The 3' Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies' association with histone locus bodies.

Non-polyadenylated mRNAs of replication-dependent histones (RDHs) are synthesized by RNA polymerase II (Pol II) at histone locus bodies (HLBs). HLBs frequently associate with Cajal bodies (CBs), in which 3'-end processing factors for RDH genes are enriched; however, this association's role in transcription termination of RDH genes remains unclear. Here, we show that Pol II pauses immediately upstream of transcript end sites of RDH genes and Mediator plays a role in this Pol II pausing through CBs' association with HLBs. Disruption of the Mediator docking site for Little elongation complex (LEC)-Cap binding complex (CBC)-Negative elongation factor (NELF), components of CBs, interferes with CBs' association with HLBs and 3' Pol II pausing, resulting in increased aberrant unprocessed RDH gene transcripts. Our findings suggest Mediator's involvement in CBs' association with HLBs to facilitate 3' Pol II pausing and subsequent 3'-end processing of RDH genes by supplying 3'-end processing factors.

Phosphorylation and dephosphorylation of Ser852 and Ser889 control the clustering, localization and function of PAR3.

Cell polarity is essential for various asymmetric cellular events, and the partitioning defective (PAR) protein PAR3 (encoded by PARD3 in mammals) plays a unique role as a cellular landmark to establish polarity. In epithelial cells, PAR3 localizes at the subapical border, such as the tight junction in vertebrates, and functions as an apical determinant. Although we know a great deal about the regulators of PAR3 localization, how PAR3 is concentrated and localized to a specific membrane domain remains an important question to be clarified. In this study, we demonstrate that ASPP2 (also known as TP53BP2), which controls PAR3 localization, links PAR3 and protein phosphatase 1 (PP1). The ASPP2-PP1 complex dephosphorylates a novel phosphorylation site, Ser852, of PAR3. Furthermore, Ser852- or Ser889-unphosphorylatable PAR3 mutants form protein clusters, and ectopically localize to the lateral membrane. Concomitance of clustering and ectopic localization suggests that PAR3 localization is a consequence of local clustering. We also demonstrate that unphosphorylatable forms of PAR3 exhibited a low molecular turnover and failed to coordinate rapid reconstruction of the tight junction, supporting that both the phosphorylated and dephosphorylated states are essential for the functional integrity of PAR3.

Shank2 Binds to aPKC and Controls Tight Junction Formation with Rap1 Signaling during Establishment of Epithelial Cell Polarity.

Epithelial cells establish apicobasal polarity by forming tight junctions (TJs) at the apical-lateral boundary, which play fundamental roles in physiological functions. An evolutionarily conserved atypical protein kinase C (aPKC)-partitioning defective (PAR) complex functions as a platform for TJ assembly during cell polarity establishment. However, how this complex converts the spatial cues into a subsequent active unit is unclear. Here, we identify an epithelial isoform of Shank2 as a mediator of the aPKC-PAR complex. Shank2 binds to and colocalizes with aPKC at apical junctional regions of polarized epithelial cells. Shank2 knockdown results in defects in TJ formation. Mechanistically, we find that the N-terminal SPN domain is required for the junctional localization of Shank2 and binds to the active form of Rap1 small GTPase, which is involved in TJ formation. Our findings suggest that a close physical and functional relationship between aPKC and Shank2-active Rap1 signaling serves as the platform for TJ assembly to regulate epithelial cell polarity.

The role of Mediator and Little Elongation Complex in transcription termination.

Mediator is a coregulatory complex that regulates transcription of Pol II-dependent genes. Previously, we showed that human Mediator subunit MED26 plays a role in the recruitment of Super Elongation Complex (SEC) or Little Elongation Complex (LEC) to regulate the expression of certain genes. MED26 plays a role in recruiting SEC to protein-coding genes including c-myc and LEC to small nuclear RNA (snRNA) genes. However, how MED26 engages SEC or LEC to regulate distinct genes is unclear. Here, we provide evidence that MED26 recruits LEC to modulate transcription termination of non-polyadenylated transcripts including snRNAs and mRNAs encoding replication-dependent histone (RDH) at Cajal bodies. Our findings indicate that LEC recruited by MED26 promotes efficient transcription termination by Pol II through interaction with CBC-ARS2 and NELF/DSIF, and promotes 3' end processing by enhancing recruitment of Integrator or Heat Labile Factor to snRNA or RDH genes, respectively.

MTCL1 crosslinks and stabilizes non-centrosomal microtubules on the Golgi membrane.

Recent studies have revealed the presence of a microtubule subpopulation called Golgi-derived microtubules that support Golgi ribbon formation, which is required for maintaining polarized cell migration. CLASPs and AKAP450/CG-NAP are involved in their formation, but the underlying molecular mechanisms remain unclear. Here, we find that the microtubule-crosslinking protein, MTCL1, is recruited to the Golgi membranes through interactions with CLASPs and AKAP450/CG-NAP, and promotes microtubule growth from the Golgi membrane. Correspondingly, MTCL1 knockdown specifically impairs the formation of the stable perinuclear microtubule network to which the Golgi ribbon tethers and extends. Rescue experiments demonstrate that besides its crosslinking activity mediated by the N-terminal microtubule-binding region, the C-terminal microtubule-binding region plays essential roles in these MTCL1 functions through a novel microtubule-stabilizing activity. These results suggest that MTCL1 cooperates with CLASPs and AKAP450/CG-NAP in the formation of the Golgi-derived microtubules, and mediates their development into a stable microtubule network.

Regulation of dendritic cell functions against harmful respiratory pathogens by a cysteinyl leukotrienes receptor antagonist.

Cysteinyl leukotriene receptor antagonist (LTRA) is a widely used medicine for asthma. Cysteinyl leukotrienes (cysLTs) are involved in the regulation of dendritic cell (DC) function. However, the effects of LTRA on DC-related antimicrobial immunity against harmful respiratory pathogens remain unknown. The purpose of this study was to examine the effects of LTRA administered in vivo on DC function against representative respiratory pathogens in vitro. Pulmonary DCs were isolated from four groups of mice: control, mite allergen sensitized (AS), and AS mice treated with the corticosteroid dexamethasone (Dex) or with the LTRA pranlukast (Prl). These DCs were incubated with mite allergen, lipopolysaccharide (LPS), Aspergillus fumigatus, or respiratory syncytial virus (RSV). IL-10 and IL-12 production was then determined. Dex treatment significantly inhibited lipopolysaccharide (LPS)-induced IL-10 and IL-12 production as well as baseline IL-12 production in AS mice. The Prl did not significantly inhibit LPS-induced IL-10 and IL-12 production in AS mice. More importantly, Prl significantly increased IL-10 and IL-12 in AS mice after RSV infection. This study shows that LTRA that is used for asthma potentially up-regulates antimicrobial immunity through modulation of DC function against some respiratory infections without immunosuppression.

[Two cases of chronic pulmonary thromboembolism diagnosed clinically as chronic obstructive pulmonary disease].

Two cases of chronic pulmonary thromboembolism accidentally diagnosed in the clinical evaluation of COPD are reported. Case 1, a 69-year-old woman who had never smoked, was given a diagnosis of COPD since she had exertional dyspnea with obstructive pulmonary dysfunction and multiple low attenuation areas on her chest CT. Pulmonary ventilation and perfusion scintigram showed mismatched defects and as chronic pulmonary thromboembolism (CPTE) was finally diagnosed. Case 2, a 70-year-old former smoking man had also been previously given a diagnosis of COPD. He had exertional dyspnea, hypoxemia and multiple low attenuation areas in chest CT. Nonetheless his pulmonary function was normal. Pulmonary ventilation and perfusion scintigram showed mismatched defects and pulmonary angiography identified a floating thrombus in the pulmonary artery. Thus he CPTE was diagnosed. The diagnosis of CPTE in COPD is not easy. Physicians should recognize that CPTE can be associated with COPD and perform further examinations including pulmonary ventilation and perfusion scintigram when patients do not show typical results and/or adequately respond to conventional therapy for COPD.

An essential role of the universal polarity protein, aPKClambda, on the maintenance of podocyte slit diaphragms.

Glomerular visceral epithelial cells (podocytes) contain interdigitated processes that form specialized intercellular junctions, termed slit diaphragms, which provide a selective filtration barrier in the renal glomerulus. Analyses of disease-causing mutations in familial nephrotic syndromes and targeted mutagenesis in mice have revealed critical roles of several proteins in the assembly of slit diaphragms. The nephrin-podocin complex is the main constituent of slit diaphragms. However, the molecular mechanisms regulating these proteins to maintain the slit diaphragms are still largely unknown. Here, we demonstrate that the PAR3-atypical protein kinase C (aPKC)-PAR6beta cell polarity proteins co-localize to the slit diaphragms with nephrin. Furthermore, selective depletion of aPKClambda in mouse podocytes results in the disassembly of slit diaphragms, a disturbance in apico-basal cell polarity, and focal segmental glomerulosclerosis (FSGS). The aPKC-PAR3 complex associates with the nephrin-podocin complex in podocytes through direct interaction between PAR3 and nephrin, and the kinase activity of aPKC is required for the appropriate distribution of nephrin and podocin in podocytes. These observations not only establish a critical function of the polarity proteins in the maintenance of slit diaphragms, but also imply their potential involvement in renal failure in FSGS.

Observational study of the additive effects of pranlukast on inflammatory markers of clinically stable asthma with inhaled corticosteroids and long-acting beta 2 agonists.

BACKGROUND: Little is understood about the additive effects of leukotriene receptor antagonists (LTRA) on asthmatics currently medicated with inhaled corticosteroids (ICS) and long-acting beta(2)-agonists (LABA). OBJECTIVES: The present study examines the anti-inflammatory effects of the LTRA pranlukast in addition to ICS and LABA, among asthmatic patients with normal pulmonary function and unremarkable clinical symptoms. METHODS: Fifteen adult asthmatics participated in a 2-month, open-label, uncontrolled, prospective, multicenter, observational trial. Patients stabilized (predicted forced expiratory volume in 1 s >80%) by medication with ICS and LABA were also given pranlukast (450 mg daily). Asthma-related symptoms, pulmonary function, blood eosinophil counts and several inflammatory markers in sputum were monitored at week 0, as well as at 4 and 8 weeks after starting therapy with pranlukast. RESULTS: Adding pranlukast did not further improve blood eosinophil counts, pulmonary function and symptoms, but significantly attenuated sputum cysteinyl leukotrienes, tumor necrosis factor-alpha and interleukin-5 concentrations. CONCLUSIONS: Although the clinical relevance remains obscure, adding an LTRA attenuates allergic airway inflammation in some asthmatics undergoing treatment with ICS and LABA.

Effects of repeated respiratory syncytial virus infections on pulmonary dendritic cells in a murine model of allergic asthma.

BACKGROUND: Primary and secondary respiratory syncytial virus (RSV) infection differentially regulates preexisting allergic airway inflammation. OBJECTIVES: The present study was designed to determine the effects of primary and secondary low-grade RSV infections on pulmonary dendritic cell (DC) functions. METHODS: Eight groups of BALB/c mice were used: one group each for control primary and secondary sensitization, primary and secondary sensitization to Dermatophagoides farinae (Derf) allergen, primary and secondary infection with RSV, and primary and secondary sensitization to Derf plus infection with RSV. CD11c+ pulmonary DCs were isolated from these mice and then transferred to naïve mice followed by intranasal Derf challenge. Furthermore, either anti-IL-12 monoclonal antibody (alphaIL-12 mAb) or anti-IL-10 (alphaIL-10) mAb were injected into donor mice after Derf challenge and during RSV infection to determine the involvement of IL-12 and IL-10. RESULTS: Primary RSV infection failed to induce polarization in DCs since it failed to induce IL-10 and IL-12 production in Derf-sensitized donor lung. In contrast, secondary RSV infection significantly enhanced IL-12 production from Derf-sensitized donor lung, thereby enhancing both Th1 and Th2 responses. During RSV infection, alphaIL-12 but not alphaIL-10 mAb treatment blocked these immunological effects. CONCLUSION: Via IL-12, DCs may play a critical role in shifting the immune response in this experimental model of repeated respiratory viral infection in allergic asthma.

[Case of idiopathic bronchiolitis obliterans successfully treated by medication].

A 53-year-old woman visited a clinic for stridor and dyspnea, and was treated with steroid and heparin for bronchial asthma and pulmonary embolism. She was later admitted to our hospital for progressive dyspnea. Blood gas analysis showed severe hypoxemia with hypercapnia. Pulmonary funtion tests revealed severe obstractive pulmonary dysfunction. Chest computed tomography showed a mosaic perfusion pattern. Ventilation-perfusion scanning showed bilateral multiple matched defects, especially in the basal region. Since specimens of Video-assisted thoracoscopic surgical (VATS) lung biopsy showed lymphocytic infiltration in membranous bronchiole and occlusion of the membranous bronchiole lumen, bronchiolitis obliterans was diagnosed. We initiated treatment with steroids, macrolides and bronchodilators and her condition stabilized. Although these therapies did not cure the BO, they did retard its progression.

Cytokine production from peripheral blood mononuclear cells of mite allergen-sensitized atopic adults stimulated with respiratory syncytial virus and mite allergen.

BACKGROUND: The interaction between viral respiratory tract infection and allergen sensitization in allergic asthma is unclear. Respiratory syncytial virus (RSV) has attracted attention as an important lower respiratory pathogen during childhood, while recent evidence indicates that RSV is also an important lower respiratory pathogen for adults. Immunity against RSV differs between children and adults. Several reports suggest that RSV infection in children results in a Th2-skewed immune response. The purpose of the present study was to determine the effects of RSV infection in adults who had previously been sensitized with a common aeroallergen. METHODS: Peripheral blood mononuclear cells (PBMCs) isolated from 9 mite-sensitized atopic subjects and 11 healthy nonatopic subjects were exposed to live or UV-inactivated RSV concomitant to incubation with or without mite allergen, and the subsequent production of cytokines - interleukin (IL)-5, interferon (IFN)-gamma, IL-10 and IL-12p70 - was measured. RESULTS: Mite allergen significantly increased IL-5 production in atopic PBMCs. RSV infection significantly increased IFN-gamma production from healthy and atopic PBMCs; the levels of IFN-gamma were significantly higher for atopic PBMCs. Live RSV infection significantly attenuated IL-5 production from mite allergen-stimulated atopic PBMCs. UV-inactivated RSV, but not live RSV, significantly enhanced allergen-specific IL-10 production in atopic PBMCs. IL-12 could not be detected in the present study. CONCLUSION: The present findings suggest that RSV infection did not simply enhance allergen-specific Th2-like response in atopic adults. Live RSV-induced IFN-gamma and RSV protein-induced IL-10 appear to play important roles in the regulation of allergic airway inflammation in atopic adults.

[Case of idiopathic chylothorax successfully treated with surgical ligation of thoracic ducts].

A 72-year-old woman visited a clinic for anorexia and general fatigue but no particular abnormality was detected by routine examination at that time. Thereafter, she experienced gradually increasing dyspnea and chest X ray showed right pleural effusion. Idiopathic chylothorax was diagnosed due to the milky effusion with a high concentration of triglyceride (2618 mg/dl) and no apparent causative disease. Irrespective of treatments including dietary restriction, drainage of the pleural space, and somatostatin injections, her effusion did not reduce. The leakage of lymph fluid from the right posterior mediastinum was identified by lymphatic scintigraphy and she was successfully treated with surgical ligation of the thoracic ducts.

Effects of respiratory syncytial virus infection on dendritic cells and cysteinyl leukotrienes in lung tissues of a murine model of asthma.

BACKGROUND: Pulmonary dendritic cells (DCs) play critical roles in both allergy and in viral infection. Levels of cysteinyl leukotrienes (cysLTs) increase after allergen sensitization and viral infection and can modulate the migration and functions of DCs. The present study examines the effects of respiratory syncytial virus (RSV) infection on numbers of DCs and cysLT concentrations in lung tissues of mice sensitized with mite allergen. METHODS: We examined Control, Dermatophagoides farinae allergen sensitized (Df), RSV infected (RSV) and Df allergen sensitized and RSV infected (Df-RSV) Balb/c mice. We then determined the number of CD11c-positive DCs and the LT concentration in lung tissues of the mice and examined lung pathology and cytokine profiles in thoracic lymph nodes. RESULTS: Infection with RSV significantly enhanced allergic airway inflammation in Df mice with concomitant increases in Th1 and Th2 immunity. The number of DCs and the cysLT concentrations were significantly increased in the lungs of Df and RSV mice and more so in Df-RSV, than in Df mice. CONCLUSIONS: The present findings suggest that RSV infection increases the number of DCs and the cysLT concentrations in lung tissues of asthma patients, both of which could result in enhanced allergic airway inflammation.

aPKC acts upstream of PAR-1b in both the establishment and maintenance of mammalian epithelial polarity.

BACKGROUND: aPKC and PAR-1 are required for cell polarity in various contexts. In mammalian epithelial cells, aPKC localizes at tight junctions (TJs) and plays an indispensable role in the development of asymmetric intercellular junctions essential for the establishment and maintenance of apicobasal polarity. On the other hand, one of the mammalian PAR-1 kinases, PAR-1b/EMK1/MARK2, localizes to the lateral membrane in a complimentary manner with aPKC, but little is known about its role in apicobasal polarity of epithelial cells as well as its functional relationship with aPKC. RESULTS: We demonstrate that PAR-1b is essential for the asymmetric development of membrane domains of polarized MDCK cells. Nonetheless, it is not required for the junctional localization of aPKC nor the formation of TJs, suggesting that PAR-1b works downstream of aPKC during epithelial cell polarization. On the other hand, aPKC phosphorylates threonine 595 of PAR-1b and enhances its binding with 14-3-3/PAR-5. In polarized MDCK cells, T595 phosphorylation and 14-3-3 binding are observed only in the soluble form of PAR-1b, and okadaic acid treatment induces T595-dependent dissociation of PAR-1b from the lateral membrane. Furthermore, T595A mutation induces not only PAR-1b leakage into the apical membrane, but also abnormal development of membrane domains. These results suggest that in polarized epithelial cells, aPKC phosphorylates PAR-1b at TJs, and in cooperation with 14-3-3, promotes the dissociation of PAR-1b from the lateral membrane to regulate PAR-1b activity for the membrane domain development. CONCLUSIONS: These results suggest that mammalian aPKC functions upstream of PAR-1b in both the establishment and maintenance of epithelial cell polarity.