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.
Highlights
The corneal endothelium, which originates from the neural crest via the periocular mesenchyme (PM), is crucial for maintaining corneal transparency
TFAP2B expression began in the PM at embryonic day 11.5 and in corneal endothelial cells (CECs) during adulthood. siRNA-mediated knockdown of TFAP2B in CECs decreased the expression of the corneal endothelium– specific proteins type VIII collagen ␣2 (COL8A2) and zona pellucida glycoprotein 4 (ZP4) and suppressed cell proliferation
It has been reported that TFAP2B is important for mouse corneal endothelial development, at E11.5 and at E15.5 [21]
Summary
The expression pattern of the AP-2 family in the human corneal endothelium was confirmed by RT-PCR. In the cell proliferation assay, the proliferative capacity of TFAP2B siRNA–treated CECs decreased compared with that of control siRNA–treated CECs (Fig. 3E) These data suggest that TFAP2B regulates the corneal endothelial cell–specific markers COL8A2 and ZP4 and promotes cell proliferation. The assay confirmed the binding of TFAP2B proteins to the candidate binding site within the promoters of the COL8A2 and ZP4 genes in the primary CECs (Fig. 4, D and H) These data indicate that TFAP2B protein directly regulates the transcriptional activities of the COL8A2 and ZP4 genes in CECs. Because ZP4 has been reported as one of the cell-surface proteins [24], we isolated ZP4-expressing CECs using an anti-ZP4 antibody by FACS. These data suggest that ZP4-expressing CECs may have a high proliferative potential
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