Autophagy Induces Expression of IL-6 in Human Periodontal Ligament Fibroblasts Under Mechanical Load and Overload and Effects Osteoclastogenesis in vitro

Abstract

<b>Objective:</b> Autophagy is an important cellular adaptation mechanism to mechanical stress. In animal experiments, inhibition of autophagy during orthodontic tooth movement triggered increased expression of inflammation-related genes and decreased bone density. The aim of this study was to investigate how autophagy affects cytokine levels of interleukin 6 (IL-6) in human periodontal ligament (hPDL) fibroblasts under mechanical pressure and the resulting influence on osteoblast communication. <b>Methods:</b> hPDL fibroblasts were subjected to physiologic mechanical load, constant overload, or rapamycin treatment for 16 to 24 h ± autophagy inhibitor 3-MA. Autophagosomes were quantified by flow cytometry. Gene expression of <i>il-6</i> as well as IL-6 levels in the supernatant were determined with rtPCR and ELISA. To investigate the influence of mechanically-induced autophagy on cell-cell communication, an osteoblast-culture was subjected to supernatant from stimulated hPDL fibroblasts ± soluble IL-6 receptor (sIL-6R). After 24 h, <i>osteoprotegerin</i> (<i>opg</i>) and <i>receptor activator of nuclear factor</i> κ<i>B ligand</i> (<i>rankl</i>) gene expressions were detected with rtPCR. Gene expression of <i>a disintegrin and metalloproteinases</i> (<i>adam</i>) <i>10</i> and <i>17</i> in stimulated hPDL fibroblasts was examined via rtPCR. <b>Results:</b> Autophagy was induced by biomechanical stress in hPDL fibroblasts in a dose-dependent manner. Mechanical load and overload increased IL-6 expression at gene and protein level. Autophagy inhibition further enhanced the effects of mechanical stimulation on IL-6 expression. Mechanical stimulation of hPDL fibroblasts downregulated <i>adam10</i> and <i>adam17</i> expressions. Inhibition of autophagy had stimulus-intensity depending effects: autophagy inhibition alone or additional application of physiological stress enhanced <i>adam10</i> and <i>adam17</i> expressions, whereas mechanical overload had adverse effects. Osteoblasts showed significantly reduced <i>opg</i> expression in the presence of supernatant derived of hPDL fibroblasts treated with autophagy inhibitor and sIL-6R. <b>Conclusion:</b> IL-6 levels were increased in response to pressure in hPDL fibroblasts, which was further enhanced by autophagy inhibition. This caused a decrease in <i>opg</i> expression in osteoblasts. This may serve as an explanatory model for accelerated tooth movement observed under autophagy inhibition, but may also represent a risk factor for uncontrolled bone loss.