피부암세포에서 IL-6/soluble IL-6 Receptor Signaling의 기능연구

Abstract

Interleukin-6 (IL-6) is a cytokine which is involved in the regulation of inflammatory and immunologic responses. IL-6-mediated signaling is initiated by its specific binding to the cognate receptor (IL-6Rα), and then this complex binds to gp130 to occur biological activities. The IL-6Rα form exists as either a membrane-bound or a soluble form. The soluble form of the IL-6R (sIL-6R) is generated by shedding of the membrane-bound forms or by mRNA alternative splicing. sIL-6R can also form a complex with IL-6, resulting in activation of signal transduction. The highly metastatic F10.9 melanoma cells, derived from a spontaneous B16 melanoma of C57B1/6 mice, have been shown to be insensitive to IL-6; however, this unresponsiveness can be restored by IL-6, in conjunction with sIL-6R. It has been shown that co-treatment of IL-6 with sIL-6R resulted in growth inhibition and morphological change of F10.9 melanoma cells through p21 accumulation. These findings suggest IL-6/sIL-6R treatment may affect the cytotoxic responsiveness of melanoma cells to other toxic stimuli. IL6RIL6 is a highly active fusion protein of the soluble form of IL-6R and IL-6; this fusion protein has been shown to be highly active in gp130 positive cells. In chapter 1 of these studies, we investigated whether the IL-6/sIL-6R system potentiates the cytotoxic responsiveness of TNF-α in TNF-α-resistant B16/F10.9 melanoma cells. We found that IL6RIL6 sensitizes TNF-α-resistant B16/F10.9 melanoma cells to TNF-α-mediated apoptosis; this effect was associated with both the upregulation of TNF-R55 expression and the reduction of Bcl-2 expression in response to IL6RIL6. These results suggest that the IL-6/sIL-6R/gp130 system, which sensitizes TNF-α-resistant melanoma cells to TNF-α-induced apoptosis, may provide a new target and for immunotherapy. Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is an important bioactive material and signaling molecule that mediates a variety of biological actions such as vasodilatation, neurotransmission, and host defense. In chapter 2, we explored whether various cytokines can modulate the expression of the iNOS gene in melanoma cells. We found that IL6RIL6 plus TNF-α potently induced iNOS gene expression. Gel shift and reporter gene analyses revealed that IL6RIL6 selectively activated AP-1; while TNF-α increased the activities of NF-kB. Stimulation of cells with IL6RIL6 plus TNF-α resulted in the activation of mitogen-activated protein kinases (MAPK) such as c-Jun N-terminal kinase (JNK), ERK1/2 and p38. In addition, we found that JNK and p38 MAPK are involved in the activation of c-Jun and c-Fos, components of AP-1, in response to IL6RIL6 plus TNF-α. These results suggest that IL-6/sIL-6R/gp130 complex signaling has an unexpected positive effect on iNOS gene expression through JNK/p38 MAPK mediated-AP-1 activation in melanoma cells. Astrocytes are the resident glial cells in the central nervous system (CNS) that support brain capillary endothelium, and are in intimate contact with brain endothelial cells via perivascular end-feet. Exposure of astrocytes to inflammatory cytokines, such as TNF-α, IL-β, and IFN-γ, induce the expression of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1). In chapter 3 of the present studies, we examined the effect of human plasma high density lipoproteins (HDL) and other lipoproteins on VCAM-1 expression in astroglioma cells. The exposure of astroglioma cells to the major plasma lipoprotein fractions (VLDL, LDL and HDL) had no effect on the VCAM-1 expression. However, TNF-α-induced VCAM-l was inhibited by HDL in a dose-dependent manner, but not by VLDL or LDL. The inhibitory effect of HDL on TNF-α-induced VCAM-l was reversed by the inclusion of apo A-I antibody, the major apolipoprotein of HDL, demonstrating the specificity of this response. Reconstituted HDL (discoidal complex of apo HDL and DMPC), but not apo HDL or DMPC, was effective in suppressing the VCAM-1 expression. RNase protection assay (RPA) revealed that TNF-α-induced VCAM-l mRNA expression was markedly inhibited by HDL (500 ㎍ cholesterol/ml). These results indicate that HDL-like particles in the CNS may function as an immunosuppressive molecule in pathologic conditions of CNS.