Effects of RNAi-mediated inhibition of aggrecanase-1 and aggrecanase-2 on rat costochondral chondrocytes in vitro.
OBJECTIVE
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-1beta (IL-1beta). 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, aggrecanase-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 alpha-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-1beta.


CONCLUSIONS
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.
Wang
Zheng-hui
ZH
Department of Oral and Maxillofacial Plastic Surgery, Stomatological Hospital, Xi-an Jiao Tong University, Xi-an 710004, China.
Yang
Zhuang-qun
ZQ
He
Xi-jing
XJ
Wang
Li
L
Li
Li-xia
LX
Tu
Jun-bo
JB
D051722:ADAM Proteins / Q000037:antagonists & inhibitors*; D000071121:ADAMTS4 Protein; D000071116:ADAMTS5 Protein; D053674:Aggrecans / Q000378:metabolism; D000818:Animals; D019902:Chondrocytes / Q000187:drug effects* / Q000201:enzymology*; D024043:Collagen Type II / Q000378:metabolism; D053583:Interleukin-1beta / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011348:Procollagen N-Endopeptidase / Q000037:antagonists & inhibitors*; D034622:RNA Interference / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection; D000511:alpha-Macroglobulins / Q000096:biosynthesis
D053674:Aggrecans; D024043:Collagen Type II; D053583:Interleukin-1beta; D000511:alpha-Macroglobulins; D051722:ADAM Proteins; D000071116:ADAMTS5 Protein; C518696:Adamts5 protein, rat; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein