Upregulation of matrix metalloproteinase‐9 in murine 5T33 multiple myeloma cells by interaction with bone marrow endothelial cells

Abstract

MM is a B-cell malignancy mainly characterized by monoclonal expansion of plasma cells in the BM, presence of paraprotein in serum and occurrence of osteolytic bone lesions. MMPs are a family of proteolytic enzymes that can contribute to cancer growth, invasion, angiogenesis, bone degradation and other processes important in the pathogenesis of MM. We investigated MMP-9 production in the 5T33MM murine model. Expression of MMP-9 protein in supernatant and cell extracts was analyzed by gelatin zymography. The in vitro, stroma-independent variant 5T33MMvt showed no protein expression of MMP-9 in contrast to in vivo growing MM cells, 5T33MMvv. However, when 5T33MMvt cells were injected into naive mice and isolated after tumor take (5T33MMvt-vv), they secreted a significant amount of MMP-9. These results were confirmed by specific staining of cytospins with an anti-MMP-9 antibody. The MMP-9 production by 5T33MMvt-vv cells disappeared when the cells were recultured in vitro. These data demonstrated that upregulation of MMP-9 occurs in vivo and that this process is dependent on the microenvironment. Key words: multiple myeloma; matrix metalloproteinase-9; bone marrow; stromal cell; endothelial cell MMPs are a family of zinc-dependent endopeptidases involved in the degradation of many components of the ECM and the basement membrane. 1,2 Depending on their substrate specificity and structure, members of the MMP family can be divided into subgroups of collagenases (MMP-1, -8, -13), stromelysins (MMP-3, -10, -11, -7), gelatinases (MMP-2, -9), membrane-type MMPs and other MMPs. 2,3 MMPs are secreted as inactive proenzymes and activated extracellularly by proteolytic cleavage. MMP production is regulated at the level of transcription, secretion and/or activation. 3,4 All MMPs are inhibited by specific TIMPs, including TIMP-1, -2, -3 and -4. TIMP-1 and TIMP-2 bind tigthly with, respectively, pro-MMP-9 and pro-MMP-2 to regulate their activity. 5 The balance between MMP and TIMP levels determines the net proteolytic activity. This equilibrium is highly regulated in physiologic conditions, e.g., wound healing and embryogenesis, 3 but is disturbed in pathologic circumstances, e.g., rheumatoid arthritis, multiple sclerosis and cancer. 6 -9 MM is a B-cell cancer mainly characterized by proliferation of malignant plasma cells in the BM, presence of a monoclonal serum immunoglobulin and occurence of osteolytic lesions. The disease occurs at older ages and remains incurable despite progress in treatment. Therefore, new approaches for therapy are necessary. The hypothesis is that MMPs are involved in a number of events underlying MM progression, e.g., transendothelial migration, invasion, osteolytic lesions and angiogenesis. 10 Barillé et al. 11 demonstrated MMP-9 production by human MM cells. MMP-9 is involved in transendothelial migration and invasion. 12,13 This suggests a role of the protease in the homing of MM cells to the BM, which implicates transendothelial migration and invasion of MM cells in the BM. Coculture of malig...

Hypoxia-inducible factor (HIF)-1α suppression in myeloma cells blocks tumoral growth in vivo inhibiting angiogenesis and bone destruction

“…CCL2 and MMP9 could be also involved in the pro-angiogenic role of HIF-1a, because both cytokines are produced by MM cells and contribute to angiogenesis and BM spreading. [41][42][43] Interestingly, our data suggest that other than angiogenic cytokines, HIF-1a suppression affects expression and production of MM-derived pro-osteoclastogenic factors that are known to be involved in osteoclast formation and bone destruction such as CCL3/MIP1a 44 and IL-7. 45 To the best of our knowledge, this is the first time that the capacity of HIF-1a has been reported to regulate pro-osteoclastogenic cytokine in MM cells, although the evidence that CCL3/MIP1a and IL-7 are potential target genes of HIF-1a has been shown in other cell types such as acute myeloid leukemia cells for CCL3/MIP1a 46 and mesenchymal/osteoblastic cells for IL-7.…”

Angiogenesis and Multiple Myeloma

“…However MMP-9 expression is similar between MM and MGUS patients [8]. MMP-9 secretion by myeloma cells is enhanced by their interaction with endothelial cells in the microenvironment [65,66]. Myeloma cells also upregulate MMP-1 secretion by BM stromal cells [67] and MMP-7 secreted by MM cells induces activation of the pro-MMP-2 [68].…”

“…Osteopontin Osteopontin (OPN) is a pro-angiogenic factor that promotes endothelial cell migration and survival [60][61][62][63][64][65][66][67][68][69][70], adhesion of both endothelial and smooth muscle cells [71], and has been associated with tumoral angiogenesis in mouse models [72,73]. OPN is produced directly by myeloma cells and is critical for the stimulation of endothelial cells.…”

Endothelial cell-driven regulation of CD9 or motility-related protein-1 expression in multiple myeloma cells within the murine 5T33MM model and myeloma patients

“…29,30 Isolation and purification of the myeloma cells in the BM was performed as described previously. 31 The murine 5T33MMvt cell line is a clonally identical, but in vitro stroma-independent growing variant of the 5T33MMvv cell line. Cells were cultured and maintained in Rosewell Park Memorial Institute (RPMI)-1640 medium (BioWhittaker, Verviers, Belgium) supplemented with 10% fetal calf serum (FCS) (Fetal clone I; Hyclone, Logan, UT, USA), 1% natriumpyruvate, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine and 1% minimum essential medium (supplements from BioWhittaker).…”