MicroRNA-144-3p targets relaxin/insulin-like family peptide receptor 1 (RXFP1) expression in lung fibroblasts from patients with idiopathic pulmonary fibrosis

Harinath Bahudhanapati,Jiangning Tan,Justin A Dutta,Stephen B Strock,John Sembrat,Diana Àlvarez,Mauricio Rojas,Benedikt Jäger,Antje Prasse,Yingze Zhang,Daniel J Kass

doi:10.1074/jbc.ra118.004910

Abstract

The hormone relaxin is considered a potential therapy for idiopathic pulmonary fibrosis (IPF). We have previously shown that a potential limitation to relaxin-based IPF therapy is decreased expression of a relaxin receptor, relaxin/insulin-like family peptide receptor 1 (RXFP1), in IPF fibroblasts. The mechanism that down-regulates RXFP1 in IPF remains unclear. To determine whether microRNAs (miRs) regulate RXFP1 gene expression, here we employed a bioinformatics approach to identify miRs predicted to target RXFP1 and identified a putative miR-144-3p target site in the RXFP1 mRNA. In situ hybridization of IPF lung biopsies revealed that miR-144-3p is expressed in fibroblastic foci. Furthermore, we found that miR-144-3p is up-regulated in IPF fibroblasts compared with lung fibroblasts from healthy donors. Transforming growth factor β increased miR-144-3p expression in both healthy and IPF lung fibroblasts in a SMAD family 2/3 (SMAD2/3)-dependent manner, and Jun proto-oncogene AP-1 transcription factor subunit (AP-1) was required for constitutive miR-144-3p expression. Overexpression of an miR-144-3p mimic significantly reduced RXFP1 mRNA and protein levels and increased expression of the myofibroblast marker α-smooth muscle actin (α-SMA) in healthy lung fibroblasts. IPF lung fibroblasts transfected with anti-miR-144-3p had increased RXFP1 expression and reduced α-SMA expression. Of note, a lentiviral luciferase reporter carrying the WT 3' UTR of RXFP1 was significantly repressed in IPF lung fibroblasts, whereas a reporter carrying a mutated miR-144-3p-binding site exhibited less sensitivity toward endogenous miR-144-3p expression, indicating that miR-144-3p down-regulates RXFP1 in IPF lung fibroblasts by targeting its 3' UTR. We conclude that miR-144-3p directly represses RXFP1 mRNA and protein expression.

Highlights

The hormone relaxin is considered a potential therapy for idiopathic pulmonary fibrosis (IPF)
We show by qRT-PCR that IPF lung fibroblasts have significantly lower basal levels of RXFP1 mRNA compared with donor lung fibroblasts, confirming our previous findings (Fig. 2B) [7]
In Western blotting and densitometry analysis for ␣-SMA levels for the experiment from Fig. 2E and Fig. S2A; we found that miR-144-3p overexpression resulted in increased levels of ␣-SMA in donor lung fibroblasts; ␣-SMA levels increased significantly in IPF lung fibroblasts over baseline (Fig. 2F)

Summary

Introduction

The hormone relaxin is considered a potential therapy for idiopathic pulmonary fibrosis (IPF). Overexpression of an miR-144-3p mimic significantly reduced RXFP1 mRNA and protein levels and increased expression of the myofibroblast marker ␣-smooth muscle actin (␣-SMA) in healthy lung fibroblasts. IPF lung fibroblasts transfected with anti-miR-144-3p had increased RXFP1 expression and reduced ␣-SMA expression. A lentiviral luciferase reporter carrying the WT 3؅ UTR of RXFP1 was significantly repressed in IPF lung fibroblasts, whereas a reporter carrying a mutated miR-144-3p– binding site exhibited less sensitivity toward endogenous miR-144-3p expression, indicating that miR-144-3p down-regulates. In further support of the importance of this pathway, we have shown that gene expression levels for the relaxin receptor, RXFP1, in IPF is directly associated with pulmonary function [7]. Because the loss of RXFP1 expression in IPF may desensitize fibroblasts from the positive effects of relaxin-like agonists [7], a strategy to increase RXFP1 expression in IPF might prove to be an effective therapeutic approach

Methods

Results

Conclusion