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Soluble TNF Regulates TACE via AP-2? Transcription Factor in Mouse Dendritic Cells.

ABSTRACT: Dendritic cells (DCs), the essential immunoregulatory and APCs, are major producers of the central mediator of inflammation, soluble TNF-? (sTNF). sTNF is generated by TNF-? converting enzyme (TACE) proteolytic release of the transmembrane TNF (tmTNF) ectodomain. The mechanisms of TACE and sTNF regulation in DCs remain elusive. This study newly defines that sTNF regulates TACE in mouse DCs by engaging the AP-2? transcription factor. We found that the expression of AP-2? was higher, whereas the expression and activity of TACE were lower, in wild-type DCs (wtDCs) than in TNF knockout (TNFko) DCs. Exogenous sTNF rapidly and simultaneously induced increases of AP-2? expression and decreases of TACE expression and activity in wtDCs and TNFko DCs, indicating that AP-2? and TACE are inversely dependent on sTNF and are functionally associated. To define this functional association, we identified an AP-2? binding site in TACE promoter and demonstrated, using EMSAs and chromatin immunoprecipitation assays, that AP-2? could bind to TACE promoter in a TNF-dependent manner. Additionally, sTNF simultaneously enhanced AP-2? expression and decreased TACE promoter luciferase activity in DCs. Similarly, transfection of AP-2? cDNA decreased TACE promoter luciferase activity, TACE expression, and TACE enzymatic activity in wtDCs or TNFko DCs. In contrast, transfection of AP-2? small interfering RNA increased TACE promoter luciferase activity, TACE expression, and TACE enzymatic activity in wtDCs. These results show that TACE is a target of, and is downregulated by, sTNF-induced AP-2? transcription factor in DCs.

SUBMITTER: Ge L

PROVIDER: S-EPMC5173404 | biostudies-other | 2017 Jan

REPOSITORIES: biostudies-other

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Soluble TNF Regulates TACE via AP-2α Transcription Factor in Mouse Dendritic Cells.

Ge Lisheng L Vujanovic Nikola L NL

Journal of immunology (Baltimore, Md. : 1950) 20161116 1

Dendritic cells (DCs), the essential immunoregulatory and APCs, are major producers of the central mediator of inflammation, soluble TNF-α (sTNF). sTNF is generated by TNF-α converting enzyme (TACE) proteolytic release of the transmembrane TNF (tmTNF) ectodomain. The mechanisms of TACE and sTNF regulation in DCs remain elusive. This study newly defines that sTNF regulates TACE in mouse DCs by engaging the AP-2α transcription factor. We found that the expression of AP-2α was higher, whereas the e ...[more]

PMID: 27852742

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Project description:ObjectiveMitochondrial transcription factor A (TFAM) is normally bound to and remains associated with mitochondrial DNA (mtDNA) when released from damaged cells. We hypothesized that TFAM, bound to mtDNA (or equivalent CpG-enriched DNA), amplifies TNF? release from TLR9-expressing plasmacytoid dendritic cells (pDCs) by engaging RAGE.Materials and methodsMurine Flt3 ligand-expanded splenocytes obtained from C57BL/6 mice were treated with recombinant human TFAM, alone or in combination with CpG-enriched DNA with subsequent TNF? release measured by ELISA. The role of RAGE was determined by pre-treatment with soluble RAGE or heparin or by employing matching RAGE (-/-) splenocytes. TLR9 signaling was evaluated using a specific TLR9-blocking oligonucleotide and by inhibiting endosomal processing, PI3K and NF-?B. Additional studies examined whether heparin sulfate moieties or endothelin converting enzyme-1 (ECE-1)-dependent recycling of endosomal receptors were required for TFAM and CpG DNA recognition.Main resultsTFAM augmented splenocyte TNF? release in response to CpGA DNA, which was strongly dependent upon pDCs and regulated by RAGE and TLR9 receptors. Putative TLR9 signaling pathways, including endosomal acidification and signaling through PI3K and NF-?B, were essential for splenocyte TNF? release in response to TFAM+CpGA DNA. Interestingly, TNF? release depended upon endothelin converting enzyme (ECE)-1, which cleaves and presumably activates TLR9 within endosomes. Recognition of the TFAM-CpGA DNA complex was dependent upon heparin sulfate moieties, and recombinant TFAM Box 1 and Box 2 proteins were equivalent in terms of augmenting TNF? release.ConclusionsTFAM promoted TNF? release in a splenocyte culture model representing complex cell-cell interactions in vivo with pDCs playing a critical role. To our knowledge, this study is the first to incriminate ECE-1-dependent endosomal cleavage of TLR9 as a critical step in the signaling pathway leading to TNF? release. These findings, and others reported herein, significantly advance our understanding of sterile immune responses triggered by mitochondrial danger signals.

Project description:In mouse blastocysts segregation of the inner cell mass (ICM) and the trophectoderm (TE) is regulated by the mutually antagonistic effects of the transcription factors Oct4 and Cdx2 expressed in the ICM and TE, respectively. In contrast, in other species such as bovine and human, Oct4 is not restricted to the ICM and continues to be expressed in the Cdx2-positive TE. A recent comparative study of the bovine and mouse Oct4 promoters revealed that additional mechanisms might act in conjunction with Cdx2 to downregulate Oct4 expression in the mouse TE lineage. For instance, the mouse Oct4 distal enhancer contains an AP-2? (Tcfap2c) binding motif that is absent in the bovine and human Oct4 distal enhancer. Nonetheless, the functional relevance of Tcfap2c in Oct4 repression during mouse preimplantation development was not tested. To elucidate the role of Tcfap2c in Oct4 expression an RNA interference approach was utilized. Depletion of Tcfap2c triggered a decrease in Oct4 expression at the 8-cell and morula stage. Remarkably, at the blastocyst stage depletion of Tcfap2c and/or its family member Tcfap2a had no effect on Oct4 repression. To test whether Tcfap2c interacts with Oct4 to positively regulate Oct4 expression, chromatin immunoprecipitation and in situ co-immunoprecipitation analyses were performed. These experiments revealed Tcfap2c and Oct4 binding were enriched at the Oct4 distal enhancer in embryonic stem (ES) cells, but were rapidly lost during differentiation into trophoblast-like cells when Oct4 became repressed. Moreover, Tcfap2c and Oct4 interactions were detected at the morula stage, but were lost during blastocyst formation. In summary, these data demonstrate that Tcfap2c is not required for Oct4 silencing in mouse blastocysts, but may be necessary for the maintenance of Oct4 expression during the 8 cell-to-morula transition. These findings support the notion Cdx2 is the predominant negative regulator of Oct4 expression during blastocyst formation in mice.

Dataset Information

Soluble TNF Regulates TACE via AP-2? Transcription Factor in Mouse Dendritic Cells.

Publications

Soluble TNF Regulates TACE via AP-2α Transcription Factor in Mouse Dendritic Cells.

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