Kondababu Kurakula

Bone Marrow–Specific Deficiency of Nuclear Receptor Nur77 Enhances Atherosclerosis

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, IFN&ggr;, and SDF-1&agr; and increased NO synthesis in (non)-stimulated Nur77−/− BMM cells. SDF-1&agr; expression in nonstimulated Nur77−/− BMM is repressed by Nur77 and the chemoattractive activity of Nur77−/− BMM is abolished by SDF-1&agr; 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-1&agr; 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-1&agr; expression and inhibits atherosclerosis.

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.

Cardiac endothelial cells express Wilms' tumor-1: Wt1 expression in the developing, adult and infarcted heart.

Myocardial infarction is the leading cause of death worldwide. Due to their limited regenerative capacity lost cardiomyocytes are replaced by a non-contractile fibrotic scar tissue. The epicardial layer of the heart provides cardiac progenitor cells during development. Because this layer regains embryonic characteristics in the adult heart after cardiac injury, it could serve as a promising source for resident cardiac progenitor cells. Wilms tumor-1 (Wt1) is associated with the activation and reactivation of the epicardium and therefore potentially important for the differentiation and regenerative capacity of the epicardium. To gain more insight into the regulation of Wt1 we examined the spatiotemporal expression pattern of Wt1 during murine development and after cardiac injury. Interestingly, we found that Wt1 is expressed in the majority of the cardiac endothelial cells within the myocardial ventricular layer of the developing heart from E12.5 onwards. In the adult heart only a subset of coronary endothelial cells remains positive for Wt1. After myocardial infarction Wt1 is temporally upregulated in the endothelial cells of the infarcted area and the border zone of the heart. In vitro experiments show that endothelial Wt1 expression can be induced by hypoxia. We show that Wt1 is associated with endothelial cell proliferation: Wt1 expression is higher in proliferating endothelial cells, Wt1 knockdown inhibits the proliferation of endothelial cells, and Wt1 regulates CyclinD1 expression. Finally, endothelial cells lacking Wt1 are not capable to establish a proper vascular network in vitro. Together, these results suggest a possible role for Wt1 in cardiac vessel formation in development and disease.

Inhibition of GTPase Rac1 in Endothelium by 6-Mercaptopurine Results in Immunosuppression in Nonimmune Cells: New Target for an Old Drug

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.

FHL2 protein is a novel co-repressor of nuclear receptor Nur77.

Background: Nur77 is an orphan nuclear receptor involved in vascular disease, of which the regulation of activity is poorly understood. Results: FHL2 binds Nur77 and represses its transcriptional activity by inhibition of Nur77 association with DNA. Conclusion: FHL2 acts as a co-repressor of Nur77. Significance: Our data suggest that interaction of FHL2 with Nur77 plays a pivotal role in vascular disease. The three members of the NR4A orphan nuclear receptor subfamily Nur77, Nurr1, and NOR-1, regulate a variety of biological functions including vascular disease and metabolism. In this study, we identified Four and a half LIM domains protein-2 (FHL2) as a novel interacting protein of NR4A nuclear receptors by yeast two-hybrid screen and co-immunoprecipitation studies. Each of the four LIM domains of FHL2 can bind Nur77, and both the amino-terminal domain and the DNA binding domain of Nur77 are involved in the interaction between FHL2 and Nur77. FHL2 represses Nur77 transcriptional activity in a dose-dependent manner, and short hairpin RNA-mediated knockdown of FHL2 results in increased Nur77 transcriptional activity. ChIP experiments on the enolase3 promoter revealed that FHL2 inhibits the association of Nur77 with DNA. FHL2 is highly expressed in human endothelial and smooth muscle cells, but not in monocytes or macrophages. To substantiate functional involvement of FHL2 in smooth muscle cell physiology, we demonstrated that FHL2 overexpression increases the growth of these cells, whereas FHL2 knockdown results in reduced DNA synthesis. Collectively, these studies suggest that association of FHL2 with Nur77 plays a pivotal role in vascular disease.

Nuclear Receptor Nur77 Attenuates Airway Inflammation in Mice by Suppressing NF-κB Activity in Lung Epithelial Cells

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.

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.

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.

The LIM-Only Protein FHL2 Reduces Vascular Lesion Formation Involving Inhibition of Proliferation and Migration of Smooth Muscle Cells

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.

Regulatory RNAs controlling vascular (dys)function by affecting TGF-ß family signalling

Cardiovascular disease (CVD) is a leading cause of morbidity and mortality worldwide. Over the last few years, microRNAs (miRNAs) have emerged as master regulators of gene expression in cardiovascular biology and disease. miRNAs are small endogenous non-coding RNAs that usually bind to 3′ untranslated region (UTR) of their target mRNAs and inhibit mRNA stability or translation of their target genes. miRNAs play a dynamic role in the pathophysiology of many CVDs through their effects on target mRNAs in vascular cells. Recently, numerous miRNAs have been implicated in the regulation of the transforming growth factor-β (TGF-β)/bone morphogenetic protein (BMP) signalling pathway which plays crucial roles in diverse biological processes, and is involved in pathogenesis of many diseases including CVD. This review gives an overview of current literature on the role of miRNAs targeting TGF-β/BMP signalling in vascular cells, including endothelial cells and smooth muscle cells. We also provide insight into how this miRNA-mediated regulation of TGF-β/BMP signalling might be used to harness CVD.