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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

Autophagy, an evolutionarily conserved process for the bulk degradation of cytoplasmic components, serves as a cell survival mechanism. The purpose of this study was to elucidate the role of autophagy in human chondrocytes and pathophysiology of osteoarthritis (OA).

Methods

Autophagy in articular cartilage and primary chondrocytes was assessed using antibodies for the autophagy markers light chain 3 and beclin 1. The states of autophagy under catabolic and nutritional stresses were examined. We also examined the effects of inhibition or induction of autophagy under stimulation with interleukin-1β. Autophagy was inhibited by small interfering RNA targeting ATG5, and autophagy was induced by rapamycin. The effects of inhibition or induction of autophagy were examined by real-time polymerase chain reaction for aggrecan, COL2A1, MMP13, and ADAMTS5 messenger RNA. To further examine the mechanism of autophagy regulation in OA human chondrocytes, we investigated whether autophagy modulates apoptosis and reactive oxygen species (ROS).

Results

Autophagy was increased in OA chondrocytes and cartilage. Catabolic and nutritional stresses increased autophagy. In addition, the inhibition of autophagy caused OA-like gene expression changes, while the induction of autophagy prevented them. Furthermore, the inhibition of autophagy increased the amount of cleaved poly(ADP-ribose) polymerase and cleaved caspase 9, while the induction of autophagy inhibited these increases. ROS activity was also decreased by induction of autophagy.

Conclusion

These observations suggested that increased autophagy is an adaptive response to protect cells from stresses, and that autophagy regulates OA-like gene expression changes through the modulation of apoptosis and ROS. Further studies about autophagy in chondrocytes will provide novel insights into the pathophysiology of OA.

Osteoarthritis (OA) is the most common disorder in joint diseases that causes joint pain and dysfunction in the affected patients. Multiple factors have been suggested to be involved in the pathogenesis of OA, including mechanical, genetic, and aging-associated factors. OA is characterized by loss of tissue cellularity and extracellular matrix (ECM) damage (1). Chondrocytes are the only resident cells found in cartilage and are responsible for both synthesis and turnover of the abundant ECM (2). Therefore, maintaining the chondrocytes in a healthy condition appears to be an important factor for maintaining the entire cartilage and preventing degeneration of cartilage.

Autophagy is a cellular self-digestion process that is evolutionarily conserved among species and generally activated under conditions of nutrient deprivation. Under conditions of nutrient deprivation, cells maintain only the essential minimum components to prevent energy loss by degrading unnecessary intracellular components with the autophagic process. Thus, autophagy has been suggested as an important cell survival mechanism under stresses (3). A growing number of studies have recently revealed that autophagy plays the important role of housekeeping in the physiologic process through the intracellular clearance of unnecessary proteins, pathogens, and damaged organelles, including mitochondria, peroxisomes, and endoplasmic reticulum (4–6). In addition to the important roles of autophagy in the physiologic process, it has been implicated in the pathogenesis of a variety of human diseases, such as neurodegenerative disease, cardiac disease, muscle disease, and inflammatory disease (4). One of the common pathologic conditions observed in such diseases is the accumulation of aggregate-prone proteins potentially harmful to the cells, which is most likely caused by impairments of autophagy (7). Therefore, autophagy has been suggested to have preventive roles against some human disease.

Judging from the broad cell-protective functions of autophagy in a variety of cells, autophagy may also play a protective role in human chondrocytes and prevent chondrocytes from undergoing OA changes. However, the roles of autophagy in the pathogenesis of OA are still unclear. In the present study, we investigated the roles of autophagy in human chondrocytes and in the pathophysiology of OA. Our observations suggest that autophagy protects human chondrocytes from stresses and may prevent chondrocytes from undergoing OA changes.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

OA and non-OA cartilage sampling and processing.

OA cartilage tissues were obtained from femoral condyles of 8 patients with medial knee OA during total knee joint replacement surgery (2 men and 6 women, mean age 74.3 years). The cartilage samples were obtained from the distal part of both the lateral and medial femoral condyles of the knee joint. Lateral condyles and medial condyles were used as cartilage with mild OA and cartilage with severe OA, respectively. Articular cartilage tissues without OA were obtained from 5 age-matched patients undergoing surgery for femoral neck fracture (1 man and 4 women, mean age 75.3 years). None of the 5 patients had a history of joint disease, and none of the samples showed macroscopically obvious progressed OA changes. For histologic experiments, the samples were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 20–24 hours at 4°C and decalcified in 20% EDTA (pH 7.2) at 4°C. Sections (7 μm) were cut. For Safranin O and fast green staining, sections were stained with hematoxylin for 4 minutes, followed by fast green staining for 5 minutes and Safranin O staining for 1 minute. All OA and non-OA cartilage samples were obtained in accordance with the World Medical Association Declaration of Helsinki ethical principles for medical research involving human subjects.

Culture of human chondrocytes.

We purchased normal human knee articular chondrocytes from Lonza. These are human primary chondrocytes isolated from donor knee joints without any abnormalities. We confirmed that these cells preserved the characteristic phenotype of normal chondrocytes as we and others have previously reported (8, 9). Chondrocytes were maintained according to the protocol of the manufacturer. Chondrocytes between passages 3 and 7 were used for the analysis. OA chondrocytes were isolated from the lateral cartilage tissues and cultured for 3 days until reaching confluence. RNA and protein were extracted from chondrocytes after confluence was reached. The expression of light chain 3 (LC3) and beclin 1 was examined by real-time polymerase chain reaction (PCR) and immunoblotting as described below.

Assessment of autophagy in normal chondrocytes under stresses.

Briefly, 24 hours after subculture, adherent cells were subjected to nutritional stress and catabolic stresses, after which the state of autophagy was examined by real-time PCR. For nutritional stress, cells were cultured with serum-starved medium (Dulbecco's modified Eagle's medium supplemented with 1% fetal bovine serum [FBS]) for 6, 12, and 24 hours. For catabolic stresses, cells were cultured with 10 ng/ml of interleukin-1β (IL-1β; R&D Systems) or 0.25 mM sodium nitroprusside (SNP; Wako) for 12 and 24 hours. SNP was used as an exogenous source of nitric oxide (NO).

Autophagy inhibition.

Autophagy was suppressed by small interfering RNA (siRNA) targeting ATG5, an autophagy-essential gene. Atg5 is a key component of the Atg12–Atg5 conjugation complex that is required for the formation of autophagosome (10). The essential role of Atg5 in autophagy has been established by the deficit of autophagy in Atg5-knockout mice (11, 12). All siRNA were purchased from Invitrogen, and 2 siRNA with distinctive sequences were used. The sense strand sequences of the RNA duplexes were as follows: for ATG5 no. 1, 5′-AAUUCGUCCAAACCACACAUCUCGA-3′; for ATG5 no. 2, 5′-UCGAGAUGUGUGGUUUGGACGAAUU-3′. The siRNA were delivered into the normal chondrocytes by lipofection according to the manufacturer's instructions (Lipofectamine 2000; Invitrogen).

Autophagy activation.

To activate autophagy, rapamycin was used as described previously (13, 14). Normal chondrocytes were pretreated with 10 μM rapamycin (Tocris) 1 hour prior to treatment with 10 ng/ml IL-1β. Twenty-four hours after the treatment with IL-1β, RNA or protein extraction was performed.

Immunohistochemistry.

Cartilage tissues were fixed in 4% paraformaldehyde in 0.1M PBS for 24 hours and embedded in paraffin wax. Each specimen was cut into 5 μm slices along the sagittal plane, and the sections were incubated with primary antibody for 1 hour at 37°C. Following this, sections were incubated with horseradish peroxidase (HRP)–conjugated goat anti-rabbit IgG polyclonal antibody (Nichirei Bioscience) for 1 hour at 37°C. The signal was developed as a brown reaction product using the peroxidase substrate 3,3′-diaminobenzidine (Nichirei Bioscience), and the sections were counterstained with methyl green and examined using a microscope. To detect LC3 and beclin 1, rabbit anti-human LC3β and anti–beclin 1 polyclonal antibody (Santa Cruz Biotechnology) were used at a 1:20 dilution (pretreated with citrate buffer for 40 minutes at 95°C).

Immunocytochemistry.

Normal human knee articular chondrocytes were seeded on 4-well chamber slides at a density of 5 × 104/well. Cells were cultured in 1% FBS or treated with 10 ng/ml IL-1β or 0.25 mM SNP. Forty-eight hours after the incubation under the above conditions, cells were fixed in 4% paraformaldehyde in 0.1M PBS for 15 minutes, and then the fixed cells were incubated in blocking solution (3% bovine serum albumin) for 1 hour. The cells were incubated with a primary antibody against human LC3β (Santa Cruz Biotechnology) overnight at 4°C followed by an Alexa Fluor 488–conjugated secondary antibody (Cell Signaling Technology) for 1 hour at room temperature. Nuclei were counterstained with DAPI. Images were obtained using a microscope (Biozero; Keyence).

Real-time PCR analysis.

Normal chondrocytes were cultured in 6-well plates, and RNA was extracted with QIAshredder homogenizers and the RNeasy Mini kit (Qiagen) according to the manufacturer's protocol. One microgram of total RNA was reverse-transcribed to first-strand complementary DNA (cDNA). The converted cDNA samples (2 μl) were amplified in triplicate by real-time PCR (using the ABI Prism 7700 sequence detection system) in a final volume of 25 μl using SYBR Green or TaqMan Master Mix reagent (Applied Biosystems). Melting curve analysis was performed using Dissociation Curves software (Applied Biosystems) to ensure that only a single product was amplified. Specificity of the reactions was confirmed by 2.5% agarose gel electrophoresis. Results were obtained using ABI Prism 7700 sequence detection software and evaluated using Excel (Microsoft). Predesigned primers for LC3, beclin1, ATG5, COL2A1, and actin were obtained from Takara Bio, and predesigned primers for MMP13, ADAMTS5, aggrecan, and GAPDH were obtained from Applied Biosystems.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blotting.

Normal chondrocytes were lysed on ice for 30 minutes in lysis buffer supplemented with proteinase and phosphatase inhibitor mix (Roche Diagnostics). The lysates were centrifuged at 15,000 revolutions per minute for 5 minutes to remove cellular debris, and the supernatants were collected. Separated proteins were transblotted electrically onto the blotting membrane (Amersham Biosciences). The membranes were probed with primary antibodies followed by an HRP-conjugated secondary antibody. Proteins were visualized with ECL Plus reagent (Amersham Biosciences) with Chemilumino analyzer LAS-3000 mini (Fujifilm). The following antibodies were used in this study: rabbit anti-human type II collagen antibody and mouse anti-human aggrecan antibody (both from Abcam); rabbit anti-human LC3B antibody, rabbit anti-human beclin 1 antibody, rabbit anti-human cleaved caspase 9 antibody, and rabbit anti-human cleaved poly(ADP-ribose) polymerase (PARP) antibody (all from Cell Signaling Technology); and HRP-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG (both from Amersham Biosciences).

LysoTracker staining for lysosomal activity.

LysoTracker staining (Molecular Probes) was performed as described previously (15) to detect functional lysosomes and an expansion of the lysosomal system which are usually observed during autophagy. Briefly, the cultures were incubated at 37°C for 20 minutes with 50 nM LysoTracker Red, and then after one rinse and replacement of the medium they were visualized using fluorescence microscopy, and representative photomicrographs were obtained.

Flow cytometric assay for reactive oxygen species (ROS).

The intracellular ROS level was assessed by flow cytometry. The dye dihydrodichlorofluorescein diacetate (DCFH-DA; Invitrogen) was used to evaluate the intracellular production of ROS (16). Briefly, normal chondrocytes were incubated for 30 minutes with 10 μM DCFH-DA. After washing, the cells were collected and visualized by flow cytometry in the FL3 channel, with fluorescence levels denoting the percentage of cells positive for ROS production.

Statistical analysis.

All data were expressed as the mean ± SD. Comparisons between 2 groups were made using one-way analysis of variance. Post hoc analysis was performed by Fisher's protected least significant difference test. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Autophagy in human chondrocytes and human articular cartilage tissues.

We used normal human knee articular chondrocytes (hereinafter called normal chondrocytes). We first examined the expression of the autophagy markers LC3 and beclin 1 in human chondrocytes and compared the expression levels between normal chondrocytes and chondrocytes from OA patients (OA chondrocytes). The expression of LC3 and beclin1 messenger RNA (mRNA) was increased in OA chondrocytes compared with normal chondrocytes (Figure 1A). To confirm autophagic activity, expression levels of LC3-II and beclin 1 protein were further confirmed by Western blotting, since the conversion of LC3-I to the lower migrating form, LC3-II, has been used as an indicator of autophagy (17). Western blotting analysis showed that LC3-II and beclin 1 levels in chondrocytes from OA cartilage tended to be more increased than in normal chondrocytes (Figure 1B).

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Figure 1. Autophagy in human chondrocytes and articular cartilage. A and B, The expression of LC3 and beclin1 mRNA was examined by real-time polymerase chain reaction (A), while the expression of light chain 3 (LC3; two migrating forms, LC3-I and LC3-II) and beclin 1 protein was examined by immunoblotting (B). Normal indicates normal human knee articular chondrocytes; OA1–OA5 indicates chondrocytes from 5 different patients with osteoarthritis. The value for normal was set to 1. Values in A are the mean. C, Safranin O and fast green staining was performed on articular cartilage of the femoral head from a 75-year-old female patient with femoral neck fracture (non-OA cartilage), on articular cartilage of the lateral condyle from a 68-year-old male patient undergoing total knee arthroplasty (cartilage with mild OA), and on articular cartilage of the medial femoral condyle from the same 68-year-old patient with OA (cartilage with severe OA). D and E, Shown are immunohistochemistry analyses of LC3 (D) and beclin 1 (E) in the same types of cartilage samples shown in C. Arrows indicate LC3-positive cells and beclin 1–positive cells. Boxed areas in images at left are shown at higher magnification in images at right. LC3 and beclin 1 were more strongly expressed in cartilage with mild OA than in non-OA cartilage and cartilage with severe OA. Representative results from repeated experiments are shown. Experiments were performed 3 times using different sections.

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We also examined LC3 and beclin 1 expression in human articular cartilage tissues. We examined LC3 and beclin 1 expression using non-OA cartilage of the femoral head from patients with femoral neck fracture, cartilage of the lateral femoral condyle from patients with mild OA, and severely degenerated cartilage of the medial femoral condyle from patients with medial-type OA. Safranin O staining showed well-stained ECM in the non-OA cartilage and slightly less-stained cartilage of the lateral condyle. The cartilage of the medial condyle was severely degenerated and was barely stained with Safranin O (Figure 1C) (for further information, please contact the corresponding author). LC3 and beclin 1 were strongly expressed in the cartilage with mild OA of the lateral compartment of the knee joint, compared with the non-OA cartilage of the femoral head and the cartilage with severe OA of the medial compartment of the knee joint (Figures 1D and E) (for further information, please contact the corresponding author). LC3-positive and beclin 1–positive cells were mainly observed in the superficial zone of the lateral cartilage samples (Figures 1D and E) (for further information, please contact the corresponding author).

State of autophagy in normal chondrocytes under nutritional and catabolic stresses.

We next examined LC3 and beclin 1 expression under various stresses. The nutritional stress induced by treatment with serum-starved medium for 24 hours significantly up-regulated LC3 and beclin1 mRNA expression levels in human chondrocytes (Figure 2A). The catabolic stresses induced by treatment with 10 ng/ml IL-1β or 0.25 mM SNP for 24 hours also resulted in increased expression levels of LC3 and beclin1 mRNA in human chondrocytes (Figures 2B and C), indicating that autophagic activity was induced by the stresses. In addition, Western blotting analysis showed that LC3-II and beclin 1 in human chondrocytes were increased by nutritional and catabolic stresses in a time-dependent manner (Figure 2D). Immunocytochemical analysis consistently showed that LC3 expression was increased in cells under nutritional and catabolic stresses compared with the control (Figure 2E) (for further information, please contact the corresponding author). These observations suggested that autophagy was increased by the stresses.

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Figure 2. The state of autophagy in human chondrocytes under stresses. AC, Significant up-regulation of LC3 and beclin1 mRNA was observed 24 hours after nutritional stress (1% fetal bovine serum [FBS]) (A), and expression of mRNA for LC3 and beclin1 was significantly up-regulated 24 hours after treatment with 10 ng/ml interleukin-1β (IL-1β) (B) or 0.25 mM sodium nitroprusside, an exogenous source of nitric oxide (NO) (C), as determined by polymerase chain reaction. Values are the mean ± SD. ∗ = P < 0.05. D, Shown is Western blotting for light chain 3 (LC3; two migrating forms, LC3-I and LC3-II) and beclin 1. LC3-II and beclin 1 were increased in a time-dependent manner by nutritional stress (top), treatment with 10 ng/ml IL-1β (middle), and treatment with 0.25 mM sodium nitroprusside (bottom). E, Shown is immunocytochemistry analysis for LC3. Stronger expression of LC3 and LC3-positive puncta was observed in the cytoplasm of chondrocytes subjected to nutritional stress than in the cytoplasm of control chondrocytes.

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Inhibition or activation of autophagy in normal chondrocytes.

To further examine the effect of autophagy in human chondrocytes, we inhibited or activated autophagy under stimulation with IL-1β. To inhibit autophagy, we used siRNA for ATG5, which is essential for autophagosome formation (18). To activate autophagy, we used rapamycin, which was reported to be a potent inducer of autophagy (13, 14). We first confirmed whether siRNA for ATG5 effectively down-regulated ATG5 mRNA and ATG5 protein. Real-time PCR analysis showed a significant reduction of ATG5 mRNA (P < 0.05). The mean expression level of ATG5 mRNA was reduced to 40% of the expression level of the control. In addition, Western blotting analysis showed that ATG5 protein was efficiently reduced by ATG5 siRNA compared with the control (Figure 3A). To confirm modulation of autophagy by ATG5 siRNA or rapamycin, we examined lysosomal activity. As expected, LysoTracker Red labeling was enhanced in chondrocytes treated with rapamycin, whereas labeling was debilitated in chondrocytes treated with ATG5 siRNA (Figure 3B).

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Figure 3. The state of autophagy in normal chondrocytes after transfection of small interfering RNA (siRNA) for ATG5 or treatment with rapamycin. A, Quantitative results of real-time polymerase chain reaction for ATG5 mRNA and Western blotting for ATG5 protein after transfection of control siRNA or ATG5 siRNA. The expression of ATG5 mRNA was significantly reduced 48 hours after the transfection of siRNA for ATG5. Values are the mean ± SD. ∗ = P < 0.05. B, LysoTracker Red labeling after transfection of siRNA for ATG5 or treatment with rapamycin. C and D, Immunoblotting for light chain 3 (LC3; two migrating forms, LC3-I and LC3-II) and beclin 1 after transfection of ATG5 siRNA (C) or treatment with rapamycin (D) and stimulation with interleukin-1β (IL-1β). Results are representative of those from 3 independent experiments.

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We next examined the effect of inhibition and activation on expression of LC3-II and beclin 1 proteins in normal chondrocytes under stimulation with IL-1β. The transfection of control siRNA alone slightly increased LC3-II (for further information, please contact the corresponding author). The transfection of siRNA for ATG5 significantly reduced the amounts of LC3-II and beclin 1 induced by treatment with IL-1β (Figure 3C), while treatment with rapamycin increased the amounts of LC3-II and beclin 1 (Figure 3D). These observations indicated that ATG5 siRNA effectively reduced autophagy while treatment with rapamycin effectively activated autophagy.

Effect of inhibition or activation of autophagy on IL-1β–induced gene expression changes.

Since IL-1β stimulation has been reported to induce OA-like gene expression changes (19, 20), we examined the effects of autophagy on chondrocyte gene expression under stimulation with IL-1β. The transfection of ATG5 siRNA alone slightly decreased the expression level of COL2A1 and aggrecan mRNA, and treatment with IL-1β significantly decreased the expression of COL2A1 and aggrecan. In addition, inhibition of ATG5 under stimulation with IL-1β further reduced COL2A1 and aggrecan mRNA (Figure 4A). The same tendency was also observed in protein levels detected by Western blotting (Figure 4C) (for further information, please contact the corresponding author). In remarkable contrast to the inhibition of autophagy by the ATG5 siRNA, treatment with rapamycin under stimulation with IL-1β recovered COL2A1 and aggrecan expression reduced by stimulation with IL-1β (Figure 4B). On the other hand, expression of the cartilage-degrading enzymes MMP13 and ADAMTS5 was increased by IL-1β, and inhibition of autophagy by ATG5 siRNA further increased expression of those genes (Figure 4D). In addition, activation of autophagy by rapamycin reduced increased MMP13 and ADAMTS5 expression induced by IL-1β (Figure 4E). These observations suggested that activation of autophagy ameliorated the OA changes induced by treatment with IL-1β, while inhibition of autophagy exacerbated them.

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Figure 4. Effects of inhibition or activation of autophagy under stimulation with IL-1β. A and B, Quantitative results of real-time polymerase chain reaction (PCR) for COL2A1 and aggrecan after transfection of siRNA for ATG5 (A) or treatment with rapamycin (B), with or without IL-1β stimulation. C, Immunoblotting for type II collagen and aggrecan after transfection of siRNA for ATG5, with or without IL-1β stimulation. D and E, Quantitative results of real-time PCR for MMP13 and ADAMTS5 after transfection of siRNA for ATG5 (D) or treatment with rapamycin (E), with or without IL-1β stimulation. Values are the mean ± SD. ∗ = P < 0.05. Cont = control (see Figure 3 for other definitions).

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Effect of inhibition or activation of autophagy on apoptosis of human chondrocytes.

Since it has been reported that increased apoptosis is a cause of OA (21, 22) and IL-1β is a known apoptosis inducer (23, 24), we also examined whether autophagy modulates IL-1β–induced apoptotic signals in human chondrocytes. The transfection of siRNA for ATG5 alone caused a slight increase in the level of cleaved PARP and cleaved caspase 9, and treatment with IL-1β alone strongly increased the level of cleaved PARP and cleaved caspase 9. In addition, ATG5 siRNA under treatment with IL-1β further increased the level of cleaved PARP and cleaved caspase 9 (Figure 5A), while rapamycin substantially inhibited the increase in cleaved PARP and cleaved caspase 9 induced by IL-1β (Figure 5B). These observations indicated that IL-1β–induced apoptosis was enhanced by inhibition of autophagy while it was reduced by activation of autophagy.

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Figure 5. Effect of autophagy on apoptosis and reactive oxygen species (ROS) under stimulation with IL-1β. A and B, Shown is immunoblotting for cleaved caspase 9 and cleaved poly(ADP-ribose) polymerase (PARP) after transfection of siRNA for ATG5 (A) or treatment with rapamycin (B), with or without IL-1β stimulation. C, The ROS level was examined by flow cytometry. Top left, Chondrocytes without stimulation (control); top right, chondrocytes treated with rapamycin; bottom left, unstimulated chondrocytes treated with IL-1β; bottom right, chondrocytes treated with rapamycin and IL-1β. Increased generation of ROS was detected in IL-1β–treated cells. In contrast, treatment with rapamycin partially reduced the increased generation of ROS induced by treatment with IL-1β. The experiments were conducted 5 times using different cultures. D, Shown is quantitative analysis of ROS levels. The level of ROS in normal chondrocytes was set at 100% for the respective comparisons. The mean relative ROS level with IL-1β was increased to 131%, and the increase was significantly reduced to 101% by treatment with rapamycin. Values are the mean ± SD. ∗ = P < 0.05. FACS = fluorescence-activated cell sorting (see Figure 3 for other definitions).

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Effect of activation of autophagy on the intracellular ROS level in human chondrocytes.

It has been reported that IL-1β induction of proteinase expression in articular chondrocytes was found to be ROS-dependent (25, 26). To further examine the mechanism of modulation of IL-1β–induced gene expression changes by autophagy, we next investigated intracellular ROS levels by flow cytometry. The generation of ROS was increased in IL-1β–treated cells. In contrast, treatment with rapamycin partially reduced increased generation of ROS induced by treatment with IL-1β (Figure 5C). When the level of ROS in normal chondrocytes without stimulation was set at 100% for the respective comparisons, the mean relative ROS level was increased to 131% by treatment with IL-1β, and the increase was significantly reduced to 101% of the control by treatment with rapamycin (Figure 5D). These observations suggest that autophagy modulates the production of intracellular ROS induced by IL-1β.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Autophagy is a well-conserved system among species and has been suggested to play important roles in various physiologic processes and pathologic events. In the present study, we examined the roles of autophagy in human chondrocytes. We first examined that state of autophagy using human chondrocytes and cartilage samples. Autophagy was more up-regulated in OA cartilage than in non-OA cartilage. In addition, the expression of autophagic markers was up-regulated in OA chondrocytes compared with normal chondrocytes. Conflicting observations were reported by Carames et al. In contrast to our observations, they reported that autophagy was decreased in cartilage with mild OA compared with normal cartilage (27). The difference between their result and ours may be attributed to the location of harvested OA cartilage samples. We examined the lateral condyle cartilage as cartilage with mild OA and the medial condyle cartilage as cartilage with severe OA. Since all our patients had varus deformity, the medial condyle cartilage was severely degenerated and had low cellularity. The degenerative change of the lateral cartilage was mild, and our observations may reflect the initial cartilage degeneration stage. Of note, we observed increased autophagy in chondrocytes under stresses, such as NO and IL-1β (Figure 2), suggesting that at least autophagy can increase during the initial degenerative phase in which chondrocytes were under such stresses.

Consistent with our observations, Bohensky et al reported that chondrocytes in OA cartilage displayed numerous autophagic LC3 puncta. In contrast, those in healthy cartilage did not show an elevation in punctate LC3 (28). In addition, Almonte-Becerril et al reported that autophagy was increased in the superficial and middle zones of articular cartilage in the rat OA model (29). Interestingly, we also observed that LC3 and beclin 1 tended to be more increased in the superficial zone of the cartilage where chondrocytes were presumably under more stresses compared with the deep zone. Thus, during the development of OA, autophagy may increase as an adaptive response to protect cells from various stresses, and failure of the adaptation may lead to further progression of degeneration. In support of this idea, autophagy was decreased in the severely damaged cartilage of the medial femoral condyle. Further studies are required to elucidate the detailed changes of autophagic activity during the progression of OA.

To further examine the role of autophagy in chondrocytes under stresses, we examined the effect of inhibition of autophagy and activation of autophagy under stimulation with IL-1β. Although inhibition or activation of autophagy without IL-1β stimulation did not significantly affect gene expression changes, inhibition of autophagy exacerbated IL-1β–induced OA-like gene expression changes and apoptotic signals, while activation of autophagy inhibited them. These observations suggested that autophagy plays protective roles predominantly in conditions where chondrocytes are under stresses. To support this notion, Carames et al also proposed protective roles in chondrocytes based on their immunohistochemical analysis (27).

We also examined possible mechanisms of the effect of autophagy on chondrocyte gene expression changes. Interestingly, activation of autophagy by rapamycin reduced the intracellular ROS levels induced by IL-1β. It has been reported that IL-1β induction of proteinase expression in articular chondrocytes was ROS-dependent (25, 26). In addition, increased ROS activity has been reported to enhance catabolic signaling pathways, thereby causing a reduction of matrix synthesis, an inhibition of growth factor expression, and increased production of matrix metalloproteinases and cytokines (28). Furthermore, it has been reported that increased release of ROS from damaged mitochondria can induce apoptosis (30). Given that one of the cytoprotective functions of autophagy is removal of damaged mitochondria (31), the inhibitory effect of activation of autophagy against IL-1β–induced OA-like gene expression changes and apoptotic signals might possibly occur through reduction of the intracellular ROS level by eliminating harmful damaged mitochondria (Figure 6).

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Figure 6. Our proposed model showing how autophagy modulates osteoarthritis (OA)–like gene expression changes in human chondrocytes. Increased release of reactive oxygen species (ROS) from damaged mitochondria induced by interleukin-1β (IL-1β) causes increased catabolic signals, increased apoptosis, and decreased anabolic signals. The activation of autophagy reduces the intracellular ROS by removing damaged mitochondria, thereby protecting chondrocytes from OA-like changes.

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In summary, we demonstrated that inhibition of autophagy enhanced IL-1β–induced OA-like gene expression changes, while induction of autophagy prevented them, possibly through modulation of the intracellular level of ROS. Our observations suggest that autophagy plays important roles in human chondrocytes to protect cells from stresses and may relate to pathogenesis of OA. Further studies about autophagy in chondrocytes will provide novel insights into the pathophysiology of OA and will lead to new therapeutic approaches.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Matsushita had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Sasaki, Takayama, Matsushita, Ishida, Kubo, Matsumoto, Fujita, Oka, Kurosaka, Kuroda.

Acquisition of data. Sasaki, Takayama, Matsushita, Ishida, Kubo, Matsumoto, Fujita, Oka, Kurosaka, Kuroda.

Analysis and interpretation of data. Sasaki, Takayama, Matsushita, Ishida, Kubo, Matsumoto, Fujita, Oka, Kurosaka, Kuroda.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We thank Ms Kyoko Tanaka, Ms Minako Nagata, Ms Maya Yasuda, and Mr. Takeshi Ueha for technical assistance and Janina Tubby for English language assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES