Yukiko Aida

IL-6 and soluble IL-6 receptor stimulate the production of MMPs and their inhibitors via JAK-STAT and ERK-MAPK signalling in human chondrocytes

Elevated concentrations of IL‐6 (interleukin‐6) and sIL‐6r (soluble IL‐6 receptor) in the synovial fluid and serum of patients with arthritis have been implicated in joint cartilage destruction. This study examined the effects of IL‐6 and sIL‐6r on the expression of MMPs (matrix metalloproteinases), TIMPs (tissue inhibitor of metalloproteinases), the plasminogen activation system including tPA (tissue‐type PA), uPA (urokinase‐type PA) and PAI‐1 (PA inhibitor type 1) using chondrocytes derived from normal human femur cartilage. The cells were cultured with or without 50 ng/ml IL‐6 and/or 30 ng/ml sIL‐6r in the presence or absence of the JAK3 (Janus kinase 3) inhibitor WHI‐P131 or the MEK [MAPK (mitogen‐activated protein kinase)/ERK (extracellular signal protein kinase) kinase] inhibitor PD98059 for up to 28 days. The expression of MMPs, TIMPs, uPA, tPA and PAI‐1 was investigated at the mRNA and protein levels. MMP protein expression and pSTAT3 (phosphorylation of signal transducer and activator of transcription 3) and pERK (phosphorylation of ERK) were also measured. Treatment with both IL‐6 and sIL‐6r markedly increased the expression of MMP‐1, MMP‐13, TIMP‐1 and PAI‐1, while significantly decreasing the expression of tPA and uPA and stimulating pSTAT3 and pERK. Adding WHI‐P131 or PD98059 decreased IL‐6 and sIL‐6r enhancement of MMP‐1, −3 and −13. The results suggest that IL‐6 and sIL‐6r stimulate the production of MMPs and their inhibitor via JAK—STAT and ERK—MAPK signalling in chondrocytes.

Effects of IL-6 and soluble IL-6 receptor on the expression of cartilage matrix proteins in human chondrocytes.

Elevated concentrations of interleukin (IL)-6 and soluble IL-6 receptor (sIL-6Rα) in synovial fluid have been implicated in joint cartilage destruction. We examined the effect of IL-6 and sIL-6Rα on cell growth, alkaline phosphatase (ALPase) activity, and the expression of Sox-9, type II collagen, aggrecan core, link protein, BMP-7, and BMP receptors in human chondrocytes. Cell proliferation increased slightly in the presence of both IL-6 and sIL-6Rα, whereas ALPase activity decreased markedly. The expression of Sox-9 and aggrecan core did not change in the presence or absence of IL-6 and sIL-6Rα, whereas the expression of type II collagen, link protein, BMP-7, and BMP receptors increased in the presence of both IL-6 and sIL-6Rα. These results suggest that IL-6 and sIL-6Rα suppress the differentiation of chondrocytes and induce the repair of arthrodial cartilage through an increase in the expression of cartilage matrix proteins, BMP-7, and BMP receptors in the cells.

IL-1 Stimulates the Expression of Prostaglandin Receptor EP4 in Human Chondrocytes by Increasing Production of Prostaglandin E2

Prostaglandin (PG) E2, which exerts its actions via the PG receptors EP1–4, is produced from arachidonic acid by cyclooxygenase (COX)-1 and COX-2. The aim of this study was to investigate the mechanisms by which interleukin (IL)-1β induces the expression of PG receptors in cultured human chondrocytes and to explore the role of PGE2 in this process. The cells were cultured with 0, 10, or 100 U/mL IL-1β with or without 1 μM celecoxib, a specific inhibitor of COX-2, for up to 28 days. Expression of the genes encoding COX-1, COX-2, and EP1–4 was quantified using real-time PCR, and expression of the corresponding proteins was examined using immunohistochemical staining. PGE2 production was determined using ELISA. IL-1β treatment caused a marked dose- and time-dependent increase in the levels of PGE2, COX-2, and EP4 as compared with the untreated control. It did not affect the expression of COX-1, and it decreased the expression of EP1 and EP2. EP3 expression was not detected in either the absence or the presence of IL-1β. When celecoxib was also present, IL-1β failed to stimulate PGE2 production and EP4 expression, but its stimulatory effect on COX-2 expression and its inhibitory effect on EP1 and EP2 expression were unchanged. IL-1β increases the production of PGE2, COX-2, and the PG receptor EP4 in cultured human chondrocytes. The increase in EP4 expression appears to be a result of the increased PGE2 production.

Interleukin-17F affects cartilage matrix turnover by increasing the expression of collagenases and stromelysin-1 and by decreasing the expression of their inhibitors and extracellular matrix components in chondrocytes.

Interleukin (IL)-17, a proinflammatory cytokine, is produced primarily by activated Th17 cells. IL-17 consists of six ligands that signal through five receptors (IL-17Rs); IL-17A and IL-17F share the highest homology in the family. Matrix metalloproteinases (MMPs) degrade the extracellular matrix during cartilage remodeling whereas tissue inhibitor of metalloproteinases (TIMPs) inhibit the action of MMPs. In the present study, we examined the effect of IL-17F on the degradation and synthesis of the extracellular matrix in cartilage using human articular chondrocytes. We examined the effect of IL-17F on the expression of IL-17Rs, MMPs, TIMPs, type II collagen, aggrecan, link protein, and cyclooxygenases (COXs), as well as on prostaglandin E2 (PGE2) production. We also examined the indirect effect of PGE2 on the above IL-17F-induced/reduced components using NS-398, a specific inhibitor of COX-2. Cells were cultured with or without IL-17F in the presence or absence of either an IL-17R antibody or NS-398 for up to 28 days. Expression of IL-17Rs, MMPs, TIMPs, type II collagen, aggrecan, link protein, and COXs at mRNA and protein levels was determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. PGE2 production was determined by ELISA. The expression of all types of IL-17Rs was detected in chondrocytes. However, IL-17RE expression was extremely low, compared with other IL-17Rs. The expression of MMP-1, MMP-3, MMP-13, and COX-2 as well as PGE2 production were increased by addition of IL-17F, whereas the expression of IL-17RD, TIMP-2, TIMP-4, type II collagen, aggrecan, link protein, and COX-1 was decreased. The expression of IL-17RA, IL-17RB, IL-17RC, MMP-2, MMP-14, TIMP-1, and TIMP-3 was unaffected by addition of IL-17F. The IL-17R antibody blocked the stimulating/reducing effect of IL-17F on the expression of MMP-1, MMP-3, MMP-13, TIMP-2, TIMP-4, type II collagen, aggrecan, and link protein. NS-398 blocked the reducing effect of IL-17F on aggrecan expression, whereas it did not completely block the stimulating/reducing effects of IL-17F on the expression of MMP-1, MMP-3, MMP-13, TIMP-2, TIMP-4, type II collagen, and link protein. Our results suggest that IL-17F stimulates cartilage degradation by increasing the expression of collagenases (MMP-1 and -13) and stromelysin-1 (MMP-3) and by decreasing expression of their inhibitors (TIMP-2 and -4), type II collagen, aggrecan, and link protein in chondrocytes. Furthermore, our results suggest that the expression of aggrecan, link protein, and TIMP-4 decrease through the autocrine action of PGE2 in chondrocytes.

IL-1β Suppresses the Formation of Osteoclasts by Increasing OPG Production via an Autocrine Mechanism Involving Celecoxib-Related Prostaglandins in Chondrocytes

Elevated interleukin (IL)-1 concentrations in synovial fluid have been implicated in joint bone and cartilage destruction. Previously, we showed that IL-1β stimulated the expression of prostaglandin (PG) receptor EP4 via increased PGE2 production. However, the effect of IL-1β on osteoclast formation via chondrocytes is unclear. Therefore, we examined the effect of IL-1β and/or celecoxib on the expression of macrophage colony-stimulating factor (M-CSF), receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG) in human chondrocytes, and the indirect effect of IL-1β on osteoclast-like cell formation using RAW264.7 cells. OPG and RANKL expression increased with IL-1β; whereas M-CSF expression decreased. Celecoxib blocked the stimulatory effect of IL-1β. Conditioned medium from IL-1β-treated chondrocytes decreased TRAP staining in RAW264.7 cells. These results suggest that IL-1β suppresses the formation of osteoclast-like cells via increased OPG production and decreased M-CSF production in chondrocytes, and OPG production may increase through an autocrine mechanism involving celecoxib-related PGs.

Thickness of the Metal Frame by Electroforming-Influence of Positioning Die in the Electroplating Solution-

目的: ゴールドエレクトロフォーミング法は, メタルコーピングを回転する電解溶液中で製作するため, 電鋳歯型の位置づけがコーピングの厚さに影響を及ぼすと考えられる. そこで, 電解槽内での水平的位置づけを変化させ, その影響を検討した.方法: フルベークタイプの陶材焼付冠製作用に形成した上顎中切歯支台歯模型 (松風) を原型とし, 複製電鋳歯型を製作した. 電解槽装置 (GAMMATeasy, Gramm) のビーカー (内径56.0mm) 内で, 垂直的に電極上縁から切縁中央までを20.0mmとし, 水平的に1.ビーカー中心, 2.中心から11.0mm, 3.中心から22.0mm (以下, P0, P1, P2) の3条件に位置づけした. その後, 厚さ200μmに設定しエレクトロフォーミングを行い試料とした. 試料を包埋し, 唇舌的に切断し, 走査型レーザー顕微鏡 (Laser microscope 1 LM 21 W, Lasertec) にてコーピングの厚さを測定した. 測定部位は唇面中央部, 切端部, 舌面中央部, 基底結節上部, 舌側軸面中央部 (以下, a, b, c, d, e) の5点とした. 試料数は各条件5個とし, 統計にはKruskal-Wallis testとBonferroni Correctionを用い, 各測定部位での比較を行った.結果: コーピングの厚さは, P0では全測定部位で最大値を示し, 設定値に近似した値を示した.そのほかは設定値より小さな値を示した. d, eにおいてはP0>P1>P2の順に大きな値を示し, bではP0>P1≧P2の順に大きな値を示した.結論: ビーカー内での電鋳歯型の位置づけがコーピングの厚さに影響を及ぼし, 設定した厚さより薄くなることが判明した.