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Diabetes and Endocrinology Research Center, Department of Internal Medicine, The University of Iowa and Veterans Administration Medical Center, Iowa City, Iowa 52246
Address all correspondence and requests for reprints to: Ngozi E. Erondu, The University of Iowa Department of Internal Medicine, ENDO 3E17 VA Medical Center, Iowa City, Iowa 52246.
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Abstract |
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Introduction |
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IGFBP-3 is the major IGFBP in the circulation, accounting for the binding of >90% of the plasma IGFs in a trimeric 150-kDa complex containing IGF, IGFBP-3, and an acid labile subunit (6). IGFBP-3 is secreted by cultured endothelial cells (7) and its mRNA has been localized in endothelial cells in a variety of human and rat tissues, in vivo (8, 9, 10). A diverse number of substances and conditions have been reported to modulate the production of IGFBP-3, acting at both transcriptional and posttranscriptional levels. We have previously shown that IGF-I and TGF-ß1 do not affect IGFBP-3 mRNA stability in cultured bovine endothelial cells (11), suggesting that the reported IGF-1 stimulation and TGF-ß1 inhibition of IGFBP-3 mRNA occur at the level of gene transcription. IGF-I stimulation of IGFBP-3 mRNA has been reported in several cell types; however, the TGF-ß1 induced inhibition of IGFBP-3 mRNA synthesis has only been demonstrated in bovine endothelial cells. To better understand the molecular basis for these observations, we have isolated and sequenced the chromosomal gene for bovine IGFBP-3 and have begun the characterization of its promoter.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cDNA library preparation and cloning of IGFBP-3 cDNA
Total RNA was isolated from bovine microvessel endothelial cells
by the CsCl gradient centrifugation method (12). Poly(A)+ RNA was
purified and oligo (dT) primed cDNA synthesized. The cDNA was ligated
into the 
Uni Zap XR cloning vector (Stratagene, La Jolla, CA) and
packaged to generate a library of 5 x 106 plaque
forming units (pfu), with approximately 99% recombinants. Aliquots of
this library containing approximately 50,000 pfu each were plated on
XL1-Blue cells to generate the amplified cDNA library subsequently used
for screening. A 480-bp PCR product corresponding to nucleotides 516 to
996 was generated from primers based on the published bovine cDNA
sequence (13). This PCR product was labeled to high specific activity
by the random priming method using 32P-deoxy CTP. The
labeled probe was used to screen the microvessel cDNA library. The cDNA
clone, BP-3.511, containing the largest insert (
2.4 kb) was chosen
for further characterization.
Isolation of the bovine IGFBP-3 chromosomal gene
A commercial bovine genomic library (Stratagene) was screened
according to the manufacturer’s instructions using radiolabeled
IGFBP-3 probe. DNA from plaque purified positive clones was digested
with a number of restriction enzymes and subcloned into
pBluescript(Stratagene). Flourescent automated DNA sequencing was
performed in the DNA core facility at the University of Iowa utilizing
PE-ABD 373 automated DNA sequencers (Perkin-Elmer, Foster City,
CA).
Southern blotting
Total cellular DNA was isolated from bovine aorta endothelial
cells (12). DNA (20 µg) was digested with restriction enzymes
(EcoRI, HindIII and XhoI) according to
the supplier’s instructions. The fragments were separated on a 0.8%
agarose gel and transferred to a nylon membrane. The IGFBP-3 PCR
product generated as described above was labeled with 32P
by the random priming method and used to probe the filter under
standard conditions (14).
Primer extension analysis
A synthetic 23 base oligonucleotide (5'-CTGGGC GGC AGC GAG CTG
AGC GA-3') complementary to nucleotides -50 to -73 relative to the
translation initiation site of the bovine cDNA was end-labeled with
[32P]ATP using T4 polynucleotide kinase (Promega,
Madison, WI). The labeled oligonucleotide was hybridized to either
bovine endothelial cell poly(A)+ RNA or yeast transfer RNA
at 65 C for 30 min and slowly cooled to room temperature. After
hybridization, the primer was extended at 42 C for 30 min. The
hybridization buffer, extension buffer and reverse transcriptase were
supplied in the Promega primer extension kit. The reaction products
were separated on a 6% polyacrylamide-7 M urea sequencing
gel in parallel with a sequencing reaction to determine the sizes of
the reaction products.
Construction of recombinant plasmids
A 3.0-kb EcoRI genomic fragment containing 
1.5 kb
of IGFBP-3 5' flanking region was subcloned into pBluescript
(pBS:3000R). This plasmid was double digested with EcoRV and
Cel II to release a 1480 bp fragment corresponding to
nucleotides -1409 to +70 relative to the transcription start site.
This fragment was subcloned into the SmaI site of the basic
luciferase reporter vector, pGL3 in sense (pGL3:1480S) and antisense
(pGL3:1480AS) orientation. To generate a larger construct, a 6-kb
EcoRI genomic fragment, immediately upstream of the 3-kb
EcoRI fragment, was subcloned into the EcoRI site
of pGL3:1480S to generate a construct that had 7.4 kb of 5' flanking
region (pGL3:7400S). Plasmid pGL3:7400S was digested with
HindIII and the 3.0 kb HindIII fragment subcloned
into the HindIII site of pGL3 in two
orientations–pGL3:3000S and pGL3:3000AS. pGL3:7400S was also double
digested with HincII and XhoI and an
approximately 4 kb 5' flanking fragment subcloned into pGL3 to yield
pGL3:4000S. Plasmid pBS:3000R was digested with RsaI to
yield a 520-bp fragment that was subcloned into the SmaI
site of pBluescript (pBS:520S). An 300 bp SacII fragment
was excised from pBS:520S and the remaining recombinant containing 220
bp of 5' flanking region religated (pBS:220S). Plasmids pBS:520S and
pBS:220S were double digested with SacI and
HindIII and the released fragments subcloned into pGL3
vector that had been predigested with SacI and
HindIII, to generate pGL3:520S and pGL3:220S. Plasmid
pGL3:220S was double digested with KpnI (3' overhang) and
Ecl 136 I (blunt end) followed by sequential incubation with
Exonuclease III and Mung bean nuclease under conditions recommended by
the manufacturer (Stratagene). A number of nested deletions were
created and one of them, pGL3:180S, contained 188 bp of 5' flanking
region. Primers were designed for specific amplication of both a 140 bp
and a 110 bp segment of the 5' flanking region of the IGFBP-3 gene. The
upstream primer for the 140 bp segment (5'-GAG AGA TAG GAG
CTC AGC CGG CGC GCC GCT-3') is a composite primer
which contains 16 bases at the 3' end (bold print) that
correspond to nt -76 to -61 relative to the CAP site. The upstream
primer for the 110 bp segment (5'-GAG AGA TAG TGA GCT
CGG CCG CCC GGC TTC-3') is also a composite primer which
contains 17 bases at the 3' end (bold print) which correspond to nt -48
to -32 relative to the CAP site. The 5' end of each primer was designed
to contain a SacI site, GAG CTC (italicized). The
downstream primer was T7 primer (5'-GTA ATACG ACTC ACTA TAGG GC-3') and
the template used in the PCR was pBS:220S. The polymerase chain
reaction was carried out under standard conditions (12). Following
purification, the product was double digested with SacI and
HindIII and the released fragment subcloned into pGL3 basic
vector to generate pGL3:140S and pGL3:110S.
Cell culture and DNA transfection
Bovine aorta endothelial cells were prepared and characterized
as previously described (11). Cells were grown to confluence in 35-mm
well plates, washed and incubated in serum free media before
transfection. Plasmids were purified using the QAIGEN (Chatsworth, CA)
midi plasmid prep kit. Cells in each well were transfected by
incubation at 37 C for 4 h in a reaction mixture containing 2 µg
test plasmid, 1 µg pCMV SEAP (Tropix, Bedford, MA) and 10 µl
Transfectam, a cationic lipid purchased from Promega. After
transfection, cells were incubated for an additional 4 h in serum
free media at 37 C. Secreted alkaline phosphatase (from pCMV SEAP) in
each well was assayed (Tropix) to check for any differences in
transfection efficiency. Subsequently, fresh serum free media ±
the indicated hormones was added to the wells and incubation continued
for 18 h. The cells were washed with
Mg2+/Ca2+ free PBS and lysed with reporter
lysis buffer (Promega). Luciferase activity was determined in duplicate
according to the manufacturer’s recommendations. The protein
concentration in the lysate was determined by the Bradford method
(15).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, begins with the last nucleotide residue (G) of the
published sequence (13). It should be noted that a stretch of 13 A
residues follows the last G in the previously published sequence and
this poly A stretch probably served as a site for oligo dT primed cDNA
synthesis to generate the 1.6 kb cDNA isolated from cow liver (13).
Underlined and in bold print are an AAATAAA
polyadenylation signal motif and an ATTTA motif which may promote mRNA
degradation when located in the 3' untranslated region of an mRNA
transcript (16).
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) that were isolated initially, lacked Exon 5 and the
library was rescreened with a 3' fragment of IGFBP-3 cDNA (a 550-bp
SacI-XhoI fragment). The third phage clone (bov
BP3–3) contained all the exons and both 5' and 3' flanking
regions(Fig. 2A). A schematic representation of the bovine IGFBP-3
gene, which depicts the relative size and location of its exons and
introns is shown in Fig. 2A. Like human IGFBP-3, the bovine gene
consists of five exons, the fifth containing the 3' untranslated region
(Fig. 2B). These exons are separated by four introns of 2954, 474,
1049, and 1544 bp; the corresponding introns in the human IGFBP-3 gene
are 3285, 544, 1595, and 941 bp, respectively (17). We have sequenced
the gene including 4 kb of the 5' flanking region and 1 kb
beyond the polyadenylation site. The sequence information is a
composite generated from the three overlapping phage clones, bov
BP3–1, -2, and -3 (Fig. 2A). A TATA box was located 162 bp 5' to the
translation initiation codon. No CAAT box was present but a GC rich
sequence element was located 5' to the TATA box.
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, hybridized to two restriction fragments in each lane.
The genomic DNA in this region has single sites for the enzymes used
(Fig. 2A
). This result is consistent with the presence of a single copy
of the IGFBP-3 gene in the bovine genome.
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[32P]ATP using T4 polynucleotide kinase (Promega). The
labeled oligonucleotide was hybridized to either poly (A+)
RNA from bovine endothelial cells or yeast transfer RNA at 65 C for 30
min, then slowly cooled to room temperature. After hybridization, the
primer was extended at 42 C for 30 min. The reaction products were
separated on a 6% polyacrylamide-7 M urea sequencing gel
in parallel with a sequencing reaction to determine the size of the
reaction product. As shown in Fig. 4, an 87 base product
was generated indicating that the mRNA cap site is 137 bp 5' to the
translation initiation code. A similar result was obtained when total
RNA from cow liver was substituted for the endothelial cell poly
(A+) RNA (data not shown).
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). In
addition, the first 130 bp of the 5' flanking region (PGL3:180S)
retained nearly 80% of the maximal promoter activity. By contrast,
none of these constructs exhibited promoter activity when the 5'
flanking regions were inserted in the antisense orientation (only data
for PGL3:1480AS is shown in Fig. 5).
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). These putative
response elements include TGF-ß activating sequence (TAE), GH and
steroid (ERE) response elements as well as binding sites for activating
protein-2 (AP-2), thyroid-specific transcription factors (TTF-1 and
-2), ATF and nuclear factor-1 (NF-1).
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have been normalized for transfection
efficiency against the secreted alkaline phosphatase and expressed as
percentage of the activity of the PGL3:1480S. QAYL IGF-I, an analog of
IGF-I that retains affinity for the type I IGF-receptor but has lower
affinity for IGFBPs, stimulated the promoter activity of the construct
2- to 3-fold, which is similar to the fold increase in IGFBP-3 mRNA
levels previously reported in bovine endothelial cells exposed to QAYL
IGF-I (11). It was of particular interest that pGL3:220S and pGL3:180S
were also stimulated by QAYL IGF-I because these constructs lack
TTF-I/TTF-2 (thyroid specific transcription factors) binding elements
that are required for IGF-I stimulation of thyroglobulin synthesis and
have been suggested to be involved in IGF-I regulation of IGFBP-3
transcription (18). However, QAYL IGF-1 had no significant effect on
the promoter activity of pGL3:140S, a construct which lacks the GC rich
sequence element that contains two overlapping putative AP-2 binding
sites. Finally, TGF-ß1 caused a 2-fold increase in the promoter
activity of pGL3:1480S but had no significant effect on the activity of
pGL3:7400S, pGL3:4000S, and pGL3:3000S (constructs containing 3kb or
more of the 5' flanking region). In these transient transfection
studies, we have been unable to demonstrate significant TGF-ß1
induced decrease in promoter activity, which indicates that the
TGF-ß1 mediated inhibition of IGFBP-3 transcription requires sequence
element(s) that are not present in any of the constructs tested.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A number of cytokines/growth factors to which the vascular endothelium
is exposed, have been shown to play a major role in the synthesis of
IGFBP-3 by the endothelium and in the pathogenesis of diabetic
complications, such as poor wound healing, retinopathy, and nephropathy
(21). We have previously shown that two such factors, IGF-1 and
TGF-ß1 regulate the transcription of IGFBP-3 in bovine endothelial
cells (11). To understand the molecular basis for this transcriptional
regulation it was necessary to clone and sequence the bovine IGFBP-3
gene. The 5' flanking region of the bovine IGFBP-3 chromosomal gene
contains several consensus hormone response elements (Fig. 6), similar
to results reported for the human and rat homologs(17, 18). Of
particular interest are the NF-1 and TAE elements known to mediate
TGF-ß activation of transcription (22, 23); TTF-1 and 2 binding
elements that have been suggested to be involved in IGF-1 regulation of
IGFBP-3 mRNA synthesis (18) as well as AP-2 binding elements shown to
contribute to the constitutively high expression of IGFBP-5 and its
cAMP responsiveness in human fibroblasts (24). Despite the cloning of
the human and rat IGFBP-3 promoters, very limited information is
available regarding IGFBP-3 mRNA regulation. In fact, attempts to
demonstrate hormonal responsiveness of the human IGFBP-3 promoter have
been unsuccessful (25).
As a first step towards identifying the cis-acting elements
that mediate the IGF-1 stimulation and TGF-ß1 inhibition of bovine
endothelial IGFBP-3 mRNA synthesis, we generated a number of luciferase
constructs containing 50 to 7400 bp of bovine IGFBP-3 5' flanking
region. Following transfection, bovine aorta endothelial cells were
exposed to serum free media in the absence or presence of IGF-I or
TGF-ß1. The promoter activity of almost all the constructs tested was
stimulated by QAYL IGF-I, an analog of IGF-I that has high affinity for
the type 1 IGF receptor, but lower affinity for IGFBPs. Interestingly,
plasmids pGL3:220S and pGL3:180S were also stimulated by QAYL IGF-I
despite the fact that they lack TTF-1 and -2 binding elements that have
been recently suggested to be involved in the regulation of IGFBP-3
transcription by IGF-I (18). This implies that IGF-I stimulation of
bovine endothelial IGFBP-3 mRNA synthesis requires novel
cis-acting sequence elements. Equally fascinating is the
fact that the IGF-1 stimulation of IGFBP-3 promoter activity was lost
with pGL3:140S, a construct lacking the GC rich sequence element. It
should be noted that the GC rich box has two overlapping putative AP-2
binding sites (Fig. 6), raising the possibility that AP-2 may
contribute to the IGF-1 responsiveness of the IGFBP-3 promoter in
bovine endothelial cells. This is interesting as AP-2 has been shown to
contribute to the consitutively high expression of IGFBP-5 and its cAMP
responsiveness in human fibroblasts (24). TGF-ß1 stimulated the
promoter activity of constructs containing 1.5 kb or less of IGFBP-3 5'
flanking region, an effect that may be mediated by the TGF-ß1
activating sequences present in this region (TAE, NF-1 Fig. 6). On the
other hand, TGF-ß1 had no effect on the promoter activity of
constructs containing 3kb or more of 5' flanking region. Although the
ability of TGF-ß1 to stimulate promoter activity was lost in
constructs containing 3 kb or more of 5' flanking region, no
significant decrease below basal promoter activity was seen in any of
the constructs. Thus, the TGF-ß mediated inhibition of IGFBP-3
transcription previously reported in cultured endothelial cells(11),
likely requires additional sequence elements(s) that are not present in
any of the constructs tested thus far.
In summary, we have cloned the bovine IGFBP-3 chromosomal gene and demonstrated both basal and hormonally responsive promoter activity in its 5' flanking region. Further studies are required to identify and characterize endothelial specific cis and trans-acting element(s)/factor(s) involved in the regulation of IGFBP-3 transcription. The data presented in this report will provide a basis for such future experiments.
Received November 1, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1(I) collagen promoter via a transforming
growth factor-ß activation element. J Biol Chem 268:12635–13631
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1.5 kb
of 5' flanking IGFBP-3 genomic fragments. The products of the
sequencing reaction (lanes G, A, T, and C) and of the primer extension
were run on the same gel. The sequence surrounding the 5' end of the
IGFBP-3 mRNA appears on the right, the circled A being the adenosine
that serves as the cap site.
