The critical role of the cellular thiol homeostasis in cadmium perturbation of the lung extracellular matrix
Introduction
Lysyl oxidase (LO, E.C.1.4.3.13), a copper (Cu)-dependent enzyme, catalyzes crosslinking of collagen and elastin essential for extracellular matrix (ECM) morphogenesis and tissue repair (Kagan and Li, 2003). The critical role of LO in the lung physiology and pathology is evidenced by findings that inhibition of LO induces emphysematous lung in the animal model (Snider et al., 1986).
Cadmium (Cd) is a toxic metal but still widely used in industries. Occupational exposure to Cd occurs mainly in the form of airborne dusts and fumes affecting an estimated 510,000 workers in the US (IARC, 1993). In addition to the occupational exposure, cigarette smoke constitutes a major source of Cd exposure for humans since tobacco leaves naturally accumulate Cd. Since it binds to cellular thiols such as metallothionein (MT) and glutathione (GSH) with high affinity, Cd can be absorbed by and accumulated in the lung with a biological half-life of 9.4 years (IARC, 1993). Long-term exposure to Cd results in emphysema. Fatal emphysema developed in Cd-poisoned patients who had survived acute lung injuries in industrial accidents (Lane and Campbell, 1954, Snider, 1992). Smokers with severe emphysema have higher Cd levels in their lungs than non-smokers (Paakko et al., 1989, Post et al., 1984).
Elevation of lung metallothionein (MT) and glutathione (GSH) as a defense response is a resultant marker for Cd exposure (Hart et al., 2001, Paakko et al., 1989). Mammalian MT consists of 61–62 amino acids of which 20 are cysteines (Cys). These Cys-SH groups provide metal binding sites for MT. One mole of MT protein can bind a total of 7 moles of Cd or 11 or 12 moles of Cu (Kagi and Schaffer, 1988, Vasak, 2005). GSH is a thiol-containing tripeptide playing a critical role in cellular metal transport, metabolism and protection (Meister, 1984). Our previous studies have shown that downregulation of LO was associated with upregulation of cellular MT and GSH in Cd-resistant cells isolated from long-term Cd exposure (Zhao et al., 2006). To probe the molecular mechanisms of Cd injury to the lung ECM, we further examined the role of cellular thiol homeostasis in LO regulation in Cd-pulsed rat fetal lung fibroblasts (RFL6) and in Cd-dosed lungs of rats. We found that Cd enhanced levels of metal scavenging thiols, e.g., MT and GSH in pulse-treated cells and in the animal model concomitant with downregulation of LO. Cd downregulation of LO was closely coupled with suppression of synthesis of collagen, a major structure component of the lung ECM. Moreover, the in vitro assays further indicated that Cd upregulation of cellular thiols played a critical role in facilitating downregulation of LO, a potential mechanism for Cd damage to the lung ECM.
Section snippets
Materials
Cadmium chloride, 99.9% pure, was from Aldrich Chemicals (Milwaukee, WI). Glutathione (GSH) monoethyl ester (GME), GSH, GSH reductase, reduced nicotinamide adenine dinucleotide phosphate (NADPH), hemoglobin, diaminopentane, and β-aminopropionitrile (BAPN) were obtained from Sigma Chemical Company (St. Louis, MO). [3H] or [14C]proline (250 mCi/mmol), [3H]lysine (100 Ci/mmol), 32P-d-CTP (3000 Ci/mmol) and carrier-free 109Cd (1.83 mCi/mg) were purchased from PerkinElmer Life and Analytical Sciences,
Cd enhancement of cellular levels of MT and GSH concomitant with inhibition of LO catalytic activity
MT and GSH are major cellular thiols regulating the metal metabolism and heavy metal detoxification (Kagi and Schaffer, 1988, Meister, 1984, Vasak, 2005). Cd is an effective inducer of cellular MT and enhancer of cellular GSH. LO is a critical ECM crosslinking enzyme using Cu as cofactor (Kagan and Li, 2003). To evaluate the critical role of cellular thiols in Cd damage to the lung ECM, we first examined Cd effects on cellular levels of MT and GSH, and LO catalytic activity in conditioned media
Discussion
Using rat lung fibroblasts (RFL6) and rat lungs as in vitro and in vivo models, we studied Cd effects on cellular thiol homeostasis and lung ECM metabolism. Results indicated that Cd exposure enhanced cellular levels of MT-I, MT-II, and GSH, major metal scavenging agents, as well as the heavy chain of γ-GCS, an enzyme for GSH biosynthesis, but inhibited the expression of LO and collagen, two major components of the lung ECM. Changes in levels of MT, GSH, LO and collagen synthesis were
Conflict of interest statement
The authors declare that there are no conflicts of interest in this work.
Acknowledgements
This work was supported by research grants from NIH 2R56-ES 011340, NIH R01-ES 011340, and the Philip Morris External Research Program.
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New address: Department of Pharmacology, Zhongshan Medical College, Sun Yat-Sen University, Guangzhou, PR China.