Investigating the synergistic signalling mechanisms underlying the development of obliterative bronchiolitis

Abstract

Therapies to limit or reverse fibrosis have thus far proved unsuccessful, highlighting the need for a greater understanding of the basic mechanisms driving fibrosis and in particular the link between fibrosis and inflammation. Obliterative Bronchiolitis (OB) is the pathological correlate of Bronchiolitis Obliterans Syndrome (BOS) a progressive disease that results in the fibrotic obliteration and blockage of the airways. Development of OB is strongly associated with elevated fibrotic, transforming growth factor beta 1 (TGF- β1), and inflammatory, tumour necrosis factor alpha (TNFα) and interleukin 1 beta, mediators; and the process of epithelial to mesenchymal transition (EMT) has been proposed as a mechanism of OB initiation and progression. Previous work in our group has demonstrated that a physiologically relevant dose of TGF-β1 is capable of driving EMT in primary human bronchial epithelial cells (PBEC) isolated from lung-transplant recipients, an effect that was accentuated by TNFα. It was hypothesized that an unknown synergistic signalling cascade may mediate this accentuation, and identifying candidates for this role is the main aim of this thesis. TGF-β1 and TNFα were used to induce EMT in PBEC cultures; the relative roles of several signalling proteins (SMAD3, IKKβ, TAK1, p38, ERK-1/2 and JNK-1/2) in the development of EMT were assessed by chemical inhibition and siRNA knockdown along with phosphorylation response, to describe a signalling cascade. The results describe a mechanism whereby TGF-β1 drives EMT through SMAD3 with a requirement for TAK1 and JNK-2. TNFα signals through TAK1, IKKβ and JNK-2 but is not capable of driving EMT alone. Upon co-stimulation, TAK1 and JNK-2 display an enhanced activation that leads to an accentuation of EMT, possibly through c-Jun activation. JNK-2 sits downstream of TAK1 therefore the results indicate that TAK1 activity plays a key role both modulating TGF-β1 SMAD3 driven EMT, and its accentuation by TNFα. TAK1 was also shown to be more strongly activated in fibrotic human airway sections compared to control, suggesting that the findings have direct disease relevance. Much further work into how TAK1 modulates SMAD3 activity after TGF-β1 stimulation and how its enhanced activation leads to accentuated EMT is required. Greater understanding of these mechanisms may lead to the discovery of novel therapeutic targets for fibrotic and inflammatory disorders.