Abstract:
Dysregulation of retinal pigment epithelium (RPE) cells is the main cause of a variety of ocular diseases. Potentially heat shock
proteins, by preventing molecular and cellular damage and modulating inflammatory disease, may exert a protective role in eye
disease. In particular, the inducible form of heat shock protein 70 (Hsp70) is widely upregulated in inflamed tissues, and in vivo
upregulation of Hsp70 expression by HSP co-inducing compounds has been shown to be a potential therapeutic strategy for
inflammatory diseases. In order to gain further understanding of the potential protective effects of Hsp70 in RPE cells, we
developed a method for isolation and culture of canine RPE cells. Identity of RPE cells was confirmed by detection of its specific
marker, RPE65, in qPCR, flow cytometry, and immunocytochemistry analysis. The ability of RPE cells to express Hsp70 upon
experimental induction of cell stress, by arsenite, was analyzed by flow cytometry. Finally, in search of a potential Hsp70 coinducer,
we investigated whether the compound leucinostatin could enhance Hsp70 expression in stressed RPE cells. Canine
RPE cells were isolated and cultured successfully. Purity of cells that strongly expressed RPE65 was over 90%. Arsenite-induced
stress led to a time- and dose-dependent increase in Hsp70 expression in canine RPE cells in vitro. In addition, leucinostatin,
which enhanced heat shock factor-1-induced transcription from the heat shock promoter in DNAJB1-luc-O23 reporter cell line,
also enhanced Hsp70 expression in arsenite-stressed RPE cells, in a dose-dependent fashion. These findings demonstrate that
leucinostatin can boost Hsp70 expression in canine RPE cells, most likely by activating heat shock factor-1, suggesting that
leucinostatin might be applied as a new co-inducer for Hsp70 expression.
