Recovery of function in osteoarthritis chondrocytes induced by p16INK4a-specific siRNA in vitro

Abstract

Objective. To demonstrate the roles of p16INK4a in the senescence of human chondrocytes and the progression of osteoarthritis (OA). Methods. Immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR) were performed to examine p16INK4a expression in fetal, normal age-matched and OA cartilage, and Western blot was used in primary cultured chondrocytes from different origins. To explore a functional p16INK4a knockdown in OA chondrocytes, the primary cultured cells were treated with p16INK4a-specific small interfering ribonucleic acids (siRNAs. Expression of p16INK4a, p14ARF and p53 was observed by Western blot and RT-PCR. The phosphorylation status of pRb, senescence-associated β-galactosidase (SA-β-gal), cell G1/S transition and cell proliferation were studied by Western blot, histological staining, 3H-thymidine incorporation and cell counts respectively. Expression of the collagen I, collagen II and aggrecan genes was measured by semiquantitative RT-PCR. To establish the response of chondrocytes to cytokines, cells were treated with transforming growth factor-β1 (TGF-β1) or interleukin-1α (IL-1α) and examined for incorporation of 3H-thymidine, 3H-proline and 35S-sulphate respectively. Results. A significant increase of p16INK4a was detected in OA chondrocytes compared with normal age-matched and fetal chondrocytes (P < 0.01) in vivo and in vitro. Treated with p16INK4a-specific siRNAs, OA chondrocytes displayed a significant decrease in p16INK4a expression with an increase of phosphorylated pRb, but no alteration of p14ARF and p53 expression, followed by decreases of senescent features and increases in the expression of some chondrocyte-specific genes and overall repair capacity. Conclusions. p16INK4a is instrumental in the senescence of human articular chondrocytes or OA. The reduction of p16INK4a by RNA interference (RNAi) contributed to the recovery of osteoarthritic chondrocytes, suggesting that p16INK4a may be a viable future therapeutic candidate. © British Society for Rheumatology 2004; all rights reserved.