Langerhans cell homeostasis in mice is dependent on mTORC1 but not mTORC2 function.

The PI3K/Akt/mTOR pathway has emerged as a critical regulator of dendritic cell (DC) development and function. The kinase mTOR is found in 2 distinct complexes, mTORC1 and mTORC2. In this study, we show that mTORC1 but not mTORC2 is required for epidermal Langerhans cell (LC) homeostasis. Although the initial seeding of the epidermis with LCs is not affected, the lack of mTORC1 activity in DCs by conditional deletion of Raptor leads to a progressive loss of LCs in the skin of mice. Ablation of mTORC2 function by deletion of Rictor results in a modest reduction of LCs in skin draining lymph nodes. In young mice Raptor-deficient LCs show an increased tendency to leave the skin, leading to a higher frequency of migratory DCs in skin draining lymph nodes, indicating that the loss of LCs results from enhanced migration. LCs lacking Raptor are smaller and display reduced expression of Langerin, E-cadherin, ß-catenin, and CCR7 but unchanged levels of MHC-II, ruling out enhanced spontaneous maturation. Ki-67 and annexin V stainings revealed a faster turnover rate and increased apoptosis of Raptor-deficient LCs, which might additionally affect the preservation of the LC network. Taken together our results show that the homeostasis of LCs strictly depends on mTORC1.

Membrane-proximal signaling events in beta-2 integrin activation.

In the immune system, integrins have essential roles in leukocyte trafficking and function. These include immune cell attachment to endothelial and antigen-presenting cells, cytotoxicity, and extravasation into tissues. The integrin leukocyte function-associated antigen-1 (LFA-1), which is exclusively expressed on hematopoietic cells, has been intensely studied since this receptor is important for many functions of the immune system. LFA-1 is involved in a) the interaction between T-cells and antigen presenting cells, b) the adhesion of cells to post-capillary high endothelial venules or to activated endothelium at sites of inflammation (extravasation), c) the control of cell differentiation and proliferation, and d) the regulation of T-cell effector functions. Therefore, a precise understanding of the spatial and temporal control of LFA-1 interaction with its cellular counter-receptors, the intercellular adhesion molecules (ICAM) -1, -2 and -3, in the various contexts, is of high interest. LFA-1 mediated adhesion is induced by several extracellular stimuli in different cell types. In T-cells, LFA-1 becomes activated upon signaling from the T-cell receptor (TCR), and upon cytokine and chemokine sensing. Adhesion of monocytes to ICAM-1 is induced by lipopolysaccharide (LPS), a component of the bacterial cell wall. To investigate the regulation of LFA-1 adhesiveness, research has focused on the identification of interaction partners of the intracellular portions of the integrin alpha and beta subunits. This review will highlight recent developments on transmembrane and intracellular signaling proteins, which have been implicated in beta-2 integrin activation.

Discriminatory aptamer reveals serum response element transcription regulated by cytohesin-2.

Cytohesins are a family of highly homologous guanine nucleotide exchange factors (GEFs) that act on ADP-ribosylation factors (ARFs). The small ARF-GEFs are involved in integrin signaling, actin cytoskeleton remodeling, and vesicle transport. Here, we selected and applied a specific inhibitor for ARF nucleotide-binding site opener (ARNO)/cytohesin-2, an RNA aptamer that clearly discriminates between cytohesin-1 and cytohesin-2. This reagent bound to an N-terminal segment of cytohesin-2 and did not inhibit ARF-GEF function in vitro. When transfected into HeLa cells, it persisted for at least 6 h without requiring stabilization. Its effect in vivo was to down-regulate gene expression mediated through the serum-response element and knockdown mitogen-activated protein kinase activation, indicating that cytohesin-2 acts by means of mitogen-activated protein kinase signaling. We conclude that the N-terminal coiled-coil and parts of the Sec7 domain of cytohesin-2 are required for serum-mediated transcriptional activation in nonimmune cells, whereas cytohesin-1 is not. Our results indicate that intramer technology can be used not only for assigning novel biological functions to proteins or protein domains but also to prove nonredundancy of highly homologous proteins.

Attenuation of cell adhesion in lymphocytes is regulated by CYTIP, a protein which mediates signal complex sequestration.

An important theme in molecular cell biology is the regulation of protein recruitment to the plasma membrane. Fundamental biological processes such as proliferation, differentiation or leukocyte functions are initiated and controlled through the reversible binding of signaling proteins to phosphorylated membrane components. This is mediated by specialized interaction modules, such as SH2 and PH domains. Cytohesin-1 is an intracellular guanine nucleotide exchange factor, which regulates leukocyte adhesion. The activity of cytohesin-1 is controlled by phospho inositide-dependent membrane recruitment. An interacting protein was identified, the expression of which is upregulated by cytokines in hematopoietic cells. This molecule, CYTIP, is also recruited to the cell cortex by integrin signaling via its PDZ domain. However, stimulation of Jurkat cells with phorbol ester results in re-localization of CYTIP to the cytoplasm, and membrane detachment of cytohesin-1 strictly requires co-expression of CYTIP. Consequently, stimulated adhesion of Jurkat cells to intracellular adhesion molecule-1 is repressed by CYTIP. These findings outline a novel mechanism of signal chain abrogation through sequestration of a limiting component by specific protein-protein interactions.