Lysyl Oxidase Gene Research Articles

Alternative promoter activation leads to the expression of a novel human lysyl oxidase variant that functions as an amine oxidase.

The lysyl oxidase (LOX) family is an emerging family of amine oxidases that is responsible for lysine-mediated crosslinks found in collagen and elastin. Several novel functions, such as tumor suppression, tumor progression, cellular senescence, chemotaxis and the modification of histones have recently been attributed to the LOX family of proteins. In the search for more human LOX paralogs, in the present study, we identified several expressed sequence tag (EST) clones that showed an alternative exon-intron splice pattern from LOX. These ESTs corresponded to the LOX transcript variant2 (LOX-v2) that was recently reported in GenBank (accessionno.NM_001178102). LOX-v2 mRNA lacks exon1 of LOX, but contains an additional 222bp sequence from the 5'-flanking intronic region of exon2. The deduced LOX-v2 polypeptide contains the characteristic C-terminal domains of the LOX family, but does not contain the N-terminal propeptide region that has been reported to have tumor suppressor activity. In peroxidase-coupled fluorometric assays, LOX-v2 showed β-aminopropionitrile-inhibitable amine oxidase activity toward collagen and elastin. RT-PCR analysis of human tissues revealed a distinct tissue specificity of LOX-v2 expression compared to that of LOX. Promoter assays indicated that an alternative promoter element present in the exon1 region of LOX was sufficient for the differential expression of LOX-v2. These findings indicate that the human LOX gene encodes 2variants, LOX and LOX-v2, both of which function as amine oxidases with distinct tissue specificities.

Lysyl oxidase genetic variants affect gene expression in cervical cancer.

Lysyl oxidase (LOX) is a copper-dependent amine oxidase that plays important roles in the homeostasis of tumors. The aim of this study was to investigate the association between LOX polymorphisms and cervical cancer, and the effect of these polymorphisms on gene expression. We evaluated two polymorphisms of LOX, rs1800449G/A (G473A) and rs2278226C/G, in 262 cervical cancer cases and 298 healthy controls in the Chinese population. Results showed that the prevalence of rs1800449AA genotype was significantly increased in cases than in controls (p=0.004). Individuals who carried the rs1800449A allele had a 1.56-fold increased risk for cervical cancer than those with the rs1800449G allele (p=0.003). The rs2278226CG genotype also revealed a significantly higher proportion in cases (20.6%) than in controls (7.7%, p<0.001). Interestingly, when analyzing these two polymorphisms with the serum level of LOX, we identified that cervical cancer patients carrying the rs2278226CG genotype had a significantly elevated level of LOX than those with rs2278226CC wild type, whereas the same phenomenon was not observed in controls. The rs1800449 polymorphism did not affect the LOX serum level in either controls or patients. These results suggest that the polymorphisms in the LOX gene may be involved in the development of cervical cancer through various mechanisms.

Lysyl oxidase-like 2 (LOXL2) controls tumor-associated cell proliferation through the interaction with MARCKSL1

Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase gene family that contributes to the invasiveness and metastasis in tumor progression. However, the role of LOXL2 in cellular signaling is incompletely understood. In this study, we investigated a possible mechanism of LOXL2 function in tumor metastases in vitro, using a human breast carcinoma cell line. Myristoylated alanine-rich C kinase substrate-like 1 (MARCKSL1), a modulator in the regulation of cellular homeostasis, was identified as a LOXL2 interacting protein. We examined the binding domains that are required for the interaction between LOXL2 and MARCKSL1. The scavenger-receptor domain of LOXL2 was shown to interact with the N-terminal domain of MARCKSL1. Luciferase activity was noticeably reduced by the transfection of MARCKSL1 in a dose-dependent manner. In addition, over-expression of LOXL2 activates cell growth by inhibiting MARCKSL1-induced apoptosis. The effect of LOXL2 on cell cycle and apoptosis-related components was also confirmed through the silencing of LOXL2 expression. LOXL2 activates the FAK/Akt/mTOR signaling pathways, and MARCKSL1 suppresses LOXL2-induced oncogenesis. These insights supply evidence that LOXL2 promotes cell proliferation and inhibits apoptotic cell death. Taken together, our results indicate an underlying mechanism for an increase of LOXL2-related activity in breast tumor cells.

MiR-29b inhibits collagen maturation in hepatic stellate cells through down-regulating the expression of HSP47 and lysyl oxidase

Altered expression of miR-29b is implicated in the pathogenesis and progression of liver fibrosis. We and others previously demonstrated that miR-29b down-regulates the expression of several extracellular-matrix (ECM) genes including Col 1A1, Col 3A1 and Elastin via directly targeting their 3′-UTRs. However, whether or not miR-29b plays a role in the post-translational regulation of ECM biosynthesis has not been reported. Heat shock protein 47 (HSP47) and lysyl oxidase (LOX) are known to be essential for ECM maturation. In this study we have demonstrated that expression of HSP47 and LOX was significantly up-regulated in culture-activated primary rat hepatic stellate cells (HSCs), TGF-β stimulated LX-2 cells and liver tissue of CCl4-treated mice, which was accompanied by a decrease of miR-29b level. In addition, over-expression of miR-29b in LX-2 cells resulted in significant inhibition on HSP47 and LOX expression. Mechanistically, miR-29b inhibited the expression of a reporter gene that contains the respective full-length 3′-UTR from HSP47 and LOX gene, and this inhibitory effect was abolished by the deletion of a putative miR-29b targeting sequence from the 3′-UTRs. Transfection of LX-2 cells with miR-29b led to abnormal collagen structure as shown by electron-microscopy, presumably through down-regulation of the expression of molecules involved in ECM maturation including HSP47 and LOX. These results demonstrated that miR-29b is involved in regulating the post-translational processing of ECM and fibril formation.

Molecular screening of lysyl oxidase G473A polymorphism in oral submucous fibrosis.

Aim:To investigate the presence of lysyl oxidase (LOX) G473A polymorphism in group 1 {Oral submucous fibrosis (OSMF) patients}, group 2 (betel quid chewers without OSMF) and group 3 (healthy individuals).Materials and Methods:A total of 60 patients were taken for the study, which included 20 OSMF patients (group 1), 20 betel quid chewers without OSMF (group 2) and 20 healthy individuals without OSMF and betel quid chewing habit (group 3). DNA was isolated using Qiagen kit. The isolated DNA was quantified using spectroscopic methods. Polymerase chain reaction (PCR) was carried out at annealing temperature of 67o C. PCR amplification was checked on 2% agarose gel. Further, the amplified PCR products were subjected to automated DNA sequencer, to assess LOX G473A polymorphism.Results:The gene sequence data generated from the automated DNA sequencer was received as colored electropherograms. These gene-sequencing results did not show LOX G473A polymorphism in any of the 3 groups.Conclusion:In our study, gene-sequencing results did not show LOX G473A polymorphism in OSMF patients. Since only one study in the literature has shown the association of LOX gene polymorphism and OSMF patients, we conclude that further studies are required to unveil the role of LOX gene polymorphism in OSMF.

Inhibition of CIN85-Mediated Invasion by a Novel SH3 Domain Binding Motif in the Lysyl Oxidase Propeptide

The lysyl oxidase gene inhibits Ras signaling in transformed fibroblasts and breast cancer cells. Its activity was mapped to the 162 amino acid propeptide domain (LOX-PP) of the lysyl oxidase precursor protein. LOX-PP inhibited the Her-2/Ras signaling axis in breast cancer cells, and reduced the Her-2-driven breast tumor burden in a xenograft model. Since its mechanism of action is largely unknown, co-affinity-purification/mass spectrometry was performed and the “Cbl-interacting protein of 85-kDa” (CIN85) identified as an associating protein. CIN85 is an SH3-containing adapter protein that is overexpressed in invasive breast cancers. The CIN85 SH3 domains interact with c-Cbl, an E3 ubiquitin ligase, via an unconventional PxxxPR ligand sequence, with the highest affinity displayed by the SH3-B domain. Interaction with CIN85 recruits c-Cbl to the AMAP1 complex where its ubiquitination activity is necessary for cancer cells to develop an invasive phenotype and to degrade the matrix. Direct interaction of LOX-PP with CIN85 was confirmed using co-immunoprecipitation analysis of lysates from breast cancer cells and of purified expressed proteins. CIN85 interaction with c-Cbl was reduced by LOX-PP. Domain specific CIN85 regions and deletion mutants of LOX-PP were prepared and used to map the sites of interaction to the SH3-B domain of CIN85 and to an epitope encompassing amino acids 111 to 116 of LOX-PP. Specific LOX-PP point mutant proteins P111A and R116A failed to interact with CIN85 or to compete for CIN85 binding with c-Cbl. Structural modeling identified a new atypical PxpxxRh SH3-binding motif in this region of LOX-PP. The LOX-PP interaction with CIN85 was shown to reduce the invasive phenotype of breast cancer cells, including their ability to degrade the surrounding extracellular matrix and for Matrigel outgrowth. Thus, LOX-PP interacts with CIN85 via a novel SH3-binding motif and this association reduces CIN85-promoted invasion by breast cancer cells.

Lack of association of Lysyl oxidase (LOX) gene polymorphisms with intracranial aneurysm in a south Indian population

Intracranial aneurysm (IA) accounts for 85 % of haemorrhagic stroke and is mainly caused due to weakening of arterial wall. Lysyl oxidase (LOX) is a cuproenzyme involved in cross linking structural proteins collagen and elastin, thus providing structural stability to artery. Using a case-control study design, we tested the hypothesis whether the variants in LOX gene flanking the two LD block, can increase risk of aSAH among South Indian patients, either independently, or by interacting with other risk factors of the disease. SNPs were genotyped by fluorescence-based competitive allele-specific PCR (KASPar) chemistry. We selected 200 radiologically confirmed aneurysmal cases and 235 ethnically and age and gender matched controls from the Dravidian Malayalam speaking population of South India. We observed marked interethnic differences in the genotype distribution of LOX variants when compared to Japanese and African populations. However, there was no significant association with any of the LOX variants with IA. This study also could not observe any significant role of LOX polymorphisms in influencing IA either directly or indirectly through its confounding factors such as hypertension and gender in South Indian population.

Lysyl oxidase secreted by tumour endothelial cells promotes angiogenesis and metastasis

Background:Molecules that are highly expressed in tumour endothelial cells (TECs) may be candidates for specifically targeting TECs. Using DNA microarray analysis, we found that the lysyl oxidase (LOX) gene was upregulated in TECs compared with its expression in normal endothelial cells (NECs). LOX is an enzyme that enhances invasion and metastasis of tumour cells. However, there are no reports on the function of LOX in isolated TECs.Methods:TECs and NECs were isolated to investigate LOX function in TECs. LOX inhibition of in vivo tumour growth was also assessed using β-aminopropionitrile (BAPN).Results:LOX expression was higher in TECs than in NECs. LOX knockdown inhibited cell migration and tube formation by TECs, which was associated with decreased phosphorylation of focal adhesion kinase (Tyr 397). Immunostaining showed high LOX expression in human tumour vessels in vivo. Tumour angiogenesis and micrometastasis were inhibited by BAPN in an in vivo tumour model.Conclusion:LOX may be a TEC marker and a possible therapeutic target for novel antiangiogenic therapy.

Interactive Effect of Bisphenol A (BPA) Exposure with -22G/C Polymorphism in LOX Gene on the Risk of Osteosarcoma

Osteosarcomas have many established risk factors, both genetic and environmental, but by themselves these explain only part of the total cancer incidence. Bisphenol A (BPA) is an environmental estrogen associated with risk of several kinds of tumour. The lysyl oxidase gene (LOX) may also contribute to risk of tumours including osteosarcomas. Here, we investigated possible interactions of BPA and a LOX polymorphism on the risk of osteosarcoma. The present hospital-based case-control study included 106 cancer patients and 112 controls from a Chinese population. Internal burden of BPA exposure was assessed using high-performance liquid chromatography-mass spectrometry (HPLC-MS) method. Genotypes were determined using PCR-RFLP methods. Compared with those in low BPA exposure group, subjects with BPA more than or equal to median value had significant increased risk of osteosarcoma among subjects who carried GC or CC genotypes. A significant interaction with BPA level and the -22 G/C polymorphism was observed for osteosarcoma overall, osteosarcoma affecting knee and osteosarcoma affecting hip, as P(forinteraction) = 0.036 for osteosarcoma overall; P(forinteraction) = 0.024 for osteosarcoma affecting knee; and P(forinteraction) = 0.017 for osteosarcoma affecting hip. The results suggest that BPA exposure interacts with the -22 G/C polymorphism of the LOX gene to increase the risk of osteosarcoma.

Lysyl oxidase genetic variants and the prognosis of glioma

Lysyl oxidase (LOX) is a copper-dependent amine oxidase that plays important roles in the development and homeostasis of primary brain tumors such as glioma. The aim of this study was to investigate whether polymorphisms in the LOX gene were associated with susceptibility to glioma. We tested two functional polymorphisms of LOX, -22G/C and 473G/A, and compared them between 466 glioma cases and 502 healthy controls in the Chinese population. Results showed that the prevalence of 473AA genotype was significantly increased in cases than in controls (p = 0.001). Individuals who carried 473A allele had a 1.44-fold of increased risk for glioma than those with 473G allele (p = 0.002). In addition, when analyzing the survival time of glioma patients with LOX 473G/A polymorphism, cases with AA genotype had significantly shorter survival time compared to the patients carrying G allele (25.0 months vs 43.0 months, p = 0.0009). These results suggested that polymorphism in LOX gene was associated with increased susceptibility to glioma and could be used as prognostic factor for this malignancy.

Gremlin utilizes canonical and non-canonical TGFβ signaling to induce lysyl oxidase (LOX) genes in human trabecular meshwork cells

The TGFβ/BMP signaling pathways are involved in glaucomatous damage to the trabecular meshwork (TM) leading to elevated intraocular pressure (IOP), which is a major risk factor for the development and progression of glaucoma. The BMP antagonist gremlin is elevated in glaucomatous TM cells and tissues and can directly elevate IOP. Gremlin utilizes the TGFβ2/SMAD pathway to induce TM extracellular matrix (ECM) proteins. The purpose of this study is to determine whether expression of the ECM cross-linking lysyl oxidase (LOX) genes is regulated by gremlin in cultured human TM cells. Human TM cells were treated with recombinant gremlin, and expression of the LOX genes was examined by quantitative RT-PCR and western immunoblotting. TM cells were pretreated with TGFBR inhibitors (LY364947 or SB431542), an inhibitor of the SMAD signaling pathway (SIS3), or with JNK (SP600125) and p38 MAPK (SB203580) inhibitors to identify the signaling pathway(s) involved in gremlin induction of LOX protein expression. All five LOX genes (LOX and LOXL1–4) were induced by gremlin. Gremlin induction of LOX genes and protein expression was blocked by TGFBR inhibitors as well as by inhibitors of the SMAD3, JNK and p38 MAPK signaling pathways. We conclude that gremlin employs both canonical TGFβ/SMAD and the non-canonical JNK and p38 MAPK signaling pathways to induce LOX genes and proteins in cultured human TM cells. Increased LOX levels may be at least partially responsible for gremlin-mediated IOP elevation and increased aqueous humor outflow resistance leading to glaucoma.

Lysyl oxidase is downregulated by the EWS/FLI1 oncoprotein and its propeptide domain displays tumor supressor activities in Ewing sarcoma cells.

Ewing sarcoma is the second most common bone malignancy in children and young adults. It is driven by oncogenic fusion proteins (i.e. EWS/FLI1) acting as aberrant transcription factors that upregulate and downregulate target genes, leading to cellular transformation. Thus, identificating these target genes and understanding their contribution to Ewing sarcoma tumorigenesis are key for the development of new therapeutic strategies. In this study we show that lysyl oxidase (LOX), an enzyme involved in maintaining structural integrity of the extracellular matrix, is downregulated by the EWS/FLI1 oncoprotein and in consequence it is not expressed in Ewing sarcoma cells and primary tumors. Using a doxycycline inducible system to restore LOX expression in an Ewing sarcoma derived cell line, we showed that LOX displays tumor suppressor activities. Interestingly, we showed that the tumor suppressor activity resides in the propeptide domain of LOX (LOX-PP), an N-terminal domain produced by proteolytic cleavage during the physiological processing of LOX. Expression of LOX-PP reduced cell proliferation, cell migration, anchorage-independent growth in soft agar and formation of tumors in immunodeficient mice. By contrast, the C-terminal domain of LOX, which contains the enzymatic activity, had the opposite effects, corroborating that the tumor suppressor activity of LOX is mediated exclusively by its propeptide domain. Finally, we showed that LOX-PP inhibits ERK/MAPK signalling pathway, and that many pathways involved in cell cycle progression were significantly deregulated by LOX-PP, providing a mechanistic explanation to the cell proliferation inhibition observed upon LOX-PP expression. In summary, our observations indicate that deregulation of the LOX gene participates in Ewing sarcoma development and identify LOX-PP as a new therapeutic target for one of the most aggressive paediatric malignancies. These findings suggest that therapeutic strategies based on the administration of LOX propeptide or functional analogues could be useful for the treatment of this devastating paediatric cancer.

Influence of interfering lysyl oxidase gene on biological features of MDA-MB-231 cells |

Objective To observe the influence of RNAi against lysyl oxidase (LOX) gene on biological features of MDA-MB-231 cells.Methods The MDA-MB-231 cells were transfected with lentiviral interference vector (LOX-RNAi-LV),and divided into three groups:the experimental group,the blank group and the negative group.The growth and proliferation of cells were determined by using methyl thiazol tetrazolium (MTT) assay,cell colony formation and flow cytometry.The migration and invasion were observed by using transwell assays.Results The cell growth rate in the experimental group,the blank group and the negative group at 24,48,72 and 96 h was (12.73 ± 3.30) ％,(33.93 ± 1.99) ％,(52.63 ±1.04)％,(63.80±0.70)％;0,0,0,0; (7.60±6.27)％,(10.30±1.49)％,(9.10±2.69)％,(6.03 ± 1.21) ％,respectively (P ＜ 0.05).Cells invading between three groups was 47 ± 2 ; 100 ± 1 ; 88 ± 2 (P ＜ 0.05).Cell migration was 63 ± 2 ; 118 ± 2 ; 96 ± 2 (P ＜ 0.05).The difference of the cell cycle was not statistically significant (P ＞ 0.05).Conclusion After the LOX gene expression restrained,the creature feature of MDA-MB-231 were effectively suppressed. Key words: Breast carcinoma; Lysyl oxidase gene; RNA interference; Biological characteristics

The expression of lysyl‐oxidase gene family members in myeloproliferative neoplasms

Myeloproliferative neoplasms (MPNs) are malignant disorders originating from clonal expansion of a single neoplastic stem cell and characteristically show an increase in bone marrow reticulin fibers. Lysyl oxidases (LOXs) are copper-dependent amine oxidases that play a critical role in the biogenesis of connective tissue by crosslinking extracellular matrix proteins, collagen and elastin. Expression of LOX gene family members is increased in disorders associated with increased fibrosis. To evaluate involvement of LOX gene family in various MPNs. In-situ hybridization was used to detect Lysyl-Oxidase family members in bone marrow biopsies from patients with different MPNs. We compared normal bone marrows and those from patients with polycythemia vera, essential thrombocythemia, chronic myeloid leukemia, and primary myelofibrosis (PMF). Serum levels of lysyl-oxidase from patients with PMF and healthy controls were also examined. LOX gene family was not detected in normal bone marrows. All members of the LOX gene family were over expressed in PMF. In other MPNs a differential pattern of expression was observed. Differences in gene expression were statistically significant (P < 0.010). The medianserum LOX levels in normal controls was 28.4 ± 2.5 ng\ml and 44.6 ± 9.44 ng\ml in PMF (P = 0.02). The varying pattern of expression of LOX genes may reflect differences in the pathophysiology of bone marrow fibrosis in these MPNs. These observations could be used as the basis for future targeted therapy directed against bone marrow fibrosis.

Effects of Lysyl Oxidase Genetic Variants on the Susceptibility to Rhegmatogenous Retinal Detachment and Proliferative Vitreoretinopathy

Proliferative vitreoretinopathy (PVR), the most common cause of failure of rhegmatogenous retinal detachment (RD) surgery, is an anomalous scarring process related to ocular inflammation. Lysyl oxidase (LOX) is a copper-dependent amine oxidase that may play important roles in ocular tissue integrity. The aim of this study was to investigate whether polymorphisms in the LOX gene were associated with susceptibility to RD and PVR. We screened the promoter region of LOX gene and tested two previously reported polymorphisms (-22 G/C and 473 G/A) in RD patients with or without PVR and healthy controls. Data showed that prevalence of the -22CC genotype and -22C allele were significantly higher in the RD cases than in the control group after adjustment for sex and age (p < 0.001 and p < 0.001, respectively). Similarly, a significant difference was observed regarding LOX 473GA genotype and 473A allele between RD patients and healthy donors after adjustment for sex and age (p = 0.005 and p = 0.012, respectively). Also, when compared to RD cases without PVR, patients who developed PVR had significantly higher numbers of -22CC genotype and -22C allele (p = 0.048 and p = 0.003, respectively). These results indicated that LOX polymorphisms were associated with increased susceptibility to RD and PVR and suggest a potential correlation between LOX and ocular inflammation.

Hypoxia-induced collagen crosslinking as a mechanism for enhancing mechanical properties of engineered articular cartilage

The focus of tissue engineering of neocartilage has traditionally been on enhancing extracellular matrix and thus biomechanical properties. Emphasis has been placed on the enhancement of collagen type and quantity, and, concomitantly, tensile properties. The objective of this study was to improve crosslinking of the collagen network by testing the hypothesis that hypoxia could promote pyridinoline (PYR) crosslinks and, thus, improve neocartilage's tensile properties. Chondrocyte expression of lysyl oxidase (LOX), an enzyme responsible for the formation of collagen PYR crosslinks, was first assessed pre- and post- hypoxia application. Then, the mechanical properties of self-assembled neocartilage constructs were measured, after 4 weeks of culture, for groups exposed to 4% O2 at different initiation times and durations, i.e., during the 1st and 3rd weeks, 3rd and 4th weeks, 4th week only, continuously after cell seeding, or never. Results showed that LOX gene expression was upregulated ∼20-fold in chondrocytes in response to hypoxia. Hypoxia applied during the 3rd and 4th weeks significantly increased PYR crosslinks without affecting collagen content. Excitingly, neocartilage tensile properties were increased ∼2-fold. It should be noted that these properties exhibited a distinct temporal dependence to hypoxia exposure, since upregulation of these properties was due to hypoxia applied only during the 3rd and 4th weeks. These data elucidate the role of hypoxia-mediated upregulation of LOX and subsequent increases in PYR crosslinks in engineered cartilage. These results hold promise toward applying hypoxia at precise time points to promote tensile integrity and direct construct maturation.

Upregulated expression of LOX is a novel independent prognostic marker of worse outcome in gastric cancer patients after curative surgery

Lysyl oxidase (LOX) initiates the enzymatic stage of collagen and elastin cross-linking. It also has intracellular functions involved in the regulation of cell differentiation, motility/migration and gene transcription. Aberrant expression of the LOX gene has been reported in multiple tumors. However, the correlation of its expression with clinicopathological parameters and its prognostic significance in gastric cancer remains largely unknown. In order to address this problem, total RNA of paired tissue samples (n=10) and a tissue microarray containing 161 paired tissues from patients with gastric cancers at different stages were collected. Quantitative real-time PCR and immunochemistry assay were conducted to investigate the expression of LOX. Based on the results, LOX mRNA was increased in gastric cancer tissues compared with the adjacent normal mucosa. Immunohistochemical detection revealed that expression of LOX was associated with depth of tumor invasion (P<0.05), lymph node status (P<0.05), TNM stage (P<0.05) and survival (P<0.05). Cox regression analysis revealed that positive expression of LOX (P=0.026) was an independent prognostic marker for survival in patients with gastric cancer.

Differential regulation of collagen, lysyl oxidase and MMP‐2 in human periodontal ligament cells by low‐ and high‐level mechanical stretching

Mechanical stretching modulates extracellular matrix (ECM) protein synthesis by periodontal ligament (PDL) cells. However, the mechanoregulation of lysyl oxidase (LOX), a key enzyme for collagen cross-linking, is not fully understood. In the present study, we hypothesized that low-level and high-level mechanical stretching differentially regulates collagen deposition and the expression of LOX and the enzymes responsible for ECM degradation, such as MMP-2 in PDL cells. Human PDL cells were cultured on flexible-bottom culture plates and subjected to cyclic mechanical stretching (3% and 10% elongation at 0.1 Hz) for 24 and 48 h in a Flexercell FX-4000 strain unit. The levels of expression of type I collagen alpha 1 (COL1A1), type III collagen alpha 1 (COL3A1), lysyl oxidase (LOX), MMP2 and TIMP2 mRNAs were analyzed using an RT-PCR technique. The cell layer and the culture medium were separately collected and processed for detection of the following ECM-related molecules: (i) total collagen content using a Sircol dye-binding method; (ii) LOX protein expression by western blotting; (iii) LOX activity using a fluorometric assay; and (iv) MMP-2 enzyme activity by gelatin zymography. Low-level (3%) mechanical stretching of PDL cells upregulated the expression of COL1A1, COL3A1 and LOX mRNAs, enhanced the production of collagen and increased the LOX activity but did not change the level of expression of MMP2 or TIMP2 mRNA. The collagen content and LOX activity showed obvious elevation in the medium, but not in the cell layer. High-level (10%) mechanical stretching downregulated COL1A1 mRNA but upregulated COL3A1 mRNA; however, the effect on COL3A1 was smaller, and occurred earlier, compared with the effect on the COL1A1 gene. High-level mechanical stretching upregulated the expression of MMP2 and TIMP2 mRNAs but did not change collagen production or LOX activity. Moreover, high-level mechanical stretching increased the level of pro-MMP-2, especially in the cell layer. This study substantiates the mechanoregulation of the expression of ECM-related molecules in PDL cells. High-level mechanical stretching upregulated the expression of MMP2 and TIMP2 mRNAs, but did not affect collagen production or LOX activity. In addition to increasing the transcription of COL1A1, COL3A1 and LOX genes, low-level mechanical stretching enhanced total collagen production and LOX activity, which should favor ECM stabilization. As an effective regulator of ECM remodeling, mechanical stretching can be exploited in periodontal regeneration and ligament tissue engineering via application of appropriate mechanical stimulation.

The expression and role of Lysyl oxidase (LOX) in dentinogenesis

To establish whether eliminating Lysyl oxidase (LOX) gene would affect dentine formation. Newborn wild-type (wt) and homo- and heterozygous LOX knock-out (Lox(-/-) and Lox(+/-) , respectively) mice were used to study developing tooth morphology and dentine formation. Collagen aggregation in the developing dentine was examined histochemically with picrosirius red (PSR) staining followed by polarized microscopy. Because Lox(-/-) die at birth, adult wt and Lox(+/-) mouse tooth morphologies were examined with FESEM. Human odontoblasts and pulp tissue were used to study the expression of LOX and its isoenzymes with Affymetrix cDNA microarray. No differences between Lox(-/-) , Lox(+/-) and wt mice developing tooth morphology were seen by light microscopy. Histochemically, however, teeth in wt mice demonstrated yellow-orange and orange-red polarization colours with PSR staining, indicating thick and more densely packed collagen fibres, whilst in Lox(-/-) and Lox(+/-) mice, most of the polarization colours were green to green-yellow, indicating thinner, less aggregated collagen fibres. Fully developed teeth did not show any differences between Lox(+/-) and wt mice with FESEM. Human odontoblasts expressed LOX and three of four of its isoenzymes. The data indicate that LOX is not essential in dentinogenesis, even though LOX deletion may affect dentine matrix collagen thickness and packing. The absence of functional LOX may be compensated by LOX isoenzymes.

Hypoxia-Response Element (HRE)–Directed Transcriptional Regulation of the Rat Lysyl Oxidase Gene in Response to Cobalt and Cadmium

Lysyl oxidase (LO) catalyzes crosslink of collagen, elastin, and histone H1, stabilizing the extracellular matrix and cell nucleus. This enzyme displays dual functions for tumorigenesis, i.e., as a tumor suppressor inactivating the ras oncogene and as a tumor promoter enhancing malignant cell metastasis. To elucidate LO transcriptional regulation, we have cloned the 804 base pair region upstream of the translation start site (ATG) of the rat LO gene with the maximal promoter activity. Computer analysis indicated that at least four hypoxia-response element (HRE) consensuses (5'-ACGTG-3') exist in the cloned LO promoter. Treatment of rat lung fibroblasts (RFL6) with CoCl2 (Co, 10-100 μM), a chemical hypoxia reagent, enhanced LO mRNA expression and promoter activities. Overexpression of LO was associated with upregulation of hypoxia-inducible factor (HIF)-1α at mRNA levels in cobalt (Co)-treated cells. Thus, LO is a hypoxia-responsive gene. Dominant negative-HIF-1α inhibited LO promoter activities stimulated by Co. Electrophoretic mobility shift, oligonucleotide competition, and in vitro translated HIF-1α binding assays indicated that only one HRE mapped at -387/-383 relative to ATG was functionally active among four consensuses. Site-directed mutation of this HRE significantly diminished the Co-induced and LO promoter-directed expression of the reporter gene. Cadmium (Cd), an inducer of reactive oxygen species, inhibited HIF-1α mRNA expression and HIF-1α binding to the LO gene in Co-treated cells as revealed by RT-PCR and ChIP assays, respectively. Thus, modulation of the HRE activity by Co and Cd plays a critical role in LO gene transactivation.