K. Takebe

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

OBJECTIVE 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. METHODS 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. RESULTS 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. CONCLUSION 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.

Regulation of p38 MAPK phosphorylation inhibits chondrocyte apoptosis in response to heat stress or mechanical stress

Activation of p38 MAPK has been associated with a stress response and with apoptotic processes. However, the function of p38 MAPK in chondrocytes is not clearly understood. In this study, we analyzed the expression of p38 MAPK in chondrocytes and investigated the function of p38 MAPK in response to heat stress and mechanical stress. Chondrocytes were isolated from human cartilage and cultured. Expression of p38 and phosphorylated p38 in cartilage of patients with osteoarthritis (OA) was compared to those in normal cartilage by immunohistochemistry and Western blotting. Human knee chondrocytes were exposed to heat stress or mechanical stress. Normal knee chondrocytes were pre-treated with SB203580 or p38 small interfering RNA (siRNA) before induction of heat stress or mechanical stress. Chondrocyte apoptosis was detected by TUNEL staining and Western blotting of cleaved caspases. OA and normal chondrocytes expressed p38; however, OA chondrocytes showed much higher phosphorylated p38 compared to normal chondrocytes. Heat stress or mechanical stress induced apoptosis and increased phosphorylated p38 in normal chondrocytes. The TUNEL positive cells and expression levels of phosphorylated p38 in response to stress decreased when chondrocytes were incubated with SB203580 or transfected with siRNA against p38. In conclusion, we have demonstrated that heat stress or mechanical stress increased chondrocyte apoptosis via phosphorylation of p38. Stress-induced chondrocyte apoptosis decreased due to inhibition of p38 MAPK activation. In contrast, the phosphorylation of p38 MAPK increased in OA chondrocytes. Our results show that down-regulation of p38 MAPK activation inhibits chondrocyte death induced by heat stress or mechanical stress.

Akt phosphorylation in human chondrocytes is regulated by p53R2 in response to mechanical stress

OBJECTIVE The p53 tumor-suppressor protein p53R2 is activated in response to various stressors that act on cell signaling. When DNA is damaged, phosphorylation of p53 at its Ser 15 residue induces p53R2 production. The role of p53R2 in chondrocytes remains poorly understood. In this study, we evaluated in chondrocytes, p53R2 expression and its regulation in response to mechanical stress. Furthermore, we investigated the function of p53R2 in relation to mechanotransduction. METHODS Osteoarthritis (OA) cartilage obtained from total knee replacements and normal cartilage obtained from femoral neck fractures was used to measure p53R2 expression by using immunohistochemistry, western blotting, and real-time polymerase chain reaction (PCR). The OA chondrocytes were subjected to a high magnitude of cyclical tensile strain by using an FX-2000 Flexercell system. Next, sulfated glycosaminoglycan (sGAG) production was quantified in these cells. Protein expression of p53R2, and phosphorylation of Akt, p38MAPK, ERK1/2, and JNK was also detected using western blotting. Moreover, Akt phosphorylation was detected after transfecting the cells with p53R2-specific small interfering RNA (siRNA). RESULTS Expression of p53R2 was significantly increased in OA chondrocytes and in chondrocytes after applying 5% tensile strain to the cells. However, Akt phosphorylation was down-regulated in OA chondrocytes after the strain, and was up-regulated after transfection of p53R2. sGAG protein as well as collagen type II and aggrecan mRNA was increased following transfection of p53R2-specific siRNA after 5% tensile strain. CONCLUSIONS p53R2 could regulate matrix synthesis via Akt phosphorylation during chondrocyte mechanotransduction. Down-regulation of p53R2 may be a new therapeutic approach in OA therapy.

Evidence for Genetic Contribution to Variation in Post‐Traumatic Osteoarthritis in Mice

Recombinant inbred mouse strains generated from an LG/J and SM/J intercross offer a unique resource to study complex genetic traits such as osteoarthritis (OA). We undertook this study to determine the susceptibility of 14 strains to various phenotypes characteristic of posttraumatic OA. We hypothesized that phenotypic variability is associated with genetic variability.

268 EXPRESSION OF P53R2 IN CHONDROCYTES IS REGULATED BY MECHANICAL STRESS

deposition and remodeling with downstream generation of collagen 2 bioactive peptides. These were able to promote nuclear localization of RUNX-2, the pivotal transcription factor in chondrocyte hypertrophy and osteoblast generation. Indeed, samples stimulated with polyamines showed an enhanced mineralization, along with increased caspase activity, indicating increased chondrocyte terminal differentiation. Conclusions: Polyamine pathway can represent a potential target to control and correct chondrocyte inappropriate maturation in osteoarthritis Acknowledgements: supported by CARISBO foundation