Background In human osteoarthritic (OA) chondrocytes, a substantial proportion of the CD44 undergoes degradation similar to cleavage observed in several tumor cell systems . This signature degradation pattern includes the cleavage of the extracellular domain of CD44 by a metalloproteinase (e.g., ADAM10, ADAM17, MT1-MMP) releasing into the extracellular matrix a shed CD44 ecto-domain, leaving the 18-20 kD C-terminal fragment within the membrane (CD44-EXT) . The CD44-EXT fragment is then cleaved within the intramembranous domain releasing a 15 kD intracellular domain (CD44-ICD) into the cytoplasm. As this signature degradation leads to lost extracellular matrix, we suspect that this degradation is one of the varieties of the cartilage degeneration. Statin family is an inhibitor for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which is an enzyme involved in cholesterol biosynthetic pathway. Previous study reported that statins suppressed the cartilage degeneration induced by proinflammtory cytokines .
Objectives The objective of this study was investigated to the effect of simvastatin to inhibit CD44 fragmentation in chondrocytes.
Methods HCS-2/8 cells, a chondrocyte cell line established from a human chondrosarcoma, were pretreated with the indicated dose of simvastatin for 48h prior to stimulation with 0.1 ng/ml IL-1βand 10 ng/ml Oncostatin M (OSM) for 24h. CD44 fragmentation was detected with western blotting anakysis. HCS-2/8 cells were transfected with siRNA targeting membrane type of MMPs that introduce cleavage of CD44. Sucrose density gradient analysis was used to identify co-localization of lipid raft and MMPs. 2 and 3 in 10 fractions that exhibited high expression of the lipid raft marker flotillin-1 were defined as lipid raft fractions. The association of CD44 and MMP that induced cleavage of CD44 was detected using Co-immunoprecipitation (Co-IP) analysis.
Results We confirmed that IL-1+OSM stimulation induced the CD44 fragmentation in HCS-2/8 cells. The enhanced fragmentation included the 18-20 kD doublet CD44-EXT bands and the 15 kD CD44-ICD bands (Fig. 1). Pre-incubation with indicated dose of simvastatin inhibited the IL-1β+OSM induced CD44 fragmentation in a dose dependent manner (Fig. 2A). Supplementation with mevalonic acid (MA) or the isoprenoids, farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP) and squalene (Sq), partially counteracted the inhibitory effect of simvastatin on CD44 fragmentation (Fig. 2B). HCS-2/8 cells were transfected with MT1-MMP, ADAM10 and ADAM17 siRNAs. Depletion of ADAM10 inhibited CD44 cleavage (Fig. 3). Sucrose density gradient analysis showed that simvastatin significantly lowered the band of the lipid raft maker of flotillin-1 (Fig. 4A). Additionally, the raft localization of ADAM10 was disrupted with pre-incubation with simvastatin. Co-IP by anti-CD44 antibody indicated that ADAM10 was associated with CD44 by stimulation. On the other hand simvastatin disrupted the association of CD44 with ADAM10 (Fig. 4B).
Conclusions This study is the first to demonstrate that simvastatin inhibits the CD44 fragmentation by ADAM10. Simvastatin would have inhibited degradation of chondrocytes by the prevention of CD44 fragmentation.
Takahashi N, et al. Arthritis and Rheum 2010, 62; 1338.
Nagano O, et al. Cancer Sci 2004, 95; 930.
Barter MJ, et al. Ann Rheum Dis, 69; 2189
Disclosure of Interest None declared