LOX-like Proteins Research Articles

β-Aminopropionitrile Induces Distinct Pathologies in the Ascending and Descending Thoracic Aortic Regions of Mice.

β-aminopropionitrile (BAPN) is a pharmacological inhibitor of LOX (lysyl oxidase) and LOXLs (LOX-like proteins). Administration of BAPN promotes aortopathies, although there is a paucity of data on experimental conditions to generate pathology. The objective of this study was to define experimental parameters and determine whether equivalent or variable aortopathies were generated throughout the aortic tree during BAPN administration in mice. BAPN was administered in drinking water for a period ranging from 1 to 12 weeks. The impacts of BAPN were first assessed with regard to BAPN dose, and mouse strain, age, and sex. BAPN-induced aortic pathological characterization was conducted using histology and immunostaining. To investigate the mechanistic basis of regional heterogeneity, the ascending and descending thoracic aortas were harvested after 1 week of BAPN administration before the appearance of overt pathology. BAPN-induced aortic rupture predominantly occurred or originated in the descending thoracic aorta in young C57BL/6J or N mice. No apparent differences were found between male and female mice. For mice surviving 12 weeks of BAPN administration, profound dilatation was consistently observed in the ascending region, while there were more heterogeneous changes in the descending thoracic region. Pathological features were distinct between the ascending and descending thoracic regions. Aortic pathology in the ascending region was characterized by luminal dilatation and elastic fiber disruption throughout the media. The descending thoracic region frequently had dissections with false lumen formation, collagen deposition, and remodeling of the wall surrounding the false lumen. Cells surrounding the false lumen were predominantly positive for α-SMA (α-smooth muscle actin). One week of BAPN administration compromised contractile properties in both regions equivalently, and RNA sequencing did not show obvious differences between the 2 aortic regions in smooth muscle cell markers, cell proliferation markers, and extracellular components. BAPN-induced pathologies show distinct, heterogeneous features within and between ascending and descending aortic regions in mice.

Lysyl Oxidase Family Proteins: Prospective Therapeutic Targets in Cancer.

The lysyl oxidase (LOX) family, consisting of LOX and LOX-like proteins 1–4 (LOXL1–4), is responsible for the covalent crosslinking of collagen and elastin, thus maintaining the stability of the extracellular matrix (ECM) and functioning in maintaining connective tissue function, embryonic development, and wound healing. Recent studies have found the aberrant expression or activity of the LOX family occurs in various types of cancer. It has been proved that the LOX family mainly performs tumor microenvironment (TME) remodeling function and is extensively involved in tumor invasion and metastasis, immunomodulation, proliferation, apoptosis, etc. With relevant translational research in progress, the LOX family is expected to be an effective target for tumor therapy. Here, we review the research progress of the LOX family in tumor progression and therapy to provide novel insights for future exploration of relevant tumor mechanism and new therapeutic targets.

SUN-169 A COPPER CHELATING DRUG INHIBITS RENAL FIBROSIS BY INHIBITING LYSYL OXIDASE AND LOSS OF PERITUBULAR CAPILLARIES

Chronic kidney disease (CKD) is one of the important global health burdens which involves interstitial fibrosis as an underlying pathological process associated with compromised renal function irrespective of etiological cause of the injury. Lysyl oxidase (LOX) is a copper-dependent enzyme that mediates cross linking of extracellular matrix (ECM) proteins including collagen and elastin. The proper LOX activity is essential for a physiologically functional ECM, while the aberrant expression of LOX and related proteins has been found in conditions with excessively deposited ECM such as organ fibrosis. We hypothesized that a copper chelator, disulfiram (DSF) which is used for alcohol deaddiction, could be repurposed to reduce the activation of LOX proteins and subsequent reduction in collagen deposition in fibrotic conditions. We used Male Swiss albino mice for unilateral ureteral obstruction (UUO)-induced animal model of renal fibrosis and investigated the effects of DSF (10 and 30 mg/kg) on LOX and LOX-like (LOXL) proteins as a strategy to treat/prevent renal fibrosis. We used a pan-LOX inhibitor, β-aminopropionitrile (BAPN, 100 mg/kg) as a standard LOX inhibitor for comparison. We performed histopathology, hydroxyproline assay, western blot, ELISA and immunofluorescence studies to assess the effect of DSF on renal injury and fibrosis. The levels of different isoforms of LOX were found to be higher in the fibrotic kidney as compared to sham/contralateral kidney control. The treatment of animals with DSF led to reduced expression of LOX and related proteins. Interestingly, the reduction in expression of LOX proteins with DSF was found to be better than BAPN. Further, the DSF reduced UUO-induced expression of fibrotic proteins (collagen I, fibronectin, TGF-β). Furthermore, DSF reduced the phenotypic conversion of epithelial cells to mesenchymal type by reducing the epithelial-mesenchymal transition (EMT) program as evident by reduced expression of mesenchymal markers such as α-SMA and vimentin along with improved expression of epithelial marker, E-cadherin. DSF prevented renal injury, reduced tubulointerstitial fibrosis and collagen deposition in fibrotic kidney. In addition to that, DSF also prevented peritubular capillary (PTC) loss in the obstructed kidneys of animals which is a hallmark of clinical and preclinical conditions of renal fibrosis. DSF, an FDA approved drug, can be repurposed for fibrotic disorders due to its modulating effects on the expression of LOX and LOXL proteins. The DSF holds a great potential to prevent organ fibrosis and it could be used as a successful strategy for amelioration of different fibrotic conditions, if translated further.

Activity and Trafficking of Copper-Transporting ATPases in Tumor Development and Defense against Platinum-Based Drugs.

Membrane trafficking pathways emanating from the Golgi regulate a wide range of cellular processes. One of these is the maintenance of copper (Cu) homeostasis operated by the Golgi-localized Cu-transporting ATPases ATP7A and ATP7B. At the Golgi, these proteins supply Cu to newly synthesized enzymes which use this metal as a cofactor to catalyze a number of vitally important biochemical reactions. However, in response to elevated Cu, the Golgi exports ATP7A/B to post-Golgi sites where they promote sequestration and efflux of excess Cu to limit its potential toxicity. Growing tumors actively consume Cu and employ ATP7A/B to regulate the availability of this metal for oncogenic enzymes such as LOX and LOX-like proteins, which confer higher invasiveness to malignant cells. Furthermore, ATP7A/B activity and trafficking allow tumor cells to detoxify platinum (Pt)-based drugs (like cisplatin), which are used for the chemotherapy of different solid tumors. Despite these noted activities of ATP7A/B that favor oncogenic processes, the mechanisms that regulate the expression and trafficking of Cu ATPases in malignant cells are far from being completely understood. This review summarizes current data on the role of ATP7A/B in the regulation of Cu and Pt metabolism in malignant cells and outlines questions and challenges that should be addressed to understand how ATP7A and ATP7B trafficking mechanisms might be targeted to counteract tumor development.

Role of the lysyl oxidase enzyme family in cardiac function and disease

Heart diseases are a major cause of morbidity and mortality world-wide. Lysyl oxidase (LOX) and related LOX-like (LOXL) isoforms play a vital role in remodelling the extracellular matrix (ECM). The LOX family controls ECM formation by cross-linking collagen and elastin chains. LOX/LOXL proteins are copper-dependent amine oxidases that catalyse the oxidation of lysine, causing cross-linking between the lysine moieties of lysine-rich proteins. Dynamic changes in LOX and LOXL protein-expression occur in a variety of cardiac pathologies; these changes are believed to be central to the associated tissue-fibrosis. An awareness of the potential pathophysiological importance of LOX has led to the evaluation of interventions that target LOX/LOXL proteins for heart-disease therapy. The purposes of this review article are: (i) to summarize the basic biochemistry and enzyme function of LOX and LOXL proteins; (ii) to consider their tissue and species distribution; and (iii) to review the results of experimental studies of the roles of LOX and LOXL proteins in heart disease, addressing involvement in the mechanisms, pathophysiology and therapeutic responses based on observations in patient samples and relevant animal models. Therapeutic targeting of LOX family enzymes has shown promising results in animal models, but small-molecule approaches have been limited by non-specificity and off-target effects. Biological approaches show potential promise but are in their infancy. While there is strong evidence for LOX-family protein participation in heart failure, myocardial infarction, cardiac hypertrophy, dilated cardiomyopathy, atrial fibrillation and hypertension, as well as potential interest as therapeutic targets, the precise involvement of LOX-family proteins in heart disease requires further investigation.

Roles of Copper-Binding Proteins in Breast Cancer.

Copper ions are needed in several steps of cancer progression. However, the underlying mechanisms, and involved copper-binding proteins, are mainly elusive. Since most copper ions in the body (in and outside cells) are protein-bound, it is important to investigate what copper-binding proteins participate and, for these, how they are loaded with copper by copper transport proteins. Mechanistic information for how some copper-binding proteins, such as extracellular lysyl oxidase (LOX), play roles in cancer have been elucidated but there is still much to learn from a biophysical molecular viewpoint. Here we provide a summary of copper-binding proteins and discuss ones reported to have roles in cancer. We specifically focus on how copper-binding proteins such as mediator of cell motility 1 (MEMO1), LOX, LOX-like proteins, and secreted protein acidic and rich in cysteine (SPARC) modulate breast cancer from molecular and clinical aspects. Because of the importance of copper for invasion/migration processes, which are key components of cancer metastasis, further insights into the actions of copper-binding proteins may provide new targets to combat cancer.

Increased Lysyl oxidase-like 2 (LOXL2) gene expression accurately designates advanced liver fibrosis induced by the Hepatitis C Virus (HCV) regardless race, blood specimen or co-infection with the Human Immunodeficiency Virus (HIV)

Background: The LOX gene encodes a member of the lysyl-oxidase (LOX) gene family, which is involved in biogenesis of connective tissue, and 4 LOX-like proteins (LOXL1 – 4) have been identified so far. This study was aimed to investigate gene expression of LOXL1 – 4 in association with fibrogenesis in Hepatitis C Virus (HCV) infected patients with and without co-infection with the Human Immundeficiency Virus (HIV) in peripheral blood mononuclear cells (PBMC), peripheral whole blood (PwB) and liver tissue (LT) samples. Materials and Methods: PwB samples from 34 therapy-naive patients (34/34 Caucasians, 18/34 males, 28/34 with HCV-genotype-1) with chronic HCV infection treated at the University Hospital Essen (Germany) and PBMC samples from 23 HIV/HCV co-infected subjects (11/23 African Americans and none Caucasian, 20/23 males, 23/23 with HCV-genotype-1) recruited at the NIAID/NIH (MD/U.S.A.) were enrolled in this study. Liver biopsies obtained at baseline were evaluated using the Batts-Ludwig staging (BL-S) system for HCV mono-infected patients and the Ishak score (IS) for HIV/HCV co-infected donors. 34 PwB and 23 PBMC samples (and 7 LT samples from HIV/HCV co-infected participants) were subjected to microarray analysis. For all statistical analyses (i.e. pair-wise group comparison) 1-way-ANOVA was performed. Overall, a P< 0.05 was considered significant. Results: PwB samples from 34 HCV mono-infected patients were grouped according to BL-S as early fibrosis (BL-S: 0 – 1, “EF”, N= 20,) and late fibrosis (BL-S: 2 – 4, “LF”, N= 14) and PBMC samples from 23 HIV/HCV co-infected patients were grouped based on IS as early fibrosis (IS: 0 – 2, “EF”, N= 20) and late fibrosis (IS: 3 – 6, “LF”, N= 14). We consistently detected significantly increased LOXL-2 gene expression levels in association with “LF” in HCV mono-infected patients (PwB, P< 0.041) and HIV/HCV co-infected subjects (PBMC, P< 0.038). In addition, linear regression analysis was performed between fibrosis grades (IS: 0 – 6) and LOXL1 – 4 gene expression levels measured in LT samples of 7 HIV/HCV co-infected subjects; here, a significant correlation was found only for LOXL1 (r= 0.8, P> 0.031). These results are currently being validated by quantitative real-time polymerase chain reaction (qRT-PCR). Conclusions: This study suggests significant involvement of LOX-like proteins, especially LOXL2, in the fibrogenic liver transformation process during chronic HCV infection. Previous research reported: 1. Association between increased mRNA levels of LOXL2 and type-III-procollagen in liver fibrosis in mice. 2. Abnormal deposition of collagen in hepatocytes obtained from humans with Wilson's disease. 3. LOXL2 involvement in fibroblast activation. These findings (1.-3.) along with the present data demonstrating consistent significance in two demographically very heterogeneous cohorts further highlight the pathogenic role suggested for LOXL in fibrogenesis and the importance of generating LOXL2-targeted therapies for the prevention/treatment of liver fibrosis.

Molecular Characterization of a Lipoxygenase from the Basidiomycete MushroomPleurotus ostreatus

The full-length cDNA of the gene PoLOX1 encoding a lipoxygenase (LOX) and its corresponding genomic DNA were isolated from the basidiomycete mushroom Pleurotus ostreatus strain H1. The deduced amino acid sequence of PoLOX1 showed similarity to a valencene dioxygenase of Pleurotus sapidus, putative LOX-like proteins from ascomycete, basidiomycete, and deuteromycete fungi, and known LOXs from plants, animals, and bacteria. PoLOX1 also contained the LOX iron-binding catalytic domain in the C-terminal region, but not the polycystin-1, lipoxygenase, alpha-toxin (PLAT) domain, which is usually found in the N-terminal region of eukaryotic LOXs. Genomic sequence analysis revealed that PoLOX1 was interrupted by one intron, and that the promoter region included TATA and CAAT boxes. Southern blot analysis indicated that PoLOX1 is a member of a small gene family comprising highly similar genes. Northern blot analysis revealed that it is transcribed more abundantly in the stipes of the fruit bodies than in the caps.

Cancer–stromal cell interactions mediated by hypoxia-inducible factors promote angiogenesis, lymphangiogenesis, and metastasis

Interactions between cancer cells and stromal cells, including blood vessel endothelial cells (BECs), lymphatic vessel endothelial cells (LECs), bone marrow-derived angiogenic cells (BMDACs) and other bone marrow-derived cells (BMDCs) play important roles in cancer progression. Intratumoral hypoxia, which affects both cancer and stromal cells, is associated with a significantly increased risk of metastasis and mortality in many human cancers. Recent studies have begun to delineate the molecular mechanisms underlying the effect of intratumoral hypoxia on cancer progression. Reduced O2 availability induces the activity of hypoxia-inducible factors (HIFs), which activate the transcription of target genes encoding proteins that play important roles in many critical aspects of cancer biology. Included among these are secreted factors, including angiopoietin 2, angiopoietin-like 4, placental growth factor, platelet-derived growth factor B, stem cell factor (kit ligand), stromal-derived factor 1, and vascular endothelial growth factor. These factors are produced by hypoxic cancer cells and directly mediate functional interactions with BECs, LECs, BMDACs and other BMDCs that promote angiogenesis, lymphangiogenesis, and metastasis. In addition, lysyl oxidase (LOX) and LOX-like proteins, which are secreted by hypoxic breast cancer cells, remodel extracellular matrix in the lungs, which leads to BMDC recruitment and metastatic niche formation.

Lysyl Oxidase and the Lysyl Oxidase–like Protein Modulate Odontoblastic Differentiation of Human Dental Pulp Cells

IntroductionThe lysyl oxidase (LOX) family is an emerging family of amine oxidases responsible for the formation of collagen fibrils in the extracellular matrix. To date, 5 LOX family genes have been identified in humans, encoding LOX and LOX-like proteins (LOXL, LOXL2, LOXL3, and LOXL4). The goal of this study was to evaluate the expression and function of the LOX family genes in odontoblastic differentiation of human dental pulp (HDP) cells. MethodsExpression of the LOX family genes was assessed by reverse transcriptase polymerase chain reaction analysis, and the amine oxidase activity of HDP cells was evaluated by peroxidase-coupled fluorometric assays. Mineral nodule formation and expression of odontoblastic marker genes were assessed in the presence and absence of specific small interfering RNAs (siRNAs) of the LOX family genes. ResultsAmong the LOX family genes, only LOX and LOXL showed prominent expression during odontoblastic differentiation of HDP cells. Suppression of LOX and LOXL expression by siRNA-induced interference substantially decreased the amine oxidase activity of the differentiating HDP cells. Furthermore, interference of LOX and LOXL expression inhibited mineral nodule formation and expression of odontoblastic marker genes during odontoblastic differentiation of HDP cells. ConclusionsThese findings show for the first time that the LOX- and LOXL-mediated organization of collagen fibrils in extracellular matrices of HDP cells might be an important regulator for odontoblastic differentiation of HDP cells.

Lysyl Oxidase: From Basic Science to Future Cancer Treatment

In this mini-review, we discuss the physiological and pathological roles of lysyl oxidase (LOX) and its family, LOX-like proteins (LOXL), in relation to prognosis of major cancers. The number of reports on LOX family is numerous. We have decided to review the articles that were recently published (i.e. past 5 years). Experimental techniques in molecular biology have advanced surprisingly in the past decade. Accordingly, the results of the studies are more reliable. Most studies reached the same conclusion; a higher LOX- or LOXL- expression is associated with a poor prognosis. Molecular experiments have already started aiming for clinical application, and the results are encouraging. Suppressing LOX or LOXL activities resulted in lower cell motility in collagen gel and, moreover, succeeded in reducing metastases in mice. LOX family members were originally recognized as molecules that cross-link collagen fibers in the extracellular matrix. Recent studies demonstrated that they are also involved in a phenomenon called Epithelial Mesenchymal Transition (EMT). This may affect cell movement and cancer cell invasiveness. LOX and LOXL2 are regulated by hypoxia, a major factor in the failure of cancer treatment. Here we discuss the molecular biology of the LOX family in relation to its role in tumor biology.

MS3-2: HIF-1, Metabolism, and Breast Cancer Metastasis.

Abstract Increased glucose uptake and metabolism is a universal characteristic of advanced solid cancers. There are two well-established mechanisms underlying the reprogramming of tumor metabolism. First, intratumoral hypoxia induces the activity of the transcriptional activator hypoxia-inducible factor 1 (HIF-1) by inhibiting the O2-dependent prolyl and asparaginyl hydroxylases that inhibit HIF-1α stability and transactivation, respectively. Second, genetic alterations increase the activity of HIF-1, thereby increasing the expression of glucose transporters (GLUT1, GLUT3), glycolytic enzymes (ALDOA, ENO1, HK2, LDHA, PKM2), pH regulators (CAR9, NHE1, MCT4), and proteins that inhibit mitochondrial metabolism (BNIP3, PDK1). Metabolites, such as the glycolytic end-product lactate, also induce HIF-1 activity, thereby providing a signal to further increase glycolytic metabolism. Recently, we have identified a novel feed-forward mechanism by which glycolytic enzyme expression leads to increased HIF-1 transcriptional activity. Pyruvate kinase isoforms PKM1 and PKM2 are alternatively spliced products of the PKM2 gene. PKM2, but not PKM1, alters glucose metabolism in cancer cells and contributes to tumorigenesis by mechanisms that are not explained by its known biochemical activity. We show that PKM2 gene transcription is activated by HIF-1. PKM2 interacts directly with the HIF-1α subunit and promotes transactivation of HIF-1 target genes by enhancing HIF-1 binding and p300 recruitment to hypoxia response elements, whereas PKM1 fails to regulate HIF-1 activity. Interaction of PKM2 with prolyl hydroxylase 3 (PHD3) enhances PKM2 binding to HIF-1α and PKM2 coactivator function. Mass spectrometry and anti-hydroxyproline antibody assays demonstrate PKM2 hydroxylation on proline-403/408. PHD3 knockdown inhibits PKM2 coactivator function, reduces glucose uptake and lactate production, and increases O2 consumption in cancer cells. Thus, PKM2 participates in a positive feedback loop that promotes HIF-1 transactivation and reprograms glucose metabolism in cancer cells (1). HIF-1 also plays critical roles in breast cancer metastasis. HIF-1 controls metastatic niche formation by activating transcription of genes encoding lysyl oxidase (LOX) and LOX-like proteins 2 and 4, which remodel collagen in the lungs, thereby recruiting bone marrow-derived cells that establish a microenvironment suitable for colonization by breast cancer cells (2). HIF-1 also promotes the extravasation of circulating breast cancer cells in the lungs by activating transcription of the genes encoding L1CAM, which encodes a cell adhesion molecule that promotes the interaction of breast cancer cells with vascular endothelial cells (ECs), and angiopoietin-like 4, which encodes a secreted factor that inhibits EC-EC interaction (3). Inhibition of HIF-1 activity by genetic or pharmacologic strategies dramatically inhibits the metastasis of breast cancer cells to the lungs in orthotopic mouse models (2, 3). (1) Luo W. et al. Cell 2011;145:732. (2) Wong CC et al. Proc Natl Acad Sci USA 2011 Sept 12. doi:10.1073/pnas.1113483108. (3) Zhang H. et al. Oncogene 2011 Aug 22. doi: 10.1038/onc.2011.365. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr MS3-2.

508 Alternative splicing of Lysyl Oxidase-Like 4 in ovarian carcinoma

Lysyl oxidase (LOX) is a copper-containing amine oxidase belonging to a heterogeneous family of enzymes that oxidize primary amine substrates to reactive aldehydes. It has been traditionally known for one function—the extracellular catalysis of lysine-derived cross-links in fibrillar collagens and elastin. Diverse roles have been attributed to lysyl oxidase and its novel activities cover a spectrum of diverse biological functions, such as developmental regulation, tumor suppression, cell motility, and cellular senescence. Lysyl oxidase has also been shown to have both intracellular and intranuclear locations. The multifunctional properties of lysyl oxidase (LOX) and the discovery of three novel members of this amine oxldase family—LOX-like (LOXL), LOXL2, and LOXL3—indicate the possibility that these varied functions are performed in both intracellular and extracellular environments by individual novel members of the LOX amine oxidase family. Structural similarity of the highly conserved copper-blnding and lysyltyrosylquinone cofactor sites among the LOX and LOX-like proteins results in similar amine oxldase activities. However, specific novel functions, such as a potential role in cell adhesion and cell growth control, is determined by other conserved domains such as the cytokine receptor-like domain that is shared by all LOXs and by multiple scavenger receptor cysteine-rich (SBCR) domains present in LOXL2 and LOXL3.

Abstract 3147: Alternative splicing of Lysyl Oxidase-like 4 in ovarian carcinoma

Abstract Introduction: Lysyl oxidase (LOX) is an amine oxidase that is usually synthesized and secreted by fibrogenic cells. Four LOX-like (LOXL) genes have been identified so far in mammalian genomes, encoding four different LOX-like proteins: LOXL1, LOXL2, LOXL3 and LOXL4. All members of the LOX family show a highly conserved C-terminus region that contains the catalytic domain. The N-terminus of the LOX isoforms is less conserved among the different members and is thought to determine the individual role and tissue distribution of each isoenzyme. LOXL4, the least studied member of the LOX-like family enzymes, undergoes a process of alternative splicing in cancer, in a site- and stage-specific manner that we have previously shown. The purpose of the current study was to uncover the splicing mechanism that is responsible for this process. Experimental procedures: I. ShRNAs for four splicing factors: SF2/ASF, SRp55, hnRNP-A1 and hnRNP-A2, were transfected in two cell lines: U-87 MG cell line (human glioblastoma) and NCI-H460 (human large-cell lung carcinoma). II. Over-expression of SF2 was performed in MST0-211H cell line (human malignant mesothelioma), HeLa cell line (human epithelial cervical cancer) and MCF10A cell line (human mammary epithelial line). III. Western blotting for SF2/ASF and tubulin. IV. RT-PCR for LOXL4 full length, splice-variant1 (splv1) and splice-variant2 (splv2) mRNA expression. Results: We examined LOXL4 expression in U-87 MG cells. When untreated, these cells express the full length and splv2, almost equally. The silencing of two factors, SF2/ASF and hnRNP-A1, resulted in a significant change in the expression pattern of LOXL4. For both silenced factors, LOXL4 full-length mRNA expression was much stronger, while the shortest variant, splv2, completely vanished. The silencing of hnRNP-A2 led to a smaller decrease in splv2, while SRp55 silencing did not seem to change LOXL4 splicing. In NCI-H460 cells, which normally express small amounts of all variants, no significant changes were found following silencing. In an attempt to further establish the splicing factor responsible for LOXL4 splicing, we over-expressed SF2/ASF in MST0-211H cells, which normally express only the full length LOXL4. Expression of SF2/ASF resulted in the appearance of splv2, while dramatically reducing the expression of the full length. Similar results were seen in HeLa cells. Over-expression of SF2/ASF in MCF10A cells, which untreated, have the unique quality of expressing splv2 alone, caused only a slight increase in the expression of splv2. Conclusions: These results demonstrate for the first time, that LOXL4 is a direct target of the splicing factor SF2/ASF. Furthermore, in concordance with our previous in-vivo findings, it can be concluded that LOXL4 splicing occurs similarly in other epithelial cancer types, such as breast cancer and mesothelioma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3147.

New Roles for Copper Metabolism in Cell Proliferation, Signaling, and Disease

Over the past 2 decades, the molecular mechanisms by which cells acquire, distribute, and utilize copper have been under intense investigation. Significant progress has been made in the identification of genes encoding copper homeostasis proteins and in fundamental aspects of their structure, function, and mechanisms of action in copper balance. A number of more comprehensive reviews of the field with respect to the genetics, structure, function, and physiology of copper metabolism have recently appeared elsewhere (1–4). Here, we review general mechanisms for eukaryotic copper metabolism at the cellular level in the context of recent discoveries in the field, identifying potential new functions for copper and copper metabolism proteins in cell signaling, gene expression, tumor cell metastasis, and resistance to anti-neoplastic drugs.

Lysyl oxidase: an oxidative enzyme and effector of cell function.

Lysyl oxidase (LOX) oxidizes the side chain of peptidyl lysine converting specific lysine residues to residues of alpha-aminoadipic-delta-semialdehyde. This posttranslational chemical change permits the covalent crosslinking of the component chains of collagen and those of elastin, thus stabilizing the fibrous deposits of these proteins in the extracellular matrix. Four LOX-like (LOXL) proteins with varying degrees of similarity to LOX have been described, constituting a family of related proteins. LOX is synthesized as a preproprotein which emerges from the cell as proLOX and then is processed to the active enzyme by proteolysis. In addition to elastin and collagen, LOX can oxidize lysine within a variety of cationic proteins, suggesting that its functions extend beyond its role in the stabilization of the extracellular matrix. Indeed, recent findings reveal that LOX and LOXL proteins markedly influence cell behavior including chemotactic responses, proliferation, and shifts between the normal and malignant phenotypes.

Lysyl Oxidase Is Essential for Normal Development and Function of the Respiratory System and for the Integrity of Elastic and Collagen Fibers in Various Tissues

Lysyl oxidases, a family comprising LOX and four LOX-like enzymes, catalyze crosslinking of elastin and collagens. Mouse Lox was recently shown to be crucial for development of the cardiovascular system because null mice died perinatally of aortic aneurysms and cardiovascular dysfunction. We show here that Lox is also essential for development of the respiratory system and the integrity of elastic and collagen fibers in the lungs and skin. The lungs of E18.5 Lox(-/-) embryos showed impaired development of the distal and proximal airways. Elastic fibers in E18.5 Lox(-/-) lungs were markedly less intensely stained and more disperse than in the wild type, especially in the mesenchyme surrounding the distal airways, bronchioles, bronchi, and trachea, and were fragmented in pulmonary arterial walls. The organization of individual collagen fibers into tight bundles was likewise abnormal. Similar elastic and collagen fiber abnormalities were seen in the skin. Lysyl oxidase activity in cultured Lox(-/-) skin fibroblasts and aortic smooth muscle cells was reduced by approximately 80%, indicating that Lox is the main isoenzyme in these cells. LOX abnormalities may thus be critical for the pathogenesis of several common diseases, including pulmonary, skin, and cardiovascular disorders.

Expression of lysyl oxidase isoforms in MC3T3-E1 osteoblastic cells

Covalent intermolecular cross-linking of collagen is initiated by the action of lysyl oxidase (LOX) on the telopeptidyl lysine and hydroxylysine residues. Recently, several LOX isoforms, i.e., LOX-like proteins 1–4 (LOXL1–4), have been identified but their specific tissue distribution and functions are still largely unknown. In this study, mRNA expression of LOX and LOXL1–4 in MC3T3-E1 osteoblastic cells was screened by RT-PCR and quantitatively analyzed by real-time PCR during cell differentiation and matrix mineralization. The results demonstrated that LOX and all LOXLs, except LOXL2, were expressed in this cell line and that the expression pattern during cell differentiation and matrix mineralization was distinct from one another. This indicates that the expression of LOX and its isoforms is highly regulated during osteoblast differentiation, suggesting their distinct roles in collagen matrix stabilization and subsequent mineralization.

Structural and functional diversity of lysyl oxidase and the LOX-like proteins

Lysyl oxidase (LOX) and four lysyl oxidase-like proteins, LOXL, LOXL2, LOXL3 and LOXL4, each contain a copper binding site, conserved lysyl and tyrosyl residues that may contribute to quinone co-factor formation, and a cytokine receptor-like domain. Each protein differs mainly in their N-terminal sequence, which may confer individual functions. Processing of the LOX proteins by BMP-1 and possibly other mechanisms may result in multiple functional forms. Splicing, reported for LOXL3, may also generate additional variants with unique functions. Each LOX, with its individual, developmentally regulated tissue and cell-specific expression and localization, results in a complex structural and functional variation for the LOX amine oxidases. The presence of only two LOX-like proteins in Drosophila, each with distinct spatial and temporal expression, allows for the assignment of individual function to one of these amine oxidases. Comparative expression analysis of each LOX protein is presented to help determine their functional significance.

Lysyl oxidases: A novel multifunctional amine oxidase family

Lysyl oxidase (LOX), a copper-containing amine oxidase, belongs to a heterogeneous family of enzymes that oxidize primary amine substrates to reactive aldehydes. LOX has been traditionally known for one function, the extracellular catalysis of lysine-derived cross-links in fibrillar collagens and elastin. More recently, diverse roles have been attributed to lysyl oxidase and these novel activities cover a spectrum of diverse biological functions such as developmental regulation, tumor suppression, cell motility, and cellular senescence. Lysyl oxidase has also been shown to have both intracellular and intranuclear locations. The multifunctional properties of lysyl oxidase (LOX) and our recent discovery of three novel members of this amine oxidase family, LOX-like (LOXL), LOXL2, and LOXL3, indicate the possibility that these varied functions are performed in both intracellular and extracellular environments by individual novel members of the LOX amine-oxidase family. Structural similarities of the highly conserved copper-binding and lysyl-tyrosylquinone cofactor sites among the LOX and LOX-like proteins may result in similar amine oxidase activities. However, specific novel functions, such as a potential role in cell adhesion and cell growth control, will be determined by other, conserved domains such as the cytokine receptor-like domain that is shared by all LOXs and by multiple scavenger receptor cysteine-rich (SRCR) domains present in LOXL2 and LOXL3. Furthermore, these functions may be carried out in a temporally and spatially regulated fashion.