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 8

Synectin is upregulated in murine pulmonary fibrosis, human pulmonary fibrosis, and human cirrhosis.

Options: View larger image (or click on image) Download as PowerPoint
Synectin is upregulated in murine pulmonary fibrosis, human pulmonary fi...
(A) mRNA was harvested from murine lungs treated with bleomycin to induce fibrosis or a vehicle control. qPCR was performed to analyze mRNA expression of synectin, PDGFR-β, or α-SMA. We observed increased synectin, PDGFR-β, and α-SMA mRNA in the bleomycin exposed samples, n = 3. (B) Pulmonary fibroblasts were isolated from patients diagnosed with Idiopathic Pulmonary Fibrosis. mRNA was harvested from these fibroblasts and analyzed by qPCR for synectin expression. qPCR revealed a statistically significant increase in synectin mRNA levels compared with control samples, n = 4. Tissue was obtained from patients with liver cirrhosis or matched controls. mRNA and cell lysates were isolated from these samples and analyzed by qPCR (C) or Western blot (D, samples were run on the same gel but were noncontiguous). Both Synectin and α-SMA were increased at the mRNA and protein level in cirrhotic patients compared with controls. (Control, n = 5; cirrhosis, n = 6 for qPCR. Control, n = 8; cirrhosis, n = 5 for Western blot.) (E) Proposed mechanism of synectin regulation of the PDGFR-α and -β isoforms is shown in the illustration. Data are expressed as mean ± SEM. Student’s unpaired t test was used to analyze the differences between groups for statistical significance (*P < 0.05, **P < 0.001, ***P < 0.0001).