Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.793180
Title: Expression and activity of WNT1-inducible signalling protein-1 in idiopathic pulmonary fibrosis
Author: Ambler, Lyndsy Jane
ISNI:       0000 0004 8501 7054
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2016
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Abstract:
Background: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease characterised by excessive extracellular matrix (ECM) deposition and disruption of lung architecture. Wnt1-inducible signalling protein1 (WISP-1) is a matricellular protein reported to play a role in aberrant epithelialmesenchymal crosstalk in fibrotic disease. Therefore, it was hypothesised that WISP-1 contributes to pro-fibrotic changes in pulmonary fibroblasts and epithelial cells, and that these effects are mediated by alternatively spliced WISP-1 variants. Methods: MRC5, A549 and primary parenchymal fibroblasts were stimulated with the pro-fibrotic cytokine TGFβ1 or the pro-inflammatory cytokine TNFα and mRNA expression of full length (FL) WISP-1 and its splice variants (v2/3/4) was measured. Antibodies were characterised for the detection of WISP-1 protein. Expression constructs for FL WISP-1 and v2, v3 and v4 were made in pcDNA3. HEK293T cells were transfected with WISP-1 constructs and cell conditioned medium (CM) tested on A549 and MRC5 cells. A549 cells were stably transfected with FL WISP-1. Time lapse microscopy, gene expression and cell proliferation analyses were performed. Results: TNFα induced WISP-1 mRNA expression in pulmonary epithelial (A549) and fibroblast (MRC5) cell lines. TGFβ1 induced epithelial to mesenchymal transition (EMT) and WISP-1 mRNA expression in A549 cells, and suppressed WISP-1 in MRC5 cells. In primary IPF fibroblasts, higher levels of WISP-1 mRNA were detected compared to control lung fibroblasts. Expression of WISP-1 mRNA in response to TGFβ1 and TNFα was variable between donors. WISP-1 variant specific qPCR assays showed differences in expression levels between donors, reflecting the overall WISP-1 expression. FL WISP-1 was detected in cell lysates from some IPF and control parenchymal fibroblast donors. When expressed in HEK293T cells, FL WISP-1 and v2, v3 and v4 could be detected in cell lysates. FL WISP-1 and v2 were also detected as secreted proteins. When CM from the different recombinant WISP-1 expressing HEK293T cells were applied to A549 or MRC5 cells, distinct responses were observed. FL WISP-1 CM increased endogenous FL WISP-1 expression in A549 cells, but the WISP-1 variants were without effect. In contrast, WISP-1v4 CM strongly induced FL WISP-1 and WISP-1v4 in MRC5 fibroblasts while WISP-1v2 and v3 only induced their own expression and FL WISP-1 had no effect. FL and variant WISP-1 CM did not stimulate ECM production by MRC5 cells, or EMT in A549 cells. Conclusions: WISP-1 is expressed in parenchymal fibroblasts and exists as several alternatively spliced forms. WISP-1 is overexpressed in IPF fibroblasts but is not uniformly increased following pro-fibrotic or pro-inflammatory stimulation in primary fibroblasts. WISP-1 expression is increased following induction of EMT in A549 cells however overexpression of WISP-1 did not induce EMT. The differential responses of epithelial cells and fibroblasts to CM from recombinant HEK293T cells expressing different WISP-1 variants suggests that further work is required to dissect the regulation and function of WISP-1 in relation to lung fibrosis.
Supervisor: Davies, Donna ; Collins, Jane ; O'Reilly, Karen Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.793180  DOI: Not available
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