Molecular changes during TGFβ-mediated lung fibroblast-myofibroblast differentiation: implication for glucocorticoid resistance

Airway remodeling is an important process in response to repetitive inflammatory-mediated airway wall injuries. This is characterized by profound changes and reorganizations at the cellular and molecular levels of the lung tissue. It is of particular importance to understand the mechanisms involved in airway remodeling, as this is strongly associated with severe asthma leading to devastating airway dysfunction. In this study, we have investigated the transforming growth factor-β (TGFβ, a proinflammatory mediator)-activated fibroblast to myofibroblast transdifferentiation pathway, which plays a key role in asthma-related airway remodeling. We show that TGFβ induces fibroblast to myofibroblast transdifferentiation by the expression of αSMA, a specific myofibroblast marker. Furthermore, Smad2/Smad3 gene and protein expression patterns are different between fibroblasts and myofibroblasts. Such a change in expression patterns reveals an important role of these proteins in the cellular phenotype as well as their regulation by TGFβ during cellular transdifferentiation. Interestingly, our data show a myofibroblastic TGFβ-mediated increase in glucocorticoid receptor (GR) expression and a preferential localization of GR in the nucleus, compared to in fibroblasts. Furthermore, the GRβ (nonfunctional GR isoform) is increased relative to GRα (functional isoform) in myofibroblasts. These results are interesting as they support the idea of a GRβ-mediated glucocorticoid resistance observed in the severe asthmatic population. All together, we provide evidence that key players are involved in the TGFβ-mediated fibroblast to myofibroblast transdifferentiation pathway in a human lung fibroblast cell line. These players could be the targets of new treatments to limit airway remodeling and reverse glucocorticoid resistance in severe asthma.This work is supported by the University of Canberra Strategic Research Funds (grant to R. Ghildyal, postdoctoral fellowship to D. Heydet) and an Early Career Grant from Centre for Research in Therapeutic Solutions (to D. Heydet)

Neuronal cilia and appetite regulation in Alms1 mutant mice

The foz/foz mouse is a murine model of Alstr{u00F6}m syndrome, a monogenetic disorder characterised in humans by childhood obesity, hearing loss, blindness, hyperinsulinaemia, early-onset type 2 diabetes and liver disease. In 2006, research from the host laboratory reported that foz/foz mice inherit a spontaneous mutation (foz) , an 11 base pair deletion in exon 8 of the Alms1 gene, and develop a similar phenotype to patients suffering from Alstr{u00F6}m syndrome. Thus, foz/foz mice are obese and exhibit high circulating insulin and leptin levels as well as fatty liver disease and metabolic syndrome. The purpose of the studies presented in this thesis was to further characterise the pathogenesis of obesity in foz/foz mice, by studying the role of Alms1 and hypothalamic appetite-regulating neuropeptide expression during the development of obesity. ALMS1 has been shown to localise at the base of primary cilia in what is termed the basal body or centrosome. Primary cilia are ubiquitously expressed hair-like organelles. Therefore, the first approach was to study primary cilia in the hypothalamus as well as hypothalamic Alms1 gene expression and Alms1 localisation in foz/foz and wildtype (WT) mice from birth until the obese phenotype is evident. At birth, foz/foz mice showed similar number of ciliated hypothalamic neurons to WT mice. However, from weaning and onwards the number of cilia was significantly decreased in foz/foz mice. In addition, while cilia were present in primary neuronal cultures from foz/foz and WT mice, Alms1 was only detected in WT neurons, appearing as two perinuclear dots at the base of cilia. After weaning, serum leptin levels become greatly elevated in foz/foz compared to WT mice. Leptin decreases appetite by acting in the hypothalamus and inducing or inhibiting the release of appetite-regulating neuropeptides. A detailed study of key hypothalamic neuropeptides demonstrated no differences in their gene and/or protein expression or localisation between foz/foz and WT mice. This failure of elevated leptin levels to decrease appetite and body weight is defined as leptin resistance. Further studies were therefore performed on hypothalamic leptin receptor (Ob-R) expression and signalling pathways to characterise leptin resistance in foz/foz mice. These results demonstrated induction of two proteins, SOCS3 and PTP1B, which negatively regulate Ob-R signalling and have been implicated in leptin resistance. Taken together, the data presented in this thesis strongly support the proposal that foz/foz mice develop leptin resistance, which correlates molecularly with over-expression of at least two negative feedback regulators of Ob-R. In addition, the post-natal reduction in ciliated hypothalamic neurons in foz/foz mice, in combination with the lack of Alms1 detection are consistent with the proposal that primary cilia stability/maintenance could be impaired as a consequence of the Alms1 mutation. In conclusion, foz/foz mice provide new opportunities for studying the role of Alms1 and neuronal cilia in appetite regulation, particularly with respect to the onset of leptin resistance. A better understanding of Alms1, cilial stability and behavioural responses that underlie obesity could provide clues to novel therapeutic approaches to combat more common forms of obesity

A fresh look at NASH pathogenesis. Part 1: The metabolic movers

The strong relationship between over-nutrition, central obesity, insulin resistance/metabolic syndrome and non-alcoholic fatty liver disease (NAFLD) suggest pathogenic interactions, but key questions remain. NAFLD starts with over-nutrition, imbalance between energy input and output for which the roles of genetic predisposition and environmental factors (diet, physical activity) are being redefined. Regulation of energy balance operates at both central nervous system and peripheral sites, including adipose and liver. For example, the endocannabinoid system could potentially be modulated to provide effective pharmacotherapy of NAFLD. The more profound the metabolic abnormalities complicating over-nutrition (glucose intolerance, hypoadiponectinemia, metabolic syndrome), the more likely is NAFLD to take on its progressive guise of non-alcoholic steatohepatitis (NASH). Interactions between steatosis and insulin resistance, visceral adipose expansion and subcutaneous adipose failure (with insulin resistance, inflammation and hypoadiponectinemia) trigger amplifying mechanisms for liver disease. Thus, transition from simple steatosis to NASH could be explained by unmitigated hepatic lipid partitioning with failure of local adaptive mechanisms leading to lipotoxicity. In part one of this review, we discuss newer concepts of appetite and metabolic regulation, bodily lipid distribution, hepatic lipid turnover, insulin resistance and adipose failure affecting adiponectin secretion. We review evidence that NASH only occurs when over-nutrition is complicated by insulin resistance and a highly disordered metabolic milieu, the same 'metabolic movers' that promote type 2 diabetes and atheromatous cardiovascular disease. The net effect is accumulation of lipid molecules in the liver. Which lipids and how they cause injury, inflammation and fibrosis will be discussed in part two

Nucleocytoplasmic transport of nucleocapsid proteins of enveloped RNA viruses

Most viruses with non-segmented single stranded RNA genomes complete their life cycle in the cytoplasm of infected cells. However, despite undergoing replication in the cytoplasm, the structural proteins of some of these RNA viruses localize to the nucleus at specific times in the virus life cycle, primarily early in infection. Limited evidence suggests that this enhances successful viral replication by interfering with or inhibiting the host antiviral response. Nucleocapsid proteins of RNA viruses have a well-established, essential cytoplasmic role in virus replication and assembly. Intriguingly, nucleocapsid proteins of some RNA viruses also localize to the nucleus/nucleolus of infected cells. Their nuclear function is less well understood although significant advances have been made in recent years. This review will focus on the nucleocapsid protein of cytoplasmic enveloped RNA viruses, including their localization to the nucleus/nucleolus and function therein. A greater understanding of the nuclear localization of nucleocapsid proteins has the potential to enhance therapeutic strategies as it can be a target for the development of live-attenuated vaccines or antiviral drugs

A truncating mutation of Alms1 reduces the number of hypothalamic neuronal cilia in obese mice

Primary cilia are ubiquitous cellular antennae whose dysfunction collectively causes various disorders, including vision and hearing impairment, as well as renal, skeletal, and central nervous system anomalies. One ciliopathy, Alström syndrome, is close