IntroductionBone is a dynamic tissue in which the synthesis of bone matrix by osteoblasts and bone resorption by osteoclasts are coupled processes. Osteoclasts differentiate from hematopoietic precursor cells under the control of humoral factors and cell-cell contact with osteoblasts or stromal cells. Key regulators of osteoclastogenesis are members of the tumor necrosis family of receptors and ligands: receptor activator of nuclear factor (NF)-B (RANK), receptor activator of NF-B ligand (RANKL), and osteoprotegerin (OPG). RANKL is expressed by activated T cells, stromal cells, and osteoblasts. [1][2][3] When RANKL binds to RANK on osteoclast precursors, maturation and differentiation of the osteoclasts are induced, leading to bone resorption. 4,5 OPG is a soluble decoy receptor that is secreted by osteoblasts and bone marrow (BM) stromal cells and that competes with RANK for binding to RANKL. Binding of OPG to RANKL inhibits the development of osteoclasts. 6,7 The importance of OPG as a negative regulator of osteoclastogenesis is evident from experiments with transgenic mice, where overexpression of OPG leads to severe osteopetrosis and reduced numbers of mature osteoclasts. 6 In contrast, OPG knockout mice are osteoporotic. 8 Multiple myeloma (MM) is a malignancy characterized by accumulation of plasma cells in the BM. Bone destruction is a common complication of the disease and is associated with severe morbidity. A number of osteoclast-activating factors are implicated in myeloma bone disease (for a review, see Callander and Roodman 9 ). However, accumulating data suggest that a disruption of the balance between RANKL and OPG is of major importance. Histologic examination of BM biopsies from patients with MM showed enhanced expression of RANKL in the BM, as well as reduced OPG expression. 10,11 Furthermore, we have recently shown that serum OPG levels are lower in myeloma patients than in healthy individuals and that myeloma patients with osteolytic lesions have reduced levels of OPG in serum compared to myeloma patients without clinical bone disease. 12 Osteoprotegerin has a highly basic heparin-binding domain, 13 making interactions with heparin and heparan sulfates possible. A feature of both normal and malignant plasma cells is the abundant expression of syndecan-1, 14,15 which is a transmembrane proteoglycan with heparan sulfate side chains. These side chains allow interactions with several macromolecules, including extracellular matrix proteins, growth factors, cytokines, and pathogens (for a review, see Tumova et al 16 ). In addition to modifying the action of its ligands, 17,18 syndecan-1 has been shown to mediate the catabolism of several proteins. 19 Patients, materials, and methods MM and control patientsBone marrow was aspirated from the iliac crest or sternum of 33 patients with MM for diagnostic purposes before treatment (median age, 65 years; 19 men, 14 women). Twenty-seven patients who underwent BM aspiration for diagnostic purposes, but who had normal BM morphology and subsequently were not ...

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.

Although the biological significance of proteoglycans (PGs) has previously been highlighted in multiple myeloma (MM), little is known about serglycin, which is a hematopoietic cell granule PG. In this study, we describe the expression and highly constitutive secretion of serglycin in several MM cell lines. Serglycin messenger RNA was detected in six MM cell lines. PGs were purified from conditioned medium of four MM cell lines, and serglycin substituted with 4-sulfated chondroitin sulfate was identified as the predominant PG. Flow cytometry and confocal microscopy showed that serglycin was also present intracellularly and on the cell surface, and attachment to the cell surface was at least in part dependent on intact glycosaminoglycan side chains. Immunohistochemical staining of bone marrow biopsies showed the presence of serglycin both in benign and malignant plasma cells. Immunoblotting in bone marrow aspirates from a limited number of patients with newly diagnosed MM revealed highly increased levels of serglycin in 30% of the cases. Serglycin isolated from myeloma plasma cells was found to influence the bone mineralization process through inhibition of the crystal growth rate of hydroxyapatite. This rate reduction was attributed to adsorption and further blocking of the active growth sites on the crystal surface. The apparent order of the crystallization reaction was found to be n ‫؍‬ 2, suggesting a surface diffusion-controlled spiral growth mechanism. Our findings suggest that serglycin release is a constitutive process, which may be of fundamental biological importance in the study of MM.

The cytokine hepatocyte growth factor (HGF) and its receptor c-Met are a ligand-receptor pair with important functions in a communicative interplay between HGF-producing, mesenchymal cells and c-Met-expressing target cells. HGF is cytoprotective and causes regeneration of parenchyma after tissue damage in several organs. The receptor c-Met was first characterized as an oncogene product being responsible for the transformation of an osteosarcoma cell line. HGF or c-Met is overexpressed in several human cancers, including various carcinomas. Some cells of hematopoietic origin also seem to be capable of c-Met expression, but the precise role of HGF in normal hematopoiesis is yet to be determined. In blood malignancies like acute myelogenous leukemia and, notably, multiple myeloma, HGF is overproduced and has implications for the prognosis of the patients. Biological significance of HGF overexpression in multiple myeloma is discussed and is likely to include effects on bone turnover and angiogenesis.

for The Nordic Myeloma Study Group 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.

Bone morphogenetic proteins (BMPs) can be isolated from organic bone matrix and are able to initiate de novo cartilage and bone formation. Here it is shown that BMP-4 inhibited DNA synthesis in a dosedependent manner in 3 IL-6-dependent multiple myeloma (MM) cell lines (OH-2, IH-1, and ANBL-6). In contrast, no effect on DNA synthesis was observed in 3 IL-6-independent MM cell lines ( IntroductionMultiple myeloma (MM) is a B-cell malignancy characterized by a low-grade proliferation of plasma cells in the bone marrow. Tumor progression is frequently accompanied by osteolysis caused by increased bone resorption and decreased bone formation. In vitro, myeloma cells can be responsive to several cytokines with proliferative and anti-apoptotic effects, of which IL-6 is the most important. 1,2 Few cytokines with the ability to induce apoptosis or to inhibit proliferation of myeloma cells have been identified. Interferon (IFN)-␥ inhibits IL-6-dependent proliferation of myeloma cell lines and patient samples. 3,4 In vitro effects of IFN-␣ are more complex because induction of proliferation, increased survival, and inhibition of proliferation have been reported. 2 IFN-␣ treatment of patients with MM may prolong the plateau phase, but it does not significantly prolong survival. 5,6 IFN-␥ was evaluated in one small clinical trial and showed no beneficial effect. 7 Activin A, a member of the transforming growth factor (TGF)-␤ superfamily, induces apoptosis and inhibits proliferation in murine plasmacytoma. [8][9][10][11] In addition, Fas ligand (FasL) and TRAIL (Apo-2 ligand) may induce apoptosis in some myeloma cells. 12,13 Bone morphogenetic proteins (BMPs) are members of the TGF-␤ superfamily and were originally identified by their unique ability to induce ectopic cartilage and bone formation in vivo. 14,15 To date, at least 15 BMPs have been characterized. 16 In bone, osteoblasts and chondrocytes synthesize BMPs, and these proteins play important roles in bone formation and bone cell differentiation by stimulating alkaline phosphatase activity and synthesis of proteoglycan, collagen, and osteocalcin. 17,18 BMPs also induce osteoblast differentiation of uncommitted bone marrow stromal precursor cells and are involved in postnatal bone remodeling. 19,20 Furthermore, in animal models, BMPs promote the healing of large segmental fractures and bone defects. 16,21 BMPs and their receptors are expressed in tissues other than bone, and it has been demonstrated that BMPs play multiple roles in the regulation of growth, differentiation, and apoptosis of various cell types. This may be exemplified by the essential role of BMP-4 in ventralization of the embryo. [22][23][24] In malignant disease, BMPs modulate cell proliferation, frequently in an inhibitory manner, and have been implicated in the osteosclerotic pattern of prostate cancer bone metastasis. [25][26][27][28][29] Recently, BMPs have emerged as proteins of significance in hematology. BMP-4 strictly controls the formation of the ventral hematopoietic islands in Xenopus emb...

We have examined whether the hepatocyte growth factor (HGF)/c-met receptor-ligand pair is expressed in freshly isolated and highly purified myeloma cells and whether HGF can be found in the sera of myeloma patients. Myeloma cells were purified with an immunomagnetic method using the syndecan 1-specific antibody B-B4. HGF and c-met mRNA in these cells were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). HGF and c-met proteins were detected by enzyme- linked immunosorbent assay (ELISA) and Western blot, respectively. Serum from 13 myeloma patients was obtained at diagnosis and the levels of HGF were determined by ELISA. HGF and c-met mRNA were expressed in all examined samples (n = 7). HGF was detected in the supernatants of 17 of 20 primary cultures of myeloma cells, whereas bone marrow mononuclear cells from normal controls did not produce detectable amounts of HGF (n = 3). The mean HGF level in serum of myeloma patients at diagnosis was more than fourfold higher than the mean level in normal controls. Possible implications of HGF/c-met expression for the pathophysiology of multiple myeloma are discussed.

Serum from 398 myeloma patients at diagnosis and serial samples from 29 patients were analysed for hepatocyte growth factor (HGF). HGF was elevated at diagnosis in 43% of myeloma patients compared with healthy controls (median 1.00 ng/mL and 0.44 ng/mL, respectively;P < .00001). In the group with elevated HGF levels 46% of the patients reached plateau phase, as compared with 60% of the patients with low HGF levels (P = .005), and the median survival time was 21 and 32 months, respectively (P = .002). In a univariate Cox regression analysis, HGF was a significant predictor of mortality (P = .02). In the subgroup of patients with β2-microglobulin levels less than or equal to 6 mg/L, high versus low HGF was a prognostic factor when a multivariate Cox regression analysis was performed. In serial samples HGF was higher at the time of diagnosis and relapse (median 0.57 ng/mL and 0.52 ng/mL, respectively; P = .0018) than at response (median 0.24 ng/mL, P = .008). We conclude that HGF may be a useful follow-up parameter in myeloma patients. Measurement of HGF may identify a group of patients with poor response to melphalan-prednisone treatment and short survival. HGF was a prognostic factor in patients with high levels of β2-microglobulin.