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.
Bibliographical noteFunding Information:
We thank Elyse Froehling, Aaron Becker, and Juan Abrahante, Ph.D., at the University of Minnesota Genomic Center for performing microarray, RNA-seq, and data analysis; Dr. Makiko Fujii at Aichi Cancer Center Research Institute, Japan, for providing p300 plasmid; and Dr. Tatiana Kisseleva at UCSD for collagen1A1-cre transgenic mouse line.
© 2019 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of American Association for the Study of Liver Diseases.