Dicer1 deficiency in the idiopathic pulmonary fibrosis fibroblastic focus promotes fibrosis by suppressing MicroRNA biogenesis

Jeremy Herrera, Daniel J. Beisang, Mark Peterson, Colleen Forster, Adam J Gilbertsen, Alexey Benyumov, Karen Smith, Christopher E. Korenczuk, Victor H Barocas, Kacey Guenther, Ryan Hite, Lin Zhang, Craig A Henke, Peter B Bitterman

Research output: Contribution to journalArticle

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Abstract

Rationale: The lung extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) mediates progression of fibrosis by decreasing fibroblast expression of miR-29 (microRNA-29), a master negative regulator of ECM production. The molecular mechanism is undefined. IPF-ECM is stiffer than normal. Stiffness drives fibroblast ECM production in a YAP (yes-associated protein)-dependent manner, and YAP is a known regulator of miR-29. Therefore,we tested the hypothesis that negative regulation of miR-29 by IPF-ECM was mediated by mechanotransduction of stiffness. Objectives: To determine how IPF-ECM negatively regulates miR-29. Methods: We decellularized lung ECM using detergents and prepared polyacrylamide hydrogels of defined stiffness by varying acrylamide concentrations. Mechanistic studies were guided by immunohistochemistry of IPF lung and used cell culture, RNAbinding protein assays, and xenograft models. Measurements and Main Results: Contrary to our hypothesis, we excluded fibroblast mechanotransduction of ECM stiffness as the primary mechanism deregulating miR-29. Instead, systematic examination of miR-29 biogenesis revealed a microRNA processing defect that impeded processing ofmiR-29 into its mature bioactive forms. Immunohistochemical analysis of the microRNA processing machinery in IPF lung specimens revealed decreased Dicer1 expression in the procollagen-rich myofibroblastic core of fibroblastic foci compared with the focus perimeter and adjacent alveolar walls. Mechanistically, IPF-ECM increased association of the Dicer1 transcript with RNA binding protein AUF1 (AU-binding factor 1), and Dicer1 knockdown conferred primary human lung fibroblasts with cell-autonomous fibrogenicity in zebrafish and mouse lung xenograft models. Conclusions: Our data identify suppression of fibroblast Dicer1 expression in the myofibroblast-rich IPF fibroblastic focus core as a central step in the mechanism by which the ECM sustains fibrosis progression in IPF.

Original languageEnglish (US)
Pages (from-to)486-496
Number of pages11
JournalAmerican journal of respiratory and critical care medicine
Volume198
Issue number4
DOIs
StatePublished - Aug 15 2018

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Idiopathic Pulmonary Fibrosis
MicroRNAs
Extracellular Matrix
Fibrosis
Fibroblasts
Lung
Heterografts
Procollagen
Proteins
RNA-Binding Proteins
Myofibroblasts
Acrylamide
Zebrafish
Protein Binding
Detergents
Cell Culture Techniques
Immunohistochemistry

Keywords

  • Extracellular matrix
  • Idiopathic pulmonary fibrosis
  • Yes-associated protein

Cite this

Dicer1 deficiency in the idiopathic pulmonary fibrosis fibroblastic focus promotes fibrosis by suppressing MicroRNA biogenesis. / Herrera, Jeremy; Beisang, Daniel J.; Peterson, Mark; Forster, Colleen; Gilbertsen, Adam J; Benyumov, Alexey; Smith, Karen; Korenczuk, Christopher E.; Barocas, Victor H; Guenther, Kacey; Hite, Ryan; Zhang, Lin; Henke, Craig A; Bitterman, Peter B.

In: American journal of respiratory and critical care medicine, Vol. 198, No. 4, 15.08.2018, p. 486-496.

Research output: Contribution to journalArticle

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abstract = "Rationale: The lung extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) mediates progression of fibrosis by decreasing fibroblast expression of miR-29 (microRNA-29), a master negative regulator of ECM production. The molecular mechanism is undefined. IPF-ECM is stiffer than normal. Stiffness drives fibroblast ECM production in a YAP (yes-associated protein)-dependent manner, and YAP is a known regulator of miR-29. Therefore,we tested the hypothesis that negative regulation of miR-29 by IPF-ECM was mediated by mechanotransduction of stiffness. Objectives: To determine how IPF-ECM negatively regulates miR-29. Methods: We decellularized lung ECM using detergents and prepared polyacrylamide hydrogels of defined stiffness by varying acrylamide concentrations. Mechanistic studies were guided by immunohistochemistry of IPF lung and used cell culture, RNAbinding protein assays, and xenograft models. Measurements and Main Results: Contrary to our hypothesis, we excluded fibroblast mechanotransduction of ECM stiffness as the primary mechanism deregulating miR-29. Instead, systematic examination of miR-29 biogenesis revealed a microRNA processing defect that impeded processing ofmiR-29 into its mature bioactive forms. Immunohistochemical analysis of the microRNA processing machinery in IPF lung specimens revealed decreased Dicer1 expression in the procollagen-rich myofibroblastic core of fibroblastic foci compared with the focus perimeter and adjacent alveolar walls. Mechanistically, IPF-ECM increased association of the Dicer1 transcript with RNA binding protein AUF1 (AU-binding factor 1), and Dicer1 knockdown conferred primary human lung fibroblasts with cell-autonomous fibrogenicity in zebrafish and mouse lung xenograft models. Conclusions: Our data identify suppression of fibroblast Dicer1 expression in the myofibroblast-rich IPF fibroblastic focus core as a central step in the mechanism by which the ECM sustains fibrosis progression in IPF.",
keywords = "Extracellular matrix, Idiopathic pulmonary fibrosis, Yes-associated protein",
author = "Jeremy Herrera and Beisang, {Daniel J.} and Mark Peterson and Colleen Forster and Gilbertsen, {Adam J} and Alexey Benyumov and Karen Smith and Korenczuk, {Christopher E.} and Barocas, {Victor H} and Kacey Guenther and Ryan Hite and Lin Zhang and Henke, {Craig A} and Bitterman, {Peter B}",
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AU - Herrera, Jeremy

AU - Beisang, Daniel J.

AU - Peterson, Mark

AU - Forster, Colleen

AU - Gilbertsen, Adam J

AU - Benyumov, Alexey

AU - Smith, Karen

AU - Korenczuk, Christopher E.

AU - Barocas, Victor H

AU - Guenther, Kacey

AU - Hite, Ryan

AU - Zhang, Lin

AU - Henke, Craig A

AU - Bitterman, Peter B

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N2 - Rationale: The lung extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) mediates progression of fibrosis by decreasing fibroblast expression of miR-29 (microRNA-29), a master negative regulator of ECM production. The molecular mechanism is undefined. IPF-ECM is stiffer than normal. Stiffness drives fibroblast ECM production in a YAP (yes-associated protein)-dependent manner, and YAP is a known regulator of miR-29. Therefore,we tested the hypothesis that negative regulation of miR-29 by IPF-ECM was mediated by mechanotransduction of stiffness. Objectives: To determine how IPF-ECM negatively regulates miR-29. Methods: We decellularized lung ECM using detergents and prepared polyacrylamide hydrogels of defined stiffness by varying acrylamide concentrations. Mechanistic studies were guided by immunohistochemistry of IPF lung and used cell culture, RNAbinding protein assays, and xenograft models. Measurements and Main Results: Contrary to our hypothesis, we excluded fibroblast mechanotransduction of ECM stiffness as the primary mechanism deregulating miR-29. Instead, systematic examination of miR-29 biogenesis revealed a microRNA processing defect that impeded processing ofmiR-29 into its mature bioactive forms. Immunohistochemical analysis of the microRNA processing machinery in IPF lung specimens revealed decreased Dicer1 expression in the procollagen-rich myofibroblastic core of fibroblastic foci compared with the focus perimeter and adjacent alveolar walls. Mechanistically, IPF-ECM increased association of the Dicer1 transcript with RNA binding protein AUF1 (AU-binding factor 1), and Dicer1 knockdown conferred primary human lung fibroblasts with cell-autonomous fibrogenicity in zebrafish and mouse lung xenograft models. Conclusions: Our data identify suppression of fibroblast Dicer1 expression in the myofibroblast-rich IPF fibroblastic focus core as a central step in the mechanism by which the ECM sustains fibrosis progression in IPF.

AB - Rationale: The lung extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) mediates progression of fibrosis by decreasing fibroblast expression of miR-29 (microRNA-29), a master negative regulator of ECM production. The molecular mechanism is undefined. IPF-ECM is stiffer than normal. Stiffness drives fibroblast ECM production in a YAP (yes-associated protein)-dependent manner, and YAP is a known regulator of miR-29. Therefore,we tested the hypothesis that negative regulation of miR-29 by IPF-ECM was mediated by mechanotransduction of stiffness. Objectives: To determine how IPF-ECM negatively regulates miR-29. Methods: We decellularized lung ECM using detergents and prepared polyacrylamide hydrogels of defined stiffness by varying acrylamide concentrations. Mechanistic studies were guided by immunohistochemistry of IPF lung and used cell culture, RNAbinding protein assays, and xenograft models. Measurements and Main Results: Contrary to our hypothesis, we excluded fibroblast mechanotransduction of ECM stiffness as the primary mechanism deregulating miR-29. Instead, systematic examination of miR-29 biogenesis revealed a microRNA processing defect that impeded processing ofmiR-29 into its mature bioactive forms. Immunohistochemical analysis of the microRNA processing machinery in IPF lung specimens revealed decreased Dicer1 expression in the procollagen-rich myofibroblastic core of fibroblastic foci compared with the focus perimeter and adjacent alveolar walls. Mechanistically, IPF-ECM increased association of the Dicer1 transcript with RNA binding protein AUF1 (AU-binding factor 1), and Dicer1 knockdown conferred primary human lung fibroblasts with cell-autonomous fibrogenicity in zebrafish and mouse lung xenograft models. Conclusions: Our data identify suppression of fibroblast Dicer1 expression in the myofibroblast-rich IPF fibroblastic focus core as a central step in the mechanism by which the ECM sustains fibrosis progression in IPF.

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KW - Yes-associated protein

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