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*Bone Cancer
Endocrinology Vol. 138, No. 10 4240-4247
Copyright © 1997 by The Endocrine Society

ARTICLES

Osteogenic Protein-1 Stimulates Production of Insulin-Like Growth Factor Binding Protein-3 Nuclear Transcripts in Human Osteosarcoma Cells1

J. M. Hayden, D. D. Strong, D. J. Baylink, D. R. Powell, T. K. Sampath and S. Mohan

Departments of Biochemistry (D.D.S., D.J.B., S.M.), Medicine (J.M.H., D.D.S., D.J.B, S.M.), Microbiology and Molecular Genetics (D.D.S.), and Physiology (S.M.), Loma Linda University and Mineral Metabolism Unit (J.M.H., D.D.S., D.J.B., S.M.), Jerry L. Pettis Veterans Administration Medical Center, Loma Linda California 92357; Baylor College of Medicine (D.R.P.), Houston, Texas 77054; and Creative Biomolecules (T.K.S.), Hopkinton, Massachusetts 01748

Address all correspondence and requests for reprints to: Subburaman Mohan, Ph.D., Mineral Metabolism (151), Pettis VA Medical Center, 11201 Benton Street, Loma Linda, California 92357.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To begin delineating molecular mechanisms by which osteogenic protein-1 (OP-1) modulates its effect on the insulin-like growth factor (IGF) system in human skeletal cells, we evaluated time-course effects of OP-1 on the expression of IGFBP-3 messenger RNA (mRNA) in human SaOS-2 osteosarcoma cells and found that 100 ng/ml of OP-1 increased (maximum 10.7-fold at 24 h; P < 0.01) the level of IGFBP-3 mRNA in a time-dependent manner (from 3–36 h; treatment x time interaction, P < 0.001). The stimulatory effect of OP-1 on IGFBP-3 mRNA was not promoted by transcript stabilization; actually, OP-1 treatment selectively increased the decay of mRNA for IGFBP-3 (T1/2 = 5 h vs. 24 h for OP-1 and controls), but not for IGFBP-4 or ß-actin. Conversely, OP-1 acutely increased IGFBP-3 nuclear transcript abundance in total RNA samples ranging between 1–24 h of treatment. After 6 h of treatment, OP-1 produced an average 4-fold increase (P < 0.02; n = 4 experiments) in the level of IGFBP-3 nuclear transcripts vs. a 3-fold increase (P < 0.01; n = 2 experiments) in mRNA abundance. The OP-1 stimulated induction of IGFBP-3 nuclear transcript and mRNA expression was dependent on de novo protein synthesis. Transient transfection experiments were undertaken to isolate putative OP-1 stimulatory cis-elements within 1.8-kb of the IGFBP-3 5'-flanking region in SaOS-2 and TE-85 osteosarcoma cells. In these experiments, OP-1 did not stimulate IGFBP-3 proximal promoter activity in either cell line, thus suggesting that OP-1 reactive domains may be located either beyond the currently established 5'-flanking region, or within internal exon/intron regions of the IGFBP-3 gene. In conclusion, OP-1 treatment stimulates IGFBP-3 expression in human osteoblastic cells by a mechanism that largely promotes the production of IGFBP-3 nuclear transcripts, a process that requires de novo protein synthesis, and overrides an OP-1-induced targeted degradation of IGFBP-3 steady-state mRNA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE BONE morphogenetic proteins (BMPs) represent a unique set of growth factors because they are the only proteins that can stimulate mesenchymal stem cells to differentiate into osteoblasts and subsequently induce the newly formed osteoblasts to deposit bone matrix and promote new bone formation (1, 2, 3, 4). In studies examining the mechanisms by which BMPs increase human bone cell proliferation (5), we and others (6, 7, 8) have made an interesting discovery that BMPs modulate many components of the insulin-like growth factor (IGF) system, a key regulatory growth factor system in bone (9, 10, 11, 12). Previously, we reported that BMP-7 (also osteogenic protein-1; OP-1) promotes production of the stimulatory components of the IGF system such as IGF-II, IGF binding protein (BP)-3 and IGFBP-5, while decreasing the production of inhibitory-acting IGFBP-4 in human osteosarcoma cells (for the role of IGFBP control on IGF action in human bone cells, see 13 . Overall, the positive modulation of these components by OP-1 may enhance IGF availability, and subsequently promote IGF bioactivity in human bone cells. Of the aforementioned IGF system components, IGFBP-3 production exhibits the greatest induction (maximum ~10-fold) by OP-1 treatment in human osteosarcoma cells (6).

To date, the molecular mechanisms by which OP-1 regulates gene expression and subsequent cellular function in human osteoblasts remain undefined. Based on the relatively simple structure of the IGFBP-3 gene (e.g. a single transcription start site, 14 , and based on the large magnitude by which OP-stimulates IGFBP-3 messenger RNA (mRNA) expression, the IGFBP-3 gene provides for an excellent model to characterize mechanisms by which BMPs regulate nuclear transcript production. The objectives of this present study were to examine whether OP-1 promotes an increase in IGFBP-3 steady-state mRNA expression via a mechanism that involves the stimulation of IGFBP-3 nuclear transcript production, modulation of IGFBP-3 mRNA stability and to examine whether putative OP-1 responsive cis-elements reside within the currently established IGFBP-3 promoter domain.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and cell culture
A subpopulation of low alkaline phosphatase producing (L)-SaOS-2 cells (ATCC H7B85) and TE-85 osteosarcoma (provided by J. Fogh, Sloan Kettering Institute for Cancer Research) cells were used in this study (15, 16). For examination of the time-course effects of OP-1 on mRNA abundance, L-SaOS-2 cells were initially plated at a density of approximately 0.5–1.0 x 106 cells per dish (100 mm x 20 mm, Corning Inc., Corning, NY), and the cells were allowed to grow to 70% confluence in DMEM (GIBCO-BRL, Grand Island, NY) supplemented with 10% calf serum (Hyclone, Logan, UT). Once the appropriate level of cell growth was attained, the media was replaced with serum-free DMEM supplemented with 0.1% BSA (Fluka, Ronkonkoma, NJ) for 24 h before the onset of each experiment. Human recombinant OP-1 was added at a concentration of 100 ng/ml as based on the results of Knutsen et al. (6), which demonstrated that this concentration of OP-1 optimally stimulated the production of IGFBP-3 in the human osteosarcoma cells that were used in this study. For the experiments that examined the time-course effect of OP-1 on IGFBP-3 mRNA abundance, total RNA was isolated at 3, 6, 12, 24, and 36 h after treatment. For the mRNA stability experiments, L-SaOS-2 cells were cultured with OP-1 for 24 h before the addition of 20 µg/ml of 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB, Calbiochem, La Jolla, CA), and control and OP-1 treated RNA samples were collected initially (0 h) and at 6, 12, 18, and 24 h after DRB administration. To examine the requirement of de novo protein synthesis on OP-1 stimulated transcript production, L-SaOS-2 cells were treated with 10 µM cycloheximide (CHX) for 12 h before the addition of OP-1 and subsequent isolation of RNA at 6 h and 24 h after BMP treatment.

Total RNA isolation and Northern blots
Total RNA was isolated from the osteosarcoma cells by the acid-guanidine thiocyanate phenol-chloroform method of Chomczynski and Sacchi (17). Thirty micrograms of total RNA were size fractionated in a gel containing 1% agarose, 1% MOPS (10x = 0.2 M 3-(N-morpholino) propanesulfonic acid 0.005 M sodium acetate, 0.005 M EDTA, pH 7.0) and 2.2 M formaldehyde. The RNA was transferred to Magnagraph nylon membranes (MSI, Westboro, MA) by capillary transfer with 10 x SSPE (20 x = 3.6 M NaCl, 0.03 M NaH2PO4, 0.02 M EDTA, pH 7.4) overnight. RNA was fixed to the membrane by UV cross-linking (UVC-508 UV Crosslinker, Ultra-Lum, Inc., Carson, CA). Methylene blue staining was employed to verify the integrity and the loading uniformity of the RNA on the nylon (18). Human complementary DNA (cDNA) for IGFBP-3 (475 bp HindIII/EcoR1 fragment, 19 , ß-actin (1.8 kb BamHI fragment, 20 and GAPDH (780 bp PstI/XbaI fragment, ATCC no. 57090, 21 were used in these experiments. Random-prime labeling and purification of the cDNA probes was described previously by Zhou et al. (22). Prehybridization of the nylon blots was conducted for at least 2 h at 42 C in a solution containing 50% deionized formamide, 5 x SSPE, 5 x Denhardt’s solution (1 x = 0.02% each of Ficoll, polyvinylpyrrolidone, and BSA), 0.1% SDS and 200 µg/ml herring sperm DNA. After prehybridization was completed, the initial solution was decanted and fresh solution (as above) containing 1–3 x 106 cpm of each respective cDNA probe was applied to the blots. Hybridization was conducted overnight at 42 C. After hybridization, the blots were washed as follows: twice with 1 x SSPE/0.1% SDS at room temperature, and once with 1 x SSPE/0.1% SDS at 55 C for IGFBP-3, or 55 C with 0.1 x SSPE/0.1% SDS for IGFBP-4 and GAPDH. Autoradiography was conducted with Biomax-MS (Eastman Kodak, Rochester, NY) film with dual intensifying screens (Quanta III, Cronex, Eastman Kodak) at -70 C. The quantity of transcripts was attained by laser densitometry (Biomed Instruments, Fullerton, CA).

RT-PCR nuclear transcript analysis
Nuclear transcripts were amplified from total RNA by use of the Gene Amp EZ rTth RNA PCR method (Perkin-Elmer, Norwalk, CT). The unique property of the rTth DNA polymerase used in these studies is the capability of this enzyme to reverse transcribe RNA to cDNA, and subsequently extend specific primers during a PCR amplification reaction within a common reaction system. A final reaction volume of 50 µl contained the following reagent concentrations: 0.5 x EZ buffer (5x = 250 mM bicine, 575 mM potassium acetate, 40% (wt/vol) glycerol, pH = 8.2), 2.5 mM Mn(OAc)2, 300 µM each of dGTP, dCTP, dATP, dTTP, 5 U rTth DNA polymerase, and 0.45 µM of each primer. The 20 mer forward and reverse primer pairs used in these experiments were designed to have similar Tm and G:C content, and to coamplify intronic IGFBP-3 (233 bp of intron 2) and internal control ß-actin (331 bp of intron 2) transcripts (Table 1Go) as based on previously published genomic sequences (14, 23). Initial quality control assays demonstrated that a linear transcript amplification range (2.5, 5.0, 7.5, 10, and 15 µg of OP-1 treated total RNA was tested) for both IGFBP-3 and ß-actin was maintained with total RNA ranging in concentration between 5 to 10 µg (maximum) after 40 amplification cycles (Perkin-Elmer Thermocycler, model 480). The actual thermocycler cycling parameters were as follows: 60 C for 30 min for the reverse transcriptase reaction; 94 C for 3 min for heat denaturation; 94 C and 60 C each 1 min for 40 cycles for the amplification reaction; and 60 C for 7 min for extension. Treatment of total RNA with or without 0.1 U DNase/µg RNA (RQ1 RNase-free DNase, Promega) yielded comparable results in these assays, thus confirming that the results presented in this study were not affected by potential contaminating sources of genomic DNA within the RNA samples. After treatment with DNase, the enzyme was deactivated by heat-treating the samples at 75 C for 10 min before the addition of the rTth DNA polymerase. Products from the RT-PCR reaction (10 µl) were separated by electrophoresis in 1% agarose gels containing 1 x TAE (50 x = 2 M Tris-acetate, 0.05 M EDTA; pH 8.0). The transcripts were further denatured and transferred to Magnagraph nylon by standard Southern-blot procedures (MSI protocols). Detection and quantitation of the IGFBP-3 and ß-actin transcripts was accomplished by hybridization with respective 32[P] end-labeled (24) 20 mer oligonucleotides that were derived from internal sequences within the expected products (Table 1Go). Additional hybridization with a random-prime 32[P]-labeled genomic clone (14) also verified that this product was amplified from a region within intron 2 of the IGFBP-3 gene. Before hybridization with the oligonucleotides, the blots were treated with a solution containing 5 x Denhardt’s solution, 0.5% SDS and 10 mM EDTA, pH 8.0 for 1–2 h at 42 C. This solution was decanted and replaced with a solution containing 5 x SSPE, 5 x Denhardt’s solution, 0.1% SDS and 250 µg/ml yeast tRNA, and 5 x 106 cpm/ml of the respective 32[P]-labeled oligonucleotide. The blots were then hybridized for at least 12 h at 42 C. After hybridization, the blots were washed as follows: twice with 6 x SSPE/0.1% SDS at 42 C for 15 min, and once at 55 C for 15 min. The blots were then sealed in heat seal pouches and subjected to autoradiography. Transcript abundance was obtained after autoradiography by laser densitometry as described above.


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  		Table 1. Oligonucleotide primers for RT-PCR amplification of IGFBP-3 and ß-actin nuclear transcripts

 
Promoter analysis
L-SaOS-2 and TE-85 osteosarcoma cells were plated at a density of 20,000 cells per dish (60 mm x 15 mm). The cells were grown to approximately 50% confluence before transfection with 2 µg of chloramphenicol acetyltransferase (CAT) reporter vectors containing regions of either 0.4-kb (RsaI fragment ranging between -431 and +73 bp) or 1.8-kb (FokI fragment ranging between -1804 and +73 bp) 5' of the putative mRNA cap site of the IGFBP-3 chromosomal gene (14), and 2 µg of a CMV containing ß-galactosidase vector (Clonetech Laboratories, Inc., Palo Alto, CA). All transient transfection of the cells was accomplished by using lipofectAMINE as per the manufacturer’s recommendation (GIBCO-BRL, Gaithersburg, MD). Cells were treated for 12 h with 16 µl of the combined plasmid complexes, before changing the media to DMEM plus 10% calf serum for an additional 12 h. At this time, the media was changed to DMEM plus 0.1% BSA and 100 ng/ml of OP-1 was added to the cultures for 48 h. The cells were rinsed with PBS, and 0.5 ml of reporter lysis buffer (Promega) was added into each dish and incubated at room temperature for 15 min. Subaliquots of the cell lysates were obtained for subsequent CAT (25) and ß-galactosidase (26) analysis. Fractions that were collected for the CAT assays were pretreated at 60 C for 10 min before storage at -70 C.

Statistical analysis
Results that were determined from multiple experiments were subjected to analysis of variance (Systat, version 5.0, Systat Inc., Evanston, IL) in which treatment and time effects were examined. Data presented are means ± SEM. Values are considered significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time course effect of OP-1 on steady-state IGFBP-3 mRNA
Northern blot analysis was conducted to examine the effect of OP-1 on IGFBP-3 mRNA expression. As demonstrated in two individual experiments, the level of IGFBP-3 mRNA rose rapidly and remained elevated after 36 h of OP-1 treatment (Fig. 1Go, A and B). The results of combined densitometric analysis from these experiments demonstrated that the increase in IGFBP-3 mRNA expression was dependent upon OP-1 treatment and time (treatment x time interaction; P < 0.001) and that the maximal stimulatory effect of OP-1 on IGFBP-3 mRNA abundance occurred after 24 h of treatment (10.7-fold, P < 0.01; Fig. 1CGo). Because the level of GAPDH and ß-actin steady-state mRNA abundance declined over time (results not shown), densitometric results for IGFBP-3 mRNA expression (Fig. 1CGo) were normalized with densitometric values obtained from corresponding 28S rRNA transcripts to correct for experimental variation associated with lane loading and capillary blot transfer.



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  		Figure 1. The effect of OP-1 (100 ng/ml) on IGFBP-3 mRNA expression. Total RNA (30 µg/lane) was isolated from L-SaOS-2 cells treated with OP-1 over a period ranging from 3–36 h. The expression of IGFBP-3 mRNA was determined by Northern blot analysis from two individual experiments (Fig. 1Go, A and B). The results of densitometric scans of these experiments are provided in Fig. 1CGo. Data presented in this figure are means ± SEM, which have been normalized for corresponding 28S rRNA transcript expression. ANOVA demonstrated an OP-1 treatment effect and a treatment x time interaction (P < 0.001) on IGFBP-3 mRNA abundance. *, P < 0.02; **, P < 0.01 vs. respective controls per each period.

 
OP-1 effects on IGFBP-3 mRNA stability
To investigate whether OP-1 treatment increased IGFBP-3 production by a mechanism that involved cytosolic transcript stabilization, we treated L-SaOS-2 cells with DRB and measured the decay of IGFBP-3 mRNA over a 24-h period. In these experiments, the SaOS-2 cells were pretreated with OP-1 for 24 h before DRB administration because OP-1 maximally stimulated IGFBP-3 mRNA expression after 24 h of treatment (Fig. 1CGo). As demonstrated by results obtained by Northern blot analysis in Fig. 2AGo, the level of IGFBP-3 mRNA in the OP-1 treated group declined more rapidly than those in the control group after DRB administration (treatment and time effect by ANOVA, P < 0.001; Table 2Go). The resulting densitometric scans of the IGFBP-3 transcripts from two independent experiments were corrected for differences in ribosomal transcript abundance of each sample because DRB specifically arrests the transcriptional activity of RNA polymerase-II (27), and thus ribosomal genes (RNA polymerase-I transcribed) are better internal controls for these experiments. As demonstrated by Fig. 2BGo, OP-1 treatment actually promoted destabilization of IGFBP-3 mRNA (T1/2 = 5 h vs. 24 h in OP-1 and controls, n = 2 experiments). This decrease in IGFBP cytosolic transcript half-life was specific for IGFBP-3 because OP-1 did not affect IGFBP-4 (Fig. 2CGo) or ß-actin (Fig. 2DGo) mRNA decay (both 32[P] random prime-labeled IGFBP-4 and ß-actin cDNA were hybridized to the same blots that were previously examined for IGFBP-3 mRNA abundance). The respective half-lives for IGFBP-4 and ß-actin mRNA were approximately 12 h and 7 h (Fig. 2Go, C and D). Furthermore, the result that these genes are differentially controlled by OP-1 also demonstrates that the destabilization of IGFBP-3 mRNA is not produced by a deleterious effect of combined OP-1 and DRB treatments on L-SaOS-2 cell viability.



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  		Figure 2. The effect of OP-1 on the decay of cytosolic transcripts for IGFBP-3, IGFBP-4, and ß-actin. L-SaOS-2 cells were pretreated with OP-1 for 24 h before the addition of 20 µg/ml of DRB. Samples were collected before and after DRB treatment over a 24-h period. For- IGFBP-3, autoradiographs from Northern blots were exposed for either 12 h for OP-1 stimulated IGFBP-3 transcripts, or 7 days for nontreated control IGFBP-3 transcripts (Fig. 2AGo). Methylene blue stained 18S ribosomal transcripts are provided to demonstrate uniform loading of total RNA (30 µg) per each lane (Fig. 2AGo). Densitometric analysis from two individual experiments examining the effects of OP-1 on IGFBP-3 (Fig. 2BGo), IGFBP-4 (Fig. 2CGo), and ß-actin (Fig. 2DGo) mRNA stability in L-SaOS-2 cells are expressed as the percentage of the initial 0 h controls for each respective OP-1 and control treatment group. Data were normalized by using values obtained from ribosomal transcripts (pooled 28 S and 18 S) for each blot (n = 2/experiment). The pooled SE of the treatment means (i.e. control and OP-1 treated groups) are presented on the control treatments for each individual time point.

 

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  		Table 2. The effect of OP-1 on the decay IGFBP-3 mRNA over time

 
RT-PCR determination of IGFBP-3 nuclear transcript production
To examine the effects of OP-1 on IGFBP-3 gene transcription, we employed nuclear run-on, and RNase protection assays to monitor changes in the expression of IGFBP-3 nuclear transcripts. However, because the sensitivity of these procedures were not adequate, we developed an alternative approach using a RT-PCR procedure to selectively coamplify IGFBP-3 and ß-actin (control) nuclear transcripts from total RNA obtained from L-SaOS-2 cells.

An autoradiograph of a Southern blot containing amplified RT-PCR products, which demonstrate the effect of OP-1 on IGFBP-3 and ß-actin transcript expression is presented in Fig. 3AGo. In this experiment, results from RT-PCR reactions using either 5 or 10 µg of total RNA from L-SaOS-2 cells that were treated with OP-1 for 6 or 24 h demonstrate a profound increase in nuclear transcript expression for IGFBP-3 but not for ß-actin. For example, in the samples in which 5 µg of RNA (a quantity that is within the RT-PCR linear amplification curve, see methods) was used, there was a 2.4- and 8.0-fold increase in IGFBP-3 nuclear transcript abundance after 6 and 24 h of OP-1 treatment (Fig. 3AGo).



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  		Figure 3. The effect of OP-1 on IGFBP-3 nuclear transcript expression. An RT-PCR procedure was employed to examine the effect of OP-1 on IGFBP-3 pre-mRNA abundance in L-SaOS-2 cells. In these assays, ß-actin was coamplified as an internal control, and 5 or 10 µg of control or OP-1 treated (6 h vs. 24 h) total RNA was used in each reaction set. An autoradiograph of a Southern blot containing PCR products that were hybridized with 32[P] end-labeled antisense oligonucleotides from the mid-region of the IGFBP-3 and ß-actin amplified products (see Table 1Go and methods for detail) is presented in A. OP-1 treatment stimulated transcript production by 2.4-fold (6 h) and 8.0-fold (24 h) in the reactions using 5 µg of total RNA. Results of densitometric scans derived from an experiment examining more detailed effects of OP-1 and time on IGFBP-3 transcript production is presented in B. In this experiment, IGFBP-3 transcript expression rose rapidly (2.2-fold within 1 h), and increased over a period of 12 h after OP-1 treatment.

 
Densitometric results of a more detailed experiment (Exp 2) examining the time course effect of OP-1 on IGFBP-3 nuclear transcript expression is provided in Fig. 3BGo. In this experiment, OP-1 profoundly stimulated IGFBP-3 nuclear transcript production by 2.2-, 6.0-, 9.4-, and 5.4-fold after 1, 3, 6, and 12 h of treatment. For statistical analysis of the OP-1 effects on IGFBP-3 nuclear transcript production, the changes in pre-mRNA abundance were examined from the 6 h treatment period obtained from four separate experiments. ANOVA revealed an average 4-fold increase in the level of IGFBP-3 nuclear transcripts by OP-1 treatment among these experiments (0.21 vs. 0.79 ± 0.13 pooled SEM, image density units; P < 0.02).

To examine whether de novo protein synthesis is required to promote the stimulatory effect of OP-1 on IGFBP-3 nuclear transcript and mRNA production, we pretreated L-SaOS-2 cells with CHX before administering OP-1 for either 6 or 24 h. Nuclear transcript levels were examined by RT-PCR in the 6 h treated samples, and the autoradiograph of the resulting Southern blot is presented in Fig. 4AGo. CHX treatment largely blocked the stimulatory effect of OP-1 on the expression of IGFBP-3 pre-mRNA (5.3-fold induction in non-CHX controls vs. 1.4-fold in the CHX treated group in 5 µg total RNA samples), whereas ß-actin nuclear transcript production remained unaltered. Similarly, the OP-1-induced increase in IGFBP-3 mRNA abundance (Fig. 4BGo) was also greatly inhibited by pretreating L-SaOS-2 cells with CHX before OP-1 treatment for 6 h (5.1- vs. 1.4-fold in non-CHX and CHX treated groups) but to a lesser extent after 24 h of treatment (6.0- vs. 4.3-fold in non-CHX and CHX treated groups). In addition, CHX treatment also selectively reduced the expression of control IGFBP-3 mRNA (Fig. 4BGo) at both 6 h (0.98 vs. 0.22 image density units) and 24 h (0.43 vs. 0.14 image density units). The finding that CHX did not markedly affect the abundance of ß-actin nuclear transcripts (Fig. 4AGo), or the expression of ß-actin and GAPDH mRNA (Fig. 4BGo), further confirms that cell viability was not compromised by the exposure or dosage of CHX that was used in the present study.



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  		Figure 4. The stimulatory effect of OP-1 on the level of IGFBP-3 nuclear transcripts and steady-state mRNA requires de novo protein synthesis. L-SaOS-2 cells were treated with 10 µM cycloheximide for 12 h before the addition of 100 ng/ml of OP-1. Total RNA was isolated after 6 and 24 h of OP-1 treatment. The change in nuclear transcript abundance for IGFBP-3 and ß-actin (6 h treated samples) was examined by RT-PCR and Southern blot analysis (Fig. 4AGo). IGFBP-3, ß-actin and GAPDH mRNA abundance (6 h and 24 h treated samples) was examined by Northern blot analysis (Fig. 4BGo).

 
Effect of OP-1 on IGFBP-3 promoter activity
To examine whether putative OP-1 responsive cis-elements may reside within the promoter domain of the IGFBP-3 gene, we conducted transient transfection experiments using CAT reporter vectors that contained either 0.4- or 1.8-kb of the 5'-flanking region. A ß-galactosidase expression vector was also cotransfected in these experiments, and corresponding ß-galactosidase activity was used to correct results for variation in cellular transfection efficiency. Previous experiments confirmed that OP-1 treatment did not alter ß-galactosidase activity. In addition, an increase (average 2.0- and 1.3-fold for 0.4-kb and 1.8-kb promoter vectors) in basal promoter activity was demonstrated in vectors that contained the promoter domains vs. the promoterless negative control (results not shown). In two individual experiments, OP-1 did not alter CAT activity from lysates obtained from either L-SaOS-2 cells (Fig. 5Go) or TE-85 cells (the TE-85 cell line is responsive to the OP-1 stimulation of IGFBP-3 but demonstrates a higher level of transfection efficiency than L-SaOS-2; data not provided).



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  		Figure 5. The effect of OP-1 on chloramphenicol acetyltransferase (CAT) reporter vectors containing the IGFBP-3 promoter. Reporter vectors containing 431 and 1804 bp of the 5'-flanking region of the IGFBP-3 gene were transiently transfected into L-SaOS-2 and TE-85 osteosarcoma cells. Results presented above are means ± SEM (n = 3 for each treatment and vector) and were corrected for ß-galactosidase activity. OP-1 did not stimulate the promoter activity of these vectors in either human L-SaOS-2 (above) or TE-85 (data not provided) osteosarcoma cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we describe for the first time molecular mechanisms by which OP-1 modulates the production of IGFBP-3 in human osteosarcoma cells. Previously, we demonstrated that OP-1 treatment markedly increases the level of IGFBP-3 mRNA and IGFBP-3 protein produced in human osteosarcoma cells that retain an osteoblastic phenotype (6), and in human bone marrow stromal cells (28). We further investigated in the present study whether the OP-1-induced increase in the level of IGFBP-3 cytosolic mRNA may involve an increase in mRNA stability and/or the stimulation of IGFBP-3 nuclear transcript production. In contrast to the anticipated increase of OP-1 on the half-life of IGFBP-3 cytosolic transcripts, treatment with this BMP actually promoted an acute and specific destabilization of IGFBP-3 mRNA. The calculated half-life value of approximately 24 h for IGFBP-3 transcripts from control L-SaOS-2 cultures closely matched previously published values obtained in human osteosarcoma cells (22) and rat liver cells (29), thus confirming that the result obtained from this present study did not result from any deleterious effects of DRB or OP-1 on the cultured cells. The aberrant disassociation between the decreased half-life in IGFBP-3 mRNA and the increase in overall mRNA expression is not unique to IGFBP-3, or this study, because it has been recently shown that retinoic acid treatment enhances IGFBP-5 mRNA stability but yet promotes a decrease in the production of IGFBP-5 cytosolic transcripts in human osteoblasts (22). Presently, the biological significance for the uncoupling between mRNA expression and cytosolic transcript stability by agents such as OP-1 and retinoic acid remains unknown. Furthermore, it also remains to be determined whether common or independent receptor/postreceptor transduction mechanisms may mediate the differential effects of OP-1 on cytosolic mRNA stability and mRNA production in human bone cells.

Based on the findings of this study, we propose that OP-1 promotes IGFBP-3 production by a mechanism that largely involves the regulation of a transcriptional mechanism. This hypothesis is supported by the observations that OP-1 treatment rapidly and selectively stimulates the expression of IGFBP-3 nuclear transcripts, while concomitantly destabilizing IGFBP-3 cytosolic transcripts. Theoretically, the level of IGFBP-3 nuclear transcripts may be governed by changes in the rate of transcription and/or processing and trafficking of the transcripts. Although the determination of nuclear transcript abundance by RT-PCR does not allow direct measurement of these processes, it has been established that changes in the level of in vivo nuclear transcripts of genes, other than IGFBP-3, are positively correlated with the rate of nuclear transcription obtained from isolated nuclei in vitro (30, 31). It is also noteworthy that the determination of pre-mRNA abundance by RT-PCR allows for the measurement of low abundant transcripts that are synthesized by intact cells (32), whereas nuclear run-on assays measures primary nacent RNA that are radiolabeled and hybridized to segments of specific genes in vitro. Aberrant observations have arisen by nuclear run-on analysis, where most problems result from high background associated with antisense transcripts that are synthesized from the wrong strand (template) DNA, or sense transcripts that are anomalously initiated at sites other than the authentic promoter (for detailed discussion, refer to Refs. 30, 33). An additional benefit of monitoring nuclear transcript abundance by the RT-PCR procedure is the technical feasibility that more samples can be analyzed within a given experiment.

The present study demonstrates that the stimulation of IGFBP-3 nuclear transcript expression by OP-1 is dependent on new protein synthesis. The inhibitory effect of IGFBP-3 mRNA expression by CHX is more pronounced in the samples that were treated with OP-1 after 6 h, whereas this effect is dampened after a prolonged treatment period of 24 h. It is not known at this time why the relative potency of CHX to effectively block the OP-1 induced increase in IGFBP-3 mRNA abundance was decreased after extended periods of OP-1 treatment. The present study also demonstrates that the overall levels of control IGFBP-3 mRNA are reduced with CHX treatment. This reduction may suggest that the components that regulate basal transcriptional activity of the IGFBP-3 gene may also require continual de novo protein synthesis to maintain a steady pool of transactivators which modulate IGFBP-3 transcription. Similar to this study, Gabbitas and Canalis (8) have demonstrated that basal expression of IGFBP-5 in primary osteoblasts from rat calvaria requires de novo protein synthesis. To date, the exact intracellular signaling mechanisms that mediate BMP effects in murine and human skeletal cells remain to be fully elucidated. In addition, it also remains to be determined whether the effects of BMPs on target genes selectively requires new protein synthesis.

After we established that OP-1 treatment enhanced IGFBP-3 nuclear transcript production, we began to examine whether OP-1 stimulatory cis-elements reside within a domain 1.8-kb 5' of the established start site of the IGFBP-3 gene (14). Transient transfection assays with CAT reporter vectors containing either 0.4- or 1.8-kb of the IGFBP-3 promoter region did not strongly indicate that active OP-1 responsive cis-elements reside within this domain of the IGFBP-3 gene. In regard to the molecular mechanisms by which BMPs modulate gene transcription, there have been two published reports in other model system that are relevant to this study. First, Harada et al. (34) have recently shown that an active OP-1 cis-element (an AP-1-like motif, TGAATCATCA) resides within 327-bp of the 5'-flanking region of the human type-X collagen gene, and interacts with a Fos-like transactivator. Second, Tamura and Noda (35) have established that BMP-2 treatment promotes the production of a transactivating protein that interacts with a specific E-box binding domain (CACATG motif) located within the proximal promoter of the rat osteocalcin gene. Based on these reports, we mapped seven putative E-box motifs (CANNTG, and exclusive of the exact rat osteocalcin CACATG motif) in the 1.8-kb 5'-flanking region of the IGFBP-3 gene. In addition, no type-X collagen AP-1-like motifs were located in this region. As judged by the results obtained from the transient transfection studies that were conducted in L-SaOS-2 and TE-85 cells, these aforementioned motifs in the context of the promoter fragments tested do not appear to confer OP-1 mediated transactivation. Presently, more detailed experiments are required to fully determine whether unique cis-elements, and/or E-box and AP1-like motifs that are located beyond the known 5'-flanking region, or within internal intronic/exonic domains, mediate OP-1-induced regulation of IGFBP-3 transcription in human osteoblast-like cells.

Currently, it has not been established whether the increase in IGFBP-3 production is required for OP-1 to fully elicit biological responses in human bone cells. It has been recently shown that BMP treatment promotes differentiation and proliferation of bone cells in a variety of experimental model systems (1, 2, 3, 4), and that IGFBP-3 may cell differentially modulate IGF-induced effects on osteosarcoma cell proliferation (36, 37, 38). Moreover, administration of IGFBP-3/IGF-I complexes greatly promotes in vivo formation of bone in rats (39, 40, 41). Although the effect of IGFBP-3 on human bone cell proliferation has not been fully established, the finding that OP-1 modulates several IGF system components (6, 7) is consistent with the theory that this system may, in part, provide a role in mediating the stimulatory actions of BMPs in bone cells. Presently, further studies are required to establish the exact cause and effect relationship between the increase in IGFBP-3 expression and the promotion of human bone cell differentiation and proliferation by OP-1.

In conclusion, the present study demonstrates that OP-1 stimulates IGFBP-3 production in human osteoblast-like osteosarcoma cells by a mechanism that predominantly involves an increase in IGFBP-3 nuclear transcript production, a process that requires new protein synthesis, and compensates for an OP-1-induced stimulation of IGFBP-3 cytosolic transcript degradation.


   Footnotes
 
1 This study was supported by funds from the NIH, Grants AR-31062, AR-07543, and DK-38773, the Veterans Administration, and the Department of Medicine, Loma Linda University. Back

Received February 3, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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