Gene network analysis for identification of microRNA biomarkers for asthma.

BACKGROUND: To date, reliable biomarkers for asthma have not been identified. MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate post-transcriptional gene expression, and they are involved in various diseases, including asthma. MiRNAs may serve as ideal biomarkers due to their ability to regulate multiple pathways. This study aims to identify miRNA biomarker signatures for asthma. METHODS: We used the house dust mite (HDM) mouse model of allergic inflammation. Mice were phenotyped by assessing lung function, allergic response, airway inflammation, and remodeling. The miRNA signature profiles in serum and lung tissue were determined by small RNA sequencing, and data were analyzed using Qiagen CLC Genomics Workbench. To identify relevant gene targets, we performed mRNA sequencing, followed by miRNA-targets analysis. These miRNAs and targets were subject to subsequent pathway and functional analyses. RESULTS: Mice exposed to HDM developed phenotypic features of allergic asthma. miRNA sequencing analysis showed that 213 miRNAs were substantially dysregulated (FDR p-value < 0.05 and fold change expression > + 1.5 and < - 1.5) in the lung of HDM mice relative to the control mice. In contrast, only one miRNA (miR-146b-5p) was significantly increased in serum. Target analysis of lung dysregulated miRNAs revealed a total of 131 miRNAs targeting 211 mRNAs. Pathway analysis showed T helper 2/1 (Th2/Th1) as the top significantly activated signaling pathway associated with the dysregulated miRNAs. The top enriched diseases were inflammatory response and disease, which included asthma. Asthma network analysis indicated that 113 of 131 miRNAs were directly associated with asthma pathogenesis. CONCLUSIONS: These findings suggest that most dysregulated miRNAs in the HDM model were associated with asthma pathogenesis via Th2 signaling. We identified a panel of 30 miRNAs as potential biomarker candidates for asthma.

Differential regulation of cytokine and chemokine expression by MK2 and MK3 in airway smooth muscle cells.

BACKGROUND: Airway smooth muscle (ASM) contributes to local inflammation and plays an immunomodulatory role in airway diseases. This is partially regulated by p38 mitogen-activated protein kinase (MAPK), which further activates two closely related isoforms of the MAPK-activated protein kinases (MKs), MK2 and MK3. The MKs have similar substrate specificities but less is known about differences in their functional responses. This study was undertaken to identify differential downstream inflammatory targets of MK2 and MK3 signaling and assess cross-talk between the MAPK pathway and NF-κB signaling relevant to ASM function. METHODS: Wild-type and kinase-deficient MK2 (MK2WT, MK2KR) and MK3 (MK3WT, MK33A) were expressed in human ASM cells stimulated for 20 h with 10 ng/ml each interleukin (IL)-1ß, tumor necrosis factor (TNF)-α and interferon (IFN)-γ. Inflammatory mediator secretion was assessed by Luminex assays and ELISA. Signaling pathway activation was monitored by Western blotting. RESULTS: Expression of these MKs and stimulation with 10 ng/ml IL-1ß, TNFα and IFNγ for 20 h did not affect secretion of multiple cytokines including IL-4, IL-5, IL-13 and monocyte chemotactic protein (MCP)-1/CCL2 but did differentially affect the secretion of regulated upon activation, normal T cell expressed and secreted (RANTES)/CCL5, IL-6 and granulocyte macrophage-colony stimulating factor (GM-CSF). RANTES/CCL5 secretion was decreased by MK2WT or MK3WT and stimulated by inhibition of MK2 or MK3 activity with expression of the kinase-deficient enzymes MK2KR or MK33A. IL-6 and GM-CSF secretion was decreased by inhibition of MK2 activity with MK2KR and while MK3WT had no effect, the kinase-deficient MK33A further decreased secretion of these mediators. Cross-talk of the MKs with other signaling pathways was investigated by examining NF-κB activation, which was inhibited by expression of MK3 but not affected by MK2. CONCLUSIONS: These results suggest an inhibitory role for MK2 and MK3 activity in RANTES/CCL5 secretion and cross-talk of MK3 with NF-κB to regulate IL-6 and GM-CSF. These findings differentiate MK2 and MK3 function in ASM cells and provide insight that may enable selective targeting of MKs in ASM to modulate local inflammation in airway disease.

Direct evidence for functional smooth muscle myosin II in the 10S self-inhibited monomeric conformation in airway smooth muscle cells.

The 10S self-inhibited monomeric conformation of myosin II has been characterized extensively in vitro. Based upon its structural and functional characteristics, it has been proposed to be an assembly-competent myosin pool in equilibrium with filaments in cells. It is known that myosin filaments can assemble and disassemble in nonmuscle cells, and in some smooth muscle cells, but whether or not the disassembled pool contains functional 10S myosin has not been determined. Here we address this question using human airway smooth muscle cells (hASMCs). Using two antibodies against different epitopes on smooth muscle myosin II (SMM), two distinct pools of SMM, diffuse, and stress-fiber-associated, were visualized by immunocytochemical staining. The two SMM pools were functional in that they could be interconverted in two ways: (i) by exposure to 10S- versus filament-promoting buffer conditions, and (ii) by exposure to a peptide that shifts the filament-10S equilibrium toward filaments in vitro by a known mechanism that requires the presence of the 10S conformation. The effect of the peptide was not due to a trivial increase in SMM phosphorylation, and its specificity was demonstrated by use of a scrambled peptide, which had no effect. Based upon these data, we conclude that hASMCs contain a significant pool of functional SMM in the 10S conformation that can assemble into filaments upon changing cellular conditions. This study provides unique direct evidence for the presence of a significant pool of functional myosin in the 10S conformation in cells.

Transgenic overexpression of α7 integrin in smooth muscle attenuates allergen-induced airway inflammation in a murine model of asthma.

Asthma is a chronic inflammatory disorder of the lower airways characterized by modulation of airway smooth muscle (ASM) function. Infiltration of smooth muscle by inflammatory mediators is partially regulated by transmembrane integrins and the major smooth muscle laminin receptor α7ß1 integrin plays a critical role in the maintenance of ASM phenotype. The goal of the current study was to investigate the role of α7 integrin in asthma using smooth muscle-specific α7 integrin transgenic mice (TgSM-Itgα7) using both acute and chronic OVA sensitization and challenge protocols that mimic mild to severe asthmatic phenotypes. Transgenic over-expression of the α7 integrin in smooth muscle resulted in a significant decrease in airway resistance relative to controls, reduced the total number of inflammatory cells and substantially inhibited the production of crucial Th2 and Th17 cytokines in airways. This was accompanied by decreased secretion of various inflammatory chemokines such as eotaxin/CCL11, KC/CXCL3, MCP-1/CCL2, and MIP-1ß/CCL4. Additionally, α7 integrin overexpression significantly decreased ERK1/2 phosphorylation in the lungs of TgSM-Itgα7 mice and affected proliferative, contractile, and inflammatory downstream effectors of ERK1/2 that drive smooth muscle phenotype in the lung. Taken together, these results support the hypothesis that enhanced expression of α7 integrin in vivo inhibits allergic inflammation and airway resistance. Moreover, we identify ERK1/2 as a potential target by which α7 integrin signals to regulate airway inflammation. We conclude that identification of therapeutics targeting an increase in smooth muscle α7 integrin expression could serve as a potential novel treatment for asthma.

Knockdown of subunit 3 of the COP9 signalosome inhibits C2C12 myoblast differentiation via NF-KappaB signaling pathway.

BACKGROUND: The COP9 signalosome (CSN) is a conserved protein complex composed of 8 subunits designated CSN1-CSN8. CSN3 represents the third subunit of the CSN and maintains the integrity of the complex. CSN3 binds to the striated muscle-specific ß1D integrin tail, and its subcellular localization is altered in differentiated skeletal muscle cells. However, the role of CSN3 in skeletal muscle differentiation is unknown. The main goal of this study was to identify whether CSN3 participates in myoblast differentiation and the signalling mechanisms involved using C2C12 cells as a skeletal muscle cell model. METHODS: Small-hairpin (shRNA) was used to knockdown CSN3 in C2C12 cells. Differentiation was evaluated by immunostaining and confocal microscopy. Markers of differentiation, NF-κB signaling and CSN subunits expression, were assessed by immunoblotting and/or immunostaining. Cell proliferation was analysed by cell counting, flow cytometry and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data were analyzed by one or two-way analysis of variance (ANOVA) followed by post-hoc testing. RESULTS: Transduction of C2C12 cells with two distinct CSN3 shRNAs led to the production of two cells lines expressing 7% of CSN3 protein (shCSN3-Low) and 43% of CSN3 protein (CSN3-Med) compared to controls. Knockdown of CSN3 was accompanied by destabilization of several CSN subunits and increased nuclear NF-κB localization. shCSN3-Med cells expressed less myogenin and formed shorter and thinner myotubes. In contrast, the shCSN3-Low cells expressed higher levels of myogenin prior and during the differentiation and remained mononucleated throughout the differentiation period. Both CSN3 knockdown cell lines failed to express sarcomeric myosin heavy chain (MHC) protein during differentiation. The fusion index was significantly higher in control cells than in shCSN3-Med cells, whereas shCSN3-Low cells showed no cell fusion. Interestingly, CSN3 knockdown cells exhibited a significantly slower growth rate relative to the control cells. Cell cycle analysis revealed that CSN3 knockdowns delayed in S phase and had increased levels of nuclear p21/Cip1 and p27/Kip1. CONCLUSIONS: This study clarifies the first step toward unrevealing the CSN3/CSN-mediated pathways that controls C2C12 differentiation and proliferation. Further in vivo characterization of CSN/CSN3 may lead to the discovery of novel therapeutic target of skeletal muscle diseases such as muscular dystrophies.

Inhibition of Aß42 oligomerization in yeast by a PICALM ortholog and certain FDA approved drugs.

The formation of small Aß42 oligomers has been implicated as a toxic species in Alzheimer disease (AD). In strong support of this hypothesis we found that overexpression of Yap1802, the yeast ortholog of the human AD risk factor, phosphatidylinositol binding clathrin assembly protein (PICALM), reduced oligomerization of Aß42 fused to a reporter in yeast. Thus we used the Aß42-reporter system to identify drugs that could be developed into therapies that prevent or arrest AD. From a screen of 1,200 FDA approved drugs and drug-like small compounds we identified 7 drugs that reduce Aß42 oligomerization in yeast: 3 antipsychotics (bromperidol, haloperidol and azaperone), 2 anesthetics (pramoxine HCl and dyclonine HCl), tamoxifen citrate, and minocycline HCl. Also, all 7 drugs caused Aß42 to be less toxic to PC12 cells and to relieve toxicity of another yeast AD model in which Aß42 aggregates targeted to the secretory pathway are toxic. Our results identify drugs that inhibit Aß42 oligomers from forming in yeast. It remains to be determined if these drugs inhibit Aß42 oligomerization in mammals and could be developed as a therapeutic treatment for AD.

Epigenetic targets for novel therapies of lung diseases.

In spite of substantial advances in defining the immunobiology and function of structural cells in lung diseases there is still insufficient knowledge to develop fundamentally new classes of drugs to treat many lung diseases. For example, there is a compelling need for new therapeutic approaches to address severe persistent asthma that is insensitive to inhaled corticosteroids. Although the prevalence of steroid-resistant asthma is 5-10%, severe asthmatics require a disproportionate level of health care spending and constitute a majority of fatal asthma episodes. None of the established drug therapies including long-acting beta agonists or inhaled corticosteroids reverse established airway remodeling. Obstructive airways remodeling in patients with chronic obstructive pulmonary disease (COPD), restrictive remodeling in idiopathic pulmonary fibrosis (IPF) and occlusive vascular remodeling in pulmonary hypertension are similarly unresponsive to current drug therapy. Therefore, drugs are needed to achieve long-acting suppression and reversal of pathological airway and vascular remodeling. Novel drug classes are emerging from advances in epigenetics. Novel mechanisms are emerging by which cells adapt to environmental cues, which include changes in DNA methylation, histone modifications and regulation of transcription and translation by noncoding RNAs. In this review we will summarize current epigenetic approaches being applied to preclinical drug development addressing important therapeutic challenges in lung diseases. These challenges are being addressed by advances in lung delivery of oligonucleotides and small molecules that modify the histone code, DNA methylation patterns and miRNA function.

HSP20 phosphorylation and airway smooth muscle relaxation.

HSP20 (HSPB6) is a small heat shock protein expressed in smooth muscles that is hypothesized to inhibit contraction when phosphorylated by cAMP-dependent protein kinase. To investigate this hypothesis in airway smooth muscle (ASM) we showed that HSP20 was constitutively expressed as well as being inducible in cultured hASM cells by treatment with 1 µM isoproterenol or 10 µM salmeterol. In contrast, a mixture of proinflammatory mediators (interleukin-1ß, tumor necrosis factor α, and interferon γ) inhibited expression of HSP20 by about 50% in 48 hours. To determine whether phosphorylation of HSP20 is sufficient to induce relaxation, canine tracheal smooth muscle was treated with a cell permeant phosphopeptide that mimics the phosphorylation of HSP20. The HSP20 phosphopeptide antagonized carbachol-induced contraction by 60% with no change in myosin light chain phosphorylation. Recombinant full length HSP20 inhibited skeletal actin binding to smooth muscle myosin subfragment 1 (S1), and recombinant cell permeant TAT-HSP20 S16D mutant reduced F-actin filaments in cultured hASM cells. Carbachol stimulation of canine tracheal smooth muscle tissue caused redistribution of HSP20 from large macromolecular complexes (200-500 kDa) to smaller complexes (<60 kDa). The results are consistent with HSP20 expression and macromolecular structure being dynamically regulated in airway smooth muscle. HSP20 is upregulated by beta agonists and downregulated by proinflammatory cytokines. HSP20 is phosphorylated in vivo in a cAMP-dependent manner and the phosphorylated form promotes airway smooth muscle relaxation, possibly through depolymerization of F-actin as well as inhibition of myosin binding to actin.

Overexpression of human Hsp27 inhibits serum-induced proliferation in airway smooth muscle myocytes and confers resistance to hydrogen peroxide cytotoxicity.

Airway smooth muscle (ASM) hypertrophy and hyperplasia are characteristics of asthma that lead to thickening of the airway wall and obstruction of airflow. Very little is known about mechanisms underlying ASM remodeling, but in vascular smooth muscle, it is known that progression of atherosclerosis depends on the balance of myocyte proliferation and cell death. Small heat shock protein 27 (Hsp27) is antiapoptotic in nonmuscle cells, but its role in ASM cell survival is unknown. Our hypothesis was that phosphorylation of Hsp27 may regulate airway remodeling by modifying proliferation, cell survival, or both. To test this hypothesis, adenoviral vectors were used to overexpress human Hsp27 in ASM cells. Cells were infected with empty vector (Ad5) or wild-type Hsp27 (AdHsp27 WT), and proliferation and death were assessed. Overexpressing Hsp27 WT caused a 50% reduction in serum-induced proliferation and increased cell survival after exposure to 100 microM hydrogen peroxide (H(2)O(2)) compared with mock-infected controls. Overexpression studies utilizing an S15A, S78A, and S82A non-phosphorylation mutant (AdHsp27 3A) and an S15D, S78D, and S82D pseudo-phosphorylation mutant (AdHsp27 3D) showed phosphorylation of Hsp27 was necessary for regulation of ASM proliferation, but not survival. Hsp27 provided protection against H(2)O(2)-induced cytotoxicity by upregulating cellular glutathione levels and preventing necrotic cell death, but not apoptotic cell death. The results support the notion that ASM cells can be stimulated to undergo proliferation and death and that Hsp27 may regulate these processes, thereby contributing to airway remodeling in asthmatics.