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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 18, 2002 as doi:10.1096/fj.02-0393fje. |
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Indiana University School of Medicine, Indianapolis, Indiana, USA; and
* The Jackson Laboratory, Bar Harbor, Maine, USA
2Correspondence: Department of Anatomy and Cell Biology, Indiana University School of Medicine, 635 Barnhill Dr., MS 5045, Indianapolis, IN 46202, USA. E-mail: arobling{at}anatomy.iupui.edu
SPECIFIC AIMS
The cross-sectional size of mouse long bones is partly under genetic control. Skeletal loading experiments conducted in different inbred strains of mice reveal that bone tissue’s sensitivity (adaptive response) to mechanical stimulation is also under genetic control. We investigated whether genetic loci governing bone size might exert their influence by modulating mechanosensitivity.
PRINCIPAL FINDINGS
1. Mechanosensitivity is greater in B6.C3H-4T than C57BL/6J mice
Although it is clear that genes are responsible for the degree of bone adaptation to mechanical stimulation, the genes contributing to skeletal mechanosensitivity have yet to be identified. Differences in mechanical adaptation observed between C3H/He (C3H) and C57BL/6 (B6) mouse strains suggest that these mice can be used as genetic tools to elucidate the genes involved in mechanosensitivity regulation. We began looking for mechanosensitivity genes in the mouse genome by focusing on bone size, based on our hypothesis that mice with larger bone size might have a genetic predisposition for greater mechanosensitivity than mice with smaller bone size, i.e., perhaps adult bones that are larger became that way because they were more sensitive to routine mechanical loading signals encountered during normal cage activities. We have mapped several quantitative trait loci (QTL) for bone density in the mouse genome. Some of these QTLs also conferred differences in bone size (external diameter and polar moment of inertia), with the most significant locus mapped to chromosome 4 (Chr. 4). To examine whether a QTL that affects bone size might influence mechanosensitivity, we subjected the right ulna of skeletally mature B6 mice and B6.C3H-4T mice (4T) to short (0.5 min) bouts of mechanical loading in vivo once a day for 3 days (Fig. 1
B). These two mouse strains are genetically identical (i.e., they are both B6), with the exception of a small region of Chr. 4 (a QTL affecting bone size) that derives from the C3H mouse stock (a less mechanosensitive mouse strain) in the 4T animals (see Fig. 2
). When normalized to mechanical strain Fig. 1A
), loading induced a greater osteogenic response and a more mechanically advantageous geometric adaptation in the 4T mice vs. B6 mice (Fig. 3
). No differences in osteogenic threshold were detected between mouse strains.
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2. Bone size and formation rates are greater in B6.C3H-4T compared with C57BL/6J mice
To further test our hypothesis that greater mechanosensitivity during growth and development would lead to larger bones in the adult, we evaluated whether the Chr. 4 QTL had an effect on bone formation rates during growth and on bone size/geometry in bones from adult animals engaged in routine mechanical loading (normal cage activity). Fluorochrome-derived bone formation rates measured in the midshaft femur and ulna of growing (6-wk-old) 4T mice were 48–98% greater than in B6 mice of the same age. Cross-sectional geometric and areal properties in adult animals were significantly greater (11–28%) in nonloaded 4T ulnae compared with nonloaded B6 ulnae (Fig. 3)
.
CONCLUSIONS AND SIGNIFICANCE
Our main objective in this study was to determine whether the presence of a small portion of C3H Chr. 4 (which is known to exert a significant influence on bone cross-sectional size) bred into the B6 genome would influence skeletal mechanosensitivity. Our results indicate that 4T mice were more responsive to loading than B6 controls. The histomorphometric results indicate that mechanically induced bone formation in the 4T ulna was roughly 68 to 75% greater than that in the B6 ulna at the moderate (2500 µ
) and high (3000 µ) strains tested. The greater responsiveness in the 4T ulna was manifest as an enhanced capacity to form new bone per unit mechanical strain, but not as a change in the minimum level of strain required to initiate a response (osteogenic threshold).
The C3H ulna is less responsive to mechanical loading in terms of osteogenic threshold and bone formation per unit strain than the B6 ulna. Note that a genetic locus derived from a less mechanosensitive mouse strain (C3H) can enhance mechanosensitivity in a mouse strain that is already highly mechanosensitive (B6). This paradox suggests that the C3H mice possess some genes that increase skeletal responsiveness to mechanical loading in addition to genes that suppress mechanosensitivity. Our findings that 4T mice have greater mechanosensitivity than B6 controls might explain why the adult 4T ulnar (and femoral) cross section is larger than that from B6 mice.
The Chr. 4 QTL is a large (from 40–80 cM) region with hundreds or perhaps thousands of genes. Our confirmation of this QTL role in bone mechanotransduction suggests a potentially lucrative locus to begin fine mapping and further probing for mechanosensitivity genes. The association between adult bone size and mechanosensitivity elucidated in our experiment suggests that a significant portion of the variation in bone size (and potentially strength) could be explained by genes governing mechanosensitivity.
A number of candidate mechanosensitivity genes reside in the Chr 4 QTL, including 2 neurotransmitter receptors (5-hydroxytryptamine [serotonin] receptor 1D [Htr1d] and an ionotropic glutamate receptor [Grik3]), the leptin receptor (Lepr), and bone morphogenic protein 8 (Bmp-8; also called osteogenic protein-2). Serotonin plays a role in development of the craniofacial skeleton, and osteoblasts express a functional serotonin signaling system (including functional 1D receptors). Bone cells also possess functional glutamate signaling machinery; ionotropic glutamate receptors and glutamate transporters have been identified in osteoblasts and osteocytes. Moreover, glutamate transporter expression appears to be regulated by mechanical loading. Although these pathways are somewhat novel to bone biology, there is considerable evidence that neurotransmitter signaling (e.g., serotonin, glutamate) plays a significant role in mechanotransduction. Leptin has been shown to regulate bone formation. Leptin signaling, which occurs through leptin receptors (Lepr) in the hypothalamic nuclei, has downstream effects that can inhibit osteoblast function. Finally, a wealth of data indicates that Bmps can stimulate bone formation in vivo, suggesting that the Bmp-8 could play a role in the response we observed.
Adequate bone mass and strength are key factors in the prevention of osteoporotic fracture. Exercise is a simple yet effective way to stimulate bone formation and retard bone loss, which can ultimately improve bone mass and strength. However, the benefits of exercise depend on the sensitivity of bone cells to mechanical stimulation. Our data suggest that mechanosensitivity in murine bone has a strong genetic component and that a gene or set of genes on mouse Chr. 4 modulates mechanosensitivity. Extrapolating our findings to humans, perhaps individuals possessing alleles for low sensitivity, does not acquire the same osteogenic returns from exercise as individual possessing high-sensitivity alleles. These observations might explain the large range of efficacy found in many exercise intervention studies. The data suggest that human Chr. 1p (the region of the human genome syntenic with the mouse Chr. 4 QTL) might contain important target genes for modulating bone mass.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0393fje; to cite this article, use FASEB J. (December 18, 2002) 10.1096/fj.02-0393fje 
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