Mechanism of cyclosporin A- induced gingival overgrowth

In cyclosporin A (CsA)-induced gingival overgrowth, collagen degradation by fibroblasts is inhibited, resulting in fibrosis. Since fibrillar collagen is the primary ligand for the discoidin domain receptor 1 (DDR1), we hypothesized that CsA perturbs DDR1 function.  Objectives: 1) Determine whether CsA affects DDR1 expression; 2) Examine whether CsA inhibits collagen binding to DDR1; 3) Assess whether the effects of CsA on DDR1 are independent of β1-integrins. Methods: Mouse 3T3 fibroblastic cells expressing DDR1 or null for DDR1, were treated with vehicle (dimethyl sulfoxide, DMSO) or with CsA (10µM in DMSO). DDR1 and β1-integrin protein levels were assessed by immunoblotting 3 days after treatments. Collagen binding was measured by flow cytometry of cells incubated with type I collagen-coated fluorescent beads. Immunostaining with 9EG7, a β1-integrin activation-specific antibody, and flow cytometry was used to measure β1-integrin activation of cells in the presence or absence of type I collagen and of DDR1. Mean and standard error were calculated for all data (student’s t test, p<0.05).  Results: CsA treatment significantly increased DDR1 protein expression (107.5+0.14% increase vs. vehicle +SEM; p<0.001) but did not significantly affect β1-integrin protein expression in DDR1-expressing cells (32.5+0.43% increase vs. vehicle; p=0.49) or in DDR1-null cells (52.3+0.31% increase vs. vehicle; p=0.17).  CsA significantly reduced collagen binding in DDR1-expressing cells (57.9+0.13% reduction vs. vehicle; p<0.01) but not in DDR1-null cells (12.2+0.08% reduction vs. vehicle; p=0.20) or in DDR1-expressing, β1-integrin-null cells (53.0+0.1% reduction vs. vehicle; p<0.01).  CsA did not significantly affect β1-integrin activation in the presence or absence of DDR1 or of fibrillar collagen in DDR1-expressing cells (mean fluorescence; vehicle: 7.7+0.82, CsA: 7.4+1.2; p=0.84) or in DDR1-null cells (mean fluorescence; vehicle: 5.0+0.32, CsA: 4.7+3.2; p=0.51). Conclusions: CsA promotes upregulation of DDR1 protein expression and inhibits collagen I binding independent of β1-integrins.