Up-regulated expression of cartilage intermediate-layer protein and ANK in articular hyaline cartilage from patients with calcium pyrophosphate dihydrate crystal deposition disease
Up-regulated expression of cartilage intermediate-layer protein and ANK in articular hyaline cartilage from patients with calcium pyrophosphate dihydrate crystal deposition disease
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DOI：
10.1002/art.10632
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发表时间：
2002-12-01
影响因子：
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通讯作者：
Masuda, I
中科院分区：
文献类型：
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作者：
Hirose, J;Ryan, LA;Masuda, I
目标。老年人软骨细胞外无机焦磷酸（ePPi）的过量积累是软骨基质中焦磷酸钙二水合物（CPPD）晶体形成的关键。epppi的两个来源是产生epppi的外酶（NTPPPH）和细胞内PPi通过ANK的胞外转运。本研究通过研究含CPPD晶体和不含CPPD晶体的骨关节炎（OA）关节软骨软骨细胞中NTPPPH酶（软骨中间层蛋白[CILP]和浆细胞膜糖蛋白1 [PC-1]）和ANK的表达，来评估NTPPPH和ANK在epi形成中的作用。关节成形术时从膝关节软骨中获取软骨细胞（含CPPD晶体的OA [CPPD]， n = 8；无晶体的OA [OA]， n = 10）。正常成人软骨细胞（n = 1）作为对照。软骨细胞用转化生长因子β 1 （tgfβ 1）和/或胰岛素样生长因子1 （IGF-1）培养，前者刺激ePPi的生成，后者抑制ePPi的生成。48 h时测定培养基中NTPPPH和epi。点免疫印迹法检测培养基CILP、PC-1、ANK。提取软骨细胞信使RNA （mRNA）进行逆转录聚合酶链反应，研究CILP、PC-1和ANK mRNA的表达。同时对新鲜冷冻软骨中NTPPPH、ANK mRNA和蛋白的表达进行了研究。与正常软骨细胞相比，条件培养基中CPPD软骨细胞的基础epi细化和NTPPPH活性升高，并倾向于高于OA软骨细胞。CPPD软骨细胞和CPPD软骨提取物中CILP（软骨细胞）和ANK（软骨）mRNA的基础表达量均高于OA和正常样品。PC-1 mRNA在CPPD软骨细胞和软骨提取物中的表达量低于OA软骨细胞和软骨提取物，但差异不显著。CPPD、OA和正常软骨细胞或软骨提取物中CILP、PC-1和ANK蛋白水平相似。所有来源的软骨细胞中，TGFbeta1均能刺激CILP和ANK mRNA的表达，并能抑制IGF-1对epi的影响。CILP和ANK mRNA表达与CPPD和OA软骨细胞周围的软骨细胞epi积累相关，并且对生长因子刺激的反应相似。这些发现表明，CILP和ANK表达的上调有助于CPPD晶体形成软骨中epppi的积累。
Objective. Excess accumulation of extracellular inorganic pyrophosphate (ePPi) in aged human cartilage is crucial in calcium pyrophosphate dihydrate (CPPD) crystal formation in cartilage matrix. Two sources of ePPi are ePPi-generating ectoenzymes (NTPPPH) and extracellular transport of intracellular PPi by ANK. This study was undertaken to evaluate the role of NTPPPH and ANK in ePPi elaboration, by investigating expression of NTPPPH enzymes (cartilage intermediate-layer protein [CILP] and plasma cell membrane glycoprotein 1 [PC-1]) and ANK in human chondrocytes from osteoarthritic (OA) articular cartilage containing CPPD crystals and without crystals.Methods. Chondrocytes were harvested from knee cartilage at the time of arthroplasty (OA with CPPD crystals [CPPD], n = 8; OA without crystals [OA], n = 10). Normal adult human chondrocytes (n = 1) were used as a control. Chondrocytes were cultured with transforming growth factor beta1 (TGFbeta1), which stimulates ePPi elaboration, and/or insulin-like growth factor 1 (IGF-1), which inhibits ePPi elaboration. NTPPPH and ePPi were measured in the media at 48 hours. Media CILP, PC-1, and ANK were determined by dot-immunoblot analysis. Chondrocyte messenger RNA (mRNA) was extracted for reverse transcriptase-polymerase chain reaction to study expression of mRNA for CILP, PC-1, and ANK. NTPPPH and ANK mRNA and protein were also studied in fresh frozen cartilage.Results. Basal ePPi elaboration and NTPPPH activity in conditioned media from CPPD chondrocytes were elevated compared with normal chondrocytes, and tended to be higher compared with OA chondrocytes. Basal expression of mRNA for CILP (chondrocytes) and ANK (cartilage) was higher in both CPPD chondrocytes and CPPD cartilage extract than in OA or normal samples. PC-1 mRNA was less abundant in CPPD chondrocytes and cartilage extract than in OA chondrocytes and extract, although the difference was not significant. CILP, PC-1, and ANK protein levels were similar in CPPD, OA, and normal chondrocytes or cartilage extracts. Both CILP and ANK mRNA expression and ePPi elaboration were stimulated by TGFbeta1 and inhibited by IGF-1 in chondrocytes from all sources.Conclusion. CILP and ANK mRNA expression correlates with chondrocyte ePPi accumulation around CPPD and OA chondrocytes, and all respond similarly to growth factor stimulation. These findings suggest that up-regulated CILP and ANK expression contributes to higher ePPi accumulation from CPPD crystal-forming cartilage.