Contribution of the Transcription Factors Sp1/Sp3 and AP-1 to Clusterin Gene Expression during Corneal Wound Healing of Tissue-Engineered Human Corneas

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.

The Impaired Wound Healing Process Is a Major Factor in Remodeling of the Corneal Epithelium in Adult and Adolescent Patients With Keratoconus

“…The presence of variants in genes involved in antigen processing and presentation (Jaskiewicz et al 2022, unpublished data), together with the eye rubbing and allergy, could contribute to chronic corneal epithelial injury and trigger constant stimulation of wound healing in KTCN, as previously suggested. 59 In line with these results, here we presented the decreased level of clusterin (CLU) protein in the peripheral region of CE, which was previously demonstrated to be repressed during closure of wounds in tissue-engineered human cornea, 60 and the increased level of keratin 12 (KRT12) protein in the central region of CE, earlier reported as an effect of repairing response after injury. 61 …”

Long Non-Coding RNAs and Proliferative Retinal Diseases

“…Ocular disorders are prevalent in both developed and developing nations. In the world, at least 2.2 billion people suffer from some form of vision impairment, with at least 1 billion cases being either preventable or unaddressed [ 1 ]. From 2020 to 2045, the global DR population is expected to grow by 55.6% (57.4 million) [ 2 ].…”