Abstract

Relaxin/insulin-like family peptide receptor 1 (RXFP1) mediates relaxin’s antifibrotic effects and has reduced expression in the lung and skin of patients with fibrotic interstitial lung disease (fILD) including idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc). This may explain the failure of relaxin-based anti-fibrotic treatments in SSc, but the regulatory mechanisms controlling RXFP1 expression remain largely unknown. This study aimed to identify regulatory elements of RXFP1 that may function differentially in fibrotic fibroblasts. We identified and evaluated a distal regulatory region of RXFP1 in lung fibroblasts using a luciferase reporter system. Using serial deletions, an enhancer upregulating pGL3-promoter activity was localized to the distal region between -584 to -242bp from the distal transcription start site (TSS). This enhancer exhibited reduced activity in IPF and SSc lung fibroblasts. Bioinformatic analysis identified two clusters of activator protein 1 (AP-1) transcription factor binding sites within the enhancer. Site-directed mutagenesis of the binding sites confirmed that only one cluster reduced activity (-358 to -353 relative to distal TSS). Co-expression of FOS in lung fibroblasts further increased enhancer activity. In vitro complex formation with a labeled probe spanning the functional AP-1 site using nuclear proteins isolated from lung fibroblasts confirmed a specific DNA/protein complex formation. Application of antibodies against JUN and FOS resulted in the complex alteration, while antibodies to JUNB and FOSL1 did not. Analysis of AP-1 binding in 5 pairs of control and IPF lung fibroblasts detected positive binding more frequently in control fibroblasts. Expression of JUN and FOS was reduced and correlated positively with RXFP1 expression in IPF lungs. In conclusion, we identified a distal enhancer of RXFP1 with differential activity in fibrotic lung fibroblasts involving AP-1 transcription factors. Our study provides insight into RXFP1 downregulation in fILD and may support efforts to reevaluate relaxin-based therapeutics alongside upregulation of RXFP1 transcription.

Highlights

  • Pulmonary fibrosis is a hallmark of fibrotic interstitial lung diseases

  • The reporter activities for the proximal promoter in both control and Idiopathic pulmonary fibrosis (IPF) fibroblasts were reduced compared with the pB vector luciferase activities from both were very low

  • We have identified a strong enhancer within the distal regulatory region of RXFP1, which had reduced activities upon introduction into fibrotic lung fibroblasts compared to controls

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Summary

Introduction

Pulmonary fibrosis is a hallmark of fibrotic interstitial lung diseases (fILD). The pathogenesis of fILD is not fully understood [1], fibroblast activation in the lungs of patients with fILD results in aberrant extracellular matrix (ECM) collagen accumulation [2]. Idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) are two of the most common types of fILD. IPF is a chronic and progressive disease associated with high morbidity and mortality [1, 2]. In patients with SSc, fILD is the disease manifestation associated with the highest mortality [3]. Despite the increasing global burden of fILD [4, 5], our understanding of the mechanisms underlying the development and progression of fibrosis and our ability to target these pathogenic pathways is lacking

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