Synectin promotes fibrogenesis by regulating PDGFR isoforms through distinct mechanisms
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
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
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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

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Figure 5

PDGFR-α undergoes selective autophagy and degradation.

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PDGFR-α undergoes selective autophagy and degradation. (A) Coimmunopreci...
(A) Coimmunoprecipitation (Co-IP) was performed from hHSC lysates using an antibody against PDGFR-α, and the recovered proteins were analyzed by Western blot, n = 4. (B) hHSC were transduced with adenoviral flag–tagged PDGFR-α and PDGFR-β constructs, followed by treatment with or without bafilomycin (10 μM) and coimmunostained using flag and p62 antibodies. Imaging of hHSC showed colocalization of p62 with FLAG–PDGFR-α, but not FLAG–PDGFR-β. Colocalization was measured using the Pearson’s coefficient, calculated by JoCIP plug-in in ImageJ, and it is displayed in the graph below. (C) Synectin was knocked down in hHSCs using shRNA, followed by treatment with PDGF-bb (10 ng/ml). Lysates were harvested and a Co-IP performed with an antibody against PDGFR-α. The association between PDGFR-α and p62 increased in cells with synectin knockdown treated with PDGF-bb, as measured by increased ratio of p62 to PDGFR-α after synectin knockdown, n = 3. (D) hHSC were transduced with adenoviral flag–tagged PDGFR-α construct and coimmunostained using flag and p62 antibodies. Representative pictures are shown. Colocalization was determined by measuring the Pearson’s Coefficient calculated by JoCIP plug-in in ImageJ, and it is displayed in the adjacent graph. (E) Synectin-knockdown cells were transduced with an adenoviral flag–tagged PDGFR-α construct and transfected with LC3b-GFP plasmid. Colocalization was determined by measuring the Pearson’s coefficient calculated by JoCIP plug-in in ImageJ, and it is displayed in the adjacent graph, n = 3. For all colocalization experiments, 2 images were obtained from 3 separate wells for a total of 6 fields taken at 63×. All data are expressed as mean ± SEM. Scale bars: 10 μm. Samples were run on the same gel but were noncontiguous in A and C. One-way ANOVA with Bonferroni’s multiple comparison test was used to analyze groups for statistical significance (*P < 0.05,**P < 0.001, ***P < 0.0001). Student’s unpaired t test was used to analyze the differences between 2 groups (*P < 0.05, **P < 0.001).