Expression and function of Wnt-inducible signalling protein 1 in idiopathic pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal fibrotic interstitial lung disease affecting older adults. It has poor prognosis and clinical interventions are limited. IPF is characterised by the build-up of fibrotic extracellular matrix in the lung interstitium, leading to progressively impaired gas exchange and eventual death. The cause of IPF is unknown but is thought to involve repeated microinjuries to lung tissue leading to chronic activation of wound healing pathways such as Wnt, TGF-β and hypoxia inducible factor (HIF) signalling. IPF tissue is characterised by the presence of fibroblastic foci – aggregates of matrix-secreting fibroblasts and myofibroblasts which drive fibrosis by producing stiffened collagen which exhibits abnormal, bone-type crosslinking.
CCN proteins are a family of proteins with similar protein structure, which affect cell signalling by interacting with other proteins to modulate their function. Wnt-inducible signalling protein 1 (WISP-1) is a Wnt-driven CCN protein that has roles in bone development and stem cell survival. While WISP-1 expression is upregulated in IPF tissue, its role in disease pathogenesis is unclear. It is hypothesised that WISP-1 is dysregulated in fibroblastic foci, and that this contributes to IPF pathogenesis by affecting protein-protein interactions. Thus, the aims of this project were to (i) characterise the localisation of WISP-1; (ii) to identify drivers of WISP-1 expression; and (iii) to identify potential functions of WISP-1 by determining what proteins it interacts with.
WISP-1 expression was found to localise to fibroblastic foci in a laser-capture microdissection (LCMD) RNAseq dataset of control and IPF alveolar septae and fibroblastic foci. This was confirmed by RNAscope in-situ hybridisation. WISP1 was identified as being expressed in two distinct fibroblast types in an IPF single cell RNAseq dataset, myofibroblasts and senescent, profibrotic HAS1-high fibroblasts. Gene signatures from these cells were identified in LCMD fibroblastic focus RNAseq data using CibersortX in-silico cell sorting. Gene expression signatures associated with HIF and TGF-β signalling were found to be upregulated in WISP1 expressing mesenchymal cells. Treatment of primary lung fibroblasts from healthy or IPF donors with HIF activators or hypoxic conditions in vitro led to induction of WISP1 which was greatly augmented in IPF lung fibroblasts, suggesting WISP-1 expression is HIF driven in IPF. Using an affinity purification-mass spectrometry (AP-MS) workflow to identify protein interaction partners of GFP-tagged WISP-1, the mitogen fibroblast growth factor 2 (FGF2) and the mitochondrial cell survival factors voltage dependent anion channel 1 (VDAC1) and prohibitin (PHB) were identified as WISP-1 interaction partners in MRC-5 cultured lung fibroblasts. Interactions of WISP-1 with FGF2 and VDAC1 were confirmed by western blotting or using binding assays.
In conclusion, WISP-1 is localised in fibroblastic foci, where its expression occurs in two distinct, profibrotic fibroblast types: myofibroblasts and IPF-specific HAS-1 high fibroblasts. In vitro studies demonstrate that WISP-1 expression in IPF fibroblasts is driven by HIF signalling and that WISP-1 interacts with proteins known to affect cell survival and proliferation. These findings are consistent with a role for WISP-1 in IPF pathogenesis.

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Identifiers

ORCID for Donna Davies: orcid.org/0000-0002-5117-2991

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