Miyahara T, Tonoyama H, Watanabe M, Okajima A, Miyajima S, Sakuma T, Nemoto N, Takayama K
Department of Toxicology, Faculty of Pharmaceutical Sciences, Toyama Medical & Pharmaceutical University, Toyama 939-0194, Japan.
Calcif Tissue Int. 2001 Mar;68(3):185-91. doi: 10.1007/s002230001216.
We have reported that cadmium (Cd) stimulates bone resorption via prostaglandin E2 (PGE2), which is mainly produced in osteoblasts. Prostaglandin (PGs) is regulated by arachidonic acid (AA) release by phospholipase A2 (PLA2) and its conversion to PGs by cyclooxygenase (COX). In the present study, we investigated the possibility that Cd-induced PGE2 synthesis was mediated through PLA2 or COX or both using primary mouse osteoblastic cells in serum-free medium. Cd at 1 microM and above stimulated 14C-AA release from 14C-AA-prelabeled osteoblastic cells. PLA2 activity of cytosolic fraction in Cd-treated cells preferentially hydrolyzed AA at the Sn2 position of phospholipids and was inhibited by arachidonyltrifluoromethyl ketone (AACOCF3), an inhibitor of cytosolic PLA2 (cPLA2). Cd at 1 microM and above increased cPLA2 activity and the level of constitutive cPLA2 mRNA. Secretory PLA2 mRNA was not detected. On the other hand, Cd at 1 microM and above stimulated PGE2 production and its production was inhibited by an inhibitor of COX-2 (NS-398). Cd at 1 microM and above markedly stimulated COX-2 mRNA expression and slightly increased the level of COX-1 mRNA. An inhibitor of COX-1 (varelylsalicylic acid) did not affect Cd-induced PGE2 production. In addition, Cd-induced PGE2 synthesis was inhibited by AA-COCF3, On the other hand, IL-1 alpha, an inducer of COX-2, did not stimulated PGE2 production in present culture system. When IL-1 alpha- or Cd-treated cells were incubated with AA for 10 minutes, IL-1 alpha-treated cells as well as Cd-treated ones caused an increase in PGE2 production. This suggests that the mechanism of Cd-induced PGE2 production is different from that of IL-1 alpha, which may require an activation of cPLA2. From these results, it was found that Cd by itself stimulated PGE2 production by two successive steps that Cd increased cPLA2 activity and then COX-2 induction.
我们曾报道，镉（Cd）通过主要在成骨细胞中产生的前列腺素E2（PGE2）刺激骨吸收。前列腺素（PGs）受磷脂酶A2（PLA2）释放花生四烯酸（AA）以及环氧化酶（COX）将其转化为PGs的调控。在本研究中，我们使用无血清培养基中的原代小鼠成骨细胞，研究了Cd诱导的PGE2合成是否通过PLA2或COX或两者介导。1 microM及以上浓度的Cd刺激了14C-AA预标记的成骨细胞释放14C-AA。Cd处理细胞的胞质部分的PLA2活性优先水解磷脂Sn2位的AA，并受到胞质PLA2（cPLA2）抑制剂花生四烯酰三氟甲基酮（AACOCF3）的抑制。1 microM及以上浓度的Cd增加了cPLA2活性和组成型cPLA2 mRNA的水平。未检测到分泌型PLA2 mRNA。另一方面，1 microM及以上浓度的Cd刺激了PGE2的产生，其产生受到COX-2抑制剂（NS-398）的抑制。1 microM及以上浓度的Cd显著刺激了COX-2 mRNA的表达，并略微增加了COX-1 mRNA的水平。COX-1抑制剂（伐地昔布）不影响Cd诱导的PGE2产生。此外，AA-COCF3抑制了Cd诱导的PGE2合成。另一方面，COX-2诱导剂IL-1α在当前培养系统中未刺激PGE2的产生。当IL-1α或Cd处理的细胞与AA孵育10分钟时，IL-1α处理的细胞以及Cd处理的细胞均导致PGE2产生增加。这表明Cd诱导PGE2产生的机制与IL-1α不同，IL-1α可能需要激活cPLA2。从这些结果发现，Cd自身通过两个连续步骤刺激PGE2产生，即Cd增加cPLA2活性，然后诱导COX-2。
