Fibrosis is a common pathology in many cardiac disorders and is driven by the activation of resident fibroblasts. The global posttranscriptional mechanisms underlying fibroblast-to-myofibroblast ...conversion in the heart have not been explored.
Genome-wide changes of RNA transcription and translation during human cardiac fibroblast activation were monitored with RNA sequencing and ribosome profiling. We then used RNA-binding protein-based analyses to identify translational regulators of fibrogenic genes. The integration with cardiac ribosome occupancy levels of 30 dilated cardiomyopathy patients demonstrates that these posttranscriptional mechanisms are also active in the diseased fibrotic human heart.
We generated nucleotide-resolution translatome data during the transforming growth factor β1-driven cellular transition of human cardiac fibroblasts to myofibroblasts. This identified dynamic changes of RNA transcription and translation at several time points during the fibrotic response, revealing transient and early-responder genes. Remarkably, about one-third of all changes in gene expression in activated fibroblasts are subject to translational regulation, and dynamic variation in ribosome occupancy affects protein abundance independent of RNA levels. Targets of RNA-binding proteins were strongly enriched in posttranscriptionally regulated genes, suggesting genes such as MBNL2 can act as translational activators or repressors. Ribosome occupancy in the hearts of patients with dilated cardiomyopathy suggested the same posttranscriptional regulatory network was underlying cardiac fibrosis. Key network hubs include RNA-binding proteins such as Pumilio RNA binding family member 2 (PUM2) and Quaking (QKI) that work in concert to regulate the translation of target transcripts in human diseased hearts. Furthermore, silencing of both PUM2 and QKI inhibits the transition of fibroblasts toward profibrotic myofibroblasts in response to transforming growth factor β1.
We reveal widespread translational effects of transforming growth factor β1 and define novel posttranscriptional regulatory networks that control the fibroblast-to-myofibroblast transition. These networks are active in human heart disease, and silencing of hub genes limits fibroblast activation. Our findings show the central importance of translational control in fibrosis and highlight novel pathogenic mechanisms in heart failure.
Long noncoding RNAs (lncRNAs) are a heterogenous group of RNAs, which can encode small proteins. The extent to which developmentally regulated lncRNAs are translated and whether the produced ...microproteins are relevant for human development is unknown. Using a human embryonic stem cell (hESC)-based pancreatic differentiation system, we show that many lncRNAs in direct vicinity of lineage-determining transcription factors (TFs) are dynamically regulated, predominantly cytosolic, and highly translated. We genetically ablated ten such lncRNAs, most of them translated, and found that nine are dispensable for pancreatic endocrine cell development. However, deletion of
diminishes insulin
cells, in a manner independent of the nearby TF
. One-by-one disruption of each of
's open reading frames suggests that the RNA, rather than the produced microproteins, is required for endocrine development. Our work highlights extensive translation of lncRNAs during hESC pancreatic differentiation and provides a blueprint for dissection of their coding and noncoding roles.
Little is known about the impact of trans-acting genetic variation on the rates with which proteins are synthesized by ribosomes. Here, we investigate the influence of such distant genetic loci on ...the efficiency of mRNA translation and define their contribution to the development of complex disease phenotypes within a panel of rat recombinant inbred lines.
We identify several tissue-specific master regulatory hotspots that each control the translation rates of multiple proteins. One of these loci is restricted to hypertrophic hearts, where it drives a translatome-wide and protein length-dependent change in translational efficiency, altering the stoichiometric translation rates of sarcomere proteins. Mechanistic dissection of this locus across multiple congenic lines points to a translation machinery defect, characterized by marked differences in polysome profiles and misregulation of the small nucleolar RNA SNORA48. Strikingly, from yeast to humans, we observe reproducible protein length-dependent shifts in translational efficiency as a conserved hallmark of translation machinery mutants, including those that cause ribosomopathies. Depending on the factor mutated, a pre-existing negative correlation between protein length and translation rates could either be enhanced or reduced, which we propose to result from mRNA-specific imbalances in canonical translation initiation and reinitiation rates.
We show that distant genetic control of mRNA translation is abundant in mammalian tissues, exemplified by a single genomic locus that triggers a translation-driven molecular mechanism. Our work illustrates the complexity through which genetic variation can drive phenotypic variability between individuals and thereby contribute to complex disease.
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Glucocorticoids such as dexamethasone (Dex) are widely used to treat both acute and chronic inflammatory conditions. They regulate immune responses by dampening cell-mediated immunity ...in a glucocorticoid receptor (GR)-dependent manner, by suppressing the expression of pro-inflammatory cytokines and chemokines and by stimulating the expression of anti-inflammatory mediators. Despite its evident clinical benefit, the mechanistic underpinnings of the gene regulatory networks transcriptionally controlled by GR in a context-specific manner remain mysterious. Next generation sequencing methods such mRNA sequencing (RNA-seq) and Ribosome profiling (ribo-seq) provide tools to investigate the transcriptional and post-transcriptional mechanisms that govern gene expression. Here, we integrate matched RNA-seq data with ribo-seq data from human acute monocytic leukemia (THP-1) cells treated with the TLR4 ligand lipopolysaccharide (LPS) and with Dex, to investigate the global transcriptional and translational regulation (translational efficiency, ΔTE) of Dex-responsive genes. We find that the expression of most of the Dex-responsive genes are regulated at both the transcriptional and the post-transcriptional level, with the transcriptional changes intensified on the translational level. Overrepresentation pathway analysis combined with STRING protein network analysis and manual functional exploration, identified these genes to encode immune effectors and immunomodulators that contribute to macrophage-mediated immunity and to the maintenance of macrophage-mediated immune homeostasis. Further research into the translational regulatory network underlying the GR anti-inflammatory response could pave the way for the development of novel immunomodulatory therapeutic regimens with fewer undesirable side effects.
Fibrosis is a common pathology in cardiovascular disease. In the heart, fibrosis causes mechanical and electrical dysfunction and in the kidney, it predicts the onset of renal failure. Transforming ...growth factor β1 (TGFβ1) is the principal pro-fibrotic factor, but its inhibition is associated with side effects due to its pleiotropic roles. We hypothesized that downstream effectors of TGFβ1 in fibroblasts could be attractive therapeutic targets and lack upstream toxicity. Here we show, using integrated imaging-genomics analyses of primary human fibroblasts, that upregulation of interleukin-11 (IL-11) is the dominant transcriptional response to TGFβ1 exposure and required for its pro-fibrotic effect. IL-11 and its receptor (IL11RA) are expressed specifically in fibroblasts, in which they drive non-canonical, ERK-dependent autocrine signalling that is required for fibrogenic protein synthesis. In mice, fibroblast-specific Il11 transgene expression or Il-11 injection causes heart and kidney fibrosis and organ failure, whereas genetic deletion of Il11ra1 protects against disease. Therefore, inhibition of IL-11 prevents fibroblast activation across organs and species in response to a range of important pro-fibrotic stimuli. These results reveal a central role of IL-11 in fibrosis and we propose that inhibition of IL-11 is a potential therapeutic strategy to treat fibrotic diseases.
BackgroundThe efficacy of CAR T cell therapy correlates with the ability of the transferred therapeutic T cells to traffic to the respective tumor niche. We observed that engineered T cells ...downregulate the lymphoid homing receptor CCR7 during the manufacturing process. Consequently, in-vitro and in-vivo migration of CAR T cells toward the corresponding chemokine ligands was severely impaired. To improve nodal CAR T cell trafficking and anti-lymphoma efficacy of B-NHL-targeting anti-CXCR5 CAR T cells, we engineered CAR T cells which co-expressed the homing receptor CCR7.MethodsThe anti-human CXCR5 CAR and anti-mouse CXCR5 CAR, encoded within a retroviral MP71 backbone, were generated essentially as described.1 In case of the human and mouse CXCR5 CAR-CCR7 vectors, hCCR7 or mCCR7 cDNAs without stop codons were introduced upstream of the CXCR5 CAR cassette via P2A linkage. To test the functionality of CCR7-engineered CAR T cells in-vitro, transwell migration, cytotoxicity, and cytokine release assays were employed. CAR T cell killing dynamics were determined by microscopy-based methods. Mouse leukemia and lymphoma models served as tools to study in-vivo CCR7-modulated CAR T cell homing to lymph nodes and target cell killing.ResultsCCR7 overexpression on human and mouse anti-CXCR5 CAR T cells restored their migratory capacity and improved lymph node homing. In addition, we observed an enhanced killing capacity of CCR7-equipped CAR T cells in-vitro, which is likely supported by the costimulatory role of CCR7 in the formation of an immunological synapse. We showed that CCR7 accumulated in mature CAR synapses, an observation that is supported by the transcriptional upregulation of genes associated with cytoskeletal rearrangement, relevant for immunological synapse formation and migration in CCR7-engineered CAR T cells.ConclusionsTherapeutically, CCR7-enforced nodal recruitment and enhanced killing kinetics of CAR T cells conferred improved lymphoma eradication in a mouse lymphoma model. Beyond improved anti-tumor responses in nodal hematological malignancies, we envisage that this CCR7-engineering approach might be useful to improve CAR T cell therapies that specifically target solid tumor entities metastasized to lymph nodes.AcknowledgementsThis work was funded by grants from the HGF ”Zukunftsthema Inflammation & Immunology”.ReferenceBunse M, Pfeilschifter J, Bluhm J, et al. CXCR5 CAR-T cells simultaneously target B cell non-Hodgkin’s lymphoma and tumor-supportive follicular T helper cells. Nat Commun. 2021;12:240.
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