Myeloma cells suppress osteoblasts through sclerostin secretion

S Colucci,A Zallone,R Rizzi,F Sardone,E Rinaldi,M Grano,G Mori,A Oranger,V Liso,G Specchia,G Passeri,P Curci,G Brunetti

doi:10.1038/bcj.2011.22

Abstract

Wingless-type (Wnt) signaling through the secretion of Wnt inhibitors Dickkopf1, soluble frizzled-related protein-2 and -3 has a key role in the decreased osteoblast (OB) activity associated with multiple myeloma (MM) bone disease. We provide evidence that another Wnt antagonist, sclerostin, an osteocyte-expressed negative regulator of bone formation, is expressed by myeloma cells, that is, human myeloma cell lines (HMCLs) and plasma cells (CD138+ cells) obtained from the bone marrow (BM) of a large number of MM patients with bone disease. We demonstrated that BM stromal cells (BMSCs), differentiated into OBs and co-cultured with HMCLs showed, compared with BMSCs alone, reduced expression of major osteoblastic-specific proteins, decreased mineralized nodule formation and attenuated the expression of members of the activator protein 1 transcription factor family (Fra-1, Fra-2 and Jun-D). Moreover, in the same co-culture system, the addition of neutralizing anti-sclerostin antibodies restored OB functions by inducing nuclear accumulation of β-catenin. We further demonstrated that the upregulation of receptor activator of nuclear factor κ-B ligand and the downregulation of osteoprotegerin in OBs were also sclerostin mediated. Our data indicated that sclerostin secretion by myeloma cells contribute to the suppression of bone formation in the osteolytic bone disease associated to MM.

Highlights

Osteoblastogenesis involves the differentiation of BM stromal cells (BMSCs) into functional OBs, implicating the activation of different transcription factors such as Cbfa1/Runx[2], which directly regulates the expression of the major OB markers such as collagen type I (COLL I), osteopontin, bone sialoprotein II (BSP II) and osteocalcin (OSTC).[9,10]
Wnt pathway is tightly regulated by several secreted antagonists, that is, soluble frizzled-related proteins, which interfere with Wnt/Frizzled receptor binding, or Dickkopf (DKK) proteins and sclerostin, which bind the co-receptor LRP5/6.19 The OB suppression occurring in MM bone disease has been related to the inhibition of the canonical Wnt signaling, through DKK1 and sFRP-2 and -3 secreted from the myeloma cells; otherwise, there are no literature data on a possible involvement of sclerostin, except for those showing high serum level of sclerostin in MM patients.[24]
By real-time PCR, the lowest level of sclerostin mRNA was of anti-sclerostin mAb at two different concentrations partially found in controls, whereas it was several folds higher in almost restored colony-forming unit-fibroblast (CFU-F) and colony-forming unit-OB (CFU-OB) formation (Figures 2a and b)

Summary

Introduction

Multiple myeloma (MM) is a hematological B-cell malignancy that results from the clonal expansion of plasma cells within bone marrow (BM), mostly with overproduction of monoclonal immunoglobulins.[1,2,3] Symptomatic MM is defined by the evidence of end-organ or tissue damage attributable to plasma cell proliferation according to the CRAB criteria, that consist of C: hypercalcemia (411.5 mg/dl); R: renal failure (serum creatinine 4173 mmol/l); A: anemia (hemoglobin o10 g/dl or 2 g/dl below the lower limit of normal); and B: bone disease (lytic lesions, severe osteopenia or pathological fractures).[2,3] Symptomatic MM needs appropriate therapy, and it is differentiated from monoclonal gammapathy of undetermined significance (MGUS) and asymptomatic (smoldering) MM basedOsteoblastogenesis involves the differentiation of BMSCs into functional OBs, implicating the activation of different transcription factors such as Cbfa1/Runx[2], which directly regulates the expression of the major OB markers such as collagen type I (COLL I), osteopontin, bone sialoprotein II (BSP II) and osteocalcin (OSTC).[9,10] Another relevant transcription factor is the activator protein 1 (AP-1), which consists of multiple dimers of proteins belonging to Fos (c-Fos, FosB, Fra-1 and Fra-2) and Jun families (c-Jun, JunB and JunD).[11]. Wnts are secreted cysteine-rich glycoproteins known as regulators of hematopoietic and mesenchymal cell differentiation as well as of embryonic development.[14,15,16] The activation of canonical Wnt signaling, induced by binding of Wnt proteins to both Frizzled receptor and low-density lipoprotein receptor-related protein (LRP-5/6) co-receptor, is followed by b-catenin translocation into the nucleus,[17,18] resulting in the activation of major OB transcription factors. Wnt pathway is tightly regulated by several secreted antagonists, that is, soluble frizzled-related proteins (sFRPs), which interfere with Wnt/Frizzled receptor binding, or Dickkopf (DKK) proteins and sclerostin, which bind the co-receptor LRP5/6.19 The OB suppression occurring in MM bone disease has been related to the inhibition of the canonical Wnt signaling, through DKK1 Wnt pathway is tightly regulated by several secreted antagonists, that is, soluble frizzled-related proteins (sFRPs), which interfere with Wnt/Frizzled receptor binding, or Dickkopf (DKK) proteins and sclerostin, which bind the co-receptor LRP5/6.19 The OB suppression occurring in MM bone disease has been related to the inhibition of the canonical Wnt signaling, through DKK1 (ref. 20) and sFRP-2 and -3 (refs 21–23) secreted from the myeloma cells; otherwise, there are no literature data on a possible involvement of sclerostin, except for those showing high serum level of sclerostin in MM patients.[24]

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Conclusion