Abstract
Purpose:
Control of proteolytic events inside the RPE cell as well as in its immediate extracellular environment is important in the maintenance of retinal homeostasis. The purpose of this study was to therefore investigate the expression and secretion of specific cysteine proteases in RPE cells.
Methods:
Protein expression was analysed by Western blotting of cell lysates and conditioned media of confluent ARPE-19 and D407 cell cultures. Immunoreactivity for cathepsin B, L and S with appropriate intra/extracellular loading controls was assessed. Quantification was carried out using densitometry analysis and data was normalised to loading controls/total protein content. In addition, transcriptional level of cathepsins was quantified by RNA sequencing in human foetal RPE cells.
Results:
Both pro and active forms of cysteine proteases cathepsin B, L and S were detected in both cell lysates of ARPE-19 and D407 cell lines. Although expression levels of these cathepsins were slightly different in the two cell lines, the active form for all cathepsins was the predominant form in all cases. Results were corroborated by high expression levels of cathepsin specific transcripts in human foetal RPE cells. The most abundantly expressed cysteine protease was cathepsin B followed by L and then S. Active secretion of pro and active forms of all these cathepsins was evident in all RPE cells except for cathepsin B which was detected only in its active form.
Conclusions:
Cysteine proteases cathepsin B, L and S are expressed and secreted at high level by RPE cells indicating a likely extracellular role. These cysteine proteases are expected molecular targets of one of the most abundantly expressed proteins by the RPE, the cysteine proteinase inhibitor cystatin C, a variant of which is associated with AMD pathogenesis. Altered RPE expression of these cathepsins and/or cystatin C could lead to a proteolytic imbalance potentially contributing to pathogenic structural and functional extracellular changes.
Keywords: 701 retinal pigment epithelium •
662 proteolysis •
519 extracellular matrix