# A novel approach to reverse the ineffectiveness of relaxin-based anti-fibrotic therapy in SSc

> **NIH NIH R21** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2020 · $172,150

## Abstract

ABSTRACT
 Skin and lung fibrosis are the hallmarks of systemic sclerosis (SSc, scleroderma), and the reversal of
fibrosis is a very challenging clinical problem. Relaxin is a circulating hormone with strong preclinical evidence
supporting its role as a potent anti-fibrotic molecule for organ fibrosis. But despite the strong evidence from
animal models, relaxin-based therapy in clinical trial failed in reducing the skin fibrosis of SSc patients. One
potential molecular explanation for the failure of relaxin in clinical trial is that cells in fibrosis may be insensitive
to relaxin. We have found that the mRNA and protein levels of relaxin receptor, RXFP1, are dramatically
decreased in the lungs and lung fibroblasts of SSc patients compared to controls. Fibroblasts from fibrotic lungs
are impaired in their in vitro sensitivity to a relaxin-like agonist, but enhancement of RXFP1 expression restores
relaxin sensitivity in these cells. However, the molecular mechanisms leading to RXFP1 down-regulation is
unknown. We have identified a lung fibroblast-specific enhancer in the distal region of the RXFP1 gene that
results in >100-fold increase of SV40 promoter-driven luciferase activity in control lung fibroblasts and the
enhancement is partially blunted in fibroblasts from SSc (<20-fold). In addition, regions of transcriptionally active
histone modifications as well as an upstream CpG island flank this novel enhancer. Therefore, we hypothesize
that abnormal function of this distal RXFP1 enhancer, in combination with altered epigenetic changes in SSc
lung fibroblasts, is a major driver of RXFP1 down-regulation and that modifications of these regulatory elements
and epigenetic events will up-regulation of RXFP1 expression and sensitize SSc fibroblasts to relaxin treatment.
We will perform fine mapping of the enhancer region and to identify transcription factors (TFs) responsible for
the lung fibroblast-specific enhancement of RXFP1 (Aim 1). The expression levels of Identified TFs will be further
correlated with endogenous RXFP1 expression in both control and SSc lungs and fibroblasts. Direct binding of
the TFs to the enhancer will be analyzed using EMSA and ChIP assays. We will also determine the role of
epigenetic modifications surrounding the enhancer in driving lung fibroblast specific RXFP1 expression (Aim 2).
Both histone modification and DNA methylation status will be analyzed in control and SSc lungs and fibroblasts
using molecular techniques including our novel method ISH-PLA, a single-cell epigenetic assay allowing
detection of histone modifications in tissue sections. The success of this study will provide a novel approach to
reverse the ineffectiveness of relaxin-based anti-fibrotic treatment in SSc by identifying potential therapeutic
targets for restoring RXFP1 expression in fibrotic lungs of these patients.

## Key facts

- **NIH application ID:** 9981632
- **Project number:** 5R21AR076024-02
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** Yingze Zhang
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $172,150
- **Award type:** 5
- **Project period:** 2019-09-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9981632

## Citation

> US National Institutes of Health, RePORTER application 9981632, A novel approach to reverse the ineffectiveness of relaxin-based anti-fibrotic therapy in SSc (5R21AR076024-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9981632. Licensed CC0.

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