Agnes Lovas

Low temperature inhibits RNA silencing-mediated defence by the control of siRNA generation

Temperature dramatically affects plant–virus interactions. Outbreaks of virus diseases are frequently associated with low temperature, while at high temperature viral symptoms are often attenuated (heat masking) and plants rapidly recover from virus diseases. However, the underlying mechanisms of these well‐known observations are not yet understood. RNA silencing is a conserved defence system of eukaryotic cells, which operates against molecular parasites including viruses and transgenes. Here we show that at low temperature both virus and transgene triggered RNA silencing are inhibited. Therefore, in cold, plants become more susceptible to viruses, and RNA silencing‐based phenotypes of transgenic plants are lost. Consistently, the levels of virus‐ and transgene‐derived small (21–26 nucleotide) interfering (si) RNAs—the central molecules of RNA silencing‐mediated defence pathways—are dramatically reduced at low temperature. In contrast, RNA silencing was activated and the amount of siRNAs gradually increased with rising temperature. However, temperature does not influence the accumulation of micro (mi) RNAs, which play a role in developmental regulation, suggesting that the two classes of small (si and mi) RNAs are generated by different nuclease complexes.

Lymphotoxin β receptor signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged ApoE−/− mice

Atherosclerosis involves a macrophage-rich inflammation in the aortic intima. It is increasingly recognized that this intimal inflammation is paralleled over time by a distinct inflammatory reaction in adjacent adventitia. Though cross talk between the coordinated inflammatory foci in the intima and the adventitia seems implicit, the mechanism(s) underlying their communication is unclear. Here, using detailed imaging analysis, microarray analyses, laser-capture microdissection, adoptive lymphocyte transfers, and functional blocking studies, we undertook to identify this mechanism. We show that in aged apoE−/− mice, medial smooth muscle cells (SMCs) beneath intimal plaques in abdominal aortae become activated through lymphotoxin β receptor (LTβR) to express the lymphorganogenic chemokines CXCL13 and CCL21. These signals in turn trigger the development of elaborate bona fide adventitial aortic tertiary lymphoid organs (ATLOs) containing functional conduit meshworks, germinal centers within B cell follicles, clusters of plasma cells, high endothelial venules (HEVs) in T cell areas, and a high proportion of T regulatory cells. Treatment of apoE−/− mice with LTβR-Ig to interrupt LTβR signaling in SMCs strongly reduced HEV abundance, CXCL13, and CCL21 expression, and disrupted the structure and maintenance of ATLOs. Thus, the LTβR pathway has a major role in shaping the immunological characteristics and overall integrity of the arterial wall.

Mouse Aorta Smooth Muscle Cells Differentiate Into Lymphoid Tissue Organizer-Like Cells on Combined Tumor Necrosis Factor Receptor-1/Lymphotoxin β-Receptor NF-κB Signaling

Objective—Mouse aorta smooth muscle cells (SMC) express tumor necrosis factor receptor superfamily member 1A (TNFR-1) and lymphotoxin &bgr;-receptor (LT&bgr;R). Circumstantial evidence has linked the SMC LT&bgr;R to tertiary lymphoid organogenesis in hyperlipidemic mice. Here, we explored TNFR-1 and LT&bgr;R signaling in cultured SMC. Methods and Results—TNFR-1 signaling activated the classical RelA NF-&kgr;B pathway, whereas LT&bgr;R signaling activated the classical RelA and alternative RelB NF-&kgr;B pathways, and both signaling pathways synergized to enhance p100 inhibitor processing to the p52 subunit of NF-&kgr;B. Microarrays showed that simultaneous TNFR-1/LT&bgr;R activation resulted in elevated mRNA encoding leukocyte homeostatic chemokines CCL2, CCL5, CXCL1, and CX3CL1. Importantly, SMC acquired features of lymphoid tissue organizers, which control tertiary lymphoid organogenesis in autoimmune diseases through hyperinduction of CCL7, CCL9, CXCL13, CCL19, CXCL16, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1. TNFR-1/LT&bgr;R cross-talk resulted in augmented secretion of lymphorganogenic chemokine proteins. Supernatants of TNFR-1/LT&bgr;R–activated SMC markedly supported migration of splenic T cells, B cells, and macrophages/dendritic cells. Experiments with ltbr−/− SMC indicated that LT&bgr;R-RelB activation was obligatory to generate the lymphoid tissue organizer phenotype. Conclusion—SMC may participate in the formation of tertiary lymphoid tissue in atherosclerosis by upregulation of lymphorganogenic chemokines involved in T-lymphocyte, B-lymphocyte, and macrophage/dendritic cell attraction.

The TATA-binding protein regulates maternal mRNA degradation and differential zygotic transcription in zebrafish

Early steps of embryo development are directed by maternal gene products and trace levels of zygotic gene activity in vertebrates. A major activation of zygotic transcription occurs together with degradation of maternal mRNAs during the midblastula transition in several vertebrate systems. How these processes are regulated in preparation for the onset of differentiation in the vertebrate embryo is mostly unknown. Here, we studied the function of TATA‐binding protein (TBP) by knock down and DNA microarray analysis of gene expression in early embryo development. We show that a subset of polymerase II‐transcribed genes with ontogenic stage‐dependent regulation requires TBP for their zygotic activation. TBP is also required for limiting the activation of genes during development. We reveal that TBP plays an important role in the degradation of a specific subset of maternal mRNAs during late blastulation/early gastrulation, which involves targets of the miR‐430 pathway. Hence, TBP acts as a specific regulator of the key processes underlying the transition from maternal to zygotic regulation of embryogenesis. These results implicate core promoter recognition as an additional level of differential gene regulation during development.

The pivotal role of the alternative NF-κB pathway in maintenance of basal bone homeostasis and osteoclastogenesis†

The alternative NF‐κB pathway consists predominantly of NF‐κB‐inducing kinase (NIK), IκB kinase α (IKKα), p100/p52, and RelB. The hallmark of the alternative NF‐κB signaling is the processing of p100 into p52 through NIK, thus allowing the binding of p52 and RelB. The physiologic relevance of alternative NF‐κB activation in bone biology, however, is not well understood. To elucidate the role of the alternative pathway in bone homeostasis, we first analyzed alymphoplasic (aly/aly) mice, which have a defective NIK and are unable to process p100, resulting in the absence of p52. We observed increased bone mineral density (BMD) and bone volume, indicating an osteopetrotic phenotype. These mice also have a significant defect in RANKL‐induced osteoclastogenesis in vitro and in vivo. NF‐κB DNA‐binding assays revealed reduced activity of RelA, RelB, and p50 and no binding activity of p52 in aly/aly osteoclast nuclear extracts after RANKL stimulation. To determine the role of p100 itself without the influence of a concomitant lack of p52, we used p100−/− mice, which specifically lack the p100 inhibitor but still express p52. p100−/− mice have an osteopenic phenotype owing to the increased osteoclast and decreased osteoblast numbers that was rescued by the deletion of one allele of the relB gene. Deletion of both allele of relB resulted in a significantly increased bone mass owing to decreased osteoclast activity and increased osteoblast numbers compared with wild‐type (WT) controls, revealing a hitherto unknown role for RelB in bone formation. Our data suggest a pivotal role of the alternative NF‐κB pathway, especially of the inhibitory role of p100, in both basal and stimulated osteoclastogenesis and the importance of RelB in both bone formation and resorption.

Differential RelA- and RelB-dependent gene transcription in LTβR-stimulated mouse embryonic fibroblasts

BackgroundLymphotoxin signaling via the lymphotoxin-β receptor (LTβR) has been implicated in biological processes ranging from development of secondary lymphoid organs, maintenance of spleen architecture, host defense against pathogens, autoimmunity, and lipid homeostasis. The major transcription factor that is activated by LTβR crosslinking is NF-κB. Two signaling pathways have been described, the classical inhibitor of NF-κB α (IκBα)-regulated and the alternative p100-regulated pathway that result in the activation of p50-RelA and p52-RelB NF-κB heterodimers, respectively.ResultsUsing microarray analysis, we investigated the transcriptional response downstream of the LTβR in mouse embryonic fibroblasts (MEFs) and its regulation by the RelA and RelB subunits of NF-κB. We describe novel LTβR-responsive genes that were regulated by RelA and/or RelB. The majority of LTβR-regulated genes required the presence of both RelA and RelB, revealing significant crosstalk between the two NF-κB activation pathways. Gene Ontology (GO) analysis confirmed that LTβR-NF-κB target genes are predominantly involved in the regulation of immune responses. However, other biological processes, such as apoptosis/cell death, cell cycle, angiogenesis, and taxis were also regulated by LTβR signaling. Moreover, LTβR activation inhibited expression of a key adipogenic transcription factor, peroxisome proliferator activated receptor-γ (pparg), suggesting that LTβR signaling may interfere with adipogenic differentiation.ConclusionMicroarray analysis of LTβR-stimulated fibroblasts provided comprehensive insight into the transcriptional response of LTβR signaling and its regulation by the NF-κB family members RelA and RelB.

p100 Deficiency Is Insufficient for Full Activation of the Alternative NF-κB Pathway: TNF Cooperates with p52-RelB in Target Gene Transcription

Background Constitutive activation of the alternative NF-κB pathway leads to marginal zone B cell expansion and disorganized spleen microarchitecture. Furthermore, uncontrolled alternative NF-κB signaling may result in the development and progression of cancer. Here, we focused on the question how does the constitutive alternative NF-κB signaling exert its effects in these malignant processes. Methodology/Principal Findings To explore the consequences of unrestricted alternative NF-κB activation on genome-wide transcription, we compared gene expression profiles of wild-type and NF-κB2/p100-deficient (p100−/−) primary mouse embryonic fibroblasts (MEFs) and spleens. Microarray experiments revealed only 73 differentially regulated genes in p100−/− vs. wild-type MEFs. Chromatin immunoprecipitation (ChIP) assays showed in p100−/− MEFs direct binding of p52 and RelB to the promoter of the Enpp2 gene encoding ENPP2/Autotaxin, a protein with an important role in lymphocyte homing and cell migration. Gene ontology analysis revealed upregulation of genes with anti-apoptotic/proliferative activity (Enpp2/Atx, Serpina3g, Traf1, Rrad), chemotactic/locomotory activity (Enpp2/Atx, Ccl8), and lymphocyte homing activity (Enpp2/Atx, Cd34). Most importantly, biochemical and gene expression analyses of MEFs and spleen, respectively, indicated a marked crosstalk between classical and alternative NF-κB pathways. Conclusions/Significance Our results show that p100 deficiency alone was insufficient for full induction of genes regulated by the alternative NF-κB pathway. Moreover, alternative NF-κB signaling strongly synergized both in vitro and in vivo with classical NF-κB activation, thereby extending the number of genes under the control of the p100 inhibitor of the alternative NF-κB signaling pathway.