Cell autonomous ANTXR1-mediated regulation of extracellular matrix components in primary fibroblasts

Abstract

Our previous studies of Antxr1 knockout mice suggested that fibrotic skin abnormalities in these mice are associated with increased VEGF signaling. Here, based on studies of primary fibroblasts isolated from skin of Antx1+/+ and Antxr1-/- mice at embryonic stage E17.5 and postnatal day P49, we conclude that increased Col1a1 and Fn1 expression in Antxr1-deficient fibroblasts is partly mediated by a cell-autonomous ANTXR1-dependent mechanism. In turn, this may act in parallel with VEGF-dependent regulation of collagen type I and fibronectin production. We demonstrate that shRNA mediated knockdown of VEGF in Antxr1-/- fibroblasts reduces Col1a1 and Fn1 expression to below control levels, and these are restored by exogenous addition of recombinant VEGF. In addition, the increase in protein levels of collagen type I and fibronectin in mutant cells is blocked by VEGF neutralizing antibody. However, expressing the longest isoform of ANTXR1 (sv1) in mutant fibroblasts decreases levels of Ctgf, Col1a1 and Fn1 transcripts, but has no effect on VEGF expression. Taken together, our data suggest that the increased matrix production in Antxr1- deficient fibroblasts primarily occurs via a CTGF-dependent pathway and that other ANTXR1-associated mechanisms contribute to VEGF-dependent increase of collagen type I and fibronectin expression. Our findings provide a basis for further studies of novel ANTXR1-dependent connective tissue homeostatic control mechanisms in healthy individuals, patients with organ fibrosis, and patients with GAPO syndrome.

Keywords: ANTXR1/TEM8 GAPO syndrome; CTGF; Collagen type I; Extracellular matrix accumulation; Fibroblasts; Infantile hemangioma; Skin fibrosis; VEGF.

Fig. 1

Loss of ANTXR1 is associated with increased transcript levels of ECM components. (A) Western blotting of fibroblasts isolated from Antxr1+/+ and Antxr1−/− mice at P49. (B, C) Bar graphs show average transcript levels (fold increase) in lysates of control and mutant cells at E17.5 and P49. (*P<0.05, n = 6 per group).

Fig. 2

Mutant fibroblasts express high VEGF levels. (A) Bar graphs show average transcript levels (fold increase) in control and mutant fibroblasts isolated from P49 mice. (*P<0.05, n = 6 per group). (B) VEGF protein levels were assessed by ELISA (*P<0.05, n = 4 per group). (C) Immunoblotting of lysates from control and mutant fibroblasts of P49 mice.

Fig. 3

VEGF induced transcript levels of collagen α1(I) and fibronectin. (A) VEGF protein levels were assessed by ELISA (*P<0.05, n = 4 per group) in shControl and shVegf–treated fibroblasts (*P<0.05, n = 4 per each group). (B) Bar graphs showing average transcript levels (fold increase) of shControl and Vegf knockdown cell lines (*P<0.05, n = 4 per group). (C) RT-PCR data show transcript levels after treatment of cells with 10 ng/ml of recombinant VEGF (rVEGF). (*P<0.05, n = 4 per group).

Fig. 4

VEGF regulates collagen α1(I) and Fn1 protein levels. (A, B) Representative immunofluorescence images and data analyses of cells treated with 1μg/ml of VEGF neutralizing antibody or with 25 ng/ml of rVEGF. (n= 3; *, ^#, P<0.01; **, ^##, P<0.05; where *,** vs. wt (Antxr1+/+) and ^{#, ##} vs. ko (Antxr1−/−)). Scale bar is100 μm; veh-vehicle. For each condition, 3–6 fields from duplicate plates were used for analysis, covering a total of 180 to 240 cells. The percentage of areas stained with antibodies against collagen α1(I) or fibronectin, was normalized to 100 cells.

Fig. 5

Re-expression of ANTXR1(sv1) restored levels of Col1a1 and Fn1 transcripts. (A) Assessment of sv1 transfection efficiency by western blotting of lysates from P49 control (wt) and Antxr1 null (ko) fibroblasts treated with vehicle or sv1pcDNA3-ANTXR1-sv1-HA (sv1) expression plasmid (ko/sv1). Numbers above the bands represent relative expression levels of ANTXR1 and ANTXR1-sv1 proteins normalized to β–actin. (B) Bar graphs showing average transcript levels (fold increase) of Ctgf, Col1a1, Fn1 and Vegf-a (*P<0.01; ns-not statistically significant, n = 4 per group). (C) Immunofluorescence staining for collagen α1(I) and fibronectin in cultures of fibroblasts from P49 control (wt) mice and fibroblasts from Antxr1 null (ko) mice treated with vehicle (ko) or sv1- containing expression plasmid (ko/sv1). Scale bar is 50 μm. Representative images are from triplicates of 2 independent experiments. (D) VEGF protein levels were assessed by ELISA in sv1 transfected and untransfected fibroblasts (**P<0.05; ns-not statistically significant, n = 4 per each group). (E) Schematic diagram of ANTXR1-dependent regulation of Col1a1 and Fn1 in fibroblasts. ANTXR1 (including all 5 splice variants sv1- sv5) negatively modulates Ctgf, Col1a1 and Fn1, Vegf-a and Hif1-α/expression (black lines) and sv1 negatively modulates Ctgf, Col1a1 and Fn1 only (red lines). ANTXR1-dependent increase of collagen α1(I) and fibronectin expression in mutant fibroblasts is primarily mediated by sv1/CTGF-dependent mechanism. Other ANTXR1 splice variants or unknown player (s), downstream of ANTXR1 may contribute to Hif1-α/VEGF-dependent matrix production in Antxr1 null fibroblasts.