Title: Internalized NG2 Regulates Oxidative Stress in Mandibular Condylar Cartilage
David Reed (Presenter)
University of Illinois
MAMORU YOTSUYA, University of Illinois
Toru Sato, Tokyo Dental College
Scott Bicknell, University of Illinois
Andrew Bertagna, The University of Illinois College of Dentistry
Nageeb Hasan, University of Illinois
Objectives: A key aspect of degenerative disorders in the temporomandibular joint (TMJ DJD) is the pro-apoptotic signaling resulting from oxidative stress. Nerve/glial antigen 2 (NG2) has been shown in some cells to protect against oxidative stress through interactions with the pro-apoptotic OMI/Htra2. Experimentally induced TMJ DJD is associated with high levels of internalized NG2 in articular cells. Here we evaluate the role of internalized NG2 in TMJ DJD as a mediator of oxidative stress.
Methods: Primary cells were collected from the mandibular condylar cartilage (MCC) of 10 day postnatal c57 BL/6 mice. At confluence, the cells were treated for four hours with and without 300 ng/ml phorbol myristate acetate (PMA) for Protein Kinase C (PKC) mediated phosphorylation of the NG2 intracellular domain, and/or 100 µM dynasore hydrate for inactivation of dynamin2 mediated endocytosis. Following treatment, cells were immunofluorescently stained for NG2, the endocytosis markers clathrin heavy chain, AP-2, and dynamin2, and the oxidative stress marker OMI/Htra2. Nitric oxide synthase 2 (NOS2) and MMP-9 expression was quantified by RT-qPCR.
Results: Internalized NG2 colocalizes with the endocytosis markers CHC, AP2, and dynamin, with the highest level of colocalization occurring near the cell membrane. PMA treatment yields significantly higher levels of NOS2 and MMP9 expression when compared with controls (p< 0.05). PMA induced NOS2 but not MMP-9 levels were partially rescued by dynasore hydrate treatment (p< 0.05; p = 0.07 respectively). Internalized NG2 colocalizes with OMI/Htra2.
Conclusions: These data illustrate that internalized NG2 is endocytosed through a clathrin-mediated pathway and could sequester pro-apoptotic OMI/Htra2 signaling in the MCC. Phosphorylation of the NG2 intracellular domain elevates oxidative stress, and can be rescued by inhibition of the dynamin dependent endocytosis pathway. Together, these data support the hypothesis that clathrin dependent endocytosis of NG2 may be a key mechanism regulating oxidative stress in TMJ DJD.