Original Article

Effects of RNAi-mediated inhibition of aggrecanase-1 and aggrecanase-2 on rat costochondral chondrocytes in vitro

Zheng-hui Wang, Zhuang-qun Yang, Xi-jing He, Li Wang, Li-xia Li, Jun-bo Tu


Aim: Failure of transplanted cartilage or allogenic chondrocytes is attributed mainly to immunological rejection and cartilage degradation. A major feature is the loss of aggrecan from the cartilage matrix, primarily due to the action of the specific proteinases aggrecanase-1 and aggrecanase-2. The aim of this in vitro study was to determine whether the specific inhibition of aggrecanase-1 and aggrecanase-2 by RNAi would mitigate aggrecan loss from cultured chondrocytes.
Methods: Expression plasmid vectors of shRNA targeting aggrecanase-1 and aggrecanase-2 were constructed and transfected into cultured rattus costochondral chondrocytes. The transfected cells were induced with interleukin-1β (IL-1β). Gene mRNA levels were analyzed by RT—PCR. Aggrecan and collagen II content were measured by immunohistochemistry and Western blotting.
Results: As the chondrocytes underwent dedifferentiation, agggrecanase-1 increased significantly. The specific inhibition of aggrecanase-1 and aggrecanase-2 by RNAi had no negative effect on the morphology and growth velocity of the chondrocytes. The mRNA of aggrecanase-1 and aggrecanase-2 decreased significantly. The α-2-macroglobulin expression level was increased by the shRNA specific for aggrecanase-1. Other genes of the chondrocytic extracellular matrix were not affected. RNAi significantly increased the aggrecan and collagen II content of chondrocytes treated with IL-1β.
Conclusion: The results suggest that inhibition of aggrecanase-1 and aggrecanase-2 by RNAi can mitigate aggrecan degradation, without interfering with chondrocytic gene phenotype recovery. RNAi technology can be a useful tool for studying degenerative processes in cartilage.