A 300 bp 5′-upstream sequence of a differentiation-dependent rabbit K3 keratin gene can serve as a keratinocyte-specific promoter

Keratinocytes of the suprabasal compartment of many stratified epithelia synthesize as a major differentiation product a keratin pair, consisting of an acidic and a basic keratin, which accounts for 10–20% of the newly synthesized proteins. While genes of several differentiation-related keratins have been cloned and studied, relatively little is known about the molecular basis underlying their tissue-specific and differentiation-dependent expression. We have chosen to study, as a prototype of these genes, the gene of K3 keratin, which has the unique property of being expressed in the majority of corneal epithelial basal cells but suprabasally in peripheral cornea, the site of corneal epithelial stem cells. Using a monoclonal antibody, AE5, specific for K3 keratin, and a fragment of human K3 gene as probes, we have isolated several cDNA and genomic clones of rabbit K3 keratin. One genomic clone has been sequenced and characterized, and the identity of its coding sequence with that of cDNAs indicates that it corresponds to the single, functional rabbit K3 gene. Transfection assays showed that its 3.6 kb 5′-upstream sequence can drive a chloramphenicol acetyl transferase (CAT) reporter gene to express in cultured corneal and esophageal epithelial cells, but not in mesothelial and kidney epithelial cells or fibroblasts, all of rabbit origin. Serial deletion experiments narrowed this keratinocyte-specific promoter to within -300 bp upstream of the transcription initiation site. Its activity is not regulated by the coding or 3′-noncoding sequences that have been tested so far. This 300 bp 5′-upstream sequence of K3 keratin gene, which can function in vitro as a keratinocyte-specific promoter, contains two clusters of partially overlapping motifs, one with an NFkB consensus sequence and another with a GC box. The combinatorial effects of these multiple motifs and their cognate binding proteins may play an important role in regulating the expression of this tissue-restricted and differentiation-dependent keratin gene.

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Regulation of K3 keratin gene transcription by Sp1 and AP-2 in differentiating rabbit corneal epithelial cells.

Molecular and Cellular Biology ◽

10.1128/mcb.17.6.3056 ◽

1997 ◽

Vol 17 (6) ◽

pp. 3056-3064 ◽

Cited By ~ 79

Author(s):

T T Chen ◽

R L Wu ◽

F Castro-Munozledo ◽

T T Sun

Keyword(s):

Epithelial Cells ◽

Gene Promoter ◽

Epithelial Differentiation ◽

Binding Motif ◽

Basal Cells ◽

High Concentration ◽

Corneal Epithelial Cells ◽

Keratin Gene ◽

Corneal Epithelial ◽

Basal Keratinocytes

Rabbit corneal epithelial cells cultured in the presence of 3T3 feeder cells undergo biochemical differentiation, as evidenced by their initial expression of K5 and K14 keratins characteristic of basal keratinocytes, followed by the subsequent expression of K3 and K12 keratin markers of corneal epithelial differentiation. Previous data established that mutations of an Sp1 site in a DNA element, E, that contains overlapping Sp1 and AP-2 motifs reduce K3 gene promoter activity by 70% in transfection assays. We show here that Sp1 activates while AP-2 represses the K3 promoter. Although undifferentiated corneal epithelial basal cells express equal amounts of Sp1 and AP-2 DNA-binding activities, the differentiated cells down-regulate their Sp1 activity slightly but their AP-2 activity drastically, thus resulting in a six- to sevenfold increase in the Sp1/AP-2 ratio. This change coincides with the activation and suppression of the differentiation-related K3 gene and the basal cell-related K14 keratin gene, respectively. In addition, we show that polyamines, which are present in a high concentration in proliferating basal keratinocytes, can inhibit the binding of Sp1 to its cognate binding motif but not that of AP-2. These results suggest that the relatively low Sp1/AP-2 ratio as well as the polyamine-mediated inhibition of Sp1 binding to the E motif may account, in part, for the suppression of the K3 gene in corneal epithelial basal cells, while the elevated Sp1/AP-2 ratio may be involved in activating the K3 gene in differentiated corneal epithelial cells. Coupled with the previous demonstration that AP-2 activates the K14 gene in basal cells, the switch of the Sp1/AP-2 ratio during corneal epithelial differentiation may play a role in the reciprocal expression of the K3 and K14 genes in the basal and suprabasal cell layers.

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Limbal Niche Cells in Three-Dimensional Matrigel Induced Dedifferentiation of Mature Corneal Epithelial Cells toward a Progenitor State

10.1101/2020.06.01.128629 ◽

2020 ◽

Author(s):

Hui Zhu ◽

Wei Wang ◽

Lingjuan Xu ◽

Menglin Jiang ◽

Yongyao Tan ◽

...

Keyword(s):

Stem Cells ◽

Epithelial Cells ◽

Single Cells ◽

Three Dimensional ◽

Stem Cell Marker ◽

Epithelial Stem Cells ◽

Corneal Epithelial Cells ◽

Corneal Epithelial ◽

Epithelial Marker

ABSTRACTPurposeTo investigate the possibility and the key factors of stably committed mature corneal epithelial cells dedifferentiate into corneal epithelial stem cells in vitro.MethodsMature cornea epithelia cell (MCEC) sheets or limbal epithelial progenitor cell (LEPC) sheets were isolated from central corneas or limbal segments by Dispase II and further digested with 0.25% trypsin/1 mM EDTA (T/E) to yield single cells. Limbal niche cells (LNC) were isolated from the limbal stroma by collagenase A and expanded on 5% Matrigel coated plastic. Single MCECs were seeded on 50% Matrigel with or without LNC culturing for 10 days, regarding as three-dimensional MCEC (3D-MCEC) group or three-dimensional MCEC+LNC (3D-MCEC+LNC) group. Expression of CK12, p63α, PCK, Vimentin were analyzed with immunofluorescence staining.ResultsThe expression of mature cornea epithelial marker (CK12) in MCEC was higher than that in LEPC (P=0.020) but epithelial stem cell marker (p63α) was lower than that in LEPC (P=0.000). When seeded in 3D Matrigel, single MCEC cells could form spheres within 72 hours, and the expression of CK12 reduced (P=0.005) and the expression of p63α also reduced to zero (P=0.000) compared to MCEC. Serial passages of LNC which were expanded in coated Matrigel could form spheres in 3D Matrigel. After mixing MCECs with LNC, rounder spheres emerged within 24 hours which consisted of both epithelia cells (PCK+/Vim-) and LNC (PCK-/Vim+). Moreover, epithelia cells in 3D-MCEC+LNC group expressed less CK12 and more p63α than those in MCEC group (P=0.043, 0.000). Besides, the diameter of spheres in 3D-MCEC+LNC group were larger than that in 3D-MCEC group (P=0.000).ConclusionHuman LNC and three-dimensional Matrigel could induce the dedifferentiation of mature corneal epithelial cells into corneal epithelial stem cells.

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Establishment of Novel Limbus-Derived, Highly Proliferative ABCG2+/ABCB5+ Limbal Epithelial Stem Cell Cultures

Stem Cells International ◽

10.1155/2017/7678637 ◽

2017 ◽

Vol 2017 ◽

pp. 1-12 ◽

Cited By ~ 3

Author(s):

Eung Kweon Kim ◽

Ga-Hyun Lee ◽

Boram Lee ◽

Yong-Sun Maeng

Keyword(s):

Stem Cell ◽

Epithelial Cells ◽

Telomerase Activity ◽

Stem Cell Markers ◽

Success Rates ◽

Epithelial Stem Cells ◽

Double Positive ◽

Corneal Epithelial Cells ◽

Corneal Epithelial

Homeostasis and regeneration of corneal epithelia are sustained by limbal epithelial stem cells (LESCs); thus, an LESC deficiency is a major cause of blindness worldwide. Despite the generally promising results of cultivated LESC transplantation, it has been limited by variations in long-term success rates, the use of xenogeneic and undefined culture components, and a scarcity of donor tissues. In this study, we identified the culture conditions required to expand LESCs in vitro and established human limbus-derived highly proliferative ABCG2+/ABCB5+ double-positive LESCs. These LESCs exhibited the LESC marker profile and differentiated into corneal epithelial cells. In addition, cultured LESCs expressed high levels of the stem cell markers Sox2, Oct4, c-Myc, and Klf4, had high telomerase activity, and had stable, normal genomes. These results suggest that our novel cultivation protocol affects the phenotype and differentiation capacity of LESCs. From the limbus, which contains a heterogenous cell population, we have derived highly proliferative ABCG2+/ABCB5+ double-positive cells with the ability to differentiate into corneal epithelial cells. This study opens a new avenue for investigation of the molecular mechanism of LESC maintenance and expansion in vitro and may impact the treatment of corneal disease, particularly corneal blindness due to an LESC deficiency.

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