Inhibition of the expression of lysyl oxidase and its substrates in cadmium-resistant rat fetal lung fibroblasts.
Inhibition of the expression of lysyl oxidase and its substrates in cadmium-resistant rat fetal lung fibroblasts.
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DOI：
10.1093/toxsci/kfj112
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发表时间：
2006-04
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影响因子：
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通讯作者：
Yinzhi Zhao;Song Gao;I. Chou;P. Toselli;P. Stone;Wande Li
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作者：
Yinzhi Zhao;Song Gao;I. Chou;P. Toselli;P. Stone;Wande Li
铜依赖赖氨酰氧化酶(LO)催化胶原和弹性蛋白的交联，稳定细胞外基质(ECM)。慢性吸入有毒金属镉(Cd)会导致肺气肿。为了探讨镉对肺损伤的机制，我们从大鼠胎肺成纤维细胞(RFL6)慢性暴露于1~40微米的氯化镉(2)中建立了耐镉(CDR)细胞，并进一步检测了它们的LO、LO底物和清除铜的硫醇的表达。CDR细胞中细胞硫醇、金属硫蛋白和谷胱甘肽的水平分别升高到亲代对照的13.0倍和3.2倍，而这些细胞的LO mRNA和蛋白水平显著降低，催化活性仅下降到亲本对照的16%。在CDR细胞提取液中出现明显的52 kDa酶原，而不是正常的50 kDa酶原，它与内质网标记[DiOC5(3)]共存于细胞内，暗示Cd诱导的52 kDa酶原是分泌过程中异常Lo加工缺陷的产物。在CDR细胞中加入铜后，细胞中LO的表达、蛋白和催化活性增加，反映了铜对细胞中LO的生物有效性有限。随着LO的抑制，CDR细胞也表现出LO的底物胶原和弹性蛋白的下调。通过将CDR细胞暴露于纯化的LO或铜来恢复胶原合成，提示Cd对LO的抑制和对铜辅因子的限制，作为关键的表型变化，加速了胶原和弹性蛋白的损伤，这是与肺气肿发病相关的关键事件。
Copper (Cu)-dependent lysyl oxidase (LO) catalyzes crosslinking of collagen and elastin stabilizing the extracellular matrix (ECM). Chronic inhalation of cadmium (Cd), a toxic metal, induces emphysema. To probe mechanisms of Cd injury to the lung, we developed Cd-resistant (CdR) cells from rat fetal lung fibroblasts (RFL6) by chronic exposure to CdCl(2) from 1 to 40 microM and further examined their expressions of LO, LO substrates, and Cu-scavenging thiols. Levels of cellular thiols, metallothionein, and glutathione in CdR cells were elevated to 13.0- and 3.2-fold of parental controls, respectively, whereas LO mRNA and protein levels were markedly reduced in these cells, with catalytic activity declining to only 16% of the parental control. A conspicuous 52 kDa species rather then the normal 50 kDa proenzyme appeared in the CdR cell extract but not in the conditioned medium, which was codistributed with the endoplasmic reticulum marker [DiOC5(3)] within the cell, implying the Cd-induced 52 kDa species as a product of an abnormal LO-processing defect in secretion. Addition of Cu into CdR cell cultures enhanced the expression of LO mRNA, protein and catalytic activities reflecting limitation of Cu bioavailability for LO in these cells. With inhibition of LO, CdR cells also displayed downregulation of collagen and elastin, substrates of LO. Restoration of collagen synthesis by exposure of CdR cells to purified LO or Cu suggests that inhibition of LO and limitation of Cu cofactor by Cd, as key phenotype changes, accelerated collagen and elastin damage, a critical event pertinent to emphysema pathogenesis.