T-cell activation decreases miRNA-15a/16 levels to promote MEK1-ERK1/2-Elk1 signaling and proliferative capacity.

While miRs have been extensively studied in the context of malignancy and tumor progression, their functions in regulating T-cell activation are less clear. In initial studies, we found reduced levels of miR-15a/16 at 3 to 18 h post-T-cell receptor (TCR) stimulation, suggesting a role for decreased levels of this miR pair in shaping T-cell activation. To further explore this, we developed an inducible miR15a/16 transgenic mouse model to determine how elevating miR-15a/16 levels during early stages of activation would affect T-cell proliferation and to identify TCR signaling pathways regulated by this miR pair. Doxycycline (DOX)-induced expression of miR-15a/16 from 0 to 18 h post-TCR stimulation decreased ex vivo T-cell proliferation as well as in vivo antigen-specific T-cell proliferation. We also combined bioinformatics and proteomics approaches to identify the mitogen-activated protein kinase kinase 1 (MEK1) (Map2k1) as a target of miR-15a/16. MEK1 targeting by miR-15a/16 was confirmed using miR mimics that decreased Map2k1 mRNA containing the 3'-UTR target nucleotide sequence (UGCUGCUA) but did not decrease Map2k1 containing a mutated control sequence (AAAAAAAA). Phosphorylation of downstream signaling molecules, extracellular signal-regulated protein kinase 1/2 (ERK1/2) and Elk1, was also decreased by DOX-induced miR-15a/16 expression. In addition to MEK1, ERK1 was subsequently found to be targeted by miR-15a/16, with DOX-induced miR-15a/16 reducing total ERK1 levels in T cells. These findings show that TCR stimulation reduces miR-15a/16 levels at early stages of T-cell activation to facilitate increased MEK1 and ERK1, which promotes the sustained MEK1-ERK1/2-Elk1 signaling required for optimal proliferation.

ERK Signaling Controls Innate-like CD8+ T Cell Differentiation via the ELK4 (SAP-1) and ELK1 Transcription Factors.

In mouse thymocyte development, signaling by the TCR through the ERK pathway is required for positive selection of conventional naive T cells. The Ets transcription factor ELK4 (SAP-1), an ERK-regulated cofactor of the SRF transcription factor, plays an important role in positive selection by activating immediate-early genes such as the Egr transcription factor family. The role of ELK4-SRF signaling in development of other T cell types dependent on ERK signaling has been unclear. In this article, we show that ELK4, and its close relative ELK1, act cell autonomously in the thymus to control the generation of innate-like αß CD8+ T cells with memory-like characteristics. Mice lacking ELK4 and ELK1 develop increased numbers of innate-like αß CD8+ T cells, which populate the periphery. These cells develop cell autonomously rather than through expansion of PLZF+ thymocytes and concomitantly increased IL-4 signaling. Their development is associated with reduced TCR-mediated activation of ELK4-SRF target genes and can be partially suppressed by overexpression of the ELK4-SRF target gene EGR2. Consistent with this, partial inhibition of ERK signaling in peripheral CD8+T cells promotes the generation of cells with innate-like characteristics. These data establish that low-level ERK signaling through ELK4 (and ELK1) promotes innate-like αß CD8+ T cell differentiation, tuning conventional versus innate-like development.

Ternary complex factors SAP-1 and Elk-1, but not net, are functionally equivalent in thymocyte development.

The ternary complex factors (TCFs; SAP-1, Elk-1, and Net) are serum response factor cofactors that share many functional properties and are coexpressed in many tissues. SAP-1, the predominant thymus TCF, is required for thymocyte positive selection. In this study, we assessed whether the different TCFs are functionally equivalent. Elk-1 deletion, but not the hypomorphic Net(delta) mutation, exacerbated the SAP-1 positive selection phenotype, but triply deficient thymocytes were no more defective than SAP-1(-/-) Elk-1(-/-) cells. Inactivation of the other TCFs did not affect SAP-1-independent processes, including beta-selection, regulatory T cell selection, and negative selection, although reduced marginal zone B cells were observed in SAP-1(-/-) Elk-1(-/-) animals. Ectopic expression of Elk-1, but not Net, rescued positive selection of SAP-1(-/-) thymocytes; thus, SAP-1 and Elk-1 are functionally equivalent in this system, and the SAP-1 null selection phenotype reflects only its high expression in the thymus. Array analysis of TCR-stimulated double-positive cells identified SAP-1-dependent inducible genes whose transcription was further impaired in SAP-1(-/-) Elk-1(-/-) cells; thus, these genes, which include Egr-1 and Egr-2, represent candidate mediators of positive selection. Chromatin immunoprecipitation revealed subtly different promoter targeting between the different TCFs. Ectopic expression of Egr-1 restored positive selection in SAP-1 null thymocytes, establishing it (and possibly other Egr family members) as the major effector for ERK-SAP-1 signaling in thymocyte positive selection.

A beta integrin subunit regulates bacterial phagocytosis in medfly haemocytes.

We have recently reported that the activation of focal adhesion kinase (FAK) and its downstream targets upon pathogen challenge regulate phagocytosis in medfly haemocytes. The goal of this study was to further explore the signalling pathway underlying the process of phagocytosis. In particular, in this report, we used flow cytometry, RNA interference, enzyme-linked immunosorbent assay, Western blot and immunoprecipitation analysis to demonstrate the haemocyte surface receptor, through which the extracellular signals in response to bacteria are transmitted intracellularly. The presented data demonstrate the expression of a beta integrin subunit in the surface of medfly haemocytes that transmits signals upon pathogen triggering to FAK and its downstream targets, Src, MAP kinases and Elk-1-like protein, for the engulfment of pathogen. Interestingly LPS is not internalized through integrins.

The BASH/BLNK/SLP-65-associated protein BNAS1 regulates antigen-receptor signal transmission in B cells.

BASH/BLNK/SLP-65 is an adaptor protein necessary for the B cell receptor (BCR) signal transduction. Here we report the identification through the yeast two-hybrid system of a novel 26-kDa protein, BASH N-terminus-associated protein 1 (BNAS1), which interacts with the conserved and functionally important N-terminal domain of BASH/BLNK/SLP-65. BNAS1 presumably contains four transmembrane domains and the leucine zipper (LZ) motif, and is expressed ubiquitously. The association of BNAS1 with BASH/BLNK/SLP-65 through its LZ motif in vertebrate cells was demonstrated by immunoprecipitation assay. Confocal microscopy revealed that exogenously expressed BNAS1 is localized to the endoplasmic reticulum (ER) and the nuclear envelope. BASH/BLNK/SLP-65 alone was present diffusely in the cytoplasm, but localized to the same position as BNAS1 when co-expressed with BNAS1. Their co-localization was dependent on the domains containing the LZ motif of both molecules. BCR-signaled transcriptional activation of Elk-1 was suppressed by over-expression of BNAS1 in DT40 chicken B cells, and conversely augmented in the BNAS1-deficient DT40 cells, which was restored by BNAS1 reconstitution. This augmentation of Elk-1 activation in the BNAS1-deficient cells was abolished selectively by Jun N-terminal kinase (JNK) inhibitor, suggesting that BNAS1 regulates Elk-1 activation through JNK. Taken together, these results suggest that BNAS1 interacts with BASH/BLNK/SLP-65 at the ER and/or the outer nuclear membrane and is involved in the regulation of the signal transmission via mitogen-activated protein kinases leading to Elk-1 activation.