OPLL is an osteogenic disorder characterized by the ectopic bone formation of the PLL in the spine. Although the histopathology of this disease has been well studied, the knowledge of the signaling pathway that regulates the development and progression of OPLL is still at its infancy. Being a secreted antagonist of the Wnt/β-catenin pathway, Dkk1 inhibits osteoblast differentiation and bone formation during skeletal development and may function as an important serological molecular marker for osteoporosis and malignant bone diseases, such as bone metastasis and osteosarcoma [9–11]. However, it remains unknown whether Dkk1 impacts the development and progression of OPLL. In this study, we examine the serum level of Dkk1 and show that this Wnt inhibitor is downregulated in OPLL patients as compared to non-OPLL patients. The expression of Dkk1 is also reduced in OPLL ligament-derived fibroblasts. These findings imply that Dkk1 may act as a negative regulator in the pathogenesis of OPLL. Notably, our results are supported by a previous study, in which a decreased serum level of Dkk1 has been documented in patients suffering from spondyloarthropathies, including ankylosing spondylitis (AS), diffuse idiopathic skeletal hyperostosis (DISH), ossification of the yellow ligament (OLY), as well as OPLL [12].
The central role of the Wnt/β-catenin signaling in bone development and fracture repair has been extensively studied. However, the implication of this critical pathway in the pathogenesis of OPLL has not been reported. DISH (also called ankylosing hyperostosis) is closely associated with OPLL, in that this disease is most commonly characterized by ossification of the anterior longitudinal ligament (ALL) on the anterolateral surface of vertebrae, eventually leading to fusion of the spine [13]. As such, DISH and OPLL have some common histopathological features such as ossification of spinal longitudinal ligament [14]. Overexpression of β-catenin in endplate cartilage cells has been reported to induce extensive osteophyte formation and fusion of adjacent vertebra in the entire spine, which is similar to the defects observed in DISH disease [15]. Interestingly, activation of the Wnt pathway also plays an important role in the process of excessive bone formation in AS, an inflammatory spine disorder that causes abnormal bony outgrowth [16]. In our study, we find that the expression of β-catenin is increased in ligament fibroblasts of OPLL, indicating the accumulation of this protein. Importantly, we report a higher TCF reporter activity and gene expression of Wnt target genes including c-myc, cyclin D1, and Axin2 in OPLL fibroblasts than in non-OPLL cells. These results support the notion that downregulation of Dkk1 in OPLL cells is associated with stabilized β-catenin, which activates TCF-dependent transcriptional activity.
Notably, our results seem different from Shi et al., who have reported that OPLL is mediated by osterix (a transcription factor for osteoblasts) via the inhibition of the β-catenin signaling, in that during dexamethasone (Dex)-induced osteogenic differentiation of PLL cells, osterix was increased whereas β-catenin was reduced in response to Dex treatment [17]. This discrepancy seems highly plausible due to the different experimental conditions used. In Shi’s study, Dex was used to initiate the osteogenic differentiation of PLL cells [17]. Although Dex can induce osteoblast differentiation in a variety of cell types, this glucocorticoid has been shown to inhibit the Wnt pathway. For instance, Dex promotes adipogenic differentiation of mesenchymal progenitor cells by inducing Wnt antagonists (including Dkk1 and WIF1) and activating GSK-3β, thus leading to the inhibition of the Wnt/β-catenin pathway [18]. Dex also inhibits chondrogenic differentiation by repressing β-catenin and TCF/LEF-mediated transcription [19]. Therefore, it is not surprising that treatment with Dex in PLL cells could lead to the downregulation of β-catenin.
Besides the observations that Dkk1 is downregulated whereas β-catenin signaling is upregulated in OPLL ligament fibroblasts, we further provide evidence that Dkk1 functionally regulates OPLL at the cellular level. Dkk1 has been shown to inhibit proliferation in diverse cell types [20–22]. This Wnt inhibitor is also known to promote apoptosis [23, 24]. In agreement with these studies, we show here that treatment with recombinant Dkk1 also inhibits proliferation but promotes the apoptotic activity of OPLL fibroblasts. As such, Dkk1 possesses a growth-inhibitory effect in ligament cells.
As a Wnt inhibitor, Dkk1 has been critically implicated in the skeletal system. Heterozygous knockout of the Dkk1 gene increases bone formation and bone mass [8]. In contrast, overexpression of Dkk1 in osteoblasts leads to severe osteopenia [7]. In our study, we provide evidence that Dkk1 inhibits BMP2-induced osteogenic differentiation of ligament cells, supported by the following observations that 1) treatment with Dkk1 reduces ALP activity; 2) Dkk1 inhibits gene expression of osteoblast markers in OPLL cells; 3) Dkk1 also represses osteoblast mineralization. Moreover, we show that Dkk1 attenuates Wnt3a-upregulated ALP activity and expression of osteoblast markers in OPLL cells, suggesting that Dkk1 inhibits osteogenic differentiation via its suppression of the Wnt pathway. All these findings support that Dkk1 functions as a negative regulator in the pathogenesis of OPLL, and downregulation of Dkk1 favors the ossification of the ligament by promoting cell growth and survival, as well as accelerating osteogenic differentiation. In addition, our results also suggest that targeting the Wnt/β-catenin pathway using Dkk1 or small molecule inhibitors may represent a potential treatment against the development and progression of OPLL.
In general, we report that Dkk1 is downregulated in OPLL patients and ligament-derived cells, which is associated with activation of the β-catenin-mediated signaling pathway. Functionally, we show that Dkk1 exerts a growth-inhibitory effect and inhibits osteogenic differentiation of ligament cells by targeting the Wnt pathway. Our results demonstrate for the first time that Dkk1 acts as a negative regulator in the pathogenesis of OPLL. Targeting the Wnt pathway using Dkk1 may serve as a therapeutic strategy against OPLL.