When expression of more than one gene is required in cells, bicistronic or
multicistronic expression vectors have been used. Among various strategies
employed to construct bicistronic or multicistronic vectors, an internal
ribosomal entry site (IRES) has been widely used. Due to the large size and
difference in expression levels between genes before and after IRES, however, a
new strategy was required to replace IRES. A self-cleaving 2A peptide could be a
good candidate to replace IRES because of its small size and high cleavage
efficiency between genes upstream and downstream of the 2A peptide. Despite the
advantages of the 2A peptides, its use is not widespread because (i) there are
no publicly available cloning vectors harboring a 2A peptide gene and (ii)
comprehensive comparison of cleavage efficiency among various 2A peptides
reported to date has not been performed in different contexts. Here, we
generated four expression plasmids each harboring different 2A peptides derived
from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea
asigna virus and porcine teschovirus-1, respectively, and evaluated
their cleavage efficiency in three commonly used human cell lines, zebrafish
embryos and adult mice. Western blotting and confocal microscopic analyses
revealed that among the four 2As, the one derived from porcine teschovirus-1
(P2A) has the highest cleavage efficiency in all the contexts examined. We
anticipate that the 2A-harboring cloning vectors we generated and the highest
efficiency of the P2A peptide we demonstrated would help biomedical researchers
easily adopt the 2A technology when bicistronic or multicistronic expression is
required.
Recent studies have suggested that the expression of interleukin-8 (IL-8) directly correlates with the vascularity of human gastric carcinomas. In this study, the effect of IL-1b on IL-8 expression in human gastric cancer TMK-1 cells and the underlying signal transduction pathways were investigated. IL-1b induced the IL-8 expression in a time-and concentration-dependent manner. IL-1b induced the activation of extracellular signalregulated kinases-1/2 and P38 mitogen-activated protein kinase (MAPK), but not the activation of c-jun aminoterminal kinse and Akt. Specific inhibitors of MEK-1 (PD980590) and P38 MAPK (SB203580) were found to suppress the IL-8 expression and the IL-8 promoter activity. Expression of vectors encoding a mutated-type MEK-1 and P38 MAPK resulted in decrease in the IL-8 promoter activity. IL-1b also induced the production of reactive oxygen species (ROS). N-acetyl cysteine (NAC) prevented the IL-1b-induced ROS production and IL-8 expression. In addition, exogenous H 2 O 2 could induce the IL-8 expression. Deletional and site-directed mutagenesis studies on the IL-8 promoter revealed that activator protein-1 (AP-1) and nuclear factor (NF)-jB sites were required for the IL-1b-induced IL-8 transcription. Electrophoretic mobility shift assay confirmed that IL-1b increased the DNA-binding activity of AP-1 and NF-jB. Inhibitor (PD980590, SB203580) and ROS scavenger (NAC) studies revealed that the upstream signalings for the transcription factors AP-1 and NF-jB were MAPK and ROS, respectively. Conditioned media from the TMK-1 cells pretreated with IL-1b could remarkably stimulate the in vitro growth of HUVEC and this effect was partially abrogated by IL-8-neutralizing antibodies. The above results suggest that MAPK-AP-1 and ROS-NF-jB signaling pathways are involved in the IL-1b-induced IL-8 expression and that these paracrine signaling pathways induce endothelial cell proliferation.
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