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J Appl Physiol 84: 2132-2137, 1998;
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Vol. 84, Issue 6, 2132-2137, June 1998

Spaceflight has compartment- and gene-specific effects on mRNA levels for bone matrix proteins in rat femur

Glenda L. Evans1, Emily Morey-Holton2, and Russell T. Turner1

1 Department of Orthopedics, Mayo Clinic, Rochester, Minnesota 55905; and 2 National Aeronautics and Space Administration Ames Research Center, Moffett Field, California 94035

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the present study, we evaluated the possibility that the abnormal bone matrix produced during spaceflight may be associated with reduced expression of bone matrix protein genes. To test this possibility, we investigated the effects of a 14-day spaceflight (SLS-2 experiment) on steady-state mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), osteocalcin, osteonectin, and prepro-alpha (1) subunit of type I collagen in the major bone compartments of rat femur. There were pronounced site-specific differences in the steady-state levels of expression of the mRNAs for the three bone matrix proteins and GAPDH in normal weight-bearing rats, and these relationships were altered after spaceflight. Specifically, spaceflight resulted in decreases in mRNA levels for GAPDH (decreased in proximal metaphysis), osteocalcin (decreased in proximal metaphysis), osteonectin (decreased in proximal and distal metaphysis), and collagen (decreased in proximal and distal metaphysis) compared with ground controls. There were no changes in mRNA levels for matrix proteins or GAPDH in the shaft and distal epiphysis. These results demonstrate that spaceflight leads to site- and gene-specific decreases in mRNA levels for bone matrix proteins. These findings are consistent with the hypothesis that spaceflight-induced decreases in bone formation are caused by concomitant decreases in expression of genes for bone matrix proteins.

bone histomorphometry; Northern analysis; bone formation; weightlessness; osteoporosis

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

RADIAL BONE GROWTH is often decreased in long bones of growing male rats during spaceflight (1, 17, 45, 47). The decrease in the periosteal bone formation rate is primarily due to a reduction in the mineral apposition rate. A decrease in labeled perimeter has not been reported, suggesting that near weightlessness results in a depression in osteoblast activity but no change in osteoblast number. Interestingly, the inhibitory effects of spaceflight on radial bone growth are not uniform; the magnitude of the inhibition varies with anatomic site (38). This perturbation in growth leads to a change in bone geometry (19, 29, 38, 39). Furthermore, the nonuniform decrease in radial growth suggests that the reduction in the ground reaction force is insufficient to fully account for the skeletal response to spaceflight. Local changes in the magnitude of the mechanical loads transmitted to bone through insertions of muscle and other soft tissues may be an important contributing factor.

Strength in the rat femur was reduced after a 19-day spaceflight compared with that in age-matched weight-bearing ground controls (29). The major part of the deficit can be accounted for by the reduction in radial bone growth and resulting change in bone geometry; however, there may also have been a degradation in the material properties of the bone (19, 39, 40). This latter speculation is supported by transmission and scanning electron microscopy studies that revealed major disturbances in the organization of the bone matrix produced during spaceflight (34). Other studies suggest that spaceflight results in delay in the quality and maturity of the bone mineral (27, 28).

The effects of orbital free fall on other bone envelopes are less certain. There is evidence for decreased cancellous bone formation in normal male rats during spaceflight (16, 35, 42). On the other hand, increased bone resorption, rather than decreased bone formation, may be responsible for bone loss in pregnant (41) and ovariectomized rats (6). Dynamic cancellous bone histomorphometry has been performed in very few spaceflight studies. This is due, in part, to inherent difficulties in applying conventional fluorochrome labeling protocols to rats during spaceflight. Additionally, conventional bone histomorphometry provides no information regarding the composition of the bone matrix. An alternative approach to identifying changes in bone matrix synthesis is to assay mRNA levels for bone matrix proteins after spaceflight. Steady-state message levels for matrix proteins, especially type I collagen, are well correlated with dynamic bone histomorphometry (31, 33). The purpose of the present study of growing rats was to ascertain the effects of spaceflight on mRNA levels for three important matrix proteins (collagen, osteocalcin, and osteonectin) in the principal bone compartments of the femur.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals. The animal procedures were reviewed and approved by the National Aeronautics and Space Administration (NASA) Institutional Animal Care and Use Committee (protocol no. 93-010A).

Twelve male Sprague-Dawley rats were obtained from Harlan (Prattsville, AL). The rats were 38 days old at launch. The ground control and flight animals (6 rats per group) were individually housed in similar cages (research animal holding facility). The rats were flown for 14 days (SLS-2 mission) aboard the space shuttle Columbia (STS-58). Because of logistical constraints associated with moving the animals from the orbiter to the laboratory, the time interval between reloading (which begins at the reentry burn to bring the orbiter out of low earth orbit) and euthanasia was 5.5-6 h. The rats were killed by anesthesia (metafane) and exsanguination.

The femurs were quickly excised and frozen. They were shipped in a package with dry ice to Mayo Foundation. The bones were stored at -84°C until the RNA was isolated.

RNA isolation. Each femur was subdivided by blunt dissection into the distal epiphysis including articular cartilage, distal metaphysis, diaphysis, and proximal metaphysis including articular cartilage. The frozen samples were homogenized in 2 ml of guanidine isothiocyanate with the use of a Spex Freezer Mill (Edison, NJ). Total cellular RNA was extracted and isolated by using a modified organic solvent method (8), and the yields were determined spectrophotometrically. The quality of the RNA was verified after separation of 1 µg of each sample by nondenaturing acrylamide gel electrophoresis (Fig. 1).


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  		Fig. 1.   Representative electrophoresis of total cellular RNA on a nondenaturing gel showing intact 18S and 28S ribosomal RNA. Of each sample of RNA isolated from the femur of 3 ground control and 3 flight rats, 1 µg was electrophoresed on a 1% acrylamide gel, and the separated bands were photographed while illuminated with ultraviolet light.

Ten micrograms of each sample were denatured by incubation at 52°C in a solution of 1 M glyoxal, 50% dimethylsulfoxide, and 0.1 M NaH2PO4. This denatured RNA was separated electrophoretically in a 1% agarose gel. The amounts of RNA transferred were assessed by methylene blue staining of the nylon filters and hybridization of the filters with a 32P-labeled cDNA to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Northern blot analysis. The RNA was transferred overnight via capillary action in 20× sodium citrate solution (SSC) buffer to an Amersham Hybond nylon membrane (Arlington Heights, IL) and cross-linked with an ultraviolet Stratalinker 1800 (Stratagene, LaJolla, CA) before hybridization. The filters were prehybridized for 6 h at 45°C in a buffer containing 50% deionized formamide, 10% dextran sulfate, 5× SSC (1× SSC = 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0), 600 µg/ml of heat-denatured single-strand salmon sperm DNA, and 2× Denhardt's solution. Hybridization was carried out for 18-24 h in a buffer containing the above ingredients in addition to a minimum of 106 counts · min-1 · ml-1 of 32P-labeled cDNA probe.

cDNA probes were labeled by random sequence hexanucleotide primer extension by using the megaprime DNA labeling kit from Amersham. The filters were washed for 30 min at 45°C in 2× SSC and for 15-60 min in 0.1× SSC at 45°C. All samples from a given compartment were transferred to a single membrane. The four membranes with RNA from the four bone envelopes were hybridized simultaneously to standardize probe-specific activity and to minimize differences in hybridization conditions. This procedure was performed to allow comparison of the relative levels of expression of a given gene in the four bone compartments normalized to 18S ribosomal RNA. The mRNA bands on the Northern blots were quantified by densitometric scanning using a Molecular Dynamics PhosphorImager (Sunnyvale, CA).

The four cDNA probes used were as follows. 1) Rat prepro-alpha -2-chain of type I precollagen (collagen) was obtained from Dr. C. Genovese (University of Connecticut, Farmington, CT) (12). This is a 1,600-base pair-length fragment inserted into the Pst I site of the plasmid vector pBR322. 2) Rat osteocalcin (OC) was a gift from Dr. S. Rossi-Langer (Genetic Institute, Cambridge, MA) (7). This full-length probe is pR 22-11, which is an EcoR I insert into p5P65. 3) Human osteonectin (ON) was a gift from Dr. G. Long (University of Vermont, Burlington, VT) (43). This is pHVON-9-2 plasmid DNA containing a 546-base pair human ON cDNA insert. 4) Rat GAPDH was a gift from Dr. P. Fort (Laboratoire de Biolgie Moleculaire, Montpellier, France) (11). This pUC18 plasmid DNA contains the full-length rat GAPDH cDNA inserted in the Pst I site.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The rats tolerated spaceflight well. There were no significant changes in either body weight or rate of weight gain between the flight and flight control rats for either experiment (data not shown).

Intact RNA, as ascertained from nondenaturing gel electrophoresis, (Fig. 1) and methylene blue staining of nylon membranes, was isolated from each bone compartment. Representative phosphor images of the Northern hybridizations of matrix proteins have been published in other studies (6, 45).

Pronounced compartment-specific differences in steady-state mRNA levels for GAPDH and bone matrix proteins were found in the normal weight-bearing rats (Figs. 2-5). GAPDH was expressed in all tissues (Fig. 2), with the highest levels of expression found in proximal femoral metaphysis. High levels of OC were expressed in all compartments, with the highest level of expression occurring at the proximal femur. Type I collagen was expressed at all sites, but the distal metaphysis and epiphysis of the femur expressed higher message levels than the shaft and proximal end of the bone. Expression of ON was below the detection limit in the shaft. ON was detected at the other sites, with the highest level measured at the distal metaphysis.


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  		Fig. 2.   Densitometry of phosphoimages of Northern hybridization with a cDNA probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), showing compartment-specific and spaceflight-induced differences in steady-state mRNA levels. Values are means ± SE; n = 3-6. C, control group; F, spaceflight group; NS, not significant.


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  		Fig. 3.   Densitometry of phosphoimages of Northern hybridization with a cDNA probe for osteocalcin (OC) showing compartment-specific and spaceflight-induced differences in steady-state mRNA levels. Values are means ± SE; n = 3-6.


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  		Fig. 4.   Densitometry of phosphoimages of Northern hybridization with a cDNA probe for osteonectin (ON) showing compartment-specific and spaceflight-induced differences in steady-state mRNA levels. Values are means ± SE; n = 3-6. ND, not detected.


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  		Fig. 5.   Densitometry of phosphoimages of Northern hybridization with a cDNA probe for type I collagen (collagen) showing compartment-specific and spaceflight-induced differences in steady-state mRNA levels. Values are means ± SE; n = 3-6.

GAPDH was significantly reduced in proximal femur after spaceflight. Therefore, message levels for the matrix proteins were normalized to 18S ribosomal RNA. Spaceflight resulted in compartment-specific alterations in steady-state mRNA levels for the bone matrix proteins. The message levels for the three matrix proteins were reduced in the proximal femur. mRNA levels for ON and type I collagen were reduced in distal metaphysis. No changes were detected in the shaft or distal epiphysis.

The selective changes in steady-state mRNA levels after spaceflight resulted in compartment-specific alterations in the abundance of the three messages relative to each other. This effect was especially pronounced in proximal femur and distal metaphysis. In the former compartment, OC message was reduced compared with the other matrix proteins; in the latter tissue, however, the messages for the other proteins were reduced compared with OC.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the present study, collagen mRNA levels were measured to evaluate site-specific changes in bone matrix synthesis after spaceflight. The results demonstrate that spaceflight decreases collagen message levels in the proximal and distal metaphyses. In contrast, spaceflight had little effect on collagen message levels in the diaphysis and distal epiphysis.

Type I collagen comprises the majority of the organic matrix of bone (24). It is not surprising that a very strong correlation between collagen mRNA levels and bone matrix synthesis determined by dynamic histomorphometry was observed for tibial periosteal, endocortical, and proximal metaphysis bone cell compartments (37). A similar strong correlation between collagen mRNA levels and matrix synthesis was observed at the periosteum when age-related decreases in bone formation were investigated (36). These findings imply that message levels for collagen provide an excellent surrogate for conventional measurements of bone matrix production when the latter are not practical, as was the case for cancellous bone sites in this spaceflight. This conclusion is further supported by the observation that collagen mRNA levels and bone matrix synthesis both increase in rat long bones after ovariectomy (6) and treatment with parathyroid hormone (9), but they both decrease during disuse (44) and after treatment with estrogen (32). In contrast, mRNA levels for OC and alkaline phosphatase were more weakly correlated and not correlated with bone matrix synthesis, respectively (36, 37).

Dynamic bone histomorphometry of the femur midshaft from rats flown on the 9-day PARE3 spaceflight revealed a tendency for decreased cortical bone formation at the periosteal surface and a tendency for increased endocortical bone formation (R. T. Turner, unpublished observations). The calculated bone formation rates may underestimate the effects of spaceflight, because the fluorochrome label was given before launch, and the increased loading at liftoff may result in a transient increase in cortical bone formation (1, 32). Nevertheless, these findings are in good agreement with the lack of a change in mRNA levels for collagen observed in this study.

The putative site-specific changes in cancellous bone formation during spaceflight have not been investigated by dynamic histomorphometry. This is largely because of the ubiquitous use of young, rapidly growing animals in which the high rate of longitudinal bone growth and bone modeling results in rapid resorption of the preflight fluorochrome label.

The fluorochrome labeling problem could in theory be reduced or prevented by flying older rats (6) or by administering two sequential fluorochrome labels during the spaceflight. Unfortunately, weight restrictions prevented flying adequate numbers of older, heavier rats. Additionally, mechanisms for administration of fluorochrome labels during spaceflight are not routinely available. Despite these major limitations, previous studies (6, 16, 35, 38, 41, 42, 45, 48) provide strong circumstantial evidence for site-specific decreases in bone formation in rat long bones during spaceflight.

The specific functions of noncollagenous bone matrix proteins are only partially understood. OC and ON are quantitatively among the most important of these proteins, with each comprising 10-20% of the total noncollagen matrix proteins (13, 23, 30). OC has been implicated as having a role in the regulation of the bone remodeling cycle. OC gene knockout in mice results in increased bone mass (10). ON binds to calcium ions and collagen and also influences in vitro mineralization (25, 26, 30). Neither protein is uniformly distributed throughout bone; profound differences for both proteins have been reported between human cancellous and cortical bone (21). The significance of these differences is unknown but is probably important to establishment of the functional differences between the bone envelopes and compartments.

Spaceflight and hindlimb elevation, a ground-based model for spaceflight, both result in bone specific changes in mRNA levels for bone matrix proteins (1-3, 17, 45). Additionally, spaceflight results in gene-specific alterations in expression of bone proteins within individual bone compartments. This is most easily illustrated by comparing the proximal and distal metaphyses. Spaceflight resulted in large decreases in mRNA levels for ON and collagen at both sites, but OC was decreased only at the proximal site. Such differential changes could lead to alterations in the composition as well as rate of matrix synthesis. This possibility should be tested in a future long-term spaceflight study by regional analyses of bone matrix composition.

The mRNA levels for bone matrix proteins were measured ~6 h after reweighting. It is unlikely that the results were influenced by reestablishment of gravitational loading. We have performed detailed time course studies after reloading following hindlimb unloading. The results revealed a transient stimulation of mRNA levels for matrix proteins, but these changes required reloading for 8-12 h for cancellous bone and nearly 24 h for cortical bone (R. T. Turner and E. Morey-Holton, unpublished observations).

The mRNAs for collagen and ON, and to a much lesser extent for OC, are expressed by nonbone cells. However, the results of localization studies evaluating [3H]proline radioautography, immunohistochemistry, and in situ hybridization indicate that the vast majority of the messages detected in tissue extracts in the present study were expressed by cells of the osteoblast lineage (4, 5, 14, 15, 20, 46). If this interpretation is correct, then the level of matrix protein expression shows pronounced compartmental specificity in bone. Although differences in message levels need not necessarily reflect proportional differences in protein levels, the weight of evidence suggests a strong association for collagen. Furthermore, it is not unreasonable to hypothesize that the known regional differences in composition of organic matrix of bone (21) are related to differences in the level of expression of the genes for the matrix proteins.

In summary, the results of this study are consistent with the hypothesis that spaceflight-induced site-specific decrease in bone formation is caused by concomitant decreases in the overall expression of genes for bone matrix proteins. Additionally, we speculate that differential changes in gene expression lead to alterations in the composition of the bone matrix, which in turn lead to the observed alterations in matrix ultrastructure and mineralization.

    ACKNOWLEDGEMENTS

These studies were supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-35651, the Mayo Foundation, and NASA Grants NAG2-896 and NCC5-289.

    FOOTNOTES

Address for reprint requests: R. T. Turner, Orthopedic Research, Rm. 3-69 Medical Science Bldg., Mayo Clinic, 200 First St. SW, Rochester, MN 55905.

Received 6 October 1997; accepted in final form 19 February 1998.

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Abstract
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Results
Discussion
References

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J APPL PHYSIOL 84(6):2132-2137
8570-7587/98 $5.00 Copyright © 1998 the American Physiological Society


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