Dkk1-induced inhibition of Wnt signaling in osteoblast differentiation is an underlying mechanism of bone loss in multiple myeloma

Abstract

Expression of the Wnt signaling inhibitor, DKK1 by multiple myeloma cells is correlated with lytic bone disease in multiple myeloma. However, the mechanism(s) by which DKK1 contributes to this process is not clear. Herein, we analyzed the functional role of canonical Wnt signaling and Dkk1 inhibition of this pathway in bone morphogenic protein (BMP)-2-induced osteoblast differentiation. Osteoblast differentiation was measured by alkaline phosphatase (ALP) activity in murine (C2C12) and human pre-osteoblast (hFOB1.19) and osteoblast-like (Saos-2 and MG63) cell lines. Cytoplasmic β-catenin protein was separated by E-cadherin–GST pull-down assay and analyzed by Western blotting. A dominant negative form of β-catenin, Dkk1 and TCF reporter constructs were transfected into C2C12 cells. C2C12 cells were also transfected with siRNA specific to LRP5/6 to knockdown receptor expression. Canonical Wnt signaling was activated in these cell lines in response to Wnt3a as assessed by increased cytoplasmic, non-phosphorylated β-catenin and TCF/LEF transcription activity. Recombinant Dkk1 and plasma from MM patients containing high levels of Dkk1 blocked Wnt3a-induced β-catenin accumulation. Importantly, Dkk1 abrogated BMP-2 mediated osteoblast differentiation. The requirement for Wnt signaling in osteoblast differentiation was confirmed by the following observations: 1) overexpression of Dkk1 decreased endogenous β-catenin and ALP activity; 2) silencing of Wnt receptor mRNAs blocked ALP activity; and 3) a dominant negative form of β-catenin eliminated BMP-2-induced ALP activity. Furthermore, Wnt3a did not increase ALP activity nor did BMP-2 treatment result in β-catenin stabilization indicating that cooperation between these two pathways is required, but they are not co-regulated by either ligand. These studies have revealed that autocrine Wnt signaling in osteoblasts is necessary to promote BMP-2-mediated differentiation of pre-osteoblast cells, while Wnt signaling alone is not capable of inducing such differentiation. Dkk1 inhibits this process and may be a key factor regulating pre-osteoblast differentiation and myeloma bone disease.

Introduction

Multiple myeloma (MM) is characterized by bone marrow infiltration of malignant plasma cells where they closely interact with osteoblasts and osteoclasts, triggering osteolytic bone lesions. In an effort to elucidate the mechanisms of bone destruction, considerable attention has been focused on increased bone resorption due to increased osteoclast formation and activity [1], [2]. While bisphosphonates block bone resorption by inhibition of osteoclastogenesis, bone formation does not occur, and osteolytic bone lesions are not repaired [3]. Thus, it appears that impaired osteoblast function also plays a role in MM bone lesion development. A number of studies have reported fewer osteoblasts and decreased bone formation in MM patients with higher plasma cell infiltration [4], [5] and unbalanced bone turnover is observed in MM patients [6].

Wnts are a family of 19 secreted glycoproteins that have a well-characterized role in stem cell maintenance and development [7]. Wnt signaling is also involved in bone formation [8] and likely in MM pathogenesis [9], [10]. Many Wnt effects are mediated through β-catenin which plays a pivotal role in the ‘canonical’ Wnt signaling pathway [7]. Upon interaction of Wnt proteins with frizzled (Fz) receptors and low-density lipoprotein-receptor-related protein 5/6 (LRP5/6) [11] co-receptors, β-catenin protein accumulates in the cytoplasm and translocates to the nucleus. Nuclear localization and association with T cell factors (TCF-1, -3, and -4) and lymphoid enhancer-binding factor1 (LEF1) lead to transcriptional activation of target genes that regulate many cellular processes, including cell cycle progression and differentiation [7]. In addition to regulating gene transcription, β-catenin performs a second major function as a structural adaptor protein at cell–cell junctions [12]. Thus, cellular levels and localization of β-catenin reflect a complex dynamics that regulates multiple cell functions.

Emerging data suggest that Wnt signaling plays an essential role in normal bone biology and deregulation of this process contributes to bone disease [13], [14]. A role for Wnt signaling in osteoblast biology was initially suggested by Bradbury et al. [15], however the first direct evidence came from observations that inactivating mutations of the LRP5 gene cause osteoporosis–pseudoglioma syndrome (OPPG) [16]. Subsequently, it was shown that a separate and distinct mutation in the same gene, presumably leading to inhibition of Dkk1 binding, results in high bone density [17], [18]. Furthermore, deletion of LRP5 in a mouse model inhibited osteoblast differentiation [19]. Expression of Wnt10b in transgenic mice increased bone mass [20] and overexpression of Wnt7b and β-catenin in C3H10T1/2 cells induced osteoblast differentiation [21], [22]. However, the exact mechanism by which Wnt signaling affects osteoblast differentiation remains to be elucidated.

The canonical Wnt pathway is regulated by a large number of antagonists, including the Dkk family and secreted frizzled-related proteins (sRFPs). To date, four Dkk proteins have been identified in mammals [23], among which Dkk1 and Dkk2 have been well characterized and found to act as antagonists to the canonical Wnt pathway by binding to LRP5/6 in combination with a second protein designated as Kremen [24], [25]. Recently, in vivo experiments revealed that transgenic overexpression of Dkk1 under the control of the ColA1 promoter leads to decreased bone mass [26]. In agreement with this observation, deletion of Dkk1 expression in mouse osteoblasts results in an increase in bone formation and mass [27]. In relation to MM, we have previously suggested that elevated expression of Dkk1 by myeloma tumor cells is involved in bone lesion formation [28]. Interestingly, the role of DKK1 in promoting bone lesion development appears not limited to MM, but has also been indicated in prostate cancer [29].

Given the role of Wnt and Dkk1 in normal bone formation and disease, we sought to understand the role of canonical Wnt-β-catenin signaling in osteoblast differentiation and to decipher the molecular mechanisms by which Dkk1 promotes osteolytic bone lesions in MM. In the present study, we demonstrate that canonical Wnt signaling in osteoblast precursors is increased in response to Wnt3a and that a steady state canonical Wnt signal is necessary for BMP-2-induced osteoblast differentiation. Importantly, Dkk1 from myeloma cells inhibits this process.