A complementary DNA expression library derived from marrow samples from myeloma patients was recently screened and human macrophage inflammatory protein-1␣ (hMIP-1␣) was identified as an osteoclastogenic factor expressed in these samples. hMIP-1␣ enhanced osteoclast (OCL) formation in human marrow cultures and by highly purified OCL precursors in a dose-dependent manner (5-200 pg/mL). Furthermore, hMIP-1␣ enhanced OCL formation induced by human interleukin-6 (IL-6), which is produced by marrow stromal cells when they interact with myeloma cells. hMIP-1␣ also enhanced OCL formation induced by parathyroid hormone-related protein (PTHrP) and receptor activator of nuclear factor B ligand (RANKL), factors also implicated in myeloma bone disease. Timecourse studies revealed that the hMIP-1␣ acted during the last 2 weeks of the 3-week culture period. Reverse transcription-polymerase chain reaction analysis showed that the chemokine receptors for hMIP-1␣ (CCR1 and CCR5) were expressed by human bone marrow and highly purified early OCL precursors. Furthermore, hMIP-1␣ did not increase expression of RANKL. These data demonstrate that hMIP-1␣ is an osteoclastogenic factor that appears to act directly on human OCL progenitors and acts at the later stages of OCL differentiation. These data further suggest that in patients with myeloma, MIP-1␣ produced by myeloma cells, in combination with RANKL and IL-6 that are produced by marrow stromal cells in response to myeloma cells, enhances OCL formation through their combined effects on OCL precursors. (Blood. 2001;97:3349-3353)

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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 ...

Extensive histological study revealed the impairment of bone remodeling caused by mechanical stress in OPN knockout mice in a tooth movement system. Analysis of OPN promoter transgenic mice showed the mechanical stress response element(s) in the 5.5-kb upstream region. These results were also obtained with the primary cultured cells.Introduction: Mechanical loading system changes the bone architecture through the stimulation of bone remodeling by the action of a numbers of molecules. Among them, we showed that osteopontin (OPN) plays an important role in response to mechanical loading in rats with an experimental system for tooth movement. The results indicate the important role of OPN in bone remodeling. However, the molecular mechanism of OPN expression in response to mechanical stress is unknown. Materials and Methods: OPN knockout mice and transgenic mice carrying green fluorescent protein (GFP) in the control of the OPN promoter were used for analysis. Orthodontic closed coil springs were bonded to the maxillary first molars and incisors for the experimental tooth movement. Spatial expression of GFP and OPN was detected by in situ hybridization. Results: In contrast to wildtype mice, a smaller number of TRACP + cells was detected in OPN knockout mice after treatment. In GFP-OPN5.5 mice, OPN and GFP mRNA-expressing cells were detected in bone cells after treatment, and the localization of GFP was consistent with that of endogenous OPN. An increase in the co-expression of GFP and OPN was detected when primary cultured osteoblastic cells derived from the transgenic mice were exposed to strain or pressure force. Significant increase in the number of OPN + osteocyte was detected in the pressure side at 48 h after treatment. At 72 h, increase in the number of TRACP + cells was detected predominantly in the pressure side. Conclusions: Bone remodeling in response to mechanical stress was suppressed in OPN knockout mice. These results indicate the critical role of OPN in the process of bone remodeling. The analysis of GFP expression in the promoter transgenic mice indicated the presence of an in vivo mechanical stress response element in the 5.5-kb upstream region of the OPN gene.

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