| DC-SIGN (DC-Specific Intercellular Adhesion Molecule-3-Grabbing Nonintegrin) is an important adhesion molecule receptor on the surface of dendritic cells (DC), which can induce DC through the endodermis, and mediate the aggregation of DC and naive T cells by binding with ICAM-3. In addition, as pattern recognition receptors (PRR), DC-SIGN can recognize and mediate the infection of a variety of pathogens in human, and induce specific immune responses to the pathogens, the most widely studied of which is the relationship between DC-SIGN and HIV-1 sexual transmission. The DC and DC-SIGN under reproductive tract mucosal can bind to HIV-1 gp120 protein with high affinity, and take HIV-1 into the cytoplasm by endocytosis, where HIV-1 is stored in intracellular bodies, maintaining the infection for a long time up to 7 days, and escaping the immune surveillance. The highly infectious HIV-1 in the intracellular bodies of DC can efficiently and abundantly infect CD4+T cells in lymph nodes through the migration of DC. Therefore, DC-SIGN may play a decisive role in sexual transmission of HIV.The important role of DC-SIGN in sexual transmission of HIV-1 may be also related with its organization and cell-specific expression. DC-SIGN expression is under strict regulation and very limited range. DC-SIGN is specifically overexpressed in the organizations and cells closely related to HIV-1 sexual transmission. Study found that DC-SIGN-specific expression in vivo is dependent on IL-4, but the regulatory mechanism of DC-SIGN-specific expression is still unclear. Given the important role of DC-SIGN in HIV-1 sexual transmission, it is very necessary to study the regulation mechanism of DC-SIGN specific expression. In eukaryotic cells, the specific expression of genes is primarily dependant on the promoter activity. In this issue, we study the role of cis-element in the DC-SIGN promoter activity; then we initially explored the regulatory mechanism of DC-SIGN-specific expression and upstream signaling pathways involved using the in vitro model of IL-4 induced the expression of DC-SIGN on THP-1 cell line; the last, we studied the relationship between the DC-SIGN promoter activation and HIV-15'LTR activation.First, DC-SIGN promoter sequence was amplified by PCR, and DC-SIGN promoters without AP-1 or Ets-1 transcription factor binding site were obtained by site-directed mutagenesis. Using pGL-3 luciferase reporter plasmid system as the main research tool, the complete DC-SIGN promoter and those without AP-1 or Ets-1 transcription factor binding site were recombined into pGL-3 vectors and then transfected into 293T, Hacat and THP-1 cells, by detecting luciferase activity to reflect the DC-SIGN promoter activity. The results showed that, DC-SIGN promoter activity was different in different cell lines, which was the most active in THP-1 cells, showing significant cellular specificity. The deletion of AP-1 binding site could partially reduce the DC-SIGN promoter activity (decrease of 11.85%-75.25% range); The deletion of Ets-1 binding site had more significant effect on the DC-SIGN promoter activity than that of AP-1 binding site, so that DC-SIGN promoter activity almost disappeared (dropped over 90%). Our study demonstrates that Ets-1 binding site plays a major role in the activation of DC-SIGN promoter.We further studied the upstream signaling pathways activating DC-SIGN promoter. We used IL-4 induced PMA stimulated DC-SIGN expression on THP-1 cells as in vitro model. The induced expression of DC-SIGN on THP-1 cells was detected on different levels of mRNA, protein in cytoplasm and expression on cell surface. It was found that IL-4 could significantly enhance the expression of DC-SIGN in THP-1 cells, and was an important regulatory factor of specific expression of DC-SIGN.We selected the common signaling pathways induced by IL-4 receptor of JAK-STAT and ERK, and NF-κB and p38 signaling pathways as the studying signaling pathways. First, the signaling pathways were blocked by specific inhibitors, and the expression of DC-SIGN was detected. Then the phosphorylation of proteins involved in the signaling pathways was detected to reflect the level of signaling pathway activation. The results showed that at the levels of mRNA, protein in cytoplasm and cell surface expression, inhibitor of ERK pathway blocked the best, almost completely blocking the induction effect of IL-4; inhibitors of JAK-STAT and NF-κB pathways had partially blocked the effect off, while the p38 pathway inhibitor had no blocking effect. The phosphorylation of signaling protein showed that, ERK, JAK-STAT and NF-κB pathways were activated. This part of the study showed that multi-signaling pathways are involved in the regulation of DC-SIGN expression. ERK signaling pathway plays a main role, and JAK-STAT and NF-κB pathways participate, to form a complex regulatory network.Finally, we carried comparative study of different signaling pathway inhibitors on DC-SIGN promoter and HIV-15'LTR of activity based on the pGL-3 luciferase reporter plasmid system. It was found that HIV-15'LTR could also be activated by IL-4 signaling pathway, and NF-κB and ERK signaling pathways were involved in the activation of HIV-15'LTR. This study provides a new way of thinking for the study of the virus activation of latent HIV-1 and the relationship between HIV-1 infection and DC-SIGN.
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