Transcription factor TFAP2B up-regulates human corneal endothelial cell-specific genes during corneal development and maintenance

Abstract

The corneal endothelium, which originates from the neural crest via the periocular mesenchyme (PM), is crucial for maintaining corneal transparency. The development of corneal endothelial cells (CECs) from the neural crest is accompanied by the expression of several transcription factors, but the contribution of some of these transcriptional regulators to CEC development is incompletely understood. Here, we focused on activating enhancer-binding protein 2 (TFAP2, AP-2), a neural crest-expressed transcription factor. Using semiquantitative/quantitative RT-PCR and reporter gene and biochemical assays, we found that, within the AP-2 family, the TFAP2B gene is the only one expressed in human CECs in vivo and that its expression is strongly localized to the peripheral region of the corneal endothelium. Furthermore, the TFAP2B protein was expressed both in vivo and in cultured CECs. During mouse development, TFAP2B expression began in the PM at embryonic day 11.5 and then in CECs during adulthood. siRNA-mediated knockdown of TFAP2B in CECs decreased the expression of the corneal endothelium-specific proteins type VIII collagen α2 (COL8A2) and zona pellucida glycoprotein 4 (ZP4) and suppressed cell proliferation. Of note, we also found that TFAP2B binds to the promoter of the COL8A2 and ZP4 genes. Furthermore, CECs that highly expressed ZP4 also highly expressed both TFAP2B and COL8A2 and showed high cell proliferation. These findings suggest that TFAP2B transcriptionally regulates CEC-specific genes and therefore may be an important transcriptional regulator of corneal endothelial development and homeostasis.

Keywords: COL8A2; TFAP2B; ZP4; cell surface protein; collagen; cornea; corneal endothelial cells; development; differentiation; eye; eye development; gene regulation; molecular cell biology; neural crest; regenerative medicine; transcriptional regulation.

Figure 1.

Expression pattern of TFAP2B in human CECs. A, gel electrophoresis images of RT-PCR of the TFAP2 family in the human corneal endothelium (CE) (upper) and positive control (lower). cDNA from human neural retina was used as a positive control for RT-PCR. B, immunofluorescence of TFAP2B (red) and Hoechst 33342 (blue) in the corneal endothelium. Green signals represent the expression of the ZO-1 protein at CEC junctions. C, relative ratio of the fluorescence intensity of TFAP2B in vivo between the central and peripheral regions of the corneal endothelium. The fluorescence intensity ratio was calculated from the images of TFAP2B and Hoechst, and the relative ratio between the peripheral and the central regions was determined. The data are shown as the mean ± S.D. (error bars) (n = 6). ***,s p < 0.001. Scale bars, 20 μm. D, immunofluorescence images of TFAP2B (red) and Hoechst 33342 (blue) in cultured human CECs.

Figure 2.

TFAP2B expression in mouse CECs during developmental stages. The photographs show TFAP2B expression (red) in embryos and adult mice by immunohistochemistry. A–D, E11.5; E–H, E15.5; I–L, adult mouse. Arrowheads indicate mouse CECs. Hoechst 33342 (blue) was used to stain the nucleus. Scale bars, 20 μm. pm, periocular mesenchyme; lv, lens vehicle; ln, lens; cs, corneal stroma; ce, corneal endothelium.

Figure 3.

Repression of TFAP2B in human cultured CECs. The siRNA-mediated knockdown in human cultured CECs was performed with control siRNA (siControl) and TFAP2B siRNA (siTFAP2B) for 48 h. A, real-time qRT-PCR of the neural crest marker SOX9; periocular mesenchyme markers PITX2, FOXC1, and FOXC2; and CEC markers TJP1 (ZO-1), Na⁺/K⁺-ATPase, COL8A1, COL8A2, and ZP4 following TFAP2B siRNA treatment. The expression levels were normalized to those of the siControl-treated CECs (n = 4). B, Western blotting of TFAP2B siRNA–treated cultured CECs. C, immunofluorescence images of COL8A2 (left panel, red) and ZP4 (right panel, green) in human corneal sections. D, whole-mount immunofluorescence images of TFAP2B (red) and ZP4 (green). The TFAP2B-positive cells strongly expressed ZP4 protein in the human corneal endothelium. Hoechst 33342 (blue) was used to stain the nucleus. E, cell proliferation assay using AlamarBlue reagent of siControl- and siTFAP2B-treated CECs (n = 6, duplicate three donors). The cells were seeded at 3,000 cells/well in a 96-well plate and analyzed after 2 days. The data are shown as the mean ± S.D. (error bars) *, p < 0.05; ***, p < 0.001. Scale bars, 20 μm. cs, corneal stroma; ac, anterior chamber; dm, Descemet's membrane; ce, corneal endothelium.

Figure 4.

Transcriptional activities of the COL8A2 and ZP4 promoters with TFAP2B. A and E, scheme of the luciferase reporter vectors of the human COL8A2 and ZP4 promoters. Mutations in the TFAP2B-binding site are shown in lowercase letters. B and F, EMSA of candidate TFAP2B-binding site of COL8A2 and ZP4 promoters. The shifted bands of the DNA–TFAP2B protein complexes were only observed in WT sequences of COL8A2 and ZP4. C and G, luciferase assays. TFAP2B-overexpressing 293T cells were transfected with the luciferase reporter vector containing the promoter regions of the COL8A2 and ZP4 genes. The luciferase activities were compared between the WT (COL8A2 or ZP4) and TFAP2B-binding sequence mutant (COL8A2 mutant and ZP4 mutant) luciferase vectors. The data were normalized to the luciferase activity of the WT. D and H, ChIP. DNA–protein complexes of cultured CECs were immunoprecipitated with IgG control or anti-TFAP2B antibody. Purified DNA was amplified using PCR and separated on an agarose gel. The input samples were used as a control. The data are shown as the mean ± S.D. (error bars) (n = 4). *, p < 0.05; **, p < 0.01. Scale bars, 20 μm.

Figure 5.

Isolation of ZP4-expressing cells in CECs. A, cultured human CECs were separated into ZP4-negative or ZP4-positive (ZP4+) populations by FACS. B, expression of corneal endothelium–related genes in the sorted cells. ZP4-positive cells (ZP4 +) highly expressed ZP4, TFAP2B, and COL8A2 compared with ZP4-negative cells (ZP4−). The expression levels were normalized to those of ZP4-negative cells. The cells collected by FACS were seeded at 20,000/per well in a 96-well plate and analyzed after 2 or 7 days. C, immunostaining of cells cultured for 7 days after cell sorting. Isolated ZP4-positive or ZP4-negative cells were cultured (D), and cell proliferation assays were performed after 2 days (E). Data are shown as the mean ± S.D. (error bars) (n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.001. Scale bars, 100 μm.