| Collagen is the predominant component of extracellular matrix(ECM),in which the major form is type Ⅰ collagen(Collagen Ⅰ).Collagen Ⅰ affects inflammatory response by regulating the production of pro-inflammatory cytokines.The influence of collagen Ⅰ on inflammation seems to be complicated without precise elucidation of the underlying mechanisms.Therefore,the present study is aimed to investigate the effect of collagen Ⅰon U937 cells,human monocyte-like histiocytic lymphoma cell line.Once stimulated by phorbol 12-myristate 13-acetate(PMA),U937 cells differentiate into macrophage-like cells,changing from non-adherent to adherent form with extended pseudopodia.PMAstimulated U937 cells to form multicellular aggregates on collagen I-coated surface,whereas PMA-unstimulated cells kept themselves away off each other.The results suggest that collagen-promoted cell aggregation exists only in PMA-treated cases.Moreover,the levels of reactive oxygen species(ROS),productions of pro-inflammatory cytokines such as IL-1ββ and TNFα,and a pro-inflammatory mediator,PGE2,were downregulated in the differentiated U937 cells cultured on collagen Ⅰ-coated dishes.Cell aggregation as well as the down-regulation of IL-1β,TNFa and PGE2 caused by collagenⅠ-coating surface was suppressed by ROS donor,tert-butylhydroperoxide(tBHP).The sizes of cell aggregates became bigger in differentiated U93 7 cells by treatment with ROS scavengers such as N-acetylcysteine(NAC),superoxide dismutase(SOD),catalase(CAT)and glutathione(GSH).Both autophagy and ROS regulate the production of proinflammatory cytokines;meantime,autophagy is highly related to ROS generation.Therefore,Ⅰ examined the effect of collagen Ⅰ on autophagy level.Ⅰ found that collagenⅠ-coated culture did not influence autophagy level in differentiated U937 cells.These results suggest that culture on collagen Ⅰ-coated dishes induces the differentiated U937 cells to form cell aggregates and decreases the production of pro-inflammatory cytokines through down-regulating ROS generation.Gelatin,the product of denatured or degraded collagen,temporarily exists in tissues and may well be pathophysiologically involved in tissue remodeling,inflammation or tissue damage.ECM fragments affect inflammation by releasing cytokines.Gelatin is a product of degraded ECM in inflammatory process.Therefore,gelatin may have a strong regulatory effect on the occurrence and progression of inflammation.However,there are only a few studies on gelatin-associated inflammatory responses.Therefore,the present study is aimed to investigate possible biological roles of gelatin by examining its effects on U937 cells.Interestingly,we found that PMA-stimulated U937 cells formed multicellular aggregates on gelatin-coated dishes,accompanying the production of IL-1β,TNFa and PGE2,whereas cell aggregation was not detected on non-coated dishes.Moreover,differentiated U937 cells on gelatin-coated dishes showed increased autophagy level and endocytosis of FITC-gelatin.Surprisingly,formation of multicellular aggregates and pro-inflammatory cytokine production were both attenuated by either down-regulation of autophagy with its inhibitors,such as 3-methyladenine(3MA)or chloroquine(CQ),or suppression of endocytosis with siRNA targeting Endo180.Endol80 is mainly responsible for the intracellular degradation of collagen and its fragments.Moreover,autophagy was inhibited by si-Endo180,and endocytosis was suppressed by 3MA,suggesting existence of a positive feedback loop between autophagy and endocytosis.The results revealed that gelatin-coated dishes induced differentiated U937 cells to form cell aggregates and promote the production of pro-inflammatory cytokines at least partially through an endocytosis-autophagy signaling pathway.It was reported that autophagy is often associated with ROS levels.Therefore,we investigate the effect of ROS levels in PMA-treated U937 cells cultured on gelatin-coated surface.We found that gelatin-coating increased ROS levels in PMA-treated U937 cells,compared with those of the cells on non-coated dishes.Meantime,gelatin-stimulated cell aggregation was attenuated by treatment with ROS scavenger NAC,but further promoted by ROS donor tBHP.Release of IL-1β,TNFα and PGE2,as well as the expressions of these molecules were all inhibited by NAC.These results suggest that gelatin promotes cell aggregation and the production of pro-inflammatory cytokines through ROS generation.Moreover,autophagy induced in differentiated U937 cells on gelatin-coated surface was eliminated by NAC treatment,but the treatment with autophagy inhibitor 3MA did not affect ROS levels,suggesting a possible ROS-autophagy regulation axis in differentiated U937 cells on gelatin-coated surface.Further study showed that tBHPenhanced up-regulation of cell aggregation as well as the production of pro-inflammatory mediators in differentiated U937 cells on gelatin was attenuated by 3MA treatment.Down-regulation of formation of cell aggregation as well as in production of proinflammatory mediators with NAC was reversed by addition of autophagy inducer rapamycin.These results suggest that autophagy exists in the downstream of ROS signaling pathway.In conclusion,PMA-treated U937 cells cultured on type Ⅰ collagen-coated dishes express lowered production of pro-inflammatory mediators through reduced ROS levels in parallel with enhanced cell aggregation.Gelatin-coating induced differentiated U937 cells to form cell aggregates and promote pro-inflammatory cytokine production through an endocytosis-autophagy/ROS-autophagy signaling pathway.Gelatin is produced by degradation,accompanying denaturation from collagen under several diseases,such as myocardial infarction,inflammatory diseases,skin burns;meantime,activated macrophages release pro-inflammatory cytokines.These suggest that the change of ECM components may be associated with the progression of the disease. |
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