Bortezomib inhibits maturation and function of osteoclasts from PBMCs of patients with multiple myeloma by downregulating TRAF6
Introduction
Over 85% of patients with multiple myeloma (MM) have osteolytic bone disease (OBD) [1], characterized by severe bone pain, pathologic fractures, spinal cord compression and hypercalcemia. These devastating events may substantially reduce functional independence and quality of life. Even in patients who are responsive to chemotherapy, progressive skeletal pathologies are not infrequent [2].
The phenomenon of enhanced bone resorption in MM due to the imbalance of bone remodeling in bone marrow microenvironment, including increased activation of osteoclasts and impairment of osteoblasts [3]. Osteoclasts and OBs are systematically affected by a number of soluble factors induced by the malignant clone within the marrow microenvironment. On the other hand, myeloma cells are susceptible to growth inhibition by osteoblasts [4] and activation of osteoclasts [3]. The vicious circle between enhanced tumor growth and increased bone resorption results in exclusively osteolytic bone lesions and development of disease [5].
The soluble factors in marrow microenvironment particularly include receptor activator of NF-κB ligand (RANKL) [3], a TNF family member, which was shown to be expressed on stromal cells, osteoblasts [6] and myeloma cells [7]. It has been reported the sRANKL/OPG ratio increased and was correlated with markers of bone resorption and osteolytic lesions [8], showing that it plays a critical role in osteoclast differentiation and regulation. RANKL acts through a trans-membrane receptor RANK which is expressed on osteoclastic lineage cells [6]. When acting with macrophage-colony stimulating factor (M-CSF), it can induce osteoclast differentiation from bone marrow cells or human PBMCs, even in the absence of osteoblastic stromal cells and other osteotropic factors [9].
As a multifunctional second messenger activated by RANKL, TRAF6 is critical for RANK-induced activation of downstream signal factors such as NF-κB. In vivo experiments showed that TRAF6 gene knocked developed severe osteopetrosis due to impaired bone resorption [10], [11]. It has been recently shown in the context of osteoclastogenesis that TRAF6 forms a signaling complex containing RANK and TAK-1-binding protein (TAB)2, resulting in TGF-β-activated kinase (TAK)1 activation [12], and then activation of NF-κB, AP-1 and p38 pathways, which are crucial for osteoclast differentiation, survival, and function [13], [14]. Ubiquitin ligase activity mediated by RING finger motif of TRAF6 (Lys-63-linked auto-ubiquitination) is important for its ability to activate downstream factors [15], [16].
As a member of the proteasome inhibitors’ family, bortezomib (VELCADE™; formerly PS-341) is the first peptide boronate to enter clinical trials. Many studies have demonstrated its antitumor activity in a variety of tumor types, especially in MM. In the ubiquitin–proteasome pathway, Lys-48-linked poly-ubiquitination marks proteins for degradation by the proteasome, which confers specificity on protein degradation in cells’ cycles. Among these proteins IκB is most notable, phosphorylation, ubiquitination and thereby degradation by proteasome of IκB can release transcription factor NF-κB. As bortezomib specifically inhibits 26S proteasome, NF-κB has been logically considered to be a key target for bortezomib-induced apoptosis [17]. However, the inhibition of NF-κB by bortezomib could not fully explain anti-tumor effects mediated by bortezomib. Compared with the specific IKK inhibitor, Hideshima et al. [18] found that bortezomib was more potent than the specific IKK inhibitor PS-1145, although both abolished NF-κB activation, suggesting that other signaling pathways or molecules may be targeted by bortezomib.
In addition to their anticancer properties, bortezomib modulate inflammatory and immune responses by affecting the function and survival of immune cells such as lymphocytes and dendritic cells [19]. In this article we hypothesized that the proteasome inhibitor bortezomib might abolish osteoclast differentiation and functioning, and attempted to find out the relationship between bortezomib and TRAF6, the upper signal factor of NF-κB in osteoclasts.
Section snippets
Cultivation of osteoclasts
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of 8 patients with MM, who had achieved complete remission (CR) or very good partial response (VGPR) after treatment, using Ficoll–Hypaque density gradient (φ = 1.007). Isolated mononuclear cells were cultured at a density of 2 × 106 cells/cm2 in 10 cm petri dishes in a-MEM (GIBCO, Grand Island, NY, USA) supplemented with 10% FCS, 100 U/ml penicillin, 100 ng/ml streptomycin, 50 ng/ml M-CSF (PerProTech, London, UK) and 50 ng/ml
Confirmation of osteoclastic lineage cells
After collection of PBMCs and purification by 0.25% trypsin/0.02% EDTA, cells were cultured in the presence of RANKL and M-CSF. In our previous test, we showed that preosteoclasts on day +3 began to exhibit TRAP positive, while on day +14 cells fused to giant, multinucleated osteoclasts, about 200–300 nm in diameter, positive for TRAP staining (Fig. 1A), and capable of bone resorption at day +28.
Designated doses of bortezomib do not reduce preosteoclast survival
In the previous experiment, we demonstrated that the apoptotic concentration of bortezomib was 20 nM.
Discussion
It has been known that osteoclasts and their activators are very important for myeloma cell growth and survival [8], and recent studies have demonstrated that bortezomib can alter interactions between myeloma cells and the marrow microenvironment [20], [21]. Terpos et al. [22] reported that bortezomib administration significantly reduced serum DKK-1, sRANKL as well as bone resorption markers including TRAP-5b and CTX, leading to increased bone formation in myeloma patients. In addition,
Acknowledgement
Thanks for technical help from Gao Jianjun, PhD, of the Bone Metabolic Department, Research Center of Geriatric Medicine, Institute of Radiation Medicine, Fudan University.
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