Matrix Metalloproteinase 13 (MMP13) Upregulation Is Essential for Multiple Myeloma Related Bone Lytic Lesion

MM cells produce several osteoclast-activating factors, which result in highly activated osteoclasts and cause dysregulated bone remodeling with excessive bone resorption. Our previous data showed that matrix metalloproteinase 13 (MMP13) is highly expressed in MM cells, and co-culture with stromal cells further increased MMP13 levels. Most importantly, exogenous MMP13 increased OCL fusion and bone resorption activity induced by nuclear factor kappa-B ligand (RANKL). Here, we further addressed the mechanism of MMP13 upregulation in MM and its role in MM-related bone disease in vivo.

Based upon the previous results that IL-6 neutralizing antibody blocks MMP13 upregulation in MM cells co-cultured with stromal cells, we further investigated the role of IL-6 on MMP13 induction in MM cells. Human RPMI8266 MM cells were serum-starved for 24 hours, then treated with IL-6 for up to 96 hours. RT-PCR and western blotting showed that MMP13 expression and secretion by MM cells were upregulated after 24h and prolonged through 96h. AP-1 binding sites were identified in the MMP13 promoter, and we found that IL-6 induced upregulation of the AP-1 members c-Jun and c-fos in MM cells by RT-PCR and western blotting, which correlated with MMP13 induction. To address the role of heightened MMP13 expression/secretion of MM on OCL formation and development of lytic lesions, we silenced MMP13 expression in MM cells by lentiviral mediated shRNA transduction. 5TGM1 mouse MM cells were infected with the pKLO.1-puro-vector (EV) or pKLO.1-puro-sh-MMP13 (MMP13 Knock down [KD]) lentivirus particles and cells were selected by puromycin. MMP13 KD was confirmed by RT-PCR and western blotting. To investigate the effects of MMP13 KD on OCL formation, we co-cultured mouse bone marrow cells (BMC) with either 5TGM1-EV or 5TGM1-MMP13 KD MM cells using a transwell system to permit only soluble molecule exchange. Co-culture with 5TGM1-EV MM cells significantly (p<0.01) induced OCL fusion, while this effect was largely impaired in the co-culture group with 5TGM1-MMP13 KD MM cells. Treatment with IL-6 further enhanced the OCL fusion activity in both groups. However, in 5TGM1-MMP13 KD group, IL-6 did not compensate for impaired OCL formation due to MMP13 silencing. This indicates that MMP13 secreted from MM cells is one of the key factors facilitating OCL formation/activity in MM. We further investigated the effects of MMP13 silencing on MM tumor progression and bone disease in vivo using the 5TGM1 intratibial tumor model. Recombination activating genes 2 (RAG2) knockout mice were intratibially injected with either 5TGM1-EV or 5TGM1-MMP13 KD MM cells, and 3 weeks later, mice were sacrificed and tumor growth and bone lytic lesion were monitored by serum IgG2b level (by Elisa) and micro-QCT respectively. Preliminary results show that MMP13 KD in MM cells inhibited tumor growth and lytic lesions in trabecular bone.

Our results demonstrate that IL-6-induced high level of MMP13 in MM cells is essential for multiple myeloma tumor growth, OCL induction and development of lytic bone lesions.

Lentzsch:Celgene: Consultancy, Research Funding.