Increase of O-glycosylated oncofetal fibronectin in high glucose-induced epithelial-mesenchymal transition of cultured human epithelial cells

Abstract

Growing evidences indicate that aberrant glycosylation can modulate tumor cell invasion and metastasis. The process termed "epithelial-mesenchymal transition" (EMT) provides a basic experimental model to shed light on this complex process. The EMT involves a striking decline in epithelial markers, accompanied by enhanced expression of mesenchymal markers, culminating in cell morphology change and increased cell motility. Few recent studies have established the participation glycosylation during EMT. Studies now come into knowledge brought to light the involvement of a site-specific O-glycosylation in the IIICS domain of human oncofetal fibronectin (onfFN) during the EMT process. Herein we show that high glucose induces EMT in A549 cells as demonstrated by TGF-β secretion, cell morphology changes, increased cellular motility and the emergence of mesenchymal markers. The hyperglycemic conditions increased onfFN protein levels, promoted an up regulation of mRNA levels for ppGalNAc-T6 and FN IIICS domain, which contain the hexapeptide (VTHPGY) required for onfFN biosynthesis. Glucose effect involves hexosamine (HBP) biosynthetic pathway as overexpression of glutamine: fructose-6-phosphate amidotransferase increases mesenchymal markers, onfFN levels and mRNA levels for FN IIICS domain. In summary, our results demonstrate, for the first time that the metabolism of glucose through HBP promotes O-glycosylation of the oncofetal form of FN during EMT modulating tumorogenesis.

Figure 1. TGF-β production and expression of mesenchymal markers in A549 cells.

(A) Quantification of TGF-β in supernatants from cells cultured for 48 h in NG (white bar), HG (black bar) or OG (gray bar). (B) Western blot of cell lysates loads analyzing expression levels of N-cad, (first lane) and Vimentin (second lane) in cells cultured in NG (white bar), HG (black bar) or OG (gray bar) conditions with or without 2 ng/mL TGF-β. Signal intensities were normalized, with GAPDH as loading control, and relative intensities of N-cad (C) and Vimentin (D) are shown. The results are representative of 3 independent experiments. Quantitative analyses are shown as mean ± standard deviation. P values were calculated using the Student's t test. *P≤0.01; **P≤0.005.

Figure 2. Analysis of cell morphology and motility.

Cell morphology (A); cell motility (B) and cell circularity (C) of A549 cells treated in NG, HG or OG conditions with (right panel) or without TGF-β (left panel). Representative photos are presented. Tracks of 50 random individual cells on gold solution (D) were measured using the Scion Image program represented as squared pixels, and are shown as mean ± SD. NG (white bar), HG (black bar) or OG (gray bar). *P≤0,005.

Figure 3. Effect of hyperglycemia onfFN biosynthesis.

Western blot of A549 cell lysates cultured for 48 h in NG (white bar), HG (black bar) or OG (gray bar) medium with (+) or without (−) TGF-β, showing expression of total FN (first lane) and onfFN (second lane) (A). Signal intensities were normalized, with GAPDH as loading control, and relative intensities of total FN (B) and onfFN (C) are shown. (D) Western blot of A549 total FN (first lane) and onfFN (second lane) immunoprecipitated from cell lysates by FDC-6 mAbs, submitted (+) or not (−) to the remotion of O-glycosylation. Human plasma FN (pFN, 0.5 µg) was used as control. qRT-PCR analysis of gene that codifies IIICS domain of onfFN (E) and GalNacT6 (F) respectively. Graph shows one of three independent experiments as mean ± SD. * P≤0.005. Effect of anti-TGF-β blocking antibody in the expression of total FN (first line) and onfFN (second line) (G).

Figure 4. Effect of GFAT2 over expression in onfFN biosynthesis and expression of mesenchymal markers.

A549 cells were transfected with expression vectors encoding GFAT2 (+) or empty expression vector (−), and cultured in NG (white bars), HG (Black bars) or NG plus Lipofectamine 2000 (CP, Scratched bars). Western blot analysis of expression of total FN (first lane), onfFN (second lane), GFAT (third lane) and GAPDH (A). The results are representative of 3 independent experiments. Signal intensities were normalized, with GAPDH as loading control, and relative intensities of total FN (B) and onfFN (C) are shown. qRT-PCR analysis of gene that codifies IIICS domain of onfFN (D) and GalNNcT6 (E). Western blot of cell lysates expression levels of N-cad, (first lane) and Vimentin (second lane) in cells cultured in NG (white bar), HG (black bar) or OG (gray bar) conditions with or without transfection of GFAT2 (+), or empty expression vector (−), or NG plus Lipofectamine 2000 (CP, Scratched bars) (F). Signal intensities were normalized, with GAPDH as loading control, and relative intensities of N-cad (G) and Vimentin (H) are shown. Quantitative analyses are shown as mean ± standard deviation. P values were calculated using the Student's t test. * P≤0.05; # P = 0.18; ## P = 0.14; ### P = 0.