In multiple myeloma (MM) increased numbers of monoclonal plasma cells in the bone marrow induce localized osteolytic lesions that rarely heal, due to increased bone resorption and suppressed bone formation. Numerous studies reported the contributions that different cell types in the MM microenvironment make to MM growth and bone disease, but the role of matrix-embedded osteocytes in MM, which comprise >95% of bone cells and are major regulators of osteoclast and osteoblast activity, is unclear. We report that osteocytes in MM-bearing bones physically interact with MM cells in vivo, undergo caspase3-dependent apoptosis, and express higher RANKL and Sclerostin levels than osteocytes from control mice. Mechanistic studies revealed that osteocyte apoptosis is initiated by activation of Notch signaling in osteocytes through direct contact with MM cells, and is further amplified by MM cell-secreted TNFα. This Notch/TNFα induced osteocyte apoptosis increases osteocytic Rankl expression, the osteocytic Rankl/Opg ratio and the ability of osteocytes to attract osteoclast precursors to induce local bone resorption. Further, osteocytes in contact with MM cells express high levels of Sost/Sclerostin that decrease Wnt signaling in osteoblasts and inhibit osteoblast differentiation. Importantly, direct contact between osteocytes and MM cells reciprocally activates Notch signaling and increases Notch receptor expression in MM cells, in particular Notch3 and 4, and stimulates MM cell growth. These studies reveal a previously unknown role for bidirectional Notch signaling between MM cells and osteocytes that enhances MM growth and bone disease, and suggest the potential of targeting osteocyte-MM cell interactions as a novel MM treatment.

Increased osteoclastogenesis and angiogenesis occur in physiologic and pathologic conditions. However, it is unclear if or how these processes are linked. To test the hypothesis that osteoclasts stimulate angiogenesis, we modulated osteoclast formation in fetal mouse metatarsal explants or in adult mice and determined the effect on angiogenesis. Suppression of osteoclast formation with osteoprotegerin dosedependently inhibited angiogenesis and osteoclastogenesis in metatarsal explants. Conversely, treatment with parathyroid hormone related protein (PTHrP) increased explant angiogenesis, which was completely blocked by osteoprotegerin. Further, treatment of mice with receptor activator of nuclear factor-B ligand (RANKL) or PTHrP in vivo increased calvarial vessel density and osteoclast number. We next determined whether matrix metalloproteinase-9 (MMP-9), an angiogenic factor predominantly produced by osteoclasts in bone, was important for osteoclast-stimulated angiogenesis. The pro-angiogenic effects of PTHrP or RANKL were absent in metatarsal explants or calvaria in vivo, respectively, from Mmp9 ؊/؊ mice, demonstrating the importance of MMP-9 for osteoclast-stimulated angiogenesis. Lack of MMP-9 decreased osteoclast numbers and abrogated angiogenesis in response to PTHrP or RANKL in explants and in vivo but did not decrease osteoclast differentiation in vitro. Thus, MMP-9 modulates osteoclast-stimulated angiogenesis primarily by affecting osteoclasts, most probably by previously reported migratory effects on osteoclasts. These results clearly demonstrate that osteoclasts stimulate angiogenesis in vivo through MMP-9. (Blood.

IntroductionMyeloma bone disease is characterized by lytic bone lesions with little or no reactive new bone formation. Up to 80% of multiple myeloma (MM) patients present with bone pain, and over 70% of the patients will develop pathologic fractures during the course of their disease (1). Bone destruction in myeloma is a local event in which lesions only occur adjacent to myeloma cells. These data suggest that MM cells produce factors or induce factors that stimulate osteoclast (OCL) formation. We have used an expression cloning approach with a cDNA library constructed from RNA obtained from freshly isolated bone marrow samples from MM patients and screened it for osteoclast-activating factors (OAFs) that induce OCL formation in human and murine marrow cultures. We identified macrophage inflammatory protein 1-α (MIP-1α) as an OAF produced by myeloma cells in vivo (2). MIP-1α induced formation of bone-resorbing OCLs in human marrow cultures, acted directly on OCL precursors, and did not upregulate RANK ligand (RANKL) expression (3). Furthermore, MIP-1α enhanced the effects of IL-6 and RANKL, cytokines present in myeloma marrow, on OCL formation (3). Previously, Kukita and coworkers (4) reported that MIP-1α induces OCL formation in rat bone marrow cultures, and Fuller and coworkers (5) have shown that MIP-1α is chemotactic for OCLs. More importantly, MIP-1α levels are increased in marrow plasma from myeloma patients with active disease, whereas MIP-1α levels are reduced to almost normal levels in patients who are in complete remission, or have inactive disease, or who have stage I myeloma (2). Furthermore, addition of a neutralizing Ab to MIP-1α blocked the OAF activity present in bone marrow plasma samples from patients with myeloma (2). The purpose of the current study was to determine the role of MIP-1α in an in vivo model of human myeloma bone disease. We reported previously that intravenous injection of the human myeloma-derived cell line, ARH, into sublethally irradiated SCID mice induces myeloma in these animals (1). These mice develop all the characteristics of myeloma bone disease, including lytic bone lesions, hypercalcemia, and increased OCL formation in areas adjacent to the myeloma cells. ARH cells produce high levels of MIP-1α. Therefore, ARH cells were stably transfected with either an antisense construct to MIP-1α or an empty vector and transplanted into SCID mice to determine the role of MIP-1α in this animal model of human myeloma bone disease. We recently identified macrophage inflammatory protein 1-α (MIP-1α) as a factor produced by multiple myeloma (MM) cells that may be responsible for the bone destruction in MM (1). To investigate the role of MIP-1α in MM bone disease in vivo, the human MM-derived cell line ARH was stably transfected with an antisense construct to MIP-1α (AS-ARH) and tested for its capacity to induce MM bone disease in SCID mice. Human MIP-1α levels in marrow plasma from AS-ARH mice were markedly decreased compared with controls treated with ARH cells transfected with empty ...

Hypoxia-inducible transcription factor-1 (HIF-1a) is overexpressed in multiple myeloma (MM) cells within the hypoxic microenvironment. Herein, we explored the effect of persistent HIF-1a inhibition by a lentivirus short hairpin RNA pool on MM cell growth either in vitro or in vivo and on the transcriptional and pro-angiogenic profiles of MM cells. HIF-1a suppression did not have a significant impact on MM cell proliferation and survival in vitro although, increased the antiproliferative effect of lenalidomide. On the other hand, we found that HIF-1a inhibition in MM cells downregulates the pro-angiogenic genes VEGF, IL8, IL10, CCL2, CCL5 and MMP9. Pro-osteoclastogenic cytokines were also inhibited, such as IL-7 and CCL3/MIP-1a. The effect of HIF-1a inhibition was assessed in vivo in nonobese diabetic/severe combined immunodeficiency mice both in a subcutaneous and an intratibial MM model. HIF-1a inhibition caused a dramatic reduction in the weight and volume of the tumor burden in both mouse models. Moreover, a significant reduction of the number of vessels and vascular endothelial growth factors (VEGFs) immunostaining was observed. Finally, in the intratibial experiments, HIF-1a inhibition significantly blocked bone destruction. Overall, our data indicate that HIF-1a suppression in MM cells significantly blocks MM-induced angiogenesis and reduces MM tumor burden and bone destruction in vivo, supporting HIF-1a as a potential therapeutic target in MM.

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