Role of Wnt-signaling inhibitors DKK-1 and sclerostin in bone fragility associated with Turner syndrome

Turner syndrome (TS) is a chromosomal disorder resulting from total or partial loss of one of the X chromosomes in phenotypically female patients. Clinical features depend on the karyotype, and usually include short stature, hypergonadotropic hypogonadism due to ovarian dysgenesis, cardiac defects and neurocognitive impairment [1]. About 45% of TS women present with low bone mineral density (BMD) and osteopenia/osteoporosis, with an earlier onset than that observed for postmenopausal osteoporosis [2]. Thus, TS subjects have an increased risk of about 25% of developing fractures compared to the normal population, particularly during infancy and after 45 years of age [3]. Typical features in TS subjects are the reduction of cortical BMD and cortical thickness, while trabecular bone is generally preserved [4, 5].

Bone health depend on the correct balance between deposition of new bone by osteoblasts (OBs) and resorption from osteoclasts (OCs); the former is mainly regulated by Wnt/β-catenin pathway, the latter by the receptor activator of NF-kB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) axis [6, 7]. Multiple factors are responsible for bone impairment in people with TS, as well as estrogen deficiency, haploinsufficiency of X chromosome genes, alterations in vitamin D metabolism, and comorbidities frequently associated with TS and other chromosomopathies, such as autoimmune diseases [8‐10]. In a previous study we demonstrated an enhanced osteoclastogenesis due to high levels of circulating FSH in TS girls before hormonal replacement therapy (HRT). FSH acts both directly by binding its specific receptor on the OCs, and indirectly by enhancing the production of TNF-α, a pro-osteoclastogenic cytokine, from bone marrow macrophages and T-cells. Conversely, the increased osteoclastogenesis observed in TS girls and women on HRT is due to high RANKL levels [11]. RANKL and OPG levels are modulated by canonical Wnt/β-catenin inhibitors, such as Dickkopf-1 (DKK-1) and sclerostin, which are the main regulators of bone mass [12]. The role of these two inhibitors has been demonstrated in several congenital and acquired diseases [13]. In particular, sclerostin and DKK-1 inhibit the differentiation and activity of OBs, decrease OPG levels, and increase RANKL expression in the cells of osteoblastic lineage, thereby shifting the OPG/RANKL ratio in favor of bone resorption [12, 14]. There are no data about the activity of OBs, the bone forming cells, in TS patients. Due to the difficulty to obtain bone biopsy from patients to isolate OBs, our research has been directed to the circulating osteoblast precursors (OCPs), which can be detected by flow cytometry in the peripheral human blood, and are related to osteoblast markers, such as alkaline phosphatase and osteocalcin [15, 16]. In postmenopausal osteoporosis, a decreased percentage of OCPs has been reported [17]. Furthermore, an involvement of this cell population in skeletal growth and fracture repair has been demonstrated [18].

The aim of this study was to deepen the pathophysiology of skeletal fragility in TS subjects by evaluating the serum levels of DKK-1 and sclerostin, as markers of possible impairment of cells of the osteoblastic lineage, as well as the percentage of OCPs.

Interestingly, sclerostin levels were indirectly related with HRT. This finding represented an important issue, because is still debated the age of beginning HRT in TS girls. Estrogen replacement therapy is essential for the induction of puberty and for the maintenance of bone health in TS as well as in other disorders of sexual development [21, 22]. Estrogens regulate epiphysis closure and decrease the growth; however, low estrogen amounts do not alter the effects of rhGH on final height [23]. Furthermore, in childhood the combined therapy with ultra-low-dose estrogen and rhGH may favor the growth [24, 25], and may offer other benefits [26]. Moreover, continuous estrogen therapy improves BMD in TS patients, and the best effects on BMD are negatively related to the starting age of adult-dose estrogen treatment [27]. A recent paper reported that the starting age of estrogen replacement therapy, and the time between the starting ages of estrogen replacement therapy and estrogen–progestin therapy negatively and independently correlated with BMAD, but not with volumetric bone mineral density, after adjustment for BMI and age [28]. Lumbar BMD negatively correlated with the starting age of estrogen–progestin therapy independent of BMI and age. Thus, the authors concluded that an early start of HRT, mainly estrogen–progestin therapy, is central to reach better lumbar BMD in TS young adults [28]. Interestingly, in our TS population, DKK-1 levels were related with BMI–SDS. This is an interesting issue as DKK-1 promotes adipocyte differentiation [29]. It is known that both sclerostin and DKK-1 can affect the expression of RANKL and OPG, pro- and anti-osteoclastogenic cytokines, respectively, in the cells of the osteoblastic lineage [13]. Both sclerostin and DKK-1 were related with the high RANKL and normal OPG levels in TS patients, suggesting that in this syndrome these molecules may directly affect osteoblast activity and indirectly osteoclastogenesis. These findings further confirm our previous results which demonstrated an enhanced osteoclastogenesis in girls and young women with TS [11]. Other researchers have reported a co-regulation between RANKL and DKK-1 expression in different diseases, such as prostate cancer [30], osteosarcoma [31], and in children with 21-hydroxylase deficiency on chronic glucocorticoid treatment [32]. This was also observed in in vitro studies using murine mesenchymal stem cells [33]. Sclerostin levels has also been related to RANKL in pathological conditions, including multiple myeloma [34], and rheumatoid arthritis [35].

With regard to rhGH treatment and its correlation with the levels of Wnt inhibitors, it is important to remember that insulin-like growth factor (IGF-1), which is secreted from hepatocytes by GH, has an anabolic effect on bone growth by suppressing osteoblast apoptosis and enhancing osteoblastogenesis through a stabilization of the Wnt/β-catenin pathway [36]. In addition, IGF-1 reduces bone resorption through the OPG and RANKL system [37].

Unfortunately, we did not find statistically significant differences between TS patients and controls for the percentage of OCPs. Previously, it has been reported that these cells are more numerous and more immature in patients affected by fragility fractures than in osteoporotic patients without fractures [18], and it has been hypothesized that it could be due to a decreased homing ability in the skeleton. In this same study, it is reported that the treatment of patients with teriparatide enhanced the maturation of OCPs, but did not alter their percentage in the circulation [18]. However, it is important to specify that as summarized by Feehan et al. there is little agreement among studies with regard to the characterization of OCPs, with individual research groups using different cellular markers and criteria to identify these cells [38]. This disagreement and contradiction have reduced the applicability to the current research and restricted the progress in this area.

Finally, multiple linear regression analysis highlighted as main predictors of sclerostin levels in TS patients bALP, PTH, 25-OH vitamin D, osteocalcin, lumbar-BMAD-Z-score, and RANKL. Differently, for DKK-1 serum levels the best predictors are PTH, 25-OH Vitamin D, lumbar-BMAD-Z-score, and RANKL.

In conclusion, our results demonstrated that TS patients show higher levels of sclerostin and DKK-1 with respect to the controls which can be related to HRT, and to reduced bone formation markers as well as the increased bone resorption activity.