LOX Protein Research Articles

Zishen Qingre Lishi Huayu Recipe May Ameliorate the Symptoms of PCOS Model Rats via Alleviating Systemic and Ovarian Inflammation.

Zishen Qingre Lishi Huayu recipe (ZQLHR) has shown significant therapeutic effects in treating sex hormone levels and follicular developmental disorders in patients with polycystic ovary syndrome (PCOS). However, little is known about the potential mechanisms of its treatment. Dehydroepiandrosterone and a high-fat diet induced the PCOS model rat. The serum of rats was collected to detect the levels of sex hormones and inflammatory cytokines by enzyme-linked immunosorbent assay, and the ovaries were collected for ovarian histopathology and qPCR assay to detect the levels of inflammatory cytokines in ovarian tissues. Granulosa cells (GCs) were collected for western blot assay to detect of IL-1β, IL-6R, and LOX protein expression levels. ZQLHR could reduce body weight, regulate estrous cycles, and improve serum sex hormone levels, follicular development, and insulin resistance (IR) in PCOS model rats. In addition, ZQLHR treatment improved the levels of inflammatory cytokines in serum and ovary, and regulated the protein expression of IL-6R, IL-1β, and LOX in GCs of PCOS model rats. The results showed that the HOMA-IR index increased with the increasing levels of IL-6, IL-1β, and CRP, and decreased with the increased IL-10. This study reveals that the treatment of endocrine disorders and ovulation disorders in PCOS with ZQLHR may be closely related to the improvement of systemic and ovarian inflammation in PCOS patients, as well as the inhibition of IL-6R, IL-1β, and LOX expression in GCs, which reemphasizes the role of reducing chronic inflammatory states in the treatment of PCOS. Moreover, this study reemphasizes the correlation between multiple inflammatory mediators and IR.

Development of analytical methods fort he quantitative analysis of the enzyme abundance and activity in the arachidonic acid cascade

The arachidonic acid (ARA) cascade is a key regulatory pathway where eicosanoids and other oxylipins are formed via the oxidation of polyunsaturated fatty acids. These oxylipins are involved in controlling physiological functions such as vascular tone, blood clotting and immune regulation. Changes in the oxylipin pattern can be caused by direct enzyme inhibition or modulated gene expression. In order to understand the effect mechanisms modulating the ARA cascade it is essential to not only investigate the metabolite, i.e. oxylipin, concentrations but also the abundances of the enzymes involved in their formation. For this reason, a quantitative targeted proteomics method for the ARA cascade was developed within this thesis.A detailed workflow for method development was established where enzyme/protein levels are measured via sequence‐specific, representative peptides chosen on the basis of several criteria. With liquid chromatography‐tandem mass spectrometry‐based analysis, the cyclooxygenase (COX) pathway of the ARA cascade was investigated in human colon carcinoma cell lines and primary macrophages where strong correlations were found between the oxylipins formed via the COX pathway and the COX‐2 abundance.The modulating effects of polyphenols on the 5‐lipoxygenase (LOX) pathway of the ARA cascade were investigated in cell‐free enzyme assays and human neutrophils. Resveratrol, its dimer ɛ‐viniferin and a resveratrol imine analogue directly inhibited the 5‐LOX with micromolar potencies and modulated the total oxylipin pattern in the neutrophils with remarkably different effects. Genistein did not act as a 5‐LOX inhibitor in the cell‐free assay but the modulated oxylipin pattern in the cells suggests a global cellular interference.The enzymes of the LOX pathway were included in the targeted proteomics method which together with an extended targeted oxylipin metabolomics platform enables a sensitive multi‐ omics analysis of the ARA cascade in a single sample. The comparison of the multiple reaction monitoring (MRM) detection and the MRM^‐based approach with an additional fragmentation step demonstrated that MRM is more favorable in targeted proteomics due to its higher sensitivity, greater linear range and higher multiplexing capacities. The analysis with the multi‐ omics approach showed that the 5 LOX protein is induced together with its product formation during the differentiation of human monocytic THP 1 cells to macrophages. M1‐like human primary macrophages were also characterized by the abundance of 5‐LOX and its activating protein (FLAP), while 15‐LOX and 15‐LOX‐2 dominated the protein pattern of the M2 like macrophages accompanied by high levels of the oxylipins formed via these enzymes. The developed methodology allows mechanistic investigation of a modulation of the ARA cascade as demonstrated by lipo‐poly‐saccharide stimulation as well as pharmaceutical treatment of the cells.Overall, with this thesis, a new quantitative proteomics strategy for the enzymes of the ARA cascade is established which can be easily extended to further proteins. The use of this technique in combination with targeted oxylipin metabolomics has provided new insights into the mechanisms involved in the modulation of the ARA cascade in human immune cells.

Copper-Binding Domain Variation in a Novel Murine Lysyl Oxidase Model Produces Structurally Inferior Aortic Elastic Fibers Whose Failure Is Modified by Age, Sex, and Blood Pressure.

Lysyl oxidase (LOX) is a copper-binding enzyme that cross-links elastin and collagen. The dominant LOX variation contributes to familial thoracic aortic aneurysm. Previously reported murine Lox mutants had a mild phenotype and did not dilate without drug-induced provocation. Here, we present a new, more severe mutant, Loxb2b370.2Clo (c.G854T; p.Cys285Phe), whose mutation falls just N-terminal to the copper-binding domain. Unlike the other mutants, the C285F Lox protein was stably produced/secreted, and male C57Bl/6J Lox+/C285F mice exhibit increased systolic blood pressure (BP; p < 0.05) and reduced caliber aortas (p < 0.01 at 100mmHg) at 3 months that independently dilate by 6 months (p < 0.0001). Multimodal imaging reveals markedly irregular elastic sheets in the mutant (p = 2.8 × 10−8 for breaks by histology) that become increasingly disrupted with age (p < 0.05) and breeding into a high BP background (p = 6.8 × 10−4). Aortic dilation was amplified in males vs. females (p < 0.0001 at 100mmHg) and ameliorated by castration. The transcriptome of young Lox mutants showed alteration in dexamethasone (p = 9.83 × 10−30) and TGFβ-responsive genes (p = 7.42 × 10−29), and aortas from older C57Bl/6J Lox+/C285F mice showed both enhanced susceptibility to elastase (p < 0.01 by ANOVA) and increased deposition of aggrecan (p < 0.05). These findings suggest that the secreted Lox+/C285F mutants produce dysfunctional elastic fibers that show increased susceptibility to proteolytic damage. Over time, the progressive weakening of the connective tissue, modified by sex and blood pressure, leads to worsening aortic disease.

Differential roles for lysyl oxidase (like), family members in chronic obstructive pulmonary disease; from gene and protein expression to function.

Chronic obstructive pulmonary disease (COPD) is characterized by long-term airflow obstruction with cigarette smoke as a key risk factor. Extracellular matrix (ECM) alterations in COPD may lead to small airway wall fibrosis. Altered collagen cross-linking, potentially mediated by the lysyl oxidase (LO) family of enzymes (LOX, LOXL1-4), orchestrates disturbed ECM homeostasis. In this study, we investigated the effects of smoking status and presence and severity of COPD on LOs gene and protein expression in the airways and the impact of LOs inhibition on airway contraction in an ex vivo mouse model. We used gene expression data from bronchial brushings, airway smooth muscle (ASM) cells in vitro and immunohistochemistry in lung tissue to assess smoke- and COPD-associated differences in LOs gene and protein expression in the small airways. We found higher LOX expression in current- compared to ex-smokers and higher LOXL1 expression in COPD compared to non-COPD patients. LOX and LOXL2 expression were upregulated in COPD ASM cells treated with cigarette smoke extract. LOXL1 and LOXL2 protein levels were higher in small airways from current- compared to non-smokers. In COPD patients, higher LOXL1 and lower LOX protein levels were observed, but no differences for LOXL2, LOXL3, and LOXL4 protein were detected in small airways. Inhibiting LOs activity increased airway contraction in murine lung slices. COPD-associated changes in LOs, in particular LOX and LOXL1, may be related to smoking and contribute to impaired airway function, providing potential novel targets for preventing or treating small airways changes in COPD.

Bacterial cyclodipeptides elicit Arabidopsis thaliana immune responses reducing the pathogenic effects of Pseudomonas aeruginosa PAO1 strains on plant development

Plants being sessile organisms are exposed to various biotic and abiotic factors, thus causing stress. The Pseudomonas aeruginosa bacterium is an opportunistic pathogen for animals, insects, and plants. Direct exposure of Arabidopsis thaliana to the P. aeruginosa PAO1 strain induces plant death by producing a wide variety of virulence factors, which are regulated mainly by quorum sensing systems. Besides virulence factors, P. aeruginosa PAO1 also produces cyclodipeptides (CDPs), which possess auxin-like activity and promote plant growth through activation of the target of the rapamycin (AtTOR) pathway. On the other hand, plant defense mechanisms are regulated through the production of phytohormones, such as salicylic acid (SA) and jasmonic acid (JA), which are induced in response to pathogen-associated molecular patterns (PAMPs), activating defense genes associated with SA and JA such as PATHOGENESIS-RELATED-1 (PR-1) and LIPOXYGENASE2 (LOX2), respectively. PR proteins are suggested to play critical roles in coordinating the Systemic Acquired Resistance (SAR). In contrast, LOX proteins (LOX2, LOX3, and LOX4) have been associated with the production of JA by producing its precursors, oxylipins. The activation of defense mechanisms involves signaling cascades such as Mitogen-Activated Protein Kinases (MAPKs) or the TOR pathway as a switch for re-directing energy towards defense or growth. In this work, we challenged A. thaliana (wild type, mpk6 or mpk3 mutants, and overexpressing TOR) seedlings with P. aeruginosa PAO1 strains to identify the role of bacterial CDPs in the plant immune response. Results showed that the pre-exposure of these Arabidopsis seedlings to CDPs significantly reduced plant infection of the pathogenic P. aeruginosa PAO1 strains, indicating that plants that over-express AtTOR or lack MPK3/MPK6 protein-kinases are more susceptible to the pathogenic effects. In addition, CDPs induced the GUS activity only in the LOX2::GUS plants, indicative of JA-signaling activation. Our findings indicate that the CDPs are molecules that trigger SA-independent and JA-dependent defense responses in A. thaliana; hence, bacterial CDPs may be considered elicitors of the Arabidopsis immune response to pathogens.

Evolution and functional diversity of lipoxygenase (LOX) genes in allotetraploid rapeseed (Brassica napus L.)

Lipoxygenase (LOX, EC 1.13.11.12) is a non-haeme iron-containing dioxygenase family that catalyzes the oxygenation of polyunsaturated fatty acids into bio-functionally fatty acid diverse (oxylipins) and plays vital role in plant growth and development and responses to abiotic and biotic stresses. Though LOX genes have been studied in many plant species, their roles in Brassicaceae species are still unknown. Here, a set of 14, 18, and 33 putative LOX genes were identified in Brassica rapa, Brassica oleracea and Brassica napus (allotetraploid rapeseed), respectively, which could be divided into 9-LOX (LOX1/5), 13-LOX type I (LOX3/4/6), and type II (LOX2) subgroups. There was an expansion of LOX2 orthologous genes in Brassicaceae. Most of the LOX genes are intron rich and conserved in gene structure, and the LOX proteins all have the conserved lipoxygenase and PLAT/LH2 domain. Ka/Ks ratio revealed that the majority of LOXs underwent purifying selection in Brassicaceae. The light-, ABA-, MeJA-related cis-elements and MYB-binding sites in the promoters of BnaLOXs were the most abundant. BnaLOXs displayed different spatiotemporal expression patterns and various abiotic/biotic stress responsive expression patterns. BnaLOX1/5 were slightly or no response to phytohormones and abiotic stresses. BnaLOX3/4/6 predominantly express in roots and were strongly up-regulated by salinity and PEG treatments, and BnaLOX3/4 were the methyl jasmonate (MeJA) and salicylic acid (SA) early response genes and strongly induced by infection of Sclerotinia sclerotiorum; while the BnaLOX2 members predominantly express in stamens, were MeJA and SA continuous response genes and strongly repressed by cold, heat and waterlogging treatments in leaves. Our results are useful for understanding the biological functions of the BnaLOX genes in allotetraploid rapeseed.

Evaluation of Important Molecular Pathways and Candidate Diagnostic Biomarkers of Noninvasive to Invasive Stages in Gastric Cancer by In Silico Analysis.

Gastric cancer affects millions of people each year; it is the fifth deadliest cancer globally. Due to failure to perform routine tests such as endoscopy, it is usually diagnosed in the invasive stages. Therefore, finding diagnostic biomarkers in blood can help to speed up the initial diagnosis of cancer. This study aimed to find appropriate diagnostic biomarkers in the extracellular matrix of noninvasive to invasive stages of gastric cancer patients, using bioinformatics analysis. First, we selected the appropriate datasets from the GEO database. We evaluated the genes' signaling pathways, biological processes, and molecular functions. More accurately, we assessed the genes, in which their protein products are released into the extracellular matrix; we evaluated their protein network. Then, we validated the candidate proteins in the GEPIA and TCGA databases. The extracellular matrix, tyrosine kinase receptors, and immune response pathways are effective factors, which are related to the highly expressed genes and metabolism; cell cycle pathways are also impressive on low-expression genes. 69 highly expressed proteins are released into the extracellular matrix. After drawing the protein network, 5 proteins were selected as more suitable candidates for further studies. These proteins' expression significantly increases in the human samples, and the survival chart showed up to about 80% mortality in the individuals over time. With integrated bioinformatics analysis, BGN, LOX, MMP-9, SERPINE1, and TGFB1 proteins have been selected as suitable diagnostic biomarkers for noninvasive to invasive stages of gastric cancer. Further studies are needed to evaluate more precise mechanisms between these proteins.

Abstract 4915: Nerve growth factor (NGF) facilitates LOX expression and promotes tumor metastasis by inhibiting miR-149-5p in human chondrosarcoma cells

Abstract Chondrosarcomas are malignant bone tumors that commonly produce cartilaginous chondroid matrix and are associated with metastases in the lung, especially in patients with high-grade chondrosarcoma. Neurotrophins such as nerve growth factor (NGF) are recognized as being drivers of neurogenesis during development and regeneration, and their expression in human tumors can stimulate cancer cell growth. Importantly, the infiltration of nerves in the tumor microenvironment actively stimulates cancer cell mitogenesis, invasiveness and metastasis. Until now, it has been unclear as to the potential involvement of NGF in human chondrosarcoma metastasis. This study reports that NGF induces migration and invasion of chondrosarcoma cell lines JJ012 and SW1353 and significantly increasing LOX expression. Significant dose-dependent increases were observed in levels of LOX mRNA and protein expression in both cell lines. In immunohistochemistry analyses, levels of NGF and LOX expression in human chondrosarcoma cartilage correlated with tumor stage and were substantially higher than NGF and LOX expression in normal cartilage. NGF-induced stimulation of metastasis and increases in LOX expression were prevented by pretreatment of the cell lines with PI3K, Akt and mTOR inhibitors, or transfection with small interfering RNAs (siRNAs). Further analysis revealed that NGF induced PI3K/Akt/mTOR signaling and dose-dependently downregulated mature microRNA-149-5p (miR-149-5p), which targets the 3' untranslated region (3'-UTR) of LOX. Furthermore, JJ012 cells overexpressing NGF induced LOX expression and increased cell migration and invasion. In summary, NGF stimulates LOX expression and promotes chondrosarcoma cell migration and invasion by inhibiting miR-149-5p via the PI3K/Akt/mTOR signaling pathway. Citation Format: Anoop Thadevoos Louis, Chih-Yang Lin, Chih-Hsin Tang. Nerve growth factor (NGF) facilitates LOX expression and promotes tumor metastasis by inhibiting miR-149-5p in human chondrosarcoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4915.

Role of the lysyl oxidase family in organ development (Review).

Lysyl oxidase proteins (LOXs) are amine oxidases, which are mainly located in smooth muscle cells and fibroblasts and serve an important role in the formation of the extracellular matrix (ECM) in a copper-dependent manner. Owing to the ability of LOX proteins to modulate crosslinking between collagens and to promote the deposition of other fibers, they serve crucially in organogenesis and the subsequent organ development, as well as disease initiation and progression. In addition, ECM formation significantly influences organ morphological formation in both cancer- and non-tumor-related diseases, in addition to cellular epigenetic transformation and migration, under the influence of LOXs. A number of different signaling pathways regulate the LOXs expression and their enzymatic activation. The tissue remodeling and transformation process shares some resemblance between oncogenesis and embryogenesis. Additionally the roles that LOXs serve appeared to be stressed during oncogenesis and tumor metastasis. It has also been indicated LOXs have a noteworthy role in non-tumor diseases. Nonetheless, the role of LOXs in systemic or local organ development and disease control remains unknown. In the present study, the essential roles that LOXs play in embryogenesis were unveiled partially, whereas the role of LOXs in organ or systematic development requires further investigations. The present review aimed to discuss the roles of members of the LOX family in the context of the remodeling of organogenesis and organ development. In addition, the consequences of the malfunction of these proteins related to the development of abnormalities and resulting diseases is discussed.

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.

Development of expressed sequenced tags (EST) to identify some pathogen resistance genes expressed in Gossypium arboreum

Cotton the most important fiber crop is facing a major threat due to a viral disease caused by cotton leaf curl virus (CLCuV). The cotton specie, Gossypium arboreum is resistant to this disease. Cotton scientists are working to find the key genes in G. arboreum that confer resistance against cotton leaf curl disease (CLCuD). Current research work is an effort to find some potential biotic stress related resistance genes from G. arboreum and the their evaluation against CLCuV infection utilizing functional genomics approaches. Leaf cDNA library was constructed from field grown G. arboreum which was further utilized to identify and isolate clones involved in resistance against CLCuD. The clone sequences were exploited to establish expressed sequence tags (EST). The EST represented some important biotic stress resistance genes like lipoxygenase, cytochrome P450, CPMMV like coat protein, serine threonine kinase, a RGA, lipid transfer protein and ubiquitin conjugating enzyme E2. As cotton is a fiber crop so some trichome development genes like aquaporin, arabinogalactans and cellulose synthase were also found. Lipoxygenases are known to be involved in apoptosis and biotic and abiotic stress responses in plants. Here the members of LOX are identified in biotic stress resistant G. arboreum. G. arboreum genome encode 13 LOX proteins. The G. arboreum LOXs are validated based on protein alignment studies. This is the first report wherein number of LOXs are identified in cotton which may help to better understand the apoptosis and responses to biotic and abiotic stresses in naturally resistant G. arboreum.

Metformin Suppresses Expression of Pre-Fibrotic Enzyme Lysyl-Oxidase Levels through Inhibition of UPR in MPN Cells

The endoplasmic reticulum (ER) is a cytoplasmic organelle required for proper protein folding and sorting. When protein load exceeds the capacity of the ER, the accumulation of the misfolded proteins triggers a quality control response known as the unfolded protein response (UPR). Recent studies have revealed that increased activation of the UPR is one of the hallmarks of hematological malignancies. In addition, excess UPR is associated with pro-fibrotic conditions, i.e., pulmonary fibrosis, renal fibrosis and kidney fibrosis.Lysyl-oxidase (LOX) is an enzyme that regulates the crosslinking of extracellular matrix proteins, such as collagen, and enhances a fibrotic phenotype. Recent studies revealed that the genes of LOX family members are upregulated in myeloproliferative neoplasms (MPN). Furthermore, megakaryocytes and platelets derived from MPN patients had increased LOX protein concentrations in comparison with healthy subjects. It is also reported that the UPR is one of the important regulators of LOX. Based on this knowledge, we speculated that the enhanced UPR might be involved in the elevation of LOX in JAK2V617F harboring MPN and may contribute to the development of myelofibrosis. To study this hypothesis, we initially confirmed that JAK2V617F-positive cell lines(HEL and SET-2) show enhanced activation of UPR, including phosphorylation of eIF2-alpha, nuclear localization of ATF6 and XBP1s and upregulation of glucose response protein 78 (GRP78). We also confirmed the presence of elevated levels of LOX in these cells. Treatment of the cells with a chemical chaperone, namely, tauroursodeoxycholic acid (TUDCA), suppressed UPR and decreased expression of LOX, suggesting that UPR is responsible for elevation of LOX in MPN cells. To analyze whether JAK2V617F is responsible for the enhanced UPR and expression of LOX in MPN cells, we treated the cells with JAK1/JAK2 inhibitor ruxolitinib. Unexpectedly, ruxolitinib did not block UPR nor expression of LOX. Previously, we reported that metformin, which is a member of the biguanide family, inhibited growth of MPN cells through activation of AMPK and PP2A (Kawashima et al. Exp.Hematol, 2016). Furthermore, a number of studies demonstrated that metformin suppressed UPR in several cancer cells through activation of AMPK. Therefore, we investigated whether metformin could block UPR and LOX levels in these cells. As expected, metformin reduced the phosphorylation levels of eIF2alpha and nuclear localization of XBP1 and ATF6. Expression of GRP78 was also blocked by metformin. These results clearly indicated that metformin suppressed UPR in MPN cells. Metformin also blocked the activation of the molecules required for proteins synthesis including p70S6kinase, mTOR and 4E-PB1. These results indicated that suggested that metformin blocked aberrant UPR through inhibition of enhanced protein synthesis. Importantly, we also confirmed that LOX expression was also suppressed by metformin in MPN cells.Our observations indicated that the UPR is enhanced in MPN cells irrespective of JAK2V617F, and metformin could block this process leading to suppression of LOX levels. Combination of ruxolitinib with metformin might be a new and attractive method to suppress myelofibrosis in MPN. DisclosuresKirito:Novartis Pharma KK: Honoraria.

Isoform-specific subcellular localization of Zea mays lipoxygenases and oxo-phytodienoate reductase 2

Plant 9- and 13-lipoxygenases (9- and 13-LOXs) function in an early step of the oxylipin biosynthesis pathway. Here we report diverse subcellular localization profiles of four maize LOX family proteins and one oxo-phytodienoate reductase 2, subcellular localization of which could not be predicted by bioinformatics tools. Fluorescent protein-tagged LOX proteins were transiently expressed in maize B73 protoplasts as well as in stably transformed Arabidopsis thaliana. Localization profiles of maize 9-LOXs were more diverse than that of reported 13-LOX isoforms and spanned cytoplasm, plastids and tonoplasts, suggesting compartmentation of different oxylipin production inside of the cells. Localization profiles of maize 9-LOX were mostly consistent between maize and Arabidopsis, suggesting subcellular targeting mechanisms of these isoforms are conserved between monocots and dicots.

Abstract 73: Pre- and Post-Translational Regulation of Lysyl Oxidase/β1-Intergrin in Aortic Vascular Smooth Cell of Hypertension

Background: An increase in aortic stiffness is a fundamental component of hypertension . We previous showed that aortic vascular smooth muscle cells (VSMCs) play an causative role in aortic stiffening not only due to the increased intrinsic stiffness in VSMC itself but also due to its interaction with extracellular matrix (ECM). However, the molecular mechanism involved is unclear. Here, we test the hypothesis that the aortic VSMCs mediates pre- and post-translational regulation of Lysyl Oxidase (Lox)/β1intergrain pathway which may be one of the mechanisms of VSMCs-ECM remodeling in hypertension. Methods and Results: 4 months old male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were studied. Aortic pressure and stiffness were measured with an invasive catheter and Doppler imaging echocardiography, respectively. Compared to WKY, SHR showed a significant higher aortic pressure (mean arterial pressure (MAP) 138.7±11.8 vs 102.7±7 mmHg, ( p &lt;0.01) and a greater aorta stiffness reflected by 2.5-fold higher arterial stiffness index (ASI) in SHR vs WKY ( p &lt;0.01). VSMCs were isolated from thoracic aorta, then cultured and measured at passages 2 to 4. Real time PCR and western blot were used to detect the gene and protein expression. Compared to WKY, VSMCs from SHR showed a significant increase in mRNA Lox, a copper-dependent amine oxidase mediating the crosslinking of collagens and elastin (2.7 folds vs WKY, p &lt;0.01). The abundance of LOX protein was significant increased not only at pro-enzyme lox level, but also at the regulatory pro-peptide lox and matured active enzyme Lox in SHR VSMCs by 3.5 folds and 1.4 folds respectively ( p &lt;0.01), indicating an increased post-translational regulation of LOX in SHR VSMCs. In addition, the glycosylated β1 integrin, a transmembrane receptor that facilitate cell-ECM adhesion, was found to be significant increase in SHR VSMCs vs WKY. Conclusion: The regulation of pre- and post-translational regulation of Lox/β1intergrain pathway in aortic VSMCs presents a novel mechanism of cell-ECM interaction, which may contribute to the increased aortic stiffness in SHR.

Molecular characterization of lysyl oxidase‐mediated extracellular matrix remodeling during mouse decidualization

The establishment of decidualization is a prerequisite of successful pregnancy. Lysyl oxidase (Lox) is a copper-containing amine oxidase which catalyzes cross-linking of collagen and elastin in the ECM. Lox is expressed in the subluminal stroma surrounding the implanting blastocyst on day 5 of pregnancy. From days 6 to 8, the signals for Lox mRNA and protein are strongly detected in the decidual cells. The expression of Lox is under the control of estrogen via the GSK-3β/β-catenin/c-myc pathway. Dtprp is decreased by the inhibition of Lox activity. Furthermore, the inhibition of Lox activity decreases stromal cell migration and embryo adhesion. Our findings highlight the crucial role of Lox in endometrial stromal cells and deepen our understanding of decidualization.

Cooperative Effect of miR-141-3p and miR-145-5p in the Regulation of Targets in Clear Cell Renal Cell Carcinoma.

BackgroundDue to the poor prognosis for advanced renal cell carcinoma (RCC), there is an urgent need for new therapeutic targets and for prognostic markers to identify high risk tumors. MicroRNAs (miRNAs) are frequently dysregulated in tumors, play a crucial role during carcinogenesis and therefore might be promising new biomarkers. In previous studies, we identified miR-141-3p and miR-145-5p to be downregulated in clear cell RCC (ccRCC). Our objective was to investigate the functional association of these miRNAs, focusing on the cooperative regulation of new specific targets and their role in ccRCC progression.MethodsThe effect of miR-141-3p and miR-145-5p on cell migration was examined by overexpression in 786-O cells. New targets of both miRNAs were identified by miRWalk, validated in 786-O and ACHN cells and additionally characterized in ccRCC tissue on mRNA and protein level.ResultsIn functional analysis, a tumor suppressive effect of miR-141-3p and miR-145-5p by decreasing migration and invasion of RCC cells could be shown. Furthermore, co-overexpression of the miRNAs seemed to result in an increased inhibition of cell migration. Both miRNAs were recognized as post-transcriptional regulators of the targets EAPP, HS6ST2, LOX, TGFB2 and VRK2. Additionally, they showed a cooperative effect again as demonstrated by a significantly increased inhibition of HS6ST2 and LOX expression after simultaneous overexpression of both miRNAs. In ccRCC tissue, LOX mRNA expression was strongly increased compared to normal tissue, allowing also to distinguish between non-metastatic and already metastasized primary tumors. Finally, in subsequent tissue microarray analysis LOX protein expression showed a prognostic relevance for the overall survival of ccRCC patients.ConclusionThese results illustrate a jointly strengthening effect of the dysregulated miR-141-3p and miR-145-5p in various tumor associated processes. Focusing on the cooperative effect of miRNAs provides new opportunities for the development of therapeutic strategies and offers novel prognostic and diagnostic capabilities.

Reduced Biosynthesis of Digalactosyldiacylglycerol, a Major Chloroplast Membrane Lipid, Leads to Oxylipin Overproduction and Phloem Cap Lignification in Arabidopsis.

DIGALACTOSYLDIACYLGLYCEROL SYNTHASE1 (DGD1) is a chloroplast outer membrane protein responsible for the biosynthesis of the lipid digalactosyldiacylglycerol (DGDG) from monogalactosyldiacylglycerol (MGDG). The Arabidopsis thaliana dgd1 mutants have a greater than 90% reduction in DGDG content, reduced photosynthesis, and altered chloroplast morphology. However, the most pronounced visible phenotype is the extremely short inflorescence stem, but how deficient DGDG biosynthesis causes this phenotype is unclear. We found that, in dgd1 mutants, phloem cap cells were lignified and jasmonic acid (JA)-responsive genes were highly upregulated under normal growth conditions. The coronative insensitive1 dgd1 and allene oxide synthase dgd1 double mutants no longer exhibited the short inflorescence stem and lignification phenotypes but still had the same lipid profile and reduced photosynthesis as dgd1 single mutants. Hormone and lipidomics analyses showed higher levels of JA, JA-isoleucine, 12-oxo-phytodienoic acid, and arabidopsides in dgd1 mutants. Transcript and protein level analyses further suggest that JA biosynthesis in dgd1 is initially activated through the increased expression of genes encoding 13-lipoxygenases (LOXs) and phospholipase A-Iγ3 (At1g51440), a plastid lipase with a high substrate preference for MGDG, and is sustained by further increases in LOX and allene oxide cyclase mRNA and protein levels. Our results demonstrate a link between the biosynthesis of DGDG and JA.

Origin and evolution of lysyl oxidases.

Lysyl oxidases (LOX) are copper-dependent enzymes that oxidize primary amine substrates to reactive aldehydes. The best-studied role of LOX enzymes is the remodeling of the extracellular matrix (ECM) in animals by cross-linking collagens and elastin, although intracellular functions have been reported as well. Five different LOX enzymes have been identified in mammals, LOX and LOX-like (LOXL) 1 to 4, showing a highly conserved catalytic carboxy terminal domain and more divergence in the rest of the sequence. Here we have surveyed a wide selection of genomes in order to infer the evolutionary history of LOX. We identified LOX proteins not only in animals, but also in many other eukaryotes, as well as in bacteria and archaea – which reveals a pre-metazoan origin for this gene family. LOX genes expanded during metazoan evolution resulting in two superfamilies, LOXL2/L3/L4 and LOX/L1/L5. Considering the current knowledge on the function of mammalian LOX isoforms in ECM remodeling, we propose that LOXL2/L3/L4 members might have preferentially been involved in making cross-linked collagen IV-based basement membrane, whereas the diversification of LOX/L1/L5 forms contributed to chordate/vertebrate-specific ECM innovations, such as elastin and fibronectin. Our work provides a novel view on the evolution of this family of enzymes.

P17 - LYSYL OXIDASES: EMERGING PROMOTERS OF SENESCENCE ESCAPE, TUMOR INITIATION AND PROGRESSION

Lysyl oxidase activity, exhibited by five lox members, has been extensively associated with tumor progression and metastasis. Notably, it is well documented that lox factors, mainly LOX and LOXL2 proteins, are implicated in invasion, cell migration, and angiogenesis in response to tumor-associated hypoxia. In the clinic, their expression correlates with bad prognosis for cancer progression and patient survival. Recently, lox factors have been observed to promote senescence escape, a critical event in tumorigenesis. In two mouse cancer susceptibility models for breast and pancreatic cancer, LOX and/or LOXL2 were found to promote tumorigenesis in cooperation with an oncogenic signal. Mechanistically, lox factors might mediate their effects via several mechanisms; from reorganizing the extracellular matrix and tumor microenvironment, promoting activation of specific pathways such as FAK and Akt pathways, to regulating gene expression by modifying the histone tails on specific target genes such as E-cadherin. This modification impacts the epithelial-mesenchymal transition, a process involved in senescence escape, tumor initiation and progression. The recent literature highlights an important role of lox factors in cancer, and an antibody directed against LOXL2 is already undergoing clinical trials. A better understanding of the biology of these factors will help the design and application of new lox-derived therapeutic tools.

Lysyl Oxidase Expression Is Decreased in the Developing Diaphragm and Lungs of Nitrofen-Induced Congenital Diaphragmatic Hernia

Malformation of the nonmuscular tissue components in congenital diaphragmatic hernia (CDH) is thought to underlie the diaphragmatic defect, causing intrathoracic herniation of abdominal viscera and thus disturbing normal lung development. It has been shown that diaphragmatic and pulmonary morphogeneses require the structural integrity of connective tissue, and developmental mutations that inhibit the formation of extracellular matrix (ECM) result in CDH with hypoplastic lungs. Lysyl oxidase (lox), an extracellular enzyme that catalyzes the cross-linking of ECM proteins, plays an essential role during diaphragmatic and pulmonary development by controlling the formation of connective tissue. Furthermore, lox (-/-) knockouts exhibit abnormal connective tissue with diaphragmatic defects and impaired airway morphogenesis. We designed this study to investigate the hypothesis that diaphragmatic and pulmonary lox expression is decreased in the nitrofen-induced CDH model. Timed-pregnant Sprague-Dawley rats were exposed to either nitrofen or vehicle on gestational day 9 (D9), and fetuses were harvested on selected time points D15 and D18. The micro-dissected fetal diaphragms (n=48) and lungs (n=48) were divided into two groups: control and nitrofen-exposed samples (n=12 per specimen and time point, respectively). Diaphragmatic and pulmonary gene expression levels of lox were analyzed by quantitative real-time polymerase chain reaction. Immunohistochemical staining was performed to evaluate lox protein expression in diaphragms and lungs. Relative mRNA expression of lox was significantly reduced in diaphragms and lungs of nitrofen-exposed fetuses on D15 (0.29 ± 0.08 vs. 0.12 ± 0.05; p<0.05 and 0.52 ± 0.44 vs. 0.20 ± 0.04; p<0.05) and D18 (0.90 ± 0.25 vs. 0.57 ± 0.23; p<0.05 and 0.59 ± 0.26 vs. 0.35 ± 0.09; p<0.05) compared with controls. Diaphragmatic and pulmonary immunoreactivity of lox was markedly decreased in nitrofen-exposed fetuses on D15 and D18 compared with controls. Decreased lox expression during diaphragmatic development and lung branching morphogenesis may interfere with normal cross-linking of ECM proteins, disrupting the integrity of connective tissue, and contributing to the diaphragmatic defect and impaired airway formation in the nitrofen-induced CDH model.