ABSTRACT
AIMS/HYPOTHESIS: The general population is ageing, involving an enhanced incidence of chronic diseases such as type 2 diabetes. With ageing, DNA methylation of FHL2 increases, as well as expression of the four and a half LIM domains 2 (FHL2) protein in human pancreatic islets. We hypothesised that FHL2 is actively involved in glucose metabolism. METHODS: Publicly available microarray datasets from human pancreatic islets were analysed for FHL2 expression. In FHL2-deficient mice, we studied glucose clearance and insulin secretion. Gene expression analysis and glucose-stimulated insulin secretion (GSIS) were determined in isolated murine FHL2-deficient islets to evaluate insulin-secretory capacity. Moreover, knockdown and overexpression of FHL2 were accomplished in MIN6 cells to delineate the underlying mechanism of FHL2 function. RESULTS: Transcriptomics of human pancreatic islets revealed that individuals with elevated levels of HbA1c displayed increased FHL2 expression, which correlated negatively with insulin secretion pathways. In line with this observation, FHL2-deficient mice cleared glucose more efficiently than wild-type littermates through increased plasma insulin levels. Insulin sensitivity was comparable between these genotypes. Interestingly, pancreatic islets isolated from FHL2-deficient mice secreted more insulin in GSIS assays than wild-type mouse islets even though insulin content and islet size was similar. To support this observation, we demonstrated increased expression of the transcription factor crucial in insulin secretion, MAF BZIP transcription factor A (MafA), higher expression of GLUT2 and reduced expression of the adverse factor c-Jun in FHL2-deficient islets. The underlying mechanism of FHL2 was further delineated in MIN6 cells. FHL2-knockdown led to enhanced activation of forkhead box protein O1 (FOXO1) and its downstream genes such as Mafa and Pdx1 (encoding pancreatic and duodenal homeobox 1), as well as increased glucose uptake. On the other hand, FHL2 overexpression in MIN6 cells blocked GSIS, increased the formation of reactive oxygen species and increased c-Jun activity. CONCLUSIONS/INTERPRETATION: Our data demonstrate that FHL2 deficiency improves insulin secretion from beta cells and improves glucose tolerance in mice. Given that FHL2 expression in humans increases with age and that high expression levels of FHL2 are associated with beta cell dysfunction, we propose that enhanced FHL2 expression in elderly individuals contributes to glucose intolerance and the development of type 2 diabetes. DATA AVAILABILITY: The human islet microarray datasets used are publicly available and can be found on https://www.ncbi.nlm.nih.gov/geo/ .
Subject(s)
Diabetes Mellitus, Type 2 , Insulin-Secreting Cells , Islets of Langerhans , Aged , Animals , Basic-Leucine Zipper Transcription Factors/metabolism , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/metabolism , Forkhead Box Protein O1/metabolism , Glucose/metabolism , Humans , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/metabolism , Islets of Langerhans/metabolism , LIM-Homeodomain Proteins/genetics , Mice , Muscle Proteins/genetics , Muscle Proteins/metabolism , Reactive Oxygen Species/metabolism , Transcription Factors/metabolismABSTRACT
OBJECTIVE: Four-and-a-Half-LIM-domain-protein 2 (FHL2) modulates multiple signal transduction pathways but has not been implicated in obesity or energy metabolism. In humans, methylation and expression of the FHL2 gene increases with age, and high FHL2 expression is associated with increased body weight in humans and mice. This led us to hypothesize that FHL2 is a determinant of diet-induced obesity. METHODS: FHL2-deficient (FHL2-/-) and wild type male mice were fed a high-fat diet. Metabolic phenotyping of these mice, as well as transcriptional analysis of key metabolic tissues was performed. Correlation of the expression of FHL2 and relevant genes was assessed in datasets from white adipose tissue of individuals with and without obesity. RESULTS: FHL2 Deficiency protects mice from high-fat diet-induced weight gain, whereas glucose handling is normal. We observed enhanced energy expenditure, which may be explained by a combination of changes in multiple tissues; mild activation of brown adipose tissue with increased fatty acid uptake, increased cardiac glucose uptake and browning of white adipose tissue. Corroborating our findings in mice, expression of FHL2 in human white adipose tissue positively correlates with obesity and negatively with expression of browning-associated genes. CONCLUSION: Our results position FHL2 as a novel regulator of obesity and energy expenditure in mice and human. Given that FHL2 expression increases during aging, we now show that low FHL2 expression associates with a healthy metabolic state.
Subject(s)
LIM-Homeodomain Proteins/genetics , Muscle Proteins/genetics , Obesity/genetics , Transcription Factors/genetics , Adipogenesis/genetics , Adipose Tissue, White/metabolism , Adult , Aged , Animals , Biomarkers/metabolism , Diet, High-Fat , Female , Genetic Predisposition to Disease , Humans , LIM-Homeodomain Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Muscle Proteins/metabolism , Obesity/diagnosis , Obesity/metabolism , Transcription Factors/metabolism , Weight Gain/geneticsABSTRACT
Bleeding disorders and thrombotic complications are major causes of morbidity and mortality with many cases being unexplained. Thrombus formation involves aberrant expression and activation of tissue factor (TF) in vascular endothelial and smooth muscle cells. Here, we sought to identify factors that modulate TF gene expression and activity in these vascular cells. The LIM-only protein FHL2 is a scaffolding protein that modulates signal transduction pathways with crucial functions in endothelial and smooth muscle cells. However, the role of FHL2 in TF regulation and thrombosis remains unexplored. Using a murine model of venous thrombosis in mesenteric vessels, we demonstrated that FHL2 deficiency results in exacerbated thrombus formation. Gain- and loss-of-function experiments revealed that FHL2 represses TF expression in endothelial and smooth muscle cells through inhibition of the transcription factors nuclear factor κB and activating protein-1. Furthermore, we observed that FHL2 interacts with the cytoplasmic tail of TF. In line with our in vivo observations, FHL2 decreases TF activity in endothelial and smooth muscle cells whereas FHL2 knockdown or deficiency results in enhanced TF activity. Finally, the FHL2 single nucleotide polymorphism rs4851770 was associated with the risk of venous thrombosis in a large population of venous thrombosis cases and control subjects from 12 studies (INVENT consortium). Altogether, our results highlight functional involvement of FHL2 in TF-mediated coagulation and identify FHL2 as a novel gene associated with venous thrombosis in humans.
Subject(s)
Thromboplastin , Venous Thrombosis , Animals , Genetic Variation , Humans , LIM-Homeodomain Proteins/genetics , Mice , Muscle Proteins/genetics , Thromboplastin/genetics , Transcription Factors/genetics , Venous Thrombosis/geneticsABSTRACT
Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the fibrillin-1 gene (FBN1), resulting in aortic aneurysm formation and dissections. Interestingly, variable aortopathy is observed even within MFS families with the same mutation. Thus, additional risk factors determine disease severity. Here, we describe a case of a 2-month-old Fbn1C1039G/+ MFS mouse with extreme aortic dilatation and increased vascular inflammation, when compared to MFS siblings, which coincided with unilateral renal cystic disease. In addition, this mouse presented with increased serum levels of creatinine, angiotensin-converting enzyme, corticosterone, macrophage chemoattractant protein-1, and interleukin-6, which may have contributed to the vascular pathology. Possibly, cystic kidney disease is associated with aneurysm progression in MFS patients. Therefore, we propose that close monitoring of the presence of renal cysts in MFS patients, during regular vascular imaging of the whole aorta trajectory, may provide insight in the frequency of cystic kidney disease and its potential as a novel indicator of aneurysm progression in MFS patients.
Subject(s)
Aorta/pathology , Aortic Aneurysm/etiology , Fibrillin-1/genetics , Kidney Diseases, Cystic/etiology , Marfan Syndrome/genetics , Animals , Aorta/metabolism , Aortic Aneurysm/blood , Aortic Aneurysm/genetics , Aortic Aneurysm/pathology , Aortitis/blood , Aortitis/etiology , Aortitis/genetics , Aortitis/pathology , Biomarkers/blood , Dilatation, Pathologic , Disease Models, Animal , Fibrillin-1/metabolism , Genetic Predisposition to Disease , Kidney Diseases, Cystic/blood , Kidney Diseases, Cystic/genetics , Kidney Diseases, Cystic/pathology , Male , Marfan Syndrome/blood , Marfan Syndrome/complications , Marfan Syndrome/diagnosis , Mice, Inbred C57BL , Mice, Transgenic , PhenotypeABSTRACT
Activation of macrophages by inflammatory stimuli induces reprogramming of mitochondrial metabolism to support the production of pro-inflammatory cytokines and nitric oxide. Hallmarks of this metabolic rewiring are downregulation of α-ketoglutarate formation by isocitrate dehydrogenase (IDH) and accumulation of glutamine-derived succinate, which enhances the inflammatory response via the activity of succinate dehydrogenase (SDH). Here, we identify the nuclear receptor Nur77 (Nr4a1) as a key upstream transcriptional regulator of this pro-inflammatory metabolic switch in macrophages. Nur77-deficient macrophages fail to downregulate IDH expression and accumulate higher levels of succinate and other TCA cycle-derived metabolites in response to inflammatory stimulation in a glutamine-independent manner. Consequently, these macrophages produce more nitric oxide and pro-inflammatory cytokines in an SDH-dependent manner. In vivo, bone marrow Nur77 deficiency exacerbates atherosclerosis development and leads to increased circulating succinate levels. In summary, Nur77 induces an anti-inflammatory metabolic state in macrophages that protects against chronic inflammatory diseases such as atherosclerosis.
Subject(s)
Gene Expression Regulation/genetics , Inflammation/metabolism , Macrophages/metabolism , Mitochondria/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1/metabolism , HumansABSTRACT
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.
Subject(s)
Bone Marrow Cells/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , Protein Domains/genetics , Animals , Bone Marrow/metabolism , Bone Marrow/pathology , Flow Cytometry , Gene Expression Regulation/genetics , Homeostasis/genetics , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/chemistry , Mice , Mice, Transgenic , Nuclear Receptor Subfamily 4, Group A, Member 1/chemistryABSTRACT
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.
Subject(s)
Coagulants/metabolism , Gene Expression , NIMA-Interacting Peptidylprolyl Isomerase/metabolism , Thromboplastin/genetics , Thromboplastin/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Multiprotein Complexes/metabolism , NF-kappa B/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Proteolysis , Thromboplastin/chemistry , Transcription Factor AP-1/metabolismABSTRACT
An abdominal aortic aneurysm (AAA) is a dilatation of the abdominal aorta leading to serious complications and mostly to death. AAA development is associated with an accumulation of inflammatory cells in the aorta including NKT cells. An important factor in promoting the recruitment of these inflammatory cells into tissues and thereby contributing to the development of AAA is angiotensin II (Ang II). We demonstrate that a deficiency in CD1d dependent NKT cells under hyperlipidemic conditions (LDLr-/-CD1d-/- mice) results in a strong decline in the severity of angiotensin II induced aneurysm formation when compared with LDLr-/- mice. In addition, we show that Ang II amplifies the activation of NKT cells both in vivo and in vitro. We also provide evidence that type I NKT cells contribute to AAA development by inducing the expression of matrix degrading enzymes in vSMCs and macrophages, and by cytokine dependently decreasing vSMC viability. Altogether, these data prove that CD1d-dependent NKT cells contribute to AAA development in the Ang II-mediated aneurysm model by enhancing aortic degradation, establishing that therapeutic applications which target NKT cells can be a successful way to prevent AAA development.
Subject(s)
Antigens, CD1d/genetics , Aortic Aneurysm, Abdominal/prevention & control , Receptors, LDL/genetics , Angiotensin II/administration & dosage , Animals , Apoptosis/immunology , Flow Cytometry , Lymphocyte Activation , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Muscle, Smooth, Vascular/immunology , Muscle, Smooth, Vascular/pathology , NIH 3T3 Cells , Natural Killer T-Cells/immunology , Real-Time Polymerase Chain ReactionABSTRACT
The energetic costs of behavioral chronic stress are unlikely to be sustainable without neuronal plasticity. Mitochondria have the capacity to handle synaptic activity up to a limit before energetic depletion occurs. Protective mechanisms driven by the induction of neuronal genes likely evolved to buffer the consequences of chronic stress on excitatory neurons in prefrontal cortex (PFC), as this circuitry is vulnerable to excitotoxic insults. Little is known about the genes involved in mitochondrial adaptation to the buildup of chronic stress. Using combinations of genetic manipulations and stress for analyzing structural, transcriptional, mitochondrial, and behavioral outcomes, we characterized NR4A1 as a stress-inducible modifier of mitochondrial energetic competence and dendritic spine number in PFC. NR4A1 acted as a transcription factor for changing the expression of target genes previously involved in mitochondrial uncoupling, AMP-activated protein kinase activation, and synaptic growth. Maintenance of NR4A1 activity by chronic stress played a critical role in the regressive synaptic organization in PFC of mouse models of stress (male only). Knockdown, dominant-negative approach, and knockout of Nr4a1 in mice and rats (male only) protected pyramidal neurons against the adverse effects of chronic stress. In human PFC tissues of men and women, high levels of the transcriptionally active NR4A1 correlated with measures of synaptic loss and cognitive impairment. In the context of chronic stress, prolonged expression and activity of NR4A1 may lead to responses of mitochondria and synaptic connectivity that do not match environmental demand, resulting in circuit malfunction between PFC and other brain regions, constituting a pathological feature across disorders.SIGNIFICANCE STATEMENT The bioenergetic cost of chronic stress is too high to be sustainable by pyramidal prefrontal neurons. Cellular checkpoints have evolved to adjust the responses of mitochondria and synapses to the buildup of chronic stress. NR4A1 plays such a role by controlling the energetic competence of mitochondria with respect to synapse number. As an immediate-early gene, Nr4a1 promotes neuronal plasticity, but sustained expression or activity can be detrimental. NR4A1 expression and activity is sustained by chronic stress in animal models and in human studies of neuropathologies sensitive to the buildup of chronic stress. Therefore, antagonism of NR4A1 is a promising avenue for preventing the regressive synaptic reorganization in cortical systems in the context of chronic stress.
Subject(s)
Mitochondria/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , Prefrontal Cortex/physiopathology , Stress, Psychological/physiopathology , Synapses/metabolism , Aged , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Animals , Cell Count , Chronic Disease , Cognition Disorders/etiology , Cognition Disorders/psychology , Dendritic Spines , Female , Gene Expression Regulation/genetics , Hindlimb Suspension , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neuronal Plasticity/genetics , Prefrontal Cortex/cytology , Pyramidal Cells/physiology , Rats , Stress, Psychological/psychologyABSTRACT
Marfan syndrome (MFS) is a connective tissue disorder in which aortic rupture is the major cause of death. MFS patients with an aortic diameter below the advised limit for prophylactic surgery (<5 cm) may unexpectedly experience an aortic dissection or rupture, despite yearly monitoring. Hence, there is a clear need for improved prognostic markers to predict such aortic events. We hypothesize that elastin fragments play a causal role in aortic calcification in MFS, and that microcalcification serves as a marker for aortic disease severity. To address this hypothesis, we analysed MFS patient and mouse aortas. MFS patient aortic tissue showed enhanced microcalcification in areas with extensive elastic lamina fragmentation in the media. A causal relationship between medial injury and microcalcification was revealed by studies in vascular smooth muscle cells (SMCs); elastin peptides were shown to increase the activity of the calcification marker alkaline phosphatase (ALP) and reduce the expression of the calcification inhibitor matrix GLA protein in human SMCs. In murine Fbn1C1039G/+ MFS aortic SMCs, Alpl mRNA and activity were upregulated as compared with wild-type SMCs. The elastin peptide-induced ALP activity was prevented by incubation with lactose or a neuraminidase inhibitor, which inhibit the elastin receptor complex, and a mitogen-activated protein kinase kinase-1/2 inhibitor, indicating downstream involvement of extracellular signal-regulated kinase-1/2 (ERK1/2) phosphorylation. Histological analyses in MFS mice revealed macrocalcification in the aortic root, whereas the ascending aorta contained microcalcification, as identified with the near-infrared fluorescent bisphosphonate probe OsteoSense-800. Significantly, microcalcification correlated strongly with aortic diameter, distensibility, elastin breaks, and phosphorylated ERK1/2. In conclusion, microcalcification co-localizes with aortic elastin degradation in MFS aortas of humans and mice, where elastin-derived peptides induce a calcification process in SMCs via the elastin receptor complex and ERK1/2 activation. We propose microcalcification as a novel imaging marker to monitor local elastin degradation and thus predict aortic events in MFS patients. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Subject(s)
Elastin/metabolism , Marfan Syndrome/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Animals , Aorta/metabolism , Aortic Aneurysm/metabolism , Aortic Aneurysm/pathology , Calcinosis/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Marfan Syndrome/pathology , Mice , Mitogen-Activated Protein Kinase Kinases/metabolism , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/pathologyABSTRACT
Bile acids are established signaling molecules next to their role in the intestinal emulsification and uptake of lipids. We here aimed to identify a potential interaction between bile acids and CD4+ Th cells, which are central in adaptive immune responses. We screened distinct bile acid species for their potency to affect T cell function. Primary human and mouse CD4+ Th cells as well as Jurkat T cells were used to gain insight into the mechanism underlying these effects. We found that unconjugated lithocholic acid (LCA) impedes Th1 activation as measured by i) decreased production of the Th1 cytokines IFNγ and TNFαα, ii) decreased expression of the Th1 genes T-box protein expressed in T cells (T-bet), Stat-1 and Stat4, and iii) decreased STAT1α/ß phosphorylation. Importantly, we observed that LCA impairs Th1 activation at physiological relevant concentrations. Profiling of MAPK signaling pathways in Jurkat T cells uncovered an inhibition of ERK-1/2 phosphorylation upon LCA exposure, which could provide an explanation for the impaired Th1 activation. LCA induces these effects via Vitamin D receptor (VDR) signaling since VDR RNA silencing abrogated these effects. These data reveal for the first time that LCA controls adaptive immunity via inhibition of Th1 activation. Many factors influence LCA levels, including bile acid-based drugs and gut microbiota. Our data may suggest that these factors also impact on adaptive immunity via a yet unrecognized LCA-Th cell axis.
Subject(s)
Adaptive Immunity/drug effects , Lithocholic Acid/pharmacology , Lymphocyte Activation/drug effects , Receptors, Calcitriol/metabolism , Th1 Cells/immunology , Animals , Bile Acids and Salts/metabolism , Cell Differentiation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation/drug effects , Humans , Interferon-gamma/genetics , Interferon-gamma/metabolism , Jurkat Cells , Mice, Inbred C57BL , Phosphorylation/drug effects , Th1 Cells/drug effectsABSTRACT
LIM-only protein FHL2 is associated with several immune and inflammatory diseases such as arthritis, influenza A virus infection, and lung inflammation. However, the role of FHL2 in macrophage differentiation and in the development of granuloma formation is unknown. Here, we show that expression of FHL2 is induced in mouse bone marrow derived macrophages (BMMs) following stimulation with M2 cytokines such as IL-4 and IL-10. FHL2-knockout (FHL2-KO) BMMs exhibit a proinflammatory M1 phenotype after LPS treatment and display a reduced anti-inflammatory M2 phenotype following IL-4 treatment. Furthermore, thioglycollate-induced migration of macrophages and B cells is enhanced in FHL2-KO mice. To evaluate the importance of FHL2 in the development of pulmonary granuloma formation, FHL2-KO mice were challenged with Schistosoma mansoni eggs. FHL2-KO mice show an enhanced number of granulomas and display decreased expression of Th2 markers and an exacerbated Th1 type of inflammation, characterized by enhanced expression of neutrophil markers and Th1 cytokines. Furthermore, the expression of barrier proteins is reduced in FHL2-KO lung compared to WT. Collectively, these data identify a previously unrecognized role for FHL2 in the pathogenesis of pulmonary granulomatous inflammation, partly through its effect on macrophage polarization, modulation of the Th1/Th2 balance and regulation of permeability in lung.
Subject(s)
Granuloma, Respiratory Tract/immunology , LIM-Homeodomain Proteins/immunology , Muscle Proteins/immunology , Schistosomiasis mansoni/immunology , Transcription Factors/immunology , Animals , Cell Movement/immunology , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Granuloma, Respiratory Tract/pathology , Immunohistochemistry , Inflammation/immunology , Inflammation/pathology , Macrophages/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Real-Time Polymerase Chain Reaction , Schistosomiasis mansoni/pathologyABSTRACT
Nuclear receptor Nur77, also referred to as NR4A1 or TR3, plays an important role in innate and adaptive immunity. Nur77 is crucial in regulating the T helper 1/regulatory T-cell balance, is expressed in macrophages and drives M2 macrophage polarization. In this study we aimed to define the function of Nur77 in inflammatory bowel disease. In wild-type and Nur77-/- mice, colitis development was studied in dextran sodium sulphate (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced models. To understand the underlying mechanism, Nur77 was overexpressed in macrophages and gut epithelial cells. Nur77 protein is expressed in colon tissues from Crohn's disease and Ulcerative colitis patients and colons from colitic mice in inflammatory cells and epithelium. In both mouse colitis models inflammation was increased in Nur77-/- mice. A higher neutrophil influx and enhanced IL-6, MCP-1 and KC production was observed in Nur77-deficient colons after DSS-treatment. TNBS-induced influx of T-cells and inflammatory monocytes into the colon was higher in Nur77-/- mice, along with increased expression of MCP-1, TNFα and IL-6, and decreased Foxp3 RNA expression, compared to wild-type mice. Overexpression of Nur77 in lipopolysaccharide activated RAW macrophages resulted in up-regulated IL-10 and downregulated TNFα, MIF-1 and MCP-1 mRNA expression through NFκB repression. Nur77 also strongly decreased expression of MCP-1, CXCL1, IL-8, MIP-1α and TNFα in gut epithelial Caco-2 cells. Nur77 overexpression suppresses the inflammatory status of both macrophages and gut epithelial cells and together with the in vivo mouse data this supports that Nur77 has a protective function in experimental colitis. These findings may have implications for development of novel targeted treatment strategies regarding inflammatory bowel disease and other inflammatory diseases.
Subject(s)
Colitis, Ulcerative/metabolism , Colitis/metabolism , Crohn Disease/metabolism , Macrophages/metabolism , NF-kappa B/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1/immunology , Animals , Cell Line , Colitis/chemically induced , Colitis/immunology , Colitis, Ulcerative/pathology , Colon/metabolism , Colon/pathology , Crohn Disease/pathology , Cytokines/biosynthesis , Cytokines/genetics , Dextran Sulfate/toxicity , Disease Models, Animal , Epithelial Cells/metabolism , Epithelial Cells/pathology , Female , Forkhead Transcription Factors/biosynthesis , Forkhead Transcription Factors/genetics , Gene Expression Regulation , Humans , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclear Receptor Subfamily 4, Group A, Member 1/deficiency , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , RAW 264.7 Cells , Trinitrobenzenesulfonic Acid/toxicityABSTRACT
Allergic asthma is characterized by persistent chronic airway inflammation, which leads to mucus hypersecretion and airway hyperresponsiveness. Nuclear receptor Nur77 plays a pivotal role in distinct immune and inflammatory cells and is expressed in eosinophils and lung epithelium. However, the role of Nur77 in allergic airway inflammation has not been studied so far. In the present study, we determined the role of Nur77 in airway inflammation using a murine model of OVA-induced allergic airway inflammation. We found that OVA-challenged Nur77 knockout (KO) mice show significantly enhanced infiltration of inflammatory cells, including eosinophils and lymphocytes, and aggravated mucus production. The infiltration of macrophages is limited in this model and was similar in wild-type and Nur77 KO mice. Higher levels of Th2 cytokines were found in bronchoalveolar lavage fluid and draining lymph node cells of Nur77-KO mice, as well as increased serum IgG1 and IgG2a levels. Knockdown of Nur77 in human lung epithelial cells resulted in a marked increase in IκBα phosphorylation, corresponding with elevated NF-κB activity, whereas Nur77 overexpression decreased NF-κB activity. Consistently, Nur77 significantly decreased mRNA levels of inflammatory cytokines and Muc5ac expression and also attenuated mucus production in lung epithelial cells. To further corroborate these findings, we searched for association of single nucleotide polymorphisms in Nur77 gene with asthma and with the severity of bronchial hyperresponsiveness. We identified three Nur77 single nucleotide polymorphisms showing association with severity of bronchial hyperresponsiveness in asthma patients. Collectively, these findings support a protective role of Nur77 in OVA-induced airway inflammation and identify Nur77 as a novel therapeutic target for airway inflammation.
Subject(s)
Alveolar Epithelial Cells/metabolism , Inflammation/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1/metabolism , Respiratory Hypersensitivity/metabolism , Alleles , Animals , Asthma/genetics , Asthma/immunology , Asthma/metabolism , Asthma/pathology , Bronchoalveolar Lavage Fluid/immunology , Cytokines/metabolism , Disease Models, Animal , Enzyme Activation , Eosinophils/pathology , Gene Expression , Immunoglobulin G/blood , Immunoglobulin G/immunology , Inflammation/genetics , Inflammation/immunology , Inflammation/pathology , Leukocyte Count , Lymph Nodes/immunology , Mice , Mice, Knockout , Mucin 5AC/genetics , Mucin 5AC/metabolism , NF-kappa B/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1/deficiency , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , Odds Ratio , Polymorphism, Single Nucleotide , Respiratory Hypersensitivity/genetics , Respiratory Hypersensitivity/immunology , Respiratory Hypersensitivity/pathology , Signal TransductionABSTRACT
The LIM-only protein FHL2, also known as DRAL or SLIM3, has a function in fine-tuning multiple physiological processes. FHL2 is expressed in the vessel wall in smooth muscle cells (SMCs) and endothelial cells and conflicting data have been reported on the regulatory function of FHL2 in SMC phenotype transition. At present the function of FHL2 in SMCs in vascular injury is unknown. Therefore, we studied the role of FHL2 in SMC-rich lesion formation. In response to carotid artery ligation FHL2-deficient (FHL2-KO) mice showed accelerated lesion formation with enhanced Ki67 expression compared with wild-type (WT)-mice. Consistent with these findings, cultured SMCs from FHL2-KO mice showed increased proliferation through enhanced phosphorylation of extracellular-regulated kinase-1/2 (ERK1/2) and induction of CyclinD1 expression. Overexpression of FHL2 in SMCs inhibited CyclinD1 expression and CyclinD1-knockdown blocked the enhanced proliferation of FHL2-KO SMCs. We also observed increased CyclinD1 promoter activity in FHL2-KO SMCs, which was reduced upon ERK1/2 inhibition. Furthermore, FHL2-KO SMCs showed enhanced migration compared with WT SMCs. In conclusion, FHL2 deficiency in mice results in exacerbated SMC-rich lesion formation involving increased proliferation and migration of SMCs via enhanced activation of the ERK1/2-CyclinD1 signaling pathway.
Subject(s)
LIM-Homeodomain Proteins/genetics , LIM-Homeodomain Proteins/metabolism , Muscle Proteins/genetics , Muscle Proteins/metabolism , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Animals , Carotid Arteries/metabolism , Carotid Arteries/pathology , Cell Movement/genetics , Cell Proliferation , Cyclin D1/metabolism , Male , Mice , Mice, Knockout , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Signal TransductionABSTRACT
Azathioprine and its metabolite 6-mercaptopurine (6-MP) are well established immunosuppressive drugs. Common understanding of their immunosuppressive properties is largely limited to immune cells. However, in this study, the mechanism underlying the protective role of 6-MP in endothelial cell activation is investigated. Because 6-MP and its derivative 6-thioguanosine-5'-triphosphate (6-T-GTP) were shown to block activation of GTPase Rac1 in T lymphocytes, we focused on Rac1-mediated processes in endothelial cells. Indeed, 6-MP and 6-T-GTP decreased Rac1 activation in endothelial cells. As a result, the compounds inhibited TNF-α-induced downstream signaling via JNK and reduced activation of transcription factors c-Jun, activating transcription factor-2 and, in addition, NF κ-light-chain-enhancer of activated B cells (NF-κB), which led to decreased transcription of proinflammatory cytokines. Moreover, 6-MP and 6-T-GTP selectively decreased TNF-α-induced VCAM-1 but not ICAM-1 protein levels. Rac1-mediated generation of cell membrane protrusions, which form docking structures to capture leukocytes, also was reduced by 6-MP/6-T-GTP. Consequently, leukocyte transmigration was inhibited after 6-MP/6-T-GTP treatment. These data underscore the anti-inflammatory effect of 6-MP and 6-T-GTP on endothelial cells by blocking Rac1 activation. Our data provide mechanistic insight that supports development of novel Rac1-specific therapeutic approaches against chronic inflammatory diseases.
Subject(s)
Endothelial Cells/drug effects , Immunosuppressive Agents/pharmacology , Mercaptopurine/pharmacology , Signal Transduction/drug effects , rac1 GTP-Binding Protein/metabolism , Blotting, Western , Cell Adhesion/drug effects , Cells, Cultured , Coculture Techniques , Endothelial Cells/enzymology , Enzyme Activation/drug effects , Humans , Microscopy, Confocal , Microscopy, Electron, Scanning , Neutrophils/drug effects , Neutrophils/metabolism , Transcriptome , Transendothelial and Transepithelial Migration/drug effectsABSTRACT
AIM: To characterize the phenotype of Akt2/low-density-lipoprotein receptor double knockout (dKO) (Akt2/LDLr dKO) mice with respect to insulin resistance and features of atherosclerotic plaque progression. METHODS AND RESULTS: Metabolic profile and atherosclerotic plaque progression were compared between LDLr KO mice and Akt2/LDLr dKO mice. Total cholesterol, glucose, and insulin levels were significantly higher and oral glucose tolerance test (GTT) was more impaired in Akt2/LDLr dKO mice than in LDLr KO mice. Although atherosclerotic plaques at both the carotid artery and the aortic root of Akt2/LDLr dKO mice were significantly smaller (P < 0.05) compared with LDLr KO controls, plaque composition in these mice was more complex, showing 34-50% reduced collagen content (P < 0.01), 1.4-fold larger necrotic cores (P < 0.05) and six-fold more TUNEL-positive cells (P < 0.01). In situ zymography revealed a more than two-fold higher gelatinolytic activity in Akt2/LDLr dKO mice (P < 0.05). In vitro analyses showed that deletion of Akt2 caused decreased migration, proliferation, and collagen content of vascular smooth muscle cells (VSMCs) and disturbed the balance of metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase (TIMP) mRNA expression in macrophages and VSMCs. CONCLUSION: Akt2/LDLr dKO mice develop insulin resistance and complex atherosclerotic lesions. These phenotypic characteristics make Akt2/LDLr dKO mice an interesting mouse model to study the effects of insulin resistance on the development and progression of atherosclerosis.
Subject(s)
Aortic Diseases/enzymology , Atherosclerosis/enzymology , Carotid Artery Diseases/enzymology , Glucose Intolerance/enzymology , Insulin Resistance , Plaque, Atherosclerotic , Proto-Oncogene Proteins c-akt/deficiency , Receptors, LDL/deficiency , Animals , Aortic Diseases/blood , Aortic Diseases/genetics , Aortic Diseases/pathology , Apoptosis , Atherosclerosis/blood , Atherosclerosis/genetics , Atherosclerosis/pathology , Biomarkers/blood , Blood Glucose/metabolism , Carotid Artery Diseases/blood , Carotid Artery Diseases/genetics , Carotid Artery Diseases/pathology , Cell Movement , Cell Proliferation , Cells, Cultured , Collagen/metabolism , Disease Models, Animal , Gelatinases/genetics , Gelatinases/metabolism , Genetic Predisposition to Disease , Glucose Intolerance/blood , Glucose Intolerance/genetics , Insulin/blood , Insulin Resistance/genetics , Lipids/blood , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Smooth, Vascular/enzymology , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/enzymology , Myocytes, Smooth Muscle/pathology , Necrosis , Phenotype , Proto-Oncogene Proteins c-akt/genetics , Receptors, LDL/genetics , Time Factors , Tissue Inhibitor of Metalloproteinases/genetics , Tissue Inhibitor of Metalloproteinases/metabolismABSTRACT
RATIONALE: Nuclear receptor Nur77, also known as NR4A1, TR3, or NGFI-B, is expressed in human atherosclerotic lesions in macrophages, endothelial cells, T cells and smooth muscle cells. Macrophages play a critical role in atherosclerosis and the function of Nur77 in lesion macrophages has not yet been investigated. OBJECTIVE: This study aims to delineate the function of Nur77 in macrophages and to assess the effect of bone marrow-specific deficiency of Nur77 on atherosclerosis. METHODS AND RESULTS: We investigated Nur77 in macrophage polarization using bone marrow-derived macrophages (BMM) from wild-type and Nur77-knockout (Nur77(-/-)) mice. Nur77(-/-) BMM exhibit changed expression of M2-specific markers and an inflammatory M1-phenotype with enhanced expression of interleukin-12, IFNγ, and SDF-1α and increased NO synthesis in (non)-stimulated Nur77(-/-) BMM cells. SDF-1α expression in nonstimulated Nur77(-/-) BMM is repressed by Nur77 and the chemoattractive activity of Nur77(-/-) BMM is abolished by SDF-1α inhibiting antibodies. Furthermore, Nur77(-/-) mice show enhanced thioglycollate-elicited migration of macrophages and B cells. The effect of bone marrow-specific deficiency of Nur77 on atherosclerosis was studied in low density lipoprotein receptor-deficient (Ldlr(-/-)) mice. Ldlr(-/-) mice with a Nur77(-/-)-deficient bone marrow transplant developed 2.1-fold larger atherosclerotic lesions than wild-type bone marrow-transplanted mice. These lesions contain more macrophages, T cells, smooth muscle cells and larger necrotic cores. SDF-1α expression is higher in lesions of Nur77(-/-)-transplanted mice, which may explain the observed aggravation of lesion formation. CONCLUSIONS: In conclusion, in bone marrow-derived cells the nuclear receptor Nur77 has an anti-inflammatory function, represses SDF-1α expression and inhibits atherosclerosis.
Subject(s)
Atherosclerosis/pathology , Atherosclerosis/physiopathology , Macrophages/metabolism , Macrophages/pathology , Nuclear Receptor Subfamily 4, Group A, Member 1/deficiency , Animals , Atherosclerosis/metabolism , Cell Adhesion/physiology , Cell Movement/physiology , Cells, Cultured , Chemokine CXCL12/metabolism , Cholesterol/metabolism , Cytokines/metabolism , Disease Models, Animal , Inflammation/metabolism , Inflammation/pathology , Inflammation/physiopathology , Macrophages/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , Nuclear Receptor Subfamily 4, Group A, Member 1/metabolism , PhenotypeABSTRACT
Chronic infection and inflammation are strongly associated with the development of atherosclerosis. To investigate whether chronic inflammation in the absence of an infectious cause also predisposes to the development of atherosclerosis, we used a mouse model in which sterile inflammation is driven by enhanced costimulation. Constitutive triggering of CD27 on T cells through overexpression of CD70 on B cells increases the numbers of IFN gamma-producing effector T cells, which reduces the numbers of B cells. However, despite these pro-atherogenic features, we found that CD70-transgenic (CD70TG) mice on an ApoE*3-Leiden background were strongly protected against the induction of atherosclerotic lesions, with a normal increase in serum cholesterol level and the absence of atheroprotective antibodies. We found that circulating monocytes in CD70TG mice were activated and increased in numbers, in particular the pool of inflammatory (Ly6C(+)) monocytes. Importantly, monocytes from CD70TG mice had no defects in phagocytosis nor in TNFalpha production, but they were more prone to apoptosis, which was IFN gamma-dependent. These data indicate that sterile pro-inflammatory conditions can be protective against atherosclerosis development, possibly due to a reduced viability of circulating monocytes. This unexpected outcome provides a new insight into the consequences of costimulatory signals and their impact on innate immunity.
Subject(s)
Atherosclerosis/immunology , CD27 Ligand/metabolism , Monocytes/immunology , Animals , Apolipoproteins E/immunology , Apoptosis , B-Lymphocytes/immunology , CD27 Ligand/immunology , Cell Differentiation/immunology , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Monocytes/cytology , Phagocytosis , T-Lymphocytes/immunology , Tumor Necrosis Factor Receptor Superfamily, Member 7/biosynthesis , Tumor Necrosis Factor Receptor Superfamily, Member 7/immunologyABSTRACT
OBJECTIVE: 6-Mercaptopurine (6-MP), the active metabolite of the immunosuppressive prodrug azathioprine, is commonly used in autoimmune diseases and transplant recipients, who are at high risk for cardiovascular disease. Here, we aimed to gain knowledge on the action of 6-MP in atherosclerosis, with a focus on monocytes and macrophages. METHODS AND RESULTS: We demonstrate that 6-MP induces apoptosis of THP-1 monocytes, involving decreased expression of the intrinsic antiapoptotic factors B-cell CLL/Lymphoma-2 (Bcl-2) and Bcl2-like 1 (Bcl-x(L)). In addition, we show that 6-MP decreases expression of the monocyte adhesion molecules platelet endothelial adhesion molecule-1 (PECAM-1) and very late antigen-4 (VLA-4) and inhibits monocyte adhesion. Screening of a panel of cytokines relevant to atherosclerosis revealed that 6-MP robustly inhibits monocyte chemoattractant chemokine-1 (MCP-1) expression in macrophages stimulated with lipopolysaccharide (LPS). Finally, local delivery of 6-MP to the vessel wall, using a drug-eluting cuff, attenuates atherosclerosis in hypercholesterolemic apolipoprotein E*3-Leiden transgenic mice (P<0.05). In line with our in vitro data, this inhibition of atherosclerosis by 6-MP was accompanied with decreased lesion monocyte chemoattractant chemokine-1 levels, enhanced vascular apoptosis, and reduced macrophage content. CONCLUSIONS: We report novel, previously unrecognized atheroprotective actions of 6-MP in cultured monocytes/macrophages and in a mouse model of atherosclerosis, providing further insight into the effect of the immunosuppressive drug azathioprine in atherosclerosis.