Differential effects of Pax3 on expression of polysialyltransferases STX and PST in TGF-β-treated normal murine mammary gland cells

Abstract

Glycosylation of certain proteins at the mammalian cell surface is an essential event in carcinogenesis. Sialylation, one type of glycosylation, can act on multiple cell-behaviors, such as migration, growth, and malignant invasion. Two polysialyltransferases, ST8Sia II (STX) and ST8Sia IV (PST), are responsible for synthesis of polysialic acid on neural cell adhesion molecule. We showed previously that STX and PST are oppositely expressed in normal murine mammary gland cells undergoing transforming growth factor-β-induced epithelial-mesenchymal transition. The molecular basis for regulation of STX and PST remained unclear. In the present study, we observed that transcription factor Pax3 upregulates STX expression, downregulates PST expression, and modulates upregulated expression of PSA, which attaches primarily to neural cell adhesion molecule to form PSA-NCAM. Overexpression of Pax3 in normal murine mammary gland cells transformed the expression of epithelial-mesenchymal transition markers E-cadherin and N-cadherin, and significantly promoted cell migration, but had no effect on cell proliferation.

Keywords: PSA-NCAM; PST; Pax3; STX; epithelial-mesenchymal transition; polysialyltransferase.

Figure 1

Pax3 is upregulated in NMuMG cells undergoing TGF-β-induced EMT. (a) Fold changes of STX and PST in control vs. treated cells were assessed by glycogene chip analysis (GlycoV4 GeneChip). (b) Expression of STX, PST, and Pax3 in control vs. treated cells was analyzed by qRT-PCR. (c) Western blot analysis of expression of EMT markers and Pax3 in cells undergoing EMT. Cells were cultured in 6-well plates, treated (or not) with TGF-β for 48 h, harvested, and lysed in RIPA buffer. Lysates (30 µg protein/well) were subjected to SDS-PAGE and analysis of E-cadherin, N-cadherin, fibronectin, vimentin, and Pax3 expression. (d) Expression of STX, PST, and Pax3 in NMuMG and 4T1 cells was analyzed by qRT-PCR, with β-tubulin as internal control. Data were analyzed by the 2^−ΔΔCt method. *P < 0.05; **P < 0.01; ***P < 0.001; NS: not significant

Figure 2

Pax3 causes upregulation of STX and downregulation of PST. (a) Western blot analysis of Pax3 expression. Mock: pcDNA3.1(+). NC: scrambled sequence control. (b) and (c) Changes in STX and PST expression levels in NMuMG/Pax3 and 4T1/si-Pax3 cells, respectively, were analyzed by qRT-PCR, with β-tubulin as internal control. Data were analyzed by the 2^−ΔΔCt method and presented as fold change. **P < 0.01; ***P < 0.001; NS: not significant. (d) Schematic summary of PST promoter sequence analysis, showing two predicted Pax3 binding sites. TSS: translation start site. (e) Luciferase activity analysis of PST promoter (−443/−162) for untreated NMuMG/pcDNA3.1(+) cells (lane 1), TGF-β-treated NMuMG/pcDNA3.1(+) cells (lane 2), and NMuMG/Pax3 cells (lane 3). Firefly luciferase activity was normalized to Renilla luciferase activity, and presented as mean ± SD from three independent experiments. (f) EMSA was performed using DIG-labeled probe P-1 and nuclear extracts from NMuMG cells. Arrow: DNA-protein complexes. Competitive assay was performed with 50-fold unlabeled probe P-1 (lane 3). Supershift analysis was performed by adding anti-Pax3 antibody (lane 4; *)

Figure 3

Effects of Pax3 on PSA-NCAM and NCAM expression. (a) Western blot analysis of PSA-NCAM and NCAM in NMuMG cells. (b) Immunofluorescence staining of PSA-NCAM and NCAM in NMuMG/Mock and NMuMG/Pax3 cells. Nuclei were visualized by DAPI staining. Bars: 50 µm. (A color version of this figure is available in the online journal.)

Figure 4

Effects of Pax3 on migration and proliferation of NMuMG cells. (a) Western blot analysis of EMT markers. Cells were cultured in 6-well plates, harvested, and lysed in RIPA buffer. Lysates (30 µg protein per lane) were subjected to SDS-PAGE and analysis of E-cadherin, N-cadherin, fibronectin, and vimentin expression. (b) Immunofluorescence staining of E-cadherin and N-cadherin in NMuMG/Mock and NMuMG/Pax3 cells, and visualization of nuclei by DAPI staining. Bars: 50 µm. (c) Representative phase-contrast images showing increased migration of NMuMG/Pax3 cells, with NMuMG/Mock cells as control. After cells reached 100% confluence, scratches were made, regions were photographed (0 h), and the same regions were photographed at 24 h to assess the degree of wound healing. (d) Results of wound healing assay were analyzed using the Scion Image software program, and expressed as mean ± SD percentage area covered by moving cells at 24 h. All experiments were performed in triplicate. ***P < 0.001. (e) Proliferation (MTT) assays. NMuMG/Mock and NMuMG/Pax3 cells were cultured, and MTT assays were performed as described in Materials and Methods. Data are presented as mean ± SD from three independent experiments. (A color version of this figure is available in the online journal.)