Overexpression of metallothionein decreases sensitivity of pulmonary endothelial cells to oxidant injury

Abstract

Metallothionein (MT) is a low-molecular-weight cysteine-rich protein with extensive metal binding capacity and potential nonenzymatic antioxidant activity. Despite the sensitivity of vascular endothelium to either heavy metal toxicity or oxidative stress, little is known regarding the role of MT in endothelial cells. Accordingly, we determined the sensitivity of cultured sheep pulmonary artery endothelial cells (SPAEC) that overexpressed MT to tert-butyl hydroperoxide ( t-BOOH), hyperoxia, or 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN; peroxyl radical generator). Nontoxic doses of 10 μM Cd increased MT levels from 0.21 ± 0.03 to 2.07 ± 0.24 μg/mg and resulted in resistance to t-BOOH and hyperoxia as determined by reduction of Alamar blue or [3H]serotonin transport, respectively. SPAEC stably transfected with plasmids containing either mouse or human cDNA for MT were resistant to both t-BOOH and hyperoxia. In addition, we examined transition metal-independent, noncytotoxic AMVN-induced lipid peroxidation after metabolic incorporation of the oxidant-sensitive fluorescent fatty acid cis-parinaric acid into phospholipids and high-performance liquid chromatography separation. SPAEC that overexpressed MT after gene transfer completely inhibited peroxyl oxidation of phosphatidylserine, phosphatidylcholine, and sphingomyelin (but not phosphatidylethanolamine) noted in wild-type SPAEC. These data show for the first time that MT can 1) protect pulmonary artery endothelium against a diverse array of prooxidant stimuli and 2) directly intercept peroxyl radicals in a metal-independent fashion, thereby preventing lipid peroxidation in intact cells.

Silencing of metallothionein-I gene in mouse lymphosarcoma cells by methylation

“…The basal expression of MT-I and MT-II genes is relatively low in most tissues except in the brain and testis, but can be induced by a variety of agents that include heavy toxic metals, steroid hormones, interleukins, phorbol esters, interferons, restraint stress and other agents that produce oxygen intermediates or free oxygen radicals (Kagi, 1991;. Overexpression of MT in cells diminishes the sensitivity of the cells to the compounds that generate free oxygen radicals (Pitt et al, 1997), DNA damaging agents such as UV radiation (Chubatsu and Meneghini, 1993), nitric oxide (Schwarz et al, 1995) and certain anticancer drugs (Kelley et al, 1988). On the contrary, highly inducible MT-I and MT-II genes are silent in some lymphoid derived tumor cell lines, W-7 and S-49, but can be induced by heavy metals after treatment with 5-azacytidine (5-AzaC) (Compere and Palmiter, 1981), which suggests a role for hypermethylation in the silencing of MT gene.…”

Altered Central Nervous System Cytokine-Growth Factor Expression Profiles and Angiogenesis in Metallothionein-I+II Deficient Mice

“…MT-I+II can protect against reactive oxygen species causing oxidative damage and stress, ionizing radiation, anti-cancer drugs, and interestingly, MT-I+II may prevent neuronal apoptosis (Aschner, 1998;Lazo et al, ,1998Penkowa et al, 1999aPitt et al, 1997;Schwarz et al, 1995;Tamai et al, 1993;Thornalley and Vasak, 1985).…”

Nitric Oxide and Zinc Homeostasis in Acute Lung Injury

“…We noted that overexpression of MT in cultured SPAEC reduced the sensitivity to exposure to 95% oxygen (81). More recently we have noted that mice in which MTI and MTII were deleted by targeted ablation (MTϪ/Ϫ) were more sensitive than wild-type controls (MTϩ/ϩ) to continuous exposure to Ͼ 99% oxygen (B. R. Pitt and coworkers, unpublished observations).…”