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Department of Animal Sciences, Rutgers, State University of New Jersey (W.S.C., B.W., P.V.), New Brunswick, New Jersey 08901; and Department of Animal Sciences, Cornell University (Y.R.B.), Ithaca, New York 14853
Address all correspondence and requests for reprints to: Wendie S. Cohick, Ph.D., Rutgers, State University of New Jersey, 108 Foran Hall, 59 Dudley Road, New Brunswick, New Jersey 08901-8520. E-mail: cohick{at}aesop.rutgers.edu
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Introduction |
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The biological activity of the IGFs is regulated by a family of high affinity binding proteins (IGFBP-1 through -6) with which they associate. IGFBP-3 has been shown to potentiate IGF-stimulated DNA synthesis in both bovine MEC (9) and breast tumor cells (10). In addition, transgenic mice with IGFBP-3 expression targeted to the mammary gland during late pregnancy exhibited a decrease in mammary involution after cessation of lactation (11). In contrast, IGFBP-3 has been shown to inhibit cellular growth and/or promote apoptosis in breast tumor cell lines via IGF-independent mechanisms (12, 13). The ability of various antiproliferative agents to inhibit cellular growth has been shown to be mediated via IGFBP-3 (13, 14, 15). These studies suggest that IGFBP-3 may differentially regulate MEC growth depending on the presence of other regulatory factors present in the extracellular environment.
Although several hormones, growth factors, and intracellular signaling agents have been shown to regulate IGFBP-3 synthesis in different cell types, little is known concerning the molecular mechanisms by which this occurs. We have shown that IGF-I specifically stimulates the synthesis of IGFBP-3 by the bovine MEC line MAC-T via an IGF-I receptor-dependent mechanism (16). A second factor that stimulates IGFBP-3 synthesis in these cells is cAMP. The combination of these two treatments induces greater increases in IGFBP-3 protein and messenger RNA (mRNA) levels than does either treatment alone. The goal of the present study was to delineate the molecular mechanisms by which IGF-I and cAMP regulate IGFBP-3 synthesis in bovine MEC.
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Materials and Methods |
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Cell culture and treatments
MAC-T bovine MEC (17) were routinely grown in DMEM
supplemented with 4.5 g/liter glucose, 10% FBS, 4 mM
glutamine, gentamicin (50 µg/ml), penicillin (100 U/ml), and
streptomycin (20 µg/ml). The bovine fibroblast cell line AG08130 was
obtained from the Coriell Institute for Medical Research (Camden, NJ)
and grown in medium similar to that used for MAC-T cells, except that
20% FBS was used. To study the regulation of IGFBP-3 mRNA levels,
cells were plated at 1.0 x 104
cells/cm2 on 50-mm dishes, grown to confluence,
rinsed twice with serum-free DMEM, then incubated in serum-free DMEM at
37 C for various times depending on the experiment. After the washout
period, media were aspirated, and treatments were added in serum-free
medium. For studies of mRNA stability, cells were plated, grown to
confluence, and rinsed twice in serum-free DMEM. After a 2-h washout
period, cells were preincubated with the appropriate treatments for
3 h at 37 C, then DRB was added to the cultures at a final
concentration of 75 µM. Cells were then incubated for
varying lengths of time before RNA isolation.
RNA analysis
Total RNA was extracted using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the
manufacturer’s recommendations. RNA concentrations and integrity were
determined by absorbance at 260 and 280 nm. After denaturation with 2.2
M formaldehyde and 12.5 M formamide at 55 C for
15 min, RNA was separated by electrophoresis on a 1.0% agarose gel
containing 2.2 M formaldehyde and transferred to nylon
membranes (Biotrans, ICN Biomedicals, Inc., Irving, CA).
The membranes were hybridized overnight at 65 C with
[32P]deoxy-CTP-labeled bovine IGFBP-3, bovine
IGFBP-2, and 18S ribosomal complementary DNA (cDNA) probes
(16) in 0.25 M sodium phosphate, 7% SDS, and
1 mM EDTA, pH 7.2. Membranes were washed twice in 2 x
SSC (standard saline citrate) plus 0.1% SDS and twice in 0.1 x
SSC plus 0.1% SDS at 65 C for 20 min/wash. Membranes were exposed to
Kodak AR film (Eastman Kodak Co., Rochester,
NY) with intensifying screens at -80 C. Differences in relative band
intensities were determined using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale CA).
Nuclear run-on analysis
MAC-T cells were plated on 100-mm plates and grown to confluence
as described above. After a 16-h incubation in serum-free medium, cells
were exposed to serum-free media with or without treatments for 2
h. Relative transcription rates were determined by a previously
reported method (18). Cells were rinsed twice with
ice-cold PBS, removed by scraping, and centrifuged at 500 x
g. The cell pellets were resuspended in ice-cold lysis
buffer [10 mM Tris-Cl (pH 7.4), 3
mM CaCl2, and 2
mM MgCl2], centrifuged,
and resuspended in lysis buffer containing 0.5% Nonidet P-40. Cells
were lysed with a Dounce homogenizer (Kontes Co., Vineland, NJ), and
nuclei were collected by centrifugation at 500 x g.
The nuclei were resuspended in 200 µl 50 mM
Tris-Cl (pH 8.3), 40% glycerol, 5 mM
MgCl2, and 0.1 mM EDTA and
stored in liquid nitrogen until the run-on assay was performed. Thawed
nuclei were incubated with 1 mM each of ATP, CTP,
and GTP; 100 µCi 
-32P-labeled UTP, and 200
µl 2 x reaction buffer [10 mM Tris-Cl
(pH 8.0), 5 mM MgCl2, 0.30
M KCl, and 5 mM
dithiothreitol] for 30 min at 30 C with shaking to label preinitiated
RNA transcripts. Reactions were treated with RNase-free
deoxyribonuclease I and proteinase K. 32P-Labeled
RNA transcripts were phenol-chloroform extracted and purified using a
Quick-Spin high capacity column (Roche, Indianapolis, MN).
RNA was precipitated and resuspended in 10 mM
N-Tris (hydrozymethyl) methyl-2-aminoethanesul-fonic
acid (pH 7.4), 10 mM EDTA, and 0.2% SDS (TES
solution). Fifteen micrograms of plasmid DNA containing IGFBP-3 or
IGFBP-2 insert or vector DNA alone were linearized, denatured with
NaOH, and immobilized on nylon membrane (Bio-Rad Laboratories, Inc., Hercules, CA) with a slot blotter. The membranes were
prehybridized in 0.25 M sodium phosphate, 7%
SDS, and 1 mM EDTA, pH 7.2. Labeled transcripts
(5 x 106 cpm) were hybridized with each
membrane in TES solution containing 300 mM NaCl
for 60 h at 55 C. The membranes were washed four times in 2
x SSC containing 0.1% SDS for 30 min, then incubated for 30 min at 37
C with 10 µg/ml RNase A in 2 x SSC before a final wash in
2 x SSC at 37 C for 1 h. Membranes were exposed to
Kodak AR film with intensifying screens at -80 C.
Differences in relative band intensities were determined using a
PhosphorImager (Molecular Dynamics, Inc.).
Cloning of the 5'-flanking region of the murine IGFBP-3 gene and
plasmid construction
A cDNA corresponding to exons 2–4 of the rat IGFBP-3 gene
(19) was used to screen a mouse genomic library (129SvJ in
Fix II (Stratagene, La Jolla, CA). One of the isolated
clones was shown by Southern analysis and sequencing to contain the
entire coding region of the mouse IGFBP-3 gene (20), the
5'-untranslated region, and approximately 1.1 kb of 5'-flanking
sequence. The region corresponding to nucleotides -1280 to -3 (ATG,
+1) was excised using the restriction endonucleases NotI and
NarI and inserted by blunt end ligation into the
SmaI site of the promoterless vector pGL3-Basic
(Promega Corp.) in both the sense and antisense
orientations.
Generation of IGFBP-3 deletion constructs
Murine IGFBP-3 promoter fragments with 5'-ends at nucleotides
-1098, -916, -691, and -416 and a common 3'-end at nucleotide -3
were generated by PCR amplification using the 1.3-kb IGFBP3/pGL3
construct as template. Amplification was carried out in a DNA thermal
cycler (Perkin-Elmer Corp.) using an initial denaturation
at 94 C for 5 min, followed by 30 cycles of denaturation at 94 C for 1
min, annealing at 55 C for 1 min, and extension at 72 C for 1 min. A
7-min incubation at 72 C was performed at the completion of the final
cycle. The sense and antisense primers incorporated unique
KpnI and HindIII restriction sites, respectively,
for subcloning into pGL3-Basic. Each construct was verified by
sequencing in both directions.
Transfection assays
MAC-T cells were plated at 2 x 104
cells/cm2 in six-well plates and grown to 85%
confluence. Media were aspirated, and cells were cotransfected using 5
µl Cytofectene transfection reagent (Bio-Rad Laboratories, Inc.) with plasmid constructs IGFBP3/pGL3 (1.5 µg) and a
vector containing the Renilla luciferase gene under control
of the simian virus 40 promoter to normalize for transfection
efficiency (50 ng pRL-SV40, Promega Corp.). After a 5-h
incubation at 37 C, medium was aspirated and replaced with complete
fresh medium. After 16 h, the cells were rinsed twice in
serum-free medium and incubated in serum-free medium with or without
treatment. Preliminary experiments indicated that basal promoter
activity decreased from 8–24 h, whereas promoter activity remained
relatively constant after exposure to IGF-I and forskolin over this
interval (data not shown). Therefore, cells were treated for 24 h
in all experiments. Cells were lysed in 250 µl lysis buffer
(Promega Corp.). The activities of firefly and
Renilla luciferase were measured sequentially from
individual samples using the dual luciferase reporter assay
(Promega Corp.).
Statistical analysis
Differences between serum-free controls and treated groups were
compared using Student’s t test. P < 0.05
was considered significant.
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Results |
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). In agreement with previous results,
treatment with IGF-I increased IGFBP-3 mRNA by 3 h, with maximum
increases observed between 8–12 h of treatment. A similar time course
of IGFBP-3 mRNA induction was observed with both forskolin and
forskolin plus IGF-I. Treatment with either forskolin or IGF-I alone
maximally increased IGFBP-3 mRNA levels 8- to 15-fold relative to those
in serum-free controls. The increase in IGFBP-3 mRNA observed at each
time point after treatment with both IGF-I and forskolin was always at
least as great as the sum of the increases observed with each treatment
alone. As IGF-I and forskolin were each tested at concentrations that
maximally induce IGFBP-3 mRNA levels, the enhanced response observed
with the combination suggests that these two factors activate separate
signaling pathways. Neither of these treatments affected IGFBP-2 mRNA
levels in MAC-T cells (16); therefore IGFBP-2 was used as
a control to illustrate the specificity of the IGFBP-3 response.
IGFBP-2 mRNA actually decreased over the 24-h time course examined,
regardless of treatment (Fig. 1).
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). When fibroblasts were incubated with
IGF-I for 8 h, IGFBP-3 mRNA levels were increased 3.3 ±
0.7-fold above serum-free control values (mean ± SE;
n = 3). In contrast, treatment with forskolin failed to stimulate
IGFBP-3 mRNA levels. The increase in IGFBP-3 mRNA levels observed after
exposure to IGF-I and forskolin tended to be less than the increase
observed with IGF-I alone. Therefore, although IGF-I increases IGFBP-3
mRNA levels in both cell types, the ability of forskolin to stimulate
IGFBP-3 mRNA is specific to the mammary epithelial cell.
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, the half-life of IGFBP-3
mRNA levels in untreated cells was approximately 11 h. Treatment
with either IGF-I or forskolin enhanced the stability of IGFBP-3 mRNA.
The half-life of IGFBP-3 mRNA in cells treated with IGF-I was increased
to 27 h, representing a 2.4-fold increase in mRNA stability. The
effect of forskolin on IGFBP-3 mRNA stability was greater than that of
IGF-I, extending the half-life to 106 h. The half-life of IGFBP-3
mRNA stability in cells treated with a combination of IGF-I and
forskolin was similar to that observed for forskolin alone (data not
shown). This would be expected given that with forskolin alone
virtually no degradation had occurred by 18 h.
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). However, when cells were
exposed to forskolin and IGF-I in combination, IGFBP-3 transcription
rates were increased 3.5 ± 0.83-fold (mean ±
SE; n = 4; P < 0.05) over IGFBP-3
transcription rates in control cells. The transcription rates of
IGFBP-2 in IGF-I- and forskolin-treated cells served as negative
controls, as IGF-I and forskolin do not increase IGFBP-2 mRNA levels
(16). The transcription rate of IGFBP-2 was not affected
by any of the treatments.
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). Exposure of MAC-T cells to IGF-I or
forskolin alone consistently produced small, but significant, increases
in the activity of the sense construct of 1.57 ± 0.12- and
1.59 ± 0.08-fold (Fig. 6; mean
± SE of seven experiments; P <
0.01). Treatment with both IGF-I plus forskolin produced additive
increases in promoter activity in every experiment, significantly
increasing luciferase activity 2.25 ± 0.14% above the control
value (mean ± SE of seven experiments;
P < 0.01). Similar increases were observed when
(Bu)2cAMP was used in place of forskolin, either
alone or in combination with IGF-I (data not shown).
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Identification of regions required for regulation of IGFBP-3
promoter activity by IGF-I and forskolin
To identify the regions of the IGFBP-3 promoter mediating the
effects of IGF-I and forskolin, MAC-T cells were transfected with
5'-deletion mutants of the IGFBP-3 promoter. As shown in Fig. 7, deletion of the region between
nucleotides -1283 and -416 of the IGFBP-3 promoter did not eliminate
the ability of IGF-I and forskolin to increase luciferase activity.
Comparison of the bovine and murine IGFBP-3 promoters revealed a highly
conserved region in the remaining sequence that includes the TATA box
and several conserved AP-2 and Sp1 binding sites (Fig. 8). One or more of these binding sites
may be involved in either basal regulation of the IGFBP-3 gene and/or
regulation by IGF-I and cAMP. Studies are presently underway to
identify the molecular mechanisms responsible for this regulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experiments using the transcriptional inhibitor DRB indicated that the degradation rate of IGFBP-3 mRNA in untreated MAC-T cells was approximately 11 h. IGFBP-3 mRNA degradation rates have been estimated at 14–18 h in human hepatocarcinoma (22) or normal rat liver cells (23), 6 h in bovine kidney epithelial cells (24), and as short as 2 h in rat Leydig cells (25). This suggests that different mechanisms may regulate IGFBP-3 degradation depending on the cell type. A number of labile mammalian mRNAs, such as c-fos, contain AU-rich elements (ARE) in their 3'-untranslated regions (UTR) that have been linked to mRNA destabilization (26). The 3'-UTR of the bovine IGFBP-3 mRNA is uridine rich and contains an ARE consisting of an AUUUA pentamer motif that is conserved in the human and the rat (24). Recently, it has been shown that this region binds a 42-kDa protein present in cytoplasmic extracts of MDBK cells. This protein may belong to a family of binding proteins that bind to AREs and regulate mRNA stability (27). However, it was shown that the AUUUA motif was not required for this protein to bind to IGFBP-3 mRNA. In contrast, two uridine stretches were shown to be critical for the RNA-protein interaction (24). The lack of a requirement for the AUUUA motif agrees with studies using the stabile ß-globin mRNA in which various mutant c-fos and synthetic ARE were inserted within its 3'-UTR. The nonamer UUAUUUAUU was found to be effective in promoting mRNA degradation, whereas the pentamer AUUUA had no effect (27).
In the present study both IGF-I and forskolin increased the stability of IGFBP-3 mRNA. The half-life of IGFBP-3 mRNA was estimated to be 27 h in IGF-I-treated cells and 101 h in forskolin-treated cells. The effect of forskolin on IGFBP-3 turnover was more pronounced than that of IGF-I, suggesting that these factors may act to stabilize IGFBP-3 message through different mechanisms. A similar effect of IGF-I on mRNA stability has been reported with cocultures of rat liver cells (23). In contrast, no effect of IGF-I on mRNA stability was observed with bovine endothelial cells (28) or human hepatocarcinoma cells (22). However, in the later study where no effect was observed, the cells were not preincubated with IGF-I before DRB treatment. We found that DRB blocks the ability of IGF-I to stabilize IGFBP-3 message levels in the absence of a preincubation period with IGF-I. Thus, when added simultaneously, DRB may block the ability of IGF-I to induce the synthesis of proteins involved in stabilizing IGFBP-3 mRNA. The mechanism by which IGF-I enhances IGFBP-3 mRNA stability has not been determined for any cell type.
Forskolin has been shown to increase steady state levels of IGFBP-3 mRNA in MAC-T cells (16), MDBK cells (24, 29), and the human breast carcinoma cell line MCF-7 (30). This effect may be specific for epithelial cells, as in the present study forskolin did not stimulate IGFBP-3 mRNA in bovine fibroblasts. Forskolin has been shown to stabilize IGFBP-3 mRNA levels in MDBK cells by decreasing the binding of the 42-kDa RNA-binding protein described above to the 3'-UTR of IGFBP-3 mRNA. This effect was shown to involve phosphorylation of the RNA binding protein via a protein kinase A-dependent mechanism (24). Whether a similar mechanism is responsible for the increase in IGFBP-3 mRNA observed in forskolin-treated MAC-T cells is unknown.
The large increase in mRNA stability induced by forskolin appeared sufficient to account for the large increases in steady state mRNA levels observed after this treatment. In contrast, the 2.4-fold increase in stability induced by IGF-I appeared less likely to fully account for the large increases in steady state mRNA levels observed after IGF treatment. Therefore, we conducted nuclear run-on assays to investigate potential regulation by transcriptional mechanisms. The results of these assays indicated that treatment of MAC-T cells for 2 h with either forskolin or IGF-I alone had no detectable effect on the rate of IGFBP-3 transcription. In contrast, stimulation of MAC-T cells transiently transfected with the 5'-flanking region of the murine IGFBP-3 promoter to drive luciferase expression resulted in small, but significant, increases in luciferase activity of approximately 50%. These increases were most likely too small to detect by nuclear run- on assay, which is inherently less sensitive. Overall, the relatively small increase in promoter activity of at least 1.5-fold after 24 h of IGF-I treatment together with the 2.4-fold increase in stability appeared sufficient to account for the 5-fold increase in mRNA observed after 24 h of treatment.
The time course of IGFBP-3 mRNA induction shown in Fig. 1
shows clearly
that a much greater increase in mRNA is obtained when the two
treatments are added together. This can only be achieved by an increase
in transcription, because the combination of forskolin and IGF-I did
not result in a longer half-life for IGFBP-3 mRNA than did forskolin
alone. The increase in promoter activity obtained using the mouse
IGFBP-3 promoter agreed well with the increase in transcription rate
observed with nuclear run-on in cells treated with both IGF-I and
forskolin. Similar to the results of the nuclear run-on assay, the
effects of IGF-I and forskolin on promoter activity were additive when
cells were treated with a combination of IGF-I plus forskolin.
Therefore, the enhanced transcriptional response to IGF-I plus
forskolin explains the greater response of IGFBP-3 mRNA observed with
the combination of treatments.
The finding that IGF-I and forskolin together induce greater increases in IGFBP-3 promoter activity than either factor does alone indicates that these two factors activate separate signaling pathways that somehow act in concert to activate IGFBP-3 gene transcription. Support for the involvement of discrete intracellular signaling pathways comes from our observations that IGF-I does not increase intracellular cAMP levels in this cell line (our unpublished data). Although we have not examined whether cAMP increases IGF-I synthesis in MAC-T cells, the similar time frame of IGFBP-3 mRNA induction observed with either IGF-I or forskolin treatment suggests that cAMP does not increase IGFBP-3 synthesis via IGF-I. IGF-I mediates its intracellular effects via the IGF tyrosine kinase receptor (31). In contrast, cAMP is generally increased in response to activation of membrane-associated, trimeric, G protein-coupled receptors (32). Recently, elevations in intracellular cAMP have been shown to potentiate the ability of epidermal growth factor to activate mitogen-activated protein kinase in LNCaP prostate carcinoma cells (33). These studies raise the possibility that cross-talk between the signaling pathways activated by cAMP and the IGF tyrosine kinase receptor may be responsible for the increase in transcriptional activation of the IGFBP-3 gene.
To identify the region containing the DNA-binding sites required for the responsiveness to IGF-I and forskolin, sequential deletion fragments of the 1.3-kb IGFBP3/pGL-3 promoter construct were generated by PCR and used in luciferase reporter assays. Promoter activity was increased to a similar extent with each deletion fragment, including the smallest fragment, which contained approximately 300 bp of 5'-flanking region. Similarly, using bovine IGFBP-3 deletion fragments, Erondu et al. (34) reported that a fragment containing only 128 bp upstream of the transcription start site was responsive to IGF-I in bovine endothelial cells.
The consensus binding sequences in the IGFBP-3 gene that are required for either basal responsiveness or stimulation by IGF-I/cAMP are unknown. Examination of published IGFBP-3 promoter sequences indicates that multiple DNA-binding sites for known transcription factors are present in the sequences of the human (35), rat (36), and bovine (34) genes. However, no studies have reported whether any of these sites are functional in terms of regulating either basal or hormonal responsiveness of the IGFBP-3 promoter. We have sequenced approximately 1 kb of the upstream 5'-flanking region of the murine IGFBP-3 gene and compared it with the bovine promoter sequence. Although both promoters contain a high number of consensus binding sequences for known transcription factors, the majority of these are not conserved between the two species. In contrast, the 200-bp region immediately upstream of the transcription start site exhibits 80% homology between the two species. This region contains several AP-2 and Sp1 binding sites that are conserved between the two genes. Especially interesting is the region between nucleotides -189 and -207 in the mouse and between -216 and -234 in the bovine that contains three overlapping AP-2 binding sites and two Sp1 sites. The sequence corresponding to the 3'-most AP-2 site in this region also represents a perfectly conserved AP-2 site in the human and rat genes. The middle site in this region is a perfect AP-2 site in the bovine and is missing one nucleotide for a perfect AP-2 consensus sequence in the rat (similar to that in the mouse).
The transcription factor AP-2 is activated by several different
signaling cascades, including the cAMP-dependent protein kinase A
pathway (37, 38). This protein binds to GC-rich regions in
a variety of promoters, and considerable variation has been found
between individual binding sequences (38, 39). AP-2 has
also been shown to regulate basal activity of the human IGFBP-5 gene,
which, like IGFBP-3, is responsive to cAMP and IGF-I (40).
IGF-I regulates expression of the chicken 
1-crystallin gene and the
rat elastin gene through GC-rich sequences that bind Sp1or Sp1-like
proteins (41, 42). Therefore, these consensus binding
sequences are likely to be involved in regulation of the IGFBP-3
promoter. Studies to further define the regulatory elements involved in
regulation of the IGFBP-3 promoter are currently in progress.
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Acknowledgments |
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Footnotes |
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Received June 2, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-crystallin gene by insulin-like
growth factor I and insulin requires DNA cis elements in
chicken. Proc Natl Acad Sci USA 87:3353–3357This article has been cited by other articles:
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