Insulin-like growth factors sustain insulin-like growth
factor-binding protein-5 expression in osteoblasts
Bari
Gabbitas1 and
Ernesto
Canalis1,2
1 Departments of Research and
Medicine, Saint Francis Hospital and Medical Center, Hartford
06105; and 2 The University of
Connecticut School of Medicine, Farmington, Connecticut 06030
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ABSTRACT |
Insulin-like growth
factors (IGFs) I and II are considered to be autocrine regulators of
bone cell function. Recently, we demonstrated that IGF-I induces
IGF-binding protein-5 (IGFBP-5) expression in cultures of
osteoblast-enriched cells from 22-day fetal rat calvariae (Ob cells).
In the present study, we postulated that IGFs play an autocrine role in
the maintenance of IGFBP-5 basal expression in Ob cells. IGFBP-2 and
-3, at concentrations that bind endogenous IGFs, decreased IGFBP-5
mRNA levels, as determined by Northern blot analysis, and protein
levels, as determined by Western immunoblots of extracellular matrix
extracts of Ob cells. IGFBP-2 and -3 in excess inhibited IGFBP-5
heterogeneous nuclear RNA levels, as determined by RT-PCR, and did not
alter the half-life of IGFBP-5 mRNA in transcriptionally arrested Ob
cells. In conclusion, blocking endogenous IGFs in Ob cells represses
IGFBP-5 expression, suggesting that IGFs are autocrine inducers of
IGFBP-5 synthesis in osteoblasts.
skeletal tissue; extracellular matrix; bone formation; transcription
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INTRODUCTION |
INSULIN-LIKE GROWTH FACTORS (IGFs) I and II are among
the most prevalent growth factors secreted by skeletal cells and are considered autocrine regulators of osteoblastic cell function (6, 18,
34). IGF-I and IGF-II have mitogenic activity for bone cells and
enhance the differentiated function of the osteoblast (23, 33).
The circulating and locally synthesized IGF-I and IGF-II bind and
interact with IGF-binding proteins (IGFBPs) (41). Skeletal cells
synthesize six of the known IGFBPs, although the pattern of their
expression varies with the cell line studied and culture conditions
used (21, 31, 38). The exact function of the six IGFBPs secreted by
skeletal cells is not known, but selected binding proteins have unique
functional properties. For example, IGFBP-4 inhibits bone formation,
whereas IGFBP-5 can be either stimulatory, enhancing the effects of
IGF-I on bone cell growth, or inhibitory (1, 12, 30). The synthesis of
IGFBP-5 by the osteoblast is tightly controlled and is dependent on the
level of cell maturation (3). The expression of IGFBP-5 is modified by
locally produced growth factors, and changes in its synthesis frequently parallel changes in the synthesis of IGF-I and IGF-II in
bone (5, 14). For instance, transforming growth factor-
(TGF-
),
platelet-derived growth factor (PDGF) BB, and basic
fibroblast growth factor (FGF) inhibit IGFBP-5 as well as IGF-I and
IGF-II expression by the osteoblast (5, 8). In addition, IGF-I increases IGFBP-5 synthesis in osteoblast cultures, and IGFs stabilize the binding protein, indicating that they play a dual role in the
maintenance of appropriate levels of IGFBP-5 in the bone
microenvironment (11, 12, 14). Because IGFBP-5 can increase the effects
of IGF-I on bone formation, its enhanced synthesis by IGF-I could be
central to the regulation of this process and to the maintenance of
bone mass.
Because IGF-I and IGF-II are secreted by the osteoblast and IGF-I
increases IGFBP-5 levels in this cell, we postulated that IGF-I and
possibly IGF-II play an autocrine role in the maintenance of IGFBP-5
expression in normal osteoblasts. To test this hypothesis, we examined
the role of the locally produced IGFs in the synthesis of IGFBP-5 in
cultures of osteoblast-enriched cells from 22-day fetal rat calvariae
(Ob cells) and determined possible mechanisms involved.
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MATERIALS AND METHODS |
Cell cultures.
Primary cultures of Ob cells were prepared as previously described
(32). Parietal bones were obtained from 22-day-old fetal rats
immediately after the mothers were killed 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 by use of
bacterial collagenase (CLS II; Worthington Biochemical, Freehold, NJ).
Cell populations harvested from the third to the fifth digestion were
cultured as a pool and were previously shown to have osteoblastic
characteristics (32). Ob cells were plated at a density of
8,000-12,000 cells/cm2,
cultured in a humidified 5% CO2
incubator at 37°C, and grown until they reached confluence
(~50,000 cells/cm2). Cells
were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; both from Summit Biotechnologies, Fort Collins, CO). At
confluence the cultures were changed to serum-free conditions for
20-24 h; the medium was replaced with fresh DMEM, and cells were
exposed to test or control medium for 2-24 h. Recombinant human
IGF-I (Austral, San Ramon, CA) was dissolved in 20 mM sodium citrate
and diluted 1:1,000 in DMEM; recombinant human IGF-II (a gift from Eli
Lilly Research Laboratories, Indianapolis, IN) was dissolved in 0.1 M
acetic acid and diluted 1:100 in DMEM; recombinant human IGFBP-2 (a
gift from Sandoz Pharma, Basel, Switzerland) was dissolved in 0.05 or
0.5 M acetic acid; and recombinant human IGFBP-3 (a gift from Celtrix
Pharmaceuticals, Santa Clara, CA) was dissolved in 50 mM sodium acetate
and 100 mM NaCl and diluted 1:100 in DMEM in the absence of carrier
proteins. 5,6-Dichlorobenzimidazole riboside (DRB; Sigma Chemical, St.
Louis, MO) was dissolved in ethanol and diluted 1:200 in DMEM. Equal
amounts of the appropriate vehicle were added to control cultures.
IGF-I- neutralizing antibody Sm 1.2 (a gift of J. J. Van Wyk, Chapel
Hill, NC), control mouse IgG-containing ascites fluid (Sigma Chemical),
IGF-I receptor-neutralizing antibody (Oncogene Research Products,
Cambridge, MA), and control IgG1,
(Sigma Chemical) were added
directly to the culture medium. At the end of the incubation, RNA or
extracellular matrix was extracted from the cell layer and stored at
80°C for transcript and protein analysis, respectively.
Northern blot analysis.
Total cellular RNA was isolated with guanidine thiocyanate, at acid pH,
followed by a phenol-chloroform (Sigma Chemical) extraction and ethanol
precipitation or by RNeasy kit per manufacturer's instructions
(Qiagen, Chatsworth, CA) (10). RNA was quantitated by
spectrophotometry, and equal amounts of RNA from control or test
samples were loaded on a formaldehyde agarose gel after denaturation. The gel was stained with ethidium bromide to visualize ribosomal RNA,
confirming equal RNA loading of the various experimental samples. The
RNA was then blotted onto Gene Screen Plus charged nylon (Du Pont,
Wilmington, DE). A 330-bp Sac
II-Hind III restriction fragment of a
rat IGFBP-5 cDNA (provided by S. Shimasaki, La Jolla, CA) was labeled
with
[
-32P]deoxycytidine
triphosphate
([
-32P]dCTP) and
[
-32P]deoxyadenosine
triphosphate (specific activity of 3,000 Ci/mmol; Du Pont) by use of
the random hexanucleotide primed second strand synthesis method (15,
44). Hybridizations were carried out at 42°C for 16-72 h and
washed at 65°C in 0.5× saline sodium citrate. The blots were
stripped and rehybridized under the same conditions with an
-32P-labeled 800-bp restriction fragment of rat
glyceraldehyde-3-phosphate dehydrogenase (GAPD) cDNA (provided by R. Wu, Ithaca, NY) (46). The bound radioactive material was visualized by
autoradiography on Kodak X-AR5 film (Eastman Kodak, Rochester, NY)
employing Cronex Lighting plus intensifying screens (Du Pont). Relative
hybridization levels were determined by densitometry. Northern analyses
shown are representative of three or more cultures.
RT/PCR.
IGFBP-5 heterogeneous nuclear RNA (hnRNA) was analyzed by RT/PCR by use
of IGFBP-5 primers spanning the junction between intron 1 and exon 1 of
the IGFBP-5 gene, in accordance with published sequences (27, 44). As
previously reported, the sense exon 1-primer
5'-AAAGCTCTGTCCATGTGTC-3' and antisense intron 1-amplimer 5'-AAACCCCAGTAGCGCTCAC-3' were synthesized (20). RNA
samples, extracted as described for Northern analysis, were treated
with DNase I according to manufacturer's instructions (Life
Technologies, Grand Island, NY) to remove potentially contaminating
DNA. One microgram of RNA was copied into cDNA using the antisense
intron 1 primer and Moloney murine leukemia virus reverse transcriptase (Life Technologies) at 42°C for 30 min (4, 5, 25). An exogenous DNA
standard that can be coamplified using the IGFBP-5 hnRNA primer set was
synthesized as described and included in the PCR to correct for
variations in amplification (20). The newly transcribed cDNA was
amplified by 25 PCR cycles of 94°C for 1 min, 63°C
for 1 min, and 72°C for 1 min after the addition of the sense exon
1 primer, 10 fg of DNA standard, Taq
DNA polymerase (Life Technologies), and 5 µCi
[
-32P]dCTP (3,000 Ci/mmol; Du Pont) (4, 5, 25). The PCR products were fractionated by
electrophoresis on a 6% polyacrylamide denaturing gel, visualized by
autoradiography, and quantitated by densitometry (29). The PCR product
increased linearly with increasing amounts of RNA. To determine the
variability of the procedure, Ob cell RNA was pooled, and independent
aliquots were reverse transcribed, amplified by PCR and IGFBP-5 hnRNA,
and quantitated by densitometry. The coefficient of
variation was 11% (n = 13) for the
assay. Data on hnRNA are representative of three or more cultures.
Western immunoblot analysis.
Extracellular matrix was prepared as described (5, 24, 26). Briefly, Ob
cells were rinsed with PBS, cell membranes were removed with 0.5%
Triton X-100 (Sigma Chemical), 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 PBS and extracted with Laemmli
sample buffer containing 2% SDS, and aliquots were fractionated by
polyacrylamide gel electrophoresis on a 12% gel (29). Proteins were
transferred to Immobilon P membranes (Millipore, Bedford, MA), blocked
with a 2% BSA, and exposed to a 1:2,000 dilution of rabbit antiserum
raised against native human IGFBP-5 (UBI, Lake Placid, NY) in 1% BSA
overnight. Blots were exposed to goat anti-rabbit IgG antiserum
conjugated to horseradish peroxidase, washed, and developed with a
horseradish peroxidase chemiluminescence detection reagent (Du Pont).
IGFBP-5 was identified by comigration with recombinant purified human IGFBP-5 (UBI).
Statistical methods.
Data are expressed as means ± SE. Statistical differences were
analyzed by analysis of variance and post hoc analysis by Dunnett's or
Ryan-Einot-Gabriel-Welch F tests using
a Crunch Statistical Package (Crunch Software, Oakland, CA). Slopes
from linear regression were analyzed by the method of Sokal and Rohlf
(45).
 |
RESULTS |
In confirmation of prior observations, IGF-I at 100 nM increased
IGFBP-5 mRNA levels, and IGF-II at 100 nM had a comparable effect (Fig.
1) (14). The effect appeared after 2 h and
was sustained for 24 h. After 6 h, IGF-I and IGF-II increased IGFBP-5 transcripts by 1.8 ± 0.2-fold (mean ± SE) (n = 8) and 2.1 ± 0.3-fold (n = 3), respectively (both
P < 0.05). To determine whether
the autocrine production of IGFs was responsible for the
maintenance of IGFBP-5 mRNA levels, we tested the effects of IGF-I and
IGF-I receptor-neutralizing antibodies and of IGFBP-2 and
IGFBP-3 in excess. Although the IGF-I-neutralizing antibody Sm 1.2 has
been shown to block IGF-I effects in bone at a 1:1,000 dilution, when tested at concentrations of 1:500-1:2,000, it had either no effect or modest intrinsic stimulatory activity for IGFBP-5 mRNA levels compared with DMEM and control IgG, which had no effect (13). This
would suggest that Sm 1.2 might contain IGFBP-5 stimulatory factors,
which are absent in the control IgG; that IGFs did not act as autocrine
upregulators of IGFBP-5; or that IGF-II was not sufficiently blocked by
the Sm 1.2 antibody and was responsible for the maintenance of IGFBP-5
transcript levels. The neutralizing antibody to the human IGF-I
receptor (
IR3) has been shown to block IGF-I effects in human cells,
but its ability to neutralize IGF-I in rodent cell systems is
controversial (28, 42). In preliminary experiments,
IR3 at 10 µg/ml did not block the known stimulatory effects of IGF-I on type I
collagen transcripts in Ob cells (not shown). Therefore, it was not
used to block the autocrine function of IGF-I. IGFBP-2 and IGFBP-3 were
then tested at concentrations known to complex endogenous levels of
IGF-I and IGF-II in osteoblasts and to block IGF effects in bone
cultures (2, 13, 16, 40, 41). Confirming these observations, IGFBP-3
prevented the stimulatory effect of IGF-I on IGFBP-5 mRNA levels.
Treatment with IGF-I at 100 nM for 6 h increased IGFBP-5 transcripts
(n = 3) by 2.1 ± 0.3-fold
(P < 0.05), and IGFBP-3 at 300 nM decreased this effect to 1.0 ± 0.1-fold of control untreated cultures (Fig. 2). Treatment with IGFBP-2
or IGFBP-3 at 1 µM for 2 or 6 h did not significantly modify IGFBP-5
mRNA levels compared with control cultures. However, after 24 h,
IGFBP-2 and IGFBP-3 decreased the level of IGFBP-5 transcript
expression from 100%, in control cultures, to (means ± SE) 50 ± 6% (n = 8) and 50 ± 11%
(n = 5), respectively (both
P < 0.05; Fig.
3, A and
B). The effect of IGFBP-2 and
IGFBP-3 on IGFBP-5 mRNA levels was dose dependent. IGFBP-2 at 100 nM
and 1 µM decreased the expression of IGFBP-5 from 100% to 57 ± 6%
and 35 ± 5% (n = 3; both
P < 0.05) after 24 h; at the same
concentrations, IGFBP-3 decreased IGFBP-5 mRNA to 73 ± 5%
(P > 0.05) and 34 ± 10%
(P < 0.05; both
n = 3; Fig.
4).

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Fig. 1.
Effect of insulin-like growth factor (IGF) I and IGF-II at 100 nM on
IGF-binding protein (IGFBP)-5 mRNA expression in confluent cultures of
osteoblast-enriched cells from 22-day fetal rat calvariae (Ob cells)
treated for 2, 6, or 24 h. Total RNA from control (C) or IGF-treated
cultures was subjected to Northern blot analysis and hybridized with a
32P-labeled IGFBP-5 cDNA. Blot was
stripped and hybridized with a
32P-labeled
glyceraldehyde-3-phosphate dehydrogenase (GAPD) cDNA to demonstrate
equal RNA loading of gel. IGFBP-5 mRNA was visualized by
autoradiography and is shown in top panel; GAPD mRNA is shown below. I,
IGF-I; II, IGF-II.
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Fig. 2.
Effect of IGF-I at 100 nM, in the presence and absence of IGF-binding
protein (IGFBP)-3 at 300 nM, on IGFBP-5 mRNA expression in confluent
cultures of Ob cells treated for 6 h. Total RNA from control (C) and
IGF-I (I)- and IGFBP-3 (BP-3)-treated cultures was subjected to
Northern blot analysis and hybridized with a
32P-labeled IGFBP-5 cDNA. Blots
were stripped and hybridized with a
32P-labeled GAPD cDNA to
demonstrate equal RNA loading of gel. IGFBP-5 mRNA was visualized by
autoradiography and is shown in top panel; GAPD mRNA is shown below.
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Fig. 3.
Effect of IGFBP-2 and IGFBP-3, both at 1 µM, on IGFBP-5 mRNA
expression in confluent cultures of Ob cells treated for 2, 6, or 24 h.
Total RNA from control (C) or IGFBP-2 (BP-2)-treated cultures
(A) and from control (C) or IGFBP-3
(BP-3)-treated cultures (B) was
subjected to Northern blot analysis and hybridized with a
32P-labeled IGFBP-5 cDNA. Blots
were stripped and hybridized with a
32P-labeled GAPD cDNA to
demonstrate equal RNA loading of gel. IGFBP-5 mRNA was visualized by
autoradiography and is shown in top panel; GAPD mRNA is shown below.
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Fig. 4.
Effect of IGFBP-2 and IGFBP-3, both at 0.01-1 µM, on IGFBP-5
mRNA expression in confluent cultures of Ob cells treated for 24 h.
Total RNA from control and IGFBP-2 (BP-2)- or IGFBP-3 (BP-3)-treated
cultures was subjected to Northern blot analysis and hybridized with an
-32P-labeled IGFBP-5 cDNA.
Blots were stripped and hybridized with an
-32P-labeled GAPD cDNA to
demonstrate equal RNA loading of gel. IGFBP-5 mRNA was visualized by
autoradiography and is shown in top panel; GAPD mRNA is shown below.
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Western immunoblot analysis of extracellular matrix extracts from Ob
cells confirmed the presence of an immunoreactive protein with a
molecular mass of 31 kDa comigrating with a human IGFBP-5 standard
(Fig. 5) (5). The Western immunoblot
revealed immunoreactive protein bands of higher molecular mass. When
Western blots were stripped and incubated with
125I-labeled IGF-II, the 31-kDa
protein was visualized, whereas the higher molecular mass proteins were
not, indicating that they were not IGFBPs, but uncharacterized
cross-reacting proteins (5). Treatment with IGFBP-2 or IGFBP-3 at 1 µM for 24 h decreased the accumulation of immunoreactive IGFBP-5 by
50-100% (Fig. 5).

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Fig. 5.
Effect of IGFBP-2 and IGFBP-3, both at 1 µM, on immunoreactive
IGFBP-5 accumulation in the extracellular matrix of Ob cells treated
for 24 h. Extracts from extracellular matrix of control (C) and IGFBP-2
(BP-2)- and IGFBP-3 (BP-3)-treated cultures were subjected to Western
immunoblot analysis. IGFBP-5 (BP-5) was detected using an anti-IGFBP-5
antibody and a chemiluminescence detection system, and its presence was
confirmed by comigration with a human recombinant IGFBP-5 standard
(STD). Migration of molecular mass markers is indicated in kDa on left
margin.
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To determine whether or not IGFBP-2 and IGFBP-3 in excess acted by
transcriptional or posttranscriptional mechanisms, we examined their
effects on IGFBP-5 hnRNA levels and transcript stability. To determine changes in transcript stability, confluent cultures of Ob cells were exposed to DMEM or to IGFBP-2 or IGFBP-3, both at 1 µM, for 60 min and then treated with the RNA polymerase-II inhibitor
DRB in the absence or presence of binding proteins for 2-24 h. The
half-life of IGFBP-5 RNA in transcriptionally arrested Ob cells was
estimated to be 12 h, and it was not changed by treatment with either
IGFBP-2 or IGFBP-3 (Fig. 6,
A and
B). Similar to the changes observed
in IGFBP-5 mRNA levels, IGFBP-2 and IGFBP-3 at 1 µM decreased IGFBP-5
hnRNA levels after 6 and 24 h, suggesting a change in RNA transcription
or processing (Fig. 7,
A and
B). After 6 h, IGFBP-2 and IGFBP-3
decreased IGFBP-5 hnRNA levels from 100%, in control cultures, to 27 ± 6% and 35 ± 10% (n = 5 to 6;
both P < 0.05); after 24 h, IGFBP-2
and IGFBP-3 decreased IGFBP-5 hnRNA levels to 13 ± 6% and 26 ± 9%
(n = 5 to 6; both P < 0.05). The signal of the
internal standard was comparable in samples from control and treated
cells, indicating uniform reaction efficiency. Furthermore, no signal
of the hnRNA product was detected in any of the samples tested when the
RT step was omitted before the PCR, eliminating the possibility of DNA
contamination.

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Fig. 6.
Effect of IGFBP-2 and IGFBP-3, both at 1 µM, on IGFBP-5 mRNA decay in
transcriptionally arrested Ob cells. Cultures were treated with control
( ) or with IGFBP-2 (A, ) or
IGFBP-3 (B, ) 1 h before and
6-24 h after addition of 5,6-dichlorobenzimidazole riboside (DRB).
RNA was subjected to Northern blot analysis and hybridized with an
-32P-labeled rat IGFBP-5 cDNA,
visualized by autoradiography, and quantitated by densitometry.
Ethidium bromide staining of ribosomal RNA was used to check uniform
loading of gels and transfer. Values are means ± SE for 3 cultures. Values were obtained by densitometric scanning and are
presented as a percentage of IGFBP-5 mRNA levels relative to time of
DRB addition. Slopes of linear regression plots were analyzed by method
of Sokal and Rohlf (45) and were not found to be statistically
different.
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Fig. 7.
Effect of IGFBP-2 and IGFBP-3, both at 1 µM, on IGFBP-5 heterogeneous
nuclear RNA (hnRNA) expression in cultures of Ob cells treated for 2, 6, or 24 h. Total RNA from control (C) or IGFBP-2 (BP-2)-treated
cultures (A) and from control (C) or
IGFBP-3 (BP-3)-treated cultures (B)
was extracted, and 1 µg was subjected to RT-PCR in the presence of
IGFBP-5 intron 1 and exon 1 primers and
[ -32P]dCTP. RT-PCR
products were fractionated by PAGE and visualized by autoradiography.
For both A and
B, internal standard is at top and
IGFBP-5 hnRNA is below.
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DISCUSSION |
Recent studies revealed that IGF-I induces the synthesis of IGFBP-5 in
rat Ob cells, acting by transcriptional mechanisms (14). The present
investigation was undertaken to determine whether IGFs play an
autocrine role in the maintenance of IGFBP-5 expression. We
demonstrated that IGFBP-2 and IGFBP-3 in excess cause a decrease in
IGFBP-5 mRNA, hnRNA, and protein levels. IGFBP-2 and IGFBP-3 were
tested at concentrations that are ~1,000 times higher than those
secreted by skeletal cells and doses that are known to complex IGF-I
and IGF-II and to prevent their biological effects in bone cell
cultures (2, 13, 16, 40, 41). Sm 1.2 neutralizing antibodies to IGF-I
had no effect or modest intrinsic stimulatory activity and did not
block IGF effects on IGFBP-5 transcripts, and, as frequently is the
case in rodent cell systems, antibodies to the human IGF-I receptor
were not effective. This could suggest that IGFs do not act as
autocrine regulators of IGFBP-5 expression. Alternatively, the lack of
an effect of the neutralizing Sm 1.2 antibody on IGFBP-5 mRNA levels could be due to intrinsic stimulatory activity absent in control IgG or
to limited neutralization of IGF-II, which could maintain IGFBP-5 mRNA
levels. Because the two binding proteins used bind IGF-I and IGF-II
with similar affinity, these results suggest that IGFs act as autocrine
downregulators of IGFBP-5 expression, and it is not possible to define
whether IGF-I or IGF-II was responsible for the effects observed. The
two forms of IGFs have similar effects in bone cell function, and this
should not alter the interpretation of the results obtained (33).
IGF-I increases IGFBP-5 mRNA and hnRNA levels and the rates of IGFBP-5
transcription in Ob cells without altering the stability of IGFBP-5
mRNA in transcriptionally arrested Ob cells (14). The current studies
confirm transcriptional regulation of IGFBP-5 by the endogenously
secreted IGF-I and IGF-II, because IGFBP-2 and IGFBP-3 in excess caused
a decrease in IGFBP-5 hnRNA levels without altering the half-life of
IGFBP-5 mRNA in transcriptionally arrested Ob cells. Although
osteoblasts synthesize and secrete IGFBP-2 and IGFBP-3, the amount of
IGFBPs secreted to the culture medium is limited, and, at these low
concentrations, the binding proteins do not block the response of Ob
cells to IGF-I and IGF-II (2, 16, 35, 40). This is supported by the
finding that the response of Ob cell cultures to IGF-I and des-IGF is
almost identical, although des-IGF-I has less affinity
for IGFBPs than intact IGF-I (7). However, addition of
exogenous IGFBP-2 or IGFBP-3 at the concentrations used in this
study would complex and neutralize the endogenously synthesized IGF-I
and IGF-II. IGFBP-3 has direct effects on cell function, independent of
its actions through IGFs, and binds to putative cell receptors (37, 47,
48). Nevertheless, its effects on IGFBP-5 synthesis are the same as
those observed with IGFBP-2, suggesting that they are not independent
actions of IGFBP-3 but are secondary to the trapping of endogenous
IGFs. Furthermore, IGFBP-3 was active only at concentrations that are
100-1,000-fold higher than those naturally occurring in the
culture medium of osteoblastic cells, indicating a pharmacological and
not a physiological effect (35).
Osteoblasts secrete a number of growth factors and cytokines that
regulate IGFBP-5 gene expression. In contrast to the stimulatory effects of IGF-I and prostaglandin
E2,
TGF-
1, PDGF-BB, and basic FGF
inhibit IGFBP-5 mRNA and protein levels (5, 14, 39). Therefore, the
amount of IGFBP-5 mRNA and protein synthesized by osteoblasts is not
only dependent on the levels of IGF-I and -II in the bone
microenvironment but probably reflects the sum of inhibitory and
stimulatory influences on IGFBP-5 gene expression. The effects of IGF-I
and IGF-II in bone are, to an extent, unique and different from those
of other growth factors. IGFs have modest mitogenic activity and
increase the differentiated function of the osteoblast and increase
IGFBP-5 expression (23, 33). In contrast, growth factors with potent
mitogenic activity for bone cells have modest effects on the
differentiated function of the osteoblast and inhibit IGF-I, IGF-II,
and IGFBP-5 synthesis (5, 8, 9, 19, 22).
IGF-I is an autocrine inhibitor of matrix metalloproteinase (MMP)-13
expression in osteoblasts, and MMPs, as well as serine proteases, are
known to degrade IGFBP-5 (13, 17, 36). An increase in MMP-13 after the
neutralization of endogenous IGF-I might affect the degradation of
IGFBP-5 with a consequent decrease in protein levels. It is possible
that other proteases are involved in the regulation of IGFBP-5 by IGF-I
in the bone microenvironment and have significant effects on the
IGF/IGFBP axis.
In conclusion, the present studies suggest that IGFs play an autocrine
role in the maintenance of IGFBP-5 transcription in skeletal cells.
Because IGFBP-5 stimulates bone cell growth and can increase the
effects of IGF-I, this may be a mechanism to enhance the action of
skeletal IGFs on bone formation.
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ACKNOWLEDGEMENTS |
The authors thank Dr. S. Shimasaki for the rat IGFBP-5 cDNA clone,
Dr. R. Wu for the rat GAPD cDNA clone, Eli Lilly Research Laboratories
for the gift of IGF-II, Sandoz Pharma LTD for the gift of IGFBP-2,
Celtrix Pharmaceuticals for the gift of IGFBP-3, and Dr. J. J. Van
Wyk for the gift of IGF-I-neutralizing antibody. The authors thank
Cathy Boucher, Deena Durant, and Susan O'Lone for technical assistance
and Margaret Nagle for secretarial help.
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FOOTNOTES |
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-42424.
Address for reprint requests: E. Canalis, Dept. of Research, Saint
Francis Hospital and Medical Center, 114 Woodland St., Hartford, CT
06105-1299.
Received 10 October 1997; accepted in final form 22 April 1998.
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