Andrew J. Halayko

Apoptosis and cancer: mutations within caspase genes

The inactivation of programmed cell death has profound effects not only on the development but also on the overall integrity of multicellular organisms. Beside developmental abnormalities, it may lead to tumorigenesis, autoimmunity, and other serious health problems. Deregulated apoptosis may also be the leading cause of cancer therapy chemoresistance. Caspase family of cysteinyl-proteases plays the key role in the initiation and execution of programmed cell death. This review gives an overview of the role of caspases, their natural modulators like IAPs, FLIPs, and Smac/Diablo in apoptosis and upon inactivation, and also in cancer development. Besides describing the basic mechanisms governing programmed cell death, a large part of this review is dedicated to previous studies that were focused on screening tumours for mutations within caspase genes as well as their regulators. The last part of this review discusses several emerging treatments that involve modulation of caspases and their regulators. Thus, we also highlight caspase cascade modulating experimental anticancer drugs like cFLIP-antagonist CDDO-Me; cIAP1 antagonists OSU-03012 and ME-BS; and XIAP small molecule antagonists 1396–11, 1396–12, 1396–28, triptolide, AEG35156, survivin/Hsp90 antagonist shephedrin, and some of the direct activators of procaspase-3.

Increased Expression of IL-33 in Severe Asthma: Evidence of Expression by Airway Smooth Muscle Cells

IL-33, a new member of the IL-1 cytokine family, promotes Th2 inflammation, but evidence on the implications of this cytokine in asthma is lacking. IL-33 would be mainly expressed by structural cells, but whether proinflammatory cytokines modulate its expression in airway smooth muscle cells (ASMC) is unknown. Endobronchial biopsies were obtained from adults with mild (n = 8), moderate (n = 8), severe (n = 9), asthma and from control subjects (n = 5). Immunocytochemistry, laser-capture microdissection, reverse transcriptase, and real-time quantitative PCR were used for determining IL-33 expression in the lung tissues. ASMC isolated from resected lung specimens were cultured with proinflammatory cytokines and with dexamethasone. IL-33 expression by ASMC was determined by PCR, ELISA, and Western blotting. Higher levels of IL-33 transcripts are detected in biopsies from asthmatic compared with control subjects, and especially in subjects with severe asthma. ASMC show IL-33 expression at both protein and mRNA levels. IL-33 and TNF-α transcript levels correlate in the lung tissues, and TNF-α up-regulates IL-33 expression by cultured ASMC in a time- and dose-dependent manner. IFN-γ also increases IL-33 expression and shows synergistic effect with TNF-α. Dexamethasone fails to abolish TNF-α-induced IL-33 up-regulation. IL-33 expression increases in bronchial biopsies from subjects with asthma compared with controls, as well as subjects with asthma severity. ASMC are a source of the IL-33 cytokine. Our data propose IL-33 as a novel inflammatory marker of severe and refractory asthma.

Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma

Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma. As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling. Anti-inflammatory therapy, however, does not “cure” asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM. In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored.

Muscarinic receptor signaling in the pathophysiology of asthma and COPD

Anticholinergics are widely used for the treatment of COPD, and to a lesser extent for asthma. Primarily used as bronchodilators, they reverse the action of vagally derived acetylcholine on airway smooth muscle contraction. Recent novel studies suggest that the effects of anticholinergics likely extend far beyond inducing bronchodilation, as the novel anticholinergic drug tiotropium bromide can effectively inhibit accelerated decline of lung function in COPD patients. Vagal tone is increased in airway inflammation associated with asthma and COPD; this results from exaggerated acetylcholine release and enhanced expression of downstream signaling components in airway smooth muscle. Vagally derived acetylcholine also regulates mucus production in the airways. A number of recent research papers also indicate that acetylcholine, acting through muscarinic receptors, may in part regulate pathological changes associated with airway remodeling. Muscarinic receptor signalling regulates airway smooth muscle thickening and differentiation, both in vitro and in vivo. Furthermore, acetylcholine and its synthesizing enzyme, choline acetyl transferase (ChAT), are ubiquitously expressed throughout the airways. Most notably epithelial cells and inflammatory cells generate acetylcholine, and express functional muscarinic receptors. Interestingly, recent work indicates the expression and function of muscarinic receptors on neutrophils is increased in COPD. Considering the potential broad role for endogenous acetylcholine in airway biology, this review summarizes established and novel aspects of muscarinic receptor signaling in relation to the pathophysiology and treatment of asthma and COPD.

S100A8/A9 induces autophagy and apoptosis via ROS-mediated cross-talk between mitochondria and lysosomes that involves BNIP3

The complex formed by two members of the S100 calcium-binding protein family, S100A8/A9, exerts apoptosis-inducing activity in various cells of different origins. Here, we present evidence that the underlying molecular mechanisms involve both programmed cell death I (PCD I, apoptosis) and PCD II (autophagy)-like death. Treatment of cells with S100A8/A9 caused the increase of Beclin-1 expression as well as Atg12-Atg5 formation. S100A8/A9-induced cell death was partially inhibited by the specific PI3-kinase class III inhibitor, 3-methyladenine (3-MA), and by the vacuole H+-ATPase inhibitor, bafilomycin-A1 (Baf-A1). S100A8/A9 provoked the translocation of BNIP3, a BH3 only pro-apoptotic Bcl2 family member, to mitochondria. Consistent with this finding, ΔTM-BNIP3 overexpression partially inhibited S100A8/A9-induced cell death, decreased reactive oxygen species (ROS) generation, and partially protected against the decrease in mitochondrial transmembrane potential in S100A8/A9-treated cells. In addition, either ΔTM-BNIP3 overexpression or N-acetyl-L-cysteine co-treatment decreased lysosomal activation in cells treated with S100A8/A9. Our data indicate that S100A8/A9-promoted cell death occurs through the cross-talk of mitochondria and lysosomes via ROS and the process involves BNIP3.

Mechanisms of inflammation-mediated airway smooth muscle plasticity and airways remodeling in asthma.

Recent evidence points to progressive structural change in the airway wall, driven by chronic local inflammation, as a fundamental component for development of irreversible airway hyperresponsiveness. Acute and chronic inflammation is orchestrated by cytokines from recruited inflammatory cells, airway myofibroblasts and myocytes. Airway myocytes exhibit functional plasticity in their capacity for contraction, proliferation, and synthesis of matrix protein and cytokines. This confers a principal role in driving different components of the airway remodeling process, and mediating constrictor hyperresponsiveness. Functional plasticity of airway smooth muscle (ASM) is regulated by an array of environmental cues, including cytokines, which mediate their effects through receptors and a number of intracellular signaling pathways. Despite numerous studies of the cellular effects of cytokines on cultured airway myocytes, few have identified how intracellular signaling pathways modulate or induce these cellular responses. This review summarizes current understanding of these concepts and presents a model for the effects of inflammatory mediators on functional plasticity of ASM in asthma.

MicroRNA expression in human airway smooth muscle cells: role of miR-25 in regulation of airway smooth muscle phenotype.

Defining mechanisms by which differentiated, contractile smooth muscle cells become proliferative and secretory in response to mechanical and environmental stress is crucial for determining the contribution of airway smooth muscle (ASM) to inflammatory responses that result in airway disease. Regulation by microRNAs (miRNAs) has emerged as an important post-transcriptional mechanism regulating gene expression that may modulate ASM phenotype, but little is known about the expression and functions of miRNA in smooth muscle. In the present study we used microarrays to determine whether miRNAs in human ASM cells are altered by a proinflammatory stimulus. In ASM cells exposed to IL-1beta, TNF-alpha, and IFN-gamma, we found 11 miRNAs to be significantly down-regulated. We verified decreased expression of miR-25, miR-140*, mir-188, and miR-320 by quantitative PCR. Analysis of miR-25 expression indicates that it has a broad role in regulating ASM phenotype by modulating expression of inflammatory mediators such as RANTES, eotaxin, and TNF-alpha; genes involved in extracellular matrix turnover; and contractile proteins, most notably myosin heavy chain. miRNA binding algorithms predict that miR-25 targets Krüppel-like factor 4 (KLF4), a potent inhibitor of smooth muscle-specific gene expression and mediator of inflammation. Our study demonstrates that inhibition of miR-25 in cytokine-stimulated ASM cells up-regulates KLF4 expression via a post-transcriptional mechanism. This provides novel evidence that miR-25 targets KLF4 in ASM cells and proposes that miR-25 may be an important mediator of ASM phenotype.

IL-17A Induces Eotaxin-1/CC Chemokine Ligand 11 Expression in Human Airway Smooth Muscle Cells: Role of MAPK (Erk1/2, JNK, and p38) Pathways

Recently, IL-17A has been shown to be expressed in higher levels in respiratory secretions from asthmatics and correlated with airway hyperresponsiveness. Although these studies raise the possibility that IL-17A may influence allergic disease, the mechanisms remain unknown. In this study, we investigated the molecular mechanisms involved in IL-17A-mediated CC chemokine (eotaxin-1/CCL11) production from human airway smooth muscle (ASM) cells. We found that incubation of human ASM cells with rIL-17A resulted in a significant increase of eotaxin-1/CCL11 release from ASM cells that was reduced by neutralizing anti-IL-17A mAb. Moreover, IL-17A significantly induced eotaxin-1/CCL11 release and mRNA expression, an effect that was abrogated with cycloheximide and actinomycin D treatment. Furthermore, transfection studies using a luciferase-driven reporter construct containing eotaxin-1/CCL11 proximal promoter showed that IL-17A induced eotaxin-1/CCL11 at the transcriptional level. IL-17A also enhanced significantly IL-1β-mediated eotaxin-1/CCL11 mRNA, protein release, and promoter activity in ASM cells. Primary human ASM cells pretreated with inhibitors of MAPK p38, p42/p44 ERK, JNK, or JAK but not PI3K, showed a significant decrease in eotaxin-1/CCL11 release upon IL-17A treatment. In addition, IL-17A mediated rapid phosphorylation of MAPK (p38, JNK, and p42/44 ERK) and STAT-3 but not STAT-6 or STAT-5 in ASM cells. Taken together, our data provide the first evidence of IL-17A-induced eotaxin-1/CCL11 expression in ASM cells via MAPK (p38, p42/p44 ERK, JNK) signaling pathways. Our results raise the possibility that IL-17A may play a role in allergic asthma by inducing eotaxin-1/CCL11 production.

Brevinin-2R1 semi-selectively kills cancer cells by a distinct mechanism, which involves the lysosomal-mitochondrial death pathway

Brevinin‐2R is a novel non‐hemolytic defensin that was isolated from the skin of the frog Rana ridibunda. It exhibits preferential cytotoxicity towards malignant cells, including Jurkat (T‐cell leukemia), BJAB (B‐cell lymphoma), HT29/219, SW742 (colon carcinomas), L929 (fibrosarcoma), MCF‐7 (breast adenocarcinoma), A549 (lung carcinoma), as compared to primary cells including peripheral blood mononuclear cells (PBMC), T cells and human lung fibroblasts. Jurkat and MCF‐7 cells overexpressing Bcl2, and L929 and MCF‐7 over‐expressing a dominant‐negative mutant of a pro‐apoptotic BNIP3 (ΔTM‐BNIP3) were largely resistant towards Brevinin‐2R treatment. The decrease in mitochondrial membrane potential (ΔΨm), or total cellular ATP levels, and increased reactive oxygen species (ROS) production, but not caspase activation or the release of apoptosis‐inducing factor (AIF) or endonuclease G (Endo G), were early indicators of Brevinin‐2R‐triggered death. Brevinin‐2R interacts with both early and late endosomes. Lysosomal membrane permeabilization inhibitors and inhibitors of cathepsin‐B and cathepsin‐L prevented Brevinin‐2R‐induced cell death. Autophagosomes have been detected upon Brevinin‐2R treatment. Our results show that Brevinin‐2R activates the lysosomalmitochondrial death pathway, and involves autophagy‐like cell death.

Role of the phosphoinositide 3‐kinase p110δ in generation of type 2 cytokine responses and allergic airway inflammation

Phosphoinositide 3‐kinases (PI3K) regulate immune activation via their roles in signal transduction of multiple classes of receptors. Here, we examined the effect of genetic inactivation of the hemopoietic cell‐restricted PI3K isoform p110δ on systemic cytokine and chemokine responses and allergic airway inflammation. We found that type 2 cytokine responses (IL‐4, IL‐5 and IL‐13) are significantly decreased in p110δ mutants, whereas type 1 cytokine responses (IFN‐γ and CXCL10) were robust. Elevated IFN‐γ production during the primary response to ovalbumin (OVA) was associated with reduced production of the regulatory cytokine IL‐10. IFN‐γ and IL‐10 production normalized after secondary OVA immunization; however, type 2 cytokine production was persistently reduced. Type 2 cytokine‐dependent airway inflammation elicited by intranasal challenge with OVA was dramatically reduced, with reduced levels of eosinophil recruitment and mucus production observed in the lungs. Induction of respiratory hyper‐responsiveness to inhaled methacholine, a hallmark of asthma, was markedly attenuated in p110δ‐inactivated mice. Adoptive transfer of OVA‐primed splenocytes from normal but not p110δ‐inactivated mice could induce airway eosinophilia in naive, airway‐challenged recipient mice. These data demonstrate a novel functional role for p110δ signaling in induction of type 2 responses in vivo and may offer a new therapeutic target for Th2‐mediated airway disease.