Transcriptional regulation of Gja8 (Cx50) promoter in lens epithelial and fiber cells

Abstract

Purpose : To elucidate molecular mechanisms that differentially regulate the expression of the Gja8 gene, which encodes connexin50 (Cx50) protein, in the lens epithelial and differentiating fiber cells to understand its important role in the regulation of lens size and transparency.

Methods : The Gja8 enhancer/promoter from a B6 mouse genome was cloned into a pEGFP-N1 vector, and subsequently subcloned into an adeno-associated virus (AAV) plasmid pTR with mRuby3 and EGFP reporters. Both lens epithelial explants and primary cultured cells were transduced with AAV-Gja8 enhancer/promoter-EGFP with or without smCBA-mRuby3, and then treated with either a differentiating dose of FGF, IGF, vitreous, or a combination of FGF with IGF. For blocking studies, inhibitors of different signaling pathways were added 2 hours before treatment of growth factors. Levels of reporter genes and additional protein markers were examined by immunolabeling and western blotting to determine the activities of the Gja8 enhancer/promoter.

Results : Multiple binding sites for FGF- and BMP-regulated transcription factors (TFs) were identified in the Gja8 enhancer/promoter. The majority of lens epithelial cells were successfully transduced with the AAV-Gja8 enhancer/promoter, and the levels of reporter genes increased in elongating and differentiating cells in response to the treatment of vitreous and other growth factors. Multiple intracellular signaling pathways that include specific TFs with cis-elements in the Gja8 enhancer seemed to mediate the upregulation of Cx50 expression.

Conclusions : In lens epithelial cells, ocular growth factors activate cascades of intracellular signaling, which consequently regulate the expression of TFs that have binding sites on the Gja8 enhancer/promoter. We reveal the contributions of these specific TFs in the regulation of Gja8 gene expression in lens epithelial cells and differentiating fiber cells, which may shed light on a potential way to control cell growth and lens size by regulating the level of Cx50.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.