Figure 3
cANGPTL4 regulates COL1A2 and COL3A1 expression in fibroblasts via a bHLH-dependent signaling mechanism. (a) Immunoblot analysis of indicated proteins from fibroblasts treated with cANGPTL4 (12 µg/mL) in the presence or absence of TGFβ1 (10 ng/mL). β-tubulin, as loading control, was from the same samples. (b,d) Chromatin immunoprecipitation (ChIP) of SBE sites in the human COL1A2 gene promoter region and E-box sites in the human COL1A2 and COL3A1 gene promoter regions using phospho-Smad3 antibodies and scleraxis (ScxA) antibodies respectively. The gene sequences spanning the SBE and E-box sites and a random control sequence were analyzed by PCR from immunoprecipitated chromatin from fibroblasts treated with TGFβ1 in the presence or absence of cANGPTL4 (cANG). Pre-immune serum was used as a control. PCR was performed using chromatin before immunoprecipitation as input. M, 100 bp DNA ladder. (c) A schematic diagram showing the relative positions of Enhancer boxes (E-Boxes) and Smad binding elements (SBE) on the human COL1A2 and COL3A1 proximal promoters. Sequences of E-Boxes and SBE are shown. ChIP PCR primers designed specifically to bind scleraxis along this proximal promoter region are indicated. (e) Relative mRNA expression of ID3 in fibroblasts treated with 12 µg/mL of cANGPTL4 and lysed for mRNA at the indicated time points post-treatment. (f) Relative mRNA expression of ID3 in control (Fctrl) and ID3-knockdown (F∆ID3) fibroblasts treated for 16 h with 10 ng/mL of TGFβ1 (control) or TGFβ1 plus 12 µg/mL of cANGPTL4. (g–i) Immunodetection of ID3, COL1A2 and COL3A1 in human dermal fibroblasts subjected to the indicated treatments. (j,k) Immunodetection of ScxA and E47 in the immunoprecipitate of ID3. β-tubulin or IgG, as loading controls, were from the same samples/immunoprecipitates. Cropped images are representative of immunoblots from n = 5 independent experiments; full-length immunoblots scanned using the Li-Cor Odyssey® CLx system can be found in Supplementary Fig. S4a–g; values represent mean ± SD from n = 5 independent experiments. *P < 0.05, **P < 0.01,***P < 0.001 (Mann-Whitney U-test).
