Osteoarthritis (OA) is a degenerative joint disease that affects an estimated 9.6 % of men and 18 % of women over 60 years of age. However, there is no chondroprotective agent that is approved for clinical application. We showed that LOXL2 is elevated in the regenerative response during fracture healing in mice and has a critical role in chondrogenic differentiation. Indeed, LOXL2 is an anabolic effector that attenuates pro-inflammatory signaling in OA cartilage of the TMJ and knee joint, induces chondroprotective and regenerative responses. The goal of the present study was to evaluate if LOXL2 adenovirus in vivo promotes protective and anabolic responses in the knee and TMJ-OA cartilage. We employed (Cho/+) OA mouse model to assess knee and TMJ-OA, which is a genetic model that develops progressive TMJ-OA due to a mutation in the Col11a1 gene. Intraperitoneal injection every 2 weeks of Adv-RFP-LOXL2 in four-month-old Cho/+ male and female mice for 16 weeks upregulated Sox9, aggrecan (Acan) and other anabolic genes in the knee and TMJ. Next, to evaluate if LOXL2 expression occurred in degenerative lesions in human clinical TMJ-OA, immunofluorescence and confocal image analysis were performed. LOXL2 has the potential to induce anabolic gene expression in the progressive knee and TMJ-OA mouse model. We showed for the first time that LOXL2-related functions could be useful for anabolic therapies for Cho/+ mice progressive knee and TMJ-OA. Thus, these data show that LOXL2 could have clinical translational applications in the future for OA-related anabolic therapies.

Lysyl oxidase (LOX) and the four lysyl oxidase like proteins, LOXL, LOXL2, LOXL3 and LOXL4, are copper-containing amine oxidases constitute a heterogeneous family of enzymes that oxidize primary amine substrates to reactive aldehydes, catalyzing the cross-linking of extracellular matrix (ECM) proteins. LOXL2 induces epithelial-to-mesenchymal transition (EMT), which is associated with hypoxia, enhanced invasion, cancer metastasis and poorer cancer prognosis. Furthermore, upregulation of LOXL2 mRNA and/ or protein levels has been detected in undifferentiated breast, colon, esophagus and larynx carcinomas. The aim here is to create and optimize a method to produce large yields of enzymatically active recombinant LOXL2 protein from mammalian cells. Two viral transductions systems were used to transfect CHO-K1 cells to overexpress LOXL2 protein. Comparing lentivirus transduction with adenovirus transduction, it was found that adenovirus transduction expressed 2.18 fold the amount of enzymatically active LOXL2 compared to lentivirus transduction (P<0.05). The average LOXL2 yield of lentivirus and adenovirus transduction systems as calculated by BCA assay was 184.5 µg and 403 µg, respectively. The average specific LOXL2 enzymatic activity were calculated using an Amplex red assay and found to be 0.443 and 0.444 nmol/μg of LOXL2 in 30 minutes in lentivirus and adenovirus methods, respectively, with no statistically significant difference (P>0.05). Expression and purification of LOXL2 were confirmed by SDS-PAGE and Western blot. Optimizing this method to purify large yields of LOXL2 is a practical aid in revealing the exact structure and function of the LOX family of proteins.

Lysyl oxidase like-2 (LOXL2) was found to be present extracellularly in primary human gingival fibroblast cells. This project has been primarily focused on investigating our hypothesis that LOXL2 may play a critical role in regulating cell proliferation and collagen accumulation in primary human gingival fibroblast cells, which may contribute to the development of fibrotic changes in human gingival tissue. LOXL2 shRNA lentivirus reduced the LOXL2 mRNA and protein expression by 90 – 95%. Knockdown of LOXL2 or inhibition of LOXL2 enzymatic activity strongly inhibited both basal and CCN2/CTGF-stimulated collagen accumulation (p<0.05). Proliferation assays demonstrated a marked decrease in cell proliferation in both the short and long term in LOXL2 shRNA knockdown cells with minimal or no stimulation of cell apoptosis. Pharmacologic inhibition of LOXL2 enzyme activity reduced basal and CCN2/CTGF-stimulated cell proliferation (40% and 50%) in short term cultures. Furthermore, there was 15-20% inhibition seen in long term assays. Recombinant active LOXL2 significantly increased collagen accumulation and cell proliferation (p<0.05). Thereby, our investigation in vitro by loss and gain of function experiments confirmed that LOXL2 is critically required for both gingival fibroblast proliferation and for collagen accumulation in the presence or absence of CCN2/CTGF. LOXL2 stimulation is critical for both proliferation and collagen accumulation in primary human gingival fibroblasts. Lastly, we found that the presence of LOXL2 extracellularly and LOXL2 may regulate cell proliferation by enhancing the phosphorylation of PDGFR.

BACKGROUND: Osteoarthritis (OA) is the most common degenerative joint disease which affects the joint structures leading to disability. Studies in the last 20 years have documented the increased prevalence of knee pain and symptomatic knee OA. Similarly, of temporomandibular joint (TMJ) disorders OA is the most common. Lysyl oxidase like-2 (LOXL2) is a copper-dependent amine oxidase. previous studies showed that LOXL2 is elevated during mouse fracture healing. Our hypothesis that LOXL2 acts as a specific anabolic factor in chondrocytes METHODS: The activity of LOXL2 in human articular and TMJ chondrocytes was assessed by cell-based assays and RT-qPCR, and LOXL2-mediated activation of NF-κB and extracellular signal-related kinase (ERK) signaling pathways was measured by western blotting. To examine LOXL2-induced effect in vivo, we implanted Matrigel-imbedded human chondrocytes into nude mice and exposed them to exogenous LOXL2 for 6 weeks. We also examined if LOXL2 induces the proliferation of OA chondrocytes. RESULTS: LOXL2 staining was detected in damaged regions of human TMJ, hip and knee joints affected by OA. Stimulation with transforming growth factor (TGF)-β1 upregulated LOXL2 expression, while pro-inflammatory cytokines IL-1β and TNF-α downregulated LOXL2, in human chondrocytes. LOXL2 expression also inhibited IL-1β-induced phospho-NF-κB/p65 and TGF-β1-induced ERK1/2 phosphorylation. Matrigel constructs of human chondrocytes from the knee joint and TMJ implanted in nude mice showed anabolic responses after LOXL2 transduction, including increased expression of SOX9, ACAN, and COL2A1. We have found that LOXL2 does not induce the proliferation of human TMJ or knee OA chondrocytes. CONCLUSIONS: We showed that LOXL2 induces differentiation and attenuates OA related catabolic signaling pathways.

The epithelial to mesenchymal transition (EMT) is a highly conserved cellular program that allows well-­‐differentiated epithelial cells to convert to motile mesenchymal cells. EMT is critical for appropriate embryogenesis and plays a crucial role in tumorigenesis and cancer progression. At this regard, it has become increasingly evident that, in addition to genetic alterations, tumour development involves the alteration of gene expression patterns owing to epigenetic changes. Taking this into account, this thesis mainly addresses the description of new molecular epigenetic mechanisms underlying one of the hallmark processes governing EMT, the Snail1-­‐mediated E-­‐cadherin repression. Indeed, our results demonstrate that both Polycomb group (PcG) proteins and the LOXL2 protein are involved in this process. Apart from providing novel insights into the significance of these proteins in tumor progression, our work uncovers the characterization of a new epigenetic modification carried out by LOXL2; H3K4 deamination. / La transició epiteli-­‐mesènquima (EMT) és un programa cel·lular molt conservat que permet a les cèl·lules epitelials convertir-­‐se en cèl·lules mesenquimals indiferenciades. La EMT és un procés crucial pel desenvolupament embrionari i per la progressió tumoral. A aquest respecte, ha esdevingut cada cop més evident que el desenvolupament tumoral no només està associat a alteracions genètiques, sinó també a l'alteració de l’expressió gènica causada per canvis epigenètics. Tenint això en compte, aquesta tesi es centra en la descripció de nous mecanismes moleculars en l’àmbit de l’epigenètica associats a un dels processos clau en la EMT, la repressió de la E-­‐ cadherina mitjançada pel factor de transcripció Snail1. De fet, els nostres resultats demostren que tant les proteïnes del grup Polycomb (PcG) com la proteïna LOXL2 estan implicades en aquest procés. A part de proporcionar nova informació respecte la importància d'aquestes proteïnes en la progressió tumoral, la nostra feina ha permès la caracterització d'una nova modificació epigenètica duta a terme per la proteïna LOXL2; la deaminació de H3K4.