Tatler, Amanda Louise
Mechanisms of transforming growth factor-β activation in airway smooth muscle cells and its role in asthma.
PhD thesis, University of Nottingham.
Asthma is a chronic inflammatory disease of the airways characterised by airway hyper-responsiveness (AHR), inflammation of the airways and reversible airway obstruction. Airway remodelling is a feature of asthma, especially in cases of severe and fatal asthma, and includes structural changes such as increased airway smooth muscle (ASM) mass, mucous gland hyperplasia, subepithelial fibrosis and angiogenesis. TGF-β is a pleiotropic cytokine that has been implicated in the development of many of these changes. However, TGF-β is released from cells in a latent complex, associated with its pro-peptide the latency associated peptide (LAP). Extracellular activation of latent TGF-β is the rate limiting step in TGF-β bioavailability. Although TGF-β activation has been investigated in airway epithelial cells, to date, no studies have investigated TGF-β activation by airway smooth muscle cells. The hypothesis of this thesis is therefore that human airway smooth muscle cells can activate TGF-β in vitro.
The hypothesis of this thesis has been tested by investigating effects of the serine protease mast cell tryptase, mechanical wounding of cell mono layers and the phospholipid lysophosphatidic acid (LPA) on TGF-β activation by primary airway smooth muscle cells in vitro. We have utilised transformed mink lung epithelial cells, a reporter cell that express a TGF-β responsive promoter driving a luciferase gene, and quantitative PCR for the TGF-β-inducible gene plasminogen activator inhibitor-1 (PAI1) to investigate TGF-β activation. Moreover, we show for the first time that TGF-β activation can be assessed in vitro by detecting the translocation of Smad 2 and 3 from the cytoplasm to the nucleus by western blotting.
The results presented in this thesis provide evidence that airway smooth muscle cells are capable of activating TGF-β in vitro. These data show that the serine protease tryptase, released from activated mast cells, can proteolytically activate TGF-β via a mechanism that is independent of the tryptase receptor protease activated receptor-2 (PAR2). This effect is not accompanied by increased expression of the latent TGF-β complex. Furthermore, these data provide evidence that airway smooth muscle cells can activate TGF-β via the integrin αβV5 in response to LPA stimulation. We have found that cells from asthmatic patients activate more TGF-β in response to LPA than cells from non-asthmatic individuals and this is not due to a difference in cell surface expression levels of the αβV5 integrin. LPA-induced TGF-β activation can be inhibited by the β2 adrenoreceptor agonist formoterol, which is a commonly used asthma therapy, and the muscarinic receptor agonist methacholine, which causes cell contraction, also causes TGF-β activation by airway smooth muscle cells. Furthermore, the data presented here show that the cytoplasmic domain of the integrin β5 subunit interacts with the cytoskeletal protein talin to mediate TGF-β activation.
Together, these data highlight two previously unreported, biologically relevant, mechanisms of TGF-β activation employed by airway smooth muscle cells in vitro, both of which could contribute to the development of airway remodelling in asthma in vivo. Data concerning a αβV5 mediated TGF-β activation has led us to hypothesise that contraction of airway smooth muscle leads to TGF-β activation in vivo. If correct, this could be vital to our understanding of how airway remodelling is initiated in asthma, and could lead to the development of new therapies aimed at inhibiting contraction-induced TGF-β activation, for the treatment of asthma.
Thesis (University of Nottingham only)
||Transforming growth factors-beta, Smooth muscle cells, Serine protease tryptase, Phospholipid lysophosphatidic acid, Mast cells
||W Medicine and related subjects (NLM Classification) > WF Respiratory system
||UK Campuses > Faculty of Medicine and Health Sciences > School of Clinical Sciences
||07 Oct 2013 09:47
||13 Sep 2016 15:04
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