Publications of Dept. Gene Regulation and Differentiation (RDIF): Recent submissions
Now showing items 61-78 of 78
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IFN-type-I-mediated signaling is regulated by modulation of STAT2 nuclear export.Signaling through the IFN type I receptor is mediated by assembly of the ISGF3 complex consisting of STAT1, STAT2 and IRF9. Whereas STAT1 is instrumentalized by many cytokines, STAT2 is specifically used by type I IFNs. Here, we report that the main regulatory mechanism of nuclear accumulation of STAT2 is nuclear export. We determined the kinetics of nucleocytoplasmic shuttling of STAT2 in living cells. In the absence of IFN, a virtually exclusive cytoplasmic localisation of STAT2 can be detected. Nevertheless, STAT2 is permanently and rapidly shuttling between the cytoplasm and the nucleus. The steady-state localization is explained by a very efficient nuclear export. Our studies indicate that at least two pathways (one of which is CRM1-dependent, the other not yet identified) are responsible for clearing the nucleus from STAT2. The constitutive nucleocytoplasmic shuttling of STAT2 does neither depend on the presence of IRF9 or STAT1, nor does it require tyrosine phosphorylation. Upon treatment with IFN type I, nuclear export of STAT2 is completely abolished in cells used within this study, whereas nuclear import is functioning. This explains the observed nuclear accumulation of STAT2. We have identified a region in the C-terminus of STAT2 that is essential for its almost exclusively cytoplasmic localization in the absence of IFN and responsible for CRM1-specific export. In comparative studies we show that nucleocytoplasmic shuttling of STAT2 is significantly different from that of STAT1. STAT1 is also shuttling in the absence of IFN, but the exchange rate in unstimulated cells is more than ten times lower. We further show that the latent STAT2 protein has stronger intrinsic nuclear-export activity than STAT1. Together, these observations lead to a model for IFN-type-I-induction in which the receptor-mediated heterodimerization overcomes the slow nuclear import of STAT1 and blocks the strong STAT2 export activity that leads to the accumulation of both signal transducers in the nucleus.
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Six-colour fluorescent imaging of lymphoid tissue based on colour addition theory.Multi-colour imaging of immunofluorescently labelled tissue using confocal microscopy was accomplished by using colour addition theory. This new technique includes several improvements for immunolabelling: (1) the co-localization of two or more markers on one cell for the identification of specific cell populations; (2) the co-localization of two fluorescent dyes from secondary reagents for the identification of the cells; (3) a multi-step staining protocol with two primary antibodies originating from the same host species or with two or three biotin-conjugated primary antibodies. After image acquisition, colour segmentation/unmixing are applied to the single multi-colour image to generate multi-pseudo-channels for individual or co-localized fluorescent dyes. With this new technique, we have been able to visualize six cell populations simultaneously in the mouse lymph node and intestine. The efficiency of this method has also been demonstrated in the three-dimensional reconstruction of thick sections from mouse ileum. Our method is simple, efficient, and may be indispensable in experimental cell and tissue studies requiring multiple immunolabelling.
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Control of smooth muscle cell proliferation by ferrous iron.This study was conducted to determine the interaction of individual corrosion products from biodegradable iron stents with cells from the adjacent tissue. The response of human umbilical venous smooth muscle cells (SMCs) to an excess of ferrous ions was investigated in a cell culture model at the phenotypic and at the molecular level. When soluble ferrous ions were added to the cell culture medium the cell growth rate was reduced. Gene expression profiling indicated a reduction in the amounts of mRNA from genes that are required for cell proliferation. In addition, mRNA was regulated from multiple genes involved in iron homeostasis, DNA replication and lipid metabolism. In conclusion, ions released from iron stents could reduce the vascular SMC proliferation rate by influencing growth-related gene expression and may therefore play a beneficial role in antagonizing restenosis in vivo.
