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Expression and activity of WNT1 - inducible signalling protein-1 in idiopathic pulmonary fibrosis

Expression and activity of WNT1 - inducible signalling protein-1 in idiopathic pulmonary fibrosis
Expression and activity of WNT1 - inducible signalling protein-1 in idiopathic pulmonary fibrosis
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.
University of Southampton
Ambler, Lyndsy Jane
d4a56df9-fde6-4461-b7f1-244ed487274c
Ambler, Lyndsy Jane
d4a56df9-fde6-4461-b7f1-244ed487274c
Davies, Donna
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Collins, Jane
be0e66f1-3036-47fa-9d7e-914c48710ba4
O'reilly, Karen
a0812526-81b1-4cc3-b1df-eab66e9e1abe

Ambler, Lyndsy Jane (2016) Expression and activity of WNT1 - inducible signalling protein-1 in idiopathic pulmonary fibrosis. University of Southampton, Doctoral Thesis, 210pp.

Record type: Thesis (Doctoral)

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.

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Available under License University of Southampton Thesis Licence.

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Published date: November 2016

Identifiers

Local EPrints ID: 434982
URI: https://eprints.soton.ac.uk/id/eprint/434982
PURE UUID: c0bc2f76-d20a-4e5a-b8ec-fc0d5ec4d3af
ORCID for Donna Davies: ORCID iD orcid.org/0000-0002-5117-2991

Catalogue record

Date deposited: 17 Oct 2019 16:30
Last modified: 18 Oct 2019 00:39

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Contributors

Author: Lyndsy Jane Ambler
Thesis advisor: Donna Davies ORCID iD
Thesis advisor: Jane Collins
Thesis advisor: Karen O'reilly

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