Apoptosis In Human Chondrocytes Research Articles

Piezo1 protein induces the apoptosis of human osteoarthritis-derived chondrocytes by activating caspase-12, the signaling marker of ER stress.

The present study was carried out to determine whether the mechanically activated cation channel Piezo1 protein plays a role as a signaling pathway which causes the apoptosis of human chondrocytes. The chondrocytes were isolated, cultured, and then subjected to mechanical stretch force for 0, 2, 12, 24 and 48 h, respectively. The expression levels of Piezo1 and the apoptosis-related protein caspase-12 were assessed by reverse transcription-quantitative polymerase chain reaction, as well as the apoptosis-related genes, B cell lymphoma/leukemia-2 (Bcl-2), Bcl-associated X protein (Bax) and Bcl-2-associated death promoter (BAD). Lactate dehydrogenase (LDH) activity was used to discern dead cells. Piezo1 expression was determined by immunofluorescence. In addition, Piezo1 inhibitor, GsMTx4, was used to block the mechanically activated (MA) cation channel Piezo1, and served as a positive control. The results showed that the osteoarthritis (OA)-derived chondrocytes showed a tendency to undergo late-stage apoptosis under compressive loading. Piezo1 and caspase-12 were significantly upregulated under static compressive stimuli and the expression was related to the rate of apoptosis of the OA-derived chondrocytes during compressive loading. The expression of caspase-12 and late-stage apoptosis of the human OA-derived chondrocytes were repressed by GsMTx4, the specific inhibitor of Piezo1, while the expression of Piezo1 and the induction of the apoptosis of the OA-derived chondrocytes during compressive loading was not totally blocked. Thus, we conclude that Piezo1 plays an important role in the apoptosis of human OA-derived chondrocytes through a caspase-12-dependent pathway. The expression of Piezo1 protein was not totally inhibited by GsMTx4.

Retraction Note to: Advanced oxidation protein products induce chondrocyte death through a redox-dependent, poly(ADP-ribose) polymerase-1-mediated pathway.

This study aimed to investigate the effect of AOPPs on apoptosis in human chondrocytes. Chondrocytes were treated with AOPPs. Cell death, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, reactive oxygen species (ROS) generation, and the expression of apoptotic proteins were detected in vitro. AOPPs levels were detected by colorimetric method. The results in vitro demonstrated that AOPPs induced cell death in human chondrocyte through a redox-dependent pathway, including RAGE-mediated, NADPH oxidase-dependent ROS generation, and poly (ADP-ribose) polymerase-1 (PARP-1) activation. Targeting AOPPs-induced cellular mechanisms might emerge as a promising therapeutic option for patients with RA.

TAR DNA-binding protein 43 inhibits inflammatory response and protects chondrocyte function by modulating RACK1 expression in osteoarthritis

BackgroundTAR DNA-binding protein-43 (TDP-43, transactive response DNA binding protein 43kDa) accumulates in the cytoplasm of affected neurons and glia, as large inclusions of stress granules (SGs). However, the mechanism of TDP-43 interaction with the target genes and its specific role in osteoarthritis (OA) progression is still unknown. The goal of this study was to identify the role of TDP-43 in OA progression by modulating its target genes. MethodsMSCs were transfected with TDP-43 gene lentivirus. The role of elevated TDP-43 expression in the differentiation of MSCs to chondrocytes was investigated. Cell function assay was used to evaluate the proliferation and apoptosis of Human chondrocytes (HCs) co-cultured with MSC. Truncated TDP-43/p35 expression and SGs formation in HCs were identified using cyto-immunofluorescence assay. Critical genes mediating apoptotic and proliferative signaling in HCs were measured using co-culturing MSC assays. The phosphorylation of key kinases was analyzed using the HTRF phosphokinase assay, and the expression of key genes in proliferative and apoptotic signaling transduction pathways was detected using qRT-PCR. ResultsThe MSCs differentiated into HCs after transfection of TDP-43 genes. The TDP-43 can degrade truncated TDP-43/p35, and promote SGs formation and HCs proliferation, and inhibit HCs apoptosis after co-culturing with MSCs. TDP-43 overexpression in MSCs promoted high expression of RACK1 and promoted phosphorylation of key kinases in HCs. Critical genes were highly expressed in P38 MAPK/MKK3 proliferative signaling, but not in P38 MAPK/JNK MAPK signaling. ConclusionsThe chondrogenically differentiation of MSCs was not influenced by transfection of TDP-43 genes, and promoted HCs growth after co-culturing with HCs. The data indicated that TDP-43/p35 contributed to SGs formation by promoting RACK1 expression. The study sheds new light on post-transcriptional regulation and apoptosis in OA by RACK1, which is a potential treatment strategy for OA.

Advanced oxidation protein products induce chondrocyte death through a redox-dependent, poly (ADP-ribose) polymerase-1-mediated pathway.

This study aimed to investigate the effect of AOPPs on apoptosis in human chondrocytes. Chondrocytes were treated with AOPPs. Cell death, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, reactive oxygen species (ROS) generation, and the expression of apoptotic proteins were detected in vitro. AOPPs levels were detected by colorimetric method. The results in vitro demonstrated that AOPPs induced cell death in human chondrocyte through a redox-dependent pathway, including RAGE-mediated, NADPH oxidase-dependent ROS generation, and poly (ADP-ribose) polymerase-1 (PARP-1) activation. Targeting AOPPs-induced cellular mechanisms might emerge as a promising therapeutic option for patients with RA.

Mechanism of Piezo1 protein induced apoptosis of osteoarthritis chondrocytes through MAPK/ERK5 signal pathway

Objective To investigate whether the new mechanically-activated (MA) cation channel Piezo1 protein can cause the apoptosis of human chondrocytes under compressive loading, using a Flexercell unit by activating MAPK/ERK5 signal pathway. Methods Primary human chondrocytes were isolated, cultured, and then subjected to the static compressive loading for 2 h,12 h, 24 h and 48 h, respectively. The GsMTx4, which is the special inhibitor of the Piezo1 protein and the BIX02188, which is the inhibitor of the ERK5 served as the inhibitor group. The immunofluorescence was used to locate the expression of the Piezo1 protein. The expressions of Piezo1 and ERK5 were assessed by reverse transcription-polymerase chain reaction (RT-PCR), as well as the apoptosis gene B cell lymphoma/leukemia-2 (Bcl-2), Bclassociated X protein (Bax) and Bclassociated death promoter (BAD). In addition, Piezo1 inhibitor, GsMTx4, was used to block the MA cation channel Piezo1, served as the inhibitor group. AV-PI was used to detect the apoptosis of the OA chondrocytes. Results The location of the Piezo1 was expressed in nucleus and cytoplasm of chondrocytes. The expression of the Piezo1, ERK5, BAD, and Bax mRNA in the OA chondrocytes is weak. The 12 h stretch force group was significant increased, and the 24 h stretch force group was the highest expression. However, the expression of the 48 h group was decreasing. The expression of the Bcl-2 in the 12 h group was decreasing, and the 12 h stretch force group was the lowest expression while the 24 h stretch force group was increasing. In the GsMTx4 group, the expression of the Piezo1, ERK5, BAD, Bax was decreasing while the Bcl-2 was increasing. In the BIX02188 group, the expression of the ERK5, BAD, Bax was increasing while the Bcl-2 was decreasing, while the expression of the Piezo1 was not change. The result of AV-PI shown that the 2 h stretch force group increased early stage of apoptosis. The 12 h stretch force group increased late stage of apoptosis, and the 24 h stretch force group's apoptotic rate was the highest. However, the apoptotic rate of the 48 h group was lower than the 24 h stretch force group. The GsMTx4 could inhibit the late stage of apoptosis. Conclusion Piezo1 plays an important role in the apoptosis of human chondrocyte through the MAPK/ERK5 signal pathway. Key words: Ion channels; Chondrocytes; Osteoarthritis; MAP kinase signaling system; Apoptosis

Hyaluronan suppresses lidocaine-induced apoptosis of human chondrocytes in vitro by inhibiting the p53-dependent mitochondrial apoptotic pathway.

Intra-articular injection of local anesthetics (LAs) is a common procedure for therapeutic purposes. However, LAs have been found toxic to articular cartilage, and hyaluronan may attenuate this toxicity. In this study we investigated whether hyaluronan attenuated lidocaine-induced chondrotoxicity, and if so, to elucidate the underlying mechanisms. Human chondrocyte cell line SW1353 and newly isolated murine chondrocytes were incubated in culture medium containing hyaluronan and/or lidocaine for 72 h. Cell viability was evaluated using MTT assay. Cell apoptosis was detected with DAPI staining, caspase 3/7 activity assay and flow cytometry. Cell cycle distributions, ROS levels and mitochondrial membrane potential (ΔΨm) were determined using flow cytometry. The expression of p53 and p53-regulated gene products was measured with Western blotting. Lidocaine (0.005%-0.03%) dose-dependently decreased the viability of SW1353 cells. This local anesthetic (0.015%, 0.025%) induced apoptosis, G2/M phase arrest and loss of ΔΨm, and markedly increased ROS production in SW1353 cells. Hyaluronan (50-800 μg/mL) alone did not affect the cell viability, but co-treatment with hyaluronan (200 μg/mL) significantly attenuated lidocaine-induced apoptosis and other abnormalities in SW1353 cells. Furthermore, co-treatment with lidocaine and hyaluronan significantly decreased the levels of p53 and its transcription targets Bax and p21 in SW1353 cells, although treatment with lidocaine alone did not significantly change these proteins. Similar results were obtained in ex vivo cultured murine chondrocytes. Hyaluronan suppresses lidocaine-induced apoptosis of human chondrocytes in vitro through inhibiting the p53-dependent mitochondrial apoptotic pathway.

Effects of sodium selenite on c-Jun N-terminal kinase signalling pathway induced by oxidative stress in human chondrocytes and c-Jun N-terminal kinase expression in patients with Kashin-Beck disease, an endemic osteoarthritis.

The c-Jun N-terminal kinases (JNK) are members of the mitogen-activated protein kinase family and are activated by environmental stress. Se plays an important role in the biological pathways by forming selenoprotein. Selenoproteins have been shown to exhibit a variety of biological functions including antioxidant functions and maintaining cellular redox balance, and compromise of such important proteins would lead to oxidative stress and apoptosis. We examined the expression levels of JNK in Kashin-Beck disease (KBD) patients, tested the potential protective effects of sodium selenite on tert-butyl hydroperoxide (tBHP)-induced oxidative injury and apoptosis in human chondrocytes as well as its underlying mechanism in this study. We produced an oxidative damage model induced by tBHP in C28/I2 human chondrocytes to test the essential anti-apoptosis effects of Se in vitro. The results indicated that the expression level of phosphorylated JNK was significantly increased in KBD patients. Cell apoptosis was increased and molecule expressions of the JNK signalling pathway were activated in the tBHP-injured chondrocytes. Na2SeO3 protected against tBHP-induced oxidative stress and apoptosis in cells by increasing cell viability, reducing reactive oxygen species generation, increasing Glutathione peroxidase (GPx) activity and down-regulating the JNK pathway. These results demonstrate that apoptosis induced by tBHP in chondrocytes might be mediated via up-regulation of the JNK pathway; Na2SeO3 has an effect of anti-apoptosis by down-regulating the JNK signalling pathway.

Role of miR-146a in human chondrocyte apoptosis in response to mechanical pressure injury in vitro.

MicroRNA (miR)-146a is known to be overexpressed in osteoarthritis (OA). However, the role of miR-146a in OA has not yet been fully elucidated. In the present study, we applied mechanical pressure of 10 MPa to human chondrocytes for 60 min in order to investigate the expression of miR-146a and apoptosis following the mechanical pressure injury. Normal human chondrocytes were transfected with an miR-146a mimic or an inhibitor to regulate miR-146a expression. Potential target genes of miR-146a were predicted using bioinformatics. Moreover, luciferase reporter assay confirmed that Smad4 was a direct target of miR-146a. The expression levels of miR-146a, Smad4 and vascular endothelial growth factor (VEGF) were quantified by quantitative reverse transcription PCR and/or western blot analysis. The effects of miR-146a on apoptosis were detected by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry. The results indicated that mechanical pressure affected chondrocyte viability and induced the early apoptosis of chondrocytes. Mechanical pressure injury increased the expression levels of miR-146a and VEGF and decreased the levels of Smad4 in the chondrocytes. In the human chondrocytes, the upregulation of miR-146a induced apoptosis, upregulated VEGF expression and downregulated Smad4 expression. In addition, the knockdown of miR-146a reduced cell apoptosis, upregulated Smad4 expression and downregulated VEGF expression. Smad4 was identified as a direct target of miR-146a by harboring a miR-146a binding sequence in the 3′-untranslated region (3′-UTR) of its mRNA. Furthermore, the upregulation of VEGF induced by miR-146a was mediated by Smad4 in the chondrocytes subjected to mechanical pressure injury. These results demonstrated that miR-146a was overexpressed in our chondrocyte model of experimentally induced human mechanical injury, accompanied by the upregulation of VEGF and the downregulation of Smad4 in vitro. Moreover, our data suggest that miR-146a is involved in human chondrocyte apoptosis in response to mechanical injury, and may contribute to the mechanical injury of chondrocytes, as well as to the pathogenesis of OA by increasing the levels of VEGF and damaging the transforming growth factor (TGF)-β signaling pathway through the targeted inhibition of Smad4 in cartilage.

Up‐regulation of the chemo‐attractive receptor ChemR23 and occurrence of apoptosis in human chondrocytes isolated from fractured calcaneal osteochondral fragments

To study the expression level of a panel of pro/anti-apoptotic factors and inflammation-related receptors in chondral fragments from patients undergoing surgical treatment for intra-articular calcaneal fractures, cartilage fragments were retrieved from calcaneal fractures of 20 patients subjected to surgical treatment. Primary cultures were performed using chondral fragments from fractured and control patients. Chondrocyte cultures from each patient of the fractured and control groups were subjected to immunofluorescence staining and quantitatively analyzed under confocal microscopy. Proteins extracted from the cultured chondrocytes taken from the fractured and control groups were processed for Western blot experiments and densitometric analysis. The percentage of apoptotic cells was determined using the cleaved PARP-1 antibody. The proportion of labelled cells was 35% for fractured specimens, compared with 7% for control samples. Quantification of caspase-3 active and Bcl-2 proteins in chondrocyte cultures showed a significant increase of the apoptotic process in fractured specimens compared with control ones. Fractured chondrocytes were positively stained for ChemR23 with statistically significant differences with respect to control samples. Densitometric evaluation of the immunoreactive bands confirmed these observations. Human articular chondrocytes obtained from patients with intra-articular calcaneal fractures express higher levels of pivotal pro-apoptotic factors, and of the chemo-attractive receptor ChemR23, compared with control cultures. On the basis of these observations, the authors hypothesize that consistent prolonged chondrocyte death, associated with the persistence of high levels of pro-inflammatory factors, could enhance the deterioration of cartilage tissue with consequent development of post-traumatic arthritis following intra-articular bone fracture.

A selective estrogen receptor modulator inhibits tumor necrosis factor-α-induced apoptosis through the ERK1/2 signaling pathway in human chondrocytes

Tumor necrosis factor α (TNF-α) is a pleiotropic cytokine mediating inflammatory as well as cell death activities, and is thought to induce chondrocytic chondrolysis in inflammatory and degenerative joint diseases. Selective estrogen receptor modulators (SERMs), such as raloxifene, which are commonly used in clinical settings act as estrogen agonists or antagonists. It is assumed that estrogens have a potential role in cartilage protection; however, the precise molecular mechanism for the protective effects of estrogens is unclear. This study was designed to examine whether raloxifene inhibits TNF-α-induced apoptosis in human chondrocytes and to clarify the mechanisms involved. We also investigated the signaling pathways responsible for the anti-apoptotic effect of raloxifene. Apoptosis in chondrocytes was determined by DNA fragmentation assay and caspase-3 activation. Raloxifene significantly inhibited TNF-α-induced caspase-3 activation and cell DNA fragmentation levels in chondrocytes. The inhibitory effect of raloxifene was abolished by the estrogen receptor antagonist ICI 182,780. Extracellular signal-regulated kinase 1/2 (ERK1/2) regulates apoptosis, acting as an apoptotic or anti-apoptotic signal. TNF-α-induced apoptosis was significantly enhanced by the ERK1/2 pathway inhibitor PD98059. Raloxifene stimulated a further increase in ERK1/2 phosphorylation in TNF-α-treated chondrocytes. Furthermore, the anti-apoptotic effects of raloxifene were inhibited by PD98059. In addition, the anti-apoptotic effects of raloxifene were completely abolished in ERK1/2 siRNA-treated chondrocytes. These results suggest that raloxifene prevents caspase-3-dependent apoptosis induced by TNF-α in human chondrocytes by activating estrogen receptors and the ERK1/2 signaling pathway.

Hsp90β inhibition modulates nitric oxide production and nitric oxide-induced apoptosis in human chondrocytes

BackgroundHsp90β is a member of the Hsp90 family of protein chaperones. This family plays essential roles in the folding, maturation and activity of many proteins that are involved in signal transduction and transcriptional regulation. The role of this protein in chondrocytes is not well understood, although its increase in osteoarthritic cells has been reported. The present study aimed to explore the role of Hsp90β in key aspects of OA pathogenesis.MethodsHuman OA chondrocytes were isolated from cartilage obtained from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with proinflammatory cytokines (IL-1β or TNF-α) and nitric oxide donors (NOC-12 or SNP). For Hsp90β inhibition, two different chemical inhibitors (Geldanamycin and Novobiocin) were employed, or siRNA transfection procedures were carried out. Gene expression was determined by real-time PCR, apoptosis was quantified by flow cytometry and ELISA, and nitric oxide (NO) production was evaluated by the Griess method. Indirect immunofluorescence assays were performed to evaluate the presence of Hsp90β in stimulated cells.ResultsHsp90β was found to be increased by proinflammatory cytokines. Inhibition of Hsp90β by the chemicals Geldanamycin (GA) and Novobiocin (NB) caused a dose-dependent decrease of the NO production induced by IL-1β in chondrocytes, up to basal levels. Immunofluorescence analyses demonstrate that the NO donors NOC-12 and SNP also increased Hsp90β. Chemical inhibition or specific gene silencing of this chaperone reduced the DNA condensation and fragmentation, typical of death by apoptosis, that is induced by NO donors in chondrocytes.ConclusionsThe present results show how Hsp90β modulates NO production and NO-mediated cellular death in human OA chondrocytes.

192 VISFATIN/NAMPT: A POTENTIAL TARGET FOR NGF-TRIGGERED PAIN IN OSTEOARTHRITIS

3. TGase 2 assay. The expression and enzyme activity of TGase 2 was examined with western blot and immunocytochemistry. Total cellular proteins were isolated and western blotted as described previously. Enzyme activity was evaluated by determining the incorporated biotinylated pentylamine using horseradish peroxidase-conjugated streptavidin and measuring the absorbance at 492 nm using a microplate spectrophotometer. Results: 1. RA up-regulated TGase 2 expression and enzyme activity in human chondrocytes. Human chondrocytes treated with RA resulted in up-regulation of TGase 2 protein as shown by the western blot and immunocytochemistry. RA-induced TGase 2 expression increased time dependently. The enzyme activity of TGase 2 was also increased in RA treated cells compared with normal human chondrocyte cells. 2. TGase 2 induced by RA results in decreased apoptosis in human chondrocytes. Apoptosis in RA-treated chondrocytes was decreased, measured by Annexin-V FACS analysis, when compared with H2O2-treated cells chondrocytes. Similar patterns were observed in 3 independent experiments using chondrocytes obtained from different patients. Conclusions: We have previously reported that endogenous TGase 2 expression was increased in human chondrocytes undergoing apoptosis. Inhibition of TGase 2 by MDC and TGase 2 siRNA was also shown to increase apoptosis and suggest a possible protective role of TGase 2 in chondrocyte apoptosis. The protective role of TGase 2 was further validated in this study as the RA-induced overexpression of TGase 2 decreased apoptosis of human chondrocytes. These results implicate a protective role of TGase 2 against apoptosis in human chondrocytes and the possibility of TGase 2 as a potential modulator of osteoarthritis.

Role of transglutaminase 2 in apoptosis induced by hydrogen peroxide in human chondrocytes

Chondrocyte apoptosis has been implicated in the pathogenesis of osteoarthritis. Transglutaminase 2 (TG2), the expression of which is higher in osteoarthritis patients, has been shown to be up-regulated during apoptosis in many experimental models. This study investigated the expression and role of TG2 in human chondrocytes undergoing apoptosis induced by hydrogen peroxide (H₂O₂). Human chondrocytes were obtained from the knee articular cartilage of patients undergoing total joint arthroplasty. Apoptosis was induced by H₂O₂ and was measured with Annexin-V flow cytometry, DNA Fragmentation ELISA and DAPI staining. Western Blot, an in situ activity assay and immunocytochemistry were used to examine TG2 expression. The role of TG2 was evaluated by TG-specific siRNA transfection and monodansylcadaverine (MDC), a competitive substrate for TG2. H₂O₂ induced apoptosis of human chondrocytes in a dose- and time-dependent manner. The level of TG2 expression was higher in the chondrocytes undergoing H₂O₂-induced apoptosis. Inhibition of TG2 by siRNA or MDC increased the level of apoptosis in the H₂O₂-treated chondrocytes. TG2 expression is higher in human chondrocytes undergoing apoptosis, and inhibition of TG2 leads to increased apoptosis. These results may raise the possibility of TG2 as a modulator of cartilage damage in osteoarthritis by offering protection against chondrocyte apoptosis.

Induced Apoptosis of Chondrocytes by Porphyromonas gingivalis as a Possible Pathway for Cartilage Loss in Rheumatoid Arthritis

The role of bacterial infections in the pathogenesis of rheumatoid arthritis (RA) has gained increasing interest. Patients with RA often exhibit periodontal disease, which is associated with pathogens like Porphyromonas gingivalis. The present study examines the direct effects of P. gingivalis on apoptosis of human chondrocytes (a feature of inflammatory joint diseases) as one can assume an interrelation of pathogenesis of RA and P. gingivalis infections. Primary chondrocytes were infected with P. gingivalis. Early apoptotic and dead cell analysis was performed using Annexin-V, 7AAD, and propidium iodide and examined by flow cytometry and fluorescence microscopy. Caspase activation and DNA fragmentation were determined by western blot analysis and TUNEL reaction. Flow cytometry and fluorescence microscopy demonstrated an increase of Annexin-V-positive early apoptotic chondrocytes after infection. Western blot showed upregulation of activated caspase-3 expression, and TUNEL reaction revealed considerable DNA fragmentation following infection. The data show that P. gingivalis promotes early and later stages of apoptosis of primary human chondrocytes, which might contribute to the joint damage seen in the pathogenesis of RA.

SIRT1 regulation of apoptosis of human chondrocytes

SIRT1 is known to inhibit apoptosis and to promote survival of various types of cells. However, the roles of SIRT1 in apoptosis of human chondrocytes have never been reported. We undertook this study to investigate the relationship of SIRT1 to apoptosis of human chondrocytes, which is a characteristic feature of osteoarthritis (OA). The expression of SIRT1 in human chondrocytes was examined by reverse transcription-polymerase chain reaction, immunoblotting, and immunohistology of human cartilage samples. The expression of SIRT1 under catabolic, mechanical, and nutritional stresses was investigated by immunoblotting. To examine the effect of SIRT1 on apoptosis, SIRT1 was inhibited by small interfering RNA (siRNA) and activated by resveratrol during nitric oxide (NO)-induced apoptosis. TUNEL staining and immunoblotting of cleaved poly(ADP-ribose) polymerase (PARP) were performed to detect apoptosis. To examine the mechanisms of apoptosis, we used immunoblotting to determine the levels of cleaved caspases and mitochondria-related apoptotic signaling proteins, Bax and Bcl-2, in the mitochondrial fraction. SIRT1 expression was confirmed in human chondrocytes and human cartilage samples. All catabolic, mechanical, and nutritional stresses inhibited SIRT1 expression. SIRT1 inhibition by siRNA for SIRT1 increased the percentage of TUNEL-positive cells and increased the amounts of cleaved PARP and cleaved caspases 3 and 9 induced by NO. In contrast, treatment with resveratrol decreased the percentage of TUNEL-positive cells and decreased the amounts of cleaved PARP and cleaved caspases 3 and 9 induced by NO. Furthermore, in the mitochondrial fraction, SIRT1 inhibition by siRNA for SIRT1 increased the amount of Bax but reduced the amount of Bcl-2, while resveratrol reduced the amount of Bax but increased the amount of Bcl-2. These results indicate that SIRT1 regulates apoptosis in human chondrocytes through the modulation of mitochondria-related apoptotic signals. Further research on SIRT1 might contribute to resolving the pathogenesis of OA.

Role of p53 in human chondrocyte apoptosis in response to shear strain

Chondrocyte apoptosis plays an important role in cartilage degeneration in osteoarthritis (OA), and mechanical injury to cartilage induces chondrocyte apoptosis. In response to DNA damage, p53 expression is up-regulated, transcription activity is increased, and apoptosis signals are initiated. The p53-regulated apoptosis-inducing protein 1 (p53AIP-1) is one of the p53-regulated genes, and is activated in response to DNA damage. This study was undertaken to analyze p53 function after induction of apoptosis by shear strain in chondrocytes. OA cartilage samples were obtained from subjects undergoing total knee replacement surgery, and normal cartilage samples were obtained from subjects undergoing surgery for femoral neck fracture. Chondrocytes were isolated from human cartilage and cultured. Expression of p53 and p53AIP in chondrocytes was detected by reverse transcriptase-polymerase chain reaction and Western blotting. Shear strain was introduced in normal human knee chondrocytes. To explore p53 function, normal human knee chondrocytes were pretreated with pifithrin-alpha or p53 small interfering RNA (siRNA) before induction of shear strain. Chondrocyte apoptosis was detected by expression of cleaved caspase 9 with Western blotting and TUNEL staining. Expression of p53 and p53AIP-1 was analyzed by Western blotting. OA and normal chondrocytes expressed p53. OA chondrocytes showed much higher expression of p53 and p53AIP-1 than did normal chondrocytes. TUNEL-positive cells and expression of p53, p53AIP-1, and cleaved caspase 9 were increased by shear strain, but chondrocyte apoptosis was suppressed after pretreatment with pifithrin-alpha or p53 siRNA. Our findings indicate that p53 and p53AIP-1 play important roles in human chondrocyte apoptosis. Down-regulation of p53 expression prevents cartilage from undergoing apoptosis introduced by shear strain.

Effect of exogenous IGF‐1 on chondrocyte apoptosis in a rabbit intraarticular osteotomy model

Insulin-like growth factor-1 (IGF-1) has been shown to protect chondrocytes from apoptosis in vitro. IGF-1 expression may also assist in maintaining a fully differentiated chondrocyte phenotype. Theoretically, posttraumatic administration of IGF-1 may inhibit chondrocyte apoptosis. This study is to determine if administration of IGF-1 after fracture inhibits apoptosis in vivo. Twenty-four mature female New Zealand white rabbits were randomized to control and IGF-1 groups. All subjects underwent standardized medial femoral condyle fracture and repair. Fibrin clot was administered in all subjects, with 25 mcg/ml IGF-1 in the clot in half the subjects. Half of the animals in each group were sacrificed at 2 weeks and half at 4 weeks, specimens were fixed and underwent TUNEL staining. Two-week controls showed significantly higher rate of apoptosis than 2-week IGF-1 subjects (21 +/- 6 vs. 12 +/- 6, p = 0.04). Likewise, 4-week controls showed significantly higher rate of apoptosis than 2-week IGF-1 subjects (23 +/- 7 vs. 10 +/- 2, p = 0.01). There was no significant administration difference between 2-week control and 4-week control subjects, or between 2-week IGF-1 and 4-week IGF-1 subjects. Intraarticular IGF-1 at the time of fracture repair appears to inhibit chondrocyte apoptosis in vivo, as judged by TUNEL staining, in this animal model. If administration of IGF-1 inhibits human chondrocyte apoptosis in vivo, this may lead to interventions that may reduce posttraumatic arthritis after fracture.

Pathogenesis of osteoarthritis: Chondrocyte replicative senescence or apoptosis?

The aim of this study was to investigate by flow cytometry cellular viability and apoptosis of human chondrocytes isolated from osteoarthritic cartilage and to correlate replicative senescence with apoptosis of these cells. To understand the mechanisms underlying the process of cell death in cartilage destruction, we investigated by flow cytometry cellular viability (Cell viability calcein-AM assay) and apoptosis (Light scattering properties of chondrocytes, study of chondrocyte death using Annexin-V-FITC and propidium iodide double-labeling, caspase-3 activity determination) of human chondrocytes isolated from osteoarthritic and nonosteoarthritic cartilage. Senescent cells were characterized using the senescence-associated-beta-galactosidase marker (SA-beta-Gal marker) by staining with chromogenic substrate (X-Gal) to produce blue coloration of SA-beta-Gal-positive cells and microscopy analysis. The results we obtained show that between 25 and 40% of chondrocytes were in apoptosis and all of them were SA-beta-Gal-positive. These results demonstrate that the death of osteoarthritic chondrocytes is an apoptotic phenomenon which is preceded by an accelerated mechanism of replicative senescence.

Apoptotic insults to human chondrocytes induced by sodium nitroprusside are involved in sequential events, including cytoskeletal remodeling, phosphorylation of mitogen‐activated protein kinase kinase kinase‐1/c‐Jun N‐terminal kinase, and Bax‐Mitochondria‐Mediated caspase activation

Nitric oxide (NO) can regulate chondrocyte activities. This study was aimed to evaluate the molecular mechanisms of NO donor sodium nitroprusside (SNP)-induced insults to human chondrocytes. Exposure of human chondrocytes to SNP increased cellular NO levels but decreased cell viability in concentration- and time-dependent manners. SNP time dependently induced DNA fragmentation and cell apoptosis. Treatment with 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide, an NO scavenger, significantly lowered SNP-induced cell injuries. Administration of SNP interrupted F-actin and microtubule cytoskeletons and stimulated phosphorylation of mitogen-activated protein kinase kinase kinase-1 (MEKK1) and c-Jun N-terminal kinase (JNK). Similar to SNP, cytochalasin D, an inhibitor of F-actin formation, disturbed F-actin polymerization and increased MEKK1 and JNK activations. Overexpression of a dominant negative mutant of MEKK1 (dnMEK1) in human chondrocytes significantly ameliorated SNP-induced cell apoptosis. Exposure to SNP promoted Bax translocation from the cytoplasm to mitochondria, but application of dnMEKK1 lowered the translocation. SNP time dependently decreased the mitochondrial membrane potential, complex I NADH dehydrogenase activity, and cellular ATP levels, but increased the release of cytochrome c from mitochondria to the cytoplasm. Activities of caspase-9, -3, and -6 were sequentially increased by SNP administration. This study shows that SNP can induce apoptosis of human chondrocytes through sequential events, including cytoskeletal remodeling, activation of MEKK1/JNK, Bax translocation, mitochondrial dysfunction, cytochrome c release, caspase activation, and DNA fragmentation.

Effect of the polyamine analogue N1,N11‐diethylnorspermine on cell survival and susceptibility to apoptosis of human chondrocytes

Chondrocyte survival is closely linked to cartilage integrity, and forms of chondrocyte apoptotic death can contribute to cartilage degeneration in articular diseases. Since growing evidence also implicates polyamines in the control of cell death, we have been investigating the role of polyamine metabolism in chondrocyte survival and apoptosis. Treatment of human C-28/I2 chondrocytes with N(1),N(11)-diethylnorspermine (DENSPM), a polyamine analogue with clinical relevance as an experimental anticancer agent, inhibited polyamine biosynthesis and induced polyamine catabolism, thus rapidly depleting all main polyamines. DENSPM did not increase significantly caspase activity, but provoked a late cell death associated to DNA fragmentation. A short treatment with DENSPM did not reduce cell viability when given alone, but enhanced caspase-3 and -9 activation in chondrocytes exposed to tumor necrosis factor-alpha (TNF) and cycloheximide (CHX). A longer treatment with DENSPM however reduced caspase response to TNF plus CHX. Depletion of all polyamines obtained by specific inhibitors of polyamine biosynthesis did not cause cell death and contrasted apoptosis by decreasing caspase activities. In conclusion, following DENSPM treatment, C-28/I2 chondrocytes are initially sensitized to caspase 9-dependent apoptosis in the presence of TNF and CHX and may eventually undergo a late and mainly caspase-independent cell death in the absence of other stimuli. Moreover, these results indicate that a reduction of polyamine levels not only leads to inhibition of cell proliferation, but also of caspase-mediated pathways of chondrocyte apoptosis.