University of Illinois at Chicago
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Cell-Matrix Interactions Regulating the Migration of Mandibular Fibrochondrocytes

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posted on 2022-05-01, 00:00 authored by Shin Young Ahn
BACKGROUND: Cell migration is critical for the injury response of the temporomandibular joint. Neuron/Glial antigen 2 (NG2/CSPG4) is a transmembrane proteoglycan that interacts with β1-integrins and influences cell migration. NG2/CSPG4 regulates the mTOR signaling pathway, an established signaling axis that coordinates cell survival, proliferation, and migration. OBJECTIVE: The objective of this study is to determine if the NG2/CSPG4 impacts cell migration in an mTOR dependent manner in mandibular fibrochondrocytes. METHODS: Primary mandibular fibrochondrocytes were collected from the condylar cartilages of wild-type (WT) and NG2/CSPG4 knockout (KO) mice. Cells were digested in a type II collagenase suspended at 3 mg/ml for 45 minutes and 1.5mg/ml overnight. Isolated cells were plated in a 3-well culture insert for cell migration (Ibidi, Gräfelfing) using 55x103 cells and grown to 70-80% confluence. To evaluate the role of mTOR signaling, WT and NG2/CSPG4 KO cells were treated with and without the mTOR modulators MHY1485 (mTOR agonist, Calbiochem) and Rapamycin (mTOR antagonist, Sigma) in low-serum media for 24 hours. To evaluate the role of NG2/CSPG4 ectodomain binding, WT cells were treated with anti-NG2/CSPG4 monoclonal antibodies. To quantify cell migrations, the inserts were removed and imaged using a live cell with phase contrast microscopy (Leica, DMI6000B). Data were analyzed in ImageJ by manually tracing the leading edges and calculating cell-free areas at 0, 1, 4, 12, and 24 hours. All values were statistically compared using a one-way ANOVA with bonferroni corrections. RESULTS: Serum starvation significantly slowed the migration rate of WT, but not NG2/CSPG4 KO cells (p<0.05; n=4). NG2/CSPG4 knockout increased the migration rate of mandibular fibrochondrocytes in low serum (p<0.05; n=4). MHY 1485 attenuated the migration of NG2/CSPG4 KO, but not WT cells. Rapamycin significantly suppressed the migration of both NG2/CSPG4 KO and WT cells at all time points (p<0.05; n=4). NG2/CSPG4 antibody treatment slightly attenuated the migration of mandibular fibrochondrocytes, but not more than the IgG control. CONCLUSION: NG2/CSPG4 negatively regulates the migration of mandibular fibrochondrocytes in an mTOR dependent manner in serum starvation conditions. This novel signaling axis could be a potentially valuable therapeutic target for the mobilization of reparative migratory cells following traumatic injury to the joint.

History

Advisor

Reed, David

Chair

Reed, David

Department

Periodontics

Degree Grantor

University of Illinois at Chicago

Degree Level

  • Masters

Degree name

MS, Master of Science

Committee Member

DiPietro, Luisa Cooper, Lyndon Han, Michael

Submitted date

May 2022

Thesis type

application/pdf

Language

  • en

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