Antibody to Epitope Tag Induces Internalization of Human Muscarinic Subtype 1 Receptor

Abstract: We have studied the effect of an antibody against the epitope EYMPME on the internalization of the human muscarinic cholinergic receptor hm1 tagged with the epitope at the amino terminus. The antibody to the tag induces internalization of the hm1 receptor within minutes after exposure of human embryonic kidney 293 cells transfected with the tagged receptor. This antibody-induced internalization is reversible following removal of the antibody. In contrast to hm1 internalization induced by the agonist carbachol, internalization induced by antibody is not blocked by the muscarinic antagonist atropine. The mechanism of antibody-mediated internalization does not appear to involve receptor dimerization by the antibody, as Fab fragments derived from the antibody also induce internalization. The pathway of antibody-induced internalization, similar to the agonist-induced process, is mediated by clathrin-coated vesicles. Furthermore, antibody treatment does not result in any second messenger production, as measured by phosphoinositide accumulation. Our data show that internalization of a G protein-coupled receptor can be triggered by interaction of the amino terminus of the receptor with an exogenous ligand and can occur independently of second messenger production. This result suggests that the receptor can exist in multiple conformations, each mediating distinct downstream events.     

Cardiac RNA Induces Inflammatory Responses in Cardiomyocytes and Immune Cells via Toll-like Receptor 7 Signaling

We have recently reported that extracellular RNA (exRNA) released from necrotic cells induces cytokine production in cardiomyocytes and immune cells and contributes to myocardial ischemia/reperfusion injury. However, the signaling mechanism by which exRNA exhibits its pro-inflammatory effect is unknown. Here we hypothesize that exRNA directly induces inflammation through specific Toll-like receptors (TLRs). To test the hypothesis, we treated rat neonatal cardiomyocytes, mouse bone marrow-derived macrophages (BMDM), or mouse neutrophils with RNA (2.5–10 μg/ml) isolated from rat cardiomyocytes or the hearts from mouse, rat, and human. We found that cellular RNA induced production of several cytokines such as macrophage inflammatory protein-2 (MIP-2), ILs, TNFα, and the effect was completely diminished by RNase, but not DNase. The RNA-induced cytokine production was partially inhibited in cells treated with TLR7 antagonist or genetically deficient in TLR7. Deletion of myeloid differentiation primary response protein 88 (MyD88), a downstream adapter of TLRs including TLR7, abolished the RNA-induced MIP-2 production. Surprisingly, genetic deletion of TLR3 had no impact on the RNA-induced MIP-2 response. Importantly, extracellular RNA released from damaged cardiomyocytes also induced cytokine production. Finally, mice treated with 50 μg of RNA intraperitoneal injection exhibited acute peritonitis as evidenced by marked neutrophil and monocyte migration into the peritoneal space. Together, these data demonstrate that exRNA of cardiac origin exhibits a potent pro-inflammatory property in vitro and in vivo and that exRNA induces cytokine production through TLR7-MyD88 signaling.     

CCN1 Induces Oncostatin M Production in Osteoblasts via Integrin-Dependent Signal Pathways

Inflammatory response and articular destruction are common symptoms of osteoarthritis. Cysteine-rich 61 (CCN1 or Cyr61), a secreted protein from the CCN family, is associated with the extracellular matrix involved in many cellular activities like growth and differentiation. Yet the mechanism of CCN1 interacting with arthritic inflammatory response is unclear. This study finds CCN1 increasing expression of oncostatin m (OSM) in human osteoblastic cells. Pretreatment of αvβ3 monoclonal antibody and inhibitors of focal adhesion kinase (FAK), c-Src, phosphatidylinositol 3-kinase (PI3K), and NF-κB inhibited CCN1-induced OSM expression in osteoblastic cells. Stimulation of cells with CCN1 increased phosphorylation of FAK, c-Src, PI3K, and NF-κB via αvβ3 receptor; CCN1 treatment of osteoblasts increased NF-κB-luciferase activity and p65 binding to NF-κB element on OSM promoter. Results indicate CCN1 heightening OSM expression via αvβ3 receptor, FAK, c-Src, PI3K, and NF-κB signal pathway in osteoblastic cells, suggesting CCN1 as a novel target in arthritis treatment.     

Gliadin Induces Neutrophil Migration via Engagement of the Formyl Peptide Receptor,FPR1

Background

Gliadin, the immunogenic component within gluten and trigger of celiac disease, is known to induce the production of Interleukin-8, a potent neutrophil-activating and chemoattractant chemokine. We sought to study the involvement of neutrophils in the early immunological changes following gliadin exposure.

Methods

Utilizing immunofluorescence microscopy and flow cytometry, the redistribution of major tight junction protein, Zonula occludens (ZO)-1, and neutrophil recruitment were assessed in duodenal tissues of gliadin-gavaged C57BL/6 wild-type and Lys-GFP reporter mice, respectively. Intravital microscopy with Lys-GFP mice allowed monitoring of neutrophil recruitment in response to luminal gliadin exposure in real time. In vitro chemotaxis assays were used to study murine and human neutrophil chemotaxis to gliadin, synthetic alpha-gliadin peptides and the neutrophil chemoattractant, fMet-Leu-Phe, in the presence or absence of a specific inhibitor of the fMet-Leu-Phe receptor-1 (FPR1), cyclosporine H. An irrelevant protein, zein, served as a control.

Results

Redistribution of ZO-1 and an influx of CD11b⁺Lys6G⁺ cells in the lamina propria of the small intestine were observed upon oral gavage of gliadin. In vivo intravital microscopy revealed a slowing down of GFP⁺ cells within the vessels and influx in the mucosal tissue within 2 hours after challenge. In vitro chemotaxis assays showed that gliadin strongly induced neutrophil migration, similar to fMet-Leu-Phe. We identified thirteen synthetic gliadin peptide motifs that induced cell migration. Blocking of FPR1 completely abrogated the fMet-Leu-Phe-, gliadin- and synthetic peptide-induced migration.

Conclusions

Gliadin possesses neutrophil chemoattractant properties similar to the classical neutrophil chemoattractant, fMet-Leu-Phe, and likewise uses FPR1 in the process.     

参麦注射液对单侧输尿管梗阻大鼠肾间质纤维化作用的研究

目的 探讨参麦注射液在单侧输尿管梗阻(UUO)大鼠肾间质纤维化进程中的可能作用.方法 成年SD大鼠54只,随机分为假手术组、模型组和参麦注射液治疗组.假手术组仅完成开腹过程,不结扎输尿管;模型组和参麦注射液治疗组行开腹左侧输尿管结扎术,术后参麦治疗组每天腹腔注射参麦注射液3 mL/(kg·d),假手术组与模型组则每天腹腔注射等量生理盐水.分别于实验的第7、14、21天各组处死动物6只,取左肾组织进行HE染色和α-平滑肌肌动蛋白(α-SMA)免疫组化检查,并测定梗阻侧肾组织中SOD和MDA含量.结果 与模型组比较,参麦注射液治疗组肾小管间质病理改变明显减轻,肾间质α-SMA表达减少;肾组织中SOD活性增加,MDA含量下降.结论 参麦注射液可通过减少氧化应激,减少肾间质α-SMA表达而抑制肾间质纤维化进程.     

Sphingosine 1-Phosphate Modulates Antigen Capture by Murine Langerhans Cells via the S1P2 Receptor Subtype

Dendritic cells (DCs) play a pivotal role in the development of cutaneous contact hypersensitivity (CHS) and atopic dermatitis as they capture and process antigen and present it to T lymphocytes in the lymphoid organs. Recently, it has been indicated that a topical application of the sphingolipid sphingosine 1-phosphate (S1P) prevents the inflammatory response in CHS, but the molecular mechanism is not fully elucidated. Here we indicate that treatment of mice with S1P is connected with an impaired antigen uptake by Langerhans cells (LCs), the initial step of CHS. Most of the known actions of S1P are mediated by a family of five specific G protein-coupled receptors. Our results indicate that S1P inhibits macropinocytosis of the murine LC line XS52 via S1P₂ receptor stimulation followed by a reduced phosphatidylinositol 3-kinase (PI3K) activity. As down-regulation of S1P₂ not only diminished S1P-mediated action but also enhanced the basal activity of LCs on antigen capture, an autocrine action of S1P has been assumed. Actually, S1P is continuously produced by LCs and secreted via the ATP binding cassette transporter ABCC1 to the extracellular environment. Consequently, inhibition of ABCC1, which decreased extracellular S1P levels, markedly increased the antigen uptake by LCs. Moreover, stimulation of sphingosine kinase activity, the crucial enzyme for S1P formation, is connected not only with enhanced S1P levels but also with diminished antigen capture. These results indicate that S1P is essential in LC homeostasis and influences skin immunity. This is of importance as previous reports suggested an alteration of S1P levels in atopic skin lesions.     

Estrogenic Regulation of Histamine Receptor Subtype H1 Expression in the Ventromedial Nucleus of the Hypothalamus in Female Rats

Female sexual behavior is controlled by central estrogenic action in the ventromedial nucleus of the hypothalamus (VMN). This region plays a pivotal role in facilitating sex-related behavior in response to estrogen stimulation via neural activation by several neurotransmitters, including histamine, which participates in this mechanism through its strong neural potentiating action. However, the mechanism through which estrogen signaling is linked to the histamine system in the VMN is unclear. This study was undertaken to investigate the relationship between estrogen and histamine receptor subtype H1 (H1R), which is a potent subtype among histamine receptors in the brain. We show localization of H1R exclusively in the ventrolateral subregion of the female VMN (vl VMN), and not in the dorsomedial subregion. In the vl VMN, abundantly expressed H1R were mostly colocalized with estrogen receptor α. Intriguingly, H1R mRNA levels in the vl VMN were significantly elevated in ovariectomized female rats treated with estrogen benzoate. These data suggest that estrogen can amplify histamine signaling by enhancing H1R expression in the vl VMN. This enhancement of histamine signaling might be functionally important for allowing neural excitation in response to estrogen stimulation of the neural circuit and may serve as an accelerator of female sexual arousal.     

The Role of Toll-Like Receptor 2 in Inflammation and Fibrosis during Progressive Renal Injury

Tissue fibrosis and chronic inflammation are common causes of progressive organ damage, including progressive renal disease, leading to loss of physiological functions. Recently, it was shown that Toll-like receptor 2 (TLR2) is expressed in the kidney and activated by endogenous danger signals. The expression and function of TLR2 during renal fibrosis and chronic inflammation has however not yet been elucidated. Therefore, we studied TLR2 expression in human and murine progressive renal diseases and explored its role by inducing obstructive nephropathy in TLR2^−/− or TLR2^+/+ mice. We found that TLR2 is markedly upregulated on tubular and tubulointerstitial cells in patients with chronic renal injury. In mice with obstructive nephropathy, renal injury was associated with a marked upregulation and change in distribution of TLR2 and upregulation of murine TLR2 danger ligands Gp96, biglycan, and HMGB1. Notably, TLR2 enhanced inflammation as reflected by a significantly reduced influx of neutrophils and production of chemokines and TGF-β in kidneys of TLR2^−/− mice compared with TLR2^+/+ animals. Although, the obstructed kidneys of TLR2^−/− mice had less interstitial myofibroblasts in the later phase of obstructive nephropathy, tubular injury and renal matrix accumulation was similar in both mouse strains. Together, these data demonstrate that TLR2 can initiate renal inflammation during progressive renal injury and that the absence of TLR2 does not affect the development of chronic renal injury and fibrosis.     

Suppression of CB1 Cannabinoid Receptor by Lentivirus Mediated Small Interfering RNA Ameliorates Hepatic Fibrosis in Rats

It is recognized that endogenous cannabinoids, which signal through CB1 receptors in hepatic stellate cells (HSCs), exert a profibrotic effect on chronic liver diseases. In this study, we suppressed CB1 expression by lentivirus mediated small interfering RNA (CB1-RNAi-LV) and investigated its effect on hepatic fibrosis in vitro and in vivo. Our results demonstrated that CB1-RNAi-LV significantly inhibited CB1 expression, and suppressed proliferation and extracellular matrix production in HSCs. Furthermore, CB1-RNAi-LV ameliorated dimethylnitrosamine induced hepatic fibrosis markedly, which was associated with the decreased expression of mesenchymal cell markers smooth muscle α-actin, vimentin and snail, and the increased expression of epithelial cell marker E-cadherin. The mechanism lies on the blockage of Smad signaling transduction induced by transforming growth factor β1 and its receptor TGF-β RII. Our study firstly provides the evidence that CB1-RNAi-LV might ameliorate hepatic fibrosis through the reversal of epithelial-to-mesenchymal transition (EMT), while the CB1 antagonists AM251 had no effect on epithelial-mesenchymal transitions of HSCs. This suggests that CB1 is implicated in hepatic fibrosis and selective suppression of CB1 by small interfering RNA may present a powerful tool for hepatic fibrosis treatment.     

Cardioprotection by Aldosterone Receptor Antagonism in Heart Failure: 1. The Role of Aldosterone in Heart Failure

In recent years, understanding of the role of aldosterone has expanded beyond the known classic effects of promoting renal sodium retention and potassium and magnesium loss. It is now well documented that aldosterone causes myocardial and perivascular fibrosis, blocks the myocardial uptake of norepinephrine, and increases plasminogen activator inhibitor levels. In conjunction with angiotensin II, aldosterone causes vascular damage, endothelial dysfunction, and decreased vascular compliance. Thus, the renin-angiotensin-aldosterone system (RAAS) plays a major role in the development of both hypertension and heart failure and is, therefore, a key target for therapeutic interventions. Commonly prescribed medications for control of hypertension and congestive heart failure are inhibitors of the RAAS, including angiotensin converting enzyme inhibitors (ACE-Is) and angiotensin II (A-II) receptor antagonists. A well-documented increase in aldosterone levels occurs over several months during chronic treatment with an ACE-I or an A-II receptor antagonist. Such suppression of circulating aldosterone, however, is transient, as exemplified by the term “escape” used to describe the phenomenon. This rebound of aldosterone even occurs when patients receive both an ACE-I and an A-II receptor antagonist. In addition, ACE-Is and A-II receptor antagonists are less effective in controlling blood pressure in the estimated 60% of hypertensive patients who are salt- (volume-) sensitive and more prone to hypertension-associated morbidity, such as black patients and type 2 diabetics. Thus, chronic and complete blockade of aldosterone action requires an aldosterone receptor antagonist. The Randomized Aldactone Evaluation Study (RALES) trial results in patients with severe heart failure (New York Heart Association class III or IV) and a left ventricular ejection fraction of no more than 35% showed that administration of a subhemodynamic dose of spironolactone (25 mg/day) as an add-on therapy to ACE-Is plus standard treatment resulted in a significant mortality reduction due to decreases in both death from progressive heart failure and sudden cardiac death. These findings support the pivotal role of aldosterone in the pathophysiology of progressive heart failure. Although it is an effective antialdosterone agent, widespread use of spironolactone in humans is limited by its tendency to produce undesirable sexual side effects. At standard doses, impotence and gynecomastia can be induced in men, whereas premenopausal women may experience menstrual disturbances. Data on a selective aldosterone receptor antagonist, eplerenone, show that it appears promising for the effective blockade of aldosterone and its harmful effects without the sexual disturbances of spironolactone. Recently, eplerenone was successfully introduced for the treatment of hypertension and heart failure. A growing number of experimental studies are finding a broader role for aldosterone in driving the pathophysiology of both heart failure and hypertension. When added to conventional therapy, aldosterone receptor blockers show benefits in addition to those conferred by ACE-Is and/or A-II receptor blockers.     

IL-5 Induces Proliferation and Activation of Microglia via an Unknown Receptor

While the effects of interleukin-3 (IL-3) and granulocyte macrophage-colony stimulating factor (GM-CSF) on microglia are well documented, very little is known about the effects of a related cytokine, interleukin-5 (IL-5). We therefore undertook studies to determine how IL-5 alters various aspects of microglial functioning. Treatment of microglia with IL-5 resulted in the induction of proliferation at levels similar to those induced by GM-CSF. IL-5 also increased cellular metabolism of microglial cells. To determine whether increased metabolism correlated with activation of microglia, we measured levels of nitrite, a breakdown product of nitric oxide. Treatment of microglial cultures with IL-5 increased nitrite levels, while GM-CSF treatment had no effect. Treatment of microglia with IL-5 did not cause activation of the signal transduction pathways linked to the classical IL-5 receptor, STAT5A/5B and ERK1 and ERK2. It is therefore likely that the effects of IL-5 on microglia are not mediated via the classical IL-5 receptor, but rather via a novel receptor.     

Finerenone Impedes Aldosterone-dependent Nuclear Import of the Mineralocorticoid Receptor and Prevents Genomic Recruitment of Steroid Receptor Coactivator-1

Aldosterone regulates sodium homeostasis by activating the mineralocorticoid receptor (MR), a member of the nuclear receptor superfamily. Hyperaldosteronism leads todeleterious effects on the kidney, blood vessels, and heart. Although steroidal antagonists such as spironolactone and eplerenone are clinically useful for the treatment of cardiovascular diseases, they are associated with several side effects. Finerenone, a novel nonsteroidal MR antagonist, is presently being evaluated in two clinical phase IIb trials. Here, we characterized the molecular mechanisms of action of finerenone and spironolactone at several key steps of the MR signaling pathway. Molecular modeling and mutagenesis approaches allowed identification of Ser-810 and Ala-773 as key residues for the high MR selectivity of finerenone. Moreover, we showed that, in contrast to spironolactone, which activates the S810L mutant MR responsible for a severe form of early onset hypertension, finerenone displays strict antagonistic properties. Aldosterone-dependent phosphorylation and degradation of MR are inhibited by both finerenone and spironolactone. However, automated quantification of MR subcellular distribution demonstrated that finerenone delays aldosterone-induced nuclear accumulation of MR more efficiently than spironolactone. Finally, chromatin immunoprecipitation assays revealed that, as opposed to spironolactone, finerenone inhibits MR, steroid receptor coactivator-1, and RNA polymerase II binding at the regulatory sequence of the SCNN1A gene and also remarkably reduces basal MR and steroid receptor coactivator-1 recruitment, unraveling a specific and unrecognized inactivating mechanism on MR signaling. Overall, our data demonstrate that the highly potent and selective MR antagonist finerenone specifically impairs several critical steps of the MR signaling pathway and therefore represents a promising new generation MR antagonist.     

口服氯化汞对大鼠肾间质纤维化的作用

目的口服氯化汞(HgCl2)造成大鼠的肾间质纤维化模型并探讨相关机制。方法用不同剂量HgCl2[(A组为5mg/(kg·bw)、B组为10mg/(kg·bw)、C组为20mg/(kg·bw)]给大鼠灌胃1周,观察大鼠一般状况、肾功能和肾组织病理变化,免疫组化法观察肾组织纤维连接蛋白(FN)和α-平滑肌肌动蛋白(α-SMA)表达。结果模型大鼠体重下降,肾体比增加,肾功能损害和肾组织Hyp含量呈剂量依赖性升高,肾间质炎性细胞浸润,肾间质胶原沉积增加,肾间质FN和α-SMA表达增强,以C组病变最重。结论20mg/(kg·bw)剂量HgCl2灌胃1周可造成大鼠的肾间质纤维化病变,其部分机制在于HgCl2促使肾间质肌成纤维细胞活化和细胞外基质的生成沉积。     

Chemokine Receptor Ccr6 Deficiency Alters Hepatic Inflammatory Cell Recruitment and Promotes Liver Inflammation and Fibrosis

Chronic liver diseases are characterized by a sustained inflammatory response in which chemokines and chemokine-receptors orchestrate inflammatory cell recruitment. In this study we investigated the role of the chemokine receptor CCR6 in acute and chronic liver injury. In the absence of liver injury Ccr6 ^-/- mice presented a higher number of hepatic macrophages and increased expression of pro-inflammatory cytokines and M1 markers Tnf-α, Il6 and Mcp1. Inflammation and cell recruitment were increased after carbon tetrachloride-induced acute liver injury in Ccr6 ^-/- mice. Moreover, chronic liver injury by carbon tetrachloride in Ccr6 ^-/- mice was associated with enhanced inflammation and fibrosis, altered macrophage recruitment, enhanced CD4⁺ cells and a reduction in Th17 (CD4⁺IL17⁺) and mature dendritic (MHCII⁺CD11c⁺) cells recruitment. Clodronate depletion of macrophages in Ccr6 ^-/- mice resulted in a reduction of hepatic pro-inflammatory and pro-fibrogenic markers in the absence and after liver injury. Finally, increased CCR6 hepatic expression in patients with alcoholic hepatitis was found to correlate with liver expression of CCL20 and severity of liver disease. In conclusion, CCR6 deficiency affects hepatic inflammatory cell recruitment resulting in the promotion of hepatic inflammation and fibrosis.     

Taurocholate Induces Cyclooxygenase-2 Expression via the Sphingosine 1-phosphate Receptor 2 in a Human Cholangiocarcinoma Cell Line

Cholangiocarcinoma (CCA) is a rare, but highly malignant primary hepatobiliary cancer with a very poor prognosis and limited treatment options. Our recent studies reported that conjugated bile acids (CBAs) promote the invasive growth of CCA via activation of sphingosine 1-phosphate receptor 2 (S1PR2). Cyclooxygenase-2 (COX-2)-derived prostaglandin E₂ (PGE₂) is the most abundant prostaglandin in various human malignancies including CCA. Previous studies have indicated that COX-2 was highly expressed in CCA tissues, and the survival rate of CCA patients was negatively associated with high COX-2 expression levels. It has also been reported that CBAs induce COX-2 expression, whereas free bile acids inhibit COX-2 expression in CCA mouse models. However, the underlying cellular mechanisms and connection between S1PR2 and COX-2 expression in CCA cells have still not been fully elucidated. In the current study, we examined the role of S1PR2 in conjugated bile acid (taurocholate, (TCA))-induced COX-2 expression in a human HuCCT1 CCA cell line and further identified the potential underlying cellular mechanisms. The results indicated that TCA-induced invasive growth of human CCA cells was correlated with S1PR2-medated up-regulation of COX-2 expression and PGE₂ production. Inhibition of S1PR2 activation with chemical antagonist (JTE-013) or down-regulation of S1PR2 expression with gene-specific shRNA not only reduced COX-2 expression, but also inhibited TCA-induced activation of EGFR and the ERK1/2/Akt-NF-κB signaling cascade. In conclusion, S1PR2 plays a critical role in TCA-induced COX-2 expression and CCA growth and may represent a novel therapeutic target for CCA.     

Testosterone-Dependent Interaction between Androgen Receptor and Aryl Hydrocarbon Receptor Induces Liver Receptor Homolog 1 Expression in Rat Granulosa Cells

Androgens play a major role in the regulation of normal ovarian function; however, they are also involved in the development of ovarian pathologies. These contrasting effects may involve a differential response of granulosa cells to the androgens testosterone (T) and dihydrotestosterone (DHT). To determine the molecular pathways that mediate the distinct effects of T and DHT, we studied the expression of the liver receptor homolog 1 (LRH-1) gene, which is differentially regulated by these steroids. We found that although both T and DHT stimulate androgen receptor (AR) binding to the LRH-1 promoter, DHT prevents T-mediated stimulation of LRH-1 expression. T stimulated the expression of aryl hydrocarbon receptor (AHR) and its interaction with the AR. T also promoted the recruitment of the AR/AHR complex to the LRH-1 promoter. These effects were not mimicked by DHT. We also observed that the activation of extracellular regulated kinases by T is required for AR and AHR interaction. In summary, T, but not DHT, stimulates AHR expression and the interaction between AHR and AR, leading to the stimulation of LRH-1 expression. These findings could explain the distinct response of granulosa cells to T and DHT and provide a molecular mechanism by which DHT negatively affects ovarian function.