Tight junction-associated signaling pathways modulate cell proliferation in uveal melanoma

Abstract

Purpose: To investigate the role of tight junction (TJ)-associated signaling pathways in the proliferation of uveal melanoma.

Methods: Human uveal melanoma cell lines overexpressing the TJ molecule blood vessel epicardial substance (Bves) were generated. The effects of Bves overexpression on TJ protein expression, cell proliferation, and cell cycle distribution were quantified. In addition, localization and transcription activity of the TJ-associated protein ZO-1-associated nucleic acid binding protein (ZONAB) were evaluated using immunofluorescence and bioluminescence reporter assays to study the involvement of Bves signaling in cell proliferation-associated pathways.

Results: Bves overexpression in uveal melanoma cell lines resulted in increased expression of the TJ proteins occludin and ZO-1, reduced cell proliferation, and increased sequestration of ZONAB at TJs and reduced ZONAB transcriptional activity.

Conclusions: TJ proteins are present in uveal melanoma, and TJ-associated signaling pathways modulate cell signaling pathways relevant to proliferation in uveal melanoma.

Figure 1.

Immunofluorescence localization of Bves and densitometric analysis of tight junction protein immunoblots in OMM2.3 cells. (A) Bves protein (red) is localized in the cytoplasm of wild-type OMM2.3 cells. (B) In OMM2.3 cells that overexpress Bves, increased localization of Bves is observed at sites of cell-cell contact (arrowheads). (C) Immunoreactivity to an isotype-matched negative control antibody was minimal. (D) Quantification of immunoblots indicates that occludin and ZO-1 proteins are upregulated in OMM2.3 cells overexpressing Bves. Data are reported as mean ± SEM (n = 4; *P = 0.018, **P = 0.027, ***P = 0.04). Results are representative of those observed for other wild-type and Bves-transfected uveal melanoma cell types. Scale bar, 20 μm.

Figure 2.

Analysis of cell proliferation and three-dimensional colony-forming ability of uveal melanoma cell lines. (A–C) Cell lines transfected with Bves exhibit reduced cell proliferation compared with empty vector–transfected or wild-type–matched cell lines as measured by XTT assay. (D) Similarly, colony-forming capacity of Bves-transfected cell lines in basement membrane matrix is reduced. (A) OM431, (B) OMM1, (C) OMM2.3. Data are reported as mean ± SEM (n = 4; *P < 0.001, **P = 0.031).

Figure 3.

Flow cytometric analysis of cell cycle progression in uveal melanoma cell lines. In all three cell lines tested, cell cycle distribution in S and G₂/M phases were reduced for Bves-transfected cells (A–C). (A) OM431, (B) OMM1, (C) OMM2.3. (D) Table of cell cycle distribution percentages for each cell type. Data are reported as mean ± SEM (n = 3; *P < 0.001).

Figure 4.

Localization of ZONAB in the OMM2.3 uveal melanoma cell line. Wild-type (A–D), empty vector control (E–H), or Bves-overexpressing cell lines (I–L) were analyzed for ZONAB nuclear association. Red: ZONAB; green: nucleus; yellow: nuclear colocalization of ZONAB. (D, H, L) High-magnification insets of overlay. Bves-overexpressing cells exhibit reduced colocalization of ZONAB with the nuclear compartment, as indicated by green nuclear fluorescence. Scale bars: 50 μm (A), 10 μm (D).

Figure 5.

Dual luciferase ZONAB reporter assay. A luminometer was used to quantify ZONAB nuclear activity in wild-type, empty vector control, or Bves-overexpressing cell lines according to the dual luciferase reporter assay described in Methods. The relatively higher luciferase activity in Bves-overexpressing cells is indicative of reduced transcriptional repression by ZONAB in these cells, and therefore, reduced nuclear activity of ZONAB. Data are reported as mean ± SEM (n = 4; *P < 0.001).