miR-92a regulates TGF-β1-induced WISP1 expression in pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is the most common and fatal form of idiopathic interstitial pneumonia. MicroRNAs (miRNAs), short, single-stranded RNAs that regulate protein expression in a post-transcriptional manner, have recently been demonstrated to contribute to IPF pathogenesis. We have previously identified WNT1-inducible signaling pathway protein 1 (WISP1) as a highly expressed pro-fibrotic mediator in IPF, but the underlying mechanisms resulting in increased WISP1 expression, remain elusive. Here, we investigated whether WISP1 is a target of miRNA regulation. We applied a novel supervised machine learning approach, which predicted miR-30a/d and miR-92a target sites in regions of the human WISP1 3′UTR preferentially bound by the miRNA ribonucleoprotein complex. Both miRNAs were decreased in IPF samples, whereas WISP1 protein was increased. We demonstrated further that transforming growth factor (TGF)-β1-induced WISP1 expression in primary lung fibroblasts in vitro and lung homogenates in vivo. Notably, miR-30a and miR-92a reversed TGF-β1-induced WISP1 mRNA expression in lung fibroblasts. Moreover, miR-92a inhibition increased WISP1 protein expression in lung fibroblasts. An inverse relationship for WISP1 and miR-92a was found in a TGF-β1 dependent lung fibrosis model in vivo. Finally, we found significantly increased WISP1 expression in primary IPF fibroblasts, which negatively correlated with miR-92a level ex vivo. Altogether, our findings indicate a regulatory role of miR-92a for WISP1 expression in pulmonary fibrosis.

Introduction

Idiopathic pulmonary fibrosis (IPF) is the most common and aggressive form of idiopathic interstitial pneumonia (Raghu et al., 2011). The etiology of IPF is unknown and treatment options are still limited (Baroke et al., 2013, du Bois, 2010). Pathological hallmarks include alveolar epithelial injury and hyperplasia, aberrant wound healing, formation of fibroblast foci, as well as excessive matrix deposition resulting in disrupted lung architecture and respiratory insufficiency (Selman et al., 2001, Wolters et al., 2014). Disturbed growth factor signaling within the epithelial–mesenchymal unit has been shown to contribute to the pathobiology of IPF. Among others, the transforming growth factor-β1 (TGF-β1) has been identified as a key pro-fibrotic mediator (Fernandez and Eickelberg, 2012). In addition, reactivation of developmental pathways, in particular Wnt signaling, has been demonstrated in both experimental and human lung fibrosis (Chilosi et al., 2003, Königshoff et al., 2008, Selman et al., 2008). Both Wnt/β-catenin and TGF-β1 signaling contribute to epithelial as well as mesenchymal cell dysfunction and/or reprogramming in pulmonary fibrosis (Königshoff and Eickelberg, 2010, Lam and Gottardi, 2011). Recently, we identified the Wnt target gene WNT1-inducible signaling pathway protein 1 (WISP1) as a novel mediator of disturbed epithelial–mesenchymal crosstalk. WISP1 is markedly increased in IPF tissue specimens and neutralization of WISP1 in experimental lung fibrosis attenuated the development of pulmonary fibrosis in vivo (Königshoff et al., 2009). WISP1 is a secreted matricellular protein belonging to the CCN protein family (Berschneider and Königshoff, 2011) and allocated as CCN4 (Brigstock et al., 2003). In addition to lung fibrosis, WISP1 has been linked to stretch-induced epithelial–mesenchymal transition in alveolar epithelial type II cells in vitro (Heise et al., 2011), and to ventilator-induced lung injury models in vivo (Li et al., 2012). However, molecular mechanisms that effect WISP1 function and regulation, particularly under diseased conditions, are largely unknown.

In this respect, microRNAs (miRNAs), small, ∼21 nt long, non-coding RNAs involved in post-transcriptional gene regulation, have been demonstrated to play pivotal roles in various biological processes, including cell signaling and function. MiRNAs have been associated with a variety of diseases, including IPF (Liu et al., 2010, Pandit et al., 2011, Xie et al., 2011). Several recent studies reported altered miRNA expression in experimental and human pulmonary fibrosis, among others let-7d, miR-21, miR-29, or miR-154 (Cushing et al., 2011, Liu et al., 2010, Milosevic et al., 2012, Pandit et al., 2010). Modulation of aberrantly regulated miRNAs like miR-29 and miR-200 had an impact on the development of pulmonary fibrosis and may serve as potential therapeutic targets (Xiao et al., 2012, Yang et al., 2012).

Here, we hypothesize that WISP1 is regulated by miRNAs in pulmonary fibrosis. Therefore, the aim of our study was to identify novel miRNAs that are predicted to bind to WISP1. In order to do so, we analyzed several published miRNA array data sets and applied a novel supervised learning approach for in silico miRNA target prediction to WISP1 3′UTR. We further demonstrated that miR-92a and family members of miR-30 as well as WISP1 are differentially expressed in experimental lung fibrosis and in lung tissue specimens of IPF patients. Modulation of candidate miR-92a and miR-30a in primary lung fibroblasts altered TGF-β1-induced WISP1 expression, thereby demonstrating a new miRNA-based regulatory mechanism for WISP1 in pulmonary fibrosis.