Reactive oxygen species are responsible for the cell aggregation and production of pro-inflammatory mediators in phorbol ester (PMA)-treated U937 cells on gelatin-coated dishes through upregulation of autophagy.

PURPOSE: Our previous studies indicate that phorbol 12-myristate 13-acetate (PMA)-treated U937 cells cultured on collagen I -coated dishes express lowered production of pro-inflammatory mediators in parallel through reduced Reactive oxygen species (ROS) levels. By contrast, PMA-treated U937 cells on gelatin, the denatured collagen, show enhanced production of pro-inflammatory mediators, mediated by up-regulating autophagy levels. The present study is aimed to investigate the effect of ROS levels in PMA-treated U937 cells cultured on gelatin-coated surface.

MATERIAL AND METHODS: MTT assay, flow cytometric analysis of ROS and autophagy, biochemical detection of antioxidant levels, ELISA assay, and western blot were used.

RESULTS: Gelatin-coating increased ROS levels in PMA-treated U937 cells. Increased ROS levels are involved in the regulation of cell aggregation and the release of pro-inflammatory mediators in gelatin-coated culture. These results lead to the query about the crosstalk between the two positive regulators, the autophagy and ROS. Autophagy induction is attenuated by NAC treatment, but the treatment with autophagy inhibitor, 3-methyladenine, does not affect ROS levels, suggesting ROS are upstream of autophagy in the regulation axis of differentiated U937 cells on gelatin-coated surface. Further study confirmed that upregulation of autophagy was responsible for ROS-induced cell aggregation and production of pro-inflammatory mediators.

CONCLUSION: The results suggest that gelatin-coating promotes the aggregation of PMA-treated U937 cells and the production of pro-inflammatory mediators by ROS-autophagy signaling pathway.