ABSTRACT
Using the rabbit corneal epithelial cell line RCE1(5T5) as a model, we analyzed three differentiation stages, distinguished on basis to the growth state of cultured cells and after studying the expression of transcription factors such as Oct4, Pax6 and ΔNp63α, selected differentiation markers, and signaling or epigenetic markers such as Notch receptors and Prdm3. Namely, proliferative non-differentiated cells, committed cells, and cells that constitute a stratified epithelium with a limbal epithelial-like structure. RNAseq based transcriptome analysis showed that 4891 genes were differentially expressed among these stages displaying distinctive gene signatures: proliferative cells had 1278 genes as gene signature, and seem to be early epithelial progenitors with an Oct4+, KLF4+, Myc+, ΔNp63α+, ABCG2+, Vimentin+, Zeb1+, VANGL1+, Krt3-, Krt12- phenotype. Committed cells had a gene signature with 417 genes and displayed markers indicative of the beginning of corneal differentiation, and genes characteristic of proliferative cells; we found the possible participation of Six3 and Six4 transcription factors along this stage. The third stage matches with a stratified corneal epithelium (gene signature comprising 979 genes) and is typified by an increase in the expression of WNT10A and NOTCH 2 and 3 signaling and Cux1 transcription factor, besides Pax6, KLF4 or Sox9. The differentiated cells express about 50% of the genes that belong to the Epidermal Differentiation Complex (EDC). Analysis of the differences between corneal epithelium and epidermis could be crucial to understand the regulatory mechanisms that lead to the expression of the differentiated phenotype.
Subject(s)
Cell Differentiation/physiology , Epithelium, Corneal/cytology , Transcriptome/physiology , Animals , Cells, Cultured , Epithelium, Corneal/metabolism , Fluorescent Antibody Technique, Indirect , Gene Expression Regulation/physiology , Octamer Transcription Factor-3/genetics , PAX6 Transcription Factor/genetics , Protein Interaction Maps , Rabbits , Real-Time Polymerase Chain Reaction , Transcription Factors/genetics , Vimentin/geneticsABSTRACT
Although tight junctions (TJ) have been extensively studied in simple epithelial cells, it is still unknown whether their organization is coupled to cell differentiation in stratified epithelia. We studied the expression of TJ in RCE1(5T5) cells, an in vitro model which mimics the sequential steps of rabbit corneal epithelial differentiation. RCE1(5T5) cells expressed TJ components which were assembled once cells constituted differentiated epithelia, as suggested by the increase of transepithelial electrical resistance (TER) which followed a similar kinetic to the expression of the early differentiation marker Pax-6. TJ were functional as indicated by the establishment of an epithelial barrier nonpermeable to ruthenium red or a biotin tracer. In immunostaining experiments, TJ were located at the superficial cells from the suprabasal layers; Western blot and RT-PCR suggested that TJ were composed of claudins (cldn) -1, -2, -4, cingulin (cgn), occludin (ocln) and ZO-1. Semi-quantitative RT-PCR and TER measurements showed that TJ became organized when cells began to form a 3-5 layers stratified epithelium; TER increased once cells reached confluence, with a time course comparable to the raise in the expression of cgn, cldn-2 and -4. Nevertheless, cldn-1, -2, ZO-1 and ocln were present in the cells from the beginning of cultivation, suggesting that TER increases mainly depend on TJ assembly. While EGF increased epithelial barrier strength, retinoic acid disrupted it, increasing paracellular flux about 2-fold; this effect was concentration dependent and completely reversible. Our results suggest that TJ assembly is tightly linked to the expression of corneal epithelial terminal phenotype.
