Human beta defensins (hBDs) are antimicrobial peptides that play an important role in innate immune responses at epithelial barriers such as the skin. However, the role that hBDs play in initiating cellular immune responses that contribute to antigen-specific adaptive immunity is not well understood. Here we show that one member of the hBD family, hBD3, can induce maturation and T helper type 1 (Th1) skewing function in human Langerhans cell-like DCs (LC-DCs). Specifically, hBD3 potently induces phenotypic maturation of LC-DCs, including increased expression of CCR7 which mediates functional chemotactic responses to CCL19 and CCL21. HBD3-stimulated LC-DCs induce strong proliferation and IFN-γ secretion by naïve human T cells. HBD3 also induces phenotypic maturation of primary human skin-migratory dendritic cells derived from human skin explants. These results suggest an important role for hBD3 in inducing DC activation, migration, and polarization. Thus hBD3 contributes to the integration of innate and adaptive immune responses in the skin and may be a useful adjuvant for skin immunization and an important factor in the pathophysiology of inflammatory skin diseases.
The production of phosphatidic acid plays a crucial role in the activation of the ERK cascade. This role was linked to the binding of phosphatidate to a specific polybasic site within the kinase domain of Raf-1. Here we show that phosphatidate promotes ERK phosphorylation in intact cells but does not activate Raf in vitro. The kinase suppressor of Ras (KSR) contains a sequence homologous to the phosphatidate binding site of Raf-1. Direct binding of phosphatidate to synthetic peptides derived from the sequences of the binding domains of Raf-1 and KSR was demonstrated by spectroscopic techniques. The specificity of these interactions was confirmed using synthetic lipids and mutated peptides in which the core of the phosphatidic acid binding domain was disrupted. Insulin and exogenous dioleoyl phosphatidate induced a rapid translocation of a mouse KSR1-EGFP construct to the plasma membrane of HIRcB cells. Mutation of two arginines located in the core of the putative phosphatidate binding site abolished dioleoyl phosphatidate-and insulin-induced translocation of KSR1. Overexpression of the mutant KSR1 in HIRcB cells inhibited insulin-dependent MEK and ERK phosphorylation. The addition of dioleoyl phosphatidate or insulin increased the co-localization of KSR1 and H-Ras and promoted the formation of plasma membrane patches enriched in both proteins and phosphatidic acid. These results, in conjunction with our previous work, suggest the formation of phosphatidate-enriched membrane microdomains that contain all components of the ERK cascade. We propose that these domains act as molecular scaffolds in the coupling of signaling events.Although the idea that phosphatidic acid (PA) 3 is an important lipid second messenger seems to be widely accepted, the actual functions of this lipid in signal transduction are still poorly understood. Several physiological roles for PA have been proposed in the past. These include regulation of protein and lipid phosphorylation (1-3), regulation of cAMP degradation (4), activation of oxidative processes (3, 5), and modulation of membrane traffic (6 -9). Many of these functions are mediated by the direct interaction of PA with specific target proteins. Thus, PA appears to function in a manner analogous to other lipid second messengers (i.e. by promoting the binding of target proteins to specific regions of the cell membrane).Direct binding of PA has been demonstrated in a small subset of proteins. The interactions of PA with Raf-1 have been mapped to a 35-amino acid stretch within the kinase domain (10 -13), whereas the binding of PA to mTOR involves Arg 2109 , a residue located in the vicinity of the rapamycin binding domain (14,15). Putative binding sites for the cyclic nucleotide phosphodiesterase PDE4D3 (4) and the protein-tyrosine phosphatase SHP-1 (16) have also been described. In general, these PA binding domains bear little sequence similarity to each other, except for the fact that they all contain at least one polybasic motif.Previous work from our laboratory has demonstrated th...
Background: Phospholipase D (PLD) is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF) family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs) of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin.
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