The Regulatory Effects Of Cripto-1 On Macrophage Cytokine Secretion, Phagocytic Activity And Skp2 Expression, And Underlying Mechanisms

Cripto-1 is an oncogenic protein belonging to the epidermal growth factor–Cripto-1/FRL-1/Cryptic family. It has important roles in tumor formation and metastasis, but its effects on the immune system are unclear. In the present study, we investigated the effects of Cripto-1 overexpression on macrophage activities, and examined the underlying mechanisms.To examine the potential effects of Cripto-1 on macrophages, we established L02 cells stably overexpressing Cripto-1. We collected the culture supernatant from this cell line and added it to cultures of macrophages. Exposure to this supernatant stimulated macrophage expression of the anti-inflammatory cytokine IL-10, and of the pro-inflammatory cytokines IL-1β, TNF-α, and IL-6. However, the expression of TGF-β, another anti-inflammatory cytokine, was unaffected. Direct exposure of macrophages to purified Cripto-1 confirmed that this protein stimulated the secretion of pro- and anti-inflammatory cytokines. Overall, as Cripto-1 upregulates the expression of pro- and anti-inflammatory cytokines in macrophages, it may affect the host’s immunity.Then,phagocytosis is an important indicator of the immune functions of macrophages and is also the basis for their specific immune responses. In this study, we investigated the effects of Cripto-1 on the phagocytic activities of mouse and human macrophages. Exposure to purified Cripto-1 or the supernatant derived from the Cripto-1–overexpressing cells significantly increased macrophage phagocytic activity. These results confirm that Cripto-1 enhanced macrophage cytokine secretion and phagocytic activity. We next investigated the molecular mechanisms involved in the effects of Cripto-1. Exposure to purified Cripto-1 significantly increased IKK phosphorylation and p65 nuclear translocation in macrophages, suggesting that Cripto-1 activates the NF-κB signaling pathway. To confirm the involvement of the NF-κB signaling pathway, macrophages were treated with PDTC, an NF-κB signaling inhibitor, before exposure to Cripto-1. Pretreatment with PDTC inhibited Cripto-1–stimulated cytokine secretion and phagocytosis, confirming the involvement of NF-κB signaling in the effects of Cripto-1.Taken together, our present findings suggest that Cripto-1 enhances macrophage phagocytic activity and upregulates the production of anti- and pro-inflammatory cytokines via the NF-κB signaling pathway.Proto-oncogene Skp2 has been considered as an F-box protein and its overexpression is frequently observed in many types of cancers. Skp2 plays important roles in the development and progression of tumorigenesis. Previous study showed that when the pCI-neo-Cripto-1 plasmid was transiently transfected into 293 T cells, Cripto-1 greatly decreased the Skp2 expression in both mRNA and protein level. And we found that overexpression of Cripto-1 inhibit p38/MAPK signal pathways markedly in 293 T cells. These results suggested that Cripto-1 could regulate the expression of Skp2 by p38/MAPK signaling. To investigate whether the Skp2 expression could be regulated by p38/MAPK signaling, we transfected Skp2-overexpressing 293 T cells with the dominant-negative plasmid DN-p38 to inhibit p38/MAPK signaling. Then we found that suppression of p-p38 activity effectively down-regulated Skp2 expression. These data suggest that p38/MAPK signaling was required for the inhibitory effect of Cripto-1 on Skp2 expression.Next, we knocked down the expression of Cripto-1 by siRNA in Huh7 cells and analysed the expression of Skp2 and p-p38 level. The result shown that when cripto-1 was knocked down by siRNA, the active p38 was increased and the expression of Skp2 was upregulated. Otherwise, when transfected the Huh7 cells with the dominant-negative plasmid DN-p38, the expression of Skp2 was decreased.These results suggest that Cripto-1 regulats the expression of Skp2 via p38/MAPK signaling.In conclusion, our results confirm that Cripto-1 plays an important role in modulating macrophage activities, including cytokine secretion and phagocytosis, these results may offer new insight into the immune escape of malignant cells and immunosuppression of tumor cells. And we also confirm that Cripto-1 inhibits oncogenic Skp2 expression via p38/MAPK signaling pathways. This study laid a important theoretical foundation for development anti-cancer drugs targeting Skp2.