Protein-lysine 6-oxidase
Proteins

Author: Isaac Giraudo Giorgio Calleris
Date: 14/02/2013

Description

Protein-lysine 6-oxydase, or Lysyl oxidase, encoded by the very well evolutionarily conserved gene LOX, located on chromosome 5 (5q23.2), is an extracellular copper enzyme (EC 1.4.3.13) belonging to the class of Oxidoreductases acting on the CH-NH 2 group with oxygen as acceptor: as the name suggests, it essentially deaminates and oxidizes lysine residues on C6, catalyzing the formation of aldehydes. In homo sapiens, it is composed by 417 AA., and weights 46 944 Da.
The aldehydes formed by LOX are highly reactive and undergo spontaneous chemical reactions: this results in collagen and elastin cross-linking. Thereby, LOX activity is crucial for an appropriate connective tissue formation.

The significance of LOX was demonstrated in the LOX knockout mouse, which could not survive at birth, due to rupture of the aorta and diaphragm from incomplete elastin cross-linking. [Maki, 2002]

LOX appears to be deeply involved in various essential cellular and tissue functions, among which can be enumerated [Sethi et al., 2012] [Atlas of Genetics and Cytogenetics in Oncology and Haematology] :

  • development of the distal and proximal airways
  • formation of alveoli in the lungs
  • notochord formation
  • development of the muscle
  • differentiation of osteoblasts, by forming cross-links in the surrounding collagen matrix.
  • morphogenesis and repair of connective tissues of the cardiovascular, respiratory, skeletal, and other organ systems.

Interestingly it has been recently noticed that LOX could be also found in the cellular nucleus: LOX can also induce cross-linking between lysines in histones. LOX has also been reported as a potent chemotactic agent for monocytes and vascular smooth muscle cells.
Finally LOX seems to be relevant as far as tumor pathogenesis and prometastatic process are concerned, with different roles between its intra and extracellular activity.

Comparing LOX, PLOD1 and JMJD6

Attention must be paid on the differences between Lysyl oxidase (LOX) and Lysyl hydroxylase (PLOD1) (chr.1p36): for instance, even though they are both involved in collagen cross-links, the former has lysyl-tyrosyl-quinone and Cu 2+ as cofactors, while the latter needs ascorbic acid and Fe 2+ in order to carry out its enzymatic activity. Bifunctional arginine demethylase and lysyl-hydroxylase JMJD6 (chr.17q25) represents another lysyl-hydroxylase which ought not to be confused with LOX: JMJD6 needs Fe 2+ ions and catalyzes 5-hydroxylation only on lysine residues of specific target nuclear proteins and perhaps mRNA, acting as a regulator of RNA splicing (UniProt).

LOX-like proteins

Four LOX-like (LOXL 1-4) proteins, with varying degrees of similarity to LOX have been described, constituting a family of related proteins [CMLS, 2006]. Recent findings reveal that LOX and LOXL proteins markedly influence cell behavior including chemotactic responses, proliferation, and shifts between the normal and malignant phenotypes, in addition to ECM stabilization, via oxidization of lysine within a variety of cationic proteins.

A highly conserved amino acid sequence at the C-terminus end appears to be sufficient for amine oxidase activity, suggesting that each family member may retain this function. The N-terminus is poorly conserved and may impart additional roles in developmental regulation, senescence, tumor suppression, cell growth control, and chemotaxis to each member of the family [NCBI Genes].

THE GENE

DatabaseLink
WikigenesLOX
GeneCardsLOX
Your Favorite Gene SigmaLOX
NCBI GenesLOX
OMIMLOX

LOX gene is located on chromosome 5 (5q23.2) and appears to be extremely evolutionarily conserved, since the lowest common taxonomy group is Opisthokonta [Brenda]. Its early naissance may explain its involment in so many different roles.

Defects in this gene are a cause of autosomal recessive cutis laxa type I (CL type I). Two transcript variants encoding different isoforms have been found for this gene. [NCBI Genes]

CHEMICAL STRUCTURE AND IMAGES

References for this part:

Bollinger et al.The Formation of lysine tyrosylquinone (LTQ) etc..2005
Mure et al.Lysine tyrosyl quinone cofactor of lysyl oxidase.2003
Ryvkin et al.A peptide model of the copper-binding region of lysyl oxidase2004

X-RAYS LOX STRUCTURE

Secondary and tertiary structure of the human lysyl oxidase (LOX) has not been well characterized yet. However, the structure of the Pichia pastoris (yeast) LOX enzyme, that appears to be a homodimeric enzyme, is known. Although it is structurally different from mammalian LOX, it resulted functionally the same.

CHARACTERISTICS OF LOX AND LOXL PROTEIN PRIMARY STRUCTURE

  • LOX primary structure: 417 aa
  • LOXL-1 primary structure: 574 aa

All LOX family proteins contain:

  • C-terminal domains of LOX (copper-binding domain with a tetragonal Cu2+)
  • CRL domain (cytokine receptor-like domain)
  • LTQ residue

The copper-binding domain (WXWHXCHXHYH; where X is any amino acid) is distinguished by the presence of four histidine residues, which have been postulated to form an octahedral coordination complex with copper ion. The binding of copper may stabilize the LTQ.

The CRL domain (C–X9–C–X–W–X34–C–X13–C; where Xn is a defined number of any amino acids) of the LOX family was also found in the extracellular domains of a number of different receptors for cytokines, prolactin, and growth hormones. The four cysteine residues sparsely located in this domain were suggested to form two disulfide bonds, which were believed to be important for ligand binding in the growth factor and cytokine receptor superfamily. However, the functional significance of this CRL domain in the LOX family, particularly in relation to the tumor suppressor function, has yet to be established.

LTQ cofactor derives from an autocatalytic reaction involving tetragonal Cu 2+ -mediated/assisted oxidation of Tyr355 followed by covalent cross-linking with the ε-amine group of Lys320. These LTQ residues along with the flanking amino-acid sequences are highly conserved in all members of the human LOX family (Lys477 and Tyr512 in human LOXL), indicating the presence of a similar LTQ cofactor. The protein environment must play an essential role in LTQ biogenesis by directing the nucleophilic addition of the ε-amino group of a lysine residue to the C4 position of a putative dopaquinone intermediate.

In the C-terminal region, ten cysteine residues are also exactly conserved at the same positions, suggesting a similar tertiary structure of the C-terminal domains within the LOX family members.

LOX SECONDARY AND TERTIARY PROTEIN STRUCTURE

The overall structure of human lox could be defined as a globular, containing six regions (25%) of β-sheets, 20% of α-helix and 65% random coil. Moreover there is a high content of tryptophan residues on the surface, exposed to solvent. There is also a freely moving random coil fragment at the N-terminal end of the structure. LOX exhibits the tendency to aggregate in aqueous solution, even if the number of the hydrophilic residues is more than 60% greater than the hydrophobic ones. The limited solubility may reflect secondary and tertiary levels of the protein structure, in which hydrophobic regions are exposed.

LOX CATALYTIC MECHANISM

References for this part:

Reiser et al.Enzymatic and nonenzymatic cross-linking of collagen and elastin1992
Jung et al.Purification of enzymatically active human lysyl oxidase etc2003
Lopez et al.Identification of the copper-binding ligands of lysyl oxidase2011
Waggett et al.Microfibrillar elements in the synovial joint.1993
Wu et al.Identification of cross-linking sites etc.1992
Yamauchi et al.Lysine post-translational modifications of collagen2012
Rosenbloom et al.Extracellular matrix 4: the elastic fiber1993
Kielty et al.Microfibrillar assemblies of foetal bovine skin etc..1993
Bailey et al.Mechanisms of maturation and ageing of collagen1998

The strict conservation of the C-terminal domains indicates that the LOX family members share similar enzymatic functions. However, LOX and LOXLs localize with different types of collagen molecules in different tissues. For instance, LOX is colocalized with fibrillar collagens, such as type I and type III, while LOXL2 is associated with collagen type IV, a basement-specific collagen, suggesting that LOX and LOXLs may have distinct substrate specificity toward different types of collagens.

In the catalytic site, three of the five histidines in the putative copper-binding domain H292, H294, H296, are the copper ligands and essential to the formation of LTQ. A fourth, H289, is not involved in LTQ formation or activity, while a fifth, H303, is suggested to be a general base in the catalytic mechanism.
The reaction catalyzed by LOX is an oxidative deamination of lysine and hydroxylysine to form the respective aldehydic forms, Lys ald (α-aminoadipic acid-δ-semialdehyde) and Hyl ald

The image below shows the role of the Cu2+ cofactor in a fictive oxidative deamination, in order to better describe its role in the LOX catalyzed reaction.

Two related cross-linking pathways can be distinguished.

  • The lysine aldehyde pathway occurs primarily in the adult skin, cornea, and sclera.
  • The hydroxylysine aldehyde pathway occurs primarily in the bone, cartilage, ligament, tendons, embryonic skin, and most major internal connective tissues of the body.

The two cofactors, one tightly bound copper ion (Cu2+) and a unique covalently integrated organic cofactor, LTQ (lysyl-tyrosyl quinone), are essential to its catalytic function.

LOX DEPENDENT COLLAGEN CROSSLINKING MECHANISM

Procollagen processing and fibril assembly begin within the confines of the cell membrane. Then procollagen molecules are secreted into the extracellular space, both N- and C-propeptide extensions are cleaved off and collagen molecules then self-assemble into fibrils.
These cleavages, especially that of C-propeptide by BMP1/Tolloid-like proteinases, is necessary for the initiation of fibrillogenesis. During fibrillogenesis, specific lysine and hydroxylysine residues in the N- and C-telopeptides are oxidatively deaminated by LOX to form the respective aldehydic forms, Lys ald (α-aminoadipic acid-δ-semialdehyde) and Hyl ald, which initiates a series of spontaneous condensation reactions that result in various bi-, tri-, and tetrafunctional covalent inter-molecular cross-links.

Lysine- and hydroxylysine-derived aldehydes can react with corresponding aldehydes on adjacent polypeptide chains forming aldol condensation products, or with unmodified lysine and hydroxylysine residues, forming bifunctional cross-links, such as lysinonorleucine and hydroxylysinonorleucine. The aldol condensation products can also react with a histidine residue to form aldol histidine, which may further react with an additional lysine residue to form the tetrafunctional cross-link histidinohydroxymerodesmosine.
In the hydroxylysine pathway, the bifunctional cross-links spontaneously undergo an Amadori rearrangement, resulting in ketoimine cross-links, which then mature further into trifunctional 3-hydroxypyridinium and lysyl pyridinium cross-links.

LOX DEPENDENT ELASTIN CROSSLINKING MECHANISM

The most important feature of elastin, crucial for its proper functioning in elastic fibers, is the high degree of cross-linking of the individual polypeptide chains. Approxymately 30 of the 40 lysine residues per 1000 amino acids present in tropoelastin, the precursor of elastin, are oxidated to aldehydes.

Tropoelastin differs from collagens in that it has no hydroxylysine or histidine residues, and therefore only lysine-derived cross-links are found in mature elastin. In addition, certain condensation products, such as desmosines and isodesmosines, are specific for elastin and are not found in collagens. The cross-links in elastin are otherwise formed in a similar manner as those in collagens. Two allysines can form an aldol condensation product, and one allysine can form lysinonorleucine with an unmodified lysine residue. Aldol condensation of the product with an unmodified lysine residue results in the trifunctional merodesmosine. Further condensation of a dehydrated aldol condensation product with dehydrolysinonorleucine, or reaction of merodesmosine with allysine results in the tetrafunctional desmosine and isodesmosine cross-links.

SYNTHESIS AND TURNOVER

References for this part:

Cronshaw et al.The proteolytic processing site of the precursor of lysyl oxidase1995

The human LOX precursor is synthesized as a 48 kDa pre-pro-protein, and after extensive intracellular and extracellular processing, the N-glycosylated proenzyme is proteolytically cleaved in its 32 kDa form, that is present in the extracellular matrix. During the post-translational processing, a unique Cu 2+ ion is added and LTQ cofactors is eventually generated.
The enzymatically active 32 kDa protein, results from extracellular proteolytic cleavage between residues Gly168 and Asp169 by procollagen C-protease, also known as bone morphogenic protein -1.

The human LOXL protein is cleaved by BMP-1 too, at two different locations, Gly134–Asp135 and Ser337–Asp338, resulting in two processed forms of LOXL. Both processed forms of LOXL contained the amine oxidase activity against elastin.

CELLULAR FUNCTIONS

DatabaseLink
BRENDA - The Comprehensive Enzyme Information SystemProtein-lysine 6-oxidase
KEGG EnzymesProtein-lysine 6-oxidase
Human Metabolome DatabaseProtein-lysine 6-oxidase

Almost all the functions, as far as it is currently acknowledged, in which LOX seems involved have already been rapidly mentioned. Of course, a complete dissertation of all these aspects would go beyond the purposes of this work: we will concentrate our attention on the role of LOX alterations in the most common corneal dystrophy, affecting at least one person out of 2000 and probably under-diagnosed: the keratoconus.

Before focusing on keratoconus, it is perhaps worth spending a few words on the “non-classical” function of LOX inside and outside the cell: its extracellular activity on the ECM (collagen and elastin) in various tissues has already been debated.

HISTONE REGULATION

Recent studies [Oleggini, 2011] have proved new evidence for histone-H1-dependent mechanism for the modulation of MMTV (mouse mammary tumor virus) promoter by LOX, in a manner which strongly modifies cellular activity [Kagan, 2003] .

ROLE IN CHEMOTACTIC SENSITIVITY

Novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR[Lucero, 2011]

ROLE IN PROMETASTATIC PROCESSES

The role of LOX in the tumour shifting to malignant and invasive phenotype is not fully understood yet. LOX has been identified [Taylor, 2011] as a potential mediator that couples mechanotransduction to oncogenic signaling by TGF-β1: inactivating LOX activity impaired TGF-β1-mediated epithelial-mesenchymal transition and invasion in breast cancer cells. This suggests that measures capable of inactivating LOX function may prove effective in diminishing breast cancer progression stimulated by TGF-β1.

LOX has also been studied as tumor suppressor gene of the RAS signalling pathway: Lysyl oxidase inhibits RAS-mediated transformation by preventing activation of NF-kappa B [Jeay, 2003] .

LOX PLAYS AN IMPORTANT ROLE IN KERATOCONUS PATHOGENESIS

References for this part:

BOOKOcular Disease, Mechanisms and Management2009
Nielsen et al.Identification of differentially expressed genes in keratoconus epithelium analyzed on microarrays2003
Bykhovskaya et al.Variation in the lysyl oxidase (LOX) gene is associated with keratoconus in family-based and case-control studies2012
Burdon et al.Insights into keratoconus from a genetic perspective2013
AnirudhThe role of tear acidity and Cu-cofactor of lysyl oxidase activity in the pathogenesis of keratoconus2012
Sethi et al.Focus on Molecules: Lysyl oxidase2012
Hewitt et al.Ancestral LOXL1 variants are associated with pseudoexfoliation in Caucasian Australians but with markedly lower penetrance than in Nordic people..2008

LOX, and LOXL1 activity, seems to be relevant in the keratoconus pathogenesis as well as in other corneal dystrophies and connective tissue pathologies (LOXL1 variants, localized in cornea, lens and ciliary body, are also associated with pseudoexfoliation syndrome), and this association may be explained by the LOX and LOXL1 mediated collagen and elastin crosslinking mechanism. Finally further LOX activities as far as gene regulation can not be excluded.

DEFINITION OF KERATOCONUS

Keratoconus is a slowly progressive, noninflammatory condition in which there is central thinning of the cornea, changing it from dome-shaped to cone-shaped. Keratoconus comes from the Greek words kerato, meaning cornea, and conus, meaning cone. Keratoconus causes the cornea to become thinner centrally or inferiorly with resultant gradual bulging outward. Patients with keratoconus initially notice blurring and distortion of vision. They may also complain about photophobia, glare, disturbed night vision, and headaches from eyestrain. As keratoconus progresses, patients are increasingly myopic and astigmatism can become more irregular. Keratoconus is a bilateral condition, though usually asymmetric in severity and progression.

In the early stages of the disease, keratoconus is not visible to the naked eye. However, in the later stages of progression, the cone-shaped cornea can be visible to an observer when the patient looks down while the upper lid is raised. The pointed cornea will push the lower lid out in the area of the cone like a V-shaped dent in the lower lid. This anterior protrusion seen in the lower lid is called Munson’s sign.

KERATOCONUS EPIDEMIOLOGY

Keratoconus is the commonest corneal dystrophy in the USA, affecting approximately 1 in 2000 people. Although keratoconus occurs sporadically in most individuals, approximately 6–10% have a hereditary component since it is reported in multiple generations of families and identical twins. If a first-degree relative has keratoconus then the prevalence of other family members developing keratoconus is approximately 3.34%, which is significantly higher than the general population. It affects people of all races and both sexes, though there is a slight female preponderance.
Keratoconus can be found in 0.5–15% of trisomy 21 (Down syndrome) patients and is less frequently associated with Ehlers–Danlos syndrome and osteogenesis imperfecta. However, the vast majority of keratoconus patients do not have other ocular or systemic diseases.

KERATOCONUS ETIOLOGY AND PATHOGENESIS

A hallmark histological feature of the keratoconus cornea is focal regions where the Bowman’s layer is absent and the epithelial cells are in direct contact with the underlying stroma. These sites also show decreased levels of fibronectin, laminin, entactin, type IV collagen, and type XII collage. In areas of active disease, the stromal extracellular matrix (ECM) demonstrates elevated levels of type III collagen, tenascin-C, fibrillin-1, and keratocan, but many of these changes are nonspecific and can also be found in general wound-healing processes. Most interestingly,the ECM abnormalities in keratoconus corneas are not uniform. The corneal stroma can lose more than half its normal thickness and have deposits of fibrotic ECM while in an adjacent, thicker region the matrix patterns are normal. In addition, there is variability in the epithelial thickness, with some areas having only 1–3 cell layers and other regions appearing completely normal.

It is generally accepted that keratoconus stromal thinning is associated with increased activities in ECM-degrading enzymes. In the early 1960s it was noted that keratoconus corneas had degraded epithelial basement membranes and increased gelatinase activities. It was subsequently demonstrated that keratoconus corneas have increased levels of lysosomal enzymes (acid esterase, acid phosphatase, acidic lipase), cathepsins G, B, and VL2, matrix metalloproteinase-2 (MMP-2) and MT1-MMP (MMP-14) which can degrade many forms of ECM. Moreover, many of the naturally occurring inhibitors for those enzyme families are found in lower levels. In addition to corneal involvement, the conjunctiva of keratoconus patients shows increased lysosomal enzyme activities.

Keratoconus usually has its onset during puberty, with a gradual and irregular progression over approximately 20 years. The rate of progression and severity of the condition are quite variable, ranging from mild astigmatism to severe corneal thinning, protrusion, and scarring. In advanced keratoconus, there may be a rupture in Descemet’s membrane, causing sudden clouding of vision due to acute stromal or epithelial edema, called acute corneal hydrops. Topical corticosteroid and 5% NaCl drops are usually used to treat the acute hydrops episode. This condition often resolves over weeks to months and may result in central corneal scarring or flattening.

The keratoconus corneas have elevated levels of leukocyte common antigen-related protein (*LAR*), a transmembrane phosphotyrosine phosphatase that stimulates apoptosis, and cathepsins G, B, and V/L2, which represent a caspaseindependent pathway for apoptosis. Cathepsins mediate apoptosis by triggering mitochondrial dysfunction, cleaving Bid and releasing cytochrome c. Furthermore, keratoconus corneas have decreased levels of tissue inhibitors of metalloproteases, TIMP-1 and TIMP-3, which can modulate apoptosis. Finally, the moderate to severe atopy and vigorous eye rubbing often found in keratoconus patients may contribute to apoptosis since studies showed that chronic, repetitive injury to the corneal epithelium stimulates anterior stromal cell apoptosis.

The cornea possesses numerous antioxidant enzymes such as superoxide dismutases (SODs), catalase, aldehyde dehydrogenases (ALDH3A1), glutathione reductase, glutathione S-transferase, and glutathione peroxidase in order to eliminate the reactive oxygen/nitrogen species (ROS/RNS) and aldehydes that aregenerated by ultraviolet light. When ROS/RNS are not eliminated, they can react with other molecules and form cytotoxic aldehydes and peroxynitrites. These antioxidant enzyme activities change with aging61 and in response to cytokines and growth factors and this can increase the susceptibility to oxidative damage. Many of the antioxidant enzymes of the lipid peroxidation and nitric oxide pathways are abnormal, suggesting their involvement in keratoconus pathology.

A defect in the SOD1 gene on chromosome 21 has also been linked to keratoconus. On the contrary it is controversial as to whether the visual homeobox gene VSX1 is associated with keratoconus. Novel mutations of VSX1 were reported in a patient with both keratoconus and posterior polymorphism dystrophy and in a series of individual keratoconus patients. However, another study reported a single nondisease-causing polymorphism of Asp144Glu and concluded that the VSX1 gene lacked association with keratoconus. The expression of VSX1 occurs during wound healing as myofibroblasts differentiate and may play a role in abnormal stromal repair processes.

LOX ROLE IN KERATOCONUS PATHOGENESIS

Lysyl oxidase dramatically affects the solubility and proteolytic resistance of its ECM protein substrates, and also the rigidity and tensile strength of the ECM, by intermolecular crosslinking. Consequently, experiments involving inhibition, silencing, downregulation, or knockout of lysyl oxidase may produce results due to changes in the nature, quantity, and/or physical properties of fibrous proteins in the ECM.
A genomewide linkage scan in keratoconus families identified a locus at 5q23.2, overlapping the gene coding for the lysyl oxidase (LOX). Then the association results were analyzed for single-nucleotide polymorphisms ( SNPs ) in the LOX gene from a Genome-Wide Association Study (GWAS) investigation in two independent panels of patients with keratoconus and controls and in keratoconus families. Results provided strong genetic evidence that LOX variants lead to increased susceptibility to developing of keratoconus. Furthermore the biochemical conditions of the middle periphery of the cornea is found to inhibit copper ions movement to the center in patients with keratoconus due to increased tear alkalinity. Low concentration of dichlorocuprate (I) ion in the center of cornea results in inactivation of lysyl oxidase, an enzyme that catalyzes collagen cross-linking, and thus promotes keratoconus.

REGULATION

References for this part:

Li et al.Localization and activity of lysyl oxidase within nuclei of fibrogenic cells1997
Saad et al.Intracellular lysyl oxidase: effect of a specific inhibitor on nuclear mass in proliferating cells2010
Rodriguez et al.Lysyl oxidase (LOX) down-regulation by TNFalpha2008
Schietke et al.The lysyl oxidases LOX and LOXL2 are necessary and sufficient to repress E-cadherin in hypoxia2009
Oleggini et al.Regulation of elastin promoter by lysyl oxidase and growth factors2007
Giampuzzi et al.beta-catenin signaling and regulation of cyclin D1 promoter in NRK-49F cells transformed by down-regulation of the tumor suppressor lysyl oxidase2005
Li et al.Up-regulation and altered distribution of lysyl oxidase in the central nervous system of mutant SOD1 transgenic mouse2004
Sethi et al.Transforming growth factor-beta induces extracellular matrix protein cross-linking lysyl oxidase (LOX) genes2011
Lee et al.Transforming growth factor-beta regulation of bone morphogenetic protein-11997
Ge et al.BMP1 controls TGFbeta1 activation via cleavage of latent TGFbeta-binding protein2006
Atsawasuwan et al.Lysyl oxidase binds transforming growth factor-beta and regulates its signaling via amine oxidase activity2008

HYPOXIA ACTIVATES LOX EXPRESSION VIA HIF-1

Gene expression of lysyl oxidase (LOX) and the related LOX-like 2 (LOXL2) is strongly induced by hypoxia being a direct transcriptional target of HIF-1. Furthermore the activation of lysyl oxidases is required and sufficient for hypoxic repression of E-cadherin, which mediates cellular transformation and takes effect in cellular invasion assays. Hypoxia, which is demonstrated to repress E-cadherin expression, could induce epithelial to mesenchymal transition, which is believed to amplify tumor aggressiveness. The molecular mechanism of E-cadherin repression is unknown, yet lysyl oxidases have been implicated to be involved.
Thus, there is a molecular pathway from hypoxia to cellular transformation that includes up-regulation of HIF and subsequent transcriptional induction of LOX and LOXL2, which repress E-cadherin and induce epithelial to mesenchymal transition. Lysyl oxidases could be an attractive molecular target for cancers of epithelial origin, in particular because they are partly extracellular.

TNFINHIBITS LOX EXPRESSION

TNFalpha decreases LOX mRNA levels in endothelial cells in a dose- and time-dependent manner. The effect of TNFalpha was observed at low concentrations (0.1-1 ng/mL) and was maximal at 2.5 ng/mL (after 21 h). In transfection assays, TNFalpha reduced LOX transcriptional activity to a similar extent than LOX mRNA. Furthermore, TNFalpha decreases endothelial LOX enzymatic activity. By using both TNF receptor (TNFR) agonist and blocking antibodies we determined the involvement of TNFR2 on LOX down-regulation. Moreover, while TRAF-2 (TNFR-associated factor-2) did not mediate signalling events leading to LOX inhibition, PKC inhibitors counteracted the TNFalpha-induced decrease of LOX mRNA levels. Finally, TNFalpha administration significantly reduced vascular LOX expression in rat aorta.

TGFINDUCES LOX AND ENHANCES ITS ENZYMATIC ACTIVATION VIA BMP1

Lysyl oxidase contributes to mechanotransduction-mediated regulation of transforming growth factor-β signaling in breast cancer cells. Transforming growth factor-beta (TGF-β) induces extracellular matrix protein cross-linking lysyl oxidase (LOX) genes. Both Smad and non-Smad signaling pathways are involved in TGFβ-mediated LOX induction. Inactivating LOX activity impaired TGF-β1-mediated epithelial-mesenchymal transition and invasion in breast cancer cells.
Moreover TGF-β1 actives BMP1 which has a procollagen C-proteinase activity. BMP1, on its part, processes procollagen types I-III and mediate processing of lysyl oxidase activation and laminin 5 too. Finally it has been noticed that BMP-1 promotes TGF-beta activity cleaving LTBP at two positions, in a process required for efficient MMP-2-mediated liberation of active TGF-beta from LAP.

EXTRACELLULAR LOX BINDS AND INHIBITS TGFACTIVITY

LOX binds to mature TGF-beta1 and enzymatically regulates its signaling in bone, suppressing TGF-beta1-induced Smad3 phosphorylation. This interaction may provides a negative feedback loop on the TGF-β1 signalling.

TGFβ AND LOX NUCLEAR ACTIVITY INDUCE BOTH COLLAGEN AND ELASTIN

Beside its enzymatic activity, LOX is able to regulate the promoter of collagen III, one of its natural substrates. LOX regulates the promoter of elastin too, inducing an important activation of its activity. Treatment with TGF-beta1 abolished completely the activation induced by LOX. Thus, there is an important cross-regulation between LOX and TGF-β1 (and the other tested growth factors). LOX and TGF alone were able to enhance most of the elastin promoter regions that we analyzed.
Interestingly, these effects were mutually exclusive, since the contemporaneous presence of LOX and TGF (or FGF) abolished or strongly impaired the effects of the single factors. Two restricted regions binding AP1 and SMAD were identified as mediators of LOX effects and of LOX and TGF-beta1 cross-inhibition.

LOX NUCLEAR ACTIVITY INHIBITS RAS MEMBRANE LOCALISATION AND PI3K AND AKT mRNA SYNTHESIS

Evidence for the presence of intracellular LOX in neurons has been found, at least in certain pathologies as the amyotrophic lateral sclerosis. Moreover this enzyme has been found active within rat vascular smooth muscle cell nuclei and within fibroblasts nuclei as well.
LOX expression opposes the effect of mutationally activated Ras, which is present in about 30% of human cancers, exhibiting a tumor suppressive effect. Therefore nuclear lysyl oxidase regulates nuclear growth, and modulates cell proliferation. Expression of LOX in Ras-transformed fibroblasts resulted in reduced colony formation and deactivation of NF-κB. Downregulation of LOX induces higher levels of active Ras, increased tumorigenicity, upregulated cyclin D1 and the Wnt pathway effector β-catenin. LOX potently downregulates the PI3K and Akt kinases, while partially inhibiting MEK kinase activity. Importantly, LOX blocks membrane localization of Akt and PDK1 in Ras-transformed cells.

LOX EXTRACELLULAR ACTIVITY ACTIVES FAK/SRC AND PI3K/AKT PATHWAYS

Paradoxically LOX actives, the PI3K-Akt signaling pathway, thereby up regulating HIF-1α protein synthesis in a manner requiring LOX-mediated hydrogen peroxide production. Finally secreted LOX seems to be responsible for the invasive properties of hypoxic cancer cells through FAK/Src activity and cell-to-matrix adhesion.

LOX NUCLEAR ACTIVITY REGULATES HISTONES H1 AND H2

Homologies between lysine-rich regions of tropoelastin and the "lysine-rich" histone H1, allows H1interactions with lysyl oxidase. LOX actually interacts specifically not only with histone H1, but also with histone H2. Histone H1 has been implicated in chromosomal relaxation and nucleosome reorganization during the genomic transcription, which makes it a possible candidate to mediate LOX control of phenotype.
The phosphorylation of H1 is related to the initiation of transcription. Indeed, the introduction of a negative charge acts as a dissociating force that detaches H1 from the DNA, allowing its opening and accessibility to enzymes and transcription factors. We suggest that LOX might have a role in regulation by either binding to H1 at lysine sites, hindering the phosphorylation of a nearby serine, or by deaminating specific lysines diminishing the overall positive charge. These two processes could have opposite regulatory effects; the former reducing phosphorylation of H1, the latter simulating it. In fact, it might produce a fine regulation of the histones and nucleosome position on the condensed DNA, allowing only some localized opening of the structure. Intriguingly, it has been shown that histone H1 phosphorylation is often increased in oncogene-induced transformations.
Furthermore, histone H2 could rescue LOX to the nucleosome and orientate it towards its putative substrate H1. In fact, H1 and H2 interact through different, sequences of LOX with the carboxy-catalytic region and the amino terminal region, respectively. Thus, H2 bind LOX leaving available the catalytic region for the interaction with H1.

DIAGNOSTIC USE

References for this part:

Sakai et al.Expression of lysyl oxidase is correlated with lymph node metastasis and poor prognosis in esophageal squamous cell carcinoma.2009

LOX has been also proposed as a predictive marker of lymph node metastasis and prognosis in esophageal squamous cell carcinoma. Overall survival rates of the patients with esophageal squamous cell carcinoma with high LOX expression are significantly lower than those of the patients with esophageal squamous cell carcinoma with low LOX expression.

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