Mistry D, Chambers M G, Mason R M
Department of Clinical Pharmacology, William Harvey Research Institute, St Bartholomew's and the Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK.
Osteoarthritis Cartilage. 2006 May;14(5):486-95. doi: 10.1016/j.joca.2005.11.015. Epub 2006 Jan 27.
To investigate the role of adenosine in chondrocyte death in murine osteoarthritis (OA).
5'-Nucleotidase (5'NT) generates adenosine. Enzyme activity was measured histochemically in normal murine and osteoarthritic STR/ort strain tibial cartilage. Adenosine-mediated cell death was investigated in MC615 chondrocyte cultures. Adenosine receptors (ARs) were assessed by reverse transcriptase polymerase chain reaction (RT-PCR). Cellular uptake of [(3)H] adenosine was measured with or without the inhibitor, nitrobenzylthioinosine (NBTI). Cell death was assessed by cell counting and DNA laddering following selective receptor stimulation, or after modulating adenosine metabolism with adenosine deaminase (ADA) or adenosine kinase (AK) inhibitors [erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and Iodotubericidin (Itub)], or with homocysteine (HC). Markers of apoptosis were assessed by Western blotting. Cell studies were validated by incubating normal murine knee joints in a medium containing adenosine and metabolic inhibitors. Apoptotic chondrocytes were identified with the TUNEL reaction.
5'NT activity in STR/ort tibial cartilage increased with development of OA, especially close to OA lesions. Adenosine induced MC615 cell death in the presence of ADA inhibition (100 microM EHNA), or 1mM HC, or both. Adenosine uptake, mediated by NBTI-sensitive adenosine transporters, was required for cell death. ARs were expressed (A2b>A2a>A1) but were not involved in mediating cell death. Cell death involved the activation of caspase-3 and DNA fragmentation and was prevented by inhibiting caspase activity. However, neither caspase-8 nor caspase-9 was detected. Adenosine+EHNA induced chondrocyte apoptosis in normal murine knee joints.
Increased adenosine production may induce chondrocyte apoptosis and play a role in OA in STR/ort mice.
研究腺苷在小鼠骨关节炎（OA）软骨细胞死亡中的作用。
5'-核苷酸酶（5'NT）可生成腺苷。采用组织化学方法检测正常小鼠和骨关节炎STR/ort品系小鼠胫骨软骨中的酶活性。在MC615软骨细胞培养物中研究腺苷介导的细胞死亡。通过逆转录聚合酶链反应（RT-PCR）评估腺苷受体（ARs）。在有或无抑制剂硝基苄硫肌苷（NBTI）的情况下测量[(3)H]腺苷的细胞摄取。在选择性受体刺激后，或在用腺苷脱氨酶（ADA）或腺苷激酶（AK）抑制剂[erythro-9-(2-羟基-3-壬基)腺嘌呤（EHNA）和碘结核菌素（Itub）]调节腺苷代谢后，或用同型半胱氨酸（HC）调节后，通过细胞计数和DNA梯状条带分析评估细胞死亡。通过蛋白质免疫印迹法评估凋亡标志物。通过在含有腺苷和代谢抑制剂的培养基中孵育正常小鼠膝关节来验证细胞研究。用TUNEL反应鉴定凋亡软骨细胞。
随着OA的发展，STR/ort胫骨软骨中的5'NT活性增加，尤其是在靠近OA病变处。在ADA抑制（100μM EHNA）、1mM HC或两者存在的情况下，腺苷诱导MC615细胞死亡。细胞死亡需要由NBTI敏感的腺苷转运体介导的腺苷摄取。ARs有表达（A2b>A2a>A1），但不参与介导细胞死亡。细胞死亡涉及caspase-3的激活和DNA片段化，并且通过抑制caspase活性得以预防。然而，未检测到caspase-8和caspase-9。腺苷+EHNA诱导正常小鼠膝关节中的软骨细胞凋亡。
腺苷生成增加可能诱导软骨细胞凋亡，并在STR/ort小鼠的OA中起作用。
