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In the United States, 5–12% of adults have at least one symptom of temporomandibular joint (TMJ) disorders, including TMJ osteoarthritis (TMJ-OA). 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, and attenuates NF-kB signaling. The specific goal of the study was to evaluate if adenoviral delivery of LOXL2 is anabolic to human and mouse TMJ condylar cartilage in vivo and evaluate the protective and anabolic effect on cartilage-specific factors. We employed two different models to assess TMJ-OA. In one model, clinical TMJ-OA cartilage from 5 different samples in TMJ-OA cartilage plugs were implanted subcutaneously in nude mice. Adenovirus LOXL2 -treated implants showed higher mRNA levels of LOXL2, ACAN, and other anabolic genes compared to the adenovirus-Empty-treated implants. Further characterization by RNA-seq analysis showed LOXL2 promotes proteoglycan networks and extracellular matrix in human TMJ-OA cartilage implants in vivo. In order to evaluate if LOXL2-induced functional and sex-linked differences, both male and female four-month-old chondrodysplasia (Cho/+) mice, which develop progressive TMJ-OA due to a point mutation in the Col11a1 gene, were subjected to intraperitoneal injection with Adv-RFP-LOXL2 every 2 weeks for 12 weeks. The data showed that adenovirus delivery of LOXL2 upregulated LOXL2 and aggrecan (Acan), whereas MMP13 expression was slightly downregulated. The fold change expression of Acan and Runx2 induced by Adv-RFP-LOXL2 was higher in females compared to males. Interestingly, Adv-RFP-LOXL2 injection significantly increased Rankl expression in male but there was no change in females, whereas VegfB gene expression was increased in females, but not in males, as compared to those injected with Adv-RFP-Empty in respective groups. Our findings indicate that LOXL2 can induce specifically the expression of Acan and other anabolic genes in two preclinical models in vivo. Further, LOXL2 has beneficial functions in human TMJ-OA cartilage implants and promotes gender-specific anabolic responses in Cho/+ mice with progressive TMJ-OA, suggesting its merit for further study as an anabolic therapy for TMJ-OA.

// Saqer F. Alsaqer 1,* , Mustafa M. Tashkandi 1,* , Vinay K. Kartha 2,3 , Ya-Ting Yang 1 , Yazeed Alkheriji 1 , Andrew Salama 4 , Xaralabos Varelas 5 , Maria Kukuruzinska 1 , Stefano Monti 2,3 and Manish V. Bais 1 1 Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, MA, USA 2 Bioinformatics Program, Boston University, Boston, MA, USA 3 Section of Computational Biomedicine, Boston University School of Medicine, Boston, MA, USA 4 Department of Oral and Maxillofacial Surgery, Boston University Henry M. Goldman School of Dental Medicine, Boston, MA, USA 5 Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA * These authors have contributed equally to this work Correspondence to: Manish V. Bais, email: // Keywords : LSD1, CTGF, OSCC, PDX, orthotopic mouse model Received : December 28, 2016 Accepted : July 14, 2017 Published : July 27, 2017 Abstract Lysine-specific demethylase 1 (LSD1) is a nuclear histone demethylase and a member of the amine oxidase (AO) family. LSD1 is a flavin-containing AO that specifically catalyzes the demethylation of mono- and di-methylated histone H3 lysine 4 through an FAD-dependent oxidative reaction. LSD1 is inappropriately upregulated in lung, liver, brain and esophageal cancers, where it promotes cancer initiation, progression, and metastasis. However, unlike other lysine-specific demethylases, the role and specific targets of LSD1 in oral squamous cell carcinoma (OSCC) pathogenesis remain unknown. We show that LSD1 protein expression was increased in malignant OSCC tissues in a clinical tissue microarray, and its expression correlated with progressive tumor stages. In an orthotopic oral cancer mouse model, LSD1 overexpression in aggressive HSC-3 cells promoted metastasis whereas knockdown of LSD1 inhibited tumor spread, suggesting that LSD1 is a key regulator of OSCC metastasis. Pharmacological inhibition of LSD1 using a specific small molecule inhibitor, GSK-LSD1, down-regulated EGF signaling pathway. Further, GSK-LSD1 attenuates CTGF/CCN2, MMP13, LOXL4 and vimentin expression but increased E-cadherin expression in pre-existing, patient-derived tonsillar OSCC xenografts. Similarly, GSK-LSD1 inhibited proliferation and CTGF expression in mesenchymal cells, including myoepithelial cells and osteosarcoma cells. In addition, gene set enrichment analysis revealed that GSK-LSD1 increased p53 expression and apoptosis while inhibiting c-myc, β-catenin and YAP-induced oncogenic transcriptional networks. These data reveal that aberrant LSD1 activation regulates key OSCC microenvironment and EMT promoting factors, including CTGF, LOXL4 and MMP13.

Background: The goal of this study was to determine if adenovirus-delivered LOXL2 protects against progressive knee osteoarthritis (OA), assess its specific mechanism of action, and determine if the overexpression of LOXL2 in transgenic mice can protect against the development of OA-related cartilage damage and joint disability. Methods: Four-month-old Cho/+ male and female mice were intraperitoneally injected with either Adv-RFP-LOXL2 or an empty vector twice a month for four months. The proteoglycan levels and the expression of anabolic and catabolic genes were examined by immunostaining and qRT-PCR. The effect of LOXL2 expression on signaling was tested via the pro-inflammatory cytokine IL1&beta, in the cartilage cell line ATDC5. Finally, the OA by monosodium iodoacetate (MIA) injection was also induced in transgenic mice with systemic overexpression of LOXL2 and examined gene expression and joint function by treadmill tests and assessment of allodynia. Results: The adenovirus treatment upregulated LOXL2, Sox9, Acan and Runx2 expression in both males and females. The Adv-RFP-LOXL2 injection, but not the empty vector injection increased proteoglycan staining and aggrecan expression but reduced MMP13 expression. LOXL2 attenuated IL-1&beta, induced phospho-NF-&kappa, B/p65 and rescued chondrogenic lineage-related genes in ATDC5 cells, demonstrating one potential protective mechanism. LOXL2 attenuated phospho-NF-&kappa, B independent of its enzymatic activity. Finally, LOXL2-overexpressing transgenic mice were protected from MIA-induced OA-related functional changes, including the time and distance traveled on the treadmill and allodynia. Conclusion: Our study demonstrates that systemic LOXL2 adenovirus or LOXL2 genetic overexpression in mice can protect against OA. These findings demonstrate the potential for LOXL2 gene therapy for knee-OA clinical treatment in the future.