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Author contributions: C.E.: carried out most of the experiments, data analysis and interpretation, collection and assembly of data; A.B., H.B., and C.B.: analysis of bone micro-architecture and biomechanical parameters of mice, manuscript writing; L.Z., M.S., and Z.T.: microarray analysis, data analysis and interpretation; F.M.: contribution to the mice study, data analysis and interpretation; V.B., G.F.C., and L.E.: provision of patients, data analysis and interpretation; N.W.: histological analysis of mice tissues, data analysis and interpretation; E.L.: provision of study material, data analysis and interpretation; C.D.: data analysis and interpretation, manuscript writing; G.A.: conception and design, data analysis and interpretation, manuscript writing; E.A.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing.
The regulation of skeletal homeostasis represents an active research area as the elderly population is steadily rising. Among its physiopathological consequences, osteoporosis represents a major health problem, affecting more than 30% of women above 50 years old [1, 2]. The resistance and integrity of bone depend upon the balance between bone resorption by osteoclasts and bone formation by osteoblasts [3, 4]. The decrease in bone mass that occurs in osteoporosis—for example in association with hypogonadism—involves an acceleration of bone turnover and a disequilibrium between resorption and formation in favor of bone resorption [5, 6]. Whereas most osteoporosis treatments target bone resorption by inhibiting osteoclasts formation and activity, only a few promote bone formation. Identification of such drugs would enable the development of alternative and/or complementary treatments. In addition to bone loss, osteoporosis is associated with an increased bone marrow adipose tissue, leading to the formation of adipocytes at the expense of osteoblasts . Osteoblasts and adipocytes share the same mesenchymal cell precursor, as illustrated by the ability of mesenchymal stem cells (MSC) isolated from different tissues to differentiate into either lineage. MSC have been considered as suitable sources of adult progenitors for cell-based therapy because of easy isolation technique, expandability, and pluripotency. As human adipose-derived MSC represent a more abundant and available source of cells for autologous cell replacement than human bone marrow-derived MSC (hBMSC), interest in human adipose-derived MSC has been growing in the recent years. Recently, we isolated MSC from human adipose tissue termed hMADS (i.e., human multipotent adipose-derived stem) cells. These cells, which exhibit both a normal karyotype and high self-renewal ability, are able to differentiate into various lineages, including adipocytes and osteoblasts, and can also support in vivo regenerative processes [8, , –11].
In view of the reciprocal and inverse relationship that exists between osteogenesis and adipogenesis, controlling the fine balance between the two pathways is of clear therapeutic significance [12, 13]. However, the interplay between the two cell types and the decision to commit to either lineage are not well characterized, and a better understanding of the two pathways would be useful for the development of new drug therapies.
To gain better insight into the early steps of osteoblast and adipocyte differentiation in MSC, we performed first a microarray RNA analysis of hMADS cells at different time points of osteogenesis or adipogenesis using a previously developed microarray , data deposited in Array Express, accession number A-MARS three and E-MARS 10. As a basis of selection, we focused our attention on genes (i) differentially expressed during osteogenesis and adipogenesis, (ii) encoding for cell surface receptors, and (iii) modulated by estrogens. Oxytocin receptor (otr) emerged as a candidate gene using these criteria.
OTR, known to be a member of the heptahelical G protein-coupled receptor family, is expressed in a variety of cell types, including osteoblasts and adipocytes [15, –17]. Its ligand, oxytocin (OT), belongs to the pituitary hormone family and regulates the function of peripheral target organs. It also modulates a wide range of behaviors, such as social recognition, love, and fear [18, , –21]. OT had been suggested to play a role in bone homeostasis and osteoporosis based on the proliferative effects of OT on osteoblasts in vitro and the modulation of blood parameters associated with bone formation of normal rats [22, –24]. As a next step, we examined whether oxytocin could modulate in vitro the osteoblast/adipocyte balance using hMADS cells and hBMSC. Subsequently, the relationship between circulating OT levels and osteoporosis was analyzed as well as the effects of OT injection on bone loss in ovariectomized mice. Finally, plasma OT levels were determined in postmenopausal women suffering or not from osteoporosis and were found to be consistent with animal data. Our results show for the first time that OT signaling is implicated in the regulation of the osteoblast/adipocyte balance and reverses osteoporosis in ovariectomized mice, suggesting the use of OT as a potential therapeutic treatment.
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- Materials and Methods
- Disclosure of Potential Conflicts of Interest
- Supporting Information
A few reports have suggested the role of OT favoring osteogenesis [22, 23] whereas OT injection in normal rats appears to improve blood parameters associated with bone formation . So far, no evidence has been reported that shows that OT controls stem cell fate and could be envisioned as a therapy for osteoporosis. Herein, our data reveal that OT plays a major role in the osteoblast/adipocyte balance of MSC. Adipose-derived MSC are commonly used to study differentiation processes because of their abundance and availability. Although human adipose-derived MSC exhibit some differences with human bone marrow stromal/stem cells, many similarities have also been reported [47, , –50]. Herein, similar results were obtained when hMADS cells and hBMSC were treated with OT or Cb during differentiation in a DLP medium, indicating that both MCS are suitable cell models for studies of osteoblast/adipocyte balance.
The effects of OT on osteoblast and adipocyte differentiation are not mediated through increase in cell number as OT treatment of hMADS cells affected neither cell number nor the doubling time (data not shown). These findings are different from those reported by Petersson et al. who showed a stimulation of DNA and protein synthesis by OT in osteoblast-like cells and some osteosarcoma cells [23, 51]. Most likely, these discrepancies are due to differences in cell models and/or culture conditions.
Several modulators of the osteoblast/adipocyte balance have been described. RhoA signaling, which may induce changes in cell shape, has been reported to be involved in the osteoblast/adipocyte balance of human mesenchymal stem cells . We have observed that OT did not affect the activation of RhoA pathway in hMADS cells differentiating to either the osteogenic or the adipogenic lineage. Recently, it has been shown that liver-enriched inhibitory protein (LIP), an isoform of CCAAT/enhancer binding protein β (C/EBPβ) lacking the transcriptional activation domain, plays an important role in the control of the balance between osteoblast and adipocyte differentiation . We have observed no significant effect of OT on the levels of LIP or both isoforms of C/EBPβ during hMADS cell differentiation toward osteoblasts or adipocytes (data not shown). However, OT induced a transient phosphorylation of ERK1/2, suggesting a potential role of this pathway in the OT effects. These data are consistent with observations showing that activation of ERK1/2 leads on one hand to phosphorylation of CBFA1, the osteoblast differentiation key transcription factor, which enhances osteogenesis, and on the other hand to phosphorylation of PPARγ, the adipocyte differentiation master gene, known to inhibit its adipogenic activity [41, 42]. Further detailed analysis of the ERK and other pathways, such as Wnt pathway, will shed light on the fine mechanisms implicated in the beneficial effects of OT in osteoporosis.
Transcription of the ot and otr genes is under the control of estrogens [53, 54]. Therefore, as the estrogen level is decreased in OVX mice and rats as well as in postmenopausal women, the level of OT is lower as well. It is thus tempting to speculate that hypogonadal-induced bone loss is linked to low OT levels, and that restoring OT levels could therefore reverse osteoporosis. Interestingly, recent reports have shown that sera from postmenopausal, but not premenopausal, women promote adipogenesis of mesenchymal stromal cells at the expense of osteogenesis [55, 56]. Our data strongly suggest that circulating OT is a key hormone that may account for these observations. Although OT-deficient mice display impairments in milk ejection and social recognition, and OTR-deficient mice exhibit disorders in several aspects of social behavior, bone defects remain to be shown in these mice [57, –59].
Furthermore, we show for the first time that OT levels are inversely correlated with the development of osteoporosis in both rodents and humans. Interestingly, injecting OT into OVX mice, an animal model of osteoporosis, reversed the disequilibrium in the osteoblast/adipocyte balance, allowing the restoration of bone loss and the recovery of bone's normal biomechanical properties. As OT favored osteogenesis in vitro, it is tempting to postulate that it improves bone formation in vivo and therefore can be considered as an anabolic hormone. This hypothesis is in agreement with a previous report showing that OT injection in normal rats improves bone remodeling in favor of bone formation as circulating levels of Receptor Activator for Nuclear factor-κB Ligand (RANKL) decreased and those of Osteprotegerin (OPG) increased, leading to a decrease in RANKL/OPG ratio . Furthermore, it has been shown that (i) OT stimulates PGE2 synthesis in both undifferentiated and differentiated human osteoblastic cells, (ii) PGE2 increases bone turnover favoring bone formation, and (iii) PGE2 inhibits adipocyte differentiation. Taken together, these observations are in favor of OT exerting its effects through PGE2 [16, 60, –62].
Increased bone resorption by osteoclasts is considered as the main cause of hypogonadism-induced bone loss. As OTR is expressed and functional in human osteoclasts , it cannot be excluded that the increased bone mass observed in OT-treated OVX mice could be mediated through the inhibition of osteoclast differentiation and/or activity.
Recent reports have suggested that bone mass and remodeling are centrally controlled [64, 65]. Consistent with this, elevated levels of follicle-stimulating hormone (FSH) resulting from ovariectomy were reported to enhance bone resorption, and this effect was reversed by hypophysectomy [66, –68]. It remains controversial, however, whether or not this effect of FSH is direct . In any case our results favor the possibility that circulating OT from the pituitary gland plays a role in the control of bone mass but does not exclude the possibility that OT may also act centrally to direct bone homeostasis.
Several drugs have been reported to contribute to the reversal of bone loss, for example, by favoring osteogenesis or through antiresorptive effects [70, –72]. Therapies developed to treat bone diseases in humans are considered to be either antiresorptive (including bisphosphonates, selective estrogen-receptor modulators, calcitonin, and vitamin D) or anabolic agents (parathyroid hormone) [5, 73, 74]. For most of these treatments, if not all, side effects have been reported, that is, osteonecrosis, dysphagia, esophagitis, headache, nausea, arthralgy, dizziness, and others [75, –77]. As OT has already been safely administered to patients for other indications , it represents a highly promising molecule for the effective treatment of osteoporosis.