(Received for publication, December 6, 1995; and in revised form, January 12, 1996)
From the
Insulin-like growth factor (IGF) action is mediated by high
affinity cell surface IGF receptors and modulated by a family of
secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of
six IGFBPs characterized to date, uniquely potentiates the anabolic
actions of IGF-I for skeletal cells. In osteoblasts, IGFBP-5 production
is stimulated by prostaglandin E (PGE
), a local
factor that mediates certain effects induced by parathyroid hormone,
cytokines such as interleukin-1 and transforming growth factor-
,
and mechanical strain. In this study, we show that transcriptional and
post-transcriptional events initiated by PGE
collaborate to
enhance IGFBP-5 gene expression in primary fetal rat osteoblast
cultures. PGE
treatment stimulated up to a 7-fold rise in
steady-state levels of IGFBP-5 mRNA throughout 32 h of incubation.
Analysis of nascent IGFBP-5 mRNA suggested that PGE
had
only a modest stimulatory effect on IGFBP-5 gene transcription, and
transient transfection studies with IGFBP-5 promoter-reporter genes
confirmed that PGE
enhanced promoter activity by
2-fold. Similar stimulatory effects were seen with forskolin. A
DNA fragment with only 51 base pairs of the 5`-flanking sequence
retained hormonal responsiveness, which may be mediated by a binding
site for transcription factor AP-2 located at positions -44 to
-36 in the proximal IGFBP-5 promoter. Incubation of osteoblasts
with the mRNA transcriptional inhibitor
5,6-dichloro-1-
-D-ribofuranosylbenzimidazole demonstrated
that PGE
enhanced IGFBP-5 mRNA stability by 2-fold,
increasing the t
from 9 to 18 h. The effects of
PGE
on steady-state IGFBP-5 transcripts were abrogated by
preincubating cells with cycloheximide, indicating that the effects of
PGE
on both gene transcription and mRNA stability required
ongoing protein synthesis. Therefore, both promoter-dependent and
-independent pathways converge to enhance IGFBP-5 gene expression in
response to PGE
in osteoblasts.
Insulin-like growth factors-I (IGF-I) ()and -II are
abundant locally produced growth regulators in skeletal
tissue(1) . While the synthesis of IGFs is hormonally
controlled, their actions are ultimately determined by way of
signal-transducing receptors. IGF binding proteins (IGFBPs) are a
family of secreted proteins that also avidly bind IGFs and modify their
actions by altering their access to cell surface
receptors(2, 3) . Of the six known IGFBPs, fetal rat
osteoblasts synthesize five, IGFBP-2, -3, -4, -5, and -6(4) .
However, IGFBP-5 is the only IGFBP with a demonstrated ability to
potentiate IGF actions in bone cells(5) . Therefore, agents
that stimulate IGFBP-5 synthesis may have an important anabolic role in
skeletal growth and the maintenance of skeletal integrity.
The initial identification of IGFBP-5 was based upon its ability to augment IGF-I activity in bone cell cultures, as well as its structural and sequence similarities to other IGFBPs(6, 7, 8, 9) . The mechanism of potentiation of IGF action by IGFBP-5 has been attributed to its association within pericellular compartments (cell membrane and matrix components), resulting in a high local concentration of IGFs in close proximity to cell surface IGF receptors(5, 10, 11) . Recent studies suggest the ability of IGFBP-5 to bind ligand decreases after its association with matrix or select polysaccharides and the actions of IGFBP-5-selective proteases(12, 13, 14, 15) . The subsequent release of highly concentrated IGF in the pericellular environment thus enhances IGF receptor binding and its biological effects. IGFBP-5 has the additional attribute of high affinity binding to the calcium phosphate component of bone, which may serve to concentrate IGFs in inorganic bone matrix for storage and subsequent activation during periods of localized bone resorption(8) .
In bone cell cultures, both IGFBP-5 and IGF-I synthesis are
regulated by prostaglandin E (PGE
), parathyroid
hormone (PTH), other agents that stimulate cAMP synthesis, or by cAMP
itself (4, 16) . PGE
is produced by
osteoblasts in response to PTH, to cytokines such as interleukin-1 and
transforming growth factor-
, and to mechanical strain, and
PGE
has been shown to mediate various biological actions on
osteoblasts initiated by these
stimuli(17, 18, 19, 20, 21, 22) .
PGE
may thus serve as a local analog of PTH. Unlike PTH,
however, its influence may be more highly focused due to its synthesis
within the skeleton. Furthermore, PGE
has demonstrated
anabolic actions in bone that depend on the cellular state of
differentiation and on dose and duration of
treatment(23, 24, 25) .
While the
molecular mechanisms by which PGE regulates IGF-I synthesis
in osteoblasts are only currently being
elucidated(26, 27) , even less is known about IGFBP-5
synthesis in skeletal tissue. Previous observations reveal that
PGE
enhances steady-state levels of IGFBP-5
mRNA(4) . We now demonstrate that transcriptional and
post-transcriptional pathways are both activated by PGE
to
stimulate IGFBP-5 gene expression. These steps appear to require
ongoing protein synthesis, indicating that promoter-dependent and
-independent processes each regulate IGFBP-5 production in response to
PGE
in primary fetal rat osteoblast-enriched cultures.
PGE increases the level of IGFBP-5 mRNA and
protein in primary fetal rat osteoblast-enriched cultures(4) .
The magnitude of the rise in transcript abundance is time-dependent. In
agreement with earlier studies(4) , IGFBP-5 mRNA levels
increased by 1.4-7-fold following 4-32 h of PGE
(1 µM) treatment (Fig. 1). A subsequent
analysis by RNase protection assay confirmed these observations and
additionally demonstrated a time-dependent rise in abundance of nascent
transcripts, first observed at 3 h after PGE
treatment,
with a
3-fold increase seen at 8 h (data not shown). These results
suggest that PGE
may stimulate IGFBP-5 transcription,
although this effect appears delayed when compared with another
PGE
-stimulated gene, IGF-I, for which nascent transcripts
increase within 30 min of PGE
treatment(26) .
Figure 1:
Time course
of IGFBP-5 mRNA induction by PGE in primary
osteoblast-enriched cultures. Confluent, serum-deprived cultures of
osteoblasts were treated with vehicle (ethanol) or PGE
(1
µM) for 4, 6, 8, 24, or 32 h, as indicated in each panel). RNA blots were prepared as described under
``Experimental Procedures,'' hybridized with
P-labeled rat IGFBP-5 cDNA and antisense 18 S rRNA,
washed, and visualized by autoradiography. On the left are
pooled data for four independent Northern blots. On the right is a representative blot with IGFBP-5 transcripts shown in the upper panel and the 18 S rRNA pattern shown below. RNA
standards (Life Technologies, Inc.) were used to determine the length
(in kilobases (kb)) of IGFBP-5 transcripts, shown in the right panel.
To
investigate potential transcriptional mechanisms influenced by
PGE treatment, gene transfer experiments were conducted. In
initial studies, segments of the murine IGFBP-5 promoter containing
various lengths of 5`-flanking DNA and 120 base pairs (bp) of exon 1
were fused to the luciferase reporter gene, transiently transfected
into osteoblast-enriched cultures, and analyzed for reporter gene
expression 48 h later. As shown in Fig. 2, IGFBP-5 promoter
constructs with 1406, 1004, and 156 bp (IGFBP5-Luc3, -Luc4, and -Luc5)
of 5`-flanking DNA directed comparably high luciferase activity (100,
95.6, and 84.3%, relative to IGFBP5-Luc3). In contrast, fusion plasmids
with 75 bp or less of 5`-flanking DNA (IGFBP5-Luc6, -Luc7, and -Luc8)
had progressively diminished activity (22.9 to 5.9%, relative to
IGFBP5-Luc3). Luciferase expression for a positive control viral
promoter-driven construct, pGL2-Control, was included for comparison.
These data indicate that regions between -156 and -51 bp
contain cis-acting regulatory element(s) needed for high level
basal promoter activity in osteoblast cultures, analogous to the areas
that we defined in Hep G2 (hepatocyte) and C2I (myoblast) cell
lines(32, 37) . Two recombinant plasmids with longer
5`-flanking segments of 4100 and 3000 bp (IGFBP5-Luc1 and -Luc2)
directed lower luciferase activity, suggesting the presence of
inhibitory elements in the 5`-flanking region of the promoter. Promoter
activity was orientation-specific. When the 1004-bp promoter fragment
was inserted into the luciferase vector in the reversed orientation
(IGFBP5-Luc4 rev, Fig. 2), reporter expression was minimal,
being comparable with the promoterless pGL2-Basic parental plasmid.
Figure 2:
Identifying regions controlling basal
IGFBP-5 promoter function in osteoblast-enriched cultures. Various
IGFBP-5 promoter-luciferase reporter plasmids (depicted in the left
panel) were co-transfected with pSV--galactosidase control
vector into osteoblast-enriched cultures (9.6 cm
) using
Lipofectin. Cultures were grown to confluence (48 h), the growth medium
was aspirated, and the cultures were rinsed with serum-free
Dulbecco's modified Eagle's medium. Cultures were exposed
to control medium (containing ethanol vehicle) for 6 h. Cytoplasmic
extracts were prepared, and luciferase activity was determined as
described under ``Experimental Procedures.'' Data were
corrected for transfection efficiency (
-galactosidase expression)
and for protein content of cytoplasmic extracts. Transfections were
performed in duplicate or triplicate, and results are pooled data for
three or more separate experiments for a total of eight or more
replicate cultures. The mean ± S.E. for pooled experiments is
shown. Luciferase activity was determined by single channel photon
counting, and background levels were
930 cpm/µg of protein.
Control transfections included an SV40 promoter/enhancer-luciferase
reporter plasmid (pGL2-Control) and promoterless pGL-2 Basic (both from
Promega Corp.). Numbers on the far right in parentheses indicate the percent maximal luciferase expression determined from
the mean value for each construct, as compared with the mean value for
IGFBP5-Luc3, which has been set at 100%.
Recombinant IGFBP-5 promoter-luciferase fusion constructs were next
used to identify promoter elements that participated in
PGE-stimulated IGFBP-5 expression. While basal luciferase
activity for IGFBP5-Luc2 through -Luc7 varied up to 14-fold (Fig. 2), 6 h of PGE
treatment enhanced their
ability to drive luciferase expression to a similar extent, ranging
from 2.3- to 1.6-fold (Fig. 3). The shortest construct
responsive to PGE
, IGFBP5-Luc 7, contained only 51 bp of
5`-flanking DNA. However, deletion of the next 20 bp (IGFBP5-Luc8)
eliminated the effect of PGE
on reporter gene expression.
Using IGFBP5-Luc 4, a comparable increase was seen after 24 h of
PGE
treatment, and these effects were duplicated by
treatment with forskolin (10 µM), a strong inducer of
adenylate cyclase activity (Fig. 4). Plasmids with internal
deletions spanning -69 to -51 bp and -52 to -32
bp in the background of the very active IGFBP5-Luc 4 construct had
diminished basal activity as shown previously(32) .
Importantly, however, each of these two deletion constructs clearly
responded to PGE
treatment, although at modestly reduced
levels (1.4- and 1.7-fold, respectively; Fig. 5).
Figure 3:
Effect of PGE on IGFBP-5
promoter activity following transient transfection into
osteoblast-enriched cultures. Various IGFBP-5 promoter-luciferase
reporter plasmids (depicted in Fig. 2) were co-transfected with
a pSV-
-galactosidase control vector into osteoblast-enriched
cultures (9.6 cm
) using Lipofectin. Cultures were grown to
confluence (48 h), the growth medium was aspirated, and the cultures
were rinsed with serum-free Dulbecco's modified Eagle's
medium. Cultures were exposed to control medium (containing vehicle) or
PGE
(1 µM) for 6 h. Cytoplasmic extracts were
prepared and luciferase activity was determined. Data are corrected for
transfection efficiency (
-galactosidase expression) and for
protein content of cytoplasmic extracts. Transfections were performed
in duplicate or triplicate, and results are pooled data for three or
more separate experiments and for a total of eight or more replicate
cultures. The mean ± S.E. for luciferase expression compared
with vehicle-treated cultures (-fold stimulation) for pooled
experiments is shown. PGE
caused a statistically
significant elevation in luciferase expression (p < 0.05 versus pGL-2 Basic) for IGFBP-5 promoter reporter plasmids
IGFBP5-Luc2, -Luc3, -Luc4, -Luc5, -Luc6, and
-Luc7.
Figure 4:
Forskolin and PGE induce
comparable increases in IGFBP-5 promoter activity in
osteoblast-enriched cultures. The IGFBP-5 promoter-luciferase reporter
plasmid, IGFBP5-Luc4, was co-transfected with a pSV-
-galactosidase
control vector into osteoblast-enriched cultures using Lipofectin, as
described in Fig. 3and under ``Experimental
Procedures.'' Cultures were grown to confluence (48 h), the growth
medium was aspirated, and the cultures were rinsed with serum-free
Dulbecco's modified Eagle's medium. Cultures were exposed
to control medium (containing vehicle), forskolin at 10
µM, or PGE
at 1 µM for 6 or 24 h,
as indicated. Cytoplasmic extracts were prepared and luciferase
activity was determined. Data are corrected for
-galactosidase
expression and for protein content of cytoplasmic extracts.
Transfections were performed in duplicate or triplicate, and results
are representative data for three separate experiments and for a total
of eight or more replicate cultures. The mean ± S.E. for
luciferase expression is shown.
Figure 5:
Effect of PGE on the activity
of mutant IGFBP-5 promoter following transient transfection into
osteoblast-enriched cultures. IGFBP-5 promoter-luciferase reporter
plasmids having deletion mutations spanning -69 to -51 bp,
or -52 to -32 bp, in the IGFBP5-Luc 4 background
(IGFBP5-Luc4
a and IGFBP5-Luc4
b, respectively) (32) were co-transfected with an pSV-
-galactosidase
control vector into osteoblast-enriched cultures using Lipofectin, as
described under ``Experimental Procedures'' and in the legend
to Fig. 2. Cultures were exposed to control medium (containing
vehicle) or PGE
(1 µM) for 6 h. Cytoplasmic
extracts were prepared and luciferase activity was determined. Data are
corrected for transfection efficiency (
-galactosidase expression)
and for protein content of cytoplasmic extracts. Transfections were
performed in duplicate or triplicate, and results are pooled data for
three or more separate experiments and for a total of eight or more
replicate cultures. The relative effect of PGE
compared to
vehicle-treated cultures (-fold stimulation) is shown for pooled
experiments.
In
aggregate, these results show that the IGFBP-5 promoter is very active
in primary osteoblast cultures but demonstrate that PGE stimulated only a 2-fold increase in IGFBP-5 gene transcription,
even after a 24-h incubation. This contrasts sharply with Northern blot
data showing up to a 6-fold rise in steady-state IGFBP-5 mRNA levels at
24 h (Fig. 1). Thus, the discrepancy between maximal promoter
activity and steady-state transcripts encoding IGFBP-5 in response to
PGE
indicates the participation of transcriptional and
post-transcriptional mechanisms in regulating IGFBP-5 gene expression.
Therefore, the RNA polymerase II selective inhibitor DRB was used to
examine the influence of PGE
on IGFBP-5 mRNA stability.
Cultures were first treated with vehicle alone or PGE
(1
µM) for 24 h, followed by DRB (75 µM) to
arrest gene transcription. PGE
treatment caused a 2-fold
rise in the half-life of IGFBP-5 mRNAs; vehicle-treated control
cultures had a t
= 9 h, while cultures
pretreated with PGE
had t
=
18 h (Fig. 6). Consequently, PGE
enhanced the
stability of IGFBP-5 transcripts. To explore further the mechanisms
involved in induction of IGFBP-5 mRNA after PGE
treatment,
we examined the effect of the protein synthesis inhibitor,
cycloheximide. At a dose of 2 µM, cycloheximide blocked
>90% of ongoing protein synthesis, as measured by incorporation of
[H]proline into trichloroacetic acid-precipitable
material in PGE
-treated cultures (-cycloheximide,
12.6 ± 0.9
10
cpm versus +cycloheximide, 1.3 ± 0.1
10
cpm).
Cycloheximide also completely inhibited the induction of IGFBP-5 mRNA
after PGE
treatment (Fig. 7). These results contrast
with the lack of an effect of cycloheximide on the rise in IGF-I
transcript abundance following PGE
treatment, (
)and demonstrate that inducible protein(s) contribute to
the transcriptional and post-transcriptional effects of PGE
on IGFBP-5 gene expression in osteoblast-enriched cultures.
Figure 6:
Effect of PGE on the stability
of IGFBP-5 mRNA in osteoblasts. Osteoblast-enriched cultures were
treated for 24 h with control (vehicle) or PGE
(1
µM). Cultures were then supplemented with 75 µM DRB for the additional time intervals indicated. RNA was isolated,
and 15 µg from each sample were fractionated by electrophoresis,
blotted, and hybridized to a
P-labeled rat IGFBP-5 cDNA,
as described under ``Experimental Procedures.'' Data are
relative IGFBP-5 abundance, as compared with transcript levels at the
time of DRB addition (percent initial). Panel A, control
treated cultures are shown in closed circles and those for the
PGE
are shown in closed squares. Data are from
three separate experiments. Panel B, a representative Northern
blot is shown with IGFBP-5 transcripts in the upper panel and
the 18 S rRNA pattern below. RNA standards (Life Technologies, Inc.)
were used to determine the length (in kilobases (kb)) of
IGFBP-5 transcripts, shown at the left of panel
B.
Figure 7:
Effect of cycloheximide on IGFBP-5
transcript levels in control and PGE-treated
osteoblast-enriched cultures. Confluent, serum-deprived cultures of
osteoblasts were treated with control (C, vehicle) or
PGE
(P, 1 µM) for 6 h, in the absence
or presence of 2 µM cycloheximide. RNA blots were prepared
as described under ``Experimental Procedures,'' hybridized
with
P-labeled rat IGFBP-5 cDNA, washed, and visualized by
autoradiography. A representative Northern blot probing is shown in the upper panel. The hybridized probe was eluted, and the blot was
hybridized with
P-labeled antisense 18 S ribosomal RNA
riboprobe (Ambion, Houston, TX; shown below). RNA standards (Life
Technologies, Inc.) were used to determine the length (in kilobases (kb)) of IGFBP-5 transcripts, shown on the left.
Parallel cultures were treated with the same cycloheximide solution and
cultures pulsed with [
H]proline to assess
effectiveness of the protein synthesis inhibitor; 90-95%
inhibition of protein synthesis was observed. The Northern blot is
representative of three independent
experiments.
IGFBP-5 expression is activated through cAMP-dependent
pathways in osteoblasts and in other cell culture
models(4, 16, 39) . While IGFBP-5 transcripts
accumulate in response to PGE and other agents that elevate
cAMP, little is known about the mechanisms of hormone-induced IGFBP-5
synthesis in osteoblasts. We now present data demonstrating stimulation
of IGFBP-5 promoter activity by PGE
and show that PGE
also enhances IGFBP-5 mRNA stability. These results indicate that
promoter-dependent and -independent mechanisms function together to
regulate IGFBP-5 gene expression.
Unstimulated fetal rat osteoblasts synthesize IGFBP-5 mRNA and protein(4, 40, 41) . Results from transient transfection experiments confirm our earlier studies that near-maximal basal promoter activity resides within the first 156 bp of 5`-flanking DNA and that over 20% of basal activity is controlled by the proximal 75 bp of the promoter (32) . Similar to our earlier evidence, promoter function is attenuated by internal deletions that eliminate nucleotides -69 to -51 or -52 to -32, encoding segments that span a DNase I-footprinted region identified with Hep G2 nuclear protein extracts(32) .
In transient transfections of
osteoblasts, PGE treatment for 6 or 24 h increased
luciferase activity driven by the IGFBP-5 promoter 1.6-2.3-fold
in constructs containing as little as 51 bp or as much as 3000 bp of
5`-flanking DNA. The shortest promoter fragments mediating
PGE
-induced gene transcription do not contain a consensus
cAMP response element. However, a potential binding site for
transcription factor AP-2 is present between nucleotides -44 and
-36, and at least six AP-2 sites are dispersed throughout the
3000 bp of the IGFBP-5 promoter. The apparent decline in response to
PGE
treatment seen with recombinant plasmids having
progressively shorter promoter segments may reflect loss of individual
AP-2 binding sites or other potential cAMP-responsive cis-elements.
While this paper was in preparation, Duan and
Clemmons (42) reported the involvement of AP-2 in basal and
cAMP-mediated IGFBP-5 transcription in human dermal fibroblast cell
lines. In their study, forskolin stimulated IGFBP-5 promoter activity
2.8-fold, a result similar in magnitude to our observation in
osteoblasts. They identified an AP-2 site within nucleotides -55
to -36 in the human IGFBP-5 promoter as the key hormone response
element(42) . An identical AP-2 site is present in a comparable
location in the murine promoter, as noted above. While deletion of this
site in construct IGFBP5-Luc4
b reduced basal promoter activity, it
caused only a modest decline in the effect of PGE
.
Therefore, additional AP-2 sites or alternative cAMP response elements
may be functional in osteoblasts.
The modest 2-fold effect of
PGE
on IGFBP-5 transcription does not account for the
6-fold increase in steady-state IGFBP mRNA seen following a 24-h
incubation. As demonstrated here, PGE
also caused a
doubling of IGFBP-5 transcript half-life, from
9 to
18 h. Of
note, the t
for IGFBP-5 mRNA in
osteoblast-enriched cultures under basal conditions,
9 h, is
similar to the 11-12-h transcript half-life measured by us in C2I
myoblasts (37) but differs somewhat from reported values of
14 and
20 h obtained under basal conditions in a similar
osteoblast culture model(40, 41) , which may be
accounted for by small differences in experimental design. Thus, both
transcriptional and post-transcriptional effects of PGE
contribute to the induction of IGFBP-5 mRNA following hormone
treatment. These dual actions on IGFBP-5 gene expression can be
distinguished from transcriptional effects of PGE
on the
IGF-I gene, which appear to be mediated through an element found in the
proximal part of promoter 1, the major IGF-I gene
promoter(27) . In addition, the actions of PGE
to
enhance IGFBP-5 gene expression require ongoing protein synthesis,
since they were obliterated by preincubation with cycloheximide, while
PGE
-stimulated IGF-I gene transcription occurs even in the
absence of new protein synthesis. (
)
Levels of IGFBP-5 in extracellular compartments are modulated not only by rates of gene expression and protein biosynthesis but also by post-translational mechanisms. The existence of IGFBP-5-selective proteases has been documented in a variety of cultured cells, including normal human osteoblasts(12, 13, 14, 43) , and IGF-mediated stabilization of IGFBP-5 abundance has been described in culture models derived from bone and other cell types(44) . Since IGFBP-5 enhances the anabolic actions of IGF-I in bone cells(5) , analysis of the multiple mechanisms involved in modifying IGFBP-5 availability within the skeleton should have direct impact in understanding how growth factors regulate skeletal cell metabolism.