Yinzhi Zhao

Lysyl oxidase oxidizes basic fibroblast growth factor and inactivates its mitogenic potential

Lysyl oxidase (LO) plays a central role in the crosslinking of collagen and elastin in the extracellular matrix. Here we demonstrate that basic fibroblast growth factor (bFGF), a polypeptide which regulates proliferation, differentiation, and migration of a variety of cell types, is a substrate of LO. The oxidation of lysine residues in bFGF by LO resulted in the covalent crosslinking of bFGF monomers to form dimers and higher order oligomers and dramatically altered its biological properties. Both the mitogenic potential and the nuclear localization of bFGF were markedly inhibited in the Swiss 3T3 cells upon its oxidation by LO. NIH 3T3 IgBNM 6‐1 cells (6‐1 cells) overexpress bFGF which participates in an autocrine mechanism accounting for the transformation of these cells into a tumorigenic state. Exposure of the 6‐1 cells to nanomolar concentrations of LO in culture oxidized lysine and generated crosslinkages in bFGF within the cell and markedly reduced proliferative rates. The lack of LO expression has been correlated with hyperproliferative cell growth, while this enzyme has been identified as a suppressor of ras‐induced tumorigenesis. The present results illustrate a mechanism by which LO can depress normal and transformed cell growth.

Lysyl Oxidase, A Critical Intra- and Extra-Cellular Target in the Lung for Cigarette Smoke Pathogenesis

Cigarette smoke (CS), a complex chemical mixture, contains more than 4,800 different compounds, including oxidants, heavy metals, and carcinogens, that individually or in combination initiate or promote pathogenesis in the lung accounting for 82% of chronic obstructive pulmonary disease (COPD) deaths and 87% of lung cancer deaths. Lysyl oxidase (LO), a Cu-dependent enzyme, oxidizes peptidyl lysine residues in collagen, elastin and histone H1, essential for stabilization of the extracellular matrix and cell nucleus. Considerable evidences have shown that LO is a tumor suppressor as exemplified by inhibiting transforming activity of ras, a proto oncogene. CS condensate (CSC), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and cadmium (Cd), major components of CS, down-regulate LO expression at such multiple levels as mRNA, protein and catalytic activity in lung cells in vitro and in vivo indicating LO as a critical intra- and extracellular target for CS pathogenesis in the lung. In view of multiple biological functions and regulation characteristics of the LO gene, molecular mechanisms for CS damage to lung LO and its role in emphysema and cancer pathogenesis are discussed in this review.

Cloning and characterization of the rat lysyl oxidase gene promoter : Identification of core promoter elements and functional nuclear factor I-binding sites

Lysyl oxidase (LO) stabilizes the extracellular matrix by cross-linking collagen and elastin. To assess the transcriptional regulation of LO, we cloned the 5′-flanking region with 3,979 bp of the rat LO gene. LO transcription started at multiple sites clustered at the region from –78 to –51 upstream of ATG. The downstream core promoter element functionally independent of the initiator predominantly activated the TATA-less LO gene. 5′ Deletion assays illustrated a sequence of 804 bp upstream of ATG sufficient for eliciting the maximal promoter activity and the region –709/–598 exhibiting strongly enhancing effects on the reporter gene expression in transiently transfected RFL6 cells. DNase I footprinting assays showed a protected pattern existing in the fragment –612/–580, which contains a nuclear factor I (NFI)-binding site at the region –594/–580 confirmed by electrophoretic mobility supershift assays. Mutations on this acting site decreased both NFI binding affinity in gel shift assays and stimulation of SV40 promoter activities in cells transfected with the NFI-binding site-SV40 promoter chimeric construct. Furthermore, at least two functional NFI-binding sites, including another one located at –147/–133, were identified in the LO promoter region –804/–1. Only NFI-A and NFI-B were expressed in rat lung fibroblasts, and their interaction with the LO gene was sensitively modulated by exogenous stimuli such as cigarette smoke condensate. In conclusion, the isolated rat LO gene promoter contains functionally independent initiator and downstream core promoter elements, and the conserved NFI-binding sites play a critical role in the LO gene activation.

Microtubules as a Critical Target for Arsenic Toxicity in Lung Cells in Vitro and in Vivo

To understand mechanisms for arsenic toxicity in the lung, we examined effects of sodium m-arsenite (As3+) on microtubule (MT) assembly in vitro (0–40 µM), in cultured rat lung fibroblasts (RFL6, 0–20 µM for 24 h) and in the rat animal model (intratracheal instillation of 2.02 mg As/kg body weight, once a week for 5 weeks). As3+ induced a dose-dependent disassembly of cellular MTs and enhancement of the free tubulin pool, initiating an autoregulation of tubulin synthesis manifest as inhibition of steady-state mRNA levels of βI-tubulin in dosed lung cells and tissues. Spindle MT injuries by As3+ were concomitant with chromosomal disorientations. As3+ reduced the binding to tubulin of [3H]N-ethylmaleimide (NEM), an -SH group reagent, resulting in inhibition of MT polymerization in vitro with bovine brain tubulins which was abolished by addition of dithiothreitol (DTT) suggesting As3+ action upon tubulin through -SH groups. In response to As3+, cells elevated cellular thiols such as metallothionein. Taxol, a tubulin polymerization agent, antagonized both As3+ and NEM induced MT depolymerization. MT–associated proteins (MAPs) essential for the MT stability were markedly suppressed in As3+-treated cells. Thus, tubulin sulfhydryls and MAPs are major molecular targets for As3+ damage to the lung triggering MT disassembly cascades.

The critical role of the cellular thiol homeostasis in cadmium perturbation of the lung extracellular matrix

Cadmium (Cd) inhalation can result in emphysema. Cd exposure of rat lung fibroblasts (RFL6) enhanced levels of metal scavenging thiols, e.g., metallothionein (MT) and glutathione (GSH), and the heavy chain of gamma-glutamylcysteine synthetase (gamma-GCS), a key enzyme for GSH biosynthesis, concomitant with downregulation of lysyl oxidase (LO), a copper-dependent enzyme for crosslinking collagen and elastin in the extracellular matrix (ECM). Cd downregulation of LO in treated cells was closely accompanied by suppression of synthesis of collagen, a major structure component of the lung ECM. Using rats intratracheally instilled with cadmium chloride (30 microg, once a week) as an animal model, we further demonstrated that although 2-week Cd instillation induced a non-significant change in the lung LO activity and collagen synthesis, 4- and 6-week Cd instillation resulted in a steady decrease in the lung LO and collagen expression. The lung MT and total GSH levels were both upregulated upon the long-term Cd exposure. Emphysematous lesions were generated in lungs of 6-week Cd-dosed rats. Increases of cellular thiols by transfection of cells with MT-II expression vectors or treatment of cells with GSH monoethyl ester, a GSH delivery system, markedly inhibited LO mRNA levels and catalytic activities in the cell model. Thus, Cd upregulation of cellular thiols may be a critical cellular event facilitating downregulation of LO, a potential mechanism for Cd-induced emphysema.

Cytoskeletal injury induced by hexavalent chromate

To understand the mechanisms of toxicity of hexavalent chromate (K(2)CrO(4), Cr(6+)), the effects of this metal ion on the organization of microtubules (MTs) and microfilaments (MFs), DNA synthesis, cytoskeletal protein synthesis, cytoskeletal protein sulphhydryls (-SH groups), and cellular glutathione (GSH) levels in 3T3 cells were examined. Exposure of cells to Cr(6+) for 16 hr resulted in a dose-dependent inhibition of DNA synthesis with 50% inhibition occurring at 16.6 mum. Treatment of cells with 3.13 mum-Cr(6+) for 16 hr resulted in a slight cell retraction and, in some cells, MT bundling, without much effect on the morphology of dense MFs. At 25 mum, Cr(6+) caused disruption of the cell sheet, depolymerization of MTs, particularly those in the peripheral areas, and redistribution of MFs, which assumed a smearing morphology. Exposure to 100 mum-Cr(6+) induced severe thinning of MTs and loss of MFs. Although doses of 3.13 mum-Cr(6+) or less slightly enhanced the level of cytoskeletal protein synthesis (e.g. 38% increase at 3.13 mum-Cr(6+)), Cr(6+) at 6.25 mum or more produced a dose-dependent inhibition of cytoskeletal protein synthesis (50% inhibition at 11.25 mum). Exposure of cells to Cr(6+) for 16 hr resulted in a dose- and time-dependent increase of cellular GSH level. Furthermore, the elevated cellular GSH induced by Cr(6+) was diminished by treatment with buthionine sulphoximine (BSO), a potent inhibitor of GSH biosynthesis. In addition, depletion of GSH by BSO increased cell sensitivity to Cr(6+) insult and aggravated Cr(6+)-induced cytoskeletal perturbation. However, Cr(6+) treatment of cells did not significantly affect the amount of cytoskeletal protein sulphhydryls. These results suggest that cytoskeletal injury may be an important part of the mechanism for Cr(6+) toxicity. The cytoskeletal damage may result directly from the inhibition of cytoskeletal protein synthesis rather than from the interaction between Cr(6+) and cytoskeletal protein sulphhydryls.

NNK, a Tobacco-Specific Carcinogen, Inhibits the Expression of Lysyl Oxidase, a Tumor Suppressor

A tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is believed to contribute to the cancer burden in cigarette smokers. To evaluate NNK effects on the expression of lysyl oxidase (LOX), a tumor suppressor, we examined this enzyme at various levels in NNK-treated rat fetal lung fibroblasts (RFL6). Exposure of cells to NNK reduced levels of steady-states LOX mRNA and new transcript synthesis. NNK inhibited all LOX protein species in a dose-dependent manner. Although 300 µM NNK markedly decreased the level in the 46 kDa preproenzyme, under same conditions, there was no detectable amounts of the 50 kDa proenzyme and the 32 kDa mature enzyme suggesting NNK perturbing the LOX protein processing to its mature form. Moreover, NNK also suppressed LOX activities in conditioned media of treated cells. At the promoter level, NNK enhanced methylation of CpG, but decreased acetylation of histone H3 at the core promoter region of the LOX gene. These results indicated that transcriptional and translational processes of LOX are major targets for NNK. Thus, inactivation of tumor suppressor gene LOX may play a critical role in NNK carcinogenesis.

Lysyl Oxidase Gene G473A Polymorphism and Cigarette Smoking in Association with a High Risk of Lung and Colorectal Cancers in a North Chinese Population.

The relationship among the lysyl oxidase (LOX) G473A single nucleotide polymorphism (SNP), cigarette smoking and lung, colorectal, colon and rectum cancer susceptibility was studied in 200 cases of lung cancer, 335 cases of colorectal cancer including 130 cases of colon cancer and 205 cases of rectum cancer, and 335 healthy people in Tangshan, China. Peripheral blood DNA samples were collected, DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) performed, followed by multivariate logistic regression analysis. In comparison to LOX473GG genotype carriers, individuals with LOX473AA exhibited a higher susceptibility to lung, colon-rectum, colon, and rectum cancers with OR values amounting to 3.84-, 2.74-, 2.75-, and 2.74-fold of the control, respectively. In the LOX 473AA-positive population, females were more susceptible than males to carcinogenesis with OR values (female vs. male): 5.25 vs. 3.23, 2.29 vs. 1.51, 2.27 vs. 1.45, and 2.25 vs. 1.53, respectively, for lung, colon-rectum combined, colon, and rectum cancers. LOX G473A polymorphism apparently elevated human sensitivity to cigarette smoking carcinogens for eliciting cancers in the lung and colon only. Thus, LOX G473A polymorphism positively correlates with carcinogenesis and it may be used as an ideal intrinsic biomarker for prediction or diagnosis of carcinogenesis in humans.