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J Appl Physiol 85: 139-147, 1998;
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Vol. 85, Issue 1, 139-147, July 1998

Spaceflight modulates insulin-like growth factor binding proteins and glucocorticoid receptor in osteoblasts

Yasuhiro Kumei1, Hitoyata Shimokawa1, Hisako Katano1, Hideo Akiyama2, Masahiko Hirano2, Chiaki Mukai3, Shunji Nagaoka3, Peggy A. Whitson4, and Clarence F. Sams4

1 Faculty of Dentistry, Tokyo Medical and Dental University, Tokyo 113-8549; 2 Toray Research Center, Kamakura 248-78555; 3 National Space Development Agency, Tsukuba Space Center, Tsukuba 305-0047, Japan; and 4 National Aeronautics and Space Administration, Johnson Space Center, Houston, Texas 77058

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Rat osteoblasts were cultured for 4 or 5 days during a Space Shuttle mission. After 20-h treatment with 1alpha ,25-dihydroxyvitamin D3, conditioned media were harvested and cellular DNA and/or RNA were fixed on board. The insulin-like growth factor binding protein (IGF BP)-3 levels in the media were three- and tenfold higher than in ground controls on the fourth and fifth flight days, as quantitated by Western ligand blotting and radioimmunoassay, respectively. The increased IGF BP-3 protein levels correlated with two- to threefold elevation of IGF BP-3 mRNA levels, obtained by reverse transcription-polymerase chain reaction. The IGF BP-5 mRNA levels in flight cultures were 33-69% lower than in ground controls. The IGF BP-4 mRNA levels in flight cultures were 75% lower than in ground controls on the fifth day but were not different on the fourth day. The glucocorticoid receptor mRNA levels in flight cultures were increased by three- to eightfold on the fourth and fifth days compared with levels in ground controls. These data suggest potential mechanisms underlying spaceflight-induced osteopenia.

bone demineralization; osteoblast; microgravity

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

SPACEFLIGHT is known to induce bone demineralization. Mechanical unloading of the skeleton during spaceflight triggers alterations in mineral flux and a net change in the formation-to-resorption ratio in bones (28). A number of studies, using humans, animals, and cell cultures, have been conducted to clarify the mechanism by which microgravity induces osteopenia (22). Histological analysis of bones of growing rats during Kosmos biosatellite flights showed decreased bone formation and defects in bone maturation (15, 30). A recent study showed that spaceflight and hindlimb elevation transiently increased the mRNA levels for insulin-like growth factor (IGF)-I and IGF-I receptor in the skeletal tissue (5). The data suggest that microgravity effects on bone metabolism are mediated in part through alterations in IGFs and their receptors. The IGF-binding proteins (IGF BPs) not only modulate the interaction of IGFs and their receptors but also perform direct action on target cells and play significant roles in bone metabolism (16, 25, 27). A recent study by Kumei et al. (18) suggests that osteoblasts function inappropriately in microgravity. IGF BPs are produced in osteoblast-like cells (12, 21, 23). However, little is known about microgravity effects on IGF BP production.

Six IGF BPs have been cloned and identified thus far (29). Human normal osteoblast-like cells produce IGF BP-3, -4, -5, and -6 (12, 23). Primary cultures of fetal rat osteoblasts expressed transcripts encoding IGF BP-2, -3, -4, -5, and -6 (21). IGF BP-3 is most abundant in serum and seems to both enhance and inhibit IGF-I action in bone (25, 27). The IGF-I-stimulated mitogenesis of osteoblasts was inhibited by IGF BP-3, whereas it is stimulated by IGF BP-5 (3). It is known that IGF BP-5 synthesis is regulated by skeletal growth factors that inhibit osteoblast differentiation (6). IGF BP-1 and -6 also are produced in some osteoblasts; however, their function remains unclear. Local and systemic factors may regulate the IGF actions by modulating the type and amount of IGF BPs produced by bone cells.

We hypothesized that microgravity modulated physiological functions of normal rat osteoblasts, resulting in bone demineralization. Part of this hypothesis involved the supposition that production of IGF BPs, particularly IGF BP-3 and -5, was affected by microgravity. Thus we sought to investigate potential microgravity effects on IGF BP expression in normal rat osteoblasts.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Animals and tissue processing before flight. Animal care and use for this Space Shuttle (Space Transport System-65) experiment complied with National Institutes of Health and the National Aeronautics and Space Administration Ames Research Center guidelines, as stated in the Ames Research Center Animal User's Guide (AHB 7180-1). Eight days before launch, three specific-pathogen-free Wistar male rats (5 wk of age, 100-120 g weight; Harlan Sprague Dawley, Indianapolis, IN) were killed under CO2 gas anesthesia. Marrow cells were obtained from dissected femurs and suspended in a modified Eagle's minimum essential medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (GIBCO), 10 nM dexamethasone (Sigma Chemical, St. Louis, MO), 2 mM beta -glycerophosphate (Sigma Chemical), and 0.5 mM L-ascorbic acid (Wako, Osaka, Japan).

Osteoblasts were cultured for 5 days, but they remained in a preconfluent stage. Cells were dissociated into a single-cell suspension with trypsin-EDTA solution (GIBCO) and inoculated into flight cell culture units (CCUs) at a density of 104 cells/cm2. Each CCU consists of two separate cell culture chambers (Fig. 1). The media were replaced with fresh media 6 h before the flight CCUs were transported to the Shuttle cabin (before launch). Four CCUs were incubated onboard at 37°C, 95% air-5% CO2, and 70% humidity. Eight corresponding ground control CCUs were incubated under identical conditions. The experiment on board was conducted according to the diagram in Fig. 2.


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  		Fig. 1.   Schematic illustration of flight cell culture unit (CCU). The CCU is composed of 2 equivalent (upper and lower) chambers which are separated by a plastic culture plate. Culture chamber is filled with medium. Cells are cultured by anchoring to both top and bottom plates in the chamber. Gas exchange occurs automatically through permeable Silastic membranes of the chambers.


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  		Fig. 2.   Diagram of experimental schedule on board. Time, no. of days or hours after launch. Launch time is adjusted to be 0 h on day 0. VD3, 1alpha ,25-dihydroxyvitamin D3 (vitamin D3).

On the third day of flight (day 3), the culture media were removed. Media in one-half of the flight CCUs (CCU-1 and CCU-2) and one-half of the ground control CCUs were replaced by media containing 0.1 nM 1alpha ,25-dihydroxyvitamin D3 (vitamin D3; Calbiochem, LaJolla, CA). Media in the remaining CCUs (flight CCU-3 and CCU-4, and the corresponding ground control CCUs) were replaced by media without vitamin D3.

On the fourth day of flight (day 4), 18 h after the initial medium exchange, cells in the CCUs treated with the vitamin D3-containing media (CCU-1 and CCU-2) were harvested. This harvest process entailed removing the vitamin D3-containing media from the CCUs with syringes, washing the cells in the CCUs with phosphate-buffered saline, and fixing the cells by filling the CCUs with 4 M guanidine thiocyanate (GTC; GIBCO) solution containing 25 mM sodium citrate, 0.5% sarcosyl, and 0.1 M beta -mercaptoethanol (all from Sigma Chemical). The harvested media and CCUs filled with GTC were stored in a freezer at -20°C. Media from the remaining CCUs (CCU-3 and CCU-4, those without vitamin D3) were exchanged with fresh media containing vitamin D3.

On the fifth day (day 5), 26 h after the second medium exchange (treatment with vitamin D3), the process from day 4 (treatment with GTC) was repeated with the remaining CCUs (CCU-3 and CCU-4). The CCUs with media containing vitamin D3 were harvested and fixed. Harvested media and fixed cells were placed in a freezer at -20°C. Ground control cultures underwent identical changes of culture conditions and operation, except for a delay of 3 h relative to flight cultures.

Radioimmunoassay of IGF BP-3. The IGF BP-3 concentrations in the harvested media were measured by radioimmunoassay with a commercially available kit (Nichols Institute, San Juan Capistrano, CA). The amounts of IGF BP-3 in the media were normalized with respect to the DNA content of the osteoblasts in each culture chamber (18). Triplicate aliquots from each syringe were assayed. Four individual samples from the flight CCUs (2 culture chambers per CCU) were assayed along with eight samples from corresponding ground controls. Statistical differences between the flight and ground samples were assessed by using Student's t-test.

Western ligand blotting. Proteins bound to IGF in conditioned media were analyzed by Western ligand blotting (14, 23). Twenty microliters of the conditioned media which were harvested on day 4 (3 flight samples and 6 ground samples) were subjected to 12% SDS-PAGE under a nonreducing condition. Separated proteins were electroblotted onto nitrocellulose filters (Hybond-C; Amersham, Arlington Heights, IL) under a constant voltage (12 V) at 4°C for 8 h. Filters were blocked with 1% BSA, and IGF BPs were identified by incubation with 0.8 µCi of 125I-labeled IGF-II (2,000 Ci/mmol, Amersham) for 24 h at 4°C. 125I-IGF-II was used as ligand, because incubation with 125I-IGF-II provides more distinct bands than with 125I-IGF-I (14). Results were visualized by autoradiography with the use of a highly sensitive film (Reflection film; DuPont, Boston, MA) and quantitated by densitometric analysis by using a transmitting scanning densitometer (Imaging Densitometer GS-670; Bio-Rad, Hercules, CA).

RNA and DNA isolation. After the culture chambers and syringes were returned to earth, RNA and DNA were isolated and purified as described previously (18). Briefly, the GTC-cellular extracts solution was sheared and then mixed with cesium trifluoroacetate (Pharmacia, Uppsala, Sweden)-EDTA solution and centrifuged at 30,000 rpm for 20 h. The isolated fractions of RNA and DNA were purified by repeated phenol-chloroform extraction and cold ethanol precipitation. The amounts of RNA and DNA were determined with optical-density measurements at 260 nm.

RT-PCR. Quantitative analysis of gene expression was performed as described previously (18). Briefly, cDNA was synthesized from equal amounts of total RNA (1 µg) in the flight and ground cultures by using Superscript reverse transcriptase (GIBCO) and a mixture of oligo(dT) and random primers (GIBCO). To determine the optimal condition for each quantitative RT-PCR, the calibration curve was obtained separately for a range of PCR cycles. PCR was performed with serial 1:2 dilutions of the first strand cDNA which was prepared from 1 µg of total RNA. Exponential amplification was observed over a range of from 1/400 to 1/10 volume of the cDNA (1). A 1/20 aliquot of the first strand cDNA was amplified in each PCR. Duplicate samples were analyzed for both flight and ground samples on days 4 and 5, respectively. A set of oligonucleotide primers that recognized the forward and reverse sequence of a specific region of cDNA for IGF BP-2, -3, -4, and -5 (11) and for glucocorticoid receptor (GCR) was synthesized with a DNA synthesizer (model 304; Applied Biosystems, Tokyo, Japan). A pair of oligonucleotides also was synthesized for the control gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The sequences of oligonucleotide primers were IGF BP-2 sense: 5'-ACTGTGACAAGCATGGCCTGT-3'

antisense: 5'-CCTCCTGCTGCTCATTGTAGA-3' IGF BP-3 sense: 5'-CCAGAACTTCTCCTCCGAGTC-3'

antisense: 5'-TATCCACACACCAGCAGAAGC-3' IGF BP-4 sense: 5'-CTGTGCCCCAGGGTTCTTGC-3'

antisense: 5'-TCACCCCTGTCTTCCGATCCA-3' IGF BP-5 sense: 5'-GTTCAAAGCCAGCCCACGCAT-3'

antisense 5'-GTCGAAGGCGTGGCACTGAA-3' GCR sense: 5'-TGCCTGGTGTGCTCCGATGAA-3'

antisense: 5'-ATCACTTGACGCCCACCTAAC-3' GAPDH sense: 5'-ACCACAGTCCATGCCATCAC-3'

antisense: 5'-TCCACCACCCTGTTGCTGTA-3' Each cDNA was amplified under the optimal amplification-cycle condition by using 32P-labeled nucleotide (3,000 Ci/mmol, Amersham), 10 pmol of a set of primers, and 1.25 U of Ex Taq polymerase (Takara, Kyoto, Japan). After the first denaturation at 94°C for 7 min, PCR cycles were performed with the sequence of 94°C for 40 s, 55°C for 40 s, and 72°C for 1 min with a thermal cycler (2,400 GAmp PCR system, Perkin-Elmer, Norwalk, CT). Amplification was performed in 26 cycles for IGF BP-2, -3, and -4 and in 30 cycles for IGF BP-5, because the optimal number of amplification cycles was determined as being at the exponentially reacting points by measuring the amounts of PCR products amplified after each cycle. For this reason, separate PCR of 24 cycles was performed for GCR, respectively, with the annealing temperature at 63 instead of 55°C. For each PCR, the paired primers for GAPDH were used simultaneously to normalize the starting amount of cDNA for each sample.

For analysis of GCR mRNA levels, a quantitative RT-PCR fluorescent method (1) was used. Briefly, 0.25 µl of fluorescent-labeled N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA)-dUTP (Perkin-Elmer) was used instead of the 32P-labeled nucleotide. The ratio of dTTP to TAMRA-dUTP was 100:1. On each PCR, the paired primers for GAPDH were used simultaneously to normalize the starting amount of cDNA for each sample.

Quantification of RT-PCR products. We have utilized the RT-PCR method (19, 24) for quantitative comparison of gene transcripts. This method is usable for measurement of relative change in mRNA levels if the following two conditions are satisfied. First, tube-to-tube variation in the actual value must be minimal so that a constant value can be assumed for it in all related PCR reactions. Second, all data must be obtained before the amplification is reaching the plateau phase. In addition, a standard curve is required to determine the level of signal that corresponds to a specific number of RNA molecules. This method is rapid and simple, with sufficient resolution to detect at least twofold differences in the amount of RNA without use of any internal standards (24).

To analyze the mRNA levels of IGF BPs, the RT-PCR products were separated by 6% PAGE, followed by autoradiography (18). The levels of the RT-PCR products were obtained by using high-sensitivity radioactivity counting, coupled with high-resolution image analysis (BAS 2000, Fuji Film, Tokyo, Japan) (13). For GCR, the fluorescent RT-PCR products together with a size-standard marker (G2500; Perkin-Elmer) were loaded to an automated DNA sequencer (model 377A, Perkin-Elmer). The products were detected in a 8 M urea-6.75% sequencing gel electrophoresis (Long Ranger, AT Biochem, Malvern, PA). The levels for GCR gene transcripts were quantified as the fluorescent peak height (1) by using GeneScan 672 software (Perkin-Elmer). The results were normalized to those of GAPDH. The value for each RT-PCR product was divided by the relative amount of GAPDH product in the same sample, with the value in cell culture chamber 1 defined as 1.00.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

IGF BP-2 mRNA levels. Gene expression of IGF BP-2 was examined in the vitamin D3-treated cultures. Exponential amplification (linearity of the calibration curve, r = 0.993) was observed in the RT-PCR with the IGF BP-2 primer set for a range of 20-30 PCR cycles (Fig. 3A). After a 26-cycle protocol was used for the PCR amplification, the RT-PCR products of IGF BP-2 gene transcripts were obtained with the expected length (168 bp) in the PAGE (Fig. 3B). The steady-state mRNA levels for IGF BP-2 were not different between flight and ground control cultures on days 4 and 5 (Fig. 3C).


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  		Fig. 3.   Insulin-like growth factor binding protein (IGF BP)-2 mRNA levels in rat osteoblast culture on board spaceflight. A: calibration curve to determine optimal condition in quantitative RT-PCR for IGF BP-2. Inset: autoradiograph for amplified RT-PCR products. Radioactivity of amplified products was measured by BAS 2000 (Fuji Film) and indicated with photostimulated luminescence (PSL). PSL shows linear relationship with radioactivity of gel band and corresponds to relative amounts of the samples (13). Arrow, no. of PCR cycles to amplify the IGF BP-2 gene transcripts for quantitative comparison between flight and ground cultures. B: autoradiograph of IGF BP-2 RT-PCR products. FD, flight day; F, flight; G, ground; arrow, RT-PCR products with expected length (168 bp) in polyacrylamide gel. C: quantitative comparisons for IGF BP-2 RT-PCR products among cell culture chambers. Radioactivity of each signal in autoradiograph was measured by BAS 2000 (Fuji Film). Relative amounts of RT-PCR products after normalization to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are indicated with PSL in ordinate of graph. Hatched bars, flight cultures; open bars, ground control cultures.

IGF BP-3 production and mRNA levels. As noted above, IGF BP-3 was quantified in the conditioned culture media that were harvested on days 4 and 5 after treatment with vitamin D3. The media from synchronous ground controls were obtained in an identical manner. The IGF BP-3 concentration normalized to the amount of cellular DNA was 29.6 ± 4.7 ng/µg DNA in flight samples of day 5 and 3.1 ± 2.0 ng/µg DNA in ground control samples. On day 5, the amounts of IGF BP-3 in flight samples were 10-fold higher than those in ground control samples. On day 4, the IGF BP-3 concentration in each sample was <1.0 ng/ml, the detection limit of the assay. The Western ligand blotting and/or densitometric analysis showed that the relative amounts of the 45-kDa IGF BP in flight samples on day 4 were threefold higher than those in ground control samples (Fig. 4).


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  		Fig. 4.   Western ligand blot for IGF BP in conditioned media of rat osteoblast culture on board spaceflight. Culture media were harvested on day 4 of flight. 125I-IGF-II was used as ligand. Right: positions of molecular mass markers (mol mass). Lanes 1, 4, and 7: flight samples; lanes 2, 3, 5, 6, 8, and 9: ground controls. Lane 10: mol mass marker (Bio-Rad). Arrow, position of 45-kDa IGF BP.

The 45-kDa IGF BP would correspond to rat IGF BP-3 for the molecular mass. These data indicate that microgravity significantly (P < 0.001) increased IGF BP-3 production in normal rat osteoblasts. However, the limited number of time points cannot resolve between altered levels or altered kinetics as a cause for altered IGF BP-3 production.

The levels for IGF BP-3 gene transcripts were assessed in the same samples used for the IGF BP-3 determination, i.e., lysed cellular samples that had been treated with vitamin D3. Exponential amplification (linearity of the calibration curve, r = 0.995) was observed for a range of 20-30 cycles in the RT-PCR for IGF BP-3 gene transcripts (Fig. 5A). The quantitative analysis was performed for the IGF BP-3 RT-PCR products that were obtained after a 26-cycle amplification with the expected length (209 bp; Fig. 5B). The steady-state mRNA levels for IGF BP-3 in flight cultures of day 4 were 2.9- and 2.1-fold higher than those of the corresponding ground control cultures. The IGF BP-3 mRNA levels in flight cultures of day 5 were also 2.7- and 3.2-fold higher than those of the ground control cultures (Fig. 5C). Thus, production and/or secretion of IGF BP-3 by rat osteoblasts was significantly increased during spaceflight, with concomitant elevation of the steady-state mRNA levels for IGF BP-3.


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  		Fig. 5.   IGF BP-3 mRNA levels in rat osteoblast culture on board spaceflight. A: calibration curve to determine optimal condition in quantitative RT-PCR for IGF BP-3. Relative amounts of amplified PCR products are shown in semilogarithmic representation. Arrow, no. of PCR cycles for quantitative comparison between F and G cultures. Inset: autoradiograph for amplified RT-PCR products. The radioactivity of the amplified products was indicated with PSL, as described in Fig. 3A. B: autoradiograph of IGF BP-3 RT-PCR products. Arrow, RT-PCR products with expected length (209 bp). C: quantitative comparisons for IGF BP-3 RT-PCR products among cell culture chambers. Radioactivity of each signal was measured by BAS 2000 (Fuji Film) and expressed with PSL, as described for Fig. 3. Hatched bars, F; open bars, G.

IGF BP-4 mRNA levels. Gene expression of IGF BP-4 was examined in the vitamin D3-treated cultures as well. Exponential amplification (linearity of the calibration curve, r = 0.994) was also observed for a range of 22-30 cycles in the RT-PCR for IGF BP-4 gene transcripts (Fig. 6A). After a 26-cycle amplification, the RT-PCR products for IGF BP-4 were obtained with the expected length (204 bp; Fig. 6B). The steady-state mRNA levels for IGF BP-4 in the flight cultures of day 4 were not different between flight and corresponding ground control cultures. However, on day 5, the steady-state mRNA levels for IGF BP-4 in flight cultures were 23 and 28% as low as those of ground control cultures (Fig. 6C).


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  		Fig. 6.   IGF BP-4 mRNA levels in rat osteoblast culture on board spaceflight. A: calibration curve to determine optimal condition in quantitative RT-PCR for IGF BP-4. Relative amounts of amplified PCR products are shown in semilogarithmic representation. Arrow, no. of PCR cycles for quantitative comparison between F and G cultures. Inset: autoradiograph for amplified RT-PCR products. The radioactivity of the amplified products was indicated with PSL, as described in Fig. 3A. B: autoradiograph of IGF BP-4 RT-PCR products. Arrow: RT-PCR products with expected length (204 bp). C: quantitative comparisons for IGF BP-4 RT-PCR products among cell culture chambers. Radioactivity of each signal was measured by BAS 2000 (Fuji Film) and expressed with PSL, as described for Fig. 3. Hatched bars, F; open bars, G.

IGF BP-5 mRNA levels. In the IGF BP-5 RT-PCR, exponential amplification (linearity of the calibration curve, r = 0.997) was also observed for a range of 28-36 cycles (Fig. 7A). The RT-PCR products for IGF BP-5 after a 30-cycle amplification were shown at the expected length (316 bp) in the polyacrylamide gel (Fig. 7B). The steady-state mRNA levels for IGF BP-5 in the flight cultures of day 4 were 42 and 67% as low as the corresponding ground controls. The IGF BP-5 mRNA levels in the flight cultures of day 5 were 31 and 45% as low as the ground controls (Fig. 7C).


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  		Fig. 7.   IGF BP-5 mRNA levels in rat osteoblast culture on board. A: calibration curve to determine optimal condition in quantitative RT-PCR for IGF BP-5. Relative amounts of amplified PCR products are shown in semilogarithmic representation. Arrow: no. of PCR cycles for quantitative comparison between F and G cultures. Inset: autoradiograph for amplified RT-PCR products. The radioactivity of the amplified products was indicated with PSL, as described in Fig. 3A. B: autoradiograph of IGF BP-5 RT-PCR products. Arrow: RT-PCR products with expected length (316 bp). C: quantitative comparisons for IGF BP-5 RT-PCR products among cell culture chambers. Radioactivity of each signal was measured by BAS 2000 (Fuji Film) and expressed with PSL, as described for Fig. 3. Hatched bars, F cultures; open bars, G cultures; FD, flight day.

GCR mRNA levels. The levels of GCR gene transcripts were examined by the fluorescent RT-PCR method (1) in the same samples that were used for analyses of mRNA levels of IGF BPs. Exponential amplification (linearity of the calibration curve, r = 0.998) was observed in the RT-PCR with the GCR primer set for a range of 22-30 PCR cycles. A 24-cycle protocol was used to amplify the GCR gene transcripts (Fig. 8A). In the present study, a split signal was observed in the amplified products of the GCR primer set. The calculated lengths of the RT-PCR products of GCR gene transcripts were 452 and 455 bp, whereas the expected length was 431 bp (Fig. 8B). The RT-PCR products of the split signal were identified by a combination of the conventional DNA sequence analysis after cloning to pUC18 vector. The sequence data of the split signal observed coincided with each strand of the PCR products of GCR (data not shown). pUC18 might be generated by conformational changes in each strand due to the base composition of the products. According to the manufacturer (Perkin-Elmer/Applied Biosystems) split peaks are observed in the denaturing gel electrophoresis, especially if the adenine-thymine content is <35% or >65%. Quantitative comparison was performed for the RT-PCR products by the peak height at 455 bp. The GCR mRNA levels in the flight cultures of day 4 were 3.4- and 2.3-fold higher than those of the corresponding ground controls. The GCR mRNA levels in the flight cultures of day 5 were 5.3- and 8.0-fold higher than those of the ground controls (Fig. 8C). Thus, spaceflight elevated the steady-state mRNA levels for GCR in normal rat osteoblasts in the presence of vitamin D3.


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  		Fig. 8.   Glucocorticoid receptor (GCR) mRNA levels in rat osteoblast culture on board spaceflight. A: calibration curve to determine optimal condition in quantitative RT-PCR for GCR. Relative amounts of GCR RT-PCR products are shown in semilogarithmic representation with fluorescent signal intensity (peak height). Arrow, no. of PCR cycles for quantitative comparison between F and G cultures. B: detection of GCR RT-PCR products. RT-PCR products amplified with fluorescent N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA)-dUTP were applied to 377A automated DNA sequencer (Perkin-Elmer) by using denaturing gel. Fluorescent-signal intensity of each lane was quantified by using GeneScan 672 software (Perkin-Elmer). Top: sizes calculated from size-standard marker (abscissa). C: quantitative comparisons for GCR RT-PCR products among cell culture chambers. Fluorescent-signal intensity (peak height) at length of 455 bp was normalized to GAPDH and used for representing relative GCR mRNA levels in original samples. Hatched bars, F; open bars, G.

GAPDH mRNA levels. Amplification of GAPDH mRNA was performed at the same time with amplification of the mRNAs for IGF BPs in each sample. Exponential amplification (linearity of the calibration curve, r = 0.987) was also observed for a range of 14-20 PCR cycles (Fig. 9A). The relatively constant amounts of GAPDH mRNA after 18-cycle amplification indicated that quantitative comparison between flight and ground samples was valid for the other genes at a given number of amplification cycles (Fig. 9, B and C). Another calibration, using the fluorescent-labeled dUTP, revealed a similar exponential curve to determine the optimal condition for RT-PCR of GCR (data not shown). These data confirm the feasibility of the quantitative comparison of each gene transcript after RT-PCR.


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  		Fig. 9.   GAPDH mRNA levels in rat osteoblast culture on board spaceflight. A: calibration curve to determine optimal condition in quantitative RT-PCR for GAPDH. Relative amounts of amplified PCR products are shown in semilogarithmic representation. Arrow, no. of PCR cycles for quantitative comparison between F and G cultures. Inset: autoradiograph for amplified RT-PCR products. The radioactivity of the amplified products was indicated with PSL, as described in Fig. 3A. B: autoradiograph of GAPDH RT-PCR products. Arrow, RT-PCR products with expected length (453 bp). C: quantitative comparisons for GAPDH RT-PCR products among cell culture chambers. Radioactivity of each signal was measured by BAS 2000 (Fuji Film) and expressed with PSL, as described for Fig. 3. Hatched bars, F; open bars, G.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Spaceflight generates an environment in which physical loading on the skeleton is extremely limited. One of the consequent physiological changes is bone demineralization. Although the mature human skeleton typically maintains a balanced state of bone turnover, removal of load-bearing stress results in bone loss. No definitive explanation has been proven as to the molecular mechanisms underlying the process of bone demineralization and osteopenia induced by microgravity. Osteoblasts and bone marrow stromal cells produce IGF BPs in response to various stimuli (25). IGF BPs modify the activity of IGF-I, which is locally produced by bone cells (including osteoblasts) and plays an essential role in bone formation (7). Many stimuli for bone formation or resorption modulate endogenous IGF BP production (12). This study focused on IGF BP expression in osteoblast cultures during spaceflight. Specifically, we have examined IGF BP expression in response to vitamin D3 stimulation.

IGF BP-3 is the most prevalent of the IGF BPs in serum and is thought to be critical for IGF transport in the circulation (8). However, studies of the biological functions of IGF BP-3 have generated controversial results; IGF BP-3 seems to have multiple functions in bone formation. For example, IGF BP-3 inhibits IGF-induced stimulation of osteoblasts (27), whereas IGF BP-3 reportedly prolongs the half-life of IGFs by serving as a local reservoir from which IGFs are continuously released into the local environment for autocrine or paracrine action (25). In our study, the IGF BP-3 protein levels in rat osteoblast cultures were significantly increased during spaceflight. The steady-state mRNA levels for IGF BP-3 in flight cultures were two- to threefold higher than those of ground control cultures. Thus, spaceflight enhanced IGF BP-3 production, mediated in part through elevation of the steady-state mRNA levels for IGF BP-3.

IGF BP-5 is abundant in bone, binding with high affinity to hydroxyapatite or with the extracellular matrix in mineralized tissue (17). IGF BP-5 is the only IGF BP known to stimulate bone growth. The production and/or secretion of IGF BP-5 by bone cells enhances attachment of locally synthesized growth factors to the newly mineralized matrix. When associated with the extracellular matrix, IGF BP-5 potentiates IGF-induced mitogenesis in osteoblasts (16). During spaceflight, the mRNA levels for IGF BP-5 were 42-67 and 31-45% of the corresponding ground control values on days 4 and 5, respectively. The reduction of IGF BP-5 mRNA levels in flight cultures suggests that changes in IGF BP-5 may be a factor in spaceflight-induced alterations in bone metabolism.

Much less is known about IGF BP-2 and IGF BP-4 (16). Limited data have shown that IGF BP-4 mRNA levels were increased by severalfold in mouse osteoblastic cells after a 24-h treatment with vitamin D3 (26). Further studies will be required to clarify the roles of IGF BP-2 and -4 in bone metabolism and to ascertain the regulatory mechanisms of gene expression.

It is well known that glucocorticoids enhance bone resorption and suppress bone formation and that excessive amounts of glucocorticoids induce osteoporosis in vivo. However, glucocorticoids have shown complex effects on bone cells in vitro. Glucocorticoids suppress the synthesis of IGF-I and also regulate the synthesis of IGF BPs (9). Three homologous sequences with the consensus glucocorticoid-responsive element (GRE) were identified in the 5'-untranslated flanking region of rat IGF BP-3 gene (2). The cis-regulatory element GRE is essential for glucocorticoid-stimulated promoter activity through specific binding with glucocorticoid receptors; this is consistent with an observation of glucocorticoid-induced increase of IGF BP-3 mRNA levels in neonatal rat hepatic cells (20). In addition, rat IGF BP-1 transcription is stimulated by glucocorticoid, mediated through a functional GRE in the promoter (10). On the other hand, the promoter of rat IGF BP-5 gene does not contain the GRE consensus sequence (31), but it contains the putative 12-O-tetradecanoyl phorbol 13-acetate-responsive element (TRE). It is well documented that the TRE-mediated transcription is repressed by GCR in promoters that contain TRE but not GRE (4). This is consistent with an observation for dexamethasone-induced reduction of IGF BP-5 mRNA levels in human osteoblasts (23).

One possible explanation for the increase of IGF BP-3 and decrease of IGF BP-5 mRNA levels in flight cultures is the increase in GCR mRNA levels during spaceflight. The GCR expression is important for transcriptional regulation by glucocorticoids. However, how cis-regulatory elements and trans-acting factors are involved in transcriptional regulation of IGF BP-3 and -5 genes remains to be elucidated. Alternatively, because vitamin D3 stimulates IGF BP-4 (26) and IGF BP-5 (27) expression, decreased vitamin D3-receptor expression could potentially account for part of this response in microgravity. We observed decreased vitamin D3-receptor expression in these samples (data not shown). Further studies are needed to determine whether either (or both) of these explanations is sufficient to account for the microgravity response.

In summary, our data indicate that IGF BP-3 expression was stimulated, whereas IGF BP-5 expression was inhibited, in normal rat osteoblasts during spaceflight. Glucocorticoid receptor mRNA levels were elevated during spaceflight. Altered IGF BP production during spaceflight would modulate the IGF action, suggesting a potential mechanism that could contribute to spaceflight-induced osteopenia. Further studies are necessary to clarify the role of IGF BPs in bone metabolism on exposure to microgravity.

    ACKNOWLEDGEMENTS

Some of this work was conducted at the Johnson Space Center's Medical Sciences Division while Y. Kumei was a Senior Research Fellow of the National Research Council. The authors thank Dr. Shunichi Shimasaki for supplying cDNAs and antibodies of IGF BPs.

    FOOTNOTES

This study was funded by a grant-in-aid from the Institute of Space and Astronautical Science of Japan (to Y. Kumei) and by National Aeronautics and Space Administration Grant 106-30-12-40 (to P. Whitson and C. Sams).

Address for reprint requests: Y. Kumei, Dept. of Biomaterials Science, Faculty of Dentistry, Tokyo Medical and Dental Univ., Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549, Japan (E-mail: kumei.det2{at}dent.tmd.ac.jp).

Received 7 October 1997; accepted in final form 13 March 1998.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

1.   Akiyama, H., S. Kanai, M. Hirano, Y. Kumei, H. Shimokawa, H. Katano, E. Hara, S. Song, C. Mukai, and S. Nagaoka. An improved quantitative RT-PCR fluorescent method for analysis of gene transcripts in the STS-65 space shuttle experiment. J. Biotechnol. 47: 325-333, 1996[Medline].

2.   Albiston, A. L., R. Saffery, and A. C. Herington. Cloning and characterization of the promoter for the rat insulin-like growth factor-binding protein-3 gene. Endocrinology 136: 696-704, 1995[Abstract].

3.   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].

4.   Bamberger, C. M., H. M. Schulte, and G. P. Chrousos. Molecular determinants of glucocorticoid receptor function and tissue sensitivity to glucocorticoids. Endocr. Rev. 17: 245-261, 1996[Abstract].

5.   Bikle, D. D., J. Harris, B. P. Halloran, and E. Morey-Holton. Altered skeletal pattern of gene expression in response to spaceflight and hindlimb elevation. Am. J. Physiol. 267 (Endocrin. Metab. 30): E822-E827, 1994[Abstract/Free Full Text].

6.   Canalis, E., and B. Gabbitas. Skeletal growth factor regulates 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].

7.   Chen, T. L., L. Y. Chang, D. A. DiGregorio, A. J. Perlman, and Y.-F. Huang. Growth factor modulation of insulin-like growth factor-binding proteins in rat osteoblast-like cells. Endocrinology 133: 1382-1389, 1993[Abstract].

8.   Cohen, P., P. J. Fielder, Y. Hasegawa, H. Frisch, L. C. Giudice, and R. G. Rosenfeld. Clinical aspects of insulin-like growth factor binding proteins. Acta Endocrinol. 124: 74-85, 1991.

9.   Delany, A. M., J. M. Pash, and E. Canalis. Cellular and clinical perspectives on skeletal insulin-like growth factor I. J. Cell. Biochem. 55: 328-333, 1994[Medline].

10.   Goswami, R., R. Lacson, E. Yang, R. Sam, and T. Unterman. Functional analysis of glucocorticoid and insulin response sequences in the rat insulin-like growth factor-binding protein-1 promoter. Endocrinology 134: 736-743, 1994[Abstract].

11.   Hahnel, A., and G. A. Schultz. Insulin-like growth factor binding proteins are transcribed by preimplantation mouse embryos. Endocrinology 134: 1956-1959, 1994[Abstract].

12.   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].

13.   Hirata, S., T. Osada, M. Hirai, K. Hagihara, and J. Kato. Expression of estrogen receptor in the rat brain: detection of its mRNA using reverse transcription-polymerase chain reaction. J. Steroid Biochem. Mol. Biol. 41: 583-587, 1992[Medline].

14.   Hossenlopp, P., D. Seurin, B. Segovia-Quinson, S. Hardouin, and M. Binoux. Analysis of serum insulin-like growth factor binding proteins using Western blotting: use of the method for titration of the binding proteins and competitive binding studies. Anal. Biochem. 154: 138-143, 1986[Medline].

15.   Jee, W. S. S., T. J. Wronski, E. R. Morey, and B. B. Kimmel. Effects of spaceflight on trabecular bone in rats. Am. J. Physiol. 244 (Regulatory Integrative Comp. Physiol. 13): R310-R314, 1983[Abstract/Free Full Text].

16.   Jones, J. I., and D. R. Clemmons. Insulin-like growth factors and their binding proteins: biological actions. Endocr. Rev. 16: 3-34, 1995[Medline].

17.   Jones, J. I., 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/Free Full Text].

18.   Kumei, Y., H. Shimokawa, H. Katano, E. Hara, H. Akiyama, M. Hirano, C. Mukai, S. Nagaoka, P. A. Whitson, and C. F. Sams. Microgravity induces prostaglandin E2 and interleukin-6 production in normal rat osteoblasts: role in bone demineralization. J. Biotechnol. 47: 313-324, 1996[Medline].

19.   Lee, B. Y., T. Huynh, L. E. Prichard, J. McGuire, and M. L. Polan. Gonadal steroids modulate interleukin-1 receptor antagonist mRNA expression in cultured human monocytes. Biochem. Biophys. Res. Commun. 209: 279-285, 1995[Medline].

20.   Luo, J., and L. J. Murphy. Regulation of insulin-like growth factor binding protein-3 expression by dexamethasone. Mol. Cell. Endocrinol. 74: 213-219, 1990[Medline].

21.   McCarthy, T. L., 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].

22.   Morey-Holton, E. R., and S. B. Arnaud. Skeletal responses to spaceflight. Adv. Space Biol. Med. 1: 37-69, 1991[Medline].

23.   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].

24.   Robinson, M. O., and M. I. Simon. Determining transcript number using the polymerase chain reaction: Pgk-2, mP2, and PGK-2 transgene mRNA levels during spermatogenesis. Nuc. Acid Res. 19: 1557-1562, 1991[Abstract/Free Full Text].

25.   Rosen, C., L. R. Donahue, and S. J. Hunter. Insulin-like growth factors and bone: the osteoporosis connection. Proc. Soc. Exp. Biol. Med. 206: 83-102, 1994[Abstract].

26.   Scharla, S. H., D. D. Strong, S. Mohan, D. J. Baylink, and T. A. Linkhart. 1,25-Dihydroxyvitamin D3 differentially regulates the production of insulin-like growth factor I (IGF-I) and IGF-binding protein-4 in mouse osteoblasts. Endocrinology 129: 3139-3146, 1991[Abstract].

27.   Schmid, C. Insulin-like growth factors. Cell Biol. Int. 19: 445-457, 1995[Medline].

28.   Schneider, V. S., A. D. LeBlanc, and L. C. Taggart. Bone and mineral metabolism. In: Space Physiology and Medicine (3rd ed.), edited by A. E. Nicogossian, C. L. Huntoon, and S. L. Pool. Philadelphia, PA: Lea and Febiger, 1994, chapt. 17, p. 327-333.

29.   Shimasaki, S., and N. Liong. Identification and molecular characterization of insulin-like growth factor binding proteins (IGFBP-1, -2, -3, -4, -5 and -6). Prog. Growth Factor Res. 3: 242-248, 1992.

30.   Wronski, T. J., and E. R. Morey. Effect of spaceflight on periosteal bone formation in rats. Am. J. Physiol. 244 (Regulatory Integrative Comp. Physiol. 13): R305-R309, 1983[Abstract/Free Full Text].

31.   Zhu, X., N. Ling, and S. Shimasaki. Cloning of the rat insulin-like growth factor binding protein-5 gene and DNA sequence analysis of its promoter region. Biochem. Biophys. Res. Commun. 190: 1045-1052, 1993[Medline].

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