RESUMEN
Gelatin, denatured collagen, temporarily exists in tissues and may well be pathophysiologically involved in tissue remodeling, inflammation or tissue damage. The present study is aimed to investigate possible biological roles of gelatin by examining its effects on monocyte-like histiocytic lymphoma cell line U937. Once stimulated by phorbol 12-myristate 13-acetate (PMA), U937 cells differentiate into macrophage-like cells, changing from non-adherent to adherent cells with extended pseudopodia. Here we pre-treated the cell dishes with gelatin solution for cell culture. Interestingly, we found that PMA-stimulated U937 cells formed multicellular aggregates on gelatin-coated dishes, accompanying NF-κB-mediated production of pro-inflammatory cytokines, whereas cell aggregation was not detected on non-coated dishes. Moreover, differentiated U937 cells on gelatin-coated dishes showed increased autophagy level and endocytosis. Surprisingly, formation of multicellular aggregates and pro-inflammatory cytokine production were both attenuated by either down-regulation of autophagy with inhibitors, such as 3-methyladenine (3MA) or chloroquine (CQ), or repression of endocytosis with siRNA targeting Endo180. Moreover, autophagy was inhibited by si-Endo180, and endocytosis was suppressed by 3MA, suggesting a positive feedback loop between autophagy and endocytosis. The results revealed that gelatin-coating induced differentiated U937 cells to form cell aggregates and promote NF-κB-mediated pro-inflammatory cytokine production at least partially through an endocytosis-autophagy pathway.
Asunto(s)
Autofagia/efectos de los fármacos , Citocinas/metabolismo , Endocitosis/efectos de los fármacos , Matriz Extracelular/metabolismo , Gelatina/metabolismo , Macrófagos/efectos de los fármacos , Acetato de Tetradecanoilforbol/análogos & derivados , Adenina/análogos & derivados , Adenina/farmacología , Animales , Beclina-1/antagonistas & inhibidores , Beclina-1/genética , Beclina-1/metabolismo , Carcinógenos/farmacología , Agregación Celular , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Cloroquina/farmacología , Gelatina/aislamiento & purificación , Humanos , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Lectinas de Unión a Manosa/antagonistas & inhibidores , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/metabolismo , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Seudópodos/efectos de los fármacos , Seudópodos/inmunología , Seudópodos/metabolismo , Interferencia de ARN , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Piel/química , Sus scrofa , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The present study is aimed to investigate the effect of collagen I on U937 cells, human monocyte-like histiocytic lymphoma cell line. Differentiation of U937 cells was induced by phorbol ester (PMA) treatment. The cells were cultured on the collagen I-coated plate. PMA-stimulated U937 cells formed multicellular aggregates on collagen I-coated surface, whereas PMA-unstimulated cells kept themselves away off each other. Moreover, the levels of reactive oxygen species (ROS) and productions of pro-inflammatory cytokines such as IL-1ß, TNFα and PGE2, pro-inflammatory mediator, were down-regulated in differentiated U937 cells cultured on collagen I-coated dishes. However, collagen I did not influence the capacity of E. coli phagocytosis. Cell aggregation as well as the down-regulation of IL-1ß, TNFα and PGE2 caused by the culture on collagen I-coated surface were suppressed by ROS donor, tert-butylhydroperoxide (tBHP). The sizes of cell aggregates became bigger in differentiated U937 cells by treatment with ROS scavengers such as N-acetylcysteine (NAC), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH). In conclusion, collagen I-coated culture induces the differentiated U937 cells to form cell aggregates and decreases the production of pro-inflammatory cytokines through down-regulating ROS generation.