Der Einfluß von Zytokinen und Zell-Zell-Kontakten auf die Expression von Matrix-Metalloproteinasen beim multiplen Myelom

Multiple myeloma is a neoplasia of terminally differentiated, immunoglobulin-producing B-lymphocytes. Characteristic for the disease are multiple disseminations in the bone marrow cavity relatively early in the course and multiple osteolytic lesions. These processes involve a massive degradation and a pathologic remodeling of the extracellular matrix (ECM). In many tumor entities, like in multiple myeloma, degradation and remodeling of extracellular matrix is induced by tumor cells, but nonmalignent stroma cells are the effector cells. The family of matrix metalloproteinases (MMPs) and its specific inhibitors (TIMPs) represent a group of proteases that can degrade collagenous as well as non-collagenous extracellular matrix. Their expression is elevated in several tumor entities, and their influence on tumor progression and dissemination has been shown. This study has examined the expression of matrix metalloproteinases in a coculture model of the multiple myelom cell line U266 and human stroma cells (dermal fibroblasts) in monoculture, after incubation with conditioned media and after cell-cell contact. By semiquantitative real-time RT-PCR and ELISA the following observations were made: the myeloma cell line U266 showed a weak expression of MMP-1, MMP-2, MMP-9 and TIMP-2, whereas fibroblasts expressed MMP-2, MMP-2, MMP-7, MMP-9, TIMP-1 and TIMP-2. The influence of myeloma cells on MMP-expression in fibroblasts was investigated in subsequent experiments. U266-conditioned cell culture media increased the expression of MMP-1 in fibroblasts on an RNA and protein level. An increased expression of MMP-2 could be shown by ELISA, but not by RT-PCR. Cocultivation and cell-cell contact of myeloma cells and fibroblasts led to a prominent increase in MMP-9 RNA expression. Factor expressed on the surface of myeloma cells and factors secreted by myeloma cells thus influenced fibroblast MMP production in our cell culture model transcriptionally as well as post-transcriptionally. To closer identify the MMP-inducing agent, inhibiting antibodies against selected cytokines (TNF-alpha, IL-1beta) were added to the U266 conditioned cell culture media. Inhibition of IL-1beta led to an increase of MMP-1 expression, whereas inhibition of TNF-alpha was without effect on MMP-expression. Specific inhibition of the mitogen actived kinase (MAPK) signaling pathway by pharmacological inhibitors suggested an involvement of the p38-Kinase in the increased MMP-expression. In conclusion, a noticeable influence of multiple myeloma cells on MMP expression in non-malignant stromal cells could be shown. The interaction of the two cell types leads to an increased expression of MMP-1, MMP-2 and MMP-9 in fibroblasts. The causative agent which regulates MMP expression in fibroblasts, though, remains the target of subsequent studies