AIMP1 downregulation restores chondrogenic characteristics of dedifferentiated/degenerated chondrocytes by enhancing TGF-β signal.

J Ahn,I Han,Y Arai,H Kumar,S-H Lee,B-H Cha,S G Park,S Park

doi:10.1038/cddis.2016.17

Abstract

Dedifferentiation and degeneration of chondrocytes critically influences the efficiency of cartilage repair. One of the causes is the defect of transforming growth factor (TGF)-β signaling that promotes chondrogenic differentiation and degeneration. In the present study, we found that aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1) negatively regulates TGF-β signaling via interactions with Smad2 and Smad3 in immunoprecipitation assay and luciferase assay. In addition, we observed that the AIMP1 expression level was significantly increased in osteoarthritis (OA) patient-derived degenerated chondrocytes compared with healthy control. So, we hypothesized that downregulation of AIMP1 using small-interfering RNA (siRNA) technology in dedifferentiated (collected at passage #6) and degenerated (obtained from OA-affected areas) chondrocytes could lead to recover TGF-β signaling in both chondrocytes. Indeed, AIMP1 downregulation restored TGF-β signaling by promoting phosphorylation of Smad2 and Smad3, which shows redifferentiated characteristics in both dedifferentiated and degenerated chondrocytes. Additionally, implantation analyses using in vivo mouse model clearly showed that AIMP1 downregulation resulted in the increased chondrogenic potential as well as the enhanced cartilage tissue formation in both dedifferentiated and degenerated chondrocytes. Histological analyses clarified that AIMP1 downregulation increased expression levels of collagen type II (Col II) and aggrecan, but not Col I expression. Taken together, these data indicate that AIMP1 downregulation using siRNA is a novel tool to restore TGF-β signaling and thereby increases the chondrogenic potential of dedifferentiated/degenerated chondrocytes, which could be further developed as a therapeutic siRNA to treat OA.

Highlights

It has been known that Transforming growth factor (TGF)-β signaling is perturbed in dedifferentiated chondrocytes
Col collagen type II (II) mRNA expression was significantly increased in cells treated with aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1) short-interfering RNA (siRNA) in the presence of TGF-β compared with control siRNA (Figure 2c)
SB431542, a potent and specific inhibitor of the TGF-β superfamily type I activin receptor-like kinase (ALK) receptor, decreased collagen type II (Col II) expression in dedifferentiated chondrocytes (Figure 2d), which suggest that the increase in Col II expression following AIMP1 downregulation is due to enhanced TGF-β signaling

Summary

Introduction

Of the several TGF-β signaling cascades, the TGF-βSmad pathway is most extensively studied in chondrocytes.[15] Upon TGF-β activation, Smad[2] and Smad[3] are phosphorylated followed by Col II upregulation, which promotes chondrogenic differentiation. We investigated the role of AIMP1 and attempted to elucidate the mechanism via which AIMP1 elicits effects in dedifferentiated and degenerated chondrocytes. On the basis of the previous reports, we hypothesized that downregulation of AIMP1 in dedifferentiated and degenerated chondrocytes restores chondrogenic characteristics by upregulating Smad[2] and Smad[3] phosphorylation followed by expression of chondrogenic markers such as Col II. We treated dedifferentiated chondrocytes with AIMP1-targeting siRNA and assessed chondrogenic redifferentiation in vitro and cartilage tissue formation in vivo.

Methods

Results

Discussion

Conclusion