DC-STAMP: :Localization and Function in Dendritic Cells

Contains fulltext : 74586.pdf (publisher's version ) (Open Access)Many studies have been performed to elucidate the functional role of the DCs in orchestrating immune responses. The main goal of this thesis is to increase our understanding of DC immunobiology at the molecular level. A molecular approach was followed to unravel the role of two novel molecules in DC function. We report characterization of DC-SCRIPT as a putative transcriptional regulator having Zinc fingers mediating DNA binding flanked by a prolin-rich region and an acidic region playing possible roles in the regulation of transcription. Furthermore, the thesis focuses on the previously characterized DC-specific gene DC-STAMP. Our data show that DC-STAMP, both mouse and human homologue, localizes to the Endoplasmic Reticulum (ER). The myeloid precursor cell differentiates into two lineages. DC-STAMP is expressed in cells of the lineage generating macrophages, DCs, and osteoclasts but not the granulocyte lineage. Our data show that DC-STAMP selectively suppresses granulocyte development suggesting that its involvement in myeloid development. To gain molecular insight into DC-STAMP function, two DC-STAMP interacting proteins were identified. The amplified in osteosarcoma 9 (OS9) is reported to interact with DC-STAMP, and both proteins co-localize in the ER. The data indicate that OS9 is critically involved in the modulation of ER-to-Golgi transport of DC-STAMP in response to TLR triggering. Furthermore, Luman was characterized as a second interacting partner of DC-STAMP. Luman is an ER-resident transcription factor, which undergoes Regulated Intramembrane Proteolysis. During this process Luman translocates from ER to the Golgi apparatus, where it is cleaved followed by translocation of the liberated active part of Luman to the nucleus. This study unravels a role for DC-STAMP in Luman activation and suggests that this pathway is activated in DCs in response to DC maturation stimuli. Taken together, the implications of the results described in the thesis towards a better understanding of the DC immunobiology are discussed.RU Radboud Universiteit Nijmegen, 11 november 2009Promotor : Adema, G.J.128 p

DC-STAMP interacts with ER-resident transcription factor LUMAN which becomes activated during DC maturation.

Item does not contain fulltextDendritic cells (DCs) are the professional antigen-presenting cells (APC) which efficiently prime the immune response or induce tolerance. We recently identified Dendritic Cell Specific TrAnsMembrane Protein (DC-STAMP), a novel 470 amino acid protein preferentially expressed by dendritic cells. Previously we demonstrated that DC-STAMP re-localizes towards the Golgi upon DC maturation. To identify proteins that interact with DC-STAMP, a yeast-2-hybrid analysis was performed. Here, we report a physically interacting partner of DC-STAMP in the endoplasmic reticulum (ER), called LUMAN (also known as CREB3 or LZIP). LUMAN was previously described as an ER-resident transcription factor with unknown function. It is activated in a process called regulated intramembrane proteolysis (RIP), which involves translocation to the Golgi and subsequent proteolytic cleavage. The proteolytically activated form of the protein then translocates to the nucleus. Our data indicate that DC-STAMP plays an important role in the modulation of LUMAN activation. Moreover, we demonstrate that LUMAN is endogenously expressed by DC and becomes activated by RIP upon DC maturation induced by various different stimuli. These data define LUMAN/DC-STAMP as a novel regulatory circuit in DC.1 juli 201

The DC-derived protein DC-STAMP influences differentiation of myeloid cells.

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OS9 interacts with DC-STAMP and modulates its intracellular localization in response to TLR ligation.

Contains fulltext : 81396.pdf (publisher's version ) (Closed access)Dendritic cell-specific transmembrane protein (DC-STAMP) has been first identified as an EST in a cDNA library of human monocyte-derived dendritic cells (DC). DC-STAMP is a multimembrane spanning protein that has been implicated in skewing haematopoietic differentiation of bone marrow cells towards the myeloid lineage, and in cell fusion during osteoclastogenesis and giant cell formation. To gain molecular insight in how DC-STAMP exerts its function, DC-STAMP interacting proteins were identified in a yeast-2-hybrid analysis. Herein, we report that amplified in osteosarcoma 9 (OS9) physically interacts with DC-STAMP, and that both proteins colocalize in the endoplasmic reticulum in various cell lines, including immature DC. OS9 has previously been implicated in ER-to-Golgi transport and transcription factor turnover. Interestingly, we now demonstrate that toll-like receptor (TLR)-induced maturation of DC leads to the translocation of DC-STAMP from the ER to the Golgi while OS9 localization is unaffected. Applying TLR-expressing CHO cells we could confirm ER-to-Golgi translocation of DC-STAMP following TLR stimulation and demonstrated that the DC-STAMP/OS9 interaction is involved in this process. Collectively, the data indicate that OS9 is critically involved in the modulation of ER-to-Golgi transport of DC-STAMP in response to TLR triggering, suggesting a novel role for OS9 in myeloid differentiation and cell fusion

The dendritic cell derived protein DC-STAMP is highly conserved and localizes to the endoplasmic reticulum

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Cross-talk between human dendritic cell subsets influences expression of RNA sensors and inhibits picornavirus infection.

Dendritic cells (DCs) are professional antigen-presenting cells that provide a link between innate and adaptive immunity. Multiple DC subsets exist and their activation by microorganisms occurs through binding of conserved pathogen-derived structures to so-called pattern recognition receptors (PRRs). In this study we analyzed the expression of PRRs responding to viral RNA in human monocyte-derived DCs (moDCs) under steady-state or pro-inflammatory conditions. We found that mRNA and protein levels for most PRRs were increased under pro-inflammatory conditions, with the most pronounced increases in the RIG-like helicase (RLH) family. Additionally, freshly isolated human plasmacytoid DCs (pDCs) displayed significantly higher levels of TLR7, RIG-I, MDA5 and PKR as compared to myeloid DCs and moDCs. Finally, we demonstrate for the first time that cross-talk between TLR-matured or virus-stimulated pDCs and moDCs leads to a type I interferon-dependent antiviral state in moDCs. This antiviral state was characterized by enhanced RLH expression and protection against picornavirus infection. These findings might represent a novel mechanism by which pDCs can preserve the function and viability of myeloid DCs that are attracted to a site with ongoing infection, thereby optimizing the antiviral immune response

Analysis of genes regulated by the transcription factor LUMAN identifies ApoA4 as a target gene in dendritic cells.

Item does not contain fulltextDendritic cells (DCs) are professional antigen presenting cells of the immune system that play a crucial role in initiating immune responses and maintaining self tolerance. Better understanding of the molecular basis of DC immunobiology is required to improve DC-based immunotherapies. We previously described the interaction of transcription factor LUMAN (also known as CREB3 or LZIP) with the DC-specific transmembrane protein DC-STAMP in DCs. Target genes of LUMAN and its role in DCs are currently unknown. In this study we set out to identify genes regulated by LUMAN in DCs using microarray analysis. Expression of a constitutively active form of LUMAN in mouse DC cell line D2SC/1 identified Apolipoprotein A4 (ApoA4) as its target gene. Subsequent validation experiments, bioinformatics-based promoter analysis, and silencing studies confirmed that ApoA4 is a true target gene of LUMAN in bone marrow-derived DCs (BMDCs).1 februari 201

MicroRNA genes preferentially expressed in dendritic cells contain sites for conserved transcription factor binding motifs in their promoters

Contains fulltext : 98097.pdf (publisher's version ) (Open Access)BACKGROUND: MicroRNAs (miRNAs) play a fundamental role in the regulation of gene expression by translational repression or target mRNA degradation. Regulatory elements in miRNA promoters are less well studied, but may reveal a link between their expression and a specific cell type. RESULTS: To explore this link in myeloid cells, miRNA expression profiles were generated from monocytes and dendritic cells (DCs). Differences in miRNA expression among monocytes, DCs and their stimulated progeny were observed. Furthermore, putative promoter regions of miRNAs that are significantly up-regulated in DCs were screened for Transcription Factor Binding Sites (TFBSs) based on TFBS motif matching score, the degree to which those TFBSs are over-represented in the promoters of the up-regulated miRNAs, and the extent of conservation of the TFBSs in mammals. CONCLUSIONS: Analysis of evolutionarily conserved TFBSs in DC promoters revealed preferential clustering of sites within 500 bp upstream of the precursor miRNAs and that many mRNAs of cognate TFs of the conserved TFBSs were indeed expressed in the DCs. Taken together, our data provide evidence that selected miRNAs expressed in DCs have evolutionarily conserved TFBSs relevant to DC biology in their promoters