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

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-beta (TGF-beta ), 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.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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,kappa (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 [alpha -32P]deoxycytidine triphosphate ([alpha -32P]dCTP) and [alpha -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 alpha -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 [alpha -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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 (alpha 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, alpha 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).


View larger version (53K):
[in this window]
[in a new window]
 
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.


View larger version (90K):
[in this window]
[in a new window]
 
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.


View larger version (76K):
[in this window]
[in a new window]
 


View larger version (84K):
[in this window]
[in a new window]
 
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.


View larger version (68K):
[in this window]
[in a new window]
 
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 alpha -32P-labeled IGFBP-5 cDNA. Blots were stripped and hybridized with an alpha -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.

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).


View larger version (84K):
[in this window]
[in a new window]
 
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.

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.


View larger version (16K):
[in this window]
[in a new window]
 


View larger version (14K):
[in this window]
[in a new window]
 
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 (bullet ) or with IGFBP-2 (A, open circle ) or IGFBP-3 (B, open circle ) 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 alpha -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.


View larger version (34K):
[in this window]
[in a new window]
 


View larger version (27K):
[in this window]
[in a new window]
 
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 [alpha -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.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-beta 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.

    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.

    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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Andress, D. L., and R. S. Birnbaum. Human osteoblast-derived insulin-like growth factor (IGF) binding protein-5 stimulates osteoblast mitogenesis and potentiates IGF action. J. Biol. Chem. 267: 22467-22472, 1992[Abstract/Free Full Text].

2.   Bach, L. A., S. Hsieh, K. Sakano, H. Fujiwara, J. F. Perdue, and M. M. Rechler. Binding of mutants of human insulin-like growth factor II to insulin-like growth factor binding proteins 1-6. J. Biol. Chem. 268: 9246-9254, 1993[Abstract/Free Full Text].

3.   Birnbaum, R. S., and K. M. Wiren. Changes in insulin-like growth factor-binding protein expression and secretion during the proliferation, differentiation, and mineralization of primary cultures of rat osteoblasts. Endocrinology 135: 223-230, 1994[Abstract].

4.   Buttice, G., and M. Kurkinen. A polyomavirus enhancer A-binding protein-3 site and Ets-2 protein have a major role in the 12-O-tetradecanoylphorbol-13-acetate response of the human stromelysin gene. J. Biol. Chem. 268: 7196-7204, 1993[Abstract/Free Full Text].

5.   Canalis, E., and B. Gabbitas. Skeletal growth factors regulate the synthesis of insulin-like growth factor binding protein-5 in bone cell cultures. J. Biol. Chem. 270: 10771-10776, 1995[Abstract/Free Full Text].

6.   Canalis, E., T. McCarthy, and M. Centrella. Isolation and characterization of insulin-like growth factor I (somatomedin-C) from cultures of fetal rat calvariae. Endocrinology 122: 22-27, 1988[Abstract].

7.   Canalis, E., T. L. McCarthy, and M. Centrella. Effects of desamino (1-3) insulin-like growth factor I on bone cell function in rat calvarial cultures. Endocrinology 129: 534-541, 1991[Abstract].

8.   Canalis, E., J. Pash, B. Gabbitas, S. Rydziel, and S. Varghese. Growth factors regulate the synthesis of insulin like growth factor I in bone cell cultures. Endocrinology 133: 33-38, 1993[Abstract].

9.   Canalis, E., J. Pash, and S. Varghese. Skeletal growth factors. Crit. Rev. Eukaryot. Gene Expr. 3: 155-166, 1993[Medline].

10.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

11.   Conover, C. A., L. K. Bale, J. T. Clarkson, and O. Torring. Regulation of insulin-like growth factor binding protein-5 messenger ribonucleic acid expression and protein availability in rat osteoblast-like cells. Endocrinology 132: 2525-2530, 1993[Abstract].

12.   Conover, C. A., and M. C. Kiefer. Regulation and biological effect of endogenous insulin-like growth factor binding protein-5 in human osteoblastic cells. J. Clin. Endocrinol. Metab. 76: 1153-1159, 1993[Abstract].

13.   Delany, A. M., S. Rydziel, and E. Canalis. Autocrine down regulation of collagenase-3 in rat bone cell cultures by insulin-like growth factors. Endocrinology 137: 4665-4670, 1996[Abstract].

14.   Dong, Y., and E. Canalis. Insulin-like growth factor I and retinoic acid induce the synthesis of insulin-like growth factor binding protein-5 in rat osteoblastic cells. Endocrinology 136: 2000-2006, 1995[Abstract].

15.   Feinberg, A. P., and B. Vogelstein. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137: 266-267, 1984[Medline].

16.   Feyen, J. H., D. B. Evans, C. Binkert, G. F. Heinrich, S. Geisse, and H. P. Kocher. Recombinant human (Cys281) insulin-like growth factor-binding protein 2 inhibits both basal and insulin-like growth factor I-stimulated proliferation and collagen synthesis in fetal rat calvariae. J. Biol. Chem. 266: 19469-19474, 1991[Abstract/Free Full Text].

17.   Fowlkes, J. L., K. M. Thrailkill, D. M. Serra, K. Suzuki, and H. Nagase. Matrix metalloproteinases as insulin-like growth factor binding protein-degrading proteinases. Prog. Growth Factor Res. 6: 255-263, 1995[Medline].

18.   Frolik, C. A., L. F. Ellis, and D. C. Williams. Isolation and characterization of insulin-like growth factor-II from human bone. Biochem. Biophys. Res. Commun. 15: 1011-1018, 1988.

19.   Gabbitas, B., J. M. Pash, and E. Canalis. Regulation of insulin-like growth factor II synthesis in bone cell cultures by skeletal growth factors. Endocrinology 135: 284-289, 1994[Abstract].

20.   Gabbitas, B., J. M. Pash, A. M. Delany, and E. Canalis. Cortisol inhibits the synthesis of insulin-like growth factor binding protein-5 in bone cell cultures by transcriptional mechanisms. J. Biol. Chem. 271: 9033-9038, 1996[Abstract/Free Full Text].

21.   Hassager, C., L. A. Fitzpatrick, E. M. Spencer, B. L. Riggs, and C. A. Conover. Basal and regulated secretion of insulin-like growth factor binding proteins in osteoblast-like cells is cell line specific. J. Clin. Endocrinol. Metab. 75: 228-233, 1992[Abstract].

22.   Hock, J. M., and E. Canalis. Platelet-derived growth factor enhances bone cell replication but not differentiated function of osteoblasts. Endocrinology 134: 1423-1428, 1994[Abstract].

23.   Hock, J. M., M. Centrella, and E. Canalis. Insulin-like growth factor I (IGF-I) has independent effects on bone matrix formation and cell replication. Endocrinology 122: 254-260, 1988[Abstract].

24.   Jones, J. J., A. Gockerman, W. H. Busby, Jr., C. Camacho-Hubner, and D. R. Clemmons. Extracellular matrix contains insulin-like growth factor binding protein-5: potentiation of the effects of IGF-I. J. Cell Biol. 121: 679-687, 1993[Abstract].

25.   Kawasaki, E. S. Amplification of RNA. In: PCR Protocols: A Guide to the Methods and Applications, edited by M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White. San Diego: Academic, 1990, p. 21-27.

26.   Knudsen, B. S., P. C. Harpel, and R. L. Nachman. Plasminogen activator inhibitor is associated with the extracellular matrix of cultured bovine smooth muscle cells. J. Clin. Invest. 80: 1082-1089, 1987[Medline].

27.   Kou, K., D. Mittanck, C. Fu, and P. Rotwein. Structure and function of the mouse insulin-like growth factor binding protein-5 gene promoter. DNA Cell. Biol. 14: 241-249, 1995[Medline].

28.   Kull, F. C., S. Jacobs, Y. F. Su, M. E. Svobuda, J. J. Van Wyk, and P. Cuatrecasas. Monoclonal antibodies to receptor for insulin and somatomedin. J. Biol. Chem. 258: 6561-6566, 1983[Abstract/Free Full Text].

29.   Laemmli, U. K. Cleavage of structural protein during the assembly of the head bacteriophage T4. Nature 277: 680-685, 1970.

30.   LaTour, D., S. Mohan, T. A. Linkhart, D. J. Baylink, and D. D. Strong. Inhibitory insulin-like growth factor-binding protein: cloning, complete sequence, and physiological regulation. Mol. Endocrinol. 4: 1806-1814, 1990[Abstract].

31.   McCarthy, T., S. Casinghino, M. Centrella, and E. Canalis. Complex pattern of insulin-like growth factor binding protein expression in primary rat osteoblast enriched cultures: regulation by prostaglandin E2, growth hormone, and the insulin-like growth factors. J. Cell. Physiol. 160: 163-175, 1994[Medline].

32.   McCarthy, T. L., M. Centrella, and E. Canalis. Further biochemical and molecular characterization of primary rat parietal bone cell cultures. J. Bone Miner. Res. 3: 401-408, 1988[Medline].

33.   McCarthy, T. L., M. Centrella, and E. Canalis. Regulatory effects of insulin-like growth factor I and II on bone collagen synthesis in rat calvarial cultures. Endocrinology 124: 301-309, 1989[Abstract].

34.   Mohan, S., J. C. Jennings, T. A. Linkhart, and D. J. Baylink. Primary structure of human skeletal growth factor: homology with human insulin-like growth factor II. Biochim. Biophys. Acta 966: 44-45, 1988[Medline].

35.   Moriwake, T., H. Tanaka, S. Kanzaki, J. Higuchi, and Y. Seino. 1,25-Dihydroxyvitamin D3 stimulates the secretion of insulin-like growth factor binding protein 3 (IGFBP-3) by cultured human osteosarcoma cells. Endocrinology 130: 1071-1073, 1992[Abstract].

36.   Nam, T. J., W. H. Busby, Jr., and D. R. Clemmons. Characterization and determination of the relative abundance of two types of insulin-like growth factor binding protein-5 proteases that are secreted by human fibroblasts. Endocrinology 137: 5530-5536, 1996[Abstract].

37.   Oh, Y., H. L. Müller, G. Lamson, and R. G. Rosenfeld. Insulin-like growth factor (IGF)-independent action of IGF-binding protein-3 in Hs578T human breast cancer cells. Cell surface binding and growth inhibition. J. Biol. Chem. 268: 14964-14971, 1993[Abstract/Free Full Text].

38.   Okazaki, R., B. L. Riggs, and C. A. Conover. Glucocorticoid regulation of insulin-like growth factor binding protein expression in normal human osteoblast-like cells. Endocrinology 134: 126-132, 1994[Abstract].

39.   Pash, J. M., and E. Canalis. Transcriptional regulation of insulin-like growth factor-binding protein-5 by prostaglandin E2 in osteoblast cells. Endocrinology 137: 2375-2382, 1996[Abstract].

40.   Raisz, G., M. Fall, B. Y. Gabbitas, T. L. McCarthy, B. E. Kream, and E. Canalis. Effects of prostaglandin E2 on bone formation in cultured fetal rat calvariae; role of insulin-like growth factor I. Endocrinology 133: 1504-1510, 1993[Abstract].

41.   Rechler, M. M. Insulin-like growth factor binding proteins. Vitam. Horm. 47: 1-114, 1993[Medline].

42.   Rohlik, Q. T., D. Adams, E. C. Kull, Jr., and S. Jacobs. An antibody to the receptor for insulin-like growth factor I inhibits the growth of MCF-7 in cells in tissue culture. Biochem. Biophys. Res. Commun. 149: 276-281, 1987[Medline].

43.   Russell, W. E., J. J. VanWyk, and W. J. Pledger. Inhibition of the mitogenic effects of plasma by a monoclonal antibody to somatomedin C. Proc. Natl. Acad. Sci. USA 81: 2389-2392, 1984[Abstract].

44.   Shimasaki, S., M. Shimonaka, H. P. Zhang, and N. Ling. Identification of five different insulin-like growth factor binding proteins (IGFBPs) from adult rat serum and molecular cloning of a novel IGFBP-5 in rat and human. J. Biol. Chem. 266: 10646-10653, 1991[Abstract/Free Full Text].

45.   Sokal, R. R., and F. J. Rohlf. Biometry (2nd ed.). San Francisco: Freeman, 1981.

46.   Tso, J. Y., X. H. Sun, T.-H. Kao, K. S. Reece, and R. Wu. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res. 13: 2485-2502, 1985[Abstract].

47.   Valentinis, B., A. Bhala, T. DeAngelis, R. Baserga, and P. Cohen. The human insulin-like growth factor binding protein-3 inhibits the growth of fibroblasts with a targeted disruption of the IGF-I receptor gene. Mol. Endocrinol. 9: 361-367, 1995[Abstract].

48.   Zadeh, S. M., and M. Binoux. The 16-kDa proteolytic fragment of insulin-like growth factor (IGF) binding protein-3 inhibits the mitogenic action of fibroblast growth factor on mouse fibroblasts with a targeted disruption of the type 1 IGF receptor gene. Endocrinology 138: 3069-3072, 1997[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 275(2):E222-E228
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society