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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.
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Research Article Hepatology

Synectin promotes fibrogenesis by regulating PDGFR isoforms through distinct mechanisms

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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 1

Synectin deletion from HSC attenuates hepatic fibrogenesis in vivo.

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Synectin deletion from HSC attenuates hepatic fibrogenesis in vivo.
Mice...
Mice with HSC selective deletion of synectin (Colcre/Synectinfl/fl) and their control littermates (Synectinfl/fl) were treated with either olive oil (vehicle) or CCl4 via i.p. injections twice a week for 6 weeks. The livers were then harvested and prepared for analysis through isolation of mRNA or protein, or fixation of liver tissue for immunostaining and Sirius red analysis. (A) qPCR for collagen-1 mRNA levels showed a significant reduction in Colcre/Synectinfl/fl mice liver after CCl4 injection, n = 4–7 per group. (B) Collagen content was reduced in Colcre/Synectinfl/fl mice after CCl4 injection, as demonstrated by assessing hepatic hydroxyproline assay, n = 4–7 per group. (C) Liver sections (5 μm) were stained with Sirius red to represent the fibrotic strands correlating with the degree of fibrosis. Additional liver sections were stained with antibodies against the fibrotic proteins collagen-1, PDGFR-α, PDGFR-β, and α-SMA (green) in conjunction with nuclear costaining with DAPI (blue), representative images shown. Staining revealed decreased expression of collagen-1, PDGFR-α, PDGFR-β, and α-SMA in Colcre/Synectinfl/fl mice after CCl4 injection compared with control littermates. Quantitation was performed using ImageJ, with fold change displayed on the micrographs and graphs located in Supplemental Figure 2, A–E; n = 3–5. Scale bars: 200 μm. (D) Lysates from whole mouse liver were used to assess protein levels of PDGFR-α, PDGFR-β, and synectin in Colcre/Synectinfl/fl mice after CCl4 injection. Samples were run on the same gel but were noncontiguous. n = 3–4. Quantification was performed using ImageJ, with the densitometric values displayed below the blots and graphs located in Supplemental Figure 2F. All data are displayed as mean ± SEM (*P < 0.05). Each dot in the scatter plot indicates an individual animal in each of the panels. One-way ANOVA with Bonferroni’s multiple comparison tests were used to analyze groups for statistical significance (*P < 0.05, **P < 0.001, ***P < 0.0001).

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