Multiple myeloma (MM) is a hematologic malignancy characterized by accumulation of plasma cells in the bone marrow (BM). Bone destruction is a complication of the disease and is usually associated with severe morbidity. The balance between receptor activator of nuclear factor-κB (NF-κB) ligand and osteoprotegerin (OPG) is of major importance in bone homeostasis. We have recently shown that serum OPG levels are lower in patients with myeloma than in healthy individuals. Here we show that myeloma cells can bind, internalize, and degrade OPG, thereby providing a possible explanation for the lower levels of OPG in the BM of patients with MM. This process is dependent on interaction of OPG with heparan sulfates on the myeloma cells. The results suggest a novel biologic mechanism for the bone disease associated with MM and that treatment of the bone disease with OPG lacking the heparin-binding domain should be considered.

Serum samples drawn at diagnosis from 174 myeloma patients were analyzed for the presence of the heparin sulfate proteoglycan, syndecan-1. Syndecan-1 was elevated in 79% of patients (median, 643 units/mL) compared with 40 healthy controls (median, 128 units/mL),P < .0001. Serum syndecan-1 correlated with the following: serum creatinine, secretion of urine M-component over the course of 24 hours, soluble interleukin-6 (IL-6) receptor, C-terminal telopeptide of type I collagen, β₂-microglobulin, percentage of plasma cells in the bone marrow, disease stage, and serum M-component concentration. In order to evaluate syndecan-1 as a prognostic marker in multiple myeloma, it was entered into a multivariate Cox regression model. Data from 138 patients were available for this analysis. As a continuous variable, syndecan-1 was an independent prognostic parameter in addition to serum β₂-microglobulin and World Health Organization performance status. When syndecan-1 was dichotomized by the best cutoff (66th percentile, 1170 units/mL), the survival difference between the groups was highly significant: “high” syndecan-1 group had a median survival of 20 months, and the “low” syndecan-1 group had a median of 44 months (P < .0001). We conclude that syndecan-1 is a new independent prognostic parameter in multiple myeloma, and its role in prognostic classification systems should be further investigated.

Interleukin-21 (IL-21) is a recently cloned cytokine with homology to IL-2, IL-4, and IL-15. In this study we examined the effects of IL-21 on human myeloma cells. We found that IL-21 induced proliferation and inhibited apoptosis of the IL-6–dependent human myeloma cell lines ANBL-6, IH-1, and OH-2. The potency of IL-21 was close to that of IL-6 in the OH-2 cell line. Neutralizing antibodies to IL-6 or the IL-6 receptor transducer chain (gp130) did not affect IL-21–induced DNA synthesis, indicating that IL-21–induced proliferation was not mediated through these proteins. Tumor necrosis factor (TNF), another stimulator of myeloma cell growth, up-regulated the expression level of IL-21 receptor (IL-21R), and combinations of TNF and IL-21 gave synergistic effects on myeloma cell proliferation. Furthermore, 4 of 9 purified samples of primary myeloma cells showed a significant increase in DNA synthesis on stimulation of the cells by IL-21. By Western blot analysis, we demonstrated that the intracellular signaling pathways of IL-21 in myeloma cells involved phosphorylation of Jak1, Stat3, and Erk1/2 (p44/42 mitogen-activated protein kinase). IL-21 is a novel growth and survival factor in multiple myeloma and may represent a target for future therapy.

The bone disease in multiple myeloma is caused by an uncoupling of bone formation from bone resorption. A key difference between patients with and patients without osteolytic lesion is that the latter have fewer and less active osteoblasts. Hepatocyte growth factor (HGF) is often produced by myeloma cells and is found at high concentrations in the bone marrow of patients with multiple myeloma. Here we show that HGF inhibited bone morphogenetic protein (BMP)–induced in vitro osteoblastogenesis. Thus, HGF inhibited BMP-induced expression of alkaline phosphatase in human mesenchymal stem cells (hMSCs) and the murine myoid cell line C2C12, as well as mineralization by hMSCs. Furthermore, the expression of the osteoblast-specific transcription factors Runx2 and Osterix was reduced by HGF treatment. HGF promoted proliferation of hMSCs, and the BMP-induced halt in proliferation was overridden by HGF, keeping the cells in a proliferative, undifferentiating state. BMP-induced nuclear translocation of receptor-activated Smads was inhibited by HGF, providing a possible explanation of how HGF inhibits BMP signaling. The in vitro data were supported by the observation of a negative correlation between HGF and a marker of osteoblast activity, bone-specific alkaline phosphatase (rho = −0.45, P = .008), in sera from 34 patients with myeloma. These observations suggest that HGF inhibits bone formation in multiple myeloma.

Osteoprotegerin (OPG), the neutralizing decoy receptor for the osteoclast activator RANK ligand, was measured in serum taken from patients with multiple myeloma at the time of diagnosis. Median OPG was lower in the patients with myeloma (7.4 ng/mL; range, 2.6-80; n = 225) than in healthy age- and sex-matched controls (9.0 ng/mL; range 5.1-130; n = 40; P = .02). Importantly, OPG levels were associated with degree of radiographically assessed skeletal destruction (P = .01). The median OPG level in patients lacking osteolytic lesions was 9.1 ng/mL, as compared with 7.6 ng/mL and 7.0 ng/mL, respectively, in patients with minor or advanced osteolytic disease. Furthermore, OPG levels were associated with World Health Organization performance status (P = .003) and correlated to serum levels of carboxy-terminal propeptide of type I procollagen (PICP; P < .001) but not with clinical stage or survival. These findings suggest impaired OPG function in myeloma and give a rationale for OPG as a therapeutic agent against myeloma bone disease.

Multiple myeloma (MM) is an incurable B-cell malignancy characterized by accumulation of malignant plasma cells in bone marrow (BM) and recurrent or persistent infections. Toll-like receptors (TLRs) are essential in the host defense against infections and today 10 human TLRs (TLR1-TLR10) and one TLR-homolog (RP105) have been characterized. B cells express several TLRs (mainly TLR1, 6, 7, 9, 10 and RP105) and TLR-initiated responses in B cells include proliferation, anti-apoptosis effect and plasma cell (PC) differentiation. The present study was designed to analyze the role of TLRs in MM. We show that frequent expressions of TLRs were detected in cell lines from MM patients (minimum six TLRs in each). In comparison, only few TLRs (mainly TLR1 and or RP105) were found expressed in PCs from BM of healthy donors. In addition, TLR-specific ligands induce increased proliferation and survival of the MM cell lines, partially due to an autocrine interleukin-6 production. Importantly, we demonstrate that also PC from MM patients proliferates in response to TLR-specific ligands. In conclusion, TLR-ligands may contribute to increased growth and survival of MM cells in MM patients.