From the
Skeletal cells secrete insulin-like growth factors (IGFs) I and
II and six known IGF binding proteins (IGFBPs). IGFBP-5 stimulates bone
formation, and its synthesis correlates with changes in osteoblast cell
growth. We tested the effects of basic fibroblast growth factor (bFGF),
transforming growth factor
Skeletal cells are known to secrete insulin-like growth factors
(IGFs)
Investigations from this and other laboratories have revealed that
skeletal growth factors inhibit the synthesis of IGFs I and II in cells
of the osteoblastic lineage and that the synthesis of IGF I and IGFBP-5
is coordinated
(9, 10, 16, 17) . Studies
using osteoblast cultures have revealed that hormones that stimulate or
inhibit IGF I synthesis have similar effects on IGFBP-5 synthesis
(16, 17) . Furthermore, IGF I enhances the synthesis and
stability of IGFBP-5
(17, 18) . We postulated that
skeletal growth factors with potent mitogenic activity not only
decrease skeletal IGFs I and II synthesis but may have analogous
effects on IGFBP-5 expression, and these changes are relevant to the
growth and differentiated function of the osteoblast.
The present
studies were undertaken to examine the effects of basic fibroblast
growth factor (bFGF), transforming growth factor
To determine changes in hnRNA, total RNA from
control and test samples was prepared as described for Northern
analysis. One µg of RNA was treated with DNase and
reverse-transcribed in the presence of the rat IGFBP-5 intron
2-specific antisense primer 5`-CTTAGGATGCACGTGGTT-3` at 42 °C for
30 min with Moloney murine leukemia virus reverse transcriptase (Life
Technologies, Inc.) The newly transcribed cDNA was amplified by 25 PCR
cycles of 94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1
min, following the addition of the IGFBP-5 intron 2-specific sense
primer 5`-GCACCATCCCAATCTGAT-3`, Taq DNA polymerase, and 5
µCi of [
Northern blot analysis of total RNA extracted from confluent
cultures of Ob cells revealed a predominant IGFBP-5 transcript of 6.0
kilobases, although in accordance with observations in other cell
systems, smaller size transcripts were also detected
(30, 31, 32, 33) . Continuous treatment
of Ob cells with bFGF, TGF
Recent studies have demonstrated that skeletal cells
synthesize IGFs I and II and six known IGFBPs. The present
investigation was undertaken to determine whether growth factors known
to be synthesized by skeletal cells modify IGFBP-5 expression in
calvarial derived Ob cells. We demonstrated that bFGF, TGF
Intact IGFBP-5 is primarily
present in the extracellular matrix of skeletal and nonskeletal cells
(27) ,
While
there are uncertainties about the physiological concentrations of bFGF,
TGF
IGFBP-5 has significant effects on bone cell growth,
and its expression is coordinated with stages of osteoblast cell
growth. In addition, its expression in myoblasts is correlated with
cell differentiation and, as in bone cells, it is inhibited by growth
factors with mitogenic properties
(40, 41) . These
observations suggest a coordinated expression of IGFBP-5 in bone and
muscle and a possible relevance to the expression of the differentiated
phenotype in the musculoskeletal system. In fact, bFGF, PDGF BB, and
(to a somewhat more variable extent) TGF
In conclusion, the present studies
demonstrate that bFGF, TGF
We thank Dr. S. Shimasaki for the rat IGFBP-5 cDNA
clone, Dr. Yu Dong for assistance with the design of IGFBP-5 intronic
primers, Cathy Boucher and Deena Kjeldsen for technical assistance, and
Beverly Faulds for expert secretarial help.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1 (TGF
1), and platelet-derived
growth factor (PDGF) BB on IGFBP-5 expression in cultures of
osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob
cells). Treatment of Ob cells with bFGF, TGF
1, and PDGF BB caused
a time- and dose-dependent decrease in IGFBP-5 mRNA levels and
inhibited IGFBP-5 polypeptide levels in the extracellular matrix. The
effects of bFGF, TGF
1, and PDGF BB on IGFBP-5 transcripts were
independent of cell division and were observed in the presence and
absence of hydroxyurea. bFGF, TGF
1, and PDGF BB did not modify
the decay of IGFBP-5 mRNA in transcriptionally arrested Ob cells, and
they inhibited IGFBP-5 heterogeneous nuclear RNA and the rate of
IGFBP-5 transcription. In conclusion, bFGF, TGF
1, and PDGF BB
inhibit IGFBP-5 expression in Ob cells independently of their mitogenic
activity and through mechanisms that involve decreased transcription.
(
)
I and II as well as the six known IGF
binding proteins (IGFBPs)
(1, 2, 3, 4, 5, 6) .
IGFs I and II are among the most important local regulators of bone
cell function because of their abundance in bone tissue as well as
their stimulatory actions on multiple aspects of bone formation
(1, 3, 7) . The synthesis and activity of IGFs
are regulated by systemic hormones, by other skeletal growth factors,
and by IGFBPs
(8, 9, 10) . Whereas the exact
function of IGFBPs in bone is not known, most of the IGFBPs have been
reported to have inhibitory activities on bone formation
(11, 12, 13) . However, IGFBP-5 has been
consistently shown to increase bone cell growth and enhance the actions
of IGF I on this process
(14) . Furthermore, the expression of
IGFBP-5 appears to be related to the stage of osteoblast growth and
differentiation
(15) . Therefore, studies to define agents that
regulate the synthesis and activity of IGFBP-5 in bone cells are
critical to our understanding of its role in bone physiology.
1 (TGF
1),
and platelet-derived growth factor (PDGF) BB on IGFBP-5 synthesis in
cultures of osteoblast-enriched cells from fetal rat parietal bone (Ob
cells) and to determine possible mechanisms involved.
Culture Technique
The culture method used was
described in detail previously
(16) . Parietal bones were
obtained from 22-day-old fetal rats immediately after the mothers were
sacrificed by blunt trauma to the nuchal area. This project was
approved by the Institutional Animal Care and Use Committee of Saint
Francis Hospital and Medical Center. Cells were obtained by five
sequential digestions of the parietal bone using bacterial collagenase
(CLS II, Worthington). Cell populations harvested from the third to the
fifth digestions were cultured as a pool and were previously shown to
have osteoblastic characteristics
(19) . Ob cells were plated at
a density of 8,000-12,000 cells/cm and cultured in a
humidified 5% CO
incubator at 37 °C until reaching
confluence (about 50,000 cells/cm
). Cells were cultured in
Dulbecco's modified Eagle's medium (DMEM) supplemented with
nonessential amino acids, 100 µg/ml L-ascorbic acid,
penicillin, streptomycin, and 20 mM HEPES (all from Life
Technologies, Inc.), and 10% fetal bovine serum (HyClone, Logan, UT).
At confluence the cells were rinsed and transferred to serum-free
medium for 20-24 h, when they were again rinsed with serum-free
medium, and then exposed to test or control medium in the absence of
serum for 2-48 h. The medium of cultures lasting 48 h was
replaced with fresh solutions after 24 h. Cycloheximide, hydroxyurea
(both from Sigma), and recombinant human PDGF BB (Austral, San Ramon,
CA) were added directly to the culture medium. Basic FGF (Austral) was
dissolved in 20 mM sodium citrate and diluted 1:5,000 or
greater in culture medium, and recombinant human TGF
1 (Austral
and a gift from Genentech, South San Francisco, CA) was either added
directly to the medium or dissolved in 5 mM HCl and diluted
1:3,000 or greater in DMEM. 5,6-dichlorobenzimidazole (DRB) (Sigma) was
dissolved in absolute ethanol and diluted 1:200 in DMEM; in experiments
where DRB was used, all experimental groups were exposed to an equal
amount of ethanol. For the nuclear run on experiment, subconfluent
cultures of Ob cells were trypsinized, subcultured at a 1:6 dilution,
and grown to confluence in DMEM supplemented with 10% fetal bovine
serum. Cells were serum-deprived for 24 h and treated for 2-24 h
in serum-free DMEM. For RNA analysis, the cell layer was extracted with
guanidine thiocyanate at the end of the incubation and stored at
-80 °C. For protein analysis, the conditioned medium was
obtained, the extracellular matrix was extracted, and both were
processed for Western blots. For the nuclear run on assay, nuclei were
isolated by Dounce homogenization.
Northern Blot Analysis
Total cellular RNA was
isolated with guanidine thiocyanate, at acid pH, followed by
phenol-chloroform (Sigma) extraction
(20) . RNA was precipitated
with isopropyl alcohol, resuspended, and reprecipitated with ethanol.
The RNA recovered was quantitated by spectrometry, and equal amounts of
RNA from control or test samples were loaded on a formaldehyde-agarose
gel following denaturation. RNA standards (BDH Ltd., Poole, U. K.) were
used to determine transcript size. The gel was stained with ethidium
bromide to visualize RNA standards and ribosomal RNA, documenting equal
RNA loading of the various experimental samples. The RNA was then
blotted onto Gene Screen Plus charged nylon (DuPont). A 300-base pair
HindIII restriction fragment of the rat IGFBP-5 cDNA (kindly
provided by Dr. S. Shimasaki, La Jolla, CA) was purified by agarose gel
electrophoresis
(21) . IGFBP-5 cDNA was labeled with
[-
P]dCTP and
[
-
P]dATP (50 µCi of each at a specific
activity of 3,000 Ci/mmol; (DuPont) using the random
hexanucleotide-primed second strand synthesis method
(22) .
Hybridizations were carried out at 42 °C for 16-72 h, and
post-hybridization washes were performed at 65 °C in 0.1
saline-sodium citrate. The bound radioactive material was visualized by
autoradiography on Kodak X-AR5 film, employing Cronex Lightning Plus
intensifying screens (DuPont). Relative hybridization levels were
determined by densitometry. Northern analyses shown are representative
of three or more cultures.
Heterogeneous Nuclear RNA (hnRNA) Analysis
To
examine changes in hnRNA, rat IGFBP-5 intron 2-specific primers were
designed following partial sequence analysis of intron 2. For this
purpose, intron 2 of IGFBP-5 was amplified from rat genomic DNA
(Promega, Madison, WI), using the sense exon 2 primer
5`-GACTCTCGGGAGCATGAGGAAC-3` and the antisense exon 3 primer
5`-AGCTTCCATGTGTCTGCGGCAG-3`, by polymerase chain reaction (PCR) using
Taq DNA polymerase in accordance with manufacturer's
instructions (Perkin Elmer). The PCR amplification was performed for 30
cycles of 94 °C for 1 min, 65 °C for 1 min, and 72 °C for 1
min. A single PCR product of approximately 780 base pairs was purified
by agarose gel electrophoresis and subcloned into pCR II
(Invitrogen, San Diego, CA), and its identity was confirmed by partial
DNA sequence analysis.
-
P]dCTP (3,000 Ci/mmol, DuPont)
as described
(23, 24) . The PCR products were
fractionated by electrophoresis on a 6% urea-polyacrylamide denaturing
gel, visualized by autoradiography, and quantitated by densitometry. An
internal DNA standard was included in the PCR and used to correct for
variations in amplification. The standard was obtained by amplification
of SV40 promoter sequences in the pGL2-P plasmid DNA using the
composite sense primer 5`-GCACCATCCCAATCTGATattagtcagcaaccatagtc-3`,
and antisense primer 5`-CTTAGGATGCACGTGGTTggttccatcctctagaggat-3`. The
capital letters indicate IGFBP-5 intron 2 sequences, while the
lowercase letters represent pGL2-P vector sequences.
Nuclear Run On Assay
To examine changes in the
rate of transcription, nuclei were isolated by Dounce homogenization in
a Tris buffer containing 0.5% Nonidet P-40. Nascent transcripts were
labeled by incubation of nuclei in a reaction buffer containing 500
µM each of ATP, CTP, and guanidine triphosphate, 150 units
of RNAsin (Promega), and 250 µCi of [P]UTP
(3,000 Ci/mmol, DuPont)
(25) . RNA was isolated by treatment
with DNase I and proteinase K, followed by phenol-chloroform extraction
and ethanol precipitation. Linearized plasmid DNA containing about 1
µg of cDNA was immobilized onto GeneScreen Plus by slot blotting
according to the manufacturer's directions (DuPont). The plasmid
vector pGL2-Basic (Promega) was used as a control for nonspecific
hybridization, and a mouse 18 S cDNA clone (ATCC, Rockville, MD) was
used to estimate loading of the gel. Equal counts/min of
[
P]RNA from each sample were hybridized to cDNAs
using the same conditions as for Northern blot analysis and were
visualized by autoradiography.
Western Blots
Extracellular matrix was prepared as
described
(26, 27) . Briefly, Ob cells were rinsed in
phosphate-buffered saline, cell membranes were removed with 0.5% Triton
X-100 (Sigma), pH 7.4, and nuclei and cytoskeleton were removed by
incubation with 25 mM ammonium acetate, pH 9.0, for 5 min. The
extracellular matrix was rinsed with phosphate-buffered saline and
scraped from the culture plates. Aliquots from the conditioned medium
or the extracellular matrix were fractionated by polyacrylamide gel
electrophoresis on 10-18% denaturing gradient gels
(28) .
For Western immunoblots, proteins were transferred to Immobilon P
membranes (Millipore, Bedford, MA), blocked with 2% bovine serum
albumin, and exposed to a 1:500 dilution of rabbit antisera raised
against native human IGFBP-5 (UBI, Lake Placid, NY), in 1% bovine serum
albumin overnight. Blots were exposed to goat anti-rabbit IgG antisera
conjugated to horseradish peroxidase, washed, and developed with a
horseradish peroxidase chemiluminescence detection reagent (DuPont).
For Western ligand blots, transferred proteins were incubated with
I-IGF II, and the blot was developed by autoradiography
as described
(29) . IGFBP-5 was identified by co-migration with
recombinant human IGFBP-5 (Austral).
1, and PDGF BB caused a time-dependent
decrease in IGFBP-5 steady state mRNA levels. Treatment of Ob cells
with bFGF at 6 nM for 6 h caused no change in IGFBP-5 mRNA,
whereas TGF
1 at 0.4 nM and PDGF BB at 3.3 nM
for 6 h caused a slight decrease in IGFBP-5 transcripts (Fig. 1).
After 24 h of treatment with bFGF, TGF
1, and PDGF BB at the
indicated doses, densitometric analysis revealed a 75% or greater
decrease in IGFBP-5 mRNA, and the inhibition was sustained for 48 h.
Because the growth factors tested increase
-actin and
glyceraldehyde-3-phosphate dehydrogenase mRNA, IGFBP-5 mRNA levels were
not corrected for changes in
-actin or glyceraldehyde-3-phosphate
dehydrogenase mRNA, and uniformity of RNA loading of the gels was
estimated by ethidium bromide staining of ribosomal RNA
(9) .
(
)
Continuous treatment of Ob cells
with bFGF, TGF
1, and PDGF BB for 48 h caused a dose-dependent
inhibition of IGFBP-5 transcript levels (Fig. 2). Densitometric
analysis revealed that bFGF at 6 nM and PDGF BB at 3.3
nM decreased IGFBP-5 mRNA by 70% and that TGF
1 at 1.2
nM decreased IGFBP-5 transcripts to virtually undetectable
levels.
Figure 1:
Effect of
basic fibroblast growth factor ( FG) at 6 nM,
transforming growth factor 1 ( T
) at 0.4 nM,
and platelet-derived growth factor ( BB) at 3.3 nM on
IGFBP-5 mRNA expression in cultures of Ob cells treated for 6, 24, or
48 h. Total RNA from control ( C) or treated cultures was
subjected to Northern blot analysis and hybridized with a
P-labeled rat IGFBP-5 cDNA. Visualization of the 18 and 28
S ribosomal RNA by ethidium bromide staining demonstrates RNA loading
of the gel. IGFBP-5 mRNA was visualized by autoradiography and is shown
in the upperpanel, while ribosomal RNA is shown
below.
Figure 2:
Effect of bFGF, transforming growth factor
1 ( TGF
), and PDGF BB, in nanomolar concentrations,
on IGFBP-5 mRNA expression in cultures of Ob cells treated for 48 h.
Total RNA from control (0) or treated cultures was subjected to
Northern blot analysis and hybridized with a
P-labeled rat
IGFBP-5 cDNA. Visualization of the 18 and 28 S ribosomal RNA by
ethidium bromide staining demonstrates RNA loading of the gel. IGFBP-5
mRNA was visualized by autoradiography and is shown in the upperpanel, while ribosomal RNA is shown
below.
Western ligand blot analysis of the extracellular matrix of
untreated Ob cells revealed that they expressed a predominant IGFBP,
which co-migrated with an IGFBP-5 standard and had a molecular mass of
31 kDa (Fig. 3 A). Western immunoblots confirmed the
presence of a major form of immunoreactive IGFBP-5 of 31 kDa and two
minor forms in the 29-30-kDa range (Fig. 3 B).
These probably represent different degrees of IGFBP-5 glycosylation
(18, 30) . bFGF at 6 nM, TGF 1 at 1.2
nM, and PDGF BB at 3.3 nM for 24 h decreased the
31-kDa form of IGFBP-5 as determined by Western ligand and immunoblots
(Fig. 3, A and B). The Western immunoblot
revealed immunoreactive protein bands of 45-50 kDa, which
increased with TGF
1 and PDGF BB treatment. These proteins were
not detected in the Western ligand blot shown in
Fig. 3A. Stripping of the immunoblot shown and
incubation with
I-IGF II as ligand visualized IGFBP-5 but
not the 45-50-kDa proteins, confirming that they were not IGFBPs
(not shown). The levels of IGFBP-5 in the culture medium were low,
making the detection of an inhibitory effect impractical (not shown).
Figure 3:
Effect of basic fibroblast growth factor
( FG) at 6 nM, transforming growth factor 1
( T
) at 1.2 nM, and platelet-derived growth
factor ( BB) at 3.3 nM on IGFBP-5 polypeptide levels
in cultures of Ob cells treated for 24 h. A, extracts from
extracellular matrix of control ( C) and treated cultures were
subjected to Western ligand blot and IGFBPs detected by incubation with
I-IGF II and autoradiography. The arrow indicates the migration of a human IGFBP-5 standard (not shown).
B, extracts from extracellular matrix were subjected to
Western immunoblot analysis, and IGFBP-5 was detected using an
anti-IGFBP-5 antibody and a chemiluminescence detection
system.
To determine whether or not the effects observed on IGFBP-5 mRNA
levels were dependent on protein synthesis, serum-deprived confluent
cultures of Ob cells were treated with the various growth factors in
the presence or absence of cycloheximide at 3.6 µM. In
earlier experiments cycloheximide at doses of 2 µM and
higher was found to inhibit protein synthesis in Ob cell cultures by
80-85%
(34) . Northern blot analysis revealed that
treatment with cycloheximide for 24-48 h caused a comparable and
marked decrease in IGFBP-5 transcript levels so that further inhibitory
effects of bFGF, TGF 1, and PDGF BB could not be detected
(Fig. 4). To determine if the changes in IGFBP-5 mRNA levels
observed were related to the mitogenic activity of the growth factors
studied, serum-deprived Ob cell cultures were treated with the various
factors in the presence or absence of hydroxyurea at 1 mM, a
dose previously shown to block DNA synthesis in Ob cells
(35) .
Northern blot analysis revealed that treatment with hydroxyurea for 48
h did not modify IGFBP-5 transcripts and did not prevent the inhibitory
effect of bFGF, TGF
1, or PDGF BB (Fig. 5).
Figure 4:
Effect of basic fibroblast growth factor
( FG) at 6 nM, transforming growth factor 1
( T
) at 1.2 nM, and platelet-derived growth
factor ( BB) at 3.3 nM in the presence (+) and
absence (-) of cycloheximide at 3.6 µM on IGFBP-5
mRNA expression in cultures of Ob cells treated for 48 h. Total RNA
from control ( C) or treated cultures was subjected to Northern
blot analysis and hybridized with a
P-labeled rat IGFBP-5
cDNA. Visualization of the 18 and 28 S ribosomal RNA by ethidium
bromide staining demonstrates RNA loading of the gel. IGFBP-5 mRNA was
visualized by autoradiography and is shown in the upper panel,
while ribosomal RNA is shown below.
Figure 5:
Effect of basic fibroblast growth factor
( FG) at 6 nM, transforming growth factor 1
( T
) at 1.2 nM, and platelet-derived growth
factor ( BB) at 3.3 nM in the presence (+) or
absence (-) of hydroxyurea at 1 mM on IGFBP-5 mRNA
expression in cultures of Ob cells treated for 48 h. Total RNA from
control ( C) or treated cultures was subjected to Northern blot
analysis and hybridized with a
P-labeled rat IGFBP-5 cDNA.
Visualization of the 18 and 28 S ribosomal RNA by ethidium bromide
staining demonstrates RNA loading of the gel. IGFBP-5 mRNA was
visualized by autoradiography and is shown in the upperpanel, while ribosomal RNA is shown
below.
To examine
whether or not the effects of the growth factors studied on IGFBP-5
were due to changes in transcript stability, confluent cultures of Ob
cells were exposed to control or growth factor-containing medium for 60
min. and then treated with the RNA polymerase II inhibitor DRB in the
absence or presence of bFGF at 6 nM, TGF 1 at 1.2
nM, and PDGF BB at 3.3 nM for 6, 16, or 24 h
(36) . The half-life of IGFBP-5 mRNA in transcriptionally
arrested Ob cells was estimated at 20 h, and it was not changed by
treatment with any of the growth factors tested (Fig. 6).
Treatment of Ob cells with bFGF at 6 nM, TGF
1 at 1.2
nM, and PDGF BB at 3.3 nM for 2, 6, or 24 h decreased
IGFBP-5 hnRNA expression as estimated by reverse transcription PCR
(Fig. 7). This would suggest a change in RNA transcription or
processing. After 2 and 6 h of treatment, bFGF and TGF
1 decreased
IGFBP-5 hnRNA by at least 90% as determined by densitometry, whereas
PDGF BB inhibited IGFBP-5 hnRNA by 30-50%. The inhibitory effect
of PDGF BB on hnRNA was not observed after 24 h of treatment, whereas
bFGF and TGF
1 were effective for up to 24 h. The growth factors
tested did not change the signal of the internal standard, indicating
uniform PCR. Furthermore, no signal of the hnRNA product was detected
in any of the samples tested when the reverse transcription step was
omitted prior to the PCR, eliminating the possibility of DNA
contamination. To confirm whether the growth factors tested modified
the transcription of the IGFBP-5 gene, nuclear run on assays were
performed on nuclei from Ob cells treated for 2-24 h. bFGF at 6
nM, TGF
1 at 1.2 nM, and PDGF BB at 3.3
nM inhibited the rate of IGFBP-5 transcription after 14 h by
20-60% and after 24 h by 70-80% (Fig. 8). The factors
were not effective after 2 h, and after 6 h of treatment only a modest
decrease in the rate of IGFBP-5 transcription was observed (not shown).
Figure 6:
Effect of bFGF at 6 nM, TGF
1 at 1.2 nM, and PDGF BB at 3.3 nM on IGFBP-5
mRNA decay in transcriptionally blocked Ob cells. Cultures were treated
with bFGF, TGF
1, or PDGF BB 1 h before and 6, 16, or 24 h after
the addition of DRB. RNA was subjected to Northern blot analysis and
hybridized with a
P-labeled rat IGFBP-5 cDNA, visualized
by autoradiography, and quantitated by densitometry. Values are means
± S.E. for three or more cultures, except for TGF
1 24 h
after DRB, which represents two cultures.
Figure 7:
Effect of basic fibroblast growth factor
( FG) at 6 nM, transforming growth factor 1
( T
) at 1.2 nM, and platelet-derived growth
factor ( BB) at 3.3 nM on heterogeneous nuclear RNA
expression in cultures of Ob cells treated for 2, 6, or 24 h. Total RNA
from control ( C) or treated cultures was extracted, and 1
µg was subjected to competitive reverse transcription-PCR in the
presence of IGFBP-5 intron 2-specific 5`-sense and 3`-antisense primers
and of 5 µCi of [
-
P]dCTP. The reverse
transcriptase-PCR products were fractionated by polyacrylamide gel
electrophoresis and visualized by autoradiography. IGFBP-5 hnRNA is
shown in the upperpanel, and the internal standard,
prepared as described under ``Materials and Methods,'' is
shown below.
Figure 8:
Effect of basic fibroblast growth factor
( FG) at 6 nM, transforming growth factor 1
( T
) at 1.2 nM, and platelet-derived growth
factor ( BB) at 3.3 nM on IGFBP-5 transcription rates
in cultures of Ob cells treated for 14 ( left panel) or 24 h
( right panel). Nascent transcripts were labeled in vitro with [
P]UTP, and the labeled RNA was
hybridized to immobilized cDNA for IGFBP-5. Murine 18 S cDNA was used
to demonstrate loading, and pGL2-Basic vector DNA ( pGL2-b) was
used as a control for nonspecific
hybridization.
1, and
PDGF BB decrease IGFBP-5 mRNA levels in Ob cells in a time- and
dose-dependent manner and that de novo protein synthesis is
required for basal IGFBP-5 transcript expression. The growth factors
tested are known to stimulate bone cell replication, but the decrease
in IGFBP-5 synthesis in Ob cells was not modified by the DNA synthesis
inhibitor hydroxyurea, suggesting that this effect was independent from
their mitogenic activity. Experiments in transcriptionally blocked Ob
cells, using the RNA polymerase II inhibitor DRB, revealed that bFGF,
TGF
1, and PDGF BB did not modify IGFBP-5 mRNA stability. This, in
conjunction with a decrease in hnRNA levels and in the rate of IGFBP-5
gene transcription caused by the three growth factors studied, suggests
that bFGF, TGF
1, and PDGF BB inhibit IGFBP-5 expression at the
level of RNA transcription. Our studies revealed that changes in the
rate of IGFBP-5 transcription were delayed and somewhat less intense
than those in hnRNA. This could be due to technical differences.
However, since changes in hnRNA levels may be due to effects on
transcription or RNA processing, it is possible that the growth factors
studied had an early effect on RNA processing in addition to their
effects on IGFBP-5 transcription.
and the three growth factors studied
decreased IGFBP-5 in this compartment. PDGF BB and TGF
1 also
increased proteins migrating with a M
of
45,000-50,000. The nature of the protein reacting with the
IGFBP-5 antibody is presently unknown. However, this protein(s) does
not appear to be an IGFBP since it did not bind radiolabeled IGF II.
The amount of IGFBP-5 secreted to the culture medium of Ob cells under
the described culture conditions is small, and peptide degradation
occurs, so inhibitory changes are difficult to detect. Modifications in
IGFBP-5 protease levels or activity is another level of regulation by
which skeletal growth factors may modify IGFBP-5 polypeptide in bone
cells
(37, 38) . It was recently shown that matrix
metalloproteinase I or interstitial collagenase degrades IGFBP-5, and
bFGF, TGF
1, and PDGF BB regulate interstitial collagenase
synthesis in skeletal and nonskeletal cells
(38, 39) .
(
)
This would suggest
that the growth factors studied have the capability of regulating
IGFBP-5 by transcriptional and post-translational mechanisms.
1, and PDGF BB in bone cultures, their effects on IGFBP-5
synthesis were observed at doses that modify other parameters of
metabolic function in Ob cells and doses that are known to inhibit IGF
I and II synthesis in bone
(8, 9, 10) . This
suggests that the inhibition of IGFBP-5 synthesis may be
physiologically relevant. A decrease in IGFs I and II as well as
IGFBP-5 synthesis might mediate selected effects of growth factors on
bone cell function. Recently, it was shown that bFGF and PDGF BB
increase the synthesis of skeletal IGFBP-4, a binding protein with
inhibitory properties in bone
(5) . This effect as well as an
inhibition of IGFBP-5, IGF I, and IGF II synthesis may be relevant to
the actions of selected growth factors on bone cell function. On the
other hand, bFGF, TGF
1, and PDGF BB have complex effects in bone,
and it is likely that they have a number of actions on bone metabolism,
which are independent of their effects on the IGF-IGFBP axis
(8) .
1 have been shown to
inhibit the differentiated expression of the osteoblastic phenotype,
and they also inhibit IGFBP-5 synthesis
(8, 42, 43) . Furthermore, factors such as IGFs
I and II, which stimulate osteoblastic differentiated function,
increase IGFBP-5 synthesis and stability in bone cells
(17, 18, 30) . The stimulatory effects of
IGFBP-5 on bone cell function are unique since IGFBP-2, -3, and -4
inhibit various parameters of bone formation
(11, 12, 13) . IGFBP-1 plays a role in glucose
homeostasis, and it has not been reported to have a specific function
in skeletal metabolism
(44, 45) . IGFBP-6 inhibits IGF
II-induced differentiation of myoblasts, but its effects in bone cells
are not known
(46) .
1, and PDGF BB inhibit IGFBP-5
transcripts and polypeptide levels in skeletal cells through mechanisms
that may involve diminished transcription. The reduced level of IGFBP-5
by local growth factors in the bone microenvironment may constitute an
important level of control of the autocrine and paracrine actions of
IGF in bone via IGFBPs.
1, transforming factor
1;
PDGF, platelet-derived growth factor; DMEM, Dulbecco's modified
Eagle's medium; DRB, 5,6-dichlorobenzimidazole; hnRNA,
heterogeneous nuclear RNA; PCR, polymerase chain reaction.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.