Depletion of tropomyosin 1.6 alleviates TGF-β1-induced fibroblast activation by promoting the α-SMA-MMP9 matrix-degrading structure

Figures

• 

  Figure 1. Actomyosin contractility is required for TGF-β1-induced myofibroblast activation under soft collagen gel (A–J) NRK-49F cells (A) and primary renal fibroblast cells (B) were cultured on collagen gel-coated dish (Co) and collagen gel (CG) with and without TGF-β1 (10 ng/mL) for 24 h. The protein levels of myofibroblast marker (α-SMA) and actomyosin-associated contractile units (CUs; myosin IIA, IIB, and Tpm1) in NRK-49F cells (C, G) and primary renal fibroblasts (E, H) were analyzed by western blotting and quantification results of western blotting are shown in (D), (F), (I), and (J). The arrow in (G) and (H) shows the different isoforms of Tpm1 in NRK-49F cells and primary fibroblasts. Data are represented as mean ± SEM; n = 5 (D), 3 (F), 8 (I), 3 (J) independent experiments were performed; two-way ANOVA; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns (not significant). Scale bars, 20 μm. (K–N) Protein levels of α-SMA, N-cadherin, β1-integrin, and Tpm1 in NRK-49F cultured on CG with or without the co-treatment of actomyosin blockers (BB-blebbistatin, ML7, Y27632) for 24 h, and the respective quantification results are shown in (M) and (N). Data are represented as mean ±…

• 

  Figure 2. Silencing of the renal fibroblast-dominant isoform, Tpm1.6, suppresses TGF-β1-induced myofibroblast activation NRK-49F cells and primary renal fibroblasts were cultured on CG with or without TGF-β1 for 24 h, and total RNA was extracted and analyzed by conventional PCR (B, C) or quantitative PCR (E, F). NRK-49F cells received liposome carried biogenic RNA interference against exon 2b and 9d of Tpm1, and then were cultured on Co or CG with or without TGF-β1 for 24 h. Total RNA was extracted and analyzed by quantitative PCR (G), whereas cell lysates were analyzed by western blotting (H, I). (A) Schematic graph displays Tpm1 isoforms derived from alternative promoters and splicing exons. (B and C) PCR results represent the levels of different transcripts of Tpm1 in NRK-49F cells (B) and primary renal fibroblasts (C). Isoform1.6/1.7 of Tpm1 (Tpm1.6/1.7) was specifically upregulated by TGF-β1 in renal fibroblasts. (D) Forward/reverse primers and Taqman probes conjugated with fluorescent reporter were used for quantitative PCR studies in order to detect transcripts of specific Tpm1.6 or Tpm1.7. To identify Tpm1.6 and 1.7, we utilized 6FAM-BHQ1-and TAMRA-BHQ2-conjugated Taqman probes…

• 

  Figure 3. Silencing Tpm1.6 through exon 2b-targeted RNA interference prevents TGF-β1-induced cell contractility but promote collagen degradation (A and B) Silenced-control and Tpm1.6-silenced NRK-49F cells were cultured in CG with or without TGF-β1 for 24 h and the collagen gels were released for indicated time to visualize the gel contraction/degradation, and the quantification results are shown in (B). Cells harboring exon 2b-silencing showed most decreased gel area without increase opaqueness at 4 h and strong degradation of gel at 48 h, indicating an active process of gel digestion is present. Data are represented as mean ± SEM; n = 3 independent experiments were performed; RM one-way ANOVA; ∗p < 0.05. (C–E) Cell contractility is measured by a micropost array detector. NRK-49F cells harboring biogenic RNA interference against Tpm1 were cultured on PDMS-based microposts with or without TGF-β1 for 24 h. Each post bends in response to the applied traction force as calculated by finite element model analysis. The force map represents the contractility distribution of silenced-control and Tpm1.6-silenced cells, and the color bar shows the force level (nN). The total cell force and force per…

• 

  Figure 4. Tpm1.6-silencing reduces TGF-β1-induced augmentation of cell contractility and promote collagen deformation, which is mediated by MMPs (A–C) Cell contractility is measured by a confocal plus atomic force microscope (AFM) system. LifeAct-expressing NRK-49F cells harboring biogenic RNA interference against Tpm1 were cultured on FITC-conjugated CG for 4 days with or without TGF-β1. Cells (LifeAct, red) and collagen fibrils (FITC, green) were visualized under a confocal microscope and then subjected to co-axis atomic force microscopic analysis to assess the structure and rigidity of cells and peri-cellular collagen fibrils. AFM-Height images show the relative height information (μm) in topography. AFM-3D images display the surface morphology in three-dimensional presentation. AFM+Confocal indicates the merge of images between the confocal microscope and AFM-Height. Quantification of the rigidity of cells and peri-cellular collagen fibrils was shown in (B) and (C), respectively. Data are represented as mean ± SEM; n = 9–72 cells or fibers; two-way ANOVA; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Scale bars, 20 μm. (D–G) Silenced-control and Tpm1.6-silenced NRK-49F cells…

• 

  Figure 5. Tpm1.6-silencing triggers the formation of canonical podosomes and α-SMA dots upon TGF-β1 induction Silenced-control and Tpm1.6-silenced NRK-49F cells were cultured on CG with or without TGF-β1 for 24 h, and the cultured cells underwent immunofluorescence studies (A-F), transmission electron microscope (TEM) inspection (H-J), and the lysates were harvested for immunoprecipitation (G). (A) Canonical podosomes were represented by cortactin (red), phalloidin (green) staining; non-canonical podosomes were shown by α-SMA dots (cyan). Tpm1.6-silenced NRK-49F cells displayed more canonical and non-canonical podosomes with TGF-β1 induction. Scale bar, 20 μm. (B and C) Quantitative results show the size (μm2) of canonical podosomes and α-SMA dots in silenced-control and Tpm1.6-silenced cells. Data are represented as mean ± SEM; n = 6–11 cells; two-way ANOVA; ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (D) Fluorescent images showed the localization of α-SMA (green) and MMP9 (red), representing MMP9 enrichment in non-canonical podosomes in Tpm1.6-silenced cells. Scale bars, 20 μm. (E) Quantitative results show colocalization of MMP9 and α-SMA in silenced-control and Tpm1.6-silenced NRK-49F cells with or…

• 

  Figure 6. Tpm1.6-silencing promotes collagen digestion upon TGF-β1 stimulation via induction of α-SMA puncta Silenced-control and Tpm1.6-silenced NRK-49F cells were cultured on CG for 24 h, and then the CG was either released (A) or was treated with MMP inhibitors (GM600 or MMP9i, B-C) for 48 h with or without TGF-β1. Silenced-control and Tpm1.6-silenced cells were transfected with shSMA (D) or siCortactin (E) and cultured on unreleased CG with or without TGF-β1 for 48 h. The culture media was harvested for Western blotting analysis to detect collagen degradation. (A and F) Culture media collected from cells cultured on unreleased or released CG. Western blotting results showed Tpm1.6-silencing markedly increased collagen fragmentation (smaller than the size of monomer) upon TGF-β1. The release of CG to reduce traction force triggered the collagen degradation, and the quantification is shown in (F). Data are represented as mean ± SEM; n = 3 independent experiments were performed; two-way ANOVA; ∗p < 0.05, ∗∗∗∗p < 0.0001. (B, C, G, H) Western blotting results showed the effects of GM6001 (B) or MMP9 inhibitor (C) on TGF-β1-induced collagen degradation in Tpm1.6-silenced cells cultured on…

• 

  Figure 7. Mesenchymal-targeted silencing of Tpm1.6 by induction of promoters of Foxd1 and Acta2 attenuates UUO-induced renal fibrosis To conditionally silence Tpm1.6 in the mesenchymal population (Foxd1-lineage), we generated a transgenic mice model by mating between mesenchymal population-driven cre mice and the floxed mice containing Acta2-driven Tpm1.6-targeted miRNA (same sequence as the exon 2b-targeted RNA interference in vitro). (A) Schematic shows the generation of FoxD1GC−/+; Acta2TpmKD mice. Acta2 promoter-driven FusionRed, SecNLuc, and Tpm1.6-targeted miRNA are achieved in Foxd1-lineage cells by the cre recombinase-mediated excision of a loxP-flanked sequence. EmGFP is expressed in Acta2-expressing cells of FoxD1GC−/−; Acta2TpmKD mice or in Acta2-positive non-Foxd1-lineage cells of FoxD1GC−/+; Acta2TpmKD mice. (B) Detection of SecNLuc (RLU), which was secreted into plasma derived from FoxD1GC−/−; Acta2TpmKD and FoxD1GC−/+; Acta2TpmKD mice with or without UUO surgery for 3 or 7 days. Data are represented as mean ± SEM; n = 3 mice. (C) Fluorescent images showed the EmGFP and FusionRed-expressed population in kidney tissues. EmGFP is expressed at small arteries in sham kidneys and…