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.

Keywords: Hepatology; Signal transduction.

Figure 1. Synectin deletion from HSC attenuates hepatic fibrogenesis in vivo.

Mice with HSC selective deletion of synectin (Col^cre/Synectin^fl/fl) and their control littermates (Synectin^fl/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 Col^cre/Synectin^fl/fl mice liver after CCl₄ injection, n = 4–7 per group. (B) Collagen content was reduced in Col^cre/Synectin^fl/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 Col^cre/Synectin^fl/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 Col^cre/Synectin^fl/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).

Figure 2. The HSC activation pathway is highly regulated by synectin through transcriptomic analysis by NextGen sequencing (mRNA-Seq).

(A) Heatmap of whole genome gene expression depicting differences in the expression profile between control and synectin-knockdown human HSC (hHSC) of all genes with a logFC > 1.5 of < –1.5. (B) Ingenuity pathway analyses (IPA, Qiagen) showed that the fibrotic pathway was differentially regulated between the groups. FDR < 0.05, P < 0.05. Regulation of specific genes within the hepatic fibrosis pathway are shown, with PDGFR-β highlighted. (C) qPCR from hHSC with and without synectin knockdown demonstrated a reduction in PDGFR-β mRNA. Results are from 3 independent experiments. Student’s unpaired, 2-tailed t test was used to analyze the differences between groups for statistical significance (*P < 0.05).

Figure 3. Knockdown of Synectin in HSC results in the downregulation of canonical profibrogenic gene expression networks via histone modifications and p300.

(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).

Figure 4. Synectin regulates PDGFR protein levels.

(A) shRNA-mediated synectin knockdown in hHSC decreases both PDGFR-α and PDGFR-β protein levels as depicted by Western blot. Densitometry located in the graph below, n = 5. (B) Control or synectin-knockdown hHSCs were treated with the proteosomal inhibitor MG132 (25 μM) for 4 hours; however, in PDGFR-α or -β, protein levels were not increased by proteosomal inhibitor MG132 (n = 3) in hHSC with synectin knockdown. (C and D) In contrast, PDGFR-α levels were increased by inhibition of autophagy by bafilomycin (10 μM, overnight) and 3-MA (1 μM, overnight) in both control and synectin-knockdown hHSC, n = 3. (E) Inhibition of autophagy by ATG5 siRNA increased PDGFR-α protein levels in hHSCs, n = 3. (F) LX-2 cells were transfected with a LacZ control plasmid, a plasmid encoding WT NRP-1 (NRP-1-WT), or a plasmid encoding a NRP-1 mutant lacking the SEA domain (NRP-1-ΔSEA), and PDGFR-α protein levels were determined by Western blot. Overexpression of WT NRP-1 enhanced PDGFR-α expression; however, the NRP-1 mutant resulted in decreased PDGFR-α protein levels, n = 3. (G) siRNA-mediated NRP-1 knockdown in hHSC showed reduction in PDGFR-α protein levels without affecting PDGFR-β protein levels as shown by Western blot, n = 7. (H) Bafilomycin (10 μM) treatment increased PDGFR-α protein levels following siRNA-mediated NRP-1 knockdown as observed by Western blot, n = 3. (I) hHSCs were transfected with NRP-1 siRNA or a control, followed by treatment with MG132 (25 μM). PDGFR-β protein levels were not increased by MG132 treatment in NRP-1–knockdown cells, n = 3. Samples were run on the same gel but were noncontiguous in B–E and I. Data are expressed as mean ± SEM. Densitometry for all experiments was analyzed by ImageJ and are depicted below their respective blots. 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).

Figure 5. PDGFR-α undergoes selective autophagy and degradation.

(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).

Figure 6. PDGFR-α autophagy requires the ubiquitination of specific lysine residues.

(A) LOGO depicting sequence homology of PDGFR-α and PDGFR-β at known PDGFR-α ubiquitination sites (Weblogo 3.5). (B–D) LX2 cells were transduced with an adenovirus encoding a PDGFR-β mutant 606/971 flag–tagged construct or pShuttle vector, and treated overnight with bafilomycin (10 μM, B and C) in serum-starved media. Overexpression of the PDGFR-β mutant 606/971 in LX2 cells resulted in a 2-fold increase of PDGFR-β mutant after incubation with bafilomycin (B) and increased colocalization with autophagosome marker LC3b (C) and p62 (D). Two images were obtained from 3 separate wells for a total of 6 fields taken at 63×. Pearson’s coefficient was calculated by JoCIP plug-in in ImageJ for panels C and D. Scale bar: 10 μm. (E) Mutation of the PDGFR-α at K971 prevented its degradation after PDGF stimulation, n = 3. Samples were run on the same gel but in noncontiguous lanes. Data are expressed as mean ± SEM. One-way ANOVA with Bonferroni’s multiple comparison test was used to analyze groups for statistical significance (**P < 0.001, ). Student unpaired t test was used to analyze the differences between 2 groups (***P < 0.0001).

Figure 7. Synectin knockdown inhibits PDGF-stimulated migration and proliferation of HSC.

(A) Ingenuity Pathway Analysis (IPA) of cellular function demonstrated a reduction in genes associated with cellular movement and proliferation in synectin-knockdown hHSC. (B and C) shRNA-mediated synectin knockdown in LX2 reduced migration as observed by scratch assay (B), and proliferation as measured by MTS assay in response to PDGF (C), n = 3. For B, 3 images were obtained from each scratch, n = 3. (D) Synectin expression was knocked down by shRNA (versus control shRNA) and migration assessed using a Boyden chamber in the presence of multiple PDGF ligands, n = 3, magnification, 10x. The number of cells per field was quantified and is displayed in the adjacent graph. All data are expressed as mean ± SEM of at least 3 independent experiments. 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).

Figure 8. Synectin is upregulated in murine pulmonary fibrosis, human pulmonary fibrosis, and human cirrhosis.

(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).