Bone Sclerostin and Dickkopf-related protein-1 are positively correlated with bone mineral density, bone microarchitecture, and bone strength in postmenopausal osteoporosis

Osteoporosis is a major bone metabolic disease characterized by low bone mass and deterioration of the bone microarchitecture, resulting in fragility and increased risk of fracture. Postmenopausal osteoporosis (PO), the most common type of osteoporosis, is mainly caused by imbalanced bone turnover, which is characterized by enhanced bone resorption exceeding bone formation following estrogen decline [1]. A large number of studies have reported the critical roles of osteoblasts and osteoclasts, which are the main executors of bone formation and bone resorption, respectively, in the pathogenesis of PO. Recent breakthrough studies highlighted the central role of osteocytes in bone remodeling and revealed that osteoblasts and osteoclasts are under the control of osteocytes [2, 3].

The canonical Wnt pathway plays a key role in determining the fate of mesenchymal stem cells, which favors the maturation and survival of osteoblasts [4]. In addition, the Wnt pathway promotes the expression of osteoprotegerin rather than RANKL, thus indirectly inhibiting osteoclast activity [5, 6]. The canonical Wnt pathway in the bone is mainly regulated by osteocytes, which act through the production of sclerostin and Dickkopf-related protein 1 (Dkk-1), [7‐10]. By preventing the binding between Wnt and specific cell surface receptors (Frizzled and lipoprotein receptor-related protein 5/6), sclerostin and Dkk-1 block the activation of the Wnt canonical pathway; furthermore, β-catenin is sequestered and degraded in the absence of Wnt. Therefore, sclerostin and Dkk-1 are potent inhibitors of bone formation [4, 11, 12].

In vitro evidence has shown that estrogen inhibits the expression of sclerostin in osteocytes, mesenchymal stromal cells, and osteoblastic cells [13‐15]. In addition, in vivo data from an ovariectomized mouse model and clinical population investigations indicated that sclerostin levels are negatively correlated with estrogen levels [16‐18]. Conversely, sclerostin antibody has been shown to significantly increase bone mineral density (BMD) in women with PO [19‐21]. Similarly, Dkk-1 antibody has been demonstrated to aid the recovery of bone formation in animal models [22]. In conclusion, sclerostin and Dkk-1 play pivotal roles in the pathogenesis of PO and have emerged as valuable treatment targets.

However, the association between sclerostin and Dkk-1 and BMD in postmenopausal women remains controversial. Reportedly, as blood sclerostin levels increase, BMD decreases in postmenopausal women, and postmenopausal women with significantly increased serum Dkk-1 exhibit more significant osteoporosis [23, 24]. Other studies have suggested paradoxical associations between circulating sclerostin and Dkk-1 and BMD, revealing that serum sclerostin and Dkk-1 are positively associated with BMD in women with PO [25‐29].

Although sclerostin is mainly secreted by osteocytes, recent studies have demonstrated that other cells and tissues, including osteoblasts, vascular smooth muscle cells, and livers, are also sources of sclerostin [30, 31]; therefore, we speculated that the serum levels of sclerostin and Dkk-1 could not reflect those in the bone. Because sclerostin and Dkk-1 levels reported in most previous studies were measured in the serum, we measured sclerostin and Dkk-1 levels directly in the bone tissue and further elucidated the associations between bone sclerostin and Dkk-1 and BMD, bone microarchitecture, and bone strength.

In this study, the levels of sclerostin and Dkk-1 in the trabecular bone were measured and were found to be positively correlated with BMD, bone microarchitecture, and bone strength. To our knowledge, this study is the first to report the bone protein levels of sclerostin and Dkk-1 in Chinese postmenopausal women and to show the association between protein levels of sclerostin, Dkk-1, and bone microarchitecture and bone strength.

Sclerostin and Dkk-1 are secreted primarily by osteocytes and inhibit bone formation by blocking the osteoblast Wnt/β-catenin signaling pathway [4]. Downregulation of sclerostin and Dkk-1 is associated with significantly increased bone formation. Moreover, it has been demonstrated in both animal models and clinical experiments that the administration of sclerostin antibody is markedly effective in the prevention of osteoporosis after the decrease in estrogen level [22, 34, 35]. In addition, estrogen has been shown to decrease sclerostin expression [13, 14, 18]. Therefore, based on the results of several studies, it is reasonable to expect that sclerostin and Dkk-1 would be inversely correlated with BMD in postmenopausal women. Although a previous study supported this notion that as blood sclerostin and Dkk-1 levels increased, BMD decreased in postmenopausal women, there is more evidence indicating that BMD is positively associated with sclerostin and Dkk-1 [23‐29, 36, 37]. Most of these studies analyzed data on serum sclerostin, Dkk-1, and BMD measured by DXA. Considering serum sclerostin and Dkk-1 are also secreted by other cells and tissues, which might not reflect the levels of sclerostin and Dkk-1 in the bone, we measured the protein levels of sclerostin and Dkk-1 in the trabecular bone directly and assessed the local BMD by micro-CT. Our results demonstrated that the serum levels of sclerostin and Dkk-1 could reflect the bone levels of sclerostin and Dkk-1. Consistent with the results from the serum data, we found that the bone protein levels of sclerostin and Dkk-1 were positively associated with BMD. Moreover, these results were also supported by the mRNA levels of sclerostin and Dkk-1 reported by Jemtland et al. [38]. More importantly, the only study that reported the bone protein levels of sclerostin and Dkk-1, published recently, also demonstrated a positive relationship between bone sclerostin and Dkk-1 and BMD [39]. Compared with this study, one advantage of our study was that the specimens were kept in storage for a relatively short time, which could prevent the degradation of the protein levels of sclerostin and Dkk-1. Another paradoxical association discovered in our study was that protein levels of sclerostin and Dkk-1 were negatively associated with age; however, another study reported that serum sclerotin increased with age in men but not in women. We believe that the natural life of osteocytes after estrogen withdrawal and aging led to the paradoxical results mentioned above. Osteocytes constitute more than 90–95% of bone cells in the adult skeleton and are extremely long-lived cells that survive for up to decades in the bone matrix [40]. The life span of osteocytes is most likely determined by the rates of bone turnover, the process by which osteoclasts resorb the bone and osteoblasts replace the resorbed bone [3]. In postmenopausal women, as estrogen declines, bone turnover accelerates, which leads to the apoptosis of osteocytes [3]. It has been shown in bone samples from aged patients with hip fracture that the number of osteocytes and osteocyte activity decreased, whereas osteocyte apoptosis increased [41]. Therefore, a reasonable explanation for the positive correlation of bone sclerostin and Dkk-1 levels with BMD may be that these two proteins are mainly produced by live osteocytes and the number of live osteocytes decrease because of increased apoptosis. Consequently, sclerostin and Dkk-1 levels would reflect the number of osteocytes. From another perspective, when women with PO received teriparatide or denosumab treatment, BMD and sclerostin increased, which was accompanied by reduced bone turnover and increased osteocyte number [42, 43]. Conversely, the number of live osteocytes decreased significantly with age, which might have resulted in the reduced levels of bone sclerostin and Dkk-1.

In addition to BMD, we also evaluated bone microarchitecture and bone strength. Higher levels of sclerostin and Dkk-1 might predict better bone microarchitecture and bone strength. This finding was in accordance with that of Szulc et al., who reported that bone microarchitectural parameters are positively correlated with sclerostin in men [28]. Osteocyte number and activity play a crucial role in determining bone strength and microarchitecture. Ablation of osteocytes rapidly results in decreased bone strength and osteoporosis [44‐46]. Hence, as sclerostin and Dkk-1 are mainly secreted by osteocytes, they could reflect the number and activity of osteocytes. Therefore, the bone levels of sclerostin and Dkk-1 could also reflect bone microarchitecture and strength.

However, this study has several limitations. First, no bone specimens were obtained from healthy women as a control group. Second, the number of patients enrolled in the study was relatively low. This might be the reason why we did not find any association between bone sclerosin and Dkk-1 and P1NP and β-CTX, whereas other studies, including a total of 100 patients, reported that serum sclerostin was negatively correlated with β-CTX and P1NP [27, 29]. Third, each patient lacked estrogen dectection, although we confirmed that every patient was postmenopausal according to the medical history; fourth, the patients enrolled in the study were all Chinese women, and our observations originating from bone specimens were consistent with the serum results that were also obtained from Chinese patients; therefore, the results might differ among different races [26, 29].

Based on the findings of previous studies and our research, bone sclerostin and Dkk-1 were positively correlated with BMD in Chinese women with PO, and higher levels of bone sclerostin and Dkk-1 might predict better BMD, bone microarchitecture, and bone strength. The potential molecular mechanisms still require further study.