By treating animals with BB-supplemented diets either for a short term prior to puberty or long term continuously from weaning through adulthood, we have discovered that bone loss in adulthood resulting from sex-steroid deficiency (herein referred as OVX-induced bone loss) could be prevented. Our results suggest that the protective effect of dietary BB against bone loss is due to its ability to increase cytoskeletal organization of osteoblastic cells, which allows cells to maintain their appropriate differentiation potential and function and which makes them resistant to senescence.
We have presented results supporting a novel mechanistic paradigm of how OVX-induced bone loss proceeds and how dietary BB stimulates bone formation through enhancing myosin expression in osteoblasts and their precursors. Our findings indicate that organization of an appropriate cytoskeletal structure may drive stem cells to differentiate into a functional and tissue-specific mature cell. In particular, motor proteins in non-muscle cells, such as myosin described in this report, may act as a shuttle between cytoplasm and nucleus for essential osteoblastic cell differentiation factors such as Runx2 (the runt domain-containing transcription factor). The presence of this shuttle in osteoblastic cell precursors in early life is particularly important to preserve the fidelity of stromal cells for their differentiation potential later in development. We have demonstrated Runx2 is a critical mechanistic factor linking dietary BB, expression of cytoskeletal proteins and lineage-specific cell fate, proliferation and growth. Interestingly, our data suggest that OVX-induced degradation of bone matrix may secondarily effect osteoblastic cell differentiation and function through down-regulation of myosin expression leading to a second phase of bone loss after OVX. This extended effect has not previously been documented, and suggests a novel mechanistic explanation for the extent of OVX-induced bone loss which may involve metabolic programming, perhaps through epigenetic modulation.
Theoretically, the lower the peak bone mass acquired by the menopause, the higher the risk of osteoporosis later in life and the earlier it begins. Accordingly, with all other factors being approximately equal (such as, general health and age at menopause), women with significantly greater peak bone mass at menopause would be expected reach osteoporotic bone conditions later in life. The age at which bone is acquired and the diet are two critical factors thought to be important in determining peak bone mass. For example, it is known that bone formation during childhood and adolescence is very critical, and nearly half of the peak bone mass is acquired during these years 
. Aside from known dietary components required for bone development and health (such as, adequate protein, calories, calcium, phosphorus, and vitamin D), certain other dietary factors such as phytochemicals may increase bone development and bone mass which ultimately reduce the risk or degree of osteoporotic bone loss later in life. We have previously reported that weaning rats with only 2 weeks of BB supplemented diet had remarkable increases in bone density and bone mineral content without affecting normal growth and there were no gender differences 
. It was concluded that dietary BB had a positive effect on building peak bone mass. The significance of such increased peak bone mass in early life by BB for buffering bone loss in an adult model of estrogen deficiency (ovariectomy) is the topic of this report.
Bone loss occurs with increasing age in females is directly linked to loss of ovarian function 
. The pathophysiology of this “ovary-related” bone loss is very complicated and cannot be simply explained by either increased bone resorption or decreased bone formation 
. Currently, treatment of postmenopausal osteoporosis has been with drugs and hormone therapy, but some of the approved treatments have specific negative side effects such as mastalgia and increased risks of breast cancer and endometrial hyperplasia. Furthermore, none of these treatments has been able to satisfactorily solve long-term problems of bone loss. The results presented in the current study suggest that early life nutrition may promote higher bone mass/quality which is maintained into adulthood and protects against sex-steroid deficiency-induced bone loss: a condition modeling postmenopausal bone loss in women. Consistent with our findings, previous clinical and animal studies have also demonstrated that increased bone mass in early life produced by either appropriate nutrition or exercise persist into early adulthood 
. A recent rodent study showing that neonatal mice treated with soy isoflavones produced significant increases in adult bone quality 
. Although the underlying mechanisms still need to be defined, the above cited studies are consistent with our findings.
Moreover, our findings indicate that bone marrow mesenchymal stromal cells can be programmed postnatally by a dietary factor to commit and differentiate into functional osteoblasts in later adult life. In adults, appropriate bone remodeling is a fundamental process in which new bone formation is tightly associated with bone resorption to support and preserve skeletal health. Ovarian dysfunction results in increases in bone resorption which trigger transient compensatory increases in bone formation to maintain bone remodeling at a set level. However, bone formation is eventually exceeded by increased bone resorption, a net bone loss results. The effects of dietary BB on osteoblastogenesis and osteoclastogenesis appear similar to that of estrogens. However, the serum estrogen levels did not differ between BB-fed rats and control rats. Therefore, it is unlikely that the effect of BB diet on bone in OVX animals is due to increased levels of estrogen or its related compounds.
One therapeutic approach has used agents with anabolic properties to increase bone formation such as PTH (parathyroid hormone) to treat ovary dysfunction-induced bone loss. Similar bioactive compounds in serum associated with consumption of a BB-containing diet are still under investigation in our laboratory. Dietary BB-induced serum phenolic acids 
may play a key role in protecting against OVX-induced bone loss by stimulating bone formation. Nonetheless, the additional ability of BB diets to prevent bone and osteoblastic cells from acutely entering senescence after OVX is an interesting and novel finding. Previous studies have suggested that osteoblast apoptosis was increased in addition to increased osteoclast activity after OVX in female rodents 
. It is not known whether osteoblast apoptosis is an important consequence of increased bone remodeling after OVX. We believe that the rapid entry into cell senescence may be an important fate of osteoblasts after OVX. As we have demonstrated in this report, the activity of senescent osteoblasts was significantly lower, and their cell shape may be changed, but they can still identified as osteoblasts. This is consistent with our histomorphometric observation which showed increased osteoblast numbers in OVX animals particularly after 1 week. BB diet may increase the life span of osteoblastic stromal stem cells and progenitor self renewal, and may also enhance osteoblast commitment and differentiation. Determining the mechanisms whereby blueberries prevent the OVX-induced osteoblast senescence requires further elucidation. Furthermore, the mechanisms by which OVX triggers osteoblasts to rapidly enter senescence may be fundamental for understanding the true pathophysiology of OVX-induced bone loss.
Microarray data analyses revealed a cluster of myofibril gene expression particularly myosin related genes that were remarkably down-regulated after OVX in total RNA isolated from bone. Dietary BB significantly inhibited OVX-induced suppression of myosin expression in bone. Furthermore the expression of myosin, particularly non-cardiovascular myosin, is broad and the spectra of their functions have not been fully described in non muscular tissue. We believe that myosin not only plays a role in supporting cell structure, but also conveys some molecular signals in cells from non muscular tissue, and bone forming osteoblasts or their precursors are unlikely to be an exception. We have shown that at least 6 myosin subtypes are highly expressed in osteogenic cells and osteoblastic stromal cells. Reduction of myosin expression in skeletal muscle after OVX has been reported 
, and interestingly, during osteoclastogenesis, myosin IIA may be temporarily suppressed 
. Moreover, recent studies have suggested that myosin may play a role in driving a cell to remodel, and eventually influence stromal cells to differentiate into tissue-specific cells 
. We have found evidence that myosin expression in osteogenic cells and mesenchymal stromal cells may be associated with maintaining Runx2 expression and shuttling this well known essential osteoblast differentiation transcription factor between cell cytoplasm and nucleus. Runx2 has been shown to establish and maintain cell identity and convey phenotypic information through successive cell division or exit in progeny cells 
, and it is required for osteoblast differentiation 
. The phenomenon of Runx2 shuttling between the cytoplasm and the nucleus has been reported and may depend on microtubules in cancer cells 
. Our data in osteogenic cells indicated an association of myosin with Runx2. Treatment of osteogenic cells with serum from OVX control diet rats decreased both myosin and Runx2 complex, but treatment with serum from BB-fed rats increased this complex. To maintain this complex at the appropriate functional level may be particularly important, because significantly reduced expression of Runx2 level will not only suppress osteoblast differentiation, but also result in a cell entering the senescent stage. This is consistent with previous published evidence showing that Runx2 interacts with histone deacetylase 6 to repress the p21 gene, known as a downstream effecter of p53 that is associated with cell senescence 
Moreover, it has been shown that myosin VI DNA damage is p53-dependent 
. Consuming BB early in life contributes to development or maintenance of the myosin-dependent Runx2 shuttle, and this may also be important to preserve the ability of mesenchymal stromal cells to differentiate into functional osteoblasts later in life under inappropriate stress. However, the mechanistic associations among specific subtypes of myosin, Runx2 and p53 in osteoblastic cells need to be further elucidated.
In conclusion, we have reported that ovariectomy triggers acute bone loss and osteoblastic cell senescence in association with decreased expression of myofibril genes. Consuming a BB diet for just a short period in prepubertal life or continuous consumption starting a weaning both prevented OVX-induced bone loss and osteoblastic cell senescence in adult female rats. We have demonstrated that early exposure of osteoblastic cells or mesenchymal stromal cells to dietary BB maintains long-term cytoskeletal stability by regulation of myosin and Runx2 genes. These results show a significant prevention of OVX-induced bone loss by early life dietary BB, and further provide insight into molecular events of mesenchymal stromal cell senescence and osteoblast differentiation associated with OVX.