Kenjiro Iwasa

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.

Oxidative stress-induced apoptosis and matrix loss of chondrocytes is inhibited by eicosapentaenoic acid

Eicosapentaenoic acid (EPA) is an antioxidant and n‐3 polyunsaturated fatty acid that reduces the production of inflammatory cytokines. We evaluated the role of EPA in chondrocyte apoptosis and degeneration. Normal human chondrocytes were treated with EPA and sodium nitroprusside (SNP). Expression of metalloproteinases (MMPs) was detected by real‐time polymerase chain reaction (PCR) and that of apoptosis‐related proteins was detected by western blotting. Chondrocyte apoptosis was detected by flow cytometry. C57BL/6J mice were used for the detection of MMP expression by immunohistochemistry and for investigation of chondrocyte apoptosis. EPA inhibited SNP‐induced chondrocyte apoptosis, caspase 3 and poly(ADP‐ribose) polymerase cleavage, phosphorylation of p38 MAPK and p53, and expression of MMP3 and MMP13. Intra‐articular injection of EPA prevented the progression of osteoarthritis (OA) by inhibiting MMP13 expression and chondrocyte apoptosis. EPA treatment can control oxidative stress‐induced OA progression, and thus may be a new approach for OA therapy.

PTEN regulates matrix synthesis in adult human chondrocytes under oxidative stress.

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was identified as an important tumor suppressor gene. PTEN functions as a negative regulator of phosphoinositol‐3‐kinase (PI3K)‐Akt and MEK/ERK signaling. The PI3K‐Akt pathway is critical for cell survival, differentiation, and matrix synthesis. Oxidative stress is considered a critical factor in the onset and progression of osteoarthritis (OA). Therefore, we investigated the function of PTEN in OA chondrocytes under oxidative stress. Chondrocytes were treated with insulin‐like growth factor‐1 (IGF‐1) and/or tert‐butyl hydroperoxide (tBHP), which causes oxidative stress. The expression levels of type2 collagen (Col2a1) and aggrecan were analyzed by real‐time PCR, and phosphorylation of Akt and ERK1/2 was analyzed by Western blotting. Chondrocytes were treated with PTEN‐specific small interfering RNA (siRNA), as well as IGF‐1 and/or tBHP. PTEN and IGF‐1 expressions in OA chondrocytes were increased. The downregulation of PTEN expression increased the expression levels of Col2a1 and aggrecan, and increased proteoglycan synthesis under oxidative stress. Oxidative stress decreased the phosphorylation of Akt and increased that of ERK1/2. The downregulation of PTEN expression increased Akt phosphorylation, but did not increase that of ERK 1/2. Our results suggest that PTEN regulates matrix synthesis via the PI3K‐Akt pathway under oxidative stress.

Cyclin‑dependent kinase inhibitor p21 does not impact embryonic endochondral ossification in mice

Endochondral ossification at the growth plate is regulated by a number of factors and hormones. The cyclin-dependent kinase inhibitor p21 has been identified as a cell cycle regulator and its expression has been reported to be essential for endochondral ossification in vitro. However, to the best of our knowledge, the function of p21 in endochondral ossification has not been evaluated in vivo. Therefore, the aim of this study was to investigate the function of p21 in embryonic endochondral ossification in vivo. Wild-type (WT) and p21 knockout (KO) pregnant heterozygous mice were sacrificed on embryonic days E13.5, E15.5 and E18.5. Sagittal histological sections of the forearms of the embryos were collected and stained with Safranin O and 5-bromo-2′-deoxyuridine (BrdU). Additionally, the expression levels of cyclin D1, type II collagen, type X collagen, Sox9, and p16 were examined using immunohistochemistry, and the expression levels of p27 were examined using immunofluorescence. Safranin O staining revealed no structural change between the cartilage tissues of the WT and p21KO mice at any time point. Type II collagen was expressed ubiquitously, while type X collagen was only expressed in the hypertrophic zone of the cartilage tissues. No differences in the levels of Sox9 expression were observed between the two groups at any time point. The levels of cyclin D1 expression and BrdU uptake were higher in the E13.5 cartilage tissue compared with those observed in the embryonic cartilage tissue at subsequent time points. Expression of p16 and p27 was ubiquitous throughout the tissue sections. These results indicate that p21 may not be essential for embryonic endochondral ossification in articular cartilage of mice and that other signaling networks may compensate for p21 deletion.

MDCT image based assessment of acetabular cup orientation in total hip arthroplasty

Total hip arthroplasty (THA) is an orthopaedic surgery which replaces the damaged hip joint with implants. The acetabular cup is implanted to the acetabulum. Some studies show that the outcome of THA is strongly correlated to the orientation of the acetabular cup. This paper proposes a fully automated method for measuring the orientation using multidetector-row computed tomography (MDCT) images. The method defines the pelvic anatomical coordinate system using anterior pelvic plane (APP), and measures angles between the cup implanting axis and the pelvic anatomical coordinate axis. The angles are inclination angle and anteversion angle. The method was applied to two phantoms in which the acetabular cup was implanted to the artificial bone. We acquired multiple set of MDCT image for the same phantom with changing the pelvic pose in the MDCT scanner to evaluate the reproducibility. The standard deviations of measured angles in multiple acquisitions were less than 2.0 deg for both of the inclination and the anteversion angles.

Cyclin-Dependent Kinase Inhibitor-1-Deficient Mice are Susceptible to Osteoarthritis Associated with Enhanced Inflammation

Osteoarthritis (OA) is a multifactorial disease, and recent data suggested that cell cycle–related proteins play a role in OA pathology. Cyclin‐dependent kinase (CDK) inhibitor 1 (p21) regulates activation of other CDKs, and recently, we reported that p21 deficiency induced susceptibility to OA induced by destabilization of the medial meniscus (DMM) surgery through STAT3‐signaling activation. However, the mechanisms associated with why p21 deficiency led to susceptibility to OA by the STAT3 pathway remain unknown. Therefore, we focused on joint inflammation to determine the mechanisms associated with p21 function during in vitro and in vivo OA progression. p21‐knockout (p21−/−) mice were used to develop an in vivo OA model, and C57BL/6 (p21+/+) mice with the same background as the p21−/− mice were used as controls. Morphogenic changes were measured using micro‐CT, IL‐1β serum levels were detected by ELISA, and histological or immunohistological analyses were performed. Our results indicated that p21‐deficient DMM‐model mice exhibited significant subchondral bone destruction and cartilage degradation compared with wild‐type mice. Immunohistochemistry results revealed p21−/− mice susceptibility to OA changes accompanied by macrophage infiltration and enhanced MMP‐3 and MMP‐13 expression through IL‐1β‐induced NF‐κB signaling. p21−/− mice also showed subchondral bone destruction according to micro‐CT analysis, and cathepsin K staining revealed increased numbers of osteoclasts. Furthermore, p21−/− mice displayed increased serum IL‐1β levels, and isolated chondrocytes from p21−/− mice indicated elevated MMP‐3 and MMP‐13 expression with phosphorylation of IκB kinase complex in response to IL‐1β stimulation, whereas treatment with a specific p‐IκB kinase inhibitor attenuated MMP‐3 and MMP‐13 expression. Our results indicated that p21‐deficient DMM mice were susceptible to alterations in OA phenotype, including enhanced osteoclast expression, macrophage infiltration, and MMP expression through IL‐1β‐induced NF‐κB signaling, suggesting that p21 regulation may constitute a possible therapeutic strategy for OA treatment.

Arthroscopic Ankle Arthrodesis for Treating Osteoarthritis in a Patient with Kashin-Beck Disease

Kashin-Beck disease (KBD) is an endemic degenerative osteoarthritis. Death of cartilage and growth plate is the pathologic feature; therefore, KBD involves skeletal deformity and often results in osteoarthritis. Deficiency of selenium, high humic acid levels in water, and fungi on storage gains are considered the cause of KBD. The most frequently involved joints are ankles, knees, wrists, and elbows and symptoms are pain and limited motions of those joints. The main treatments for KBD are rehabilitation and osteotomy to correct the deformities because preventive treatment has not been established. In this report, we present a case of ankle osteoarthritis due to KBD and first describe arthroscopic ankle arthrodesis for treating osteoarthritis of KBD.

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