Abstract
In order to reduce the need for donor corneas, understanding of corneal wound healing and development of an entirely tissue-engineered human cornea (hTECs) is of prime importance. In this study, we exploited the hTEC to determine how deep wound healing affects the transcriptional pattern of corneal epithelial cells through microarray analyses. We demonstrated that the gene encoding clusterin (CLU) has its expression dramatically repressed during closure of hTEC wounds. Western blot analyses confirmed a strong reduction in the expression of the clusterin isoforms after corneal damage and suggest that repression of CLU gene expression might be a prerequisite to hTEC wound closure. Transfection with segments from the human CLU gene promoter revealed the presence of three regulatory regions: a basal promoter and two more distal negative regulatory regions. The basal promoter bears DNA binding sites for very potent transcription factors (TFs): Activator Protein-1 (AP-1) and Specificity protein-1 and 3 (Sp1/Sp3). By exploiting electrophoretic mobility shift assays (EMSA), we demonstrated that AP-1 and Sp1/Sp3 have their DNA binding site overlapping with one another in the basal promoter of the CLU gene in hCECs. Interestingly, expression of both these TFs is reduced (at the protein level) during hTEC wound healing, thereby contributing to the extinction of CLU gene expression during that process. The results of this study contribute to a better understanding of the molecular mechanisms accounting for the repression of CLU gene expression during corneal wound healing.
Highlights
Visual acuity is directly correlated with the integrity of the anatomical structures of the eyes
We examined whether formation of both the Activator Protein-1 (AP-1) and Specificity Protein 1 (Sp1)/Sp3 complexes was altered in scratch-wounded relative to unwounded human corneal epithelial cells (hCECs) grown as monoloyers
In order to reduce the needs for donor corneas, the use of an entirely tissue-engineered human cornea that can be used as a model to study the mechanisms of wound healing is of prime importance, as it may help develop alternative solutions to the need of graftable, donor corneas
Summary
Visual acuity is directly correlated with the integrity of the anatomical structures of the eyes. We demonstrated that CLU gene expression was severely repressed during hTEC wound healing and that both positive and negative regulatory elements that contribute to its transcription in hCECs are present in both the basal promoter and 5 -flanking sequence of the CLU gene. The CLU average miroarray linear signals drops from 18,624 in unwounded hTECs to only 1146 in wounded hTECs (corresponding to a 16-fold repression) Consistent with these results, CLU is known to be involved in multiple physiological mechanisms linked to wound healing processes (including apoptosis, cell adhesion, migration and proliferation, tissue remodeling, etc.) [21,22,23,24,25,26]. Microarray data for B2M and GOLGA1 are shown as controls
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