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

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.

Articular cartilage is an important supportive tissue that covers the ends of bones, reducing the mechanical stress on active joints. Chondrocytes are an unique type of cell that resides in articular cartilage. 1 Chondrocytes highly synthesize collagen type II (Col II), proteoglycans, and other types of collagens in articular cartilage, and form an integral component of the extracellular matrix (ECM). 2 Chondrocytes in articular cartilage can be damaged as a result of either traumatic mechanical destruction (automobile accidents and sports injuries) or progressive mechanical degeneration. 3 Unfortunately, damaged articular cartilage cannot be repaired due to several factors including restricted supply of blood, oxygen, and nutrients 4,5 and restricted movement of neighboring chondrocytes to the defect area. 6 Therefore, several efforts, including cell-based therapies using chondrocytes or mesenchymal stem cells, have been made to repair cartilage defects. 7,8 Autologous chondrocyte transplantation (ACT) has gained considerable attention and has renewed interest in cartilage repair. ACT is a promising technique for cartilage repair because autologous chondrocytes are homogeneous and can form hyaline cartilage without immune rejection. In ACT, after the sufficient expansion of chondrocytes by in vitro culture, they are implanted into cartilage lesions. 7 Unfortunately, the number of autologous chondrocytes isolated from a donor is extremely limited; therefore, obtaining a large quantity of chondrocytes that maintain chondrogenic characteristics is an important challenge for ACT. Isolated chondrocytes begin to lose their phenotype upon regular sub-culturing, and this results in serious impairment of their inherent characteristics (from round or polygonal phenotypes to bipolar and fibroblastic phenotypes). Dedifferentiation is a unique feature in chondrocyte biology and is characterized by a comprehensive change in chondrocyte synthetic profile. 9 Furthermore, a biphasic chondrocyte phenotype shift during dedifferentiation could be observed with a decline in Col II, aggrecan, fibromodulin, and Sox9 expression 10,11 and an increase in versican, decorin, and fibronectin expression. 9,12,13 Gene therapy and cartilage tissue engineering have been cooperatively used in cartilage repair. 11,14 Transforming growth factor (TGF)-β regulates a wide variety of cellular activities and maintains the standard properties of chondrocytes. Of the several TGF-β signaling cascades, the TGF-β-Smad pathway is most extensively studied in chondrocytes. 15 Upon TGF-β activation, Smad2 and Smad3 are phosphorylated followed by Col II upregulation, which promotes chondrogenic differentiation. However, transduction of TGF-β1 signals through Smad2 and Smad3 phosphorylation is perturbed in dedifferentiated chondrocytes due to the decreased sensitivity of TGF-β receptors. 16 Aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1, also known as p43) is ubiquitously expressed and has a significant role in many cellular processes including inflammation, 17,18 angiogenesis, 19 would healing, 20 and glucose homeostasis. 21 AIMP1 inhibits Smad2 and Smad3 phosphorylation and downregulates TGF-β signaling. [22][23][24] Interestingly, AIMP1 is reportedly a component of a negative feedback loop of TGF-β signaling. Mouse embryonic fibroblast (MEF) of AIMP1-knockout mice reportedly exhibit increased phosphorylation of Smad2 and Smad3, and knockdown of AIMP1 using short-interfering RNA (siRNA) promotes phosphorylation of Smad2 in human lung adenocarcinoma A549 cells. 22 The role of AIMP1 in the regulation of chondrocytes has not been studied. In this study, 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 Smad2 and Smad3 phosphorylation followed by expression of chondrogenic markers such as Col II. To test our hypothesis, we treated dedifferentiated chondrocytes with AIMP1-targeting siRNA and assessed chondrogenic redifferentiation in vitro and cartilage tissue formation in vivo. Furthermore, we assessed the role of AIMP1 in degenerated chondrocytes isolated from osteoarthritis (OA) patient.

Results
AIMP1 negatively regulates TGF-β signaling via its interactions with Smad2 and Smad3. In a previous report, AIMP1 was shown to downregulate TGF-β signaling via stabilization of smurf2. 22 In this study, we further Figure 1 AIMP1 inhibits TGF-β1 signaling by associating with SMAD2 and SMAD3. (a) HEK293 cells were transfected with FLAG-VN173-AIMP1 and HA-VC155-SMAD2/3. Cell lysates were subjected to immunoprecipitation using an anti-FLAG antibody and blotted with an anti-HA or anti-FLAG antibody. EV, empty vector; WCL, whole-cell lysate. (b and c) For BiFC analysis, HEK293 cells were transfected with FLAG-VN173-AIMP1 and HA-VC155-SMAD2/3 in the presence or absence of myc-AIMP1. Fluorescence images were captured by confocal microscopy (*, versus myc-AIMP1-/HA-VC155-/VN173-AIMP1+). Data represent mean ± S.D. of three independent experiments. (d) HEK293 cells were transfected with an empty vector (EV) or myc-AIMP1 for 24 h. For knockdown experiment, control or AIMP1 siRNA (20 nM) was transfected for 48 h. Then, TGF-β1 (1 ng/ml) was added as indicated. Cell lysates (30 μg) were subjected to SDS-PAGE and blotted with the indicated antibodies. Tubulin was used as an internal loading control. (e) SBE4-Luc and the Renilla luciferase vector were co-transfected into HEK293 cells in the presence or absence of the AIMP1 for 12 h. To assess knockdown effect of AIMP1, HEK293 cells were transfected with SBE4-Luc and the Renilla luciferase vector in the presence or absence or AIMP1 siRNA.Then, cells were treated with TGF-β1 (1 ng/ml) for 24 h. Luciferase activity was measured with the Dual-Luciferase Reporter Assay System and normalized against Renilla luciferase activity (*, versus AIMP1-/TGF-β1; **, versus si-con/TGF-β1). Data represent mean ± S.D. of three independent experiments. *Po0.05 and **Po0.05 assessed the direct interaction of AIMP1 with SMADs. Co-immunoprecipitation assay showed that AIMP1 associates with Smad2 and/or Smad3 (Figure 1a). To further confirm, a bimolecular fluorescence complementation (BiFC) assay was performed as previously described. 25 This clearly showed that AIMP1 mainly associated with Smad2 and Smad3 in the cytoplasm (Figure 1b). Competition assay using myc-AIMP1 decreased the interaction between FLAG-VN173-AIMP1 and HA-VC155-Smad2/3 in a dosedependent manner, further confirming the association of AIMP1 with Smad2 and Smad3 (Figure 1c). To unveil the functional relationship, we examined whether the expression level of AIMP1 affected the phosphorylation level of Smad2 via stimulation of TGF-β. In the presence of TGF-β, overexpression of AIMP1 and knockdown of AIMP1 using siRNA decreased and increased phosphorylation of Smad2, respectively ( Figure 1d). In addition, we investigated whether the AIMP1 expression level could affect the transcriptional activity of pSmad2 by stimulation of TGF-β. We performed the luciferase activity assay using SBE4-Luc, which contains aSmad-binding element (SBE). 26 Overexpression and downregulation of AIMP1 decreased and increased TGF-β-mediated luciferase activity, respectively ( Figure 1e). These data illustrate that AIMP1 negatively regulates TGF-β signaling by inhibiting phosphorylation of Smad2 and Smad3.
AIMP1 downregulation increases phosphorylation of Smad2 and Smad3 in dedifferentiated chondrocytes. It has been known that TGF-β signaling is perturbed in dedifferentiated chondrocytes. So, we assessed the feasibility that AIMP1 downregulation using siRNA could restore TGF-β signaling in dedifferentiated chondrocytes. Downregulation of AIMP1 indeed increased phosphorylation of Smad2 in dedifferentiated chondrocytes (Figure 2a and Supplementary Figure 1A). In addition, the Col II expression level was increased after treatment with AIMP1 siRNA (Figure 2b and Supplementary Figure 1B). To examine whether the increase in Col II expression was enhanced at the transcription level, we performed quantitative reverse transcription (RT)-PCR analysis. Col II mRNA expression was significantly increased in cells treated with AIMP1 siRNA in the presence of TGF-β compared with control siRNA (Figure 2c). In addition, we examined whether enhanced Col II expression was due to increased phosphorylation of Smad2 and Smad3. SB431542, a potent and specific inhibitor of the TGF-β superfamily type I activin receptor-like kinase (ALK) receptor, decreased 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. To test the feasibility of the chondrogenic differentiation of dedifferentiated chondrocytes by AIMP1 downregulation, the intracellular glycosaminoglycan (GAG) content was examined by Alcian blue staining. AIMP1 knockdown distinctly increased GAG matrix formation in dedifferentiated chondrocytes, but not in passage #3 (P3, population doubling (PD) of 6) chondrocytes (Figure 2e and Supplementary Figure 1C and D). The quantitation using ImageJ exhibited that GAG matrix formation was significantly increased in cells treated with AIMP1 siRNA than in cells treated with control siRNA, an increase of approximately 1.6-fold ( Figure 2f). These results suggest that AIMP1 silencing is critical to restore TGF-β-Smad signaling and favors chondrogenic marker expression in dedifferentiated chondrocytes.
AIMP1 downregulation increases the chondrogenic potential of dedifferentiated chondrocytes via enhanced nuclear localization of phospho-Smads. Since AIMP1 suppresses Smad2 phosphorylation by TGF-β in the cytoplasm as shown in Figure 1, we examined whether subcellular localization of AIMP1 between normal and dedifferentiated chondrocytes shows a different pattern. The expression level of AIMP1 was not changed during the process of dedifferentiation of normal chondrocytes (from P2 to P10) (Supplementary Figure 2A and B). Immunofluorescence staining showed that AIMP1 is mainly localized in the nucleus of normal chondrocytes (P2) (Supplementary Figure 2C). Interestingly, localization of AIMP1 was changed from nucleus to cytoplasm with increasing dedifferentiation (Supplementary Figure 2C and D).
To further demonstrate the effect of AIMP1 downregulation on phosphorylation of Smad2 and Smad3, we performed immunoblot and immunostaining analyses of nuclear and cytoplasmic extracts. In dedifferentiated chondrocytes (P6), combined treatment with TGF-β and AIMP1 siRNA increased nuclear translocation of Smad4, phosphorylated Smad2 and Smad3 compared with control siRNA/TGF-β (Figures 3a and  c). Additionally, we determined the area and nuclear diameter of dedifferentiated chondrocytes. The cell area and the nuclear diameter were significantly decreased in the AIMP1 siRNA-treated group in the presence of TGF-β ( Figure 3d and Supplementary Figure 3A and C). Furthermore, we assessed whether the AIMP1 overexpression could inhibit TGF-β signaling in normal chondrocytes. AIMP1 overexpression using adenovirus resulted in the reduced phosphorylation of Smad2/3 by TGF-β, and decreased nuclear translocation of Smads (Supplementary Figure 4A and C). Taken together, these results imply that inhibition of AIMP1 expression using siRNA could promote chondrogenic potential of dedifferentiated chondrocytes by restoring TGF-β signal.
AIMP1 downregulation improves cartilage tissue formation of dedifferentiated chondrocytes in vivo mouse subcutaneous model. To validate whether AIMP1 downregulation could promote the redifferentiation potential of dedifferentiated chondrocytes and cartilage tissue formation, we subcutaneously injected P6 chondrocytes (1 × 10 6 ) per site of each nude mouse after transfection of control or AIMP1 siRNA in the presence or absence of TGF-β, and implants were allowed to develop for 5 weeks in vivo. Then, we evaluated the expression of chondrogenic markers and GAG matrix formation. Alcian blue staining, Masson's trichrome staining, and immunohistochemistry for Col II exhibited the enhanced cartilage characteristics of implants when AIMP1 expression was downregulated with siRNA ( Figure 4a). Especially, AIMP1 downregulation by siRNA and treatment of TGF-β had a synergistic effect because AIMP1 siRNA/TGF-β+ (G-IV) showed the highest expression level of GAG matrix formation ( Figure 4b) and collagen fiber formation ( Figure 4c) compared with siRNA/TGF-β − group (G-I), siRNA/TGF-β+ group (G-II), and AIMP1 siRNA/TGFβ − group (G-III). Interestingly, AIMP1 downregulation using siRNA itself (G-III) showed the increased GAG matrix formation and collagen fiber formation compared with control siRNA/TGF-β+ group (G-II) (Figures 4b and c). Immunohistochemical staining of Col II shows agreement with the findings of enhanced GAG matrix and collagen fiber formation ( Figure 4d). In addition, RT-PCR analysis showed that AIMP1 siRNA/TGF-β+ group significantly increased expression of Col II and Aggrecan, suggesting that the expression AIMP1 knockdown enhanced expression of chondrogenic markers in OA patient-derived degenerated chondrocytes. Next, we also investigated whether the AIMP1 downregulation could have any effect of regeneration in degenerated cartilage tissues isolated from OA patients. First, we isolated healthy and degenerated chondrocytes from OA patients, and characterized. Cartilage tissues obtained from OA-affected areas had inflammatory regions in comparison with healthy white cartilage tissues obtained from the unaffected area ( Figure 5a). Isolated OA chondrocytes were significantly larger (2-fold) than healthy chondrocytes (Figures 5b and c). However, the proliferation of cartilage cells isolated from OA-affected areas did not show any difference compared with that of healthy cartilage cells (Figure 5d). Alcian blue staining and Col II immunostaining showed that the expression of GAG and Col II was remarkably reduced in OA-damaged tissue (Figures 5e and f). In particular, Alcian blue staining revealed that GAG matrix formation was decreased about 5-fold in OA cartilage compared with healthy cartilage (Figure 5f). It has been known that interleukin (IL)-1β, an inflammatory cytokine, is highly expressed in OA tissues. 27 IL-1β expression was increased at degenerated chondrocyte of OA cartilage tissue compared with healthy cartilage (Figure 5e), in agreement with the previous report. 27 Interestingly, immunohistochemical staining using AIMP1 antibody showed that AIMP1 was predominantly localized in the nuclei of healthy cartilage, whereas AIMP1 expression was not only increased but also delocalized in both the nucleus and the cytoplasm of degenerated chondrocyte (Figures 5e and g), which shows agreement with the above results (Supplementary Figure 2C and D).
To substantiate the role of AIMP1 in pathological conditions, we compared the expression level of chondrogenic markers and AIMP1 between healthy and degenerated chondrocytes by immunoblot analyses. As expected, expression of the osteogenic marker Col I was increased in degenerated chondrocytes compared with healthy chondrocytes, whereas there was no marked difference in Col II expression ( Figure 6a). In particular, AIMP1 expression was significantly increased in degenerated chondrocytes (Figures 6a and b). In accordance with dedifferentiated chondrocyte (Figures 1a and 3a and Supplementary Figure 1A), TGF-β stimulation with AIMP1 siRNA increased phosphorylation of Smad2 and Smad3 compared with TGF-β alone in OA patient-derived degenerated chondrocytes (Figure 6c). To verify the effect of AIMP1 downregulation in degenerated chondrocytes, we examined Col I and Col II expression by immunoblot analysis. In both healthy and degenerated chondrocytes, treatment with AIMP1 siRNA increased the expression level of Col II and decreased the expression level of Col I (Figure 6d).   Figure 7c) were significantly increased in AIMP1 siRNA treatment group (G-III) compared with control siRNA treatment group. Consistent with our findings in dedifferentiated chondrocytes, Col II mRNA expression in degenerated chondrocytes obtained from OA tissue was significantly increased upon AIMP1 downregulation (Figure 7d), whereas the expression of Col I, a chondrocyte dedifferentiation marker, was significantly decreased (Figure 7e).

Discussion
Current cell-based strategies for ACT require in vitro expansion of isolated autologous cells. 28 The present challenge in ACT is the dedifferentiation of chondrocytes during culturing. Recently, several attempts have been made to prevent the dedifferentiation of chondrocytes, thereby enhancing the efficiency of ACT. 11,29,30 In addition, several growth factors or cytokines can enhance the redifferentiation of dedifferentiated chondrocytes, accompanied by ECM formation. [31][32][33] Nonetheless, the short half-life and high cost of cytokines restrict their use in ACT.
In the present study, we have shown that AIMP1 inhibits TGF-β signal through association with Smad2 and Smad3 (Figure 1). Smad2 and Smad3 are phosphorylated by TGF-β stimulation, which promotes chondrogenic differentiation via an increase in Col II expression. However, transduction of TGF-β signals is perturbed in dedifferentiated chondrocytes. 16 Therefore, we hypothesized that AIMP1 silencing might influence the redifferentiation of dedifferentiated chondrocytes and increase their chondrogenic potential. Dedifferentiation shifts the production of ECM proteins from chondrogenic (Col II and Agg) to fibroblast specific (Col I). 11,34,35 Additionally, some small proteoglycans such as decorin, biglycan, and the large fibroblast type proteoglycan versican are upregulated during dedifferentiation. 12,14,[36][37][38] In accordance with the previous reports, we observed analogous effects in dedifferentiated chondrocytes after P6 (Supplementary Figure 5). Previous studies reported that TGF-β has a noteworthy role in all phases of chondrogenesis, chondrocyte proliferation, and finally terminal differentiation. 39-41 TGF-β signals are essential for the repression of articular chondrocyte hypertrophic differentiation. 42 Furthermore, Smad2 and Smad3 are critical mediators of the inhibitory effect of TGF-β on chondrocyte terminal differentiation. 43 A lack of TGF-β or disruption of its signaling pathways results in an cartilage phenotype closely resembling that observed in pathological OA tissue. 44 Although TGF-β has pro-chondrogenic properties, 45 its presence is not sufficient to completely maintain the articular chondrocyte phenotype and is probably detrimental in cell-  44,46,47 Along similar lines, phosphorylation of Smad2 and Smad3 and Col II expression in dedifferentiated chondrocytes were remarkably increased by AIMP1 siRNA with TGF-β compared with AIMP1 siRNA or TGF-β alone. Furthermore, GAG expression and cartilage formation were significantly increased in dedifferentiated chondrocytes treated with TGF-β and AIMP1 siRNA, validating the chondrogenic effects (Figures 2 and 4). Specific receptors, namely, type I and II, are involved in TGF-β signal transduction. Among type I receptors, ALK1 and ALK5 have opposite functions in human chondrocytes; Smad-driven effects are inhibited by the former and potentiated by the latter. 48 In the present study, SB431542, a general ALK5 inhibitor, reduced Col II expression through inhibition of Smad2 and Smad3 phosphorylation, suggesting the importance of Smad2 and Smad3 in chondrogenesis ( Figure 2d). Next, we questioned the relationship between AIMP1 and Smads in chondrocytes. Thus, we investigated the mechanism by which decreased AIMP1 expression increased phosphorylation of Smad2 and Smad3 in dedifferentiated chondrocytes. Previous reports suggest that TGF-β signals are transduced into nuclei by Smads. 49,50 The localization of AIMP1 changed from the nucleus to the cytoplasm in dedifferentiated chondrocytes (Supplementary Figure 2C and D). This suggests that the role of AIMP1 changes according to its localization in chondrocytes. In particular, AIMP1 is localized in the cytoplasm with prolonged culture, which may affect signaling functions related to the maintenance of chondrocyte characteristics. Treatment with a combination of AIMP1 siRNA and TGF-β significantly increased the nuclear, rather than the cytosolic, levels of phosphorylated Smad2 and Smad3 in dedifferentiated chondrocytes ( Figure 3). Nuclear localization of Smad2 and Smad3 is important to maintain chondrogenesis. 43 Therefore, our results suggest that activation of Smads by downregulation of AIMP1 restores chondrogenic characteristics in dedifferentiated chondrocytes (Figures 2-4).
TGF-β signaling is essential for the repression of chondrocyte hypertrophic differentiation and required for maintaining articular cartilage. 42 Injection of TGF-β into the periosteum of rat 51 or mouse 52 femur induces chondrocyte differentiation and cartilage formation. These reports suggest the importance of TGF-β signaling for chondrogenesis in vivo. Despite the pro-chondrogenic properties of TGF-β, its presence alone cannot sustain the chondrocyte phenotype. Moreover, TGF-β administration during ex vivo expansion of human articular chondrocytes redirects the cell phenotype toward hypertrophy. 53 Previous studies also reported that subsequent to three or four passages, canine, 54 pig, 55 and human 56 chondrocytes reportedly lose their ability to produce cartilage matrix in a nude mouse implantation model. In this study, our results demonstrated that TGF-β-mediated phosphorylation of Smad2 and Smad3 is increased in dedifferentiated chondrocytes by downregulation of AIMP1 expression (Figures 2a and  3a). In addition, we confirmed the chondrogenic effects of AIMP1 downregulation in dedifferentiated chondrocytes using an in vivo mouse subcutaneous model. Co-treatment of dedifferentiated chondrocytes with AIMP1 siRNA and TGF-β not only promoted cartilage formation in vivo but also reduced hypertrophy ( Figure 4). Thus, our results suggest that AIMP1 downregulation is a novel tool to induce cartilage formation in vivo and to resolve the serious problem of hypertrophy.
AIMP1 overexpression suppressed TGF-β-mediated phosphorylation of Smad and its nuclear translocation (Figure 1d and Supplementary Figure 4A and C). Interestingly, AIMP1 expression was increased at degenerated chondrocyte of OA cartilage (Figures 5e, g and 6b). It has been known that AIMP1 expression is increased by stimulation of a variety of inflammatory cytokines including TNFα. 20 TNFα as well as IL-1β are two major cytokines in the physiopathology of OA. 57 Therefore, the increased expression of AIMP1 by inflammatory cytokine in OA may induce its translocation from nucleus to cytoplasm to inhibit TGF-β signal. However, it needs further study to prove its molecular mechanism. AIMP1 is mainly localized in nucleus of normal chondrocyte, whereas its localization was changed from the nucleus to the cytoplasm with increasing passage number during dedifferentiation process of normal chondrocytes (Supplementary Figure 2C and D). In addition, AIMP1 is mainly localized in the nucleus of OA-derived healthy chondrocytes, whereas its expression was delocalized in both cytoplasm and nuclei of OA-derived degenerated chondrocytes as shown in dedifferentiated chondrocytes (Figure 5e and Supplementary Figure 2C and D). Translocation of AIMP1 from the nucleus to the cytoplasm suggests that AIMP1 inhibits TGF-β signaling by interacting with Smad2/3 in cytoplasm as shown in Figure 1. Thus, the increased expression of AIMP1 and its translocation from nucleus to cytoplasm in degenerated chondrocytes provide direct confirmation of the involvement of AIMP1 in the pathogenesis of OA (Figures 5 and 6). Therefore, we assessed whether redifferentiation potential of dedifferentiated chondrocytes by AIMP1 downregulation could be applied to OA-derived degenerated chondrocytes. Col I is typically produced when cells undergo fibroblastic or osteoblastic differentiation. 58 We found that Col I expression was increased in degenerated chondrocytes compared with healthy chondrocytes (Figure 6a). Treatment with AIMP1 siRNA decreased Col I expression in degenerated chondrocytes both in vitro and in vivo, suggesting the prospective benefit of AIMP1 downregulation (Figures 6 and 7).
A deficiency of Smad3 leads to OA with terminal hypertrophic differentiation of chondrocytes. 42 Overexpression of both Smad2 and Smad3 blocks the spontaneous maturation of Smad3-deficient chondrocytes. 42,59 In the current study, treatment with AIMP1 siRNA and TGF increased phosphorylation of Smad2 and Smad3 in both degenerated and healthy chondrocytes (Figure 6c). Therefore, analogous to dedifferentiated chondrocytes, we confirmed the chondrogenic  (Figure 7). In conclusion, the present study suggests that AIMP1 downregulation using siRNA is a novel tool to restore TGF-β signaling, and thus increases redifferentiation and chondrogenic potential of both dedifferentiated and degenerated chondrocytes, which could be further developed as therapeutics to treat OA (Figure 8).

Conclusions
AIMP1 is mainly localized in the nucleus of normal and healthy chondrocytes. Dedifferentiation and degeneration of chondrocytes induces translocation of AIMP1 from nucleus to cytoplasm, and then AIMP1 inhibits TGF-β signaling by associating with Smad2 and Smad3. AIMP1 knockdown using specific siRNA increased the expression of chondrogenic marker and decreased osteogenic marker in both dedifferentiated and OA patient-derived degenerated chondrocytes. Therefore, further development of AIMP1 siRNA may produce an effective drug for the treatment of OA. RT-PCR and quantitative real-time PCR analysis. Total RNA was extracted from transfected cells using TRIzol reagent (Invitrogen), and 1 μg of total RNA was used for cDNA synthesis with RT-PreMix (Bioneer, Daejeon, Korea). PCR was performed with PCR-PreMix (Bioneer) under standard PCR conditions. The primers used to amplify Col II, ColX, Agg, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are provided below. PCR consisted of an initial denaturation step at 94°C for 1 min, followed by 27 amplification cycles consisting of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, with a final extension at 72°C for 10 min. PCR products were analyzed by ultraviolet irradiation of a 1.2% agarose gel stained with ethidium bromide. For quantitative real-time PCR analysis, gene-specific primers were designed to amplify Col II, Agg, ColX, and the GAPDH as follows : Col II (forward  primer, 5′-CAC GTA CAC TGC CCT GAA GGA-3′; reverse primer, 5′-CGA TAA  CAG TCT TGC CCC ACT T-  Immunofluorescence. Cells grown on circular glass coverslips (VWR LabShop, Batavia, IL, USA) in 24-well plates were fixed with 4% paraformaldehyde for 5 min and then permeabilized with PBS supplemented with 0.1% Triton X-100 for 5 min. After washing with PBS with Tween (PBST) three times for 5 min, fixed cells were incubated for an additional 30 min in PBST containing 1% BSA to prevent nonspecific binding of antibodies, and then incubated with specific primary antibodies for 1 h. Cells were washed three times with PBST and then incubated with Alexa 488-and Alexa 594-conjugated goat anti-mouse or anti-rabbit secondary antibodies (Molecular Probes, Eugene, OR, USA). DNA was counterstained with 4', 6'-diamidino-2-phenylindole (DAPI, 0.1 μg/ml prepared in PBST).
Chondrocyte implantation and in vivo cartilage formation. Sevenweek-old female athymic mice (BALB/c-nude, Orientbio, Seoul, Korea) were anesthetized by intraperitoneal injection of Zoletil 50 (10 mg/kg; Virbac, Carros, France). A total of 1 × 10 6 dedifferentiated chondrocytes were implanted per site into the dorsal subcutaneous spaces of mice (n = 4 per group) with 200 μl fibrin gel (Greenplast Kit, Green Cross, Chungbuk, Korea). Each sample was injected into four groups of four mice. Five weeks after implantation, all mice were killed and all implants were retrieved for histological analysis. All procedures performed on animals followed the guidelines of an approved protocol from the CHA University Institutional Animal Care and Use Committee. Half of each sample was fixed in 4% formalin overnight at room temperature, embedded in paraffin, and sectioned transversely at a thickness of 5 μm. Cartilage tissue formation was evaluated by Alcian blue staining and immunohistochemical staining of Col II. Alcian blue stains GAG in cartilage tissues. The area of cartilage was measured using the NIH ImageJ version 1.34e software (http://rsbweb.nih.gov/ij/) coupled to a light microscope and then quantified as the percentage area (blue staining) of the available pore space and other tissues ((cartilage area/pore and other tissue areas) × 100%). Half of each unfixed specimen was used for RT-PCR analysis of chondrogenic gene expression.
Histology and immunohistochemistry. Dedifferentiated chondrocytes implanted into the dorsal subcutaneous spaces of mice were fixed with 4% formalin. Blocks of specimens were embedded in paraffin and cut into 5-μm-thick slices. Sections were stained with 0.5% Alcian blue prepared in 0.1 M HCl (pH 1.0) for 1 h and rinsed with tap water for microscopy analyses of GAG in cartilage tissue. For immunohistochemical staining, sections were deparaffinized with xylene for 10 min, followed by sequential hydration in ethanol. After being washed with PBS for 5 min, sections were blocked with 5% BSA for 30 min. Samples were incubated overnight at 4°C with a primary antibody against Col II (Millipore) and then with an FITCconjugated goat anti-rabbit antibody (Applied Biological Materials Inc., Richmond, BC, Canada) for 1 h at room temperature. Samples were examined with a Zeiss LSM 510 Meta laser scanning confocal microscope (Carl Zeiss Microimaging Inc., Göttingen, Germany).
Construction of plasmids and adenoviruses. Expression vectors encoding AIMP1, SMAD2, and SMAD3 were constructed by subcloning the corresponding cDNAs into HA-or FLAG-tagged BiFC plasmids (pFLAG-VN173-CMV or pHA-VC155-CMV) containing fragments derived from the newly engineered fluorescent proteinVenus, which were kindly provided by Chang-Deng Hu (Perdue University, IN, USA). Adenoviruses encoding human AIMP1 (Ad-AIMP1) were generated by insertion of the AIMP1 ORF into pAdTrack-CMV expressing GFP (Addgene, Cambridge, MA, USA).
Co-immunoprecipitation and western blot analysis. HEK293 cells were transfected with the indicated vectors using Lipofectamine Plus (Invitrogen). Cells were lysed with RIPA buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM AIMP1 role in chondrocytes J Ahn et al