Fibulin-6 regulates pro-fibrotic TGF-β responses in neonatal mouse ventricular cardiac fibroblasts

Figure 1. Attenuated stress fiber formation in fibulin-6 KD cells after TGF-β stimulation. Fibroblasts isolated from neonatal mouse hearts and stimulated with TGF-β were stained with fluorescently labelled phalloidin for stress fibers. (a) scr-siRNA transfected nCF display increased stress fiber formation after TGF-β stimulation compared to non stimulated cells. In fibulin-6 KD cells stress fiber formation after TGF-β stimulation is almost absent (scale bar = 100 μm). (b) Total % of cells expressing stress fibers normalized to total number of cells. (n = 4, p < 0.0001) (c) Quantitative analysis of fluorescence intensity displayed by the stress fibers in the representative cells shown in panel (d) where each peak value correspond to 3 regions of the same cell with 6 cells analyzed per picture (n = 5, p < 0.0001). Differences in the mean value between groups are determined by two tailed, non-parametric Mann-Whitney U test. (d) Fluorescence intensities of the line drawn across the cell shown in corresponding panels below were quantified using Image J software. Asterisks demarcate cells being quantified and their corresponding line graphs below.

Figure 2. TGF-β mediated collagen contraction is reduced in fibulin-6 KD CF. Collagen gels carrying either scr-siRNA or fibulin-6 KD 3T3 CF were exposed to TGF-β to induce contraction. (a) A representative picture of collagen gels 48 h after TGF-β stimulation is shown. The area of the gels (marked by the dotted circle around the collagen gel) before and after stimulation was measured using Image J and is represented in the corresponding graph. After TGF-β stimulation the size of collagen gels carrying control cells is significantly reduced while collagen gels carrying fibulin-6 KD cells display no contraction (n = 3, p < 0.001, nonparametric Mann-Whitney U test). The experiment was repeated three times in quadruplicates. (b) Photographs of collagen gels stained for phalloidin after 48 hours of TGF-β stimulation (scale bar = 100 μm). In scr-siRNA transfected 3T3 CF the formation of cellular networks during contraction can be observed, while actin cytoskeletal network and cell-cell contact in fibulin-KD CF are absent.

Figure 3. Pro-fibrotic gene expression in TGF-β induced fibroblasts. nCF transfected with scr siRNA or Fibulin-6 siRNA, after being exposed to TGF-β were subjected to RNA or protein extraction for real-time PCR or western blotting respectively. (a) Collagen I α1 and (b) CTGF mRNA expression is increased after TGF-β stimulation in scr- but not in fibulin-6-siRNA transfected nCF (n = 7, p < 0.05, nonparametric Mann-Whitney U test). Real-time RT-PCR signals were normalized to hypoxanthine phosphoribosyl-transferase (HPRT) gene expression and data are expressed as 2−ΔΔCT. (c and d) Densitometric analysis of western blots revealed upregulation of (c) collagen I α1 and (d) CTGF upon TGF-β stimulation in scr-siRNA transfected nCF. In fibulin-KD nCF no upregulation could be observed (n = 5, p < 0.05, nonparametric Mann-Whitney U test).

Figure 4. Nuclear localization of SMAD3/4 in TGF-β stimulated CF. nCF transfected with scr siRNA or Fibulin-6 siRNA, with and without TGF-β stimulation are fixed and stained for SMAD3 expression or are processed for nuclear lysate preparation. (a) Cells are stained with SMAD3 (red) and DAPI (blue) (Scale bar = 100 μm). In scr siRNA transfected CF translocation of SMAD3 into the nucleus can be observed upon TGF-β stimulation. Arrows indicate SMAD3 positive nuclei. In fibulin-6 KD CF SMAD3 staining is almost absent in nuclei. High magnification inserts show nuclear SMAD3 staining in scr siRNA transfected CF and cytoplasmic SMAD3 staining in fibulin-6 siRNA transfected CF. A corresponding graph shows quantitative measurement of SMAD3 positive nuclei compared to the total number of cells (n = 3, p < 0.0001, nonparametric Mann-Whitney U test). The experiment was repeated three times with 10 pictures analyzed each time. Western blot showing (b) SMAD3 and (c) SMAD4 nuclear signal in TGF-β stimulated nuclear lysates compared to the non-stimulated set. Westerm blot and corresponding densitometric analysis show increased localization of (b) SMAD3 and (c) SMAD4 in nuclear extracts after TGF-β…

Figure 5. Effect of fibulin-6 KD on Non-SMAD pathways. Fibulin-6 siRNA and scr-siRNA transfected cells with and without TGF-β treatment for 24 h are processed for WB analysis. (a) pERK compared to total ERK, which represents a constitutive increase upon KD of fibulin-6 in comparison to control cells (n = 4, p < 0.05, nonparametric Mann-Whitney U test) and (b) pAKT compared to total AKT (n = 4, p < 0.05, nonparametric Mann-Whitney U test), which doesn’t show any significant increase upon TGF-β induction in KD cells compared to control.

Figure 6. TGFRI and TGFRII association in fibulin-6 KD cells upon TGF-β stimulus. scr siRNA or fibulin-6 siRNA transfected nCF, after TGF-β stimulation are subjected to (a) FACS analysis. No difference in the amount of TGFβRI and TGFβRII on the surface of control and fibulin-6 KD cells was observed. (b) Immuno-precipitation of TGFRII from cell lysates of scr transfected and TGF-β stimulated cells, shows more accumulation of TGFRI after western blotting compared to non TGF-β treated nCF. (c) Immuno-precipitation of TGFRII followed by WB for TGFRI from cell lysates of scr transfected or fibulin-6 KD cells after TGF-β stimulation display reduced association of receptors in fibulin-6 KD condition. The control input lanes shows no difference and IgG control is also clean (n = 4, p < 0.05). (d) Phosphorylation status of TGFRI was analyzed by western blot using phospho-TGFRI specific antibody. Densitometric analysis of western blot display decreased phosphorylation of TGFRI in fibulin-6 KD cells after TGF-β stimulation compared to control cells (n = 5, p < 0.01, nonparametric Mann-Whitney U test).