Paul A. Whittaker

RNA interference: from gene silencing to gene-specific therapeutics

Abstract In the past 4 years, RNA interference (RNAi) has become widely used as an experimental tool to analyse the function of mammalian genes, both in vitro and in vivo. By harnessing an evolutionary conserved endogenous biological pathway, first identified in plants and lower organisms, double-stranded RNA (dsRNA) reagents are used to bind to and promote the degradation of target RNAs, resulting in knockdown of the expression of specific genes. RNAi can be induced in mammalian cells by the introduction of synthetic double-stranded small interfering RNAs (siRNAs) 21–23 base pairs (bp) in length or by plasmid and viral vector systems that express double-stranded short hairpin RNAs (shRNAs) that are subsequently processed to siRNAs by the cellular machinery. RNAi has been widely used in mammalian cells to define the functional roles of individual genes, particularly in disease. In addition, siRNA and shRNA libraries have been developed to allow the systematic analysis of genes required for disease processes such as cancer using high throughput RNAi screens. RNAi has been used for the knockdown of gene expression in experimental animals, with the development of shRNA systems that allow tissue-specific and inducible knockdown of genes promising to provide a quicker and cheaper way to generate transgenic animals than conventional approaches. Finally, because of the ability of RNAi to silence disease-associated genes in tissue culture and animal models, the development of RNAi-based reagents for clinical applications is gathering pace, as technological enhancements that improve siRNA stability and delivery in vivo, while minimising off-target and nonspecific effects, are developed.

Curcumin Restores Corticosteroid Function in Monocytes Exposed to Oxidants by Maintaining HDAC2

Oxidative stress as a result of cigarette smoking is an important etiologic factor in the pathogenesis of chronic obstructive pulmonary disease (COPD), a chronic steroid-insensitive inflammatory disease of the airways. Histone deacetylase-2 (HDAC2), a critical component of the corticosteroid anti-inflammatory action, is impaired in lungs of patients with COPD and correlates with disease severity. We demonstrate here that curcumin (diferuloylmethane), a dietary polyphenol, at nanomolar concentrations specifically restores cigarette smoke extract (CSE)- or oxidative stress-impaired HDAC2 activity and corticosteroid efficacy in vitro with an EC(50) of approximately 30 nM and 200 nM, respectively. CSE caused a reduction in HDAC2 protein expression that was restored by curcumin. This decrease in HDAC2 protein expression was reversed by curcumin even in the presence of cycloheximide, a protein synthesis inhibitor. The proteasomal inhibitor, MG132, also blocked CSE-induced HDAC2 degradation, increasing the levels of ubiquitinated HDAC2. Biochemical and gene chip analysis indicated that curcumin at concentrations up to 1 muM propagates its effect via antioxidant-independent mechanisms associated with the phosphorylation-ubiquitin-proteasome pathway. Thus curcumin acts at a post-translational level by maintaining both HDAC2 activity and expression, thereby reversing steroid insensitivity induced by either CSE or oxidative stress in monocytes. Curcumin may therefore have potential to reverse steroid resistance, which is common in patients with COPD and asthma.

PPAR‐α and ‐γ but not ‐δ agonists inhibit airway inflammation in a murine model of asthma: in vitro evidence for an NF‐κB‐independent effect

Peroxisome proliferator‐activated receptors (PPARs) are ligand‐activated transcription factors that have been proposed to regulate inflammation by antagonising the nuclear factor‐κB (NF‐κB) signalling pathway. We investigated the role of PPARs using synthetic agonists in murine models of airway inflammation, and addressed the possible effect on NF‐κB signalling in vitro using a human epithelial cell line, A549. Sensitised BALB/c mice exposed to an aerosol solution of ovalbumin had an increased number of airway eosinophils, neutrophils and lymphocytes. When given intranasally an hour before the aerosol challenge, a PPAR‐α (GW 9578) and PPAR‐γ (GI 262570) selective agonist as well as a dual PPAR‐α/γ (GW 2331) agonist selectively inhibited allergen‐induced bronchoalveolar lavage eosinophil and lymphocyte but not neutrophil influx. In contrast, a PPAR‐δ agonist (GW 501516) was inactive. When given intranasally an hour before challenge, PPAR‐α and PPAR‐γ selective agonists as well as a dual PPAR‐α/γ agonist did not inhibit lipopolysaccharide‐induced bronchoalveolar lavage neutrophil influx or tumour necrosis factor‐α (TNF‐α) and KC production. In A549 cells, selective agonists for PPAR‐α, ‐γ and ‐δ did not inhibit intracellular adhesion molecule‐1 expression following stimulation with proinflammatory cytokines. In addition, IL‐8 release and the activation of an NF‐κB‐responsive reporter gene construct were inhibited only at micromolar concentrations, suggesting that these effects were not PPAR‐mediated. Our in vivo data show that agonists of PPAR‐α and ‐γ, but not ‐δ, inhibit allergen‐induced bronchoalveolar lavage eosinophil and lymphocyte influx. In vitro data suggest that this effect might not be mediated by antagonism of the NF‐κB pathway.

Identification of PCDH1 as a novel susceptibility gene for bronchial hyperresponsiveness.

RATIONALE Asthma is a chronic inflammatory airway disease that affects more than 300 million individuals worldwide. Asthma is caused by interaction of genetic and environmental factors. Bronchial hyperresponsiveness (BHR) is a hallmark of asthma and results from increased sensitivity of the airways to physical or chemical stimulants. BHR and asthma are linked to chromosome 5q31-q33. OBJECTIVES To identify a gene for BHR on chromosome 5q31-q33. METHODS In 200 Dutch families with asthma, linkage analysis and fine mapping were performed, and the Protocadherin 1 gene (PCDH1) was identified. PCDH1 was resequenced in 96 subjects from ethnically diverse populations to identify novel sequence variants. Subsequent replication studies were undertaken in seven populations from The Netherlands, the United Kingdom, and the United States, including two general population samples, two family samples, and three case-control samples. PCDH1 mRNA and protein expression was investigated using polymerase chain reaction, Western blotting, and immunohistochemistry. MEASUREMENTS AND MAIN RESULTS In seven out of eight populations (n = 6,168) from The Netherlands, United Kingdom, and United States, PCHD1 gene variants were significantly associated with BHR (P values, 0.005-0.05) This association was present in both families with asthma and general populations. PCDH1 mRNA and protein were expressed in airway epithelial cells and in macrophages. CONCLUSIONS PCDH1 is a novel gene for BHR in adults and children. The identification of PCDH1 as a BHR susceptibility gene may suggest that a structural defect in the integrity of the airway epithelium, the first line of defense against inhaled substances, contributes to the development of BHR.

Oxidative stress modulates theophylline effects on steroid responsiveness

Oxidative stress is a central factor in many chronic inflammatory diseases such as severe asthma and chronic obstructive pulmonary disease (COPD). Oxidative stress reduces the anti-inflammatory corticosteroid action and may therefore contribute to the relative corticosteroid insensitivity seen in these diseases. Low concentrations of theophylline can restore the anti-inflammatory action of corticosteroids in oxidant exposed cells, however the mechanism remains unknown. Here, we demonstrate that a low concentration of theophylline restores corticosteroid repression of pro-inflammatory mediator release and histone acetylation in oxidant exposed cells. Global gene expression analysis shows that theophylline regulates distinct pathways in naïve and oxidant exposed cells and reverses oxidant mediated modulated of pathways. Furthermore, quantitative chemoproteomics revealed that theophylline has few high affinity targets in naive cells but an elevated affinity in oxidant stressed cells. In conclusion, oxidative stress alters theophylline binding profile and gene expression which may result in restoration of corticosteroid function.

Genes for asthma: much ado about nothing?

The identification of genes that make individuals susceptible to developing asthma is an area of increasing focus by both academic and industrial groups. The complexity of asthma genetics has made the identification of susceptibility genes difficult; however, genome-wide screens followed by positional cloning have started to identify such genes for both asthma and asthma-related traits. In addition, evidence from candidate gene studies indicates that gene-gene interactions also play an important role in conferring genetic risk for asthma development. Although progress in terms of identifying new therapeutic targets has not been dramatic, our understanding of the complexity of the genetic basis of asthma has greatly increased. There can be little doubt that, in the future, this knowledge will lead to fundamental insights into the molecular pathophysiology of asthma and the development of new therapies.

The role of bioinformatics in target validation

Bioinformatics is being increasingly used to support target validation by providing functionally predictive information mined from databases and experimental datasets using a variety of computational tools. The predictive power of these complementary approaches is strongest when information from several techniques is combined, including experimental confirmation of predictions. The aim of this review is to highlight and discuss the key approaches available in this rapidly developing area to facilitate selection of the appropriate tools and databases.:

From symptomatic treatments to causative therapy

The search for genes that predispose individuals to develop common chronic diseases such as asthma, diabetes and Alzheimers promises to give insights into their molecular pathogenesis. This will lead to the development of therapies that modulate the pathology, rather than the physiology of these diseases. As academia and the pharmaceutical industry increasingly focus on this challenge, the genetic dissection of Alzheimers is spearheading attempts to shift the therapeutic paradigm away from symptomatic to curative treatments.

Can pharmacology possibly have a role for bioinformatics

In today’s information-driven culture, there is virtually no walk of life that is not impacted on by computing. As a bridging discipline in the health sciences with activities that span both basic science and clinical interests, modern pharmacology is no exception. As the plethora of data and databases spawned by the ‘omics’ generation expand in number and complexity, bioinformatics is necessary to manage, integrate and exploit this cohort of data so that the appropriate links to molecular pathology and therapeutic response can be made. Bioinformatics is now an integral part of drug discovery and development. This article reviews the use of bioinformatics in this process, from target identification and validation, to pharmacogenomics, toxicogenomics and systems biology.