Synectin promotes fibrogenesis by regulating PDGFR isoforms through distinct mechanisms
Mary C. Drinane, … , Sheng Cao, Vijay H. Shah
Mary C. Drinane, … , Sheng Cao, Vijay H. Shah
Published December 21, 2017
Citation Information: JCI Insight. 2017;2(24):e92821. https://doi.org/10.1172/jci.insight.92821.
View: Text | PDF
Research Article Hepatology

Synectin promotes fibrogenesis by regulating PDGFR isoforms through distinct mechanisms

Abstract

The scaffold protein synectin plays a critical role in the trafficking and regulation of membrane receptor pathways. As platelet-derived growth factor receptor (PDGFR) is essential for hepatic stellate cell (HSC) activation and liver fibrosis, we sought to determine the role of synectin on the PDGFR pathway and development of liver fibrosis. Mice with deletion of synectin from HSC were found to be protected from liver fibrosis. mRNA sequencing revealed that knockdown of synectin in HSC demonstrated reductions in the fibrosis pathway of genes, including PDGFR-β. Chromatin IP assay of the PDGFR-β promoter upon synectin knockdown revealed a pattern of histone marks associated with decreased transcription, dependent on p300 histone acetyltransferase. Synectin knockdown was found to downregulate PDGFR-α protein levels, as well, but through an alternative mechanism: protection from autophagic degradation. Site-directed mutagenesis revealed that ubiquitination of specific PDGFR-α lysine residues was responsible for its autophagic degradation. Furthermore, functional studies showed decreased PDGF-dependent migration and proliferation of HSC after synectin knockdown. Finally, human cirrhotic livers demonstrated increased synectin protein levels. This work provides insight into differential transcriptional and posttranslational mechanisms of synectin regulation of PDGFRs, which are critical to fibrogenesis.

Authors

Mary C. Drinane, Usman Yaqoob, Haibin Yu, Fanghong Luo, Thomas Greuter, Juan P. Arab, Enis Kostallari, Vikas K. Verma, Jessica Maiers, Thiago Milech De Assuncao, Michael Simons, Debabrata Mukhopadhyay, Tatiana Kisseleva, David A. Brenner, Raul Urrutia, Gwen Lomberk, Yandong Gao, Giovanni Ligresti, Daniel J. Tschumperlin, Alexander Revzin, Sheng Cao, Vijay H. Shah

×

Figure 3

Knockdown of Synectin in HSC results in the downregulation of canonical profibrogenic gene expression networks via histone modifications and p300.

Options: View larger image (or click on image) Download as PowerPoint
Knockdown of Synectin in HSC results in the downregulation of canonical ...
(A and B) The histone modification H3K27ac is associated with activation of gene expression and was measured at the PDGFR-β gene locus by ChIP using H3K27ac antibody (A) and Western blot (B). A reduction in acetylation of histone H3 at lysine 27 was observed in synectin-knockdown hHSCs compared with control cells, n = 6 (A), n = 3 (B). The efficiency of shRNA-mediated knockdown of synectin was also shown. (C) shRNA-mediated knockdown of p300 decreased the protein expression of PDGFR-β and H3K27ac in hHSCs as shown by Western blot, n = 3. (D) hHSC were stained using p300 antibody (green) with background DAPI stain to show nucleus (blue). Decreased nuclear localization of p300 was observed in synectin-knockdown hHSCs. White broken line was used to define the outline of the cell. (E) Cell lysates and nuclear fractions from synectin-knockdown hHSC showed a reduction in p300 protein levels by Western blot in the nuclear fraction only. Densitometry was analyzed using ImageJ and is depicted in the graph below, n = 3. (F and G) ChIP using H3K4me3 and H3K27me3 antibodies showed decreased methylation of histone H3 at lysine 4 and increased methylation of histone H3 at lysine 27 of PDGFR-β promoter after synectin knockdown, further indicative of repressed gene transcription. All data are expressed as mean ± SEM. Scale bar: 20 μm. One-way ANOVA with Bonferroni’s multiple comparison test were used to analyze groups for statistical significance (**P < 0.001, ***P < 0.0001).