High-resolution promoter map of human limbal epithelial cells cultured with keratinocyte growth factor and rho kinase inhibitor

Abstract

An in vitro model of corneal epithelial cells (CECs) has been developed to study and treat corneal disorders. Nevertheless, conventional CEC culture supplemented with epidermal growth factor (EGF) results in a loss of CEC characteristics. It has recently been reported that limbal epithelial cells (LECs) cultured with keratinocyte growth factor (KGF) and the rho kinase inhibitor Y-27632 could maintain the expression of several CEC-specific markers. However, the molecular mechanism underlying the effect of culture media on LECs remains to be elucidated. To elucidate this mechanism, we performed comprehensive gene expression analysis of human LECs cultured with EGF or KGF/Y-27632, by cap analysis of gene expression (CAGE). Here, we found that LECs cultured with KGF and Y-27632 presented a gene expression profile highly similar to that of CECs in vivo. In contrast, LECs cultured with EGF lost the characteristic CEC gene expression profile. We further discovered that CEC-specific PAX6 promoters are highly activated in LECs cultured with KGF and Y-27632. Our results provide strong evidence that LECs cultured with KGF and Y-27632 would be an improved in vitro model in the context of gene expression. These findings will accelerate basic studies of CECs and clinical applications in regenerative medicine.

Figure 1

Study design. Distinct limbal epithelial cells (LECs) derived from 4 donors were divided into 2 groups: LECs cultured with EGF (blue) and LECs cultured with KGF and Y-27632 (K+Y, pink). RNA was extracted from LECs and CAGE analysis was performed.

Figure 2

Hierarchical clustering analysis of EGF-treated LECs and K+Y-treated LECs. In total, 31,406 promoters that had more than 10 CAGE tags in at least one sample were examined. Hierarchical clustering analysis was performed using the average linkage algorithm with a Spearman correlation distance matrix. The same numbers correspond to the same donors. LECs in each condition showed a similar expression pattern. K+Y: KGF and Y-27632.

Figure 3

Volcano plot of statistical significance against log 2 fold-change between EGF-treated LECs and K+Y-treated LECs. (a) Promoters highly expressed in LECs cultured with EGF are shown in blue (adjusted P < 0.01. log 2 fold-change <−1), and promoters highly expressed in LECs cultured with K+Y are shown in red (adjusted P < 0.01. log 2 fold-change >1). Promoters that were not differentially expressed between these two culture conditions are shown in gray. The most differentially expressed promoters were p1@CRTAC1 and p2@CRTAC1, which were quite highly expressed in K+Y. The promoter of CEC-specific miR-184 was also highly expressed in LECs cultured with K+Y. K+Y: KGF and Y-27632. (b) Expression level of miR-184 quantified by RT-PCR. Each dot represents the expression level in each sample, and each line indicates binding to samples from the same donors. An asterisk represents statistical significance.

Figure 4

Venn diagram of differentially expressed genes overlapping with cornea and conjunctiva-specific genes. Genes highly expressed in LECs cultured with K+Y are shown in red, and genes highly expressed in LECs cultured with EGF are shown in blue. Genes specifically expressed in the cornea and conjunctiva based on the microarray analysis are shown in yellow and green, respectively. The 82 cornea-specific genes highly expressed in LECs cultured with K+Y are listed, and 33 of them that are considered to be regulated by PAX6 are shown in red characters. K+Y: KGF and Y-27632.

Figure 5

CEC-specific genes regulated by PAX6 are upregulated in K+Y-treated LECs. Expression levels of CEC-specific genes regulated by PAX6 quantified by (a) CAGE and (b) RT-PCR. Each dot represents the expression levels of distinct promoters or genes in each sample, and each line indicates binding to samples from the same donors. Asterisks represent statistical significance. K+Y: KGF and Y-27632.

Figure 6

Genome browser view of PAX6 alternative promoters. (a) (Top) EGF represents CAGE tags of LECs cultured with EGF (blue). K+Y represents CAGE tags of LECs cultured with KGF and Y-27632 (red). CAGE tags of the 4 LEC samples cultured in distinct conditions were merged and normalized. p3 and p9@PAX6, indicated by a red rectangle, were highly expressed in K+Y-treated LECs and lens epithelial cells, whereas their expression levels were low in EGF-treated LECs and the cerebellum. p1 and p2@PAX6 were highly expressed in the cerebellum. (Bottom) Zoomed-out view to show the full structure of the PAX6 gene. Coding exons are represented by thick blocks, whereas untranslated exons are represented by relatively thin blocks. Arrows indicate the direction of transcription. (b) Expression levels of two types of PAX6 transcripts quantified by RT-PCR. The primer positions of each transcript are shown in (a). PAX6-short reflects the activity of p1@PAX6, and PAX6-long reflects the activities of p3 and p9@PAX6. Each dot represents the expression level in each sample, and each line indicates binding to samples from the same donors. An asterisk represents statistical significance.