Endogenous Regulatory Pathways Research Articles

Soluble epoxide hydrolase inhibition enhances production of specialized pro-resolving lipid mediator and promotes macrophage plasticity.

Epoxyeicosatrienoic acids (EETs) and other epoxy fatty acids (EpFA) are lipid mediators that are rapidly inactivated by soluble epoxide hydrolase (sEH). Uncontrolled and chronic inflammatory disorders fail to sufficiently activate endogenous regulatory pathways, including the production of specialized pro-resolving mediators (SPMs). Here, we addressed the relationship between SPMs and the EET/sEH axis and explored the effects of sEH inhibition on resolving macrophage phenotype. Mice were treated with a sEH inhibitor, EETs, or sEH inhibitor + EETs (combination) before ligature placement to induce experimental periodontitis. Using RT-qPCR, gingival samples were used to examine SPM receptors and osteolytic and inflammatory biomarkers. Maxillary alveolar bone loss was quantified by micro-CT and methylene blue staining. SPM levels were analysed by salivary metabolo-lipidomics. Gingival macrophage phenotype plasticity was determined by RT-qPCR and flow cytometry. Effects of sEH inhibition on macrophage polarization and SPM production were assessed with bone marrow-derived macrophages (BMDMs). Pharmacological inhibition of sEH suppressed bone resorption and the inflammatory cytokine storm in experimental periodontitis. Lipidomic analysis revealed that sEH inhibition augmented levels of LXA4, RvE1, RvE2, and 4-HDoHE, concomitant with up-regulation of LTB4R1, CMKLR1/ChemR23, and ALX/FPR2 SPM receptors. Notably, there is an impact on gingival macrophage plasticity was affected suggesting an inflammation resolving phenotype with sEH inhibition. In BMDMs, sEH inhibition reduced inflammatory macrophage activation, and resolving macrophages were triggered to produce SPMs. Pharmacological sEH inhibition increased SPM synthesis associated with resolving macrophages, suggesting a potential target to control osteolytic inflammatory disorders.

ANGIOTENSIN PROFILES IN PATIENTS WITH HYPERTENSION AND TYPE 2 DIABETES WITH COMBINATION THERAPY OF EMPAGLIFLOZIN AND LINAGLIPTIN VERSUS METFORMIN AND INSULIN GLARGINE

Objective: The renin-angiotensin system (RAS) plays a key role in the physiological regulation of blood pressure. Patients with type-2-diabetes-mellitus (T2DM) and hypertension are known to have increased RAS activity. However, the precise role of RAS activation after SGLT2-inhibitor therapy is unknown. The classic RAS comprises the ACE-AngII-AT1R-axis (classic-axis) that promotes vasoconstriction. The Angiotensin-converting-enzyme-2-angiotensin-(1–7)-Mas axis (Mas-axis) with its components Angiotensin-(1–7) (Ang-1–7) and Angiotensin-(1–5) (Ang-1–5) is an endogenous negative regulatory pathway of the classic-axis, which can reduce its harmful effects. Design and method: Angiotensin profiles were determined in 55 patients with hypertension and T2DM from our double blind, randomized, placebo controlled clinical trial “Effects of Empagliflozin+Linagliptin (E+L) vs Metformin+Insulin Glargine (M+I) on Renal and Vascular Changes in Type 2 Diabetes’’ (ELMI) at baseline and three months after application of either E+L or M+I. An LC-MS/MS based approach was used to simultaneously quantify individual angiotensin metabolites (Attoquant Diagnostics, Vienna, Austria). Results: In the E+L group medication induced a rise in Ang-I, Ang-(1–7) and a trend to a rise in Ang-II between baseline and treatment, which was not present in the M+I group (table). Comparison between the E+L and M+I group revealed that change between baseline and treatment was significantly different for Ang I (p = 0.002) and Ang-(1–7) (p = 0.002) between the groups. No difference in change between baseline and treatment was found for Ang II (p = 0.182) and Ang-(1–5) (p = 0.224) between the E+L and M+I group. Conclusions: In patients with hypertension and T2DM three months of therapy with E+L led to a rise of classic and protective components of the RAS, which was not present in the M+I group. The biological consequence of this complex RAS activation remains to be elucidated.

Long non-coding RNA SNHG1 activates HOXA1 expression via sponging miR-193a-5p in breast cancer progression.

Breast cancer is the leading cause of cancer death in women worldwide. Long non-coding RNA small nucleolar RNA host gene 1 (SNHG1) has been reported to be involved in human diseases, including cancer. Here, we found that SNHG1 expression was significantly upregulated in human breast cancer tissues and cell lines. We explored the function of SNHG1 in breast cancer cells using in vitro and in vivo experiments and found that SNHG1 promotes breast cancer metastasis and proliferation. The potential molecular mechanism of SNHG1 in breast cancer cells may involve SNHG1 acting as a sponge of miR-193a-5p to activate the expression of the oncogene HOXA1. In summary, our study reveals a novel SNHG1/miR-193a-5p/HOXA1 competing endogenous RNA regulatory pathway in breast cancer progression and may provide new strategies for breast cancer therapy.

LincRNA-ROR is activated by H3K27 acetylation and induces EMT in retinoblastoma by acting as a sponge of miR-32 to activate the Notch signaling pathway.

Recent studies have suggested that lincRNA-ROR is involved in the tumorigenesis of different types of cancers. However, the role of lincRNA-ROR in retinoblastoma has not been determined. We investigated lincRNA-ROR levels in 58 retinoblastoma and adjacent non-tumor tissues by quantitative reverse transcription PCR. Recurrence-free survival was analyzed using Cox regression analyses. Cell migration and invasion abilities were detected by wound-healing, Transwell invasion, and bioluminescence imaging assays. Western blotting was performed to detect epithelial-mesenchymal transition markers. Interactions between lincRNA-ROR, miR-32-5p, and Notch1 were confirmed by Luciferase, RNA pull-down, and RIP assays. Histone acetylation was detected by chromatin immunoprecipitation assays. We showed that lincRNA-ROR was significantly upregulated in retinoblastoma tissues, and overexpression of lincRNA-ROR was significantly correlated with optic nerve invasion, nodal or distant metastasis, and recurrence. We also showed that lincRNA-ROR is a critical promoter of retinoblastoma cell metastasis, both in vivo and in vitro. Further, we demonstrated that lincRNA-ROR activates the Notch signaling pathway by acting as a sponge of miR-32-5p. Upregulation of lincRNA-ROR was attributed to the CBP-mediated H3K27 acetylation at the promoter region. Our results reveal a potential competing endogenous RNA regulatory pathway, in which lincRNA-ROR modulates the epithelial-mesenchymal transition program by competitively binding to endogenous miR-32-5p and regulating Notch signaling pathway activity in retinoblastoma cells, which may provide new insights into novel molecular therapeutic targets for retinoblastoma.

Regulation of angiotensin-converting enzyme 2-angiotensin (1-7)-Mas axis provides a new target for the treatment of cardiac remodeling and heart failure

Cardiac remodeling is a common pathological manifestation of various end-stage cardiovascular diseases, which leads to myocardial diastolic and systolic dysfunction, low ejection fraction which cannot meet the needs of systemic tissue and organ metabolism, and ultimate progress into heart failure. Excessive activation of the classical renin angiotensin system (RAS), which is the angiotensin-converting enzyme-angiotensin II-type 1 angiotensin II receptor axis (ACE2-Ang II-AT1R axis), plays a key role in the pathological process of cardiac remodeling and heart failure. Angiotensin-converting enzyme 2-angiotensin (1-7)-Mas axis [ACE2-Ang (1-7)-Mas axis] is an endogenous negative regulatory pathway of classical RAS, which can reduce its harmful effects. ACE2 is a monocarboxypeptidase that can hydrolyse Ang II and produce Ang (1-7), which has cardio-protective effects. Ang (1-7), via endogenous receptor Mas, plays the role of vasodilating, anti-proliferation and anti-differentiation, anti-fibrosis, anti-thrombosis and reversing myocardial remodeling. In recent years, with increasingly growing studies on the ACE2-Ang (1-7)-Mas axis, there are more understanding about their metabolic characteristics and mechanism of action. This article describes the role of ACE2 and Ang (1-7) in cardiac remodeling and heart failure and the related mechanisms, and discusses the potential benefits by regulating ACE2 activity and Ang (1-7) levels in clinical and experimental studies, hopefully providing potential therapeutic strategies.

L-Phenylalanine Restores Vascular Function in Spontaneously Hypertensive Rats Through Activation of the GCH1-GFRP Complex

Reduced nitric oxide (NO) bioavailability correlates with impaired cardiovascular function. NO is extremely labile and has been challenging to develop as a therapeutic agent. However, NO bioavailability could be enhanced by pharmacologically targeting endogenous NO regulatory pathways. Tetrahydrobiopterin, an essential cofactor for NO production, is synthesized by GTP cyclohydrolase-1 (GCH1), which complexes with GCH1 feedback regulatory protein (GFRP). The dietary amino acid l-phenylalanine activates this complex, elevating vascular BH4. Here, the authors demonstrate that l-phenylalanine administration restores vascular function in a rodent model of hypertension, suggesting the GCH1-GFRP complex represents a rational therapeutic target for diseases underpinned by endothelial dysfunction.

The emergence of the nicotinamide riboside kinases in the regulation of NAD+ metabolism

The concept of replenishing or elevating NAD+ availability to combat metabolic disease and ageing is an area of intense research. This has led to a need to define the endogenous regulatory pathways and mechanisms cells and tissues utilise to maximise NAD+ availability such that strategies to intervene in the clinical setting are able to be fully realised. This review discusses the importance of different salvage pathways involved in metabolising the vitamin B3 class of NAD+ precursor molecules, with a particular focus on the recently identified nicotinamide riboside kinase pathway at both a tissue-specific and systemic level.

Oxytocin Modulates Nociception as an Agonist of Pain-Sensing TRPV1

Oxytocin is a hormone with various actions. Oxytocin-containing parvocellular neurons project to the brainstem and spinal cord. Oxytocin release from these neurons suppresses nociception of inflammatory pain, the molecular mechanism of which remains unclear. Here, we report that the noxious stimulus receptor TRPV1 is an ionotropic oxytocin receptor. Oxytocin elicits TRPV1 activity in native and heterologous expression systems, regardless of the presence of the classical oxytocin receptor. In TRPV1 knockout mice, DRG neurons exhibit reduced oxytocin sensitivity relative to controls, and oxytocin injections significantly attenuate capsaicin-induced nociception in invivo experiments. Furthermore, oxytocin potentiates TRPV1 in planar lipid bilayers, supporting a direct agonistic action. Molecular modeling and simulation experiments provide insight into oxytocin-TRPV1 interactions, which resemble DkTx. Together, our findings suggest the existence of endogenous regulatory pathways that modulate nociception via direct action of oxytocin on TRPV1, implying its analgesic effect via channel desensitization.

Improved insulin-secreting properties of pancreatic islet mesenchymal stem cells by constitutive expression of Pax4 and MafA.

For long-term treatment of diabetes type 1, transplantation of insulin-producing beta cells may be a promising method, but the limited number of islets for transplantation requires the development of different approaches. In this study, we aimed to generate betalike insulin-producing cells. For this purpose, MafA, Pax4, and Ngn3 genes were transferred into pancreatic islet-derived mesenchymal stem cells, and the effect of their ectopic expressions on differentiation efficiency was examined. Stemness properties of pancreatic islet stem cells were characterized. The 3 genes were transfected by electroporation and expressed constitutively. The transfected cells were further stimulated to differentiate by using chemical induction. Pax4 expression had significant effects on differentiation into insulin-producing cells. Although it caused morphological alterations in cells, similar to epithelial cells, the insulin secretion levels remained lower than those of the cell line cotransfected with MafA and Pax4. Cotransfection of the 3 transcription factors did not further improve the beta-like cell generation. MafA and Pax4 ectopic expression resulted in improved differentiation efficiency into insulin-secreting cells. However, support of this differentiation process using additional chemical induction may sufice to overcome control by endogenous regulatory pathways.

SiRNA Mediated Gene Silencing: Hurdles, Strategies and Applications

Background: RNA interference (RNAi) has emerged as one of the most extensively explored areas of research that has promising applications in therapeutics. As an endogenous regulatory pathway, RNAi can be used for silencing targeted genes involved in the progression of diseases. Small interfering RNAs (siRNA) are 21-25 nucleotides in length and work on the principle of RNA interference. Owing to their high degree of safety, specificity, efficacy, easy synthesis and unrestricted choice of target, siRNAs are considered appropriate for gene silencing. Objective: This review discusses siRNA gene silencing mechanism, the challenges posed in siRNA delivery and the strategies developed to overcome some of these hurdles using nanocarriers. A few applications of siRNA in therapeutics are presented to highlight the utility of these strategies. Conclusion: In spite of advantages of siRNA technology, several shortcomings such as off-target effects, hurdles in delivering, endosomal trapping, stimulation of immune response and short half-life limit the practical use of siRNA. However, the situation has been improved considerably using nanotechnology based tools. Keywords: Nanocarriers, RNA interference, siRNA, challenges, delivery strategies, non-viral vectors.

Multiple Sclerosis and the LIF/IL-6 Axis: Use of Nanotechnology to Harness the Tolerogenic and Reparative Properties of LIF.

Leukaemia inhibitory factor (LIF) plays a critical role in “stemness” versus “differentiation”, a property that underpins the core value of LIF as a therapeutic for both the treatment of autoimmune disease and for promoting tissue repair. This value can be realized using nano-engineering technology, where a new generation of tools can, with unprecedented ability, manipulate biological functions. One striking example is the treatment of multiple sclerosis (MS). The underpinning biology is the newly identified LIF/IL-6 axis in T lymphocytes, which can tilt the behaviour between immune tolerance versus immune attack. This LIF/IL-6 axis is ideally suited to nanotherapeutic manipulation, given its inherent mechanistic simplicity of two mutually opposing feed-forward loops that determine either tolerogenic (LIF) or inflammatory (IL-6) immunity. Using LIF that is formulated in biodegradable nanoparticles (LIF-NP) and targeted to CD4+ T cells, the axis is harnessed towards immune tolerance. This has implications for the treatment of autoimmune diseases, where the clinical burden is immense. It encompasses more than 100 diseases and, in the USA alone, costs more than $100 billion in direct health care costs annually. Other properties of LIF include the promotion of healthy neuro-glial interactions within the central nervous system (CNS), where, in addition to MS, LIF-NP therapy is relevant to inflammatory neurodegenerative diseases that represent a large and increasing need within aging populations. Thirdly, LIF is a reparative growth factor that can maintain genomic plasticity. LIF-NP supports the use of stem cell-based therapies in regenerative medicine plus augment therapeutic benefits within the patient. These core properties of LIF are greatly amplified in value by the advantage of being formulated as nanoparticles, namely (i) targeted delivery, (ii) exploitation of endogenous regulatory pathways and (iii) creation of surrogate micro-stromal niches. We discuss LIF-NP as a means to harness endogenous pathways for the treatment of MS, both to reset immune self-tolerance and to promote repair of myelin that is required to support health within the nervous system.

A cellular model of amyloid precursor protein processing and amyloid-β peptide production

BackgroundA hallmark pathologic feature of Alzheimer's disease (AD) is accumulation of neuritic senile plaques in the brain parenchyma. Neurotoxic plaque cores are composed predominantly of amyloid-β (Aβ) peptides of 40 and 42 amino acids in length, formed by sequential cleavage of amyloid precursor protein (APP) by β-, and γ-secretases. There is a great interest in approaches to modulate Aβ peptide production and develop therapeutic interventions to reduce Aβ levels to halt or slow the progression of neurodegeneration. New methodWe characterized and present the BE(2)-M17 human neuroblastoma cell line as a novel in vitro model of the APP-cleavage cascade to support future (1) functional studies of molecular regulators in Aβ production, and (2) high-throughput screening assays of new pharmacotherapeutics. ResultsIn BE(2)-M17 cells, both RNA (i.e., RT-PCR, RNA sequencing) and protein analyses (i.e., Western blots, ELISA), show endogenous expression of critical components of the amyloidogenic pathway, APP-cleavage intermediates CTF83 and CTF99, and final cleavage products Aβ40 and Aβ42. We further report effects of retinoic acid-mediated differentiation on morphology and gene expression in this cell line. Comparison with existing method(s)In contrast to primary isolates or other cell lines reported in current literature, BE(2)-M17 not only sustains baseline expression of the full contingent of APP-processing components, but also remains stably adherent during culture, facilitating experimental manipulations. ConclusionsOur evidence supports the use of BE(2)-M17 as a novel, human, cell-based model of the APP processing pathway that offers a potential streamlined approach to dissect molecular functions of endogenous regulatory pathways, and perform mechanistic studies to identify modulators of Aβ production.

Pharmacokinetics and Pharmacokinetic–Pharmacodynamic Correlations of Therapeutic Peptides

Peptides, defined as polymers of less than 50 amino acids with a molecular weight of less than 10 kDa, represent a fast-growing class of new therapeutics which has unique pharmacokinetic characteristics compared to large proteins or small molecule drugs. Unmodified peptides usually undergo extensive proteolytic cleavage, resulting in short plasma half-lives. As a result of their low permeability and susceptibility to catabolic degradation, therapeutic peptides usually have very limited oral bioavailability and are administered either by the intravenous, subcutaneous, or intramuscular route, although other routes such as nasal delivery are utilized as well. Distribution processes are mainly driven by a combination of diffusion and to a lesser degree convective extravasation dependent on the size of the peptide, with volumes of distribution frequently not larger than the volume of the extracellular body fluid. Owing to the ubiquitous availability of proteases and peptidases throughout the body, proteolytic degradation is not limited to classic elimination organs. Since peptides are generally freely filtered by the kidneys, glomerular filtration and subsequent renal metabolism by proteolysis contribute to the elimination of many therapeutic peptides. Although small peptides have usually limited immunogenicity, formation of anti-drug antibodies with subsequent hypersensitivity reactions has been described for some peptide therapeutics. Numerous strategies have been applied to improve the pharmacokinetic properties of therapeutic peptides, especially to overcome their metabolic instability, low permeability, and limited tissue residence time. Applied techniques include amino acid substitutions, modification of the peptide terminus, inclusion of disulfide bonds, and conjugation with polymers or macromolecules such as antibody fragments or albumin. Application of model-based pharmacokinetic-pharmacodynamic correlations has been widely used for therapeutic peptides in support of drug development and dosage regimen design, especially because their targets are often well-described endogenous regulatory pathways and processes.

A Molecular Framework of Light-Controlled Phytohormone Action in Arabidopsis

Environmental changes strongly affect plant growth and development. Phytohormones, endogenous plant-made small molecules such as ethylene, regulate a wide range of processes throughout the lifetime of plants. The ability of plants to integrate external signals with endogenous regulatory pathways is vital for their survival. Ethylene has been found to suppress hypocotyl elongation in darkness while promoting it in light. How ethylene regulates hypocotyl elongation in such opposite ways is largely unknown. In particular, how light modulates and even reverses the function of ethylene has yet to be characterized. Here we show that the basic-helix-loop-helix transcription factor phytochrome-interacting factor 3 (PIF3) is directly activated by ETHYLENE-INSENSITIVE 3 (EIN3) and is indispensible for ethylene-induced hypocotyl elongation in light. Ethylene via EIN3 concomitantly activates two contrasting pathways: the PIF3-dependent growth-promoting pathway and an ethylene response factor 1 (ERF1)-mediated growth-inhibiting pathway. In the light, growth-promoting PIFs are limiting due to light-dependent destabilization, and thus ethylene stimulates growth under these conditions. In contrast, ERF1 is destabilized, and thus limiting, under dark conditions, explaining why ethylene inhibits growth in the dark. Our findings provide a mechanistic insight into how light modulates internal hormone-regulated plant growth.

Abstract IA11: Control of gene expression in the nucleus by small RNAs

Abstract Double-stranded RNAs are widely used to control gene expression and are a promising class of therapeutic agent. Most studies have focused on recognition of mRNA in the cytoplasm and inhibition of translation. My presentation focuses on the action of small RNAs in the nucleus to modulate transcription or redirect alternative splicing. In 2005, my laboratory observed that synthetic RNAs complementary to the promoter region at the transcriptional start site of human progesterone receptor (PR) could block PR expression [1]. Inhibition was potent and robust, consistent with the suggestion that the synthetic RNAs were exploiting an endogenous mechanism for controlling gene expression. Promoter-targeted synthetic miRNAs were also capable of robust gene silencing [2], further supporting the existence of endogenous regulatory pathways. Modulation required complete seed sequence complementarity to the PR promoter, consistent with a mechanism that involves RNA-RNA recognition through Watson-Crick base-pairing. In 2007, we observed that small RNAs could also target the PR gene promoter and activate PR expression [3]. Activation was observed in cells that had a low, but detectable, level of PR expression, while gene silencing had been observed in cells with a high level of PR expression. Inactive and active RNAs compete for recognition, demonstrating that small changes in target site location affect activity. These results are reminiscent of protein transcription factors, which bind to specific sequences, are sensitive to target site location or small conformation changes, and can activate gene expression in some contexts but not others. Our promoter-targeted RNAs bind to long noncoding RNA transcripts at the PR promoter [4]. Use of antisense oligonucleotides to reduce transcript levels also reduces RNA-mediated modulation of gene expression. The small RNAs recruit AGO2 to the noncoding transcript, and inhibition of AGO2 expression reverses gene silencing or activation [5]. While AGO2 is normally expected to cleave fully complementary targets in the cytoplasm, our data suggests that AGO2 does not cleave transcripts associated with gene promoters in the nucleus. A noncoding RNA transcript also overlaps the 3′ terminus of the PR gene [6]. Small RNAs complementary to this noncoding transcript modulate PR expression, depending on the cell line used. The PR gene loops, juxtaposing its 3′ and 5′ termini and providing a direct path for a signal transmitting a signal from the 3′ terminus of the gene to the promoter. We have investigated whether RNAs can modulate other nuclear events. We designed small RNAs to be complementary to sequence within exons or introns near splice sites. These RNAs redirect alternative splicing and AGO2 was recruited to pre-mRNA [7]. These data provide more support for the action of small RNA in cell nuclei and for the conclusion that AGO2 may not always cause RNA cleavage when it encounters complementary targets in the nucleus. Our data suggest that small RNAs can bind noncoding transcripts and modulate gene expression. Binding occurs in proximity to the gene promoter, in cis relative to the target gene. The RNA-AGO complex acts like a transcription factor to affect the transcription machinery and tip the balance of inducible genes like PR towards repression or activation. Citation Format: David Corey. Control of gene expression in the nucleus by small RNAs [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr IA11.

New Cardiovascular and Pulmonary Therapeutic Strategies Based on the Angiotensin-Converting Enzyme 2/Angiotensin-(1–7)/Mas Receptor Axis

Angiotensin (Ang)-(1–7) is now recognized as a biologically active component of the renin-angiotensin system (RAS). The discovery of the angiotensin-converting enzyme homologue ACE2 revealed important metabolic pathways involved in the Ang-(1–7) synthesis. This enzyme can form Ang-(1–7) from Ang II or less efficiently through hydrolysis of Ang I to Ang-(1–9) with subsequent Ang-(1–7) formation. Additionally, it is well established that the G protein-coupled receptor Mas is a functional ligand site for Ang-(1–7). The axis formed by ACE2/Ang-(1–7)/Mas represents an endogenous counter regulatory pathway within the RAS whose actions are opposite to the vasoconstrictor/proliferative arm of the RAS constituted by ACE/Ang II/AT1 receptor. In this review we will discuss recent findings concerning the biological role of the ACE2/Ang-(1–7)/Mas arm in the cardiovascular and pulmonary system. Also, we will highlight the initiatives to develop potential therapeutic strategies based on this axis.

Editorial [Hot Topic:Cross-Talk Between Endogenous Compounds and Regulatory Pathways of Drug Metabolism Guest Editor: Zdenek Dvorak

Editorial [Hot Topic:Cross-Talk Between Endogenous Compounds and Regulatory Pathways of Drug Metabolism Guest Editor: Zdenek Dvorak

Immune Inhibitory Ligand CD200 Induction by TLRs and NLRs Limits Macrophage Activation to Protect the Host from Meningococcal Septicemia

Macrophage activation is essential for protection against bacterial pathogens but needs to be regulated to prevent damage to the host. We show a key role for the immune inhibitory receptor CD200R and its ligand CD200 in the context of infection with the Gram-negative human pathogen Neisseria meningitidis. N. meningitidis induced CD200 but downregulated CD200R on macrophages in a manner dependent on Neisserial lipopolysaccharide, Toll-like receptor-4 (TLR-4), and the MyD88 pathway but independent of a known Neisserial receptor, scavenger receptor A (SR-A). Agonists of the pattern-recognition receptors nucleotide oligomerization domain 2 (NOD2) and NACHT-LRR protein 3 (NALP3) also induced CD200. The NF-κB member c-Rel was essential for TLR-, NOD2-, and NALP3-mediated induction of CD200. CD200(-/-) animals showed higher lethality in response to experimental meningococcal septicemia, induced higher levels of proinflammatory cytokines, and recruited increased numbers of activated leukocytes, despite comparable bacterial clearance. Thus CD200 is induced by TLR-, NOD2-, and NALP3-mediated pathways, limiting their function and protecting the host from excessive inflammation.

Ceramide-dependent PP2A regulation of TNFα-induced IL-8 production in respiratory epithelial cells

IL-8 is a key mediator in the pathophysiology of acute lung injury. TNFalpha stimulates IL-8 production in respiratory epithelial cells by activating both the NF-kappaB and MAP kinase pathways. The precise mechanism by which these pathways are downregulated to terminate IL-8 production remains unclear. We studied the regulatory role of the serine/threonine phosphatase, PP2A, on the signaling pathways involved in IL-8 production from respiratory epithelial cells. Inhibition of PP2A using okadaic acid or gene knockdown using siRNA resulted in an augmentation of TNFalpha-induced IL-8 production. We also found that PP2A inhibition resulted in prolonged activation of JNK, p38, and ERK resulting in both increased transcriptional activation of the IL-8 promoter and posttranscriptional stabilization of IL-8 mRNA. Because TNFalpha had been shown to activate ceramide accumulation, and separate studies had linked ceramide with activation of PP2A, we hypothesized the pathway of TNFalpha-inducing ceramide to activate PP2A comprised an endogenous regulatory pathway. Inhibition of the immediate sphingomyelinase-dependent pathway as well as the de novo synthesis pathway of ceramide production reduced serine/threonine phosphatase activity and augmented IL-8 production. These data suggest that ceramide plays a role in activating PP2A to terminate ongoing IL-8 production. In summary, our data suggest that in respiratory epithelium, TNFalpha induces ceramide accumulation, resulting in subsequent activation of PP2A, which targets those kinases responsible for transcriptional activation of IL-8.

Mammalian Mirtron Genes

Mirtrons are alternative precursors for microRNA biogenesis that were recently described in invertebrates. These short hairpin introns use splicing to bypass Drosha cleavage, which is otherwise essential for the generation of canonical animal microRNAs. Using computational and experimental strategies, we now establish that mammals have mirtrons as well. We identified 3 mirtrons that are well conserved and expressed in diverse mammals, 16 primate-specific mirtrons, and 46 candidates supported by limited cloning evidence in primates. As with some fly and worm mirtrons, the existence of well-conserved mammalian mirtrons indicates their relatively ancient incorporation into endogenous regulatory pathways. However, as worms, flies, and mammals each have different sets of mirtrons, we hypothesize that different animals may have independently evolved the capacity for this hybrid small RNA pathway. This notion is supported by our observation of several clade-specific features of mammalian and invertebrate mirtrons.