Novel role of LOXL2 in TMJ and knee OA cartilage in vitro and in vivo

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

Anabolic role of lysyl oxidase like-2 in cartilage of knee and temporomandibular joints with osteoarthritis

Abstract Background Lysyl oxidase like-2 (LOXL2) is a copper-dependent amine oxidase. Our previous studies showed that LOXL2 is elevated during mouse fracture healing. The goal of this study was to evaluate the potential of LOXL2 to act as an anabolic agent in cartilage affected by osteoarthritis (OA). Methods LOXL2 was visualized in tissues from human knee and hip joints and temporomandibular joints (TMJ) by immunofluorescence. The activity of LOXL2 in human articular and TMJ chondrocytes was assessed by cell-based assays, microarray analysis, 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. Finally, LOXL2-induced effects on collagen type 2 α1 (COL2A1) and phospho-SMAD2/3 were evaluated by immunofluorescence analysis. 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. Viral transduction of LOXL2 in OA chondrocytes increased the mRNA levels of chondroitin sulfate proteoglycan (CSPG4), aggrecan (ACAN), sex determining region Y-box containing gene 9 (SOX9), and COL2A1 but reduced the levels of extracellular matrix (ECM)-degrading enzymes matrix metalloproteinase (MMP)1, MMP3, and MMP13. Further, forced expression of LOXL2 promoted chondrogenic lineage-specific gene expression, increased the expression of COL2A1 in the presence of TNF-α, and inhibited chondrocyte apoptosis. 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. Finally, immunofluorescence staining revealed co-localization of LOXL2 with SOX9 in the nuclei of cells in the implants, decreased phospho-SMAD2/3, and increased COL2A1 staining. Conclusion Our results suggest that although LOXL2 is upregulated in cartilage affected by OA, this may be a protective response that promotes anabolism while inhibiting specific catabolic responses in the pathophysiology of OA

Additional file 1: Figure S1. of Anabolic role of lysyl oxidase like-2 in cartilage of knee and temporomandibular joints with osteoarthritis

LOXL2 pull-down assay: HAC-OA cells were transduced with Adv-EV or Adv-LOXL2 vector for overnight. The next day, transduced cells were replenished with fresh chondrocyte growth media and cultured for 24 h, and extracted into non-denaturing cell lysis RIPA buffer. The cell lysates were incubated with cobalt chelate NT beads overnight at 4 °C to pull down LOXL2. The beads were washed and eluted according to kit instructions (Pull-Down PolyHis Protein: Protein Interaction Kit Thermo Scientific, Waltham, MA, USA). Eluted samples were analyzed by western blotting on denaturing SDS-PAGE probed with an LOXL2 (Genetex) or SOX9 (Abcam) antibody, and the input (5% aliquots) of initial extracts taken before the immunoprecipitation was analyzed on the same gels. The figure shows pull-down of LOXL2 in IP analysis; however, co-IP with SOX9 does not show any corresponding band. (TIF 50990 kb