(Received for publication, May 16, 1995; and in revised form, August 21, 1995)
From the
Insulin-like growth factor binding protein-5 (IGFBP-5) is an important modulator of IGF actions. IGFBP-5 mRNA is abundant in human fibroblasts and is regulated by cAMP. To understand the molecular mechanism underlying this cell type-specific expression and regulation, we isolated the 5`-flanking region of the human IGFBP-5 gene and fused it to a promoter-less reporter plasmid encoding luciferase. Transient transfection of the construct into fibroblasts displayed both constitutive and cAMP-induced promoter activity in an orientation-specific manner. Sequence analysis revealed the existence of distal and proximal consensus AP-2 recognition sites located 5` from the TATA box. Both sequences bound specifically to human AP-2 in vitro by gel shift mobility assay. The possible role of AP-2 was examined by cotransfection of AP-2-deficient HepG2 cells with the IGFBP-5 promoter construct and a human AP-2 expression construct. Cotransfection with AP-2 significantly elevated IGFBP-5 promoter activity. This trans-activation was IGFBP-5 promoter and AP-2 specific. In AP-2 abundant fibroblasts, expression of AP-2B, a dominant-negative inhibitor of AP-2, suppressed IGFBP-5 promoter activity. In HepG2 cells, AP-2B alone had no significant effect, but the AP-2-induced activation of promoter activity was inhibited by AP-2B in a dose-dependent manner. The relative functional importance of the putative AP-2 binding sites was examined using a number of deletion mutants and point mutations. When the first two distal CCCCACCC-like putative AP-2 sites were deleted or mutated, there was no change in AP-2-induced trans-activation. Deletion or mutation of the proximal GCCNNNGGC-like sequences, however, abolished the AP-2-induced activation. These results suggest that AP-2 regulates the IGFBP-5 gene expression through the proximal GCCNNNGGC-like sequences. This AP-2-mediated trans-activation contributes at least in part to the constitutively high expression of IGFBP-5 in fibroblasts and to the cAMP responsiveness of this gene.
Insulin-like growth factor I and II (IGF-I and IGF-II) ()are multifunctional growth factors required for normal
development and growth in vertebrate animals. IGF-I mediates many of
the growth-promoting effects of growth hormone during postnatal life (1) and both IGF-I and -II have been shown to be important for
fetal growth in gene targeting experiments (2) .
In extracellular fluids the IGFs are bound to one of the members of a family of soluble, high affinity binding proteins(3) . These IGF binding proteins (IGFBPs) act as carrier proteins in plasma to control the efflux of IGFs from the vascular space and prolong their half-lives. More importantly, since they bind to IGFs with higher affinities than the IGF receptors, IGFBPs provide a means of localizing IGFs on target cells. Furthermore, they can alter the biological activity of IGFs by modulating their interaction with IGF receptors (1) . Six distinct IGFBPs, designated as IGFBP-1 to 6, have been isolated and cloned and each represents an individual gene product. They share relatively high amino acid sequence similarity but each has distinct structural and biochemical properties and each is subject to differential tissue-specific expression, developmental and hormonal regulation(4) . This suggests that each IGFBP may play a specific role in regulating the biological actions of IGFs in defined tissues.
IGFBP-5 is the most conserved of the six known IGFBPs. This 252-amino acid protein is more than 97% identical among human, rat, and mouse (5) . IGFBP-5 has the unique property of associating with cell surfaces and adhering to extracellular matrix(1) . When associated with the extracellular matrix, it has been shown to potentiate the effect of IGFs on fibroblast growth(6) . In addition, IGFBP-5 may also be involved in cell differentiation. The expression of IGFBP-5 is greatly increased during terminal differentiation of the mouse C2 myoblast cell line in vitro and overexpression of IGFBP-5 alters the differentiation of these cells(5, 7) . IGFBP-5 mRNA is abundant in several tissues in adult animals, including kidney, brain, gut, uterus, and cardiac and skeletal muscle but is minimal in liver(5, 8, 9) . In the rat embryo, IGFBP-5 mRNA is detected as early as embryonic day 10.5 and is mainly distributed at the surface ectoderm, the notochord, the floor plate, limb buds, precursor cells for neuronal cells, and specific muscle cells(10) . In vitro, the level of expression of IGFBP-5 varies between cell types and is regulated by specific factors such as RA and IGF-1 in a cell type-specific manner (11, 12, 13, 14) . Previously we reported that IGFBP-5 mRNA was significantly induced by forskolin in human fetal skin fibroblasts, suggesting that IGFBP-5 is regulated by intracellular cAMP levels(15) . A similar increase was also reported in rat osteoblast-like cells by parathyroid hormone through a cAMP-mediated mechanism(16) . The molecular mechanisms underlying the tissue-specific expression and hormonal regulation of the IGFBP-5 gene are undefined.
This study was undertaken to characterize cis-acting sequences and their corresponding trans-acting factors that are responsible for the constitutively high expression in connective tissues and for the cAMP responsiveness of the human IGFBP-5 gene. We have cloned the 5`-flanking region of the human IGFBP-5 gene from a genomic DNA library and confirmed the structure published recently by Allander et al.(17) . We report here that the developmentally regulated transcription factor AP-2 stimulates transcription of the IGFBP-5 gene and that this trans-activation is at least in part responsible for the constitutively high expression in fibroblasts and for the cAMP responsiveness of this gene. We found that AP-2 regulates IGFBP-5 gene expression through a 5`-GCCNNNGGC-3`-like sequence but not 5`-CCCCACCC-3` sequences in vivo, although both sequences have been proposed as consensus AP-2 binding sites (18, 19) and both were capable of binding to AP-2 in vitro.
In the second
set of mutants, points mutants of pBP5P/Luc were generated by
site-directed mutagenesis as described previously(6) . Plasmid
DNA was transfected into Escherichia coli strain CJ236. A
60-ml culture was inoculated and cultured with a fresh colony of CJ236
and was then infected with helper phage R408. After 5 h the bacteria
were pelleted. Phagemid particles were precipitated in 16% polyethylene
glycol and ammonium acetate, and single strand phagemid DNA was
isolated by binding to glassmilk following the manufacturer's
protocol (Bio 101, La Jolla, CA). The following complementary
oligonucleotides containing mutagenic mismatches were used.
Oligonucleotide 5`-TCACACGGGGTGGGCTTTGGAGAGGCCTTCTA-3` mutated the
first distal putative AP-2 site (CCCCACCC, located at bp -147 to
-140) to CCAAAGCC, generating mutant MU1. Oligonucleotide
5`-AAACTCACAGGTGTAGGCTATGGAGAGGCCTTC-3` mutated the two overlapping
distal putative AP-2 sites (CCCCACCCCCACCC, located at bp -147 to
-134) to CCAAAGCCTCACAC, generating mutant MU2. Oligonucleotide
5`- TTAAATAGCCGGACAATGTCTGCCAGCCAG-3` mutated the proximal putative
AP-2 site (GCCAGGGGC, located at bp -47 to -39) to
TACAGTGTC, generating mutant MU3. These oligonucleotides were
phosphorylated with T4 polynucleotide kinase (Promega) for 1 h at 37
°C. An aliquot of the synthesis mixture was used to transform E. coli strain DH5 F` and the positive colonies were
selected by ampicillin resistance. DNA from the resulting colonies was
amplified and DNA sequencing was used to determine the clones
containing the correct sequences.
Figure 1:
Northern blot analyses of IGFBP-5 mRNA
levels. Total cellular RNA was prepared as described under
``Materials and Methods'' from different cell lines.
Ten-µg RNA aliquots were electrophoresed and transferred to a nylon
membrane and then hybridized with a P-labeled human
IGFBP-5 cDNA probe. The amounts of RNA in each lane were verified with
a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA probe.
The arrows denote the 6-kb IGFBP-5 message and 1.4-kb
glyceraldehyde-3-phosphate dehydrogenase message. A,
expression of IGFBP-5 mRNA in several human cell lines; B,
effects of forskolin on IGFBP-5 mRNA expression in GM-10 fibroblasts.
Confluent fibroblast monolayers were incubated in Eagle's minimal
essential medium (lane 1) or Eagle's minimal essential
medium plus forskolin (10 µM, lane 2) for 18
h.
Figure 2:
Effect of
forskolin on human IGFBP-5 promoter activity in HepG2 (A),
A673 (B), and GM-10 (C) cells. A 1278-bp DNA fragment
of the human IGFBP-5 gene 5`-flanking region was fused to a luciferase
reporter gene (pGL2-Basic) in a sense or antisense orientation to
generate pBP5P/Luc (sense) and pBP5P/Luc (antisense),
respectively. These constructs and the promoter-less vector
(pGL2-Basic) were transiently transfected into cells. After growing in
complete medium for 48 h, cells were incubated in serum-free medium
with or without forskolin (10 µM) for another 18 h. The
cellular extracts were prepared and the luciferase activity was
measured as described under ``Materials and Methods.'' The
relative luciferase activities represent the relative fold value versus the promoter-less pGL2-Basic plasmid.
pSV--galactosidase was used as an internal control. For all
transfection experiments the results reported were obtained from at
least three independent experiments each carried out in
duplicate.
Figure 3:
Binding of human AP-2 to the putative AP-2
binding sites of the human IGFBP-5 promoter. Gel-shift analysis of
bacterially expressed human AP-2 was performed with (A) P-labeled A1 or (B)
P-labeled A2
double-stranded oligonucleotides as probes. The probes were incubated
with (lane 1) or without AP-2 (lanes 2-5).
Competitive binding was shown by inclusion of 50-fold excess of
unlabeled wild type oligonucleotide (lane 3, A1WT or A2WT),
mutated oligonucleotide (lane 5, A1MU or A2MU), or consensus
AP-2 oligonucleotide (lane 4, cAP-2). Supershift assays for
AP-2
A1 (C) or AP-2
A2 complexes (D) were
performed without (lane 1) or with 1 µl of anti-AP-2
antibody (lane 2) or 1 µl of anti-IGFBP-5 antibody (lane 3).
Figure 4: Effects of AP-2 on human IGFBP-5 promoter activity in HepG2 (A) and GM-10 (B) cells. Human hepatoma cells (HepG2) or fibroblasts (GM-10) were transiently transfected with either pGL2-Basic or pBP5P/Luc and cotransfected with either a human AP-2 expression plasmid pSV-AP-2 (AP-2), the pSV vector without AP-2 sequence (vector) or pBluescript DNA control (control). Cellular extract preparation and the luciferase assays were performed as described for Fig. 2.
AP-2 binds DNA as a homodimer with the binding and dimerization domain located at the C-terminal region of the protein(19) . AP-2B, an alternatively spliced product from the AP-2 gene, has the activation domain of AP-2 but lacks the dimerization and DNA binding domain and can specifically interfere with endogenous AP-2 activity in a dominant-negative manner(20) . We therefore cotransfected construct pBP5P/Luc with a AP-2B expression construct (pSG5-AP-2B) (20) in GM-10 fibroblasts. Expression of AP-2B significantly suppressed the basal IGFBP-5 promoter activity in these cells (Fig. 5A). Similar inhibition of IGFBP-5 promoter activity by AP-2B expression was also obtained with T98G cells (data not shown). In HepG2 cells, expression of AP-2B had no significant effect on the basal promoter activity, but it inhibited the AP-2 induced activation in a dose-dependent manner (Fig. 5B). Since it is known that SV40 T-antigen inhibits the trans-activation activity of AP-2(21) , we tested whether SV40 T-antigen-induced cellular transformation could suppress the constitutively high expression of endogenous IGFBP-5 mRNA in fibroblasts. As shown in Fig. 6, two untransformed human fetal lung fibroblast cell lines, MRC-5 and IMR-90, both had high levels of IGFBP-5 mRNA (lanes 1, 2, 5, and 6) similar to GM-10 fibroblasts (lane 9). When these cells were transformed by SV40 T-antigen, the IGFBP-5 mRNA expression was almost completely abolished (lanes 3, 4, 7, and 8). This inhibition by SV40 T-antigen-induced transformation was specific for IGFBP-5, since no significant difference was seen in glyceraldehyde-3-phosphate dehydrogenase expression. These results indicate that AP-2 regulates transcription of the human IGFBP-5 gene and contributes at least in part to its constitutively high expression in human fibroblasts.
Figure 5: A, inhibition of AP-2 activity by AP-2B on IGFBP-5 promoter activities in GM-10 fibroblasts. Human fibroblasts (GM-10) were transiently transfected with the pBP5P/Luc construct (2 µg) and an AP-2B expression plasmid. B, effect of AP-2B on AP-2-induced trans-activation in HepG2 cells. Human hepatoma cells (HepG2) were transiently transfected with pBP5P/Luc and cotransfected with a human AP-2 expression plasmid pSV-AP-2 (+AP-2) or pBluescript plasmid DNA (-AP-2). The cells were cotransfected with various amounts of AP-2B. The micrograms of transfected AP-2 B expression plasmid DNA are shown at the bottom. The differences in AP-2B DNA amount were compensated for by using pBluescript DNA.
Figure 6:
Northern blot analyses of IGFBP-5 mRNA
levels in normal (UT) and SV40 T-transformed (T)
human fibroblasts. Ten µg RNA aliquots were electrophoresed and
transferred to a nylon membrane and then hybridized with a P-labeled IGFBP-5 cDNA probe and a
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe. The arrows denote the 6-kb IGFBP-5 message and 1.4-kb
glyceraldehyde-3-phosphate dehydrogenase message. The RNA samples
loaded in lanes 1 and 2, 5 and 6 are from
normal human fetal lung fibroblast lines MRC-5 and IMR-90,
respectively. Lanes 3 and 4, 7 and 8 are RNA samples isolated from their corresponding transformed cell
lines, AG 2804 and AG10076. RNA samples isolated from GM-10 or HepG2
cells were used as positive and negative controls.
Figure 7: Effect of varying amount of pSV-AP-2 plasmid DNA on human IGFBP-5 promoter activity in HepG2 (A), A673 (B), and GM-10 (C) cells. Cells were transiently transfected with pBP5P/Luc (2 µg) and cotransfected with pSV-AP-2 (AP-2) or pBluescript DNA control (control). The cellular extracts preparation and the luciferase assay were performed as described in the legend to Fig. 2. The differences in AP-2 DNA amount were compensated for by using pBluescript DNA.
Figure 8: Mapping of the functional AP-2 binding sites in the human IGFBP-5 promoter. The schematic diagram shows the pBP5P/Luc construct and a series of 5` and internal deletion mutants fused to pGL2-Basic reporter gene. The putative AP-2 binding sites are indicated with open circles (CCCCACCC) or solid circles (GCCNNNGGC), respectively. The TATA box is indicated as an open square. These constructs were cotransfected into HepG2 cells with a human AP-2 expression plasmid (pSV-AP-2) or a control plasmid (pSV). The luciferase activities induced by AP-2 represent averages of three independent experiments of the relative fold increase as compared to the control lacking AP-2.
Figure 9: A, schematic diagram showing the pBP5P/Luc construct and a series of point mutant constructs. The putative AP-2 binding sites are indicated with open circles (CCCCACCC) or solid circles (GCCNNNGGC), respectively. The TATA box is indicated as an open square. The mutated site is indicated as a cross. B, effects of point mutations in the putative AP-2 binding sites of human IGFBP-5 promoter. The wild type and mutant constructs were cotransfected into HepG2 cells with a human AP-2 expression plasmid (pSV-AP-2) or a control plasmid (pSV). The luciferase activities stimulated by AP-2 represent averages of the relative fold increases compared to the control lacking AP-2 in four independent experiments.
The results from this study have, for the first time, examined the role of specific cis-acting sequences and a transcription factor in the regulation of IGFBP-5 gene expression. We have identified the promoter/regulatory region of this gene that showed constitutive promoter activity and cAMP responsiveness when transfected in human fibroblasts. We were able to demonstrate that transcription factor AP-2 regulates IGFBP-5 gene expression through a proximal 5`-GCCNNNGGC-3`-like sequence of this promoter region. This AP-2-induced trans-activation contributes at least in part to the constitutively high expression in fibroblasts and to the cAMP responsiveness of this gene.
The IGFBP-5 gene has been recently cloned and characterized from rat, mouse, and human(23, 9, 17) . A DNA fragment containing the 5`-flanking region of the human gene has been shown to be able to direct expression of a reporter gene in human breast cancer cells(17) . A more in-depth analysis using the mouse promoter in human hepatoma HepG2 cells suggests that the IGFBP-5 promoter has a simple structure, requiring the TATA box and no more than 125 nucleotides of additional 5` DNA to generate primary promoter activity(24) . The cis-acting sequences and their corresponding trans-acting factors responsible for the developmental- and cell type-specific regulation of IGFBP-5 expression have not been reported.
Several lines of evidence point to a role for AP-2 in regulating human IGFBP-5 gene transcription. Elevation of intracellular cAMP levels increases IGFBP-5 mRNA levels in human skin fibroblasts ( (15) and this study). Since the IGFBP-5 promoter activity was induced to a similar extent by forskolin treatment (Fig. 2C), this cAMP-induced increase in mRNA is a transcriptionaly regulated event. Sequence analysis had indicated the presence of several consensus AP-2 binding sites in the promoter region of the human IGFBP-5 gene. AP-2, originally purified from HeLa cells, is a 52-kDa nuclear protein that functions as dimer(21) . AP-2 exerts its function in mediating regulation of gene expression in response to a number of diverse signal transduction pathways. Elevation of AP-2 transactivity can be elicited by RA(25) . In addition to RA, AP-2 mediates transcriptional activation in response to two other signal pathways, the cAMP-dependent protein kinase A pathway and the phorbol ester/diacylglycerol-inducible protein kinase C pathway(26, 27) . This induction of AP-2 activity is mediated by a mechanism independent of increased AP-2 mRNA and independent of protein synthesis. A similar effect by forskolin was observed with IGFBP-5 expression in this study and IGFBP-5 expression has been reported to be regulated by RA(11, 12, 14) . The spatial and temporal expression pattern of IGFBP-5 is similar to that of AP-2, the latter is known to be expressed primarily in neural crest cells and their major derivatives(28) . IGFBP-5 mRNA expression like AP-2 is restricted to a subset of ectodermal and mesodermal tissues during rodent embryogenesis(10) . Neither AP-2 nor IGFBP-5 is significantly expressed in endodermally derived tissues such as liver. This overlapping expression is also reflected in vitro. As shown in this study, IGFBP-5 mRNA is abundant in fibroblasts and glioblastoma cells, which are known to have high levels of endogenous AP-2(28) . In the AP-2 deficient HepG2 cells, IGFBP-5 mRNA levels are very low. This similarity in regulation and pattern of expression is suggestive of a role for AP-2 in regulating IGFBP-5 expression.
The potential role of AP-2 in regulating IGFBP-5
expression was addressed by DNA binding studies and transfection
studies. Gel shift mobility assay data suggest that the regions from
-152 to -127 and from -57 to -30 in the IGFBP-5
promoter were capable of binding to human AP-2 and mutation of these
regions reduced binding, suggesting that AP-2 may be involved in
binding to these regions and regulating this gene. Evidence supporting
this conclusion is that cotransfection of HepG2 cells or fibroblasts
with a human AP-2 expression plasmid increased IGFBP-5 transcription
level. Northern analysis showed that the levels of IGFBP-5 mRNA were
extremely suppressed in the SV40 T-transformed human fibroblasts (Fig. 6), suggesting SV40 T-induced cellular transformation
inhibits the expression of IGFBP-5. This could occur through the
inhibition of AP-2, since SV40 large T-antigen has been shown to
inhibit the trans-activating activity of AP-2 by directly
binding to AP-2 and thereby preventing the formation of an
AP-2DNA complex(21) . Furthermore, specific interference
of the trans-activation activity of AP-2 in human fibroblasts
by expression of AP-2B, a dominant-negative inhibitor of AP-2, resulted
in reduced IGFBP-5 promoter activity. These results suggest that AP-2
is involved in regulating human IGFBP-5 gene expression. The extent to
which AP-2 might be central in controlling IGFBP-5 expression is still
not clear. Our transient transfection studies with HepG2 cells,
however, have shown that expression of exogenous AP-2 can impart to
hepatoma cells the ability to express IGFBP-5 promoter driven
transgenes, and AP-2B counteracts this activation. These data strongly
argue that AP-2 plays an important role in regulating human IGFBP-5
gene expression.
DNase I footprinting experiments have shown that
AP-2 binds to GC-rich sequences that are present in a variety of
cellular and viral promoters and sequences of individual AP-2 binding
sites can vary substantially (21, 26) . Based on the
sequence comparison of AP-2 binding sites identified by DNase I
footprinting in a number of cellular and viral promoters, sequences
such as 5`-CCCCA(G/C)(G/C)C-3` have been proposed as consensus
sites(18) . DNase I footprinting, however, does not generate
information regarding the contribution of individual nucleotides within
the binding sites. Using methylation interference assays and missing
contact probing assays, Williams and Tjian (19) identified a
palindromic sequence, 5`GCCNNNGGC-3`, as the core recognition sequence
for AP-2 binding. In the promoter region of the human IGFBP-5 gene,
both consensus sequences for AP-2 binding are present. The DNA sequence
5`-CCCCACCCCCACCC-3` at position -147 to -134 contains two
overlapping AP-2 elements 5`-CCCCACCC-3` found in the porcine
plasminogen activator gene and the rat tyrosine aminotransferase
gene(18) . The DNA sequence at position -55 to -35
contains sequences identical or close to the consensus AP-2 element
5`-GCCNNNGGC-3`(19) . Although both sequences were capable of
binding to AP-2 in vitro, the promoter activity data of
serially 5`-deleted IGFBP-5 promoter constructs showed that the region
between positions -52 and -37 contains determinants
important for the AP-2-induced activation of transcription. Using a
number of point mutants, we were able to assign the AP-2-induced
activation of the promoter to the proximal sequences which contain
sequences identical or close to the 5`-GCCNNNGGC-3` consensus AP-2
binding sequences. The finding that the proximal 5`-GCCNNNGGC-3`
sequence but not the distal 5`-CCCCACCC-3` sequences is the functional
site that mediates the trans-activation activity of AP-2 in vivo is interesting in the context of AP-2 binding sites.
It also suggests that being able to bind to a DNA sequence in vitro does not necessarily impart functional involvement in
vivo. In the human immunodeficiency virus type I gene, AP-2 and
NF-B have been shown to be able to bind to the same region in a
mutually exclusive manner(29) . It should be noted that the
sequence 5`-CCCCACCC-3` contains sequences identical to the
retinoblastoma control element, 5`-CCACCC-3`. The retinoblastoma
control element motif has been identified as being important for
retinoblastoma-induced trans-activation(30) .
AP-2 has been implicated to play a crucial role in the control of gene expression in response to cell differentiation signals within neural crest and epidermal cell lineages(28) . In the human tetracarcinoma cell line PA-1, constitutive suppression of AP-2 transactivator function by stably expressing AP-2B resulted in a RA-resistant phenotype and these cells became tumorigenic, suggesting that normal function of AP-2 is required for these cells to respond to this differentiation signal(20) . Similarly, the neuronal differentiation of human SH-SY5Y neuroblastoma cells, induced by activation of the protein kinase C signal pathway, is accompanied by increased AP-2 trans-activator activity(31) . Intriguingly, this protein kinase C-induced SH-SY5Y neuronal differentiation is dependent on IGF-I. Pahlman et al.(32) reported that while IGF-I is mitogenic for proliferative SH-SY5Y cells, it loses its mitogenic response and strongly enhances the neuronal differentiation when added with phorbol 12-myristate 13-acetate. This ``switch'' from a mitogenic effect to a differentiation role for IGF-I is puzzling since both mitogenic and differentiation effects are mediated through the IGF-I receptor and this receptor is expressed and functional in both proliferative and differentiated SH-SY5Y cells. We speculate that this switch may be related to the potential changes in IGFBPs, since our finding that AP-2 regulates IGFBP-5 expression raises the possibility that the production of IGFBP-5 may be activated during the protein kinase A-induced differentiation in SH-SY5Y cells. Neuroblastoma cells have been shown to synthesize a number of IGFBPs(33, 34) , and an increase in IGFBP-5 production is observed during differentiation of myoblasts(7) .
The mechanism underlying the biphasic effect of trans-activation of human IGFBP-5 gene transcription by AP-2 remains to be understood, but it is probably related to the transcriptional self-interference of AP-2 reported previously. Kannan et al.(22) found that overexpression of AP-2 causes inhibition of AP-2 trans-activation activity mediated by its activation domain using an artificial promoter construct. Our data with a functional promoter are similar in that overexpression of AP-2 results in reduced AP-2 trans-activation activity. It has been speculated that the AP-2 activity results from a complex of proteins and that there are cofactors necessary for the AP-2 trans-activation activity, some of which are present in limiting amounts. Excess AP-2 molecules may interact with one or more of these putative cofactor(s), making them unavailable for the AP-2 molecules that are bound to the target site and therefore causing autointerference(22) . Such autointerference has been reported with GAL4-VP16(35) . In support of the concept of transcriptional self-interference of AP-2, our studies with cell lines expressing different levels of endogenous AP-2 showed that greater amounts of AP-2 plasmid are required for the autoinhibitory effect in AP-2-deficient cells such as HepG2 cells.
In summary, we have demonstrated that AP-2 regulates transcription of the human IGFBP-5 gene. We have identified the proximal 5`-GCCNNNGGC-3`-like sequences in the human IGFBP-5 gene where AP-2 is able to bind and trans-activate the gene. It should be noted that the structure of the IGFBP-5 promoter is highly conserved, since proximal 200 bp of human mouse and rat IGFBP-5 gene promoters are more than 90% identical(9, 17, 23) . In particular, the proximal AP-2 binding sequence found in human gene is identical to the analogous part of the rat and mouse genes. In addition, cAMP induces IGFBP-5 mRNA in other mammalian species(16) . Therefore, the regulation of IGFBP-5 gene expression by the transcriptional factor AP-2 is likely to be a general mechanism for mammals. The identification of the role of AP-2 and of a functional binding motif in the IGFBP-5 gene has provided necessary information to warrant future interest. Normal AP-2 function is involved in the control of a number of cellular events including cell growth and differentiation(36) . The involvement in these events is an interesting characteristic AP-2 has in common with the IGF system. Further studies will focus on these specific cellular events and how they work together to control IGFBP-5 levels and cell growth and differentiation.