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Identification of a distal RXFP1 gene enhancer with differential activity in fibrotic lung fibroblasts involving AP-1

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 f...

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Autores principales: Chen, Ting-Yun, Li, Xiaoyun, Goobie, Gillian C., Hung, Ching-Hsia, Hung, Tin-Kan, Hamilton, Kyle, Bahudhanapati, Harinath, Tan, Jiangning, Kass, Daniel J., Zhang, Yingze
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8719731/
https://www.ncbi.nlm.nih.gov/pubmed/34972106
http://dx.doi.org/10.1371/journal.pone.0254466
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author Chen, Ting-Yun
Li, Xiaoyun
Goobie, Gillian C.
Hung, Ching-Hsia
Hung, Tin-Kan
Hamilton, Kyle
Bahudhanapati, Harinath
Tan, Jiangning
Kass, Daniel J.
Zhang, Yingze
author_facet Chen, Ting-Yun
Li, Xiaoyun
Goobie, Gillian C.
Hung, Ching-Hsia
Hung, Tin-Kan
Hamilton, Kyle
Bahudhanapati, Harinath
Tan, Jiangning
Kass, Daniel J.
Zhang, Yingze
author_sort Chen, Ting-Yun
collection PubMed
description 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.
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spelling pubmed-87197312022-01-01 Identification of a distal RXFP1 gene enhancer with differential activity in fibrotic lung fibroblasts involving AP-1 Chen, Ting-Yun Li, Xiaoyun Goobie, Gillian C. Hung, Ching-Hsia Hung, Tin-Kan Hamilton, Kyle Bahudhanapati, Harinath Tan, Jiangning Kass, Daniel J. Zhang, Yingze PLoS One Research Article 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. Public Library of Science 2021-12-31 /pmc/articles/PMC8719731/ /pubmed/34972106 http://dx.doi.org/10.1371/journal.pone.0254466 Text en © 2021 Chen et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Chen, Ting-Yun
Li, Xiaoyun
Goobie, Gillian C.
Hung, Ching-Hsia
Hung, Tin-Kan
Hamilton, Kyle
Bahudhanapati, Harinath
Tan, Jiangning
Kass, Daniel J.
Zhang, Yingze
Identification of a distal RXFP1 gene enhancer with differential activity in fibrotic lung fibroblasts involving AP-1
title Identification of a distal RXFP1 gene enhancer with differential activity in fibrotic lung fibroblasts involving AP-1
title_full Identification of a distal RXFP1 gene enhancer with differential activity in fibrotic lung fibroblasts involving AP-1
title_fullStr Identification of a distal RXFP1 gene enhancer with differential activity in fibrotic lung fibroblasts involving AP-1
title_full_unstemmed Identification of a distal RXFP1 gene enhancer with differential activity in fibrotic lung fibroblasts involving AP-1
title_short Identification of a distal RXFP1 gene enhancer with differential activity in fibrotic lung fibroblasts involving AP-1
title_sort identification of a distal rxfp1 gene enhancer with differential activity in fibrotic lung fibroblasts involving ap-1
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8719731/
https://www.ncbi.nlm.nih.gov/pubmed/34972106
http://dx.doi.org/10.1371/journal.pone.0254466
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