The RNA binding proteins TIA1 and TIAL1 promote Mcl1 mRNA translation to protect germinal center responses from apoptosis.

Germinal centers (GCs) are essential for the establishment of long-lasting antibody responses. GC B cells rely on post-transcriptional RNA mechanisms to translate activation-associated transcriptional programs into functional changes in the cell proteome. However, the critical proteins driving these key mechanisms are still unknown. Here, we show that the RNA binding proteins TIA1 and TIAL1 are required for the generation of long-lasting GC responses. TIA1- and TIAL1-deficient GC B cells fail to undergo antigen-mediated positive selection, expansion and differentiation into B-cell clones producing high-affinity antibodies. Mechanistically, TIA1 and TIAL1 control the transcriptional identity of dark- and light-zone GC B cells and enable timely expression of the prosurvival molecule MCL1. Thus, we demonstrate here that TIA1 and TIAL1 are key players in the post-transcriptional program that selects high-affinity antigen-specific GC B cells.

The splicing regulators TIA1 and TIAL1 are required for the expression of the DNA damage repair machinery during B cell lymphopoiesis.

B cell lymphopoiesis requires dynamic modulation of the B cell transcriptome for timely coordination of somatic mutagenesis and DNA repair in progenitor B (pro-B) cells. Here, we show that, in pro-B cells, the RNA-binding proteins T cell intracellular antigen 1 (TIA1) and TIA1-like protein (TIAL1) act redundantly to enable developmental progression. They are global splicing regulators that control the expression of hundreds of mRNAs, including those involved in DNA damage repair. Mechanistically, TIA1 and TIAL1 bind to 5' splice sites for exon definition, splicing, and expression of DNA damage sensors, such as Chek2 and Rif1. In their absence, pro-B cells show exacerbated DNA damage, altered P53 expression, and increased cell death. Our study uncovers the importance of tight regulation of RNA splicing by TIA1 and TIAL1 for the expression of integrative transcriptional programs that control DNA damage sensing and repair during B cell development.

The RNA regulatory programs that govern lymphocyte development and function.

Lymphocytes require of constant and dynamic changes in their transcriptome for timely activation and production of effector molecules to combat external pathogens. Synthesis and translation of messenger (m)RNAs into these effector proteins is controlled both quantitatively and qualitatively by RNA binding proteins (RBPs). RBP-dependent regulation of RNA editing, subcellular location, stability, and translation shapes immune cell development and immunity. Extensive evidences have now been gathered from few model RBPs, HuR, PTBP1, ZFP36, and Roquin. However, recently developed methodologies for global characterization of protein:RNA interactions suggest the existence of complex RNA regulatory networks in which RBPs co-ordinately regulate the fate of sets of RNAs controlling cellular pathways and functions. In turn, RNA can also act as scaffolding of functionally related proteins modulating their activation and function. Here we review current knowledge about how RBP-dependent regulation of RNA shapes our immune system and discuss about the existence of a hidden immune cell epitranscriptome. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

The RNA-binding protein HuR is required for maintenance of the germinal centre response.

The germinal centre (GC) is required for the generation of high affinity antibodies and immunological memory. Here we show that the RNA binding protein HuR has an essential function in GC B cells to sustain the GC response. In its absence, the GC reaction and production of high-affinity antibody is severely impaired. Mechanistically, HuR affects the transcriptome qualitatively and quantitatively. The expression and splicing patterns of hundreds of genes are altered in the absence of HuR. Among these genes, HuR is required for the expression of Myc and a Myc-dependent transcriptional program that controls GC B cell proliferation and Ig somatic hypermutation. Additionally, HuR regulates the splicing and abundance of mRNAs required for entry into and transition through the S phase of the cell cycle, and it modulates a gene signature associated with DNA deamination protecting GC B cells from DNA damage and cell death.

Dose-dependent effects of prostaglandin E2 in macrophage adhesion and migration.

Macrophage migration to the focus of infection is a hallmark of the innate immune response. Macrophage spreading, adhesion, and migration through the extracellular matrix require dynamic remodeling of the actin cytoskeleton associated to integrin clustering in podosomes and focal adhesions. Here, we show that prostaglandin E2 (PGE2 ), the main prostaglandin produced by macrophages during inflammation, promote the distinctive dose-dependent formation of podosomes or focal adhesions in macrophages. Low concentrations of PGE2 increased p110γ PI3K expression, phosphorylation of actin-related protein 2, and formation of podosomes, which enhanced macrophage migration in response to chemokines. However, high doses of PGE2 increased phosphorylation of paxillin and focal adhesion kinase, the expression of serine/threonine protein kinase 1, and promoted focal adhesion formation and macrophage adhesion, reducing macrophage chemotaxis. In summary, we describe the dual role of PGE2 as a promoter of macrophage chemotaxis and adhesion, proposing a new model of macrophage migration to the inflammatory focus in the presence of a gradient of PGE2 .

Cyclooxygenase-2 deficiency in macrophages leads to defective p110γ PI3K signaling and impairs cell adhesion and migration.

Cyclooxygenase (Cox)-2 dependent PGs modulate several functions in many pathophysiological processes, including migration of immune cells. In this study, we addressed the role of Cox-2 in macrophage migration by using in vivo and in vitro models. Upon thioglycolate challenge, CD11b(+) F4/80(+) macrophages showed a diminished ability to migrate to the peritoneal cavity in cox-2(-/-) mice. In vivo migration of cox-2(-/-) macrophages from the peritoneal cavity to lymph nodes, as well as cell adhesion to the mesothelium, was reduced in response to LPS. In vitro migration of cox-2(-/-) macrophages toward MCP-1, RANTES, MIP-1α, or MIP-1ß, as well as cell adhesion to ICAM-1 or fibronectin, was impaired. Defects in cell migration were not due to changes in chemokine receptor expression. Remarkably, cox-2(-/-) macrophages showed a deficiency in focal adhesion formation, with reduced phosphorylation of paxillin (Tyr(188)). Interestingly, expression of the p110γ catalytic subunit of PI3K was severely reduced in the absence of Cox-2, leading to defective Akt phosphorylation, as well as cdc42 and Rac-1 activation. Our results indicate that the paxillin/p110γ-PI3K/Cdc42/Rac1 axis is defective in cox-2(-/-) macrophages, which results in impaired cell adhesion and migration.

Involvement of PGE2 and the cAMP signalling pathway in the up-regulation of COX-2 and mPGES-1 expression in LPS-activated macrophages.

PG (prostaglandin) E2 plays an important role in the modulation of the immune response and the inflammatory process. In the present study, we describe a PGE2 positive feedback for COX (cyclo-oxygenase)-2 and mPGES-1 [microsomal PGES (PGE synthase)-1] expression in the macrophage cell line RAW 264.7. Our results show that PGE2 induces COX-2 and mPGES-1 expression, an effect mimicked by dbcAMP (dibutyryl-cAMP) or forskolin. Furthermore, the cAMP signalling pathway co-operates with LPS (lipopolysaccharide) in the induction of COX-2 and mPGES-1 transcriptional activation. Analysis of the involvement of PGE receptors [EPs (E-prostanoids)] showed that incubation with EP2 agonists up-regulated both COX2 and mPGES-1 mRNA levels. Moreover, EP2 receptor overexpression enhanced the transcriptional activation of COX2 and mPGES-1 promoters. This induction was repressed by the PKA (protein kinase A) inhibitor H89. Activation of the PGE2/EP2/PKA signalling pathway induced the phosphorylation of CREB [CRE (cAMP-response element)-binding protein] in macrophages and stimulated the specific binding of this transcription factor to COX2 and mPGES-1 promoters. Deletion or mutation of potential CRE sites in both promoters diminished their transcriptional activity. In summary, the results of the present study demonstrate that activation of PKA/CREB signalling through the EP2 receptor by PGE2 plays a key role in the expression of COX-2 and mPGES-1 in activated macrophages.

Coordinated up-regulation of cyclooxygenase-2 and microsomal prostaglandin E synthase 1 transcription by nuclear factor kappa B and early growth response-1 in macrophages.

Prostaglandin (PG) E(2) is a potent lipid mediator that plays an essential role in inflammation, fever and pain. It is produced from arachidonic acid (AA) by a cascade of enzymatic reactions involving cyclooxygenases (COX-1 and -2) and prostaglandin E synthases (cPGES, mPGES-1 and -2). Functional coupling of the inducible enzymes COX-2 and mPGES-1 has been proposed for increased production of PGE(2) in different cell types. PGE(2) produced by macrophages plays an essential role in the pathogenesis of inflammatory diseases. Here, we have investigated the mechanisms involved in the regulation of COX-2 and mPGES-1 expressions in murine macrophages upon bacterial lipopolysaccharide (LPS) treatment. LPS stimulation induced the coordinated synthesis of COX-2 and mPGES-1 that resulted in an enhanced production of PGE(2) in RAW 264.7 macrophages. Furthermore, we show the involvement of NF-kappaB and Egr-1 transcription factors in the transcriptional induction of these enzymes. LPS treatment promoted specific binding of NF-kappaB to both COX-2 and mPGES-1 promoters. Site-directed mutagenesis, electrophoretic mobility shift assays and ChIP assays allowed the identification of a sequence acting as a NF-kappaB recognition site in the murine mPGES-1 promoter. Furthermore, LPS induced the expression of Egr-1 that cooperated with NF-kappaB in the up-regulation of COX-2 and mPGES-1. Inhibition of Egr-1 expression reduced substantially LPS-mediated induction of COX-2 and mPGES-1 expression, resulting in a decrease in PGE(2) production. Our findings point out to Egr-1 and NF-kappaB cooperation as determinant for PGE2 synthesis by macrophages in inflammatory processes through the coordinated regulation of COX-2 and mPGES-1.