The LOX gene is composed of seven exons and six introns, distributed through approximately 14.5 kb of genomic DNA (Hamalainen et al., 1993; Boyd et al., 1995). Three transcripts of sizes 2.0 kb, 3.8 kb and 4.8 kb are produced (Mariani et al., 1992), as a consequence of differential use of several polyadenylation signals within the 3 UTR (Boyd et al., 1995). There is a heritable restriction fragment length polymorphism within a PstI restriction site in the first exon (Csiszar et al., 1993). Three additional polymorphisms were also identified: Ala75Ala, Arg103Pro, Arg158Gln (Kaneda et al., 2004). There are no microsatellites within the LOX gene, but three have been described in the LOX gene locus within 20 kb and 5 kb centromeric, and 7 kb telomeric to the LOX gene (Csiszar et al., 2002). The 5 region of LOX also contains a CpG island which extends into exon 1 (Kaneda et al., 2002; Kaneda et al., 2004).

The rat LOX promoter contains a metal-response element, a hypoxia-response element, an antioxidant-response element, at least four positive regulatory segments and 2 negative regulatory segments. Multiple transcriptional start sites have been described (Gao et al., 2007).
The regulation of LOX gene expression has been described in different tissues and cells from several species, and has revealed multiple complex mechanisms that regulate the expression and activity of LOX. Many of these effectors are reviewed in Csiszar, 2001, and encompass cytokines and growth factors, such as fibroblast growth factor, basic fibroblast growth factor, insulin-like growth factor-1, interferon-gamma and transforming growth factor-beta; hormones and mediators, such as testosterone, progestin and prostaglandin E2; signaling molecules, such as cAMP, interferon regulatory factor-1 and ras; and drugs, such as adriamycin, bleomycin and hydralazine. Additional effectors have since been described, including follicle stimulating hormone (Slee et al., 2001; Harlow et al., 2003), hyperosmotic solution (Omori et al., 2002), secretory leukocyte protease inhibitor (Zhang et al., 2002), interleukin-1alpha (Rae et al., 2004), cigarette smoke condensate (Chen et al., 2005; Gao et al., 2005), tumor necrosis factor-alpha (Rodriguez et al., 2008), granulocyte macrophage colony-stimulating factor (Weissen-Plenz et al., 2008), hyaluronan with insulin-like growth factor-1 (Kothapalli and Ramamurthi, 2008) and parathyroid hormone (Lowry et al., 2008).
LOX expression is promoted in MCF-7 breast cancer cells by contact with fibroblast-conditioned media, collagen I matrix conditioned by fibroblasts (Kirschmann et al., 2002), and in HK-2 renal proximal tubule cells co-cultured with HMEC-1 microvascular endothelial cells (Aydin et al., 2008).
LOX expression is also induced by hypoxia. Hypoxia-inducible factor-1alpha (HIF-1alpha) stimulates LOX mRNA transcription (Erler et al., 2006; Higgins et al., 2007). LOX expression in hypoxia can be modulated by pH (Sorensen et al., 2007). The recruitment of HIF-1alpha to the LOX promoter is potentiated by Notch, which also increases Snail-1 expression (Sahlgren et al., 2008).
Transcription of the LOX gene is also affected by the methylation status of its CpG island (Kaneda et al., 2002; Kaneda et al., 2004).

No known pseudogene.

The lysyl oxidase transcript encodes for a 417-amino acid protein, including a signal peptide of 21 amino acids (Hamalainen et al., 1991; Mariani et al., 1992). Removal of the signal sequence and N-glycosylation within the propeptide region produces a 50 kDa proenzyme (Trackman et al., 1992). Incorporation of copper is thought to occur either in the endoplasmic reticulum or in the Golgi in prior to and independently of glycosylation (Kosonen et al., 1997). After secretion into the extracellular space, the mature, non-glycosylated 32 kDa protein (Trackman et al., 1992) is generated by proteolytic cleavage of the propeptide region between Gly-168 and Asp-169 by C-proteinase (Cronshaw et al., 1995), encoded by the bone morphogenic protein-1 (BMP-1), and the related tolloid-like-1 and -2 (Kessler et al., 1996; Uzel et al., 2001). LOX may also be a substrate of human meprin, an astacin-like metallopeptidase belonging to the same subfamily as BMP-1 and mammalian tolloid (Ambort et al., 2008).

As LOX is necessary for the assembly and tensile strength and mechanical stability of collagen fibrils, and for the assembly and repetitive and reversible deformation of elastin, it is highly expressed in tissues containing fibrillar collagen and/or elastic fibers. These include skin, lung, cartilage, the cardiovascular system and the fibrous laminia propria in the small intestine. LOX was also detected in the liver, kidney, stomach, retina, and brain (Hayashi et al., 2004). In the eye, LOX has been identified in the vitreous, iris/ciliary body, lens, choroid/retinal pigment epithelium and retina (Coral et al., 2008). LOX has also been described in the mesencephalon, corpus callosum, cerebral cortex and cerebellum of the brain (Laczko et al., 2007).
Protein expression of LOX may be influenced by microRNAs. The 3 UTR of LOX mRNA contains a binding site for mir-145, which is down-regulated in many cancers (Dalmay and Edwards, 2006).

LOX has been classically characterized as an extracellular matrix enzyme (Kagan and Trackman, 1991). However, less is known about intracellular LOX. The LOX protein has been localized within the nuclei of various cells and tissues (Li et al., 1997; Hayashi et al., 2004; Li et al., 2004), and retains its catalytic activity inhibited by beta-aminopropionitrile (bAPN) (Li et al., 1997). Nuclear LOX has been shown to originate from extracellular LOX that enters the cytosol and concentrates within the nucleus (Nellaiappan et al., 2000). Histones have been reported as substrates for lysyl oxidase (Kagan et al, 1983; Giampuzzi et al, 2003), and transfection of LOX yielded less tightly packed chromatin (Mello et al., 1995). Electron microscopy confirmed association of LOX with condensed chromatin in the nucleus (Kagan and Li, 2003).
In addition to its nuclear localization, LOX has also been identified in the cytoplasm of numerous cells and tissues (Wakasaki and Ooshima, 1990; Kobayashi et al., 1994; Hayashi et al., 2004; Jansen and Csiszar, 2007). This cytoplasmic LOX was localized to cytoskeletal filaments and microtubule networks (Wakasaki and Ooshima, 1990; Guo et al., 2007).
The propeptide of LOX (LOX-PP), which has been described to have a different function than the LOX enzyme, differs in its localization compared to the active LOX enzyme, and is dependent on cell stage. LOX-PP, which is generated extracellularly, is able to reenter the cells. In differentiating osteoblasts, both LOX-PP and LOX enzyme localized to the cytoplasm associated with tubulin and the microtubule network, while in proliferating osteoblasts, LOX-PP localized perinuclearly in the Golgi complex and endoplasmic reticulum while LOX enzyme was mainly in the nucleus (Guo et al., 2007).

Lysyl oxidase oxidizes peptidyl lysine and hydroxylysine residues in collagen and lysine residues in elastin to produce peptidyl alpha-aminoadipic-delta-semialdehydes. These aldehyde residues can spontaneously condense with vicinal peptidyl aldehydes or with epsilon-amino groups of peptidyl lysine to form the covalent cross-links that stabilize and insolubilize several fibrillar collagen types and elastin fibers (reviewed in Lucero and Kagan, 2006). This catalytic reaction can be irreversibly inhibited by bAPN, a specific inhibitor that binds to the active site of LOX (Tang et al., 1983). Semicarbazide is a partial inhibitor of LOX (Mercier et al., 2007), as is 2-mercaptopyridine-N-oxide, although it acts through a different mechanism than bAPN (Anderson et al., 2007). Pathological concentrations of homocysteine also inhibit LOX activity (Raposa et al., 2004). The activity of LOX in the extracellular matrix may be coordinated by its interaction with fibronectin (Fogelgren et al, 2005).
The critical nature of LOX enzyme activity was demonstrated in the LOX "knockout" mouse, which expires immediately after birth due to rupture of the aorta and diaphragm from incomplete cross-linking of elastin (Maki et al., 2002; Hornstra et al., 2003). Local administration of LOX resulted in inhibition of abdominal aortic aneurysm development in a mouse model (Yoshimura et al., 2006). LOX is also essential for development of the distal and proximal airways, and alveolarization in the lungs (Maki et al., 2005), and is increased in preterm lamb lungs compared to full-term (Bland et al., 2007). In zebrafish, LOX is critical for notochord formation and muscle development (Anderson et al., 2007; Gansner et al., 2007; Reynaud et al., 2008), a process that may involve the fibrillin-2 gene or alpha1 chain of type VIII collagen (Gansner et al., 2008; Gansner and Gitlin, 2008). In sea urchins, inhibition of LOX caused developmental arrest at the mesenchymal blastula state (Butler et al., 1987), and in Xenopus laevis, LOX was shown to antagonize p21-Ha-Ras-induced and progesterone-dependent oocyte maturation (Di Donato et al., 1997).

LOX also has a role in cell differentiation. LOX was identified as an early marker in adipocyte differentiation responsive to retinoic acid (Dimaculangan et al., 1994). Increased LOX may affect osteoblastic differentiation through cross-link formation in the surrounding collagen matrix (Kaku et al., 2007; Turecek et al., 2008). LOX may also modulate cartilage growth (Asanbaeva et al., 2008).

Alterations in LOX expression were observed in development and aging of the skin, as well as physiological and pathological processes of the skin, including wound healing, fibrosis, hypertrophic scarring, keloids, and scleroderma (reviewed in Szauter et al., 2005). Inhibition of LOX activity resulted in inhibition of skin graft contraction in a human skin model (Harrison et al., 2006) Decreased LOX expression was noted in pelvic organ prolapse (Klutke et al., 2008), in pregnant mouse vagina and cervix compared to non-pregnant and post-partum tissues (Drewes et al., 2007), in proliferative diabetic retinopathy and rhegmatogenous retinal detachment (Coral et al., 2008), and early atherosclerosis (reviewed in Rodriguez et al., 2008). Induction of LOX was reported in inflamed oral tissue (Trackman et al., 1998) and gingival atrophy from experimental occlusal hypofunction (Ishida et al., 2008); rheumatoid arthritis (Kaufmann et al., 2003); inflammatory bowel disease (Rivera et al., 2006); liver stiffness preceding liver fibrosis (Georges et al., 2007); fibrosis of the liver (reviewed in Kagan, 1994), lung (Counts et al., 1981; Almassian et al., 1991; Peyrol et al., 1997), kidney (Di Donato et al., 1997; Goto et al., 2005; Higgins et al., 2007), oral submucosa (reviewed in Tilakaratne et al., 2006) and heart (Lopez et al., 2008; Sivakumar et al., 2008; Spurney et al., 2008; Urashima et al., 2008); systemic sclerosis (Meyringer et al., 2007); amyotrophic lateral sclerosis (Malaspina et al., 2001; Li et al., 2004); senile plaque development in Alzheimers and non-Alzheimers dementia (Gilad et al., 2005) and stromal reactions in cancer, which are described in more detail below.

Besides collagen and elastin, other substrates have been identified for LOX. The oxidation of lysine residues in basic fibroblast growth factor (bFGF) caused covalent crosslinking of bFGF monomers to form dimers and higher order oligomers, leading to reduced mouse fibroblast proliferation (Li et al., 2003). LOX also oxidizes the platelet-derived growth factor (PDGF) receptor beta, which increases its binding affinity for PDGF-BB and decreases the turnover of PDGF receptor beta signal transduction pathway (Lucero et al., 2008). LOX also interacts with mature transforming growth factor-beta (TGF-b). LOX and TGF-b colocalize to mineral associated bone matrix, and LOX was able to suppress TGF-b1-induced Smad3 phosphorylation (Atsawasuwan et al., 2008).

LOX has been shown to regulate the promoters of collagen III (COL3A1) and elastin (Giampuzzi et al., 2000; Oleggini et al., 2007; Lelievre et al., 2008). LOX also interacts with histones H1 and H2, and may be able to modulate the condensation status of chromatin to affect transcription of other genes as well (Giampuzzi et al., 2003).

LOX has chemokinetic and chemotactic effects on human blood monocytes (Lazarus et al., 1995), a predominantly chemotactic effect on rat vascular smooth muscle cells and mouse embryonic fibroblasts (Li et al., 2000; Lucero et al., 2008), and has been demonstrated to regulate breast cancer cell migration and adhesion and astrocytoma migration through a hydrogen-peroxide mediated mechanism (Payne et al., 2005; Laczko et al., 2007). The same mechanism may also play a role in the promotion of normal breast epithelial cell proliferation and migration by the interaction of LOX and hormone placental lactogen (PL), although PL is not a substrate for LOX (Polgar et al., 2007). The catalytic domain of LOX is able to interact with Snail-1 in vitro, a transcription factor crucial to EMT (Peinado et al., 2005). LOX is responsible for increased migratory ability of renal tubular epithelial cells induced by hypoxia (Higgins et al., 2007). Smooth muscle migration may be modulated through the interaction with VE-statin/egfl7 to inhibit LOX enzyme activity (Lelievre et al., 2008). LOX also interacts and oxidizes PDGF receptor beta to modulate chemokine activity (Lucero et al., 2008).

LOX also has multiple roles in cancer, including its opposing effect on ras-transformation, tumor suppression, stromal reaction in cancer, and the promotion of cancer cell adhesion, migration, invasion and metastases, and these are described in more detail in the following sections.

In the human lysyl oxidase protein family, there are five members, named LOX, LOXL1, LOXL2, LOXL3 and LOXL4. They all contain a lysine tyrosylquinone (LTQ), the only mammalian cofactor derived from the cross-linking of two amino acid side chains (reviewed in Anthony, 1996), and which is unique to the LOX family. The other highly conserved motif that is unique to the LOX family is the copper-binding domain, which contains four histidines (Krebs and Krawetz, 1993). All LOX family members also contain a cytokine receptor-like (CRL) domain, which has part of the consensus sequence of Class 1 cytokine receptors (Bazan, 1990). LOX has closest homology to LOXL1, and both seem to be found exclusively in vertebrates.

Despite the implication of LOX in many diseases and disorders, including inflammation and inflammatory diseases, fibrosis of distinct organs and fibrotic disorders, and cancer promotion and progression, there are only sparse reports of any mutations or epigenetic alterations in the LOX gene.

Loss of heterozygosity has been documented in colon and gastric cancers. In 42 colon tumors informative for the microsatellites flanking the LOX gene, 38% demonstrated loss of heterozygosity (Csiszar et al., 2002). In gastric cancer, 33% of 27 informative tumors demonstrated loss of heterozygosity (Kaneda et al., 2004).
Somatic mutations of the LOX gene have been documented in colon cancer and possibly, an ovarian cancer cell line. One nonsense mutation that affected codon 332, a 3 rearrangement affecting exons 5-7, and six 5 intragenic alterations or deletions, were detected out of the 8 colon tumors that demonstrated both loss of heterozygosity and reduced LOX expression (Csiszar et al., 2002). Of 96 gastric cancer samples and 58 gastric, lung, colon, ovarian and pancreatic cancer cell lines, only one somatic mutation or rare polymorphism was found in an ovarian cancer cell line: Ala147Gly (Kaneda et al., 2004).
The polymorphism Arg158Glu is associated with earlier clinical stage, lower tumor grade and decreased lymph node metastases in oral squamous cell carcinomas. The polymorphism also did not induce anchorage independent growth as did the wild type LOX (Shieh et al., 2007).
As point mutations and deletions appear to be infrequent, the loss of LOX expression may be due to a combination of the more frequent loss of heterozygosity and epigenetic regulation. Indeed, some gastric cancers demonstrated biallelic methylation or loss of heterozygosity of one allele with methylation of the other allele (Kaneda et al., 2004).

The CpG island of LOX was described to be methylated in 27% of a primary gastric cancer panel. Silencing of LOX expression by methylation was also observed in gastric, colon, lung and ovarian cancer cell lines (Kaneda et al., 2002, Kaneda et al., 2004). Our group has also noted increase in LOX expression following treatment with the demethylating agent, 5-aza-2-deoxycytidine, in breast cancer cell lines (unpublished data). Methylation is thought to be the mechanism of LOX suppression after ras-transformation (Contente et al., 1999).