p300 Acetyltransferase Is a Cytoplasm-to-Nucleus Shuttle for SMAD2/3 and TAZ Nuclear Transport in Transforming Growth Factor β–Stimulated Hepatic Stellate Cells

Yuanguo Wang, Kangsheng Tu, Donglian Liu, Luyang Guo, Yunru Chen, Qing Li, Jessica L. Maiers, Zhikui Liu, Vijay H. Shah, Changwei Dou, Daniel Tschumperlin, Luke Voneschen, Rendong Yang, Ningling Kang

Research output: Contribution to journalArticle

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Abstract

Nuclear translocation of mothers against decapentaplegic homolog 2/3 (SMAD2/3), core transcription factors of transforming growth factor β (TGF-β) signaling, is critical for hepatic stellate cell (HSC) differentiation into metastasis-promoting myofibroblasts. SMAD2/3 have multiple coactivators, including WW domain-containing transcription regulator protein 1 (WWTR1 or TAZ) and p300 acetyltransferase. In the nucleus, TAZ binds to SMAD2/3 to prevent SMAD2/3 nuclear export. However, how TAZ and SMAD2/3 enter the nucleus remains poorly understood because neither contains a nuclear localization signal (NLS), an amino acid sequence tagging proteins for nuclear transport. p300 is an NLS-containing large scaffold protein, so we hypothesized that SMAD2/3 and TAZ may undergo nuclear import through complexing with p300. Coimmunoprecipitation, immunofluorescence, and nuclear fractionation assays revealed that TGF-β1 promoted binding of SMAD2/3 and TAZ to p300 and that p300 inactivation disrupted TGF-β1-mediated SMAD2/3 and TAZ nuclear accumulation. Deleting the p300 NLS blocked TGF-β1-induced SMAD2/3 and TAZ nuclear transport. Consistently, p300 inactivation suppressed TGF-β1-mediated HSC activation and transcription of genes encoding tumor-promoting factors, such as connective tissue growth factor, Tenascin C, Periostin, platelet-derived growth factor C, and fibroblast growth factor 2, as revealed by microarray analysis. Chromatin immunoprecipitation-real-time quantitative PCR showed that canonical p300-mediated acetylation of histones also facilitated transcription in response to TGF-β1 stimulation. Interestingly, although both TGF-β1-mediated and stiffness-mediated HSC activation require p300, comparison of gene expression data sets revealed that transcriptional targets of TGF-β1 were distinct from those of stiffness-p300 mechanosignaling. Lastly, in tumor/HSC coinjection and intrasplenic tumor injection models, targeting p300 of activated-HSC/myofibroblasts by C646, short hairpin RNA, or cre-mediated gene disruption reduced tumor and liver metastatic growth in mice. Conclusion: p300 facilitates TGF-β1-stimulated HSC activation by both noncanonical (cytoplasm-to-nucleus shuttle for SMAD2/3 and TAZ) and canonical (histone acetylation) mechanisms. p300 is an attractive target for inhibiting HSC activation and the prometastatic liver microenvironment.

Original languageEnglish (US)
Pages (from-to)1409-1423
Number of pages15
JournalHepatology
Volume70
Issue number4
DOIs
StatePublished - Oct 1 2019

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Hepatic Stellate Cells
Cell Nucleus Active Transport
Transforming Growth Factors
Cytoplasm
Nuclear Localization Signals
Myofibroblasts
Acetylation
Histones
Transcriptional Activation
Neoplasms
Transcription Factor 3
Smad Proteins
Connective Tissue Growth Factor
Tenascin
p300-CBP-associated factor
Chromatin Immunoprecipitation
Liver
Fibroblast Growth Factor 2
Protein Transport
Microarray Analysis

ASJC Scopus subject areas

  • Hepatology

Cite this

p300 Acetyltransferase Is a Cytoplasm-to-Nucleus Shuttle for SMAD2/3 and TAZ Nuclear Transport in Transforming Growth Factor β–Stimulated Hepatic Stellate Cells. / Wang, Yuanguo; Tu, Kangsheng; Liu, Donglian; Guo, Luyang; Chen, Yunru; Li, Qing; Maiers, Jessica L.; Liu, Zhikui; Shah, Vijay H.; Dou, Changwei; Tschumperlin, Daniel; Voneschen, Luke; Yang, Rendong; Kang, Ningling.

In: Hepatology, Vol. 70, No. 4, 01.10.2019, p. 1409-1423.

Research output: Contribution to journalArticle

Wang, Yuanguo ; Tu, Kangsheng ; Liu, Donglian ; Guo, Luyang ; Chen, Yunru ; Li, Qing ; Maiers, Jessica L. ; Liu, Zhikui ; Shah, Vijay H. ; Dou, Changwei ; Tschumperlin, Daniel ; Voneschen, Luke ; Yang, Rendong ; Kang, Ningling. / p300 Acetyltransferase Is a Cytoplasm-to-Nucleus Shuttle for SMAD2/3 and TAZ Nuclear Transport in Transforming Growth Factor β–Stimulated Hepatic Stellate Cells. In: Hepatology. 2019 ; Vol. 70, No. 4. pp. 1409-1423.
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title = "p300 Acetyltransferase Is a Cytoplasm-to-Nucleus Shuttle for SMAD2/3 and TAZ Nuclear Transport in Transforming Growth Factor β–Stimulated Hepatic Stellate Cells",
abstract = "Nuclear translocation of mothers against decapentaplegic homolog 2/3 (SMAD2/3), core transcription factors of transforming growth factor β (TGF-β) signaling, is critical for hepatic stellate cell (HSC) differentiation into metastasis-promoting myofibroblasts. SMAD2/3 have multiple coactivators, including WW domain-containing transcription regulator protein 1 (WWTR1 or TAZ) and p300 acetyltransferase. In the nucleus, TAZ binds to SMAD2/3 to prevent SMAD2/3 nuclear export. However, how TAZ and SMAD2/3 enter the nucleus remains poorly understood because neither contains a nuclear localization signal (NLS), an amino acid sequence tagging proteins for nuclear transport. p300 is an NLS-containing large scaffold protein, so we hypothesized that SMAD2/3 and TAZ may undergo nuclear import through complexing with p300. Coimmunoprecipitation, immunofluorescence, and nuclear fractionation assays revealed that TGF-β1 promoted binding of SMAD2/3 and TAZ to p300 and that p300 inactivation disrupted TGF-β1-mediated SMAD2/3 and TAZ nuclear accumulation. Deleting the p300 NLS blocked TGF-β1-induced SMAD2/3 and TAZ nuclear transport. Consistently, p300 inactivation suppressed TGF-β1-mediated HSC activation and transcription of genes encoding tumor-promoting factors, such as connective tissue growth factor, Tenascin C, Periostin, platelet-derived growth factor C, and fibroblast growth factor 2, as revealed by microarray analysis. Chromatin immunoprecipitation-real-time quantitative PCR showed that canonical p300-mediated acetylation of histones also facilitated transcription in response to TGF-β1 stimulation. Interestingly, although both TGF-β1-mediated and stiffness-mediated HSC activation require p300, comparison of gene expression data sets revealed that transcriptional targets of TGF-β1 were distinct from those of stiffness-p300 mechanosignaling. Lastly, in tumor/HSC coinjection and intrasplenic tumor injection models, targeting p300 of activated-HSC/myofibroblasts by C646, short hairpin RNA, or cre-mediated gene disruption reduced tumor and liver metastatic growth in mice. Conclusion: p300 facilitates TGF-β1-stimulated HSC activation by both noncanonical (cytoplasm-to-nucleus shuttle for SMAD2/3 and TAZ) and canonical (histone acetylation) mechanisms. p300 is an attractive target for inhibiting HSC activation and the prometastatic liver microenvironment.",
author = "Yuanguo Wang and Kangsheng Tu and Donglian Liu and Luyang Guo and Yunru Chen and Qing Li and Maiers, {Jessica L.} and Zhikui Liu and Shah, {Vijay H.} and Changwei Dou and Daniel Tschumperlin and Luke Voneschen and Rendong Yang and Ningling Kang",
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TY - JOUR

T1 - p300 Acetyltransferase Is a Cytoplasm-to-Nucleus Shuttle for SMAD2/3 and TAZ Nuclear Transport in Transforming Growth Factor β–Stimulated Hepatic Stellate Cells

AU - Wang, Yuanguo

AU - Tu, Kangsheng

AU - Liu, Donglian

AU - Guo, Luyang

AU - Chen, Yunru

AU - Li, Qing

AU - Maiers, Jessica L.

AU - Liu, Zhikui

AU - Shah, Vijay H.

AU - Dou, Changwei

AU - Tschumperlin, Daniel

AU - Voneschen, Luke

AU - Yang, Rendong

AU - Kang, Ningling

PY - 2019/10/1

Y1 - 2019/10/1

N2 - Nuclear translocation of mothers against decapentaplegic homolog 2/3 (SMAD2/3), core transcription factors of transforming growth factor β (TGF-β) signaling, is critical for hepatic stellate cell (HSC) differentiation into metastasis-promoting myofibroblasts. SMAD2/3 have multiple coactivators, including WW domain-containing transcription regulator protein 1 (WWTR1 or TAZ) and p300 acetyltransferase. In the nucleus, TAZ binds to SMAD2/3 to prevent SMAD2/3 nuclear export. However, how TAZ and SMAD2/3 enter the nucleus remains poorly understood because neither contains a nuclear localization signal (NLS), an amino acid sequence tagging proteins for nuclear transport. p300 is an NLS-containing large scaffold protein, so we hypothesized that SMAD2/3 and TAZ may undergo nuclear import through complexing with p300. Coimmunoprecipitation, immunofluorescence, and nuclear fractionation assays revealed that TGF-β1 promoted binding of SMAD2/3 and TAZ to p300 and that p300 inactivation disrupted TGF-β1-mediated SMAD2/3 and TAZ nuclear accumulation. Deleting the p300 NLS blocked TGF-β1-induced SMAD2/3 and TAZ nuclear transport. Consistently, p300 inactivation suppressed TGF-β1-mediated HSC activation and transcription of genes encoding tumor-promoting factors, such as connective tissue growth factor, Tenascin C, Periostin, platelet-derived growth factor C, and fibroblast growth factor 2, as revealed by microarray analysis. Chromatin immunoprecipitation-real-time quantitative PCR showed that canonical p300-mediated acetylation of histones also facilitated transcription in response to TGF-β1 stimulation. Interestingly, although both TGF-β1-mediated and stiffness-mediated HSC activation require p300, comparison of gene expression data sets revealed that transcriptional targets of TGF-β1 were distinct from those of stiffness-p300 mechanosignaling. Lastly, in tumor/HSC coinjection and intrasplenic tumor injection models, targeting p300 of activated-HSC/myofibroblasts by C646, short hairpin RNA, or cre-mediated gene disruption reduced tumor and liver metastatic growth in mice. Conclusion: p300 facilitates TGF-β1-stimulated HSC activation by both noncanonical (cytoplasm-to-nucleus shuttle for SMAD2/3 and TAZ) and canonical (histone acetylation) mechanisms. p300 is an attractive target for inhibiting HSC activation and the prometastatic liver microenvironment.

AB - Nuclear translocation of mothers against decapentaplegic homolog 2/3 (SMAD2/3), core transcription factors of transforming growth factor β (TGF-β) signaling, is critical for hepatic stellate cell (HSC) differentiation into metastasis-promoting myofibroblasts. SMAD2/3 have multiple coactivators, including WW domain-containing transcription regulator protein 1 (WWTR1 or TAZ) and p300 acetyltransferase. In the nucleus, TAZ binds to SMAD2/3 to prevent SMAD2/3 nuclear export. However, how TAZ and SMAD2/3 enter the nucleus remains poorly understood because neither contains a nuclear localization signal (NLS), an amino acid sequence tagging proteins for nuclear transport. p300 is an NLS-containing large scaffold protein, so we hypothesized that SMAD2/3 and TAZ may undergo nuclear import through complexing with p300. Coimmunoprecipitation, immunofluorescence, and nuclear fractionation assays revealed that TGF-β1 promoted binding of SMAD2/3 and TAZ to p300 and that p300 inactivation disrupted TGF-β1-mediated SMAD2/3 and TAZ nuclear accumulation. Deleting the p300 NLS blocked TGF-β1-induced SMAD2/3 and TAZ nuclear transport. Consistently, p300 inactivation suppressed TGF-β1-mediated HSC activation and transcription of genes encoding tumor-promoting factors, such as connective tissue growth factor, Tenascin C, Periostin, platelet-derived growth factor C, and fibroblast growth factor 2, as revealed by microarray analysis. Chromatin immunoprecipitation-real-time quantitative PCR showed that canonical p300-mediated acetylation of histones also facilitated transcription in response to TGF-β1 stimulation. Interestingly, although both TGF-β1-mediated and stiffness-mediated HSC activation require p300, comparison of gene expression data sets revealed that transcriptional targets of TGF-β1 were distinct from those of stiffness-p300 mechanosignaling. Lastly, in tumor/HSC coinjection and intrasplenic tumor injection models, targeting p300 of activated-HSC/myofibroblasts by C646, short hairpin RNA, or cre-mediated gene disruption reduced tumor and liver metastatic growth in mice. Conclusion: p300 facilitates TGF-β1-stimulated HSC activation by both noncanonical (cytoplasm-to-nucleus shuttle for SMAD2/3 and TAZ) and canonical (histone acetylation) mechanisms. p300 is an attractive target for inhibiting HSC activation and the prometastatic liver microenvironment.

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