.9), 3 mM MgCl(2), 40 mM KCL, and 10 mM beta-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(2), and 0.1% Triton X-100. Hybridization of the blot with 2.5 ng/ml radiolabeled EFE 5/6 (2 times 10^5 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.


RESULTS

Binding of Nuclear Factors to the EFE 5/6 Sequence

Since we have already shown that IGF-I addition to SMC results in a disruption of DNA-protein complexes footprinted to -165 to -137 bp of the human elastin gene (Wolfe et al., 1993), the first experiment was designed to test whether this sequence alone would mimic the results obtained on larger fragments of the promoter. Fig. 1provides gel shift analysis of the EFE 5/6 sequence incubated with nuclear proteins isolated from control and IGF-I-treated SMC (leftpanel) and competitive gel shift analysis reactions to establish specificity (rightpanel). The incubation of the radiolabeled probe with control SMC nuclear extract resulted in the formation of three major complexes denoted as I, II, and III. The largest complex, i.e. complex I, is sometimes visible as a doublet and most likely consists of two discrete complexes (Ia and Ib) whose distinctiveness will be obvious in other figures. Pertinent to the objectives of the present study was the finding that incubation of radiolabeled EFE 5/6 with nuclear proteins isolated from IGF-I-treated SMC resulted in a significant loss of complexes I and II, whereas complex III remained unchanged. This disruption of binding complexes found with nuclear proteins isolated from IGF-I-treated SMC agrees with our previous studies and verifies our use of this synthetic oligomer to examine proteins associated with the IGF-I-sensitive complexes. The specificity of binding is shown in DNA competition assays where the binding to nuclear proteins was competed with either unlabeled EFE 5/6 or a nonspecific 28-bp sequence. The 28-bp nonspecific sequence has the same base composition as the EFE 5/6 sequence, but in a scrambled sequence, and does not form any complexes with proteins extracted from control SMC (Fig. 1). The binding of proteins found in complexes I and II is competed in the presence of a 50-fold molar excess EFE 5/6, whereas the binding of proteins in complex III are not completely competed until a 100-fold excess. The nonspecific DNA oligomer, at 100-fold molar excess, does not compete for binding of any of the complexes. In an independent study, we have determined that the formation of complexes I and II is preferentially favored when the amount of poly(dI-dC) was decreased from 10 to 1 µg (Terpstra, 1994), which suggested that the proteins contained in these complexes may recognize a more GC-enriched region on the DNA.


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.



Zn(II) Is Required for Formation of Complexes I and II

To begin characterization of those proteins associated with the EFE 5/6 sequence, their dependence on metal for DNA binding was examined. Fig. 2provides a composite result in which radiolabeled EFE 5/6 was incubated with nuclear proteins isolated from control SMC with the addition of either EDTA or the divalent chelator 1,10-phenanthroline. The results demonstrated that formation of complexes I and II is sensitive to both EDTA (Fig. 2A) and 1,10-phenanthroline (Fig. 2B) chelation, whereas complex III formation is unaffected. Furthermore, restoration of complex I and II binding after 1,10-phenanthroline treatment was fully achieved by the addition of Zn, whereas Mg only partially restored binding (Fig. 2B). These results showed that the proteins comprising complexes I and II are dependent upon Zn(II) for binding and suggest that they are members of a class of transcription factors possessing zinc finger domains.


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(2) or ZnCl(2) to the binding mixture after incubation with 1,10-phenanthroline.



Identification of Sp1 as a Component of Complex I

Since the EFE 5/6 sequence possesses a sequence enriched in GC, we first investigated whether the transcription factor Sp1, which is a ubiquitous, zinc finger protein, was a component of complex I and/or II. Although the EFE 5/6 element does not contain the classic Sp1 consensus sequence, i.e. GGGCGG, Sp1 is capable of recognizing many GC-rich divergent sequences including NGGNGN, GT boxes, and CACC motifs (Gloss and Bernard, 1990; Li et al., 1991; Briggs et al., 1986; Yu et al., 1991). The identification of Sp1 was approached directly through an immunological technique (Fig. 3) and indirectly through a comparative biochemical analysis (Fig. 4). Incubation of control SMC nuclear extract and radiolabeled EFE 5/6 with rabbit antibody directed toward human Sp1 resulted in a new low mobility complex with a concomitant diminishment of complex Ia. Further competition of antibody binding with the cognate peptide antigen, i.e. amino acid residues 520-538 of human Sp1, specifically blocked the supershifted complex. As other controls, Sp1 antibody was incubated directly with the labeled probe, and a rabbit antibody directed against rat elastin fragments (Rich et al., 1992) was substituted for Sp1 antibody. These results demonstrated that Sp1 or a Sp1-like protein containing a peptide sequence very similar if not identical to amino acid residues 520-538 of human Sp1 is present in complex I. The fact that only one band associated with complexes I and II is supershifted does not necessarily mean that Sp1 is not associated with complex Ib or II. It may be that these latter complexes contain subpopulations of Sp1 that are conformationally inaccessible to the antibodies due to a post-transcriptional modification (Jackson and Tjian, 1988; Jackson et al., 1990). It is interesting to note in this regard that other studies aimed at identification of Sp1 binding to putative binding sites have noted a similar multiplicity of bands, which are apparently not supershifted with antibody prepared to human recombinant Sp1 (Sykes and Kaufman, 1990; Gloss and Bernard, 1990; Chen et al., 1994). On the other hand, it is possible that the proteins present in complexes Ib and II may be other members of a multigene Sp1 family, including Sp2 and Sp3 (Kingsley and Winoto, 1992).


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 50times 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(2).


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(2) and MgCl(2) was performed as described in Fig. 2B.



IGF-I Results in a General Loss of Sp1 Binding within the SMC although the Protein Levels and DNA Binding Capacity of Sp1 Are Not Altered

Since the above data pointed to the fact that the binding of Sp1 and other Zn(II) requiring proteins to the EFE 5/6 sequence was lost upon IGF-I treatment, we examined whether this loss of binding was specific to the EFE 5/6 sequence. The Sp1 consensus and the EFE 3 oligomers were subjected to gel shift analysis with nuclear proteins isolated from control and IGF-I-treated SMC (Fig. 7). Significantly, both oligomers exhibited a loss of Sp1 binding when incubated with nuclear extracts obtained from IGF-I-treated SMC, demonstrating that the addition of IGF-I resulted in a general inactivation of Sp1 binding within the SMC.


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. (^2)


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^2 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.



Rb Appears to Mediate the Loss of Sp1 Binding

One possible explanation for the lack of Sp1 binding in the total extract of IGF-I-treated cells is that Sp1 binding is negated by association with another protein. Evidence for this mechanism of controlling Sp1 binding was recently reported by Chen et al.(1994). These investigators demonstrated that Sp1-mediated transcription of c-Jun is regulated indirectly by Rb through sequestration or release of an Sp1 inhibitor protein. We investigated the possibility that Rb was playing a similar role in the regulation of Sp1 binding in the SMC. A gel shift analysis was performed (Fig. 9) where radiolabeled EFE 5/6 and nuclear proteins isolated from control SMC were incubated individually with antibody against egr-1, egr-3 (both zinc finger transcriptional factors), Rb, or Sp1. As a reference point, EFE 5/6 was incubated with nuclear extract isolated from IGF-I-treated SMC. Most significant was the finding that Rb antibody specifically abrogated formation of complexes I and II, thereby mimicking the pattern found in the IGF-I-treated cells, whereas neither egr antibody preparation had any effect and the Sp1 antibody supershifted a portion of complex I as previously shown. This result suggests the possibility that Rb may be mediating the effects of IGF-I on Sp1 binding in the SMC in some indirect manner through protein-protein interactions.


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.




DISCUSSION

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. (^3)

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL13262. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by Training Grant HL07429.

To whom correspondence should addressed: Dept. of Biochemistry, Boston University School of Medicine, 80 East Concord St., Boston, MA 02118. Tel.: 617-638-4361; Fax: 617-638-5339.

(^1)
The abbreviations used are: IGF-I, insulin-like growth factor I; bp, base pair(s); BSA, bovine serum albumin; EFE, elastin functional element (see Wolf et al., 1993); SMC, smooth muscle cell.

(^2)
A. J. Terpstra and J. A. Foster, manuscript in preparation.

(^3)
C. B. Rich, H. D. Goud, and J. A. Foster, unpublished data.


ACKNOWLEDGEMENTS

We thank Gail Sonenshein for critical evaluation of the manuscript. We acknowledge the superb technical assistance of Valerie Verbitzki, Rosemarie Moscaritolo, Daniel Pine, and Kevin Kelliher for isolating and maintaining smooth muscle cells and for plasmid DNA preparation.


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