Lysyl Oxidase Isoforms Research Articles

Pan-lysyl oxidase inhibition disrupts fibroinflammatory tumor stroma, rendering cholangiocarcinoma susceptible to chemotherapy.

Cholangiocarcinoma (CCA) features highly desmoplastic stroma that promotes structural and functional resistance to therapy. Lysyl oxidases (LOX, LOXL1-4) catalyze collagen cross-linking, thereby increasing stromal rigidity and facilitating therapeutic resistance. Here, we evaluate the role of lysyl oxidases in stromal desmoplasia and the effects of pan-lysyl oxidase (pan-LOX) inhibition in CCA. Resected CCA and normal liver specimens were analyzed from archival tissues. Spontaneous and orthotopic murine models of intrahepatic CCA (iCCA) were used to assess the impact of the pan-LOX inhibitor PXS-5505 in treatment and correlative studies. The functional role of pan-LOX inhibition was interrogated through in vivo and ex vivo assays. All 5 lysyl oxidases are upregulated in CCA and reduced lysyl oxidase expression is correlated with an improved prognosis in resected patients with CCA. Spontaneous and orthotopic murine models of intrahepatic cholangiocarcinoma upregulate all 5 lysyl oxidase isoforms. Pan-LOX inhibition reversed mechanical compression of tumor vasculature, resulting in improved chemotherapeutic penetrance and cytotoxic efficacy. The combination of chemotherapy with pan-LOX inhibition increased damage-associated molecular pattern release, which was associated with improved antitumor T-cell responses. Pan-LOX inhibition downregulated macrophage invasive signatures in vitro, rendering tumor-associated macrophages more susceptible to chemotherapy. Mice bearing orthotopic and spontaneously occurring intrahepatic cholangiocarcinoma tumors exhibited delayed tumor growth and improved survival following a combination of pan-LOX inhibition with chemotherapy. CCA upregulates all 5 lysyl oxidase isoforms, and pan-LOX inhibition reverses tumor-induced mechanical forces associated with chemotherapy resistance to improve chemotherapeutic efficacy and reprogram antitumor immune responses. Thus, combination therapy with pan-LOX inhibition represents an innovative therapeutic strategy in CCA.

Crosstalk of lysyl oxidase-like 1 and lysyl oxidase prolongs their half-lives and regulates liver fibrosis through Notch signal.

Lysyl oxidase (LOX) family members (LOX and LOXL1 to 4) are crucial copper-dependent enzymes responsible for cross-linking collagen and elastin. Previous studies have revealed that LOX and LOXL1 are the most dramatically dysregulated LOX isoforms during liver fibrosis. However, the crosstalk between them and the underlying mechanisms involved in the profibrotic behaviors of HSCs, as well as the progression of liver fibrosis, remain unclear. pCol9GFP-HS4,5Tg mice, Loxl1fl/flGfapCre mice, human HSC line, and primary HSCs were enrolled to study the dysregulation pattern, profibrotic roles, and the potential mechanisms of LOX and LOXL1 interaction involved in the myofibroblast-like transition of HSCs and liver fibrogenesis. LOX and LOXL1 were synergistically upregulated during liver fibrogenesis, irrespective of etiology, together orchestrating the profibrotic behaviors of HSCs. LOX and LOXL1 coregulated in HSCs, whereas LOXL1 dominated in the coregulation loop. Interestingly, the interaction between LOXL1 and LOX prolonged their half-lives, specifically enhancing the Notch signal-mediated myofibroblast-like transition of HSCs. Selective disruption of Loxl1 in Gfap+ HSCs deactivated the Notch signal, inhibited HSC activation, and relieved carbon tetrachloride-induced liver fibrosis. Our current study confirmed the synergistic roles and the underlying mechanisms of LOXL1 and LOX crosstalk in the profibrotic behaviors of HSCs and liver fibrosis progression, providing experimental evidence for further clear mechanism-based anti-LOXL1 strategy development in the therapy of liver fibrosis.

Immunoblotting identification of jumbo squid (Dosidicus gigas) LOX isoforms and in vitro crosslinking assay over selected collagenous materials

Jumbo squid (Dosidicus gigas) muscle hardness has been related to hydroxylysyl-pyridinoline (HP) formation (collagen fibre stabilization) via condensation of oxidized ε-amino groups of lysine and/or hydroxylysine by lysyl oxidase (LOX). Previously published literature has suggested the presence of LOX isoforms in squid muscle. Thus, the objective of the present research was to test the hypothesis that squid LOX isoforms exist in fresh mantle muscle. A semi-pure LOX extract (SPLE) was used to perform an immunoblotting assay using two commercial human-LOX antibodies. Furthermore, SPLE (specific activity of 20 mU/mg of protein) was tested in vitro for possible HP formation using several collagenous materials. Immunoblotting assay confirmed the presence of squid LOX at 32 kDa and its isoforms at 34 kDa and 24 kDa. Although LOX oxidized ε-amino groups on collagenous materials, no HP formation was detected. However, the latter could promote interesting collagen functional modifications.

Evaluation of Fluorine-18-Labeled α1(I)-N-Telopeptide Analogs as Substrate-Based Radiotracers for PET Imaging of Melanoma-Associated Lysyl Oxidase

Accumulating evidence suggests an unequivocal role of lysyl oxidases as key players of tumor progression and metastasis, which renders this enzyme family highly attractive for targeted non-invasive functional imaging of tumors. Considering their function in matrix remodeling, malignant melanoma appears as particularly interesting neoplasia in this respect. For the development of radiotracers that enable PET imaging of the melanoma-associated lysyl oxidase activity, substrates derived from the type I collagen α1 N-telopeptide were labeled with fluorine-18 using N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) as prosthetic reagent. With regards to potential crosslinking to tumor-associated collagen in vivo, their interaction with triple-helical type I collagen was studied by SPR. A mouse model of human melanoma was established on the basis of the A375 cell line, for which the expression of the oncologically relevant lysyl oxidase isoforms LOX and LOXL2 was demonstrated in Western blot and immunohistochemical experiments. The radiopharmacological profiles of the peptidic radiotracers were evaluated in normal rats and A375 melanoma-bearing mice by ex vivo metabolite analysis, whole-body biodistribution studies and dynamic PET imaging. Out of three 18F-labeled telopeptide analogs, the one with the most favorable substrate properties has shown favorable tumor uptake and tumor-to-muscle ratio. Lysyl oxidase-mediated tumor uptake was proven by pharmacological inhibition using β-aminopropionitrile and by employing negative-control analogs of impeded or abolished targeting capability. The latter were obtained by substituting the lysine residue by ornithine and norleucine, respectively. Comparing the tumor uptake of the lysine-containing peptide with that of the non-functional analogs indicate the feasibility of lysyl oxidase imaging in melanoma using substrate-based radiotracers.

ZEB1 induces LOXL2-mediated collagen stabilization and deposition in the extracellular matrix to drive lung cancer invasion and metastasis.

Lung cancer is the leading cause of cancer-related death, primarily due to distant metastatic disease. Metastatic lung cancer cells can undergo an epithelial-to-mesenchymal transition (EMT) regulated by many transcription factors, including double-negative feedback loop between the microRNA-200 (miR-200) family and ZEB1, but the precise mechanisms by which ZEB1-dependent EMT promotes malignancy remain largely undefined. While the cell-intrinsic effects of EMT are important for tumor progression, the reciprocal dynamic crosstalk between mesenchymal cancer cells and the extracellular matrix (ECM) is equally critical in regulating invasion and metastasis. Investigating the collaborative effect of EMT and ECM in the metastatic process reveals increased collagen deposition in metastatic tumor tissues as a direct consequence of amplified collagen gene expression in ZEB1-activated mesenchymal lung cancer cells. Additionally, collagen fibers in metastatic lung tumors exhibit greater linearity and organization as a result of collagen crosslinking by the lysyl oxidase (LOX) family of enzymes. Expression of the LOX and LOXL2 isoforms is directly regulated by miR-200 and ZEB1, respectively, and their upregulation in metastatic tumors and mesenchymal cell lines is coordinated to that of collagen. Functionally, LOXL2, as opposed to LOX, is the principle isoform that crosslinks and stabilizes insoluble collagen deposition in tumor tissues. In turn, focal adhesion formation and FAK/SRC signaling is activated in mesenchymal tumor cells by crosslinked collagen in the ECM. Our study is the first to validate direct regulation of LOX and LOXL2 by the miR-200/ZEB1 axis, defines a novel mechanism driving tumor metastasis, delineates collagen as a prognostic marker, and identifies LOXL2 as a potential therapeutic target against tumor progression.

Lysyl oxidase isoforms in gastric cancer.

Gastric cancer (GC) is the fifth most frequent cancer in the world and shows the highest incidence in Latin America and Asia. An increasing amount of evidence demonstrates that lysyl oxidase isoforms, a group of extracellular matrix crosslinking enzymes, should be considered as potential biomarkers and therapeutic targets in GC. In this review, we focus on the expression levels of lysyl oxidase isoforms, its functions and the clinical implications in GC. Finding novel proteins related to the processing of these extracellular matrix enzymes might be helpful in the design of new therapies, which, in combination with classic pharmacology, could be used to delay the progress of this aggressive cancer and offer a wider temporal window for clinical intervention.

Abstract A27: Zeb1 induces LOXL2-mediated collagen stabilization and deposition in the extracellular matrix to drive lung cancer invasion and metastasis

Abstract Introduction: Lung cancer is the leading cause of cancer-related death, primarily due to distant metastatic disease. Metastatic cancer cells undergo an epithelial-to-mesenchymal transition (EMT) regulated by a double-negative feedback loop between the microRNA-200 (miR-200) family and Zeb1, but the precise mechanisms of Zeb1-dependent EMT in promoting malignancy remain largely undefined. While the cell-intrinsic effects of EMT are important for tumor progression, the reciprocal dynamic crosstalk between mesenchymal cancer cells and the extracellular matrix (ECM) is equally critical in regulating invasion and metastasis. This study investigates the collaborative effect of EMT and ECM in the metastatic process. Methods: Bioinformatic analysis of TCGA dataset was done to correlate ECM-associated gene expression with EMT gene signature scores. Western blotting and qPCR analysis of epithelial and mesenchymal lung cancer cell lines were performed to determine expression levels of collagen, LOX, and LOXL2. Lung tumor tissues from non-metastatic KrasG12D and metastatic KrasG12D;p53R172H mutant mice were analyzed by immunohistochemistry, Masson's trichrome staining, and second harmonics generation for collagen, LOX, and LOXL2 expression as well as collagen fiber organization. Syngeneic primary tumors generated by subcutaneous injection of murine lung cancer cell lines were analyzed in a similar fashion. Promoter and 3′-UTR luciferase reporter assays were performed to determine direct regulation LOXL2 and LOX by Zeb1 and miR-200, respectively. Results: Our results reveal increased collagen deposition in metastatic tumor tissues as a direct consequence of amplified collagen gene expression in Zeb1-activated mesenchymal lung cancer cells. Additionally, collagen fibers in metastatic lung tumors exhibit greater linearity and organization as a result of collagen crosslinking by the lysyl oxidase (LOX) family of enzymes. Expression of the LOX and LOXL2 isoforms is directly regulated by miR-200 and Zeb1, respectively, and their upregulation in metastatic tumors and mesenchymal cell lines is coordinated to that of collagen. Functionally, LOXL2, as opposed to LOX, is the principle isoform driving lung cancer metastasis by crosslinking and stabilizing insoluble collagen deposition in primary tumor tissues. Conclusions: Our study demonstrates that mesenchymal lung cancer cells metastasize by modulating the compositional and structural properties of the ECM through LOXL-mediated collagen crosslinking and deposition. We are the first to validate direct regulation of LOX and LOXL2 by the miR-200/Zeb1 axis, delineate collagen as a prognostic marker for lung cancer, and identify LOXL2 as a potential therapeutic target against tumor progression. Citation Format: David H. Peng, Pan Tong, Lauren A. Byers, Jing Wang, Chad J. Creighton, Don L. Gibbons. Zeb1 induces LOXL2-mediated collagen stabilization and deposition in the extracellular matrix to drive lung cancer invasion and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A27.

Matrix cross-linking lysyl oxidases are induced in response to myocardial infarction and promote cardiac dysfunction.

After myocardial infarction (MI), extensive remodelling of the extracellular matrix contributes to scar formation. While aiming to preserve tissue integrity, this fibrotic response is also associated with adverse events, including a markedly increased risk of heart failure, ventricular arrhythmias, and sudden cardiac death. Cardiac fibrosis is characterized by extensive deposition of collagen and also by increased stiffness as a consequence of enhanced collagen cross-linking. Members of the lysyl oxidase (LOX) family of enzymes are responsible for the formation of collagen cross-links. This study investigates the contribution of LOX family members to the heart response to MI. Experimental MI was induced in C57BL/6 mice by permanent ligation of the left anterior descending coronary artery. The expression of LOX isoforms (LOX and LOXL1-4) was strongly increased upon MI, and this response was accompanied by a significant accumulation of mature collagen fibres in the infarcted area. LOX expression was observed in areas of extensive remodelling, partially overlapping with α-smooth muscle actin-expressing myofibroblasts. Tumour growth factor-β as well as hypoxia-activated pathways contributed to the induction of LOX expression in cardiac fibroblasts. Finally, in vivo post-infarction treatment with the broadband LOX inhibitor β-aminopropionitrile or, selectively, with a neutralizing antibody against the canonical LOX isoform attenuated collagen accumulation and maturation and also resulted in reduced ventricular dilatation and improved cardiac function. LOX family members contribute significantly to the detrimental effects of cardiac remodelling, highlighting LOX inhibition as a potential therapeutic strategy for post-infarction recovery.

Isolation of Lysyl Oxidase from Human Umbilical Cord

Five isoforms of human lysyl oxidases (LOX, LOXL1‐4) catalyze oxidative deamination of various primary amines and ε‐amino groups of lysine residues in proteins. LOX expression is up‐regulated by hypoxia in normal and cancer tissues. Usually, LOX is isolated from bovine aorta. However, authentic human enzyme should be preferred for pharmacological studies. We have optimized the aorta LOX protocol for human umbilical cord. The procedure includes homogenization, differential washes, urea extraction, absorption on hydroxyapatite and sulfoethylcellulose, followed by chromatography on DEAE‐cellulose and gel filtration. Simultaneously, semicarbazide sensitive amine oxidase (SSAO) can be also isolated using triton extraction followed by concanavalin A and ion exchange chromatography. Amine oxidase activities were measured as hydrogen peroxide release coupled to oxidation of 10‐acetyl‐3,7‐dihydrophenoxazine by horseradish peroxidase with methylamine as a SSAO substrate and free lysine or polyallylamine for LOX. In contrast with placenta, the cord lacks monoamine oxidase and diamine oxidase. In comparison with aorta, umbilical cord is poor in SSAO, whereas the yield of active lysyl oxidase is several folds higher. Immunoblotting using polyclonal antibodies against the five lysyl oxidase isoforms indicate that the isolated preparation contains predominantly LOX. In conclusion, the human umbilical cord is an excellent source of lysyl oxidase. Supported by RFBR grant 13‐04‐01413.

LOXL1 Deficiency in the Lamina Cribrosa as Candidate Susceptibility Factor for a Pseudoexfoliation-Specific Risk of Glaucoma

To test the hypothesis that a primary disturbance in lysyl oxidase-like 1 (LOXL1) and elastin metabolism in the lamina cribrosa of eyes with pseudoexfoliation syndrome constitutes an independent risk factor for glaucoma development and progression. Observational, consecutive case series. Posterior segment tissues obtained from 37 donors with early and late stages of pseudoexfoliation syndrome without glaucoma, 37 normal age-matched control subjects, 5 eyes with pseudoexfoliation-associated open-angle glaucoma, and 5 eyes with primary open-angle glaucoma (POAG). Protein and mRNA expression of major elastic fiber components (elastin, fibrillin-1, fibulin-4), collagens (types I, III, and IV), and lysyl oxidase crosslinking enzymes (LOX, LOXL1, LOXL2) were assessed in situ by quantitative real-time polymerase chain reaction, (immuno)histochemistry, and light and electron microscopy. Lysyl oxidase-dependent elastin fiber assembly was assessed by primary optic nerve head astrocytes in vitro. Expression levels of elastic proteins, collagens, and lysyl oxidases in the lamina cribrosa. Lysyl oxidase-like 1 proved to be the major lysyl oxidase isoform in the normal lamina cribrosa in association with a complex elastic fiber network. Compared with normal and POAG specimens, lamina cribrosa tissues obtained from early and late stages of pseudoexfoliation syndrome without and with glaucoma consistently revealed a significant coordinated downregulation of LOXL1 and elastic fiber constituents on mRNA and protein level. In contrast, expression levels of collagens and other lysyl oxidase isoforms were not affected. Dysregulated expression of LOXL1 and elastic proteins was associated with pronounced (ultra)structural alterations of the elastic fiber network in the laminar beams of pseudoexfoliation syndrome eyes. Inhibition of LOXL1 interfered with elastic fiber assembly by optic nerve head astrocytes in vitro. The findings provide evidence for a pseudoexfoliation-specific elastinopathy of the lamina cribrosa resulting from a primary disturbance in LOXL1 regulation and elastic fiber homeostasis, possibly rendering pseudoexfoliation syndrome eyes more vulnerable to pressure-induced optic nerve damage and glaucoma development and progression.

Lysyl Oxidase-like-2 (LOXL2) Is a Major Isoform in Chondrocytes and Is Critically Required for Differentiation

The lysyl oxidase family is made up of five members: lysyl oxidase (LOX) and lysyl oxidase-like 1-4 (LOXL1-LOXL4). All members share conserved C-terminal catalytic domains that provide for lysyl oxidase or lysyl oxidase-like enzyme activity; and more divergent propeptide regions. LOX family enzyme activities catalyze the final enzymatic conversion required for the formation of normal biosynthetic collagen and elastin cross-links. The importance of lysyl oxidase enzyme activity to normal bone development has long been appreciated, but regulation and roles for specific LOX isoforms in bone formation in vivo is largely unexplored. Fracture healing recapitulates aspects of endochondral bone development. The present study first investigated the expression of all LOX isoforms in fracture healing. A remarkable coincidence of LOXL2 expression with the chondrogenic phase of fracture healing was found, prompting more detailed analyses of LOXL2 expression in normal growth plates, and LOXL2 expression and function in developing ATDC5 chondrogenic cells. Data show that LOXL2 is expressed by pre-hypertrophic and hypertrophic chondrocytes in vivo, and that LOXL2 expression is regulated in vitro as a function of chondrocyte differentiation. Moreover, LOXL2 knockdown studies in vitro show that LOXL2 expression is required for ATDC5 chondrocyte cell line differentiation through regulation of SNAIL and SOX9, important transcription factors that control chondrocyte differentiation. Taken together, data provide evidence that LOXL2, like LOX, is a multifunctional protein. LOXL2 promotes chondrocyte differentiation by mechanisms that are likely to include roles as both a regulator and an effector of chondrocyte differentiation.

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.

Myocardial lysyl oxidase regulation of cardiac remodeling in a murine model of diet-induced metabolic syndrome

Metabolic syndrome (MetS) represents an increased risk of cardiovascular disease. Although its individual components adversely affect cardiac structure and function, the extent to which multiple components of MetS affect the cardiac extracellular matrix (ECM) has not been well characterized. Lysyl oxidase (LOX) is one of the cardiac ECM-modifying enzymes that catalyze the formation of collagen cross-linking. Our objective was to define the effect of diet-induced MetS on the LOX enzyme. MetS was induced in male C57BL/6 mice by administrating a high-fat, high-simple carbohydrate diet for 6 mo. Gene expression was determined by real-time PCR. The cardiac protein expression and enzymatic activity of LOX were measured. The severity of fibrosis was assessed by histology and hydroxylproline assay. Cardiac diastolic function was assessed by in vivo analysis of the pressure-volume relationship. LOX, matrix metalloproteinases, and their tissue inhibitors were analyzed, and of these three, LOX was most significantly changed in the MetS mice. Despite the blunted gene expression of LOX isoforms, MetS mice demonstrated a significant upregulation of bone morphogenetic protein-1. Correspondingly, there was an increase in the ratio of protein expression of mature to proenzyme LOX by 25.9%, enhanced LOX activity by 50.0%, and increased cardiac cross-linked collagen compared with the controls. This fibrotic response coincided with a marked increase in end-diastolic pressure, increased left ventricular stiffness, and impaired diastolic filling pattern. Our data signify that diet-induced MetS alters the remodeling enzymes, mainly LOX, thereby altering ECM structure by increasing the amount of cross-linking and inducing diastolic dysfunction.

Morpholino knockdown of lysyl oxidase impairs zebrafish development, and reflects some aspects of copper metabolism disorders

Lysyl oxidase (LOX), a copper-dependent amine oxidase known in mammals to catalyze the cross-linking of collagen and elastin in the extracellular matrix, is a member of a multigenic family. Eight genes encoding lysyl oxidase isoforms have been identified in zebrafish. Recent studies have revealed a critical role for two zebrafish lysyl oxidases-like in the formation of the notochord. We now present the role of Lox in zebrafish development. lox morpholino-mediated knockdown results in a mildly undulated notochord, truncated anterior–posterior axis, tail bending and smaller head. Analyses of morphants show a complete disorganization of muscle somites and neural defects, in accordance with the lox expression pattern. Lox inhibition also induces pigment defects and pharyngeal arch deformities consistent with neural crest dysfunction. Taken together, these data reveal a role for Lox in early morphogenesis, especially in muscle development and neurogenesis, and resume some aspects of physiopathology of copper metabolism.

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.