Purpose: Wound healing of the corneal epithelium is highly influenced by regulation of integrin gene expression. A recent study demonstrated that laminin (LM), a major constituent of the extracellular matrix (ECM), reduces expression of the human alpha6 integrin subunit gene by altering the properties of the transcription factor (TF) Sp1. In this work, a target site was identified for the TF nuclear factor I (NFI) on the human alpha6 gene, and its regulatory influence was characterized in corneal epithelial cells.
Methods: Plasmids bearing the alpha6 promoter fused to the CAT gene were transfected into human (HCECs) and rabbit (RCECs) corneal epithelial cells grown on LM. The DNA-binding site for NFI in the alpha6 promoter was identified by DNase I footprinting. Expression and DNA binding of NFI was monitored by Western blot, RT-PCR, and electrophoretic mobility shift assays (EMSAs), and its function was investigated through RNAi and NFI overexpression assays.
Results: All NFI isoforms were found to be expressed in HCECs and RCECs. Transfection analyses revealed that NFI is a repressor of alpha6 expression in both types of cells. LM increases expression of NFI, whereas inhibition of each NFI isoform increases promoter activity suggesting that NFI is a key repressor of alpha6 transcription. In addition, the negative influence of NFI appears to be potentiated by the degradation of Sp1 when cells are grown on LM.
Conclusions: Repression of alpha6 expression therefore contributes to the final steps of corneal wound healing by both reducing proliferation and allowing attachment of the epithelium to the basal membrane.