Stereoselective Synthesis, Pro-resolution, and Anti-inflammatory Actions of RvD5n-3 DPA.

The methyl ester of resolvin D5n-3 DPA, a lipid mediator biosynthesized from the omega-3 fatty acid n-3 docosapentaenoic acid, was stereoselectively prepared in 8% yield over 12 steps (longest linear sequence). The key steps for the introduction of the two stereogenic secondary alcohols were an organocatalyzed oxyamination and the Midland Alpine borane reduction. For the assembly of the carbon chain, the Sonogashira cross-coupling reaction and the Takai olefination were utilized. The physical properties, including retention time in liquid chromatography and tandem mass spectra, of the synthetic material were matched against material from human peripheral blood and mouse infectious exudates. Synthetic RvD5n-3 DPA, obtained just prior to biological experiments, displayed potent leukocyte-directed activities, upregulating the ability of neutrophils and macrophages to phagocytose bacteria, known as hallmark bioactions of specialized pro-resolving endogenous mediators.

DLC1 is a direct target of activated YAP/TAZ that drives collective migration and sprouting angiogenesis.

Endothelial YAP/TAZ (YAP is also known as YAP1, and TAZ as WWTR1) signaling is crucial for sprouting angiogenesis and vascular homeostasis. However, the underlying molecular mechanisms that explain how YAP/TAZ control the vasculature remain unclear. This study reveals that the focal adhesion protein deleted-in-liver-cancer 1 (DLC1) is a direct transcriptional target of the activated YAP/TAZ-TEAD complex. We find that substrate stiffening and VEGF stimuli promote expression of DLC1 in endothelial cells. In turn, DLC1 expression levels are YAP and TAZ dependent, and constitutive activation of YAP is sufficient to drive DLC1 expression. DLC1 is needed to limit F-actin fiber formation, integrin-based focal adhesion lifetime and integrin-mediated traction forces. Depletion of endothelial DLC1 strongly perturbs cell polarization in directed collective migration and inhibits the formation of angiogenic sprouts. Importantly, ectopic expression of DLC1 is sufficient to restore migration and angiogenic sprouting in YAP-depleted cells. Together, these findings point towards a crucial and prominent role for DLC1 in YAP/TAZ-driven endothelial adhesion remodeling and collective migration during angiogenesis.This article has an associated First Person interview with the first author of the paper.

Nur77 variants solely comprising the amino-terminal domain activate hypoxia-inducible factor-1α and affect bone marrow homeostasis in mice and humans.

Gene targeting via homologous recombination can occasionally result in incomplete disruption of the targeted gene. Here, we show that a widely used Nur77-deficient transgenic mouse model expresses a truncated protein encoding for part of the N-terminal domain of nuclear receptor Nur77. This truncated Nur77 protein is absent in a newly developed Nur77-deficient mouse strain generated using Cre-Lox recombination. Comparison of these two mouse strains using immunohistochemistry, flow cytometry, and colony-forming assays shows that homologous recombination-derived Nur77-deficient mice, but not WT or Cre-Lox-derived Nur77-deficient mice, suffer from liver immune cell infiltrates, loss of splenic architecture, and increased numbers of bone marrow hematopoietic stem cells and splenic colony-forming cells with age. Mechanistically, we demonstrate that the truncated Nur77 N-terminal domain protein maintains the stability and activity of hypoxia-inducible factor (HIF)-1, a transcription factor known to regulate bone marrow homeostasis. Additionally, a previously discovered, but uncharacterized, human Nur77 transcript variant that encodes solely for its N-terminal domain, designated TR3ß, can also stabilize and activate HIF-1α. Meta-analysis of publicly available microarray data sets shows that TR3ß is highly expressed in human bone marrow cells and acute myeloid leukemia samples. In conclusion, our study provides evidence that a transgenic mouse model commonly used to study the biological function of Nur77 has several major drawbacks, while simultaneously identifying the importance of nongenomic Nur77 activity in the regulation of bone marrow homeostasis.

Structural and cellular mechanisms of peptidyl-prolyl isomerase Pin1-mediated enhancement of Tissue Factor gene expression, protein half-life, and pro-coagulant activity.

Tissue Factor is a cell-surface glycoprotein expressed in various cells of the vasculature and is the principal regulator of the blood coagulation cascade and hemostasis. Notably, aberrant expression of Tissue Factor is associated with cardiovascular pathologies such as atherosclerosis and thrombosis. Here, we sought to identify factors that regulate Tissue Factor gene expression and activity. Tissue Factor gene expression is regulated by various transcription factors, including activating protein-1 and nuclear factor-κ B. The peptidyl-prolyl isomerase Pin1 is known to modulate the activity of these two transcription factors, and we now show that Pin1 augments Tissue Factor gene expression in both vascular smooth muscle cells and activated endothelial cells via activating protein-1 and nuclear factor-κ B signaling. Furthermore, the cytoplasmic domain of Tissue Factor contains a well-conserved phospho-Ser258-Pro259 amino-acid motif recognized by Pin1. Using co-immunoprecipitation and solution nuclear magnetic resonance spectroscopy, we show that the WW-domain of Pin1 directly binds the cytoplasmic domain of Tissue Factor. This interaction occurs via the phospho-Ser258-Pro259 sequence in the Tissue Factor cytoplasmic domain and results in increased protein half-life and pro-coagulant activity. Taken together, our results establish Pin1 as an upstream regulator of Tissue Factor-mediated coagulation, thereby opening up new avenues for research into the use of specific Pin1 inhibitors for the treatment of diseases characterized by pathological coagulation, such as thrombosis and atherosclerosis.

Protein-protein interactions of the LIM-only protein FHL2 and functional implication of the interactions relevant in cardiovascular disease.

FHL2 belongs to the LIM-domain only proteins and contains four and a half LIM domains, each of which are composed of two zinc finger structures. FHL2 exhibits specific interaction with proteins exhibiting diverse functions, including transmembrane receptors, transcription factors and transcription co-regulators, enzymes, and structural proteins. The function of these proteins is regulated by FHL2, which modulates intracellular signal transduction pathways involved in a plethora of cellular tasks. The present review summarizes the current knowledge on the protein interactome of FHL2 and provides an overview of the functional implication of these interactions in apoptosis, migration, and regulation of nuclear receptor function. FHL2 was originally identified in the heart and there is extensive literature available on the role of FHL2 in the cardiovascular system, which is also summarized in this review.

Nur77-deficiency in bone marrow-derived macrophages modulates inflammatory responses, extracellular matrix homeostasis, phagocytosis and tolerance.

BACKGROUND: The nuclear orphan receptor Nur77 (NR4A1, TR3, or NGFI-B) has been shown to modulate the inflammatory response of macrophages. To further elucidate the role of Nur77 in macrophage physiology, we compared the transcriptome of bone marrow-derived macrophages (BMM) from wild-type (WT) and Nur77-knockout (KO) mice. RESULTS: In line with previous observations, SDF-1α (CXCL12) was among the most upregulated genes in Nur77-deficient BMM and we demonstrated that Nur77 binds directly to the SDF-1α promoter, resulting in inhibition of SDF-1α expression. The cytokine receptor CX3CR1 was strongly downregulated in Nur77-KO BMM, implying involvement of Nur77 in macrophage tolerance. Ingenuity pathway analyses (IPA) to identify canonical pathways regulation and gene set enrichment analyses (GSEA) revealed a potential role for Nur77 in extracellular matrix homeostasis. Nur77-deficiency increased the collagen content of macrophage extracellular matrix through enhanced expression of several collagen subtypes and diminished matrix metalloproteinase (MMP)-9 activity. IPA upstream regulator analyses discerned the small GTPase Rac1 as a novel regulator of Nur77-mediated gene expression. We identified an inhibitory feedback loop with increased Rac1 activity in Nur77-KO BMM, which may explain the augmented phagocytic activity of these cells. Finally, we predict multiple chronic inflammatory diseases to be influenced by macrophage Nur77 expression. GSEA and IPA associated Nur77 to osteoarthritis, chronic obstructive pulmonary disease, rheumatoid arthritis, psoriasis, and allergic airway inflammatory diseases. CONCLUSIONS: Altogether these data identify Nur77 as a modulator of macrophage function and an interesting target to treat chronic inflammatory disease.

NR4A nuclear receptors are orphans but not lonesome.

The NR4A subfamily of nuclear receptors consists of three mammalian members: Nur77, Nurr1, and NOR-1. The NR4A receptors are involved in essential physiological processes such as adaptive and innate immune cell differentiation, metabolism and brain function. They act as transcription factors that directly modulate gene expression, but can also form trans-repressive complexes with other transcription factors. In contrast to steroid hormone nuclear receptors such as the estrogen receptor or the glucocorticoid receptor, no ligands have been described for the NR4A receptors. This lack of known ligands might be explained by the structure of the ligand-binding domain of NR4A receptors, which shows an active conformation and a ligand-binding pocket that is filled with bulky amino acid side-chains. Other mechanisms, such as transcriptional control, post-translational modifications and protein-protein interactions therefore seem to be more important in regulating the activity of the NR4A receptors. For Nur77, over 80 interacting proteins (the interactome) have been identified so far, and roughly half of these interactions has been studied in more detail. Although the NR4As show some overlap in interacting proteins, less information is available on the interactome of Nurr1 and NOR-1. Therefore, the present review will describe the current knowledge on the interactomes of all three NR4A nuclear receptors with emphasis on Nur77.

Nuclear receptor Nur77 and Yin-Yang 1 synergistically increase mitochondrial abundance and activity in macrophages.

Mitochondrial biogenesis requires precise regulation of both mitochondrial-encoded and nuclear-encoded genes. Nuclear receptor Nur77 is known to regulate mitochondrial metabolism in macrophages and skeletal muscle. Here, we compared genome-wide Nur77 binding site and target gene expression in these two cell types, which revealed conserved regulation of mitochondrial genes and enrichment of motifs for the transcription factor Yin-Yang 1 (YY1). We show that Nur77 and YY1 interact, that YY1 increases Nur77 activity, and that their binding sites are co-enriched at mitochondrial ribosomal protein gene loci in macrophages. Nur77 and YY1 co-expression synergistically increases Mrpl1 expression as well as mitochondrial abundance and activity in macrophages but not skeletal muscle. As such, we identify a macrophage-specific Nur77-YY1 interaction that enhances mitochondrial metabolism.

Dual function of Pin1 in NR4A nuclear receptor activation: enhanced activity of NR4As and increased Nur77 protein stability.

Nur77, Nurr1 and NOR-1 form the NR4A subfamily of the nuclear receptor superfamily and have been shown to regulate various biological processes among which are cell survival and differentiation, apoptosis, inflammation and metabolism. These nuclear receptors have been proposed to act in a ligand-independent manner and we aim to gain insight in the regulation of NR4A activity. A yeast two-hybrid screen identified the peptidyl-prolyl isomerase Pin1 as a novel binding partner of NR4As, which was confirmed by co-immunoprecipitation. Pin1 enhances the transcriptional activity of all three NR4A nuclear receptors and increases protein stability of Nur77 through inhibition of its ubiquitination. Enhanced transcriptional activity of NR4As requires the WW-domain of Pin1 that interacts with the N-terminal transactivation domain and the DNA-binding domain of Nur77. Most remarkably, this enhanced activity is independent of Pin1 isomerase activity. A systematic mutation analysis of all 17 Ser/Thr-Pro-motifs in Nur77 revealed that Pin1 enhances protein stability of Nur77 in an isomerase-dependent manner by acting on phosphorylated Nur77 involving protein kinase CK2-mediated phosphorylation of the Ser(152)-Pro(153) motif in Nur77. Given the role of Nur77 in vascular disease and metabolism, this novel regulation mechanism provides perspectives to manipulate Nur77 activity to attenuate these processes.

Deletion of macrophage Gpr101 disrupts their phenotype and function dysregulating host immune responses in sterile and infectious inflammation.

We recently found that the G protein coupled receptor GPR101 mediates the phagocyte-directed pro-resolving activities of RvD5n-3 DPA (n-3 docosapentaenoic acid-derived Resolvin D5). Herein, we investigated the endogenous role of this pro-resolving receptor in modulating macrophage biology using a novel mouse line where the expression of Gpr101 was conditionally deleted in macrophages (MacGpr101KO). Peritoneal macrophages obtained from naïve MacGpr101KO mice displayed a marked shift in the expression of phenotypic and activation markers, including the Interleukin (IL)-10 and IL-23 receptors. Loss of Gpr101 on macrophages was also associated with a significant disruption in their cellular metabolism and a decreased ability to migrate towards the chemoattractant Mcp-1. The alterations in macrophage phenotype observed in Gpr101 deficient macrophages were maintained following inflammatory challenge. This was linked with an increased inflammatory response in the Gpr101 deficient animals and a reduced ability of phagocytes, including macrophages, to clear bacteria. Loss of Gpr101 on macrophages disrupted host pro-resolving responses to zymosan challenge with MacGpr101KO mice exhibiting significantly higher neutrophil numbers and a delay in the resolution interval when compared with control mice. These observations were linked with a marked dysregulation in peritoneal lipid mediator concentrations in Gpr101 deficient mice, with a downregulation of pro-resolving mediators including MaR2n-3 DPA, Resolvin (Rv) D3 and RvE3. Together these findings identify Gpr101 as a novel regulator of both macrophage phenotype and function, modulating key biological activities in both limiting the propagation of inflammation and expediting its resolution.

MCTR3 reprograms arthritic monocytes to upregulate Arginase-1 and exert pro-resolving and tissue-protective functions in experimental arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a progressive degenerative disorder that leads to joint destruction. Available treatments only target the inflammatory component with minimal impact on joint repair. We recently uncovered a previously unappreciated family of pro-resolving mediators, the maresin conjugate in tissue regeneration (MCTR), that display both immunoregulatory and tissue-protective activities. Thus, we queried whether the production of these autacoids is disrupted in RA patients and whether they can be useful in treating joint inflammation and promoting joint repair. METHODS: Using a highly phenotyped RA cohort we evaluated plasma MCTR concentrations and correlated these to clinical markers of disease activity. To evaluate the immunoregulatory and tissue reparative activities we employed both in vivo models of arthritis and organ culture models. FINDINGS: Herein, we observed that plasma MCTR3 concentrations were negatively correlated with joint disease activity and severity in RA patients. Evaluation of the mechanisms engaged by this mediator in arthritic mice demonstrated that MCTR3 reprograms monocytes to confer enduring joint protective properties. Single cell transcriptomic profiling and flow cytometric evaluation of macrophages from mice treated with MCTR3-reprogrammed monocytes revealed a role for Arginase-1 (Arg-1) in mediating their joint reparative and pro-resolving activities. Arg-1 inhibition reversed both the anti-arthritic and tissue reparative actions of MCTR3-reprogrammed monocytes. INTERPRETATION: Our findings demonstrate that circulating MCTR3 levels are negatively correlated with disease in RA. When administered to mice in vivo, MCTR3 displayed both anti-inflammatory and joint reparative activities, protecting both cartilage and bone in murine arthritis. These activities were, at least in part, mediated via the reprogramming of mononuclear phagocyte responses. FUNDING: This work was supported by funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant no: 677542) and the Barts Charity (grant no: MGU0343) to J.D. J.D. is also supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (grant 107613/Z/15/Z).

USP25 promotes pathological HIF-1-driven metabolic reprogramming and is a potential therapeutic target in pancreatic cancer.

Deubiquitylating enzymes (DUBs) play an essential role in targeted protein degradation and represent an emerging therapeutic paradigm in cancer. However, their therapeutic potential in pancreatic ductal adenocarcinoma (PDAC) has not been explored. Here, we develop a DUB discovery pipeline, combining activity-based proteomics with a loss-of-function genetic screen in patient-derived PDAC organoids and murine genetic models. This approach identifies USP25 as a master regulator of PDAC growth and maintenance. Genetic and pharmacological USP25 inhibition results in potent growth impairment in PDAC organoids, while normal pancreatic organoids are insensitive, and causes dramatic regression of patient-derived xenografts. Mechanistically, USP25 deubiquitinates and stabilizes the HIF-1α transcription factor. PDAC is characterized by a severely hypoxic microenvironment, and USP25 depletion abrogates HIF-1α transcriptional activity and impairs glycolysis, inducing PDAC cell death in the tumor hypoxic core. Thus, the USP25/HIF-1α axis is an essential mechanism of metabolic reprogramming and survival in PDAC, which can be therapeutically exploited.

Dysregulated Maresin Concentrations in Plasma and Nasal Secretions From Patients With Chronic Rhinosinusitis.

The mechanisms that lead to disease onset and propagation in patients with chronic rhinosinusitis (CRS) are not fully elucidated. Maresins (MaR) are a family of essential fatty acid-derived lipid mediators that play a central role in the regulation of inflammation with several studies demonstrating that these mediators display protective activities in airway inflammation. Therefore, in the present studies we evaluated whether concentrations of these mediators were altered in both peripheral blood and nasal secretions from CRS patients. Herein, we focused on patients with CRS that also develop nasal polyps (CRSwNP), given that therapeutic options for the treatment of these patients are limited. Thereby, insights into disease mechanisms in these patients may help design more effective treatments. For this purpose, we compared maresin concentrations from CRSwNP patients with those found in healthy volunteers or patients with an upper respiratory tract infection (URTI), as a self-resolving inflammatory condition. Using liquid chromatography tandem mass spectrometry, we found that MaR concentrations were significantly decreased in plasma from patients with CRSwNP when compared to healthy volunteers. MaR concentrations were observed to be significantly upregulated in nasal secretions from patients with CRSwNP when compared with both healthy volunteers and URTI subjects. Concentration of these mediators in both plasma and nasal secretions from CRSwNP patients were positively correlated with quality-of-life scores in these patients. Assessment of the concentrations of other pro-resolving and pro-inflammatory lipid mediators (LM) demonstrated that there was a general shift in LM levels in both plasma and nasal secretions from CRSwNP when compared with healthy volunteers and URTI subjects. Of note, incubation of peripheral blood cells from CRSwNP patients with MaR1 downregulated the expression of activation markers on peripheral blood phagocytes, including CD41 and CD62P, markers of platelet-leukocyte heterotypic aggregates. Together these findings demonstrate that both local and systemic LM concentrations, in particularly those of the MaR family, become altered in patients with CRSwNP. They also suggest that therapeutics designed around MaR1 may be useful in regulating the activation of phagocytes in patients with CRSwNP thereby potentially also limiting the local inflammatory response in these patients.

GPR101 mediates the pro-resolving actions of RvD5n-3 DPA in arthritis and infections.

N-3 docosapentaenoic acid-derived resolvin D5 (RvD5n-3 DPA) is diurnally regulated in peripheral blood and exerts tissue-protective actions during inflammatory arthritis. Here, using an orphan GPCR screening approach coupled with functional readouts, we investigated the receptor(s) involved in mediating the leukocyte-directed actions of RvD5n-3 DPA and identified GPR101 as the top candidate. RvD5n-3 DPA bound to GPR101 with high selectivity and stereospecificity, as demonstrated by a calculated KD of approximately 6.9 nM. In macrophages, GPR101 knockdown limited the ability of RvD5n-3 DPA to upregulate cyclic adenosine monophosphate, phagocytosis of bacteria, and efferocytosis. Inhibition of this receptor in mouse and human leukocytes abrogated the pro-resolving actions of RvD5n-3 DPA, including the regulation of bacterial phagocytosis in neutrophils. Knockdown of the receptor in vivo reversed the protective actions of RvD5n-3 DPA in limiting joint and gut inflammation during inflammatory arthritis. Administration of RvD5n-3 DPA during E. coli-initiated inflammation regulated neutrophil trafficking to the site of inflammation, increased bacterial phagocytosis by neutrophils and macrophages, and accelerated the resolution of infectious inflammation. These in vivo protective actions of RvD5n-3 DPA were limited when Gpr101 was knocked down. Together, our findings demonstrate a fundamental role for GPR101 in mediating the leukocyte-directed actions of RvD5n-3 DPA.

Nur77 protects against adverse cardiac remodelling by limiting neuropeptide Y signalling in the sympathoadrenal-cardiac axis.

Aims: Cardiac remodelling and heart failure are promoted by persistent sympathetic activity. We recently reported that nuclear receptor Nur77 may protect against sympathetic agonist-induced cardiac remodelling in mice. The sympathetic co-transmitter neuropeptide Y (NPY) is co-released with catecholamines and is a known cardiac modulator and predictor of heart failure mortality. Recently, transcriptome analyses revealed NPY as a putative target of Nur77. In this study, we assess whether Nur77 modulates adverse cardiac remodelling via NPY signalling. Methods and results: Nur77 represses NPY expression in the PC12 adrenal chromaffin cell line. Accordingly, NPY levels are higher in adrenal gland, plasma, and heart from Nur77-KO compared to wild-type mice. Conditioned medium from Nur77-silenced chromaffin cells and serum from Nur77-KO mice induce marked hypertrophy in cultured neonatal rat cardiomyocytes, which is inhibited by the NPY type 1 receptor (NPY1R) antagonist BIBO3304. In cardiomyocytes from Nur77-KO mice, intracellular Ca2+ is increased partially via the NPY1R. This is independent from elevated circulating NPY since cardiomyocyte-specific Nur77-deficient mice (CM-KO) do not have elevated circulating NPY, but do exhibit BIBO3304-sensitive, increased cardiomyocyte intracellular Ca2+. In vivo, this translates to NPY1R antagonism attenuating cardiac calcineurin activity and isoproterenol-induced cardiomyocyte hypertrophy and fibrosis in full-body Nur77-KO mice, but not in CM-KO mice. Conclusions: The cardioprotective action of Nur77 can be ascribed to both inhibition of circulating NPY levels and to cardiomyocyte-specific modulation of NPY-NPY1R signalling. These results reveal the underlying mechanism of Nur77 as a promising modifier gene in heart failure.

LIM-only protein FHL2 is a positive regulator of liver X receptors in smooth muscle cells involved in lipid homeostasis.

The LIM-only protein FHL2 is expressed in smooth muscle cells (SMCs) and inhibits SMC-rich-lesion formation. To further elucidate the role of FHL2 in SMCs, we compared the transcriptomes of SMCs derived from wild-type (WT) and FHL2 knockout (KO) mice. This revealed that in addition to the previously recognized involvement of FHL2 in SMC proliferation, the cholesterol synthesis and liver X receptor (LXR) pathways are altered in the absence of FHL2. Using coimmunoprecipitation experiments, we found that FHL2 interacts with the two LXR isoforms, LXRα and LXRß. Furthermore, FHL2 strongly enhances transcriptional activity of LXR element (LXRE)-containing reporter constructs. Chromatin immunoprecipitation (ChIP) experiments on the ABCG1 promoter revealed that FHL2 enhances the association of LXRß with DNA. In line with these observations, we observed reduced basal transcriptional LXR activity in FHL2-KO SMCs compared to WT SMCs. This was also reflected in reduced expression of LXR target genes in intact aorta and aortic SMCs of FHL2-KO mice. Functionally, the absence of FHL2 resulted in attenuated cholesterol efflux to both ApoA-1 and high-density lipoprotein (HDL), in agreement with reduced LXR signaling. Collectively, our findings demonstrate that FHL2 is a transcriptional coactivator of LXRs and points toward FHL2 being an important determinant of cholesterol metabolism in SMCs.