Immune-checkpoint Protein VISTA Critically Regulates the IL-23/IL-17 Inflammatory Axis

V-domain Immunoglobulin Suppressor of T cell Activation (VISTA) is an inhibitory immune-checkpoint molecule that suppresses CD4+ and CD8+ T cell activation when expressed on antigen-presenting cells. Vsir−/− mice developed loss of peripheral tolerance and multi-organ chronic inflammatory phenotypes. Vsir−/− CD4+ and CD8+ T cells were hyper-responsive towards self- and foreign antigens. Whether or not VISTA regulates innate immunity is unknown. Using a murine model of psoriasis induced by TLR7 agonist imiquimod (IMQ), we show that VISTA deficiency exacerbated psoriasiform inflammation. Enhanced TLR7 signaling in Vsir−/− dendritic cells (DCs) led to the hyper-activation of Erk1/2 and Jnk1/2, and augmented the production of IL-23. IL-23, in turn, promoted the expression of IL-17A in both TCRγδ+ T cells and CD4+ Th17 cells. Furthermore, VISTA regulates the peripheral homeostasis of CD27− γδ T cells and their activation upon TCR-mediated or cytokine-mediated stimulation. IL-17A-producing CD27− γδ T cells were expanded in the Vsir−/− mice and amplified the inflammatory cascade. In conclusion, this study has demonstrated that VISTA critically regulates the inflammatory responses mediated by DCs and IL-17-producing TCRγδ+ and CD4+ Th17 T cells following TLR7 stimulation. Our finding provides a rationale for therapeutically enhancing VISTA-mediated pathways to benefit the treatment of autoimmune and inflammatory disorders

The LIM Protein Ajuba Influences Interleukin-1-Induced NF-κB Activation by Affecting the Assembly and Activity of the Protein Kinase Cζ/p62/TRAF6 Signaling Complex

The Zyxin/Ajuba family of cytosolic LIM domain-containing proteins has the potential to shuttle from sites of cell adhesion into the nucleus and thus can be candidate transducers of environmental signals. To understand Ajuba's role in signal transduction pathways, we performed a yeast two-hybrid screen with the LIM domain region of Ajuba. We identified the atypical protein kinase C (aPKC) scaffold protein p62 as an Ajuba binding partner. A prominent function of p62 is the regulation of NF-κB activation in response to interleukin-1 (IL-1) and tumor necrosis factor signaling through the formation of an aPKC/p62/TRAF6 multiprotein signaling complex. In addition to p62, we found that Ajuba also interacted with tumor necrosis factor receptor-associated factor 6 (TRAF6) and PKCζ. Ajuba recruits TRAF6 to p62 and in vitro activates PKCζ activity and is a substrate of PKCζ. Ajuba null mouse embryonic fibroblasts (MEFs) and lungs were defective in NF-κB activation following IL-1 stimulation, and in lung IKK activity was inhibited. Overexpression of Ajuba in primary MEFs enhances NF-κB activity following IL-1 stimulation. We propose that Ajuba is a new cytoso lic component of the IL-1 signaling pathway modulating IL-1-induced NF-κB activation by influencing the assembly and activity of the aPKC/p62/TRAF6 multiprotein signaling complex

The LIM Protein Ajuba Regulates Phosphatidylinositol 4,5-Bisphosphate Levels in Migrating Cells through an Interaction with and Activation of PIPKIα

The phosphoinositide phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] regulates the activity of many actin-binding proteins and as such is an important modulator of cytoskeleton organization during cell migration, for example. In migrating cells actin remodeling is tightly regulated and localized; therefore, how the PI(4,5)P2 level is spatially and temporally regulated is crucial to understanding how it controls cell migration. Here we show that the LIM protein Ajuba contributes to the cellular regulation of PI(4,5)P2 levels by interacting with and activating the enzymatic activity of the PI(4)P 5-kinase (PIPKIα), the predominant enzyme in the synthesis of PI(4,5)P2, in a migration stimulus-regulated manner. In migrating primary mouse embryonic fibroblasts (MEFs) from Ajuba(−/−) mice the level of PI(4,5)P2 was decreased with a corresponding increase in the level of the substrate PI(4)P. Reintroduction of Ajuba into these cells normalized PI(4,5)P2 levels. Localization of PI(4,5)P2 synthesis and PIPKIα in the leading lamellipodia and membrane ruffles, respectively, of migrating Ajuba(−/−) MEFs was impaired. In vitro, Ajuba dramatically activated the enzymatic activity of PIPKIα while inhibiting the activity of PIPKIIβ. Thus, in addition to its effects upon Rac activity Ajuba can also influence cell migration through regulation of PI(4,5)P2 synthesis through direct activation of PIPKIα enzyme activity

Phosphatidylinositol 3-Kinase Activation Is Required To Form the NKG2D Immunological Synapse▿

The receptor NKG2D allows natural killer (NK) cells to detect virally infected, stressed, and tumor cells. In human cells, NKG2D signaling is mediated through the associated DAP10 adapter. Here we show that engagement of NKG2D by itself is sufficient to stimulate the formation of the NK immunological synapse (NKIS), with recruitment of NKG2D to the center synapse. Mutagenesis studies of DAP10 revealed that the phosphatidylinositol 3-kinase binding site, but not the Grb2 binding site, was required and sufficient for recruitment of DAP10 to the NKIS. Surprisingly, we found that in the absence of the Grb2 binding site, Grb2 was still recruited to the NKIS. Since the recruitment of Grb2 was dependent on phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), we explored the possibility that recruitment to the NKIS is mediated by a pleckstrin homology (PH) domain-containing binding partner for Grb2. We found that the PH domain of SOS1, but not that of Vav1, was able to be recruited by PIP3. These results provide new insights into the mechanism of immunological synapse formation and also demonstrate how multiple mechanisms can be used to recruit the same signaling proteins to the plasma membrane

LIM-domain proteins, LIMD1, Ajuba, and WTIP are required for microRNA-mediated gene silencing

In recent years there have been major advances with respect to the identification of the protein components and mechanisms of microRNA (miRNA) mediated silencing. However, the complete and precise repertoire of components and mechanism(s) of action remain to be fully elucidated. Herein we reveal the identification of a family of three LIM domain-containing proteins, LIMD1, Ajuba and WTIP (Ajuba LIM proteins) as novel mammalian processing body (P-body) components, which highlight a novel mechanism of miRNA-mediated gene silencing. Furthermore, we reveal that LIMD1, Ajuba, and WTIP bind to Ago1/2, RCK, Dcp2, and eIF4E in vivo, that they are required for miRNA-mediated, but not siRNA-mediated gene silencing and that all three proteins bind to the mRNA 5' m7GTP capprotein complex. Mechanistically, we propose the Ajuba LIM proteins interact with the m7GTP cap structure via a specific interaction with eIF4E that prevents 4EBP1 and eIF4G interaction. In addition, these LIM-domain proteins facilitate miRNA-mediated gene silencing by acting as an essential molecular link between the translationally inhibited eIF4E-m7GTP-5'cap and Ago1/2 within the miRISC complex attached to the 3'-UTR of mRNA, creating an inhibitory closed-loop complex