 was significantly higher in the 50 mg/kg β-AET dose group (p<0.05, vs. vehicle; Fig. 4C). The observation is consistent with β-AET opposition to GC-induced suppression of chondrocyte proliferation [43]. Since chondrogenesis is responsible for forming a template upon which osteoblasts deposit bone [44], [45], the GC-induced suppression and the integrity of the proximal tibial epiphyseal growth plate by thermal trauma may have been alleviated by β-AET.
Male BALB/c mice (n = 10 per group) were subjected to 20% total body surface area burn and treated (sc injection) with vehicle alone, or with β-AET (25 or 50 mg/kg) immediately after thermal trauma. An identical treatment was given 48 hours later and then 3 times per week for 4 weeks. Cancellous bone morphometry and histomorphometry were measured at the proximal tibial metaphysis of mice (A, B). The endochondral growth rate was measured at the tibial epiphyseal growth plate (C) Endocortical eroded surface was measured at the surface of the mid-diaphyseal shaft of the femur (D), as an indicator of osteoclastic bone. ‘#’, ‘b’ and ‘*’ indicate significant differences from the sham control, baseline and vehicle group, respectively, p<0.05. Data are expressed as means ± SEM.
β-AET reduces endocortical surface bone erosion
To assess osteoclast-mediated bone erosion, we measured endocortical surface erosion. The thermal trauma increased erosion by over two-fold at the mid-diaphyseal region of femur (∼6% to 15%, ‘sham vs. ‘vehicle’ control, p<0.05) and β-AET prevented bone erosion (Figure 4D). This observation is consistent with attenuation of the glucocorticoid-mediated osteoclast activation by β-AET.
Levels of β-AET in human plasma decrease with age
The concentration of β-AET in human plasma ranged from 2 to 162 pg/mL in males (Figure 5 Top) and from 6 to 249 pg/mL in females in our study (Figure 5 Bottom). Levels of β-AET in human plasma decrease with age. Linear regression analysis revealed significant non-zero slopes for males (p = 0.02; r2 = 0.06) and females (p<0.0001; r2 = 0.12). There was no significant difference in age between males (46.5 +/− 13.6) and females (45.6 +/− 12.5) in our sample, however, females had a significantly (p = 0.0002) higher BMI than males (32.0 +/− 6.4 versus 28.7 +/− 5.6, respectively). Serum levels of DHEA were available for 75 males and 68 females (data not shown). In these subjects, serum levels of DHEA also declined with age in both males and females (p<0.0001 for each; r2 = 0.21 and 0.22, respectively). As reported elsewhere (Auci et al., submitted) serum levels of AET were highly correlated with serum levels of DHEA in both the males (p = 0.0008; r2 = 0.15) and the females (p<0.0001; r2 = 0.51). Serum levels of β-AET were also positively correlated with body mass index (BMI) and linear regression analysis of β-AET versus BMI revealed significant non-zero slope for males (p = 0.005; r2 = 0.076) and females (p<0.0001; r2 = 0.172).
β-AET levels were measured in plasma samples taken from 102 males (aged 20–80) and 150 females (aged 20–73) by reverse phase LC-MS/MS. Linear regression analysis revealed significantly non-zero slope for males (p = 0.02; r2 = 0.06) and females (p<0.0001; r2 = 0.12).
Discussion
We have shown that β-AET reverses GC-induced suppression of IL-6, IL-8 and OPG expression by human osteoblast-like MG-63 cells and induced osteoblast differentiation in human bone marrow derived MSC. In the mouse thermal injury model, β-AET treatment blunted the loss of bone mineralization, restored chondrocyte-mediated endochondral bone growth and slowed osteoclast-mediated cortical bone erosion. Whole body bone mineral content, but not density, was restored to normal levels. Finally, we showed that in men and women, levels of β-AET decline with age. These observations relevant to the prevention of osteopenia and may have therapeutic implications for the treatment of osteoporosis.
Osteoporosis is estimated to already affect 10 million Americans and almost 34 million more are estimated to have low bone mass, placing them at increased risk for osteoporosis. Bisphosphonates, such as Fosamax, which inhibit bone re-absorption, are commonly prescribed, despite side effects including nausea, abdominal pain, difficulty swallowing and the risk of an inflamed esophagus or esophageal ulcers. Although infrequent, serious side effects with bisphosphonates include osteonecrosis of the jaw, irregular heartbeats and visual disturbances. The drug inhibits bone-resorption and has been reported to produce brittle bones that result in an alarming frequency of spontaneous fractures [46], [47]. Selective estrogen receptor modulators (SERMs) such as Raloxifene mimic estrogen's beneficial effects on bone mineral density in postmenopausal women without the more serious side effects associated with estrogen replacement. However, these drugs also act by inhibiting bone reabsorption. The thyroid hormone calcitonin, given as a nasal spray, is less often prescribed because of its low potency compared to the bisphosphonates. Teriparatide is a powerful analog of parathyroid hormone used to treat osteoporosis in postmenopausal women and men who are at high risk of fractures. In contrast to other treatments, teriparatide acts by stimulating new bone growth. However, it must be given once a day by injection under the skin on the thigh or abdomen and the long-term effects are unknown. Thus, the development of safe, convenient and cost effective treatment options remain a significant medical and economic imperative.
The potential role of β-AET in bone formation relevant to the treatment of osteoporosis may be highlighted by our in vitro findings related to β-AET –induced OPG expression in Dex-treated MG-63 osteoblasts. OPG is a soluble decoy RANK receptor, secreted by osteoblasts, that binds surface RANK ligand (RANKL) on osteoblasts. This decreases the pool of RANKL available for RANK receptor binding, decreasing activation of RANK receptor on pre-osteoclasts and ultimately osteoclast commitment and differentiation [48]. Since bone formation and resorption can be uncoupled and are independent processes, the upregulation of OPG alone might tip the balance towards bone formation [49]. β-AET also attenuated GC-induced suppression of IL-6. Although high expression of IL-6 has been proposed to contribute to bone loss in GIOP, the clinical data is conflicting and it appears that the up-regulation of soluble IL-6 receptor (sIL-6Rα and its ratio to soluble gp130 co-receptor governs the effects on bone during inflammation [50], [51], [52], [53]. Local hyper-production of IL-8 has been suggested to play a specific role in subchondral bone loss, since it suppresses osteoblast activity and promotes osteoclast recruitment [54]. While not reaching statistical significance in this study, β-AET opposed GC-induced attenuation of IL-8 signaling.
The present studies are in agreement with recent work of Urban et al., that also suggested a beneficial effect of -AET on bone formation. Those studies used the immortalized human fetal osteoblast cell line hFOB-9 [55] and showed that β-AET suppressed PPARγ activation. PPARγ is essential for adipogenesis, endocrine function of adipose tissue and stem cell fate decisions that lead to the adipogenic commitment at the expense of the osteoblastic lineage [56]. The activation of PPARγ, as in diabetic patients managed with the thioglitazones, leads to higher incidence of bone loss. Conversely, PPARγ suppression shifts mesenchymal stem cell differentiation towards the osteoblastic lineage that supports bone formation. [56], [57], [58], [59], [60], [61], [62].
Our findings extend observations of Loria and colleagues [31], [63] to cells other than lymphocytes and macrophages and suggest β-AET attenuates the effects of GC in bone physiology. Mice that received a 20% TBSA full thickness scald injury (equivalent to third degree burn) exhibited dramatically increased bone erosion mediated by high osteoclast activity, IL-6 and GC levels, that leads to lower cancellous bone formation parameters 4–7 days later [34], [35]. Significant short-term osteoclast-mediated bone surface erosion was prevented by β-AET in a dose-dependent manner. In a feed-forward mechanism, the endocrine and cytokine signals initiated by thermal trauma may converge at the level of the hypothalamic-pitutary-adrenal axis and amplify both direct and indirect GC effects on the bone. These may include the disruption of normal osteoimmunological interactions that involve osteoblasts as well as marrow stromal cells (reviewed in [64]). Functional GC receptors have been reported on bone chondrocytes, mononuclear cells and endothelial cells, suggesting they may also be involved [65]. Even though β-AET protected BMC, it showed no effect on BMD after one month. Other factors, besides stress-induced increases in GC contribute to bone loss in thermally